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Gonadotropic factors affecting the onset of reproductive maturity in the female fur seal : Callorhinus… Aman, Allison Craig 1972

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I5W  GONADOTROPIC FACTORS AFFECTING THE ONSET OF REPRODUCTIVE MATURITY IN THE FEMALE FUR SEAL, CALLORHINUS URSINUS  by  ALLISON CRAIG AMAN B.Sc, McGill University, 1959 M,Sc., University of British Columbia, 1966  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Zoology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December 1 9 7 2  In presenting this thesis in partial  fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives.  It  is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of  Z . O ( % - 0 & \  The University of B r i t i s h Columbia Vancouver 8, Canada  Date  ABSTRACT  The investigation was designed to determine, by means of different i a l adenohypophysial c e l l counts of gonadotropins and bioassay of adenohypophysial gonadotropins, the sequence of gonadotropic events which affect reproductive maturity in the female fur seal, Callorhinus ursinus. Previous investigation (Craig 1963, 1966) has established that ovarian follicular cycles are initiated in March by 2- and 3-year old immature females;  the cycle is annovulatory, and maintained until September, when  the ovaries become quiescent. Four-year-old females initiate a follicular cycle in March which culminates in ovulation and reproductive maturity in late August. Five cells were identified morphologically and histochemically in the pars anterior. proteins;  Two were serous c e l l s , with granules composed of simple  by comparison and analogy to cells similarly identified in the  adenohypophyses of other mammals, these cells are considered to be the somatotrop and the luteotrop. posed of mucoproteins;  Three are mucoid c e l l s , with granules com-  by comparison and analogy to cells similarly iden-  tified in other mammalian adenohypophyses, these cells are considered to be the thyrotrop, the folliculotrop, and the interstitiotrop. Differential c e l l counts were made of folliculotrops and interstitiotrops in the pars anteriors of immature, early proestrous, and estrous female seals.  The data obtained suggest minimal gonadotropic hormone re-  lease among immature females, extensive release of gonadotropic hormones at early proestrus in preparation for estrus, and the further release of ii  ovulatory amounts of F o l l i c l e Stimulating Hormone (FSH) and Luteinizing Hormone (LH) at estrus. Adenohypophyses from 2- and 3-year-old immature females and estrous females were assayed for quantification of F o l l i c l e Stimulating Hormone (FSH) and Luteinizing Hormone (LH), using female fur seal pups 6-8 weeks old as assay animals for FSH, and immature females 2 and 3 years old, as well as pups, as assay animals for LH. The endpoint measured for FSH quantification was ovarian antral f o l l i c l e s 1.0-2.0 mm in diameter. The concentration and content of adenohypophysial FSH among immature females is approximately double that of estrous females;  the evidence is commen-  surate with the results of the differential adenohypophysial c e l l counts of gonadotrops, and suggests minimal gonadotropin release among immature females, and the release of ovulatory amounts of FSH among estrous females. The bioassays for LH were only partly successful, but demonstrated the ovulatory capabilities of 2- and 3-year-old immature females, and the presence of ovulatory amounts of LH in the adenohypophyses of 2- and 3year-old immature females. Adenohypophyses from 2- and 3-year-old immature females, early proestrous females, late proestrcus females, and estrous females were assayed for concentration.and content of FSH and LH, using intact, immature female rats as assay animals. Adenohypophysial FSH content decreases from a peak 0.8 mg FSH among 2-year-old females to 0.5 mg FSH among 3-year-old females, 0.4 mg FSH among early proestrous females, 0.35 mg FSH among late proestrous females, and 0.18 mg FSH among estrous females;  FSH concen-  tration similarly decreases, except among early proestrous females, where a iii  relatively high concentration suggests FSH synthesis at a rate greater than rate of release.  Adenohypophysial LH content is highest among 2-year-old  immature females, 34 y g , and decreases to 19 jig among 3-year-old immature females, and to 10 jig among early proestrous females;  LH content is  increased to 28 ,ug among late proestrous females, and decreases to 10 jig among estrous females. pattern.  Adenohypophysial LH concentration follows the same  The evidence suggests minimal release of FSH and LH among 2- and  3-year-old females, sufficient to initiate the annovulatory ovarian f o l l i c ular cycle in March and support i t to its conclusion in September; the evidence of differential adenohypophysial c e l l counts and assay of adenohypophysial FSH in fur seal pups supports this conclusion. Increased release of FSH and LH at early proestrous probably initiates the ovarian ovulatory follicular development and estrogen synthesis characteristic of late proestrus and estrus.  The apparent storage of LH at late proestrous is prob-  ably necessary for the ovulatory release at estrus. Recent investigation has established that in the female rat, reproductive maturity is dependent on the differential maturation of 2 areas of the hypothalamus. The "hypophysiotropic area" differentiates f i r s t , and controls, through the synthesis and release of neurohormonal gonadotropic releasing factors, the release of adenohypophysial FSH and LH; the hypophysiotropic area is responsive to negative estrogen feedback.  The anterior  hypothalamus becomes functional after the hypophysiotropic area, just prior to ovulatory maturity, and is responsive to positive estrogen feedback for the release of ovulatory amounts of FSH and LH.  By analogy, a sequence of  hypothalamic events similar to that described for the rat is inferred as the determining factor for the onset of reproductive maturity in the female fur seal. iv  TABLE OF CONTENTS  Page I. II.  III.  INTRODUCTION  1  THE MORPHOLOGY OF THE HYPOPHYSIS AND THE CYTOLOGY OF THE PARS ANTERIOR  4  A. Introduction . . . . . . . . . .  4  B. Material and methods  5  C. Morphology of the hypophysis  8  D. Histology and cytology of the hypophysis  15  1. Nomenclature 2. Pars tuberajis . 3; Zona tuberalis 4. Pars intermedia 5. Pars anterior E. Comparison of tropic cells identified in the pars anterior of the fur seal to cells similarly identified in the adenohypophyses of other mammals  15 16 16 21 22 31  CHANGES IN MUCOID CELLS OF THE FUR SEAL PARS ANTERIOR IN RELATION TO REPRODUCTIVE CONDITION A. Introduction  34 . . . . . .  34  B. Material and methods .  36  C. Changes in mucoid cells of the pars anterior  38  1. Cell types counted 2. Immature females 3. Females in early proestrus 4. Females in estrus D. Discussion  38 38 41 42 43  IV. BIOASSAY OF FOLLICLE STIMULATING HORMONE AND LUTEINIZING HORMONE IN THE ADENOHYPOPHYSES OF FEMALE FUR SEALS, USING FEMALE FUR SEAL PUPS AS ASSAY ANIMALS A. Introduct ion  47 47  v.  Page B. Material and methods  49  1. The collection, treatment, and classification of hypophyses from female fur seals . . . . . . . . . . . 2. Reproductive classification of donor females 3. Maintenance of female fur seal pups 4. Bioassay for F o l l i c l e Stimulating Hormone a. First year b. Second year . 5. Bioassay for Luteinizing Hormone a. First year b. Second year . . . . . . . . . C. Response of female fur seal pups to exogenous Follicle Stimulating Hormone 1. First year a. Saline controls b. Human Chorionic Gonadotropin controls c. Standardizing F o l l i c l e Stimulating Hormone . . . . d. F o l l i c l e Stimulating Hormone activity of adenohypophyses from immature female fur seals e. F o l l i c l e Stimulating Hormone activity of adenohypophyses from newly ovulated females f. Quantification of F o l l i c l e Stimulating Hormone . .  63 63 63 63 63 69 77 78  2. Second year a. Controls . . . . . b. Standardizing F o l l i c l e Stimulating Hormone . . . . c. F o l l i c l e Stimulating Hormone activity of adenohypophyses from 2- and 3-year-old immature female fur seals d. F o l l i c l e Stimulating Hormone activity of adenohypophyses from estrous females preparing to ovulate for the first time e. Quantification of F o l l i c l e Stimulating Hormone . .  86 91  3. Discussion  93  D. Response of female fur seal pups and immature female fur seals to exogenous Luteinizing Hormone 1. First year 2. Second year 3. Discuss ion  82 83 83 85  100 100 100 103  V. BIOASSAY OF FOLLICLE STIMULATING HORMONE AND LUTEINIZING HORMONE IN THE ADENOHYPOPHYSES OF FEMALE FUR SEALS, USING IMMATURE RATS AS ASSAY ANIMALS A.  49 51 54 55 56 58 59 60 61  Introduction  105 105  B. Material and methods  106 vi.  Page 1. The collection and classification of hypophyses from female fur seals 2. The treatment of hypophyses from female seals 3. Bioassay for F o l l i c l e Stimulating Hormone 4. Bioassay for Luteinizing Hormone a. Basis for the assay b. Method of the assay  106 Ill Ill 112 112 114  C. Adenohypophysial content of f o l l i c l e stimulating Hormone and Luteinizing Hormone  116  1. Two-year-old immature females 2. Three-year-old immature females 3. Females in early proestrus 4. Females in late proestrus 5. Females in estrus  116 116 117 132 132  D. Discussion  134  1. Two-year-old immature females . . 2. Three-year-old immature females 3. Females in early proestrus 4. Females in late proestrus 5. Females in estrus VI.  135 136 . 137 138 139  DISCUSSIONS AND CONCLUSIONS A. Introduction  . ,  141 141  B. Evidence for the dual hypothalamic control of adenohypophysial gonadotropins in the adult cyclic rat . . . . . .  142  C. Cyclic adenohypophysial gonadotropin release in the adult female rat .  145  D. Cyclic adenohypophysial gonadotropin release in the adult ewe, pig, and cow  148  E. Dual hypothalamic control of the onset of reproductive maturity in the female rat  150  F. Gonadotropic factors affecting the onset of reproductive maturity in the female pig, cow, and sheep  156  G. Gonadotropic factors affecting the onset of reproductive maturity in the female fur seal  157  1. Pups 2. Two-year-old immature females . . vii.  158 . . . . . 159  Page 3. Three-year-old immature females 4. Females in early proestrus, late proestrus, and estrus VII.  . 162 163  SUMMARY  167  LITERATURE CITED  171  viii.  LIST OF TABLES  Table 1.  2. 3.  Page Gross and histological analysis of reproductive tracts associated with hypophyses with which morphological and cytological studies were made.  6  Size of proteinaceous and mucoproteinaceous c e l l s , u in longest diameter, * standard error.  27  The average number of granulated, partly granulated, and degranulated folliculotrops and interstitiotrops counted in any cross section, ± standard error.  39a 39b  4.  Relative total percentages of mucoproteinaceous cells  5.  Gross and histological analyses of reproductive tracts associated with hypophyses collected on St. Paul Island, Alaska, for bioassay in female fur seal pups.  52  Response of the reproductive tracts of fur seal pups to Human Chorionic Gonadotropin (HCG) and F o l l i c l e Stimulating Hormone (FSH).  64  Response of the reproductive tracts of fur seal pups to adenohypophyses from 2-year-old immature female fur seals. First bioassay series.  70  Responses of the reproductive tracts of female fur seal pups to adenohypophyses from 4- and 5-year-old ovulated females. First bioassay series.  74  Concentration and content of adenohypophysial F o l l i c l e Stimulating Hormone, determined in the f i r s t bioassay series, using fur seal pups as assay animals. The end point measured is the number of antral ovarian f o l l i c l e s 1.0-2.0 mm in diameter.  79  Bioassay for FSH of adenohypophyses from 2-year-old immature female fur seals. The end point measured is the number of antral ovarian f o l l i c l e s 1.0-2.0 mm in diameter in the ovaries of recipient fur seal pups.  80  Bioassay for FSH of adenohypophyses from 4- and 5-year-old ovulated female fur seals. The end point measured is the number of antral ovarian f o l l i c l e s 1.0-2.0 mm in diameter.  81  6.  7.  8.  9.  10.  11.  ix.  Table 12.  13.  14.  15.  16.  17. 18.  19.  20.  21.  22.  Page Response of the reproductive tracts of fur seal pups to Human Chorionic Gonadotropin (HCG), F o l l i c l e Stimulating Hormone (FSH), and adenohypophyses from immature females and females i n estrus. Second bioassay series.  84  Concentration and content of adenohypophysial F o l l i c l e Stimulating Hormone, determined in the second bioassay series using fur seal pups as assay animals. The end point measured is the number of antral ovarian f o l l i c l e s 1.0-2.0 mm in diameter.  87  Bioassay for FSH of adenohypophyses from 2- and 3-year-old immature female fur seals. The end point measured is the number of antral ovarian f o l l i c l e s 1.0-2.0 mm in diameter in the ovaries of recipient fur seal pups.  88  Bioassay for FSH of adenohypophyses from estrous female fur seals. The end point measured is the number of antral f o l l icles 1.0-2.0 mm in diameter in the ovaries of recipient fur seal pups.  89  Comparison of FSH potency in adenohypophyses from immature and estrous female fur seals. The end point measured is the number of antral f o l l i c l e s 1.0-2.0 mm in diameter in the ovaries of recipient fur seal pups.  90  Response of the reproductive tracts of fur seal pups to Pregnant Mare Serum (PMS). Second bioassay series.  99  Response of the reproductive tracts of 2-, 3-, and 4-year-old immature female fur seals to Pregnant Mare Serum (PMS), Luteinizing Hormone (LH), and adenohypophyses from estrous females, early proestrous females, and 2- and 4-year-old immature females. Second bioassay series.  101  Gross and histological analyses of reproductive tracts associated with hypophyses collected on St. Paul Island, Alaska, for bioassay in immature, intact female rats.  107  Gross and histological analyses of reproductive tracts from mature females taken 24 hours post-partum or immediately post-coitem, on St. Paul Island, Alaska.  108  Bioassay for FSH of adenohypophyses from 2-year-old immature female fur seals. The assay animal is the immature rat; the end point measured is ovarian weight.  118  Bioassay for FSH of adenohypophyses from 3-year-old immature female fur seals. The assay animal is the immature female rat; the end point measured is ovarian weight.  119  x.  Table  Page  23. Bioassay for FSH of adenohypophyses from early proestrous female fur seals. The assay animal is the immature female rat; the end point measured is ovarian weight.  2 20  24. Bioassay for FSH of adenohypophyses from late proestrous female fur seals. The assay animal is the immature female rat; the end point measured is ovarian weight.  121  25. Bioassay for FSH of adenohypophyses from estrous female fur seals. The assay animal is the immature female rat; the end point measured is ovarian weight.  122  26. Concentration and content of adenohypophysial F o l l i c l e Stimulating Hormone, quantified in bioassay, using intact, immature female rats as bioassay animals.  123  27. Bioassay for LH of adenohypophyses from 2-year-old immature female fur seals. The assay animal is the immature female rat; the end point measured is number of ovulations.  125  28. Bioassay for LH of adenohypophyses from 3-year-old immature female fur seals. The assay animal is the immature female rat; the end point measured is the number of ovulations. 126 29. Bioassay for LH of adenohypophyses from early proestrous female fur seals. The assay animal is the immature female rat; the end point measured is the number of ovulations.  127  30. Bioassay for LH of adenohypophyses from late proestrous female fur seals. The assay animal is the immature female rat; the end point measured is the number of ovulations.  128  31. Bioassay for LH of adenohypophyses from estrous female fur seals. The assay animal is the immature female rat; the end point measured is the number of ovulations.  129  32. Concentration and content of Luteinizing Hormone, quantified in bioassay, using intact, immature female rats as assay animals.  130  x i .  LIST OF FIGURES Figure 1.  2. 3. 4.  5. 6.  Page  A. The diagram illustrates the morphology of the female fur seal hypophysis. B. The diagram illustrates the planes of coronal hypophysial sectioning.  9  The diagram illustrates the zonation of serous and mucoid cells in the pars anterior of the female fur seal.  10  The diagram illustrates the method used to count thyrotrops and gonadotrops in the pars anterior of the female fur seal.  35  The percentage of folliculotrops and interstitiotrops granulated, partly granulated, and degranulated among immature, early proestrous, and estrous female fur seals.  40  The graph illustrates adenohypophysial FSH content and concentration.  124  The graph illustrates adenohypophysial LH concentration and content.  131  xii.  LIST OF PLATES Plate I.  Page A saggital section of the hypophysis from a female fur seal.  11  II.  A corortal section of the hypophysis from a female fur seal.  12  III.  A. Interstitiotrops stained with aniline blue. B. Folliculotrops stained with aniline blue. C. A folliculotrop, 4 interstitiotrops, and a somatotrop. D. A degranulated folliculotrop, surrounded by degranulated interstitiotrops. E. Five thyrotrops. F. Two thyrotrops surrounded by fully granulated interstitiotrops. G. SomatDbcps and a luteotrop. H. Four somatotrops and a luteotrop.  17  A. The typical f o l l i c u l a r structure found in the pars tuberalis. B. The cells of the pars tuberalis are separated by connective tissue septae. C. Pars tuberalis tissue forming large f o l l i c l e s on the ventral aspect of the pars anterior.  18  IV.  V.  A. The residual lumen between the pars anterior and the pars intermedia is marked by colloid deposits. B. The pars intermedia, although contiguous with the pars nervosa, is separated from i t by connective tissue elements. C. The granulation of the pars intermedia c e l l is flocculant and indistinct. D. To show the relationship of the zona tuberalis, pars anter9 i o r , and pars tuberalis. *  VI.  A. The zona tuberalis, near the pars nervosa, showing the conjunction of the zona tuberalis and pars intermedia. B. A patch of the large, coarsely granulated cells of the zona tuberalis. C. Two large, coarsely granulated cells of the zona tuberalis, showing the peripheral position of the nucleus and the central 20 position of the Golgi apparatus.  VII.  A. Three thyrotrops to illustrate the irregular polygon shape of the c e l l , the peripheral nucleus, and the central Golgi apparatus. B. Three partly granulated thyrotrops. C. To illustrate the peripheral distribution and grouping of the thyrotrops.  xiii.  2 4  Plate VIII.  Page A. Fully granulated, partly granulated, and degranulated folliculotrops. B. A fully granulated folliculotrop. C. Fully granulated interstitiotrops. D. Partly granulated interstitiotrops.  IX. A. B. C. X.  XI.  XII.  XIII.  XIV.  XV.  Partly granulated and degranulated folliculotrops. Partly granulated and degranulated folliculotrops. A very large nuclear inclusion.  25  26  A. The ovary from a fur seal pup not given exogenous gonadotropins. B. The uterine endometrium from a fur seal pup not given exogenous gonadotropins. C. The glandular and rugal mucosa from (B). D. The glandular and rugal mucosa from a fur seal pup given HCG.  65  A. The ovary from a fur seal pup which received HCG and 1.0 mg FSH. B. The associated uterine endometrium from the fur seal pup of (A). C. Rugal and glandular mucosa from the fur seal pup of (A). D. The rugal and glandular epithelia of a fur seal pup receiving 0.5 mg FSH. •  66  A. 2.0 B. C.  The ovary from a fur seal pup which received HCG and mg FSH. The antral f o l l i c l e s are greatly enlarged. The glandular and rugal mucosa from the pup of (a). The uterine endometrium from the pup of (A).  67  A. 3.0 B. C.  The ovary from a fur seal pup which received HCG and adenohypophyses from immature females. The uterine endometrium from the pup of (A). The rugal epithelium from the pup of (A).  71  A. The endometrial rugal epithelium from a fur seal pup which received HCG and 1.0 adenohypophysis from a 2-yearold immature female. B. The endometrial rugal and glandular epithelia from a fur seal pup which received HCG and 4.0 adenohypophyses from 2-year-old immature females. C. The glandular epithelium from a fur seal pup which received 5.0 adenohypophyses from 2-year-old immature females.  72  A. The ovary from a fur seal pup which received 1.5 adenohypophyses from ovulated females. B. The endometrial and glandular epithelia from the pup of (A). C. The uterine endometrium from the pup of (A).  75  xiv.  Plate XVI.  Page A. The ovary from a fur seal pup which received 2.5 adenohypophyses from ovulated females. B. The uterine endometrium from the pup of (A.)C. The glandular and rugal epithelia from the pup of (A.).  xv.  76  ACKNOWLEDGEMENTS  I am indebted to the patience, tact, and moral and financial support of my supervisor, Dr. H. Dean Fisher, during the extended course of this degree. The collection of material, and f a c i l i t i e s on St. Paul Island, Alaska, were made available by the National Marine Fisheries Service, Marine Mammal Biological Laboratory, with the authorization and continuous cooperation of Mr. Ford Wilke, Director, Division of Marine Mammals (retired).  I am particularly indebted to Dr. Mark Keyes, DVM, of the  Marine Mammal Biological Laboratory, for his support, suggestions and help during this study. The Fisheries Research Board of Canada supported this investigation with financial grants. The F.R.B.C. Biological Station, Nanaimo, British Columbia, was generous in the use of its f a c i l i t i e s ;  the Marine  Mammal Investigation provided material which would not otherwise have been available, with the cooperation of Mr. Ian B. MacAskie. The moral and financial support of my husband during the last four years has made the completion of this investigation possible.  A. C. A.  xv i .  I. INTRODUCTION  There are 2 breeding populations of northern fur seal, Callorhinus ursinus, the Pribilof Islands population of the eastern Pacific, and the Robben and Commander Islands populations of the western Pacific.  Females  of the Robben and Commander Islands attain reproductive maturity and ovulate for the f i r s t time at the end of their third year;  females of the  Pribilof Islands attain reproductive maturity and ovulate for the f i r s t time at the end of their fourth year (Craig 1966). The disparity in the age of reproductive maturity between the famales of the 2 populations is apparently based on a physiological delay of one year in the maturation of the endocrine system controlling reproduction among Pribilof Island females (Craig 1966). The present investigation was designed to determine the sequence of gonadotropic events which affect reproductive maturity among Pribilof females, for the eventual clarification of the physiological disparity in the age of reproductive maturity between the 2 breeding populations of northern fur seals.  Previous investigation (Craig 1963,1966) has established  that ovarian follicular cycles are initiated in March by 2- and 3-yearold immature females of Pribilof origin;  the cycle is annovulatory, and  maintained until September, when the ovaries become quiescent.  Four-year-  old females initiate a f o l l i c u l a r cycle in March which culminates in ovulation and reproductive maturity in late August. The pattern of gonadotropic  (Follicle Stimulating Hormone and Lut-  einizing Hormone) hormone synthesis which affects reproductive maturity has been closely defined in some species of female laboratory animals, notably  2  the rat (Ramirez and Sawyer 1965; Ramirez and Sawyer 1966; Campbell and Gallardo 1966; Corbin and Daniels 1967; Lawton and Sawyer 1968). Less definitive gonadotropic patterns affecting reproductive maturity have been described for the female pig (Hollandbeck et al 1956), cow (Hansel 1959; Howe et al 1964; Jainudeen et al 1966), and sheep (Robinson 1959; Lamond 1962). There has been l i t t l e definitive investigation of reproductive maturity among wild animals. The most precise definition of reproductive maturity is based on neurohormonal mechanisms, the hypothalamo-hypophysial-gonad axis.  Recent  investigation has established that in the female rat, reproductive maturity is dependent on the differential maturation of 2 areas in the hypothalamus. The "hypophysiotropic"area differentiates f i r s t (Flerko et al 1969; Presl et al 1970), and controls, through the synthesis and release of neurohormonal gonadotropic releasing factors, the release of adenohypophysial gonadotropins for ovarian antral follicular development;  the  hypophysiotropic area is responsive to negative.estrogen feedback (Ramirez and McCann 1962; Ramirez and Sawyer 1967). The anterior hypothalamus becomes functional after the hypophysiotropic area, just prior to ovulatory maturity (Presl et al 1970) and is responsive to positive estrogen feedback (Labhsetwar 1970 a and b) for the release of ovulatory amounts of gonadotropins (Goldman and Mahesh 1968, 1969; Everett 1956; Markee et al 1952). No such definitive investigation has been made for any other mammals, but by analogy with gonadotropic hormone patterns, a similar sequence of hypothalamic events may be inferred for the female pig (Hollandbeck et al 1956), cow (Hansel 1959), and sheep (Robinson 1959). In the present investigation, adenohypophysial content and concentration of the gonadotropic hormones, F o l l i c l e Stimulating Hormone and  3  Luteinizing Hormone, have been determined with bioassay and differential adenohypophysial c e l l counts among 2- and 3-year-old immature female fur seals, and among 4-year-old females in early proestrus, late proestrus, and estrus.  By analogy, a sequence of hypothalamic events, similar to  that described for the rat, is inferred, as the determining factor for the onset of reproductive maturity in the female northern fur seal, Callorhinus urs inus.  4  II.  THE MORPHOLOGY OF THE HYPOPHYSIS AND THE CYTOLOGY OF THE PARS ANTERIOR OF THE FEMALE FUR SEAL, CALLORHINUS URSINUS  A.  Introduction  Investigation of the pinniped hypophysis has been limited.  The mor-  phology and cytology of the hypophysis and supraopticohypophysial tract, and the pars anterior of the Common seal (Phoca vitulina) are described by Amoroso et al (1965). The gross morphology of the hypophysis of the North Pacific fur seal (C. ursinus), the South American sea lion (Otaria byronica). and the walrus (Odobenus rosmarus) is briefly described by Hanstrom (1966). Racadot (in Anderson 1969) describes the adenohypophysial cytology of the Weddel seal (Leptochynotes weddeli). In the present study, a complete investigation of the morphology of the hypophysis and the cytology of the pars anterior of the female fur seal (C. ursinus) has been made. In conjunction with the physiological evaluation of adenohypophysial gonadotropic hormone content of immature and maturing females, the emphasis of the cytological examination has been placed on glands from these females.. In recent years, a number of papers reviewing hypophysial morphology and cytology have been published. The most comprehensive are those of Purves (1961, 1966) and Herlant (1964). Purves deals exhaustively with comparative hypophysial morphology and adenohypophysial cytology. Herlant emphasizes special adenohypophysial cytology, particularly the tinctorial affinities and histochemical reactions of mammalian adenohypophysial c e l l s .  Further review, except where i t is applicable to the pres-  ent investigation, would be superfluous.  5  B.  Material and Methods  Hypophyses collected for examination are listed in Table 1, with the reproductive analyses of the females from which the glands were taken. The females were taken during a commercial k i l l on St. Paul Island, Alaska, during August, at the peak of the ovulatory period for females ovulating for the f i r s t time (Craig 1966), and included 2-year-old immature females, 4-year-old early proestrous females, and 4-year-old estrous females. Hypophyses were removed whole from the sella turcica as soon as possible, in no case more than 30 minutes after death. Formol sublimate, buffered with sodium acetate, was used for fixation, to insure irreversible precipitation of the mucoid hormones (Barnett et al 1956). Glands were fixed whole for 48 hours and stored in 707o isopropyl alcohol until the material was paraffin embedded;  an optimum thickness of 6 u was used  for serial sectioning of a l l glands. Two glands were sagitally sectioned for anatomical studies;  the remainder were sectioned in the coronal  plane to facilitate differential c e l l counting. One stain and 3 histochemical reactions were used in a standard routine: Crossmon's (1937) modification of Mallory's trichrome stain; the periodic acid-Schiff-orange G (PAS-OG) reaction for the demonstration of mucoproteins (Pearse 1950);  the oxidative potassium permanganate alde-  hyde thionin-periodic acid-Schiff-orange G (AT-PAS-OG) reaction for the simultaneous demonstration of cystine-and mucoprotein-containing granules (Paget 1959; Paget and Eccleston 1960); and the oxidative performic acid alcian blue-periodic acid-Schiff-orange G (A1B-PAS-0G) reaction for the simultaneous demonstration of cystein-and mucoprotein-containing granules (Adams and Swettenham 1958). A l l 4 procedures were used in each gland.  TABLE 1. GROSS AND HISTOLOGICAL ANALYSIS OF REPRODUCTIVE TRACTS ASSOCIATED WITH HYPOPHYSES WITH WHICH MORPHOLOGICAL AND CYTOLOGICAL STUDIES WERE MADE  Number of hypophyses examined  Ovarian weight Gms/ovary  Immature  2*  4.38  53.0  33.5  9  4.25  10.9  9.5  Early proestrus  4 (2)*  5.18  50.9  35.5  9  4.75  18.3  13.9  Estrus  4 (2)*  5.0  40.9  15.8  3.3  33.0  18.1  Reproductive condition  Numbers of antral f o l l i c l e s /ovary Largest 1mm 1-2+mm 2.5-3mm (mm diam)  11.0  Mucosal epithelial cell height u Rugal Glandular  * 2 hypophyses were used for differential adenohypophisial cell counts.  7  The AT-PAS-OG reaction demonstrated 6 c e l l types in the hypophysis, and was used for 75-807o of a l l sections; made from these sections.  quantitative measurements were  The A1B-PAS-0G reaction was used as a paral-  lel check on AT-PAs-OG sections;  Crossmon's trichrome stain and the  PAS-OG reaction were used for morphological studies in saggital sections and in ventral, medial and dorsal areas of coronally sectioned glands. Individual cells were measured in the longest diameter with a visible nucleus;  consequently, the measurements are biased on the  large size, but are standardized and comparable.  8  C. Morphology of the Hypophysis  The neural component of the hypophysis of the female fur seal consists of a neural or median eminence, and a well-marked neural stalk continuous with the pars nervosa (Plate I.A., F i g . I.A.).  The pars tuber-  alis encloses the median eminence and neural stalk, extending internally to the base of the pars nervosa, where i t is sharply demarcated from the pars intermedia (Plate I.A., F i g . I.A.);  externally, the pars tuberalis  covers the median eminence and extends in a sheet on the ventro-lateral surface of the pars anterior (Plate I.B., F i g . I.A.). The pars intermedia surrounds the pars nervosa, and is contiguous with, but separated from i t by connective tissue elements. Penetration of intermedia tissue into the pars nervosa reinforces the contiguity of the 2 elements (Fig. I.A., Plate I I . ) . The pars anterior also encloses the pars nervosa;  a thin layer  of adenohypophysial tissue is continuous on the ventral hypophysial surface (Plate I.B., F i g . I.A.). The residual lumen of Rathke's pouch is partially obliterated;  pars anterior and pars intermedia are contiguous  at points, but separated by vascular and connective tissue elements. The position of the cleft is marked by colloid deposits (Plate V.A.). The pars anterior separates mechanically from the rest of the hypophysis along the position of the c l e f t . The zonation of the pars anterior is illustrated in Figure l.B, 2. A zone contains a predominant c e l l type or types, but the c e l l type may not be exclusive to the zone. Zonation in the pars anterior of the female fur seal, is particularly well marked;  since the definition is  9  FIGURE 1  A.  The diagram illustrates the morphology of the female fur seal hypophysis.  B.  The diagram illustrates the plane of coronel hypophysial sectioning,  i  '  FIGURE 1  10  FIGURE 2  The diagram illustrates the zonatibn of serous and mucoid cells in the pars anterior of the female fur seal.  FIGURE:  %  11  PLATE I  A.  A saggital section of the hypophysis from a female fur seal, showing the median eminence (ME), neural stalk (NS), pars nervosa (PN), pars intermedia (PI), pars anterior (PA) and pars tuberalis (PT). Note the distinct separation of pars tuberalis and pars intermedia (1) and the area of the zona tuberalis (2). The pars tuberalis extends onto the ventral aspect of the pars anterior (3). chrome stain).  B.  (Crossmon's t r i -  Mag. x 25.  A saggital section of the hypophysis of a female fur seal similar to that in A;  the section is more lateral, and does not include the  neural stalk.  Note that the pars anterior extends to cover the  neural stalk, and to surround the pars nervosa. penetrates into the pars neuralis (4). Mag. x 25.  The pars intermedia  (Crossmon's trichrome stain).  PLATE  I  PLATE II  A coronal section of the hypophysis from a female fur seal, showing the pars nervosa (PN), pars intermedia (PI), pars anterior (PA), zona tuberalis (ZT), pars tuberalis (PT), neural stalk (NS), and median eminence (ME).  Mag. x 50.  ME  Z T  NS  PN  PLATE  n  13  made histochemically the zones are termed "mucoid" to describe those areas containing cells whose granules are mucoproteinaceous, and "serous" to describe those areas containing cells whose granules are simple proteins. the serous zones predominate the pars anterior, extending laterally through the pars anterior, and at the midpoint extending into the most anterior tissue surrounding the pars nervosa. medial and peripheral.  The mucoid zones are  At the most ventral aspect, the mucoid zone pre-  dominates medially, and extends around the lateral surface. At the midpoint of the pars anterior, the mucoid zones are lateral and anterior. The zona tuberalis is weli marked, following the line of the pars tuberalis and extending laterally into the pars anterior.  Similar c e l l zonat-  ion is described in the adenohypophysis of the Weddel seal (Leptochynotes weddelli) by Racadot et al (in Anderson 1969). The fur seal hypophysis resembles that of the common seal, Phoca vitulina (Amoroso et al 1965). As . in the former species, pars anterior and pars intermedia surround the pars nervosa, and the position of the cleft is marked by colloid;  the pars tuberalis encloses the neural stalk  and median eminence. There are 2 morphological differences:  the neural  stalk of the fur seal hypophysis is long in comparison to that of the common seal;  the pars tuberalis extends in a sheet on the exterior pos-  tero-ventral surface of the pars anterior of the fur seal hypophysis, and does not on the hypophysis of the common seal. Hanstrom (1966) briefly describes the gross hypophysial morphology of the South American sea lion (Otaria byronia) and the walrus (Odobenus rosmarus):  the hypophysial cleft is narrow but distinct in both species;  the pars tuberalis is collar-shaped and surrounds the median eminence and  i  '  '  .  14  neural stalk.  The pars nervosa of the walrus hypophysis is surrounded  by both pars intermedia and pars anterior. Thus, four species of pinnipeds share a common hypophysial morphology.  The parts of the hypophysis are separate and distinct.  The  residual lumen is reduced but present, and may be marked by colloid deposits. stalk.  The pars tuberalis surrounds the median eminence and neural  The pars intermedia and pars anterior surround the pars nervosa.  15  D.  Histology and Cytology of the Hypophysis  1. Nomenclature Ideally, a nomenclature applied to the adenohypophysial cells of any species serves three purposes: observed; tion;  the identification of each c e l l type  the relation of specific c e l l types to their endocrine func-  and a means of comparing c e l l types of one species to functionally  homologous c e l l types of another species.  The hormone secreting cells  of the mammalian adenohypophysis have historically been named acidophils, basophils and chromophobes;  the nomenclature is based on the tinctorial  a f f i n i t y , or lack or affinity of the c e l l classes for acid and basic dyes.  The advent of histochemical methods identified 2 main classes of  chromophil cells:  those whose granules are composed of simple proteins,  the serous c e l l s , and those whose granules are composed of mucoproteins, the mucoid c e l l s . Further, biochemical investigation has identified 7 adenohypophysi a l hormones among mammals: growth or somatotropic hormone (STH); luteotropic or lactotropic hormone (LTH); adrenocorticotropic hormone (ACTH); thyrotropic or thyroid stimulating hormone (TSH);  f o l l i c l e stimulating  or folliculotropic hormone (FSH); luteinizing or interstitiotropic hormone (LH);  and melanocyte stimulating or melanotropic hormone (MSH).  Each hormone may be the product of a separate c e l l type:  in some mammals,  notably the rat, 7 morphologically distinct, hormone-secreting hypophysi a l cells have been identified;  correlation of physiological function  with changes in specific c e l l types add evidence for cellular specificity. Consequently Purves (1961, 1966), Herlant (1964) and Van Oordt (1965) ad-  16  vocate the use of a functional nomenclature for mammalian hypophysial cells;  each of 7 c e l l types, as i t is identified, would have the name  of its hormone, followed by the suffix -trop or -tropic. tification may be morphological or tinctorial;  Initial iden-  the validity of the iden-  tification must be established with correlative physiological evidence for endocrine function.  In either case, analagous comparisons between  species may be made. The system f u l f i l l s the qualifications for the ideal nomenclature, and has been used in the present investigation.  2. Pars tuberalis The cells of the pars tuberalis are grouped by connective tissue septae (Plate IV, B., D.). IV, A.);  Acinar formations are characteristic (Plate  the diameter of the acinae are variable, and the luminae are  f i l l e d with PAS+ve material.  The acinar formation is conspicuous in  tissue from the posterior ventral surface of the pars anterior, where tuberalis tissue has extended lateral and anterior;  occasionally colloid  cysts form which are macroscopically visible on the posterior surface of the pars anterior (Plate IV, C ) . The cells of the pars tuberalis are round or oval, and average 8.2 ^ i in diameter.  The nucleus varies in size and is usually vesicular  with a large nucleolus, and is peripheral or central in the c e l l (Plate IV, A.). The cells are agranular, negative to the histochemical procedures used, and pale bluish-grey following Crossmon's trichrome stain.  3. Zona tuberalis The zona tuberalis occupies an area adjacent to the pars tuberalis,  PLATE III  A.  Interstitiotrops  stained with  B. Folliculotrops  stained with  aniline blue in Crossmon's trichrome  aniline blue in Crossmon's t r i -  stain.  chrome stain.  A fully (F) and a partly  The cells are  (P) granulated, and a degranulated  partly granulated.  (D) c e l l are shown. Mag. x 1000  ation is coarser than that of  (oil).  the interstitiotrops. i  The granul-  Mag. x  1000 ( o i l ) .  C. A folliculotrop (blue), 4 inter-  D.  stitiotrops^ (pink) and a somatotrop  (blue) surrounded by degranul-  (yellow) after the oxy-AlB-PAS-OG  ated interstitiotrops (pink) af-  procedure. Mag. x 1000 ( o i l ) .  ter the oxy-AT-PAS-OG procedure.  i•  A degranulated folliculotrop  The gland was taken from an estrus female. Mag. x 1000 ( o i l ) .  E.  Five thyrotrops (blue) to show  F.  Two thyrotrops (blue) sur-  characteristic peripheral granular  rounded by fully granulated in-  clumping, and grouping of c e l l s , and  terstitiotrops (pink), to i l l u s -  the characteristic deep blue gran-  trate the distinctive granular  ular colour following the oxy-AT-  colour following the oxy-AT-PAS-  PAS-OG procedure. Mag. 1000 ( o i l ) .  OG procedure. Mag. x 1000 ( o i l ) .  G.  H. Four somatotrops (red) and a  Somatotrops (yellow) and a luteo-  trop (orange) to illustrate the dis-  luteotrop (magenta) to illustrate  tinctive granule colour following the  the distinctive granular colour  PAS-OG procedure. Mag. x 1000 ( o i l ) .  following Crossmon's trichrome stain.  Mag. x 1000 ( o i l ) .  18  A.  The typical follicular struc-  B.  The follicles (f) of the pars  ture found in the pars tuberalis;  tuberalis are separated by con-  agranular cells  nective tissue septae.  (a) surrounding  the lumen (1). Mag. x 1000  250.  (oil).  C.  Pars tuberalis tissue (PT) form-  ing large f o l l i c l e s on the ventral aspect of the pars anterior (PA). Mag. x 100.  Mag. x  PLftTE  IV  19  PLATE V.  A. The residual lumen (RL) between  B. The pars intermedia (PI),  the pars anterior (PA) and the pars  although contiguous with the  intermedia (PI) is marked by col-  pars nervosa (PN), is sepa-  loid deposits (c). Mag. x 250.  rated from i t by connective tissue (ct). Mag. x 250.  C. The granulation of the pars in-  D.  termedia c e l l is flocculent and  of the pars tuberalis (PT),  indistinct.  zona tuberalis (ZT), and pars  The cells may be part-  ly (P) or fully (F) granulated, or degranulated (D). Mag. x 1000 (oil).  To show the relationship  anterior (PA). Mag. x 100.  PLATE V  20  PLATE VI  A.  The zona tuberalis (ZT), showing  B.  A patch of the large, coarsely  the conjunction of the zona tuberal-  granulated cells (CG) of the zona  is and the pars intermedia (PI).  tuberalis.  PA: pars anterior;  partly granulated.  PN: pars ner-  The cells are fully or  vosa. Mag. x 100.  C. Two large, coarsely granulated cells of the zona tuberalis, showing the peripheral position of the nucleus (n) and the central position of the Golgi apparatus (g). Mag. x 1000 ( o i l ) .  Mag. x 450.  PLATE VI  21  extending laterally into the pars anterior, and anterior the length of the neural stalk (Plate I, A; F i g . I,A). The zone is characterized by pars tuberalis cells and mucoid cells of the pars anterior. type is localized in and restricted to the zona tuberalis.  One c e l l The c e l l has  an average diameter of 18.7 y., and is round or oval, with a distinct c e l l boundary;  i t is the largest c e l l type in the adenohypophysis. The nuc-  leus is large, vesicular, and peripheral in the c e l l . tus  The Golgi appara-  is large, circular and immediately below the nucleus (Plate VI, C).  The cytoplasmic granules are very large and distinct, homogenous in the cytoplasm, and are PAS+ve, AT+ve, AlB+ve, and dark blue after Crossmon's trichrome stain;  after the AT-PAS-OG or A1B-PAS-0G procedure, the gran-  ules are purple. The cells always contain identifiable granulation, varying from dense to sparse, and the cells tend to be grouped adjacent to blood vessels (Plate VI, B).  In relation to other mucoid, c e l l types  in the zona tuberalis and in the pars anterior, this c e l l type is not numerous.  4. Pars intermedia The pars intermedia c e l l is round, averaging 15.1p in diameter; the c e l l boundary is distinct;  The nucleus is vesicular and central;  the small, indistinct, Golgi apparatus is adjacent to the nucleus. The granules are indistinct, flocculant, and homogeneously distributed in the cytoplasm;  they are PAS+ve, AT+ve, AlBt-ve, and bluish-grey after  Crossmon's trichrome stain (Plate V,C).  Identifiable granules are a l -  ways present, but granulation varies from heavy to sparse. The junctions between pars intermedia, pars tuberalis, and pars anterior are distinct;  22  there is no mingling of c e l l types except at the zona tuberalis (Plate V, D.; VI, A.).  i  •  5. Pars anterior Five c e l l types have been identified in the pars anterior of the fur seal in this investigation. cells.  Two are serous c e l l s , three are mucoid  Each is distinct in morphology and distribution within the gland,  and in tinctorial and histochemical response. The f i r s t serous c e l l is round or oval, with a distinct c e l l i t averages 11.5 ji in diameter (Table 2). The fine granules  boundary;  are dense and refractile, and f i l l the cells; indistinct.  individual granules are  The granules stain red with acid fuchsin and bright yellow  with orange-G (Plate III, G and H).  There is no constant association  with blood vessels. The second serous c e l l is round or oval, with a c e l l boundary less distinct than that of the f i r s t serous c e l l type;  i t averages 13.1  in  diameter (Table 2). The nucleus is round and vesicular, and central in the c e l l .  The granules are large and tend to aggregate, with a homogen-  eous distribution in the cytoplasm;  individual granules are indistinct.  Granulation tends to be heavy. The granules are faintly PAS+ve, and stain faintly blue with aniline blue, but are AT-ve and Alb-ve.  The cells are  nevertheless serous, the granules retaining more orange-G than Schiff reagent or aniline blue, and in a f u l l AT-PAS-OG or A1B-PAS-0G procedure, the granules are orange (Plate III, G and H); following Crossmon's trichrome stain, the granules are magenta. The cells occur in groups, associated with blood vessels.  23  The f i r s t , or yellow serous c e l l is the predominant type, approximately 75% of the serous cells in the glands examined, and the most numerous of the pars anterior c e l l types. The second, or orange serous c e l l lis always present, but the relative number is dependent on the reproductive condition of the female. The f i r s t mucoid c e l l type is an angular polygon with an indistinct c e l l boundary (Plate VII, A.); i t averages 11.8 ji in diameter (Table 2).  The nucleus is irregular in shape, and peripheral in the c e l l . The  irregularly shaped Golgi apparatus is central in the c e l l .  The granules  are fine, and aggregate in peripheral clumps which outline the cells (Plate VII, C ) . Identifiable granules are always present; or fully granulated c e l l is rarely seen.  a sparsely  The granules stain dark blue  with aniline blue and are PAS+ve, but selectively retain thionin and Al!  cian blue in the AT-PAS-OG or A1B-PAS-0G procedures for a dark blue color (Plate III, E and F ) . The cells occur in small, loosely associated groups adjacent to. portal vessels.  The cells are concentrated in a peri-  pheral zone (Plate VII, B.), but occur in a l l the mucoid zones and occasionally in the lateral serous zones. The second mucoid c e l l is round or oval, averaging 16.4ji in diameter (Table 2);  the c e l l boundary is distinct.  The nucleus is large,  round or oval, and vesicular, with one or more large nucleoli; peripheral in the c e l l . (Plate VIII, B.).  i t is  The Golgi apparatus is central in the c e l l  The granules vary in size, and are homogeneously dis-  tributed in the cytoplasm (Plate IX, A.). The granules are lavender in color, with occasional large fuchsinophil granules after Crossmon's trichrome stain (Plate IX, B.), and a strong magenta after the PAS pro-  24  PLATE VII  A.  Three thyrotrops to illustrate  B.  Three partly granulated thy-  the irregular polygon shape of  rotrops to illustrate the char-  the c e l l (p), the peripheral nuc-  acteristic peripheral granular  leus (n), and the central Golgi  clumping.  apparatus (g). Mag. x 1000  C.  Mag. x 1000  (oil).  To illustrate the peripheral  distribution and grouping of the thyrotrops.  The cells occur in  small, loosely associated groups, on or near blood vessels, and are surrounded by serous cells (s). Mag. x 250.  (oil).  PLATE  vn  25  PLATE VIII  A.  Fully granulated ( f ) , partly  B.  A fully granulated f o l l i c -  granulated (p) and degranulated  ulotrop, illustrating the per-  (d) folliculotrops.  ipheral nucleus (n) and central  Mag. x 450.  Golgi apparatus (g). Mag. x 1000  C.  Three fully granulated (f)  interstitiotrops;  the nucleus  D.  (oil).  Six partly granulated (p)  interstitiotrops;  the nucleus  (n) is central in the c e l l .  (n) is central in the c e l l .  Mag. x 1000  Mag. x 1000  (oil).  (oil).  PLATE  VIII  26  PLATE IX  A.  Partly granulated (p) and degran-  ulated (d)'folliculotrops. 1000  (oil).  Mag. x  B.  Partly granulated f o l l i c -  ulotrops showing the characteristic fuchsinophil granules (g) following Crossmon's trichrome stain.  C.  Mag. x 1000  A very large inclusion (i) within  the nucleus of an interstitiotrop. The crennelated nuclei (nc) contain nucleoli (nl). Mag. x 1000  (oil).  (oil).  PL ATX IX  TABLE 2. SIZE OF PROTEINACEOUS AND MUCOPROTEINAGEOUS CELLS, u IN LONGEST DIAMETER, ^STANDARD ERROR  Somatotrop  ll.olo.l  Thyrotrop P<0.05 =  P<0.05 11.8±0.2 =  Interstitiotrop  12.0l0.3  Luteotrop P<0.05 < 13.Li0.3  Folliculotrop P<0.05 <  16.4±0.2  28  cedure.  Following the AT-PAS-OG or A1B-PAS-0G procedure, the granules  are purple, retaining Schiff reagent as well as aldehyde thionin and Alcian blue (Plate III, B, C and D).  Identifiable granules are usually  present. The cells occur in large groups, and although the cells are associated with blood vessels, the association is not as close as for the f i r s t mucoid c e l l type. Zonation of these cells is medial and posterior (Fig. 2). The third mucoid c e l l type is round or oval, averaging 12.0 ji in diameter (Table 2);  the c e l l boundary is indistinct.  The nucleus is  round or oval, and vesicular, with a prominent nucleolus, and peripheral in the c e l l (Plate VIII, C and D). flocculant;  The granules are fine, aggregate, and  they are homogeneously distributed in the cytoplasm. The  granules are dull blue after Crossmon's trichrome stain, pink following the PAS procedure, and pink following the AT-PAS-OG and AlB-PAS-OG procedures (Plate III, A, C and D).  Identifiable granulation is usually  present. The cells occur in large groups, with portal vessel association similar to that of the second mucoid c e l l type.  Zonation of the third  mucoid c e l l type is medial and anterior (Fig. 2). Inclusions are seen in the nuclei of a l l five c e l l types of the pars anterior;  the size of the inclusions vary from small to large  (Plate IX, C). The inclusion invariably has the color of the cytoplasm surrounding the nucleus;  i t may or may not be granulated.  In one in-  stance, a distinct Golgi apparatus was seen within the inclusion. The presence of nucleoli as well as inclusions within a nucleus suggests that the inclusions are not nucleoli and may be cellular invaginations into crennalated nuclei (Plate IX, C).  Similar inclusions are described in the  29  serous cells of the rabbit adenohypophysis (Foster et al 1965) and in the gonadotropic cells of the hamster adenohypophysis (Serber 1961).  30  Ei  Comparison of Tropic Cells Identified in the Pars Anterior of the Fur Seal to Cells Similarly Identified in the Adenohypophyses of Other Mammals  Purves (1966) notes that staining reactions of the specific granules of functionally homologous cells are variable.  Staining or histo-  chemical reactions which distinguish between the secretory products of different hypophysial cells in one vertebrate may give different results when applied to other species. Nevertheless, comparison between species allows tentative conclusions about the tropic nature of morphologically and cytologically identified c e l l s .  The present comparison is limited  to seven species in which morphological, tinctorial or histochemical identification of hypophysial cells has been confirmed with physiological evidence, and the 5 tropic cells identified in the pars anterior of the fur seal.  The analogies are made solely on the basis of similar-  ities in morphology and cytology, and are presumptive and tentative. In the adenohypophyses of rat (Knigge 1958), mouse (Barnes 1962), rabbit (Allanson et a l 1966), dog (Purves and Griesbach 1957), ferret (Holmes 1960, 1963), bat (Herlant 1964), and cow (Nyak et a l 1968), the somatotrop is a round or oval c e l l , compactly f i l l e d with fine granules which stain bright yellow with orange-G, and is zonated laterally in the adenohypophysis;  the f i r s t serous c e l l in the fur seal pars anterior is  exactly similar, and is identified as the somatotrop. The lactotrop or luteotrbp in a l l 7 species is also a round or oval, laterally distributed c e l l , whose relative number depends on sex and reproductive condition; the granules are larger than those of the somatotrop, and in the rat (Emmart et a l 1968), and mouse (Sano 1962), stain yellow following orange-  31  G, carminophil in the rabbit (Salazar 1963), and bat (Herlant 1964), fuchsinophil in the dog (Goldberg and Chaikoff 1952), and with slight PAS-positivity, orange in ferret (Holmes 1963) and cow (Nyak et a l 1968) following PAS-OG. The second serous c e l l in the fur seal pars anterior, identified as the luteotrop, is similar in morphology and distribution, with PAS pbsitivity which, following PAS-OG, produces orange coloration;  its relative number is also dependent on reproduct-  ive condition. The thyrotrop in a l l 7 species is an irregular polygon with distinct c e l l boundaries, containing PAS+ve mucoid granules which selectively stain dark red with aldehyde fuchsin, or dark blue with aldehyde thionin or alcian blue, in any oxidative procedure which includes PASOG. Thyrotrop granulation varies from coarse to fine;  the granules  are peripherally clumped in the rabbit (Foster 1963), dog (Goldberg and Chaikoff 1952) and cow (Jubb and McEntee 1955), and homogeneously distributed in the rat (Purves and Griesbach 1951), mouse (Barnes 1962), ferret (Holmes 1963) and bat (Siegal 1955). Similarly, the f i r s t mucoid c e l l of the fur seal pars anterior, identified as the thyrotrop, is an irregular polygon, containing peripherally clumped granules which stain selectively with aldehyde thionin or Alcian blue. The folliculotrop in a l l but the bovine adenohypophysis is a large, round or oval c e l l , medially distributed, containing coarse, homogeneously distributed, PAS+ve granulation;  the grouped cells are  usually in close association with portal vessels.  The granules are PAS+-  ve but AF-ve in the rat (Purves and Griesbach 1955), mouse (Barnes 1962) and rabbit (Cameron et a l 1966);  in the bat, slight AF positivity results  32  in purple coloration following the AF-PAS-OG procedure.  In dog (Purves  and Griesbach 1957) and ferret (Holmes 1963) the folliculotrop granules are slightly Alb+ve, and purple following the Alb-PAS-OG procedure. The second mucoid c e l l of the fur seal pars anterior, a round or oval c e l l whose homogeneously distributed coarse granules retain aldehyde thionin or Alcian blue as well as Schiff reagent for a purple coloration, is identified as the folliculotrop. In a l l but the bovine adenohypophysis, the interstitiotrop is smaller than the folliculotrop, with finer granulation, round or oval, and medially distributed in groups.  In the rat (Purves and Griesbach  1955) mouse (Barnes 1962) and rabbit (Cameron et al 1966), the mucoid granules are PAS+ve, with no affinity for aldehyde fuchsin, aldehyde thionin or Alcian blue.  In dog (Goldberg and Chaikoff 1952; Purves and  Griesbach 1957), ferret (Holmes 1963), and bat (Herlant 1964), the granules are similarly PAS ve and negative to aldehyde  fuchsin, aldehyde  thionin and Alcian blue, but retain some orange-G, and are brick red following the PAS-OG procedure.  The third mucoid c e l l type of the fur  seal pars anterior, smaller than the folliculotrop, with fine granulation responsive only to PAS, and medially grouped in association with portal vessels in the gland, is identified as the interstitiotrop. Racadot et a l (in Anderson 1969) have described 6 c e l l types in the pars anteriores of 2 lactating Weddel seals (Leptochynotes weddeli). The cells identified as somatotrops, folliculotrops and thyrotrops are similar in size, morphology and histochemical response as those similarly identified in the pars anterior of the northern fur seal.  Two orangeophil  cells are identified as "epsilon" and "eta" c e l l s , possibly synthesizing  33  adrenocorticotropic  and lactotropic hormone respectively;  similar in morphology and tinctorial response;  the cells are  both resemble the c e l l  identified in the fur seal pars anterior as the luteotrop.  The "gamma" i  c e l l of the Weddell seal pars anterior, which tentatively synthesizes LH, resembles the interstitiotrop of the fur seal pars anterior. No analogy may be made to the distinctive c e l l of the zona tuberalis in the fur seal pars anterior;  the temptation is to identify i t by  elimination as the corticotrop, but its morphology is so dissimilar to the finely granulated stellate cells identified as corticotrops in the adenohypophyses of rat (Purves 1966) or to the finely granulated, round corticotrop of the dog (Purves and Griesbach 1957) that this comparison is precluded. No c e l l similar to the corticotrops described has been seen in the pars anterior of the fur seal.  34  III.  CHANGES IN MUCOID CELLS OF THE FUR SEAL PARS ANTERIOR IN RELATION TO THE REPRODUCTIVE CONDITION OF IMMATURE, MATURING, AND OVULATORY FEMALES  A. Introduction  Craig (1966) has shown that among 2- and 3-year-old immature female fur seals (C. ursinus) annovulatory ovarian follicular cycles are initiated in March, reach a peak in August, and decline in September; among 2-year-olds, the associated uterine mucosae are minimally active throughout the cycle, but endometrial development begins in June among 3-year-olds. The follicular cycle, and associated uterine growth, culminates in the f i r s t ovulation during August among 4-year-old females, and reproductive maturity is established. For the purposes of the present investigation, three of the mucoid Cells of the pars anterior were examined in relation to reproductive condition, to establish which cells were gonadotropic, and the pattern of gonadotropic events resulting in each reproductive condition. A l l the females were taken in August, at the peak of the ovarian f o l l i c ular cycle.  Three reproductive conditions were examined:  annovulatory 2-year-old females; proestrus, prior to ovulation;  immature,  maturing 4-year-old females in early and ovulatory 4-year-old females, in  estrus and with ovulation imminent.  35  i  '  FIGURE 3  The diagram illustrates the method used to count thyrotrops and gonadotrops in the pars anterior of the female fur seal.  FIGURE 3  36  B.  Material and Methods  The hypophyses from which differential c e l l counts were made are listed in Table 1, with the reproductive analyses of the females from which the glands were taken, and are the same as those from which hypophysial c e l l types were identified.  Collection and treatment of the  glands are described in "Material and Methods" of Section I I . Since the AT-PAS-OG procedure clearly differentiated the 5 c e l l types of the pars anterior, a l l c e l l counts were made from these sections. The method used to count 3 mucoid c e l l types is an adaptation of the method developed by Montemurro (1964) for the rat adenohypophysis. The c e l l types observed in the fur seal pars anterior have a marked zonation, ventral to dorsal, and anterior to posterior (Fig. 2). A crossline grid was imposed over each section from which counts were to be made; counts were made along the length of each crossline, using a rectangular format ocular to delimit the area within which cells were counted at 400x magnification, and to prevent overlapping of counted areas (Fig. 3). Counts were made on coronal sections of the pars anterior, beginning at the most ventral aspect, to the level of the pars nervosa (Fig. 2). Each mucoid c e l l type within the format was classified as fully granulated, partly granulated, or degranulated, and counted.  To make a positive identification, some granulation had to be  observed, and no completely degranulated cells were counted;  further,  the arbitrary classification necessitates subjective judgement, so that the statistics used in analysis are not completely unbiased.  The same  c e l l count was made in each section of any gland, so that c e l l counts  37  between glands could be considered standardized and comparable, and analyses of variance and orthogonal contrasts were used to test for significant differences and differences between means respectively, for c e l l counts within and between, glands.  Results of differential c e l l counts  are summarized in Tables 3 and 4 and Figure 4.  i i  38  C.  1.  Changes in Mucoid Cells of the Pars Anterior in Relation to Reproductive Condition  Cell types counted  Of the five c e l l types described in the pars anterior of the fur seal, the 2 serous cells were eliminated from consideration as gonadotrops affecting the reproductive conditions examined. Granulation of the somatrop did not vary in relation to reproductive condition.  The  luteotrop is conspicuously scarce in the pars anterior of the immature female;  numbers are increased in the pars anterior of the early pro-  estrous female, but are low in relation to other c e l l s , and granulation is typically partial.  Only in estrous (and ovulated) females is the  luteotrop present in appreciable numbers, and granulation is partial. Granulation of the large mucoid c e l l restricted to the zona tuberalis did not vary in relation to reproductive condition. The mucoid cells identified as thyrotrop, folliculotrop, and interstitiotrop remained in consideration, and were classified and counted.  2.  Immature females  The ovaries of the immature 2-year-old females contained numerous small, healthy antral f o l l i c l e s , the largest less than 5 mm in diameter. The associated uterine mucosae were inactive, with l i t t l e glandular coiling, fibrocytic connective tissue stroma, and cuboidal glandular and rugal epithelia (Table 1). Granulation in the thyrotrop did not vary; were partially granulated;  most of the cells  only rarely were degranulated or fully gran-  ulated cells seen. Granulation in the presumptive folliculotrops varied  TABLE 3. TOE AVERAGE NUMBER OF GRANULATED, PARTLY GRANULATED, AND DEGRANULATED FOLLICULOTROPS AND INTERSTITIOTROPS COUNTED IN ANY CROSS SECTION,! STANDARD ERROR  Folliculotrops  P<0.05 <  P<0.05  1. Fully granulated  0.04±0.05  2. Partly granulated  3.7±0.2  3.  0.4±0.04  0.4±0.6  1=3; 2 1,3  13; 1 2 3  Degranulated  Significant differences (P 0.05)  Interstitiotrops 1. Fully granulated  2 . 4±0. 2  2. Partly granulated  5.1±0.2  3.  2.1±0.1  Degranulated  Significant differences (P 0.05)  1=3; 1 2 3  Estrus  Early Proestrus  Immature  >  > > <  0.8±0.08  >  1. zio.i  1.5±0.2  0.6±0.1 4. L±0. 3  0. i±o. 01  <  1.4±0.1  0 . 8±0.1  >  3.0i0.2  6.9±0.3  5.0l0.2  12 3  12 3  TABLE 4. RELATIVE TOTAL PERCENTAGES OF MUCOPROTEINACEOUS CELLS  Thyrotrops  Folliculotrops  InterstitIotrops  Number  Percent  Number  Percent  Number  Percent  Immature  4320  36.69  2133  12. 59  4521  41.53  10974  Early proestrus  3581  47. 55  747  9. 91  3203  42.53  7531  18266  52.63  4473  12.89  11962  34.47  34701  Estrus  Total Cells  53206  w to  40  FIGURE4 THE PERCENTAGE OF FOLUCULOTROPS AND INTERSTITIOTROPS GRANULATED, PARTLY GRANULATED, AND DEGRANULATED AMONG IMMATURE, EARLY PROESTROUS,AND ESTROUS FEMALES.  FOLLICULOTROPS  100 . o r  80l  I-  V) 3  o r  Set  '0  40_  t  I< Z z  20FULLY GRANULATED  3  3  UJQ. h-  0>  PARTLY GRANULATED  INTERSTITIOTROPS  4535-  0/  UJ  cC 3  n  5 i  '0 25-  r  55-  s  UJ  . * I  3  CC  15 _ 5  Z  FULLY GRANULATED  DEGRANULATED  •TRUS  0/ eol  Ok 3  S  J X  (A UJ  2 3  <3 to Ul  PARTLY DEGRANULATED GRANULATED  41  significantly among the 3 classifications (Table 3);  9.57o of the cells  counted were fully granulated, 82.47» were partly granulated, and 8.07, were fully granulated (Fig. 4).  Granulation in the presumptive inter-  stitiotrop also varied significantly (Table 3);  24.67P were fully gran-  ulated, 53.57o were partly granulated, and 21.87» were degranulated (Fig. 4).  The thyrotrops comprised 36.77» of the total cells counted, the  folliculotrops 19.67., and the interstitiotrops 41.57., (Table 4).  3.  Early proestrous females  The ovaries of the maturing females contained healthy Graafian f o l l i c l e s , similar in number and size to those in the ovaries of immature females.  The associated uterine mucosae were estrogenic, with  glandular coiling, fibroblastic and edematous connective tissue stroma, and low columnar glandular and rugal epithelia (Table 1).  Both females  were pre-ovulatory, in the earliest stages of proestrus. Granulation of the thyrotrop was the same as that found in immature females.  Granulation in the presumptive folliculotrop varied  significantly (Table 3); 29.7% of the cells were fully granulated, 56.07» partly granulated, and 14.17., degranulated  (Fig. 4).  Of the in-  terstitiotrops, 5.17o were fully granulated, 32.57.. were partly granulated, and 59.67., were degranulated (Figure 4);  the differences were  significant (Table 3). The thyrotrop comprised 47.67« of the total cells counted, the folliculotrop 9.97o, and the interstitiotrop 42.57o (Table 4).  42  4.  Estrous females  The ovaries of the ovulatory females contained fewer Graafian f o l l i c l e s than those of immature or maturing females, and most were atretic; istic.  a single, large, healthy ovulatory f o l l i c l e was characterThe mucosae were estrogenic;  the connective tissue stroma was  fibroblastic and edematous, glandular coiling marked, and glandular and rugal epithelia were high columnar, (Table 1).  Both females were in  estrus. Granulation of the thyrotrop did not vary.  Of the presumptive  folliculotrops, 18.170 were fully granulated, 53.77» were partly granulated, and 44.57o were degranulated  (Fig. 4);  the differences were sig-  nificant (Table 3).  Granulation in the interstitiotrop varied signif-  icantly (Table 3);  5.77o were fully granulated, 37.37o were partly gran-  ulated, and 57.07o were degranulated  (Fig. 4 ) .  The thyrotrop comprised 56.27o of the total cells counted, the folliculotrop 12.97o, and the interstitiotrop 34.57» (Table 4).  1  43  D.  Discussion  The release of adenohypophysial FSH results in ovarian f o l l i c ular growth;  the secretion of follicular f l u i d , mitotic proliferation  of granulosa c e l l s , and the development of thecal layers are the main actions of this gonadotropic hormone (Gamzell and Roos 1966). On the background of FSH activity, the release of adenohypophysial LH promotes f u l l maturation of ovarian f o l l i c l e s and thecal tissue, and the secretion of estrogen. Following a sudden release or "surge" of LH, ovulatory f o l l i c l e s are stimulated to rupture (Harris and Campbell 1966);  recent evidence (Goldman and Mahesh 1968, 1969) indicates that  the ovulatory surge of LH is immediately preceded by a similar precipitous release of FSH.  Thus, the patterns of secretion and the actions  of FSH and LH f a l l into 2 groups:  secretion of LH in conjunction with  FSH in a relatively basal, steady pattern;  and a sudden release of  FSH and LH which is responsible for ovulation. Observable adenohypophysial cellular granulation represents  the  difference between rate of cellular synthesis of tropic hormone, and rate of release, at the time the gland is taken from the animal. A fully granulated c e l l represents a rate of synthesis greater than rate of release, and storage.  A partly granulated c e l l represents more syn-  thesis than release of tropic hormones. A degranulated c e l l represents a rate of release equal to or greater than rate of synthesis. More cells partially granulated than degranulated or fully granulated suggests a steady, but relatively low level, of release, such as the secretion of LH and FSH in a basal pattern for the maintenance of ovarian f o l -  44  l i c l e s and steroidogenesis.  A preponderance of degranulated cells sug-  gests a greater rate of release than synthesis, as in the massive release of FSH and LH for ovulation.  Large numbers of fully granulated  cells may represent a reserve supply of the tropic hormone. The ovaries of the immature 2-year-old females contain healthy, antral f o l l i c l e s ;  the associated uterine mucosa is quiescent, indicat-  ing a failure of adequate ovarian estrogen secretion. Speculatively, FSH sufficient for ovarian follicular development is released from the pars anterior during the ovarian follicular cycle (March-August); the amount of LH released is insufficient for follicular maturation and estrogen synthesis.  The evidence of differential c e l l counts from the  pars anterior of the immature female confirms the speculation.  Of the  folliculotrops, more are partly granulated than in either of the other reproductive conditions, and fewer are fully granulated or degranulated, indicating a steady release of FSH at a rate less than the rate of synthesis.  More of the interstitiotrops are fully or partly granulated  than in either of the other reproductive conditions, and fewer are degranulated, suggesting a minimum release of LH. The ovarian follicular development of the maturing, early proestrous females is similar in size and numbers of antral f o l l i c l e s to that of the immature females, but the uterine mucosa is estrogenic, indicating f u l l maturation of, and estrogen secretion by, the ovarian follicles.  Speculatively, the same amount, or more FSH is released  during the follicular cycle, relative to adenohypophysial FSH release of the immature female, and more LH is released.  The evidence of dif-  ferential c e l l counts tends to confirm the speculation, but may be more  45  representative of the proestrus condition than of cyclic gonadotropin release.  There is a dramatic change from the immature condition of  interstitiotrop granulation: are degranulated;  more than half of a l l the cells counted  the pronounced degranulation was not noticeable in  counting, and did not appear as such until analyses were made, suggesting a continuous rate of LH release equal to synthesis.  More f o l l i c u -  lotrops are degranulated, and fewer partly granulated than in the immature condition;  more folliculotrops are fully granulated than in  either of the other reproductive conditions.  The degranulated and  partly granulated folliculotrops suggest a release of FSH which is equal to that of the immature female;  the fully granulated  folliculo-  trops suggest FSH storage, possibly preparatory for late proestrus and estrus release. The interstitiotrop degranulation, suggestive of continuous LH release, is commensurate with ovarian steroidogenesis obvious since June in the estrogenic development of uterine mucosae, and approaching maximum development. The ovaries of estrous females are characterized by a single ovulatory f o l l i c l e , decreased numbers of smaller antral f o l l i c l e s , and marked atresia of a l l but the ovulatory f o l l i c l e ; rium is markedly estrogenic.  the uterine endomet-  There is a dramatic increase in the number  of degranulated folliculotrops, far greater than in either of the other reproductive conditions, and a decrease in the number of fully granulated folliculotrops in comparison to the proestrus condition;  the  evidence suggests a sudden accelerated release of FSH from the early proestrus storage condition. The granular condition of the interstitiotrops is similar to that of the proestrus condition, but the pattern of release is different, and obvious on examination:  large patches of  46  interstitiotrops were degranulated, and the cells appear shrunken, suggesting a sudden, accelerated release of LH.  The pattern of folliculo-  trop and interstitiotrop degranulation suggests the sudden, accelerated ! j release of FSH and LH necessary for ovulation. The pattern of secretory events evidenced by granular variations in the presumptive gonadotropic cells tends to confirm the analagous identification of folliculotrops and interstitiotrops by tinctorial and morphological c r i t e r i a .  The use of differential c e l l counts as  direct evidence for patterns of tropic hormone release is at best limited:  the method is of necessity biased by subjective judgement;  complete evidence is unavailable, since a l l cells are not counted; and the results are interpolative, based on an educated guess at an exact physiological condition.  In the present investigation, evidential in-  terpretation is further hampered by an incomplete sequence of reproductive events from early proestrus to estrus.  Further physiological  evidence for adenohypophysial patterns of gonadotropic release w i l l be presented which confirms the identification of the gonadotrops; the results of differential c e l l counts are supportive to the physiological evidence.  47  IV.  BIOSSAY OF FOLLICLE STIMULATING HORMONE (FSH) AND LUTEINIZING HORMONE (LH) IN THE ADENOHYPOPHYSES OF FEMALE FUR SEALS, USING FEMALE FUR SEAL PUPS AS ASSAY ANIMALS.  A.  Introduction  Differential analyses of adenohypophysial c e l l counts suggests variations in tropic hormone levels; ible.  no quantitative analysis is poss-  Accurate quantification of adenohypophysial tropic hormones is  possible only with bioassay, either by direct assay of the gland, or, most accurately, by assay for blood levels of tropic hormones. Parlow (1964) states that the concentration or content of tropic hormone stored in the adenohypophysis is a guide for the evaluation of the status of hormone secretion.  He further notes the desirability of differential assay  for adenohypophysial gonadotropins:  a non-specific assay, such as the  uterine weight method in intact, immature female rats, does not differentiate between FSH and LH, since uterine responses induced by exogenous gonadotropins could be due to either or both hormones. In a standard assay, using white rats as recipient animals, a known quantity of a purified tropic hormone from a given species is used to establish a base response;  an unknown quantity of tropic hormone from  another species produces a response which is measured in terms of the base response:  2 tropic hormones from 2 different species are quantified in.a  third species.  Parlow and Reichert (1963), in testing species differences  in FSH from porcine, ovine, human, rat and equine sources, found a qualitative difference between porcine FSH and FSH from the other species, based on the Steelman-Pohley assay for FSH.  This biological difference suggests  48  that the quantification of a tropic hormone from one species, using another species as the recipient assay animal, may  limit quantification to  an estimate of the activity of the hormone in the recipient animal. i To avoid such sources of Inaccuracy, bioassays for FSH and LH content in the adenohypophyses of immature, early proestrus, estrus and ovulated female fur seals were made, using female fur seal pups as recipient assay animals.  Using a known quantity of gonadotropic hormone from  a given species to establish a base response, the unknown quantities of gonadotropic hormones from female fur seals could be measured in the usual manner. In addition, the results of the bioassays could be analysed in terms of the donor females:  quantification of more than one end  point, duplication of reproductive conditions, and in general, more information than would result from the usual bioassay. The ovaries of fur seal pups (4-6 weeks of age) are characterized by numerous antral f o l l i c l e s less than 1 mm in diameter (Craig 1966). Since ovarian follicular capability is present, 2 assays dependent on this capacity in immature rats were modified for use with fur seal pups. Assays performed in 1964 (first year) were of necessity more exploratory than those of 1965 (second year), since fur seal pups had not been previously used as assay animals; ing, the former qualifying.  the latter could be considered quantify-  49  B.  1.  Material and Methods  The collection, treatment, and classification of hypophyses  from female fur seals. A l l donor females were taken during the annual 2-3 day commercial k i l l of immature females in August on St. Paul Island, Alaska. The k i l l occurs within the interval during which females ovulating for the f i r s t time do so;  this interval includes the peak of the annovulatory f o l l i c -  ular cycle of immature females (Craig, 1966).  Thus a l l females, regard-  less of reproductive classification, were taken at the height of gonadotropic activity. Reproductive tracts were examined briefly on the k i l l i n g field to ensure that the females were immature, maturing, ovulatory or recently ovulated, with no previous pregnancies. The reproductive tracts were removed from these females, tagged with a number, and fixed and stored in 107o buffered formalin for subsequent reproductive analysis;  a canine  tooth was removed from each female for annular ring aging. Hypophyses were collected whole as quickly as possible, usually within 15 minutes of death.  On the k i l l i n g f i e l d , a gland was placed in  a plastic bag with the same number as its associated reproductive tract and canine tooth;  the bags were transported to a -45 degree C freezer in  a bucket of ice water. The glands were shipped to Vancouver in dry ice, then stored at -45 degrees C for varying times.  On completion of reprod-  uctive analyses and aging, the hypophyses were classified by age and reproductive condition. The greatest concern was to maintain adenohypophysial gonadotropic potency over extended time and distance.  Acetone drying and storage has  50  been used for the preservation of tropic hormones, but the method has been shown to significantly reduce LH potency (Barnett et al 1956; Desjardins et al 1966).  Desjard ins et a l (1966) established that bovine  adenohypophysial potency of FSH and LH is not significantly affected by freezing and storage at -20 degrees C and -79 degrees C, followed by lyophilization (freeze-drying) or by alternate freezing and thawing f o l lowed by lyophilization.  F a c i l i t i e s for lyophilization were not avail-  able on St. Paul Island.  Since arrangement for low temperature transport  and storage could be made, i n i t i a l low-temperature freezing and subsequent lyophilization were used as the best available method to maintain gonadotropic potency. Lyophilized adenohypophyses were transported back to St. Paul Island for assay in recipient fur seal pups in 1964 and 1965. Glands collected in 1963 for assay in 1964 (first year) were treated singly before completion of reproductive analyses. Hypophyses were thawed from -45 degrees C by standing in cold water;  the pars an-  terior of each gland was then detached from the neural component and pars intermedia.  Each pars anterior was homogenized individually in 2 cc cold  d i s t i l l e d water, using a variable speed lab motor, and a glass-embedded Teflon stirring rod in a stainless steel tube sunk in a beaker of ice water.  The homogenate was pipetted into a 4 cc capacity glass bulb and  lyophilized.  The bulbs were heat sealed at the completion of lyophiliz-  ation, so the dried homogenates, maintained in vacuum, could be stored at room temperature. Glands collected in 1964 for assay in 1965 (second year) were treated in groups of 4 according to reproductive condition, after the completion of reproductive analyses. Four glands were homogenized in 8 cc  51  cold d i s t i l l e d water, using the Teflon stirring rod;  the homogenate was  pipetted into a 15 cc capacity glass bulb and lyophilized, the bulbs heat sealed at the completion of lyophilization, and the homogenates stored at room temperature.  2.  Reproductive classification of donor females.  Gross and histological analyses of reproductive tracts associated with hypophyses collected for bioassay in fur seal pups are given in Table 5.  I n i t i a l classification was made by age.  Each reproductive  tract was then examined for ovarian weight, number and size of ovarian Graafian f o l l i c l e s , number of endometrial glands in a histological cross section (an index of glandular growth and coiling), and the cellular height of rugal and glandular epithelia;  the latter 2 indices are indic-  ators for ovarian estrogen synthesis and release. Final reproductive classification, and distribution of associated hypophyses was made on the basis of statistical analysis for mean and standard error within each reproductive classification.  Analysis of variance and orthogonal  contrasts were used to establish significant differences between means, between reproductive classifications. Immature females 2 and 3 years of age have small ovaries, each containing an average 24 Graafian f o l l i c l e s , the largest 2.6-4.2 mm in diameter. Glandular coiling is minimum, averaging 31.3 in any cross section of endometrium examined; cuboidal.  the rugal and glandular epithelia are  The analysis indicates an annovulatory f o l l i c u l a r cycle,  characterized by minimal ovarian estrogen synthesis and release. Females 3-4 years of age, in early proestrus, are those females  TABLE 5. GROSS AND HISTOLOGICAL ANALYSES OF REPRODUCTIVE TRACTS ASSOCIATED WITH HYPOPHYSES COLLECTED ON ST. PAUL ISLAND, ALASKA, FOR BIOASSAY IN FEMALE FUR SEAL PUPS  Number of females Ovarian weight, average gms/ovary Number of antral foll i c l e s , 1-3+mm diameter, average/ovary Largest antral foll i c l e , mm diameter  Early Immature Immature Estrus(2) proestrus 2- and 3- year 2-year-old (1 and 2) old females(2) females(1)* P<0.( )5*** P<;0.05 P<C).05 33 33 30 23 2.7-0.21  2.li0.13  23.7±2.3  2.6±0.51  21.3±2.7  <  4.2±0.24  <;  <'  <  4.9i0.24  38.2i6.9  6.0i0.34  Number of uterine glands (index of glandular coiling)  31.3±8.7  Endometrial rugal l i n ing cell height, u  10.8i0.5  10.4^0.4  Endometrial gland cell height, u  8.2+0.3  9.3±0.4  Corpus Luteum Diameter, u  * Number in parentheses indicates hypophyses used in first or second bioassay series.  59.1±2.9  <[  107.4*8.1  <;  i5.9ii.o  r  12.9i0.8  <  =  5.ll:0.22  I>  27.-2±2.4  <\  9.9i0.4  <; 165.9*18.0  <'  [  Ovulated(1) 20  5.oi:o.i7 1.7+0.. 1  2.oio.i -  33.3±2.2  45.2+0.1  18.9+3..3  23.0i0.7  **_ standard error, *** Significant difference between means.  7.8±1.3  53  approaching the f i r s t ovulation. immature condition;  Ovarian weight is doubled from the  the average number of ovarian Graafian f o l l i c l e s ,  38.2, is significantly greater than that of the immature female, as is the size of the largest f o l l i c l e , averaging 6.0 mm in diameter.  Gland-  ular coiling is doubled from the immature condition, and the glandular and rugal epithelia are columnar, significantly higher than those of the immature female. Females 4-5 years of age and in estrus, are those females in a state of imminent ovulation for the f i r s t time.  Ovarian weight is not  greater than that of the early proestrous female, but the number of ovarian Graafian f o l l i c l e s is significantly decreased to an average 27.2, and most of these are atretic.  The largest f o l l i c l e averages 9.9 mm in  diameter, and is ovulatory. The number of uterine glands is significantly greater than that of the maturing female;  the glandular and rugal  epithelia are high columnar and significantly higher than those of the maturing female. Ovulated females are those females which have ovulated for the f i r s t time. The ovaries are characterized by a small, new corpus luteum, and a drastic reduction of Graafian f o l l i c l e s , averaging 1.7 in each ovary, approximately 2.0 mm in diameter.  Glandular coiling is massive,  and the glandular and rugal epithelia are very high columnar; dometrium is typically progesteronic.  The en-  Because these females had ovul-  ated, and the predominant steroid influence was progesterone, the classification was not included in the analyses of variance, which were intended to classify females which had not ovulated.  54  3.  Maintenance and treatment of female fur seal pups used as  recipient bioassay animals. Female pups, 4-6 weeks of age and weighing 5-8 kg were collected from rookeries.  Animals which had recently fed, recognizable by their  distended stomachs, were chosen. The pups were caged in pairs' in numbered wire cages;  they were additionally tagged on one fore flipper  with a numbered metal tag, and a patch of fur was shaved from the heads of odd-numbered pups.  No feeding or watering was necessary: a recently  fed pup does not normally nurse again for 4-5 days, and experimental animals were held no longer than 4 days. A daily fresh water hosing kept pups, cages and floor clean;  the pups were held in a well-ventil-  ated, unheated, and unlit garage. The pups were calm, sleeping or resting most of the time;  there was l i t t l e activity in the cages. Forcible  handling was necessary during injection periods.  This was more damaging  to the handlers than to the pups, which subsided immediately restraints were released;  nevertheless, handling time was minimum, never more than  2 minutes per pup.  In a l l the bioassays, there was only one mortality,  the result of a localized septicemia from a misplaced metal tag which pierced a blood vessel in the fore flipper to which i t was attached. At autopsy, the pups were in good condition, with milk solids remaining in the stomachs. Assay injections in 1964 (first year) were given intrathorically, into the pleural cavity.  The injection route was suggested by Keyes  (personal communication), whose work with fur seal pups indicated this method provided an absorption rate intermediate between those of intramuscular and intravenous routes;  subcutaneous injections were considered  55  inadvisable because of the absorptive layer of fat beneath the skin. A patch of fur at the middle rib was shaved to mark the injection site; the rib was palpated, and as the pup exhaled, the needle was inserted just below the r i b , aimed lateral and anterior, and the injection made. At autopsy, no diaphragm, lung, heart or stomach punctures were found, and there were no lesions or adhesions at the injection sites.  Aseptic con-  ditions were maintained to the extent of disinfecting the injection site with hexachlorophene (Phisohex) and 707o ethanol, and using a disposable sterile syringe and needle for each injection. Several injection routes were used in the series of assays performed in 1965 (second year). scribed.  The intrathoracic route was used as de-  Intramuscular injections were made into the gastrocnemius muscle  of the hind flipper; hind flipper;  intravenous injections were made into a vein of the  and subcutaneous injections were made below the shaved  skin of the back at shoulder level. Aseptic conditions were maintained as described.  4.  Bioassay for F o l l i c l e Stimulating Hormone (FSH)  The Steelman-Pohley assay for FSH (Steelman and Pohley 1955) is based on the fact that mammalian ovarian response to exogenous FSH is augmented by the concurrent administration of Human Chorionic Gonadotropin (HCG), which mimics the effect of LH, and acts synergistically with FSH for the development of mature ovarian f o l l i c l e s .  The assay is commonly  used, with intact immature female rats as recipient animals, and ovarian weight as the end point. fur seals.  The assay was adapted for use in intact female  There are no precedents for its use in large mammals.  56  (a).  First year  Human Chorionic Gonadotropin, "Antuitrin" brand, was supplied by Parke-Davis. F o l l i c l e Stimulating Hormone (NIH-FSH-P1 porcine) was generously donated by.the United States National Institutes of Health, Endocrinology Study Section. each set of injections.  Doses of HCG and FSH were made up fresh for  The adenohypophysial homogenates were pooled by  reproductive condition and weighed in total;  the weight of one adenohy-  pophysis was 15.0 mg adenohypophysial homogenate for 2-year-old immature females, and 15.0 mg for females which had ovulated for the f i r s t time, the 2 reproductive classifications tested in bioassay in the f i r s t year. The homogenate powder, released from vacuum, was kept in closed jars in a freezer at -45 degrees C; doses were weighed out daily for suspension in sterile physiological saline. Forty female fur seal pups were used as assay animals. Six control females received 1.0 ml sterile physiological saline intrathoracically twice a day (9:00 AM and 9:00 PM) for 3 days.  Six control pups re-  ceived a total 150 IU HCG each, given as 25 IU HCG in 1.0 ml saline twice a day for 3 days. ten pups were paired for 5 dose levels of FSH:  a total 150 IU  HCG and 0.5 mg FSH given to each of 2 pups as 25 IU HCG and 0.08 mg FSH in 1.0 ml saline twice a day for 3 days;  a total 150 IU HCG and 1.0 mg  FSH given to each of 2 pups as 25 IU HCG and 0.16 mg FSH in 1.0 ml saline twice a day for 3 days;  a total 150 IU HCG and 1.5 mg FSH given to  each of 2 pups as 25 IU HCG and 0.25 mg FSH in 1.0 ml saline twice a day for 3 days;  a total 150 IU HCG and 2.0 mg FSH given to each of 2 pups as  25 IU HCG and 0.33 mg FSH in 1.0 ml saline twice a day for 3 days;  and a  57  total 150 IU HCG and 3.0 mg FSH given to each of 2 pups as 25 IU HCG and 0.5 mg FSH in 1.0 ml saline twice a day for 3 days. Ten pups were paired for 5 dose levels of HCG and adenohypophysi a l homogenate, 1.0, 2.0, 3.0, and 4.0 adenohypophysial equivalents from 2-year-old immature females. Doses were given to pairs of animals in 1.0 or 2.0 ml sterile physiological saline twice a day for 3 days as 25 IU HCG and 2.5, 5.0, 7.5. 10.0 or 12.5 mg adenohypophysial homogenate for each injection. Eight pups were paired for 4 dose levels of HCG and adenohypophysi a l homogenate, 1.0, 1.5, 2.5, and 3.0 adenohypophysial equivalents from females which had ovulated for the f i r s t time. Doses were given to pairs of pups in 1.0 or 2.0 ml sterile physiological saline twice a day for 3 days as 25 IU HCG and 2.5, 3.75, 6.25, and 7.5 mg adenohypophysial homogenate for each injection. Autopsies were performed on half the pups 24 hours after the last injection; ion.  the remainder were autopsied 48 hours after the last inject-  A l l pups were killed by cranial fracture.  Ovaries and uteri were  trimmed of fat and connective tissue and weighed in gms.  A l l tissue was  fixed and stored in 10% buffered formalin for further analysis. Statistical analyses of results were i n i t i a l l y made of five end points to determine which would be suitable for quantification of gonadotropic potency. Analyses of variance and orthogonal contrasts were used for significant differences and differences between means. The regression analysis suggested by Burn (in Burn et al 1950), and used by Steelman and Pohley (1955), was used to quantify gonadotropic potency.  58  (b)  Second year  Human Chorionic Gonadotropin, "Antuitrin" brand, was supplied by Parke-Davis;  F o l l i c l e Stimulating Hormone (NTJH-FSH-Pl porcine) was  donated by the United States National Institutes of Health, Endocrinology Study Section. Doses of FSH and HCG were made up as described for the bioassays of the f i r s t year.  The adenohypophysial homogenates were  pooled by reproductive condition and weighed in total;  the equivalent  weight of one adenohypophysis was 21.2 mg adenohypophysial homogenate for 2- and 3-year old immature females and 22.2 mg for estrous females. Doses of HCG and adenohypophysial homogenate were made up as described for the bioassays of the f i r s t year. Thirty-two female pups were divided into 8 groups of 4 each. Two control groups received physiological saline or 150 IU HCG.  Two groups  received 2 dose levels of standardizing FSH, 1.0 and 2.0 mg, concurrently with 150 IU HCG.  Two groups received 2 dose levels of adenohypophys-  i a l homogenates, 2 and 4 adenohypophysial equivalents from 2- and 3year old immature females, concurrently with 150 IU HCG.  Two groups re-  ceived 2 dose levels of adenohypophysial homogenate, 2 and 4 adenohypophysial equivalents from estrous females. equal parts:  Total doses were given in 6  each injection, in 1.0 or 2.0 ml sterile physiological  saline, was given intramuscular twice a day (9:00 AM and 9:00 PM) for 3 days. Autopsies were performed 24 hours after the last injection; a l l pups were killed by cranial fracture.  Ovaries and uteri were trimmed of  fat and connective tissue and weighed in gms; a l l tissue was fixed and stored in 10% formalin for further analysis. the same as those made in the f i r s t year.  Statistical analyses were  59  5.  Bioassay for Luteinizing Hormone  The bioassay used to attempt quantification of adenohypophysial LH from immature and maturing, early proestrus fur seals was based on the bioassay for LH suggested by Zarrow et al (1959), superovulation in the  intact, immature rat.  Pregnant Mare Serum (PMS), which has the  physiological activity of FSH, is used to promote the growth of ovulatory  follicles;  subsequent doses of ovulating hormone result in ovulat-  ion, the number of ovulations dependent on the amount of ovulating hormone given. Superovulation in the beef cow (Hafez et al 1963; Rowson 1950) and in sheep is commonly induced in this manner, for the production of viable eggs prior to mating. Howe et al (1964) induced ovulation in prepuberal heifers 1-6 months of age with a single injection of PMS, f o l lowed by a single injection of HCG as the ovulating hormone. Jainudeen et al (1966) attempted to develop a reliable method for superovulation in the c a l f , using a similar method. Dose levels of PMS and LH used in recipient female fur seal pups aid 2- and 3-year-old immature females were derived from used in prepuberal heifers (Howe et al 1964) and in cattle (Hafez et a l 1963).  "Wellcome" brand Pregnant Mare  Wellcome Ltd.  Serum was supplied by Burroughs-  Luteinizing Hormone (NIH-LH-S5 ovine) was donated by the  United States National Institutes of Health Endocrinology Study Section. Adenohypophysial homogenates were pooled by reproductive condition and weighed in total immediately prior to administration;  individual doses  were weighed from totals for suspension in sterile physiological saline.  60  (a)  First year  Forty-two female fur seal pups were used. A l l experimental animals received 150 IU PMS, administered intrathoracically as 3 equal doses of 50 IU PMS in 1.0 ml sterile physiological saline on each of 3 successive days at 9:00 AM.  Ovulating hormone, either LH or adenohypo-  physial homogenate, was administered intrathoracically 24 hours after the last PMS injection, in total doses, suspended in sterile physiological saline.  One control groupjof 6 pups received physiological saline only,  on the same schedule;  one control group of 6 pups received 150 IU PMS,  with 1.0 ml saline replacing the ovulating dose;  and a third control  group of 6 pups received 150 IU PMS with 50 IU PMS in 1.0 ml saline replacing the ovulating dose. One group of 8 pups was paired;  4 dose levels of standardizing  LH, 1.0 mg, 1.5 mg, 2.0 mg and 3.0 mg were administered in 1.0 ml saline as ovulating doses. A second group of 8 pups were paired, and 4 dose levels, 1.0, 1.5, 2.5 and 3.5 adenohypophyses from 2-year-old immature females were used as ovulating doses;  the adenohypophysial equivalent  weight of homogenate was 23.3 mg, and each total dose was suspended in 1.0, 1.5, 2.5 or 3.5 ml physiological saline. A third group of 8 paired pups received 1.0, 1.5, 2.5 or 3.5 adenohypophyses from maturing, early proestrus females as the ovulating doses;  the adenohypophysial equiv-  alent weight of homogenate was 23.3 mg, and each total dose was suspended in 1.0, 1.5, 2.5 or 3.5 ml physiological saline. Half the animals were autopsied 24 hours after the administration of ovulating hormones;  half were autopsied 48 hours after the adminis-  tration of ovulating hormones. A l l pups were killed by cranial fracture.  61  Ovaries and uteri were trimmed of fat and connective tissue and weighed in gms; further  a l l tissue was fixed and stored in 10% buffered formalin for analysis. (b).  Second year  The effects of various doses of PMS and injection routes were examined. PMS was given in a single injection; 120 hours after the administration of PMS. in twelves;  autopsies were performed  Thirty-six pups were grouped  each pup of the 3 groups received 300, 500 or 700 IU PMS in  1.0 ml physiological saline.  Within a group of 12, 4 pups received in-  tramuscular injections, 4 received intrathoracic injections, and 4 received subcutaneous injections.  At autopsy, ovaries and uteri were  trimmed of fat and connective tissue and weighed in gms; fixed and stored in 10% buffered formalin for further  a l l tissue was  analysis.  A final experiment was designed to induce f o l l i c l e s of ovulatory size and condition with PMS in the ovaries of immature (2- and 3-year old) female fur seals, preparatory to using this age group in the assay of LH. Six cows, chosen for their small size, were taken from a commercial k i l l drive, and caged singly in numbered wire cages. A single injection was given intramuscular into the gastrocnemius muscle of the hind flipper.  Two cows were used as controls, receiving 1.0 ml saline;  2 cows received 700 IU PMS in 1.0 ml physiological saline, and 2 cows received 1000 IU PMS in i.O ml saline. after the administration of PMS.  Autopsies were performed 96 hours  F o l l i c l e s of ovulatory size and condit-  ion developed in the ovaries of cows at both dose levels, suggesting the practicability of a bioassay.  62  Twenty-two immature females 2-4 years of age, chosen for their small size, were taken from a commercial k i l l drive and caged singly in numbered wire cages. These were divided into 4 groups of 4 each, and 2 groups of 3 each. A l l females received a single intramuscular injection of 1000 IU PMS in 1.0 ml physiological saline.  Seventy-two hours later,  ovulating hormone, either a standardizing preparation of LH or the experimental material was administered in total doses intravenously into a hind flipper.  Two dose levels of LH, 4 mg or 8 mg in 1.0 ml saline,  were administered to 2 groups of 4 females. Two dose levels of adenohypophysial homogenate from 2- and 3-year-old immature females, 2 and 3 adenohypophyses, with a homogenate equivalent weight of 21.1 mg homogenate per adenohypophysis, were administered in 2.0 and 3.0 ml saline to 2 groups of 4 females. Two dose levels of adenohypophysial homogenate from early proestrus females, 2 and 3 adenohypophyses, with a homogenate equivalent weight of 22.1 mg per adenohypophysis, were administered to 2 groups of 3 females.  63  C. Response of Female Fur Seals to Exogenous F o l l i c l e Stimulating Hormone  1. First year (a)  Saline controls (Table 6).  The ovaries of pups receiving saline contain an average 530.2 antral f o l l i c l e s , less than 1.0 mm in diameter in each ovary;  the f o l l i c l e s are  fully developed, with thecal layers, granulosa, and apparently normal oocytes (Plate X, A); approximately half these f o l l i c l e s are atretic. The associated endometria are quiescent:  the few glands show l i t t l e  coiling (Plate X, B); the glandular and rugal epithelia are cuboidal and non-secretory  (Plate X, C); the connective tissue stroma is dense and  fibrccytic. (b)  Human Chorionic Gonadotropin (Table 6)  There are no gross ovarian changes from those of saline-treated pups at the dose level used, 150 IU.  Ovarian weight is unchanged from an av-  erage 1.5 gms. There is no significant change in the number of antral f o l l i c l e s less than 1.0 mm in diameter; les.  and there are no larger f o l l i c -  Approximately half the antral f o l l i c l e s are atretic.  small but significant effect on the associated endometria:  There is a the index of  glandular coiling is increased 21.07« from saline control values, and the height of glandular and rugal epithelia is increased 32.17a. The rugal epithelia are slightly secretory, and the connective tissue stroma less dense and more fibroblastic than that of saline controls (Plate X, D); there are no mitotic figures. (c)  Standardizing F o l l i c l e Stimulating Hormone (Table 6)  The dose level of 0.5 mg FSH per animal was ineffective in inducing  TABLE 6. RESPONSE OF THE REPRODUCTIVE TRACTS OF FUR SEAL PUPS TO HUMAN CHORIONIC GONADOTROPIN (HCG) AND FOLLICLE STIMULATING HORMONE (FSH). FIRST BIOASSAY SERIES. Saline control  HCG control  12  12 P<0.05**  Ovarian weight average gms/ovary Average/ovary number of antral follicles 1mm diameter l-2mm diameter  0. 5 mg FSH  P<0.05  P<0.05  P<0.05  1.08*0.23* = 1.52±0.37= 1.13±0.21 <3.30±0.82  P<0.05  > 2.30±0.09  3.0 mg FSH  P<0.05  <3.33±0.38 = 3.28±0.5  530.2+170.6 = 503±212.1 ^232.5^68.2 =260 . 5±42. 3 981. 3±300. 6 = 1094±392. 3 485. 3+2. 4 14.5*0.7 = 15.0±2.4 J- 63.0*4.5 = 46.3*18.2  Number of uterine glands (index of glandular coiling)  49.0±6.6  < 62.1±8.9  Endometrial rugal cell height yx  8.4±0.3  < 12.5±0.8 < 15.2*2.1 " 17.4±0.9  Endometrial gland cell height y\  2.0 mg FSH  1.5 mg FSH  1.0 mg FSH  8.610.5  = 56.0^1.0 ^89.4^8.8  12.410.8 "7  to. 3 11.9:  * ± standard error.  +  ^18.6± 2.4  =82.3-12.3  <  y 133.5±4.9 = 207.3±18  = 16.9+0.4  19.0±2.3 = 18.9±0.8  ~ 17.4±1.5  18.2±1.2 " 16.7±0.5  ** Significant difference between means.  65  PLATE X  A.  The ovary from a fur seal pup  B.  The uterine endometrium from  not given exogenous gonadotropins.  a fur seal pup not given exogen-  The antral f o l l i c l e s (a) are  ous gonadotropins.  small, but complete, and contain  (g) are few and straight.  oocytes.  x 25.  C.  Mag. x 25.  The glandular and rugal uterine  D.  The glands Mag.  The glandular and rugal ut-  mucosae associated with A and B.  erine mucosa from a fur seal pup  The stroma (s) is dense and fibro-  which received HCG.  cytic;  (s) is more edematous and fibro-  the glandular (g) and  The stroma  rugal (r) epithelia are cuboidal.  blastic than that of C;  Mag. x 250.  rugal epithelium (r) is columnar Mag. x 250.  the  66  A.  The ovary from a fur seal pup  B.  The associated uterine en-  which received HCG and 1.0 mg. FSH.  dometrium from the pup of A.  The antral f o l l i c l e s (a) are en-  The number of glands (g) in  larged . Mag. x 25  cross section (index of glandular coiling) is increased from that of pups receiving HCG alone.  Mag. x 25.  C. The rugal and glandular muc -  D.  The rugal and glandular  osae associated with A. and B.  uterine mucosae from a fur seal  The stroma (s) is fibroblastic;  pup receiving 0.5 mg FSH. Al-  the rugal (r) and glandular  though the stroma is fibroblas-  (g) epithelia are low columnar.  t i c , the rugal (r) and glandular (g) epithelia are cuboidal. Mag. x 250.  P L A m KE  67  PLATE XII  A.  The ovary from a fur seal pup  13. The glandular and rugal  receiving HCG and 2.0 mg FSH. The  uterine mucosae from the pup of  number of antral f o l l i c l e s (a) is  A.  increased over that of pups re-  pseudostratified columnar.  ceiving 1.0 mg. FSH. Mag. x 25.  Mag. x 250.  C.  The rugal epithelium (r) is  The uterine endometrium from the pup  of A.  Glandular (g) coiling is increased  from that of pups receiving 1.0 mg. FSH. Mag. x 25.  PLATE XU  68  ovarian or uterine development. The number of antral f o l l i c l e s less than 1.0 mm in diameter is significantly decreased 46.1% from control values. The associated endometria are not significantly different in any parameter from those of HCG-treated pups except that the height of the rugal epithelia is increased an average 17.8%  (Plate XI, D).  The response of reproductive tracts to individual dose levels of 1.0 and 1.5 mg FSH did not vary significantly from each other, and are considered as equivalents.  Ovarian weight is significantly increased  from control values by 61.3%, to an average 2.8 gms, and there are an average 14.5 mature antral f o l l i c l e s 1.0 - 2.0 mm in diameter in each ovary (Plate XI, A).  The associated endometria are estrogenic:  the con-  nective tissue stroma is fibroblastic; . glandular and rugal epithelia are columnar (Plate XI, B and C), an average 5.0^ control values, and secretory;  greater in height than HCG  and the index of glandular coiling is an  average 21.7% greater than HCG control values.  A few mitotic figures are  seen. The responses of reproductive tracts to individual dose levels of 2.0 and 3.0 mg FSH did not differ significantly from each other and are considered equivalent.  Ovarian weight is not increased from that of the  1.0 - 1.5 mg FSH dose level, averaging 0.5 gms greater. The response is dramatic:  follicular  numbers of mature antral f o l l i c l e s 1.0 - 2.0 mm in  diameter are increased approximately 70.07» from the previous dose levels, to an average 54.6 f o l l i c l e s in each ovary (Plate XII, A). r i a l response is equally dramatic.  The endomet-  Glandular coiling is.significantly in-  creased an average 49.7% over that of the previous dose levels (Plate XII, B and C).  The glandular and rugal epithelia are the same height as those  69  of the previoujs dose levels.  Both columnar epithelia are secretory;  the rugal epithelia are pseudostratified;  and many mitotic figures are  seen. The connective tissue stroma is fibroblastic and edematous. The reproductive tracts approximate, in f o l l i c u l a r and endometrial development, that of a proestrous female (Table 5). The number of ovarian antral f o l l i c l e s less than 1.0 mm in diameter is significantly decreased an average 270.3 in each ovary from control values at dose levels of 0.5 and 1.0 mg FSH, but significantly increased an average 520.8 at 1.5 and 2.0 mg FSH dose levels;  at the  3.0 mg FSH dose level, the number of small antral f o l l i c l e s is not significantly different from control values.  There is a significant decrease,  19.27» in the percentage of atretic antral f o l l i c l e s at a l l FSH dose levels from control values. (d)  F o l l i c l e Stimulating Hormone activity of adenohypophyses  from immature female fur seals (Table 7). The physiological response of the reproductive tracts from recipient pups to adenohypophyses from 2-year-old immature females is qualitatively the same as that to response-producing dose levels of standardizing FSH (Table 6). Ovarian weights at a l l dose levels are significantly greater than control values by an average 56.77»;  ovarian weights do not differ sig-  nificantly from each other at any dose level, and the average ovarian weight is 3.04 Igms. Numbers of antral f o l l i c l e s less than 1.0 mm in diameter are significantly less at a l l dose levels than the control values for saline or HCG by an average 34.77»;  the numbers of these f o l -  licles in each ovary do not differ from each other at any dose level, and  TABLE 7. RESPONSE OF THE REPRODUCTIVE TRACTS OF FUR SEAL PUPS TO ADENOHYPOPHYSES FROM 2-YEAR-OLD IMMATURE FEMALE FUR SEALS. FIRST BIOASSAY SERIES.  Saline control n Ovarian weight average gms/ovary Number of antral follicles average/ovary 1mm diameter  HCG control  12 P<0.05**  1.0 adenohypophys is 4  12 P<0.05  2.0 adenohypophyses 4 05 P<0.05  1.08*0.23* = 1.52*0.37 == 3.10*0.3 =  2.18*0.24 =  3.0 4.0 5.0 adenohyadenohyadenohypophyses hypophyses pophyses 4 4 4 P<0.05 P<0.05 2.90*0.49 =  3.15*0.36 = 3.75*0.15  530.2±170.6 = 503.5*212.1= 320.0*28.6 ==378.0*33.9 = 338.5*118.1 = 286.5*33.3= :357.0*20.6 •  l-2mm diameter  •  0  28.8*8.6 =  0  32.3+4.8 <£ 47.8±4.7  64.3*6.2 <:L20.0*7.8  Number of uterine glands (index of glandular coiling)  49.0+6.6  < ^62.1+8.9  = 92.2+0.4 = < 110.3+18.1 =  93.3±7.1  Endometrial rugal cell height y\  8.4±0.3  < ' 12.5*0.8  < ' 20.4±1.8 =  19.6*1.8 =  19.5*1.1  20.0*1.1 = 20.3*0.5  Endometrial gland cell height, yx  8.6+0.5  <'12.4*0.8  <C 16.7*0.5 =  17.3*0.5 =  18.2*0.7  18.1*0.1 = 17.6*1.4  * * standard error.  < ^159.7+0.3 <:.87.5±31.4  ** Significant difference between means.  71  PLATE XIII  A.  The ovary from a fur seal pup  B.  The uterine endometrium  which received HCG and 3.0 adeno-  from the pup of A.  hypophyses from immature females.  coiling is increased from  The number and size of the antral  control values.  follicles (a) approximates that of a pup receiving 2.0 mg FSH. Mag. x 25.  C.  The rugal mucosa from B.  The stroma  (s) is fibroblastic and edemetous;  the  rugal epithelium (r) is pseudostratified, columnar, and secretory.  Mag. x 450.  Glandular  Mag. x 25.  PLATE  x m  72  PLATE XIV  A.  The endometrial rugal epithel-  Bl  The endometrial rugal (r)  ium (r) from a fur seal pup which  and glandular (g) epithelia  received HCG and 1.0 adenohypoph-  from a fur seal pup which re-  yses from, a 2-year-old immature  ceived HCG and 4.0 adenohypoph-  female is columnar and secretory.  yses from 2-year-old immature  Mag. x 250.  females.  Both epithelia are  columnar;  the rugal epithel-  ium (r) is pseudostratified and secretory.  Both epithelia ex-  hibit mitotic divisions (m). Mag. x 450.  C.  The endometrial glandular (g) epithelium  from a fur seal pup which received 5.0 adenohypophyses from 2-year-old immature females. The epithelium is columnar, secretory, and exhibits mitotic figures. Mag. x 250.  PLATE XIV  73  average 336.0. . The development of antral f o l l i c l e s 1.0-2.0 mm in diameter is least at dose levels of 1.0 and 2.0 adenohypophyses, as is uterine response. Although the number of antral f o l l i c l e s 1.0-2.0 mm in diameter is significantly greater by approximately 50%, averaging 30.5 in each ovary, en•dometrial parameters do not vary significantly from those at dose levels of 1.0 and 1.5 mg FSH (Table 6);  glandular coiling is evident, the low  columnar glandular and rugal epithelia are secretory, but there are few mitotic figures (Plate XIV, A ) . The numbers of antral f o l l i c l e s 1.0-2.0 mm in diameter at the dose levels of 3.0 and 4.0 adenohypophyses are not significnatly different from each other, but are significantly greater than at the 1.0 or 2.0 adenohypophyses dose levels, by an average 46.67o.  nevertheless there are  an average 17.0 more f o l l i c l e s in each ovary at 4.0 adenohypophyses (an average 64.3) than at 3.0 adenohypophyses (an average 47.8) (Plate XIII, A).  The index of endometrial glandular coiling at the dose level of 3.0  adenohypophyses does not differ from that of 1.0 and 2.0 adenohypophyses, but  i t is significantly greater at the dose level of 4.0 adenohypophyses  by an average 32.770.  The height of the glandular and rugal epithelia do  not differ from each other at the dose levels of 3.0 and 4.0 adenohypophyses, or from the 1.0 and 2.0 adenohypophyses dose levels.  The epithelia  are secretory, with few mitotic figures, at the dose level of 3.0 adenohypophyses, but at 4.0 adenohypophyses, the rugal epithelia is pseudostratified, and mitotic figures are frequent in both epithelia (Plate XIII, B and C; Plate XIV, B ) . Ovarian and uterine parameters at the dose level of 4.0 adenohypophyses do not differ significant^ from the same parameters at the dose levels  RESPONSE OF THE REPRODUCTIVE TRACTS OF FUR SEAL PUPS TO ADENOHYPOPHYSES FROM 4 - AND 5-YEAR-OLD OVULATED FEMALES. FIRST BIOASSAY SERIES.  TABLE 8.  Saline control . n  HCG control  12 P<0.05**  Ovarian weight average gms/ovary  1.08±0.23  1.0 1.5 2.5 3.0 adenohyadenohyadenohyadenohypophysis pophyses pophyses pophyses 12 4 4 4 4 P<0.0 5 P<0.05 P<0.05 P<0 05  1.52±0.37  2.25±0.27 =  2.10±0.44 =  530.2^170.6 =r 503.5^212.1 <' 209.8-15.2 =  271.3-86.5 =  <  '  2.90±0.21  ^  1.23±0.05  Number of antral follicles average/ovary 1mm diameter  0  0  Number of uterine glands (index of glandular coiling)  49.0^6.6  62.1^8.9  ^.  79.5-3.3  Endometrial rugal cell height p  8.4+0.3  <'  12.5+0.8  <'  15.7+1.1  Endometrial gland cell height, y  8.6±0.5  <'  12.4i0.8  =  13.6±0.9  l-2mm diameter  25.3±0.9  <"  =  50.3^6.3  <'  252.0^44.1 1 > 58.5-12.1 81.0±3.0  *  standard error.  13.3i6.0  89.6-11.8 I >  60.7-10.9 =  60.9^9.9  15.5±0.9  17.0±0.7  15.8+0.5  =• 13.5±0.6  =  =  14.2±0.7  • +  >  ** Significant difference between means.  13.3+0.3  75  PLATE XV  A.  The ovary from a fur seal pup  B.  The endometrial rugal (r) and  which received 1.5 adenohypophyses  glandular (g) epithelia from the  from ovulated females. The'antral  pup of A;  f o l l i c l e s (a) are enlarged and  columnar and secretory.  numerous. Mag. x 25.  250.  C.  both epithelia are low  The uterine endometrium from the pup  of A. ,Mag. x 25.  Mag. x  76  PLATE XVI  A.  The ovary from a fur seal pup  which received 2.5 adenohypophyses from ovulated females.  B.  The uterine endometrium from  the pup of A.  The antral  f o l l i c l e s (a) are enlarged and more numerous than those of the pups receiving 1.5 ovulated adeno-. hypophyses. Mag. x 25.  C.  The endometrial rugal (r) and gland-  ular (g) epithelia from the pup of A; both are low columnar and secretory. 250.  Mag. x  Mag. x 25.  PLATE, y y i  77  of 2.0 and 3.0 mg FSH (Table 6). The ovarian response to the dose level of 5.0 adenohypophyses is massive; ' •  the number of antral f o l l i c l e s 1.0-2.0 mm in diameter is i  double the number of the 4.0 adenohypophyses dose level, averaging 120.0 in each ovary. The uterine parameters do not differ significantly from those at the 4.0 adenohypophyses dose level (Plate XIV, B and C). The percentage of atretic antral f o l l i c l e s at a l l dose levels of adenohypophyses is significantly less than at either control level by an average 24.27c (e)  F o l l i c l e Stimulating Hormone activity of adenohypophyses  from newly ovulated females (Table 8). Ovarian weights of recipient pups are not significantly different from each other at dose levels of 1.0, 1.5, and 2.5 adenohypophyses from newly matured and ovulated females, but a l l are significantly greater than saline or HCG control values by an average 1.1 gms, or an increase of 45.97o.  Ovarian weight at the dose level of 3.0 adenohypophyses is  the same as the control values. Antral f o l l i c l e s less than 1.0 mm in diameter are equal in number to each other but are significantly less in number at dose levels of 1.0, 1.5, and 2.5 adenohypophyses than at saline and HCG control levels by an average 52.57, or 272.5 f o l l i c l e s ;  the number of these f o l l i c l e s at the  3.0 adenohypophyses dose level is very small, 11.57o of the average control value, an average 58.6 f o l l i c l e s in each ovary. Numbers of antral f o l l i c l e s 1.0-2.0 mm in diameter increase 25.07o at each dose level, with an average 25.3 f o l l i c l e s in each ovary at the dose level of 1.0 adenohypophysis (Plate XV, A), except at the dose level of 3 adenohypophyses,  78  where the number of f o l l i c l e s is significantly less than at any other dose level, averaging 13.3 f o l l i c l e s in each ovary. Follicular response at the dose level of 1.0 adenohypophysis equals that of the same dose level of adenohypophyses from immature females; at dose levels of 1.5 and 2.5 adenohypophyses, the follicular response is equal to the response at dose levels of 3.0 and 4.0 adenohypophyses from immature females, and to the response at the dose level of 2.0 and 3.0 mg FSH (Tables 6 and 7). The index of glandular coiling is significantly greater than HCG and saline control values at dose levels of 1.0 and 1.5 adenohypophyses by an average 34.47<>: the parameter does not differ significantly from control values at dose levels of 2.5 and 3.0 adenohypophyses. The rugal epithelia are significantly higher than the HCG control values at a l l dose levels by an average 21.97», or 3.5  but are not significantly  different from each other. The glandular epithelia are not significantly higher than the value for HCG controls at any dose level. Endometr i a l growth is minimal at a l l dose levels, significantly less in a l l parameters than any dose level of adenohypophyses from immature females (Table 7), and approximating the 1.0 mg FSH dose level (Table 6). The connective tissue stroma is fibroblastic, the epithelia minimally secretory, and mitotic figures are infrequent (Plate XV, B and C, Plate XVI, B and C). (f)  Quantification of adenohypophysial F o l l i c l e Stimulating  Hormone (Table 9). The development of antral f o l l i c l e s 1.0-2.0 mm in diameter in the ovaries of recipient pups in response to exogenous gonadotropins was used as the parameter to quantify FSH potency. The follicular response  79  TABLE 9.  CONCENTRATION AND CONTENT OF ADENOHYPOPHYSIAL FOLLICLE STIMULATING HORMONE, DETERMINED IN THE FIRST BIOASSAY SERIES USING FUR SEAL PUPS AS ASSAY ANIMALS. THE ENDPOINT IS THE NUMBER OF ANTRAL OVARIAN FOLLICLES 1.02.0 1MM IN DIAMETER. i < :  Adenohypophys i a l dry weight, mg  FSH concentration, mg equivalents NIH-FSH  FSH content mg equivalents NIH-FSH  Adenohypophyses from 2-year-old immature females  15.0  0.03700+0.003*  0.56550+0.045  Adenohypophyses from 4-r and 5-year-old ovulated females  15.0  0.08520+0.0027  1.2780^0.0400  * + standard error  80  \H1.V 10  BIOASSAY FOR FSH OF ADENOHYPOPHYSES FROM 2-YEAR-OLI) FEMALE Ff'R SEALS. THE END POINT MEASURED IS THE NUMBER OF GRAAFIAN FOLLICLES l-2mm IN DIAMETER IN THE OVARIES OF RECIPIENT FUR SEAL PIPS. n  Mean response±SE A  B  Total variance of reaponse  Slope b  Index of precision  7 rea t ment 1.0.2.0 mg FSH  4  2,4 adeno4 hypophyses  14.5*0.6  63.0±4.5  82.33  30.09  R±SF.  (b test b standard)  0.330 1.1307 1.0878  32.3-4.8  64.3-6.2  243.83  35.04  0.842  81  TABLE 11.  BIOASSAY FOR FSH OF ADENOHYPOPHYSES FROM 4- AND 5-YEAR-OLD FEMALE FUR SEALS. THE END POINT MEASURED IS THE NUMBER OF GRAAFIAN FOLLICLES l-2ua IN DIAMETER IN THE OVARIES OF RECIPIENT FUR SEAL PUPS. n  Total variance of response  Mean reap onse-SE A  B  Slope b  Index of precision  Treataent 1.0, 2.0 •C FSH  4  1.0, 2.5 adenohypophyses  4  14.5±0.7  63.0±4.5  82.33  33.63  R^SE (b test/ b standard)  0.274 1.2780^ 0.0399  25.3±0.9  81.0±6.5  43.00  40.24  0.421  80., 70. 60. X x  127.801339 OF FSH  %  7 50. co w 40. 3  • 30. e • FSH • ADENOHYPOPHYSES  fc 2 0 . m 10.  DOSE LEVEL  82  to the standardizing preparation of FSH was not a dose-response relationship (Table 6), but the significant difference between dose levels of 1.0 and 2.0 mg FSH allowed a significant dose-response regression, and 1  these dose levels were used for convenience. A dose-response relationship was obtained for development of f o l l i c l e s 1.0-2.0 mm in diameter in response to 2.0, 3.0, 4.0 and 5.0 adenohypophyses from immature females (Table 7), and from ovulated females at dose levels of 1.0, 1.5, and 2.5 adenohypophyses (Table 8). For the quantifying regression analyses, dose levels of 2.0 and 4.0 adenohypophyses from immature females, and 1.5 and 2.5 adenohypophyses from ovulated females were used.  The  results of the regression analyses are summarized in Tables 10.and 11. The index of precision was less than unity for each assay. An adenohypophysis from a 2-year-old immature female, with an average dry weight of 15.0 mg, contained 0.5655 mg equivalents of standardizing FSH at a concentration of 0.037 mg equivalents of FSH per mg adenohypophysial dry weight.  An adenohypophysis from an ovulated female, with  with an average dry weight of 15.0 mg, contained 1.2780 mg equivalents of stnadardizing FSH, at a concentration of 0.0852 mg equivalents of FSH per mg adenohypophysial dry weight;  the values are approximately double those  of adenohypophyses from immature females. The 2 sets of values are significantly different (P<0.05). The data suggest that the post-ovulatory condition is associated with increased adenyhypophysial content of FSH.  2.  Second year.  The quantification of adenohypophysial FSH in recipient female fur seal pups was repeated. Two dose levels of standardizing FSH, 1.0 and 2.0  83  mg, were used, and 2 dose levels of 2.0 and 4.0 adenohypophyses from immature 2- and 3-year-old females, and from 4- and 5-year-old estrous females preparing to ovulate for the f i r s t time. The same response parameters were measured:  ovarian weight and antral f o l l i c l e develop-  ment, endometrial glandular coiling, and rugal and glandular epithelial c e l l height. The results are summarized in Table 12. (a)  Controls.  Parameter values for control levels of saline and HCG did not differ from those obtained in the f i r s t bioassay (Table 6). The ovaries are small, averaging 2.0 gms each, and contain an average 240.5 antral f o l l i c l e s less than 1.0 mm in diameter; atretic.  an average 51.8% of these are  The endometria of pups treated with saline are quiescent and  non-secretory;  the glandular and rugal epithelia are cuboidal, and the  index of glandular coiling averages 39.3. The endometria of HCG-treated pups are minimally estrogenic:  the rugal and glandular epithelia are  low columnar, and the index of glandular coiling averages 70.1. (b)  Standardizing F o l l i c l e Stimulating Hormone.  Antral f o l l i c l e s 1.0-2.0 mm in diameter develop in the ovaries of recipient pups in response to dose levels of 1.0 and 2.0 mg FSH. The number of f o l l i c l e s is significantly less at both dose levels, an averag 8.0 and 27.3 f o l l i c l e s in each ovary at 1.0 mg and 2.0 mg FSH respective l y , than the same values in the f i r s t bioassay, an average 14.5 and 63.0 antral f o l l i c l e s in each ovary at 1.0 and 2.0 mg FSH respectively (Table 6).  Ovarian weight at these dose levels does not vary significantly  from control values and is less than the same values in the f i r s t bio-  TABLE  RESPONSE  12.  HCRHOKE.  THE REPRODUCTIVE TRACTS OF FUR SEAL PUPS TO HUNAN CHORIONIC GONADOTROPIN (HCG), FOLLICLE STDflJLATINC AND ADENCHTPOPWTSES FROM BMAIURE FEMALES AND FEMALES IN ESTRUS. SECOND BIOASSAY SERIES.  OF  HO control  Saline c o n t r o l  *  4  n  PO.OS** Ovarian  weight  average Number  gms/ovary of  1  ft  * KO.05  P<0.05  4 . 0 adanohypophyiaa 2-and 3-yaaro l d lnaatura fanalaa  2 . 0 adanohypophyiaa 2-and 3-yaaro l d lnaatura famalas  2.0 mg FSH  U O Bg FSH  ft P<O.OS  4 P<0.05  2.tt3>.18  241*18.6  2*0.31*1.7  -  4 . 0 adenohy pophyaaa •atroua  (aulu  4 K0.05  1  «.  1.734>.l5*--  2 . 0 adanohypophyaaa ••trout famalai  4 KO.OS  1  2.05*0.25  i  2.83*0.48  1  2.93*0.18  •  2.83*0.27  '  3.23*0.36  208.5*15.1  •  222.0*20.0  -  194.3*25.2  .  191.1*21.4  <',  3*9.0*40.6  <  37.3*3.8  <  53.5-11.3 53.5:11.5  76.0*15.3  >  32.0*9.8  <  56.5*4.2  ^  123.0X1.0  <  >  147.2tl0.2  <  92.2*3.6  >  <  ...... 20.6^0.6  I ...... i 20.6+0.6  >  >  ...... 2 4 . 8 . 0 . 4 24.sto.4  J > >  ...... 2 0 . 2 . 0 20.2*0.7  1 25.9tl.O <j  ^  16.2*0.4  j  f-  17.3^0.3  -  «  1.715). 15  a n t r a l  f o l l i c l e s average/ovary 1.0  n o d Lataeter  1 . 0 - 2 . 0  o n  -  >  8.0*2.5  e t e r  Number  of  glands  (Index  r u g a l  height  Endometrial c e l l  of  39.3*4.4  <  70.1*1.*  y  56.5*0.8  U.sto.7  <  15.5*0.5  f-. 1.3.7^*0..5  77.9+4.5  146.4*5.5  c o l l i n g )  Endometrial  ular  <<  u t e r i n e  glandular  c e l l  123.6*19.0 ^  d i a n -  >i  g l a n d height  >i  8 . 1 -t0o . l  t  <  U.7tl.l  * .standard e r r o r .  + —  i> l l . S ± 0 . 7  f  16.5*0.4  * * S l g a l f leant d i i f a r a o c * batvsan •aana.  I „18.010.8 ,.,  -  17.210.9  85  assay (Table 6).  Numbers of antral f o l l i c l e s less than 1.0 mm in dia-  meter, averaging 123.6 in each ovary in response to 1.0 mg FSH, and 208.5 in each ovary in response to 2.0 mg FSH, are significantly less than control values, and significantly less than the same values in the f i r s t bioassay, an average 260.5 f o l l i c l e s in each ovary at 1.0 mg FSH, and an average 1094.0 f o l l i c l e s at 2.0 mg FSH (Table 6).  Follicular  atresia at both dose levels averages 40.27«, not significantly different from control values, or from the same values in the f i r s t bioassay. The uterine endometria at the dose level of 1.0 mg FSH do not differ significantly in any parameter from the same values for HCG controls.  At the dose level of 2.0 mg FSH, the index of glandular coiling  is an average 123.0, significantly greater than control values and the response to 1.0 mg FSH; the columnar rugal and glandular epithelia are secretory, with infrequent mitotic figures;  the values are not differ-  ent from the same values at the dose level of 2.0 mg FSH in the f i r s t bioassay (Table 6). (c)  Follicle  Stimulating Hormone activity of adenohypophyses  from 2- and 3-year-old immature female fur seals (Table 12). Ovarian weights at dose levels of 2.0 and 4.0 adenohypophyses from immature females do not differ from each other, and average 2.9 gms per ovary, significantly greater by 30.67o than the control values, and the same as the corresponding values in the f i r s t bioassay (Table 7). The number of antral f o l l i c l e s  1.0-2.0 mm in diameter at the 2.0 adeno-  hypophyses dose level average 53.5 in each ovary, and 76.0 in each ovary at the 4.0 adenohypophyses dose level;  the values are significantly  86  different from each other, greater than the same values at 1.0 and 2.0 mg FSH, and do not differ from the same values at dose levels of 2.0 and 4.0 adenohypophyses from immature females in the f i r s t bioassay (Table 7).  Numbers of antral f o l l i c l e s less than 1.0 mm in diameter at  both dose levels are not significantly different, and average 214.2 in each ovary, equal to control values, and significantly less than corresponding values in the f i r s t bioassay (Table 7).  The percentage of at-  retic f o l l i c l e s in each ovary is the same at both dose levels, averaging 32.37o, significantly less than control or standardizing FSH values, and the same as the corresponding values in the f i r s t bioassay. The endometrial response to 2.0 adenohypophyses is similar to that at 2.0 mg FSH, although the glandular coiling index, averaging 77.9, is significantly less;  the columnar glandular and rugal epithelia are  secretory, but mitotic figures are infrequent. The endometrial values are the same as the corresponding values in the f i r s t bioassay (Table 7). The endometrial response to 4.0 adenohypophyses is significantly greater in a l l parameters than to 2.0 adenohypophyses: index is increased 47.470;  the glandular coiling  the glandular and rugal epithelia are columnar  and secretory, the rugal epithelia pseudostratified, and mitotic figures are frequent in both epithelia.  The endometrial values are the same as  the corresponding values in the f i r s t bioassay (Table 7). (d) F o l l i c l e Stimulating Hormone activity of adenohypophyses from estrous females preparing to ovulate for the f i r s t time (Table 12). The ovarian weight at dose levels of 2.0 and 4.0 adenohypophyses from estrous females do not differ from each other, and average 3.0 gms, the same as ovarian weights in response to adenohypophyses from immature  87  TABLE 13. CONCENTRATION AND CONTENT OF ADENOHYPOPHYSIAL FOLLICLE STIMULATING HORMONE, DETERMINED IN THE SECOND BIOASSAY SERIES USING FUR SEAL PUPS AS ASSAY ANIMALS. THE ENDPOINT MEASURED IS THE NUMBER OF ANTRAL OVARIAN FOLLICLES 1.0-2.0 MM IN DIAMETER.  Adenohypophys ial dry weight, mg  FSH concentration, mg equivalents, NIH-FSH  FSH content mg equivalents NIH-FSH  Adenohypophyses from 2- and 3-year-old immature females  21.20  0.066±0.037*  1.403±0.79  Adenohypophyses from 4-year-old estrus females  22.16  0.036±0.006  0.803±0.15  * -standard error.  88  TABLE 14. BIOASSAY FOR FAH OF ADENOHYPOPHYSES FROM 2-AND 3-YEAR-OLD IMMATURE FEMALE FUR SEALS. THE END POINT MEASURED IS THE NUMBER OF GRAAFIAN FOLLICLES l-2am IN DIAMETER IN THE OVARIES OF RECIPIENT FUR SEAL PUPS. n  Mean response-SE A  B  Total variance of response  Slope b  Index of precision  Treatment 1.0, 2.0 mg FSH  4  2,4 adeno- 4 hypophyses  8.0*2.5  53.5-11.5  37.3*3.8  76.0±15.3  80.91  13.58  .910  147.29  38.18  .318  A DOSE LEVEL  RISE  (b test/ b standard)  2.812* 1.579  B  89  TABLE 15. BIOASSAY FOR FSH OF ADENOHYPOPHYSES FROM ESTROUS FEMALE FUR SEALS THE END POINT MEASURED IS THE NUMBER OF GRAAFIAN FOLLICLES I-2am IN DIAMETER IN THE OVARIES OF RECIPIENT FUR SEAL PUPS. n  Mean resp•onse-^SE A  B  Total variance of response  Slope b  Index of precision  Treatment 1.0, 2.0 •« FSH  4  2,4 adenohypophyses 4  8.0*2.5  37.3*3.8  80.91  20.63  K*SE (b test/ b standard)  .604 1.606* 0.30  32.0*9.8  56. 5*4. 2  455.00  33.13  .844  SQL. £ 70.  leo. X50-.  160.60*300% OF FSH  I (0  40-  h  • FSH • ADENOHYPOPHYSES  io. 1  A DOSE LEVEL  *1  B  90  TABLE 16.  COMPARISON OF FSH POTENCY IN ADENOHYPOPHYSES FROM IMMATURE AND ESTROUS FEMALES. THE END POINT MEASURED IS THE NUMBER OF GRAAFIAN FOLLICLES l - 2 » IN DIAMETER IN THE OVARIES OF RECIPIENT FUR SEAL PUPS. Mean response A  B  Total variance of response  Slope b  Index of precision  Treataent 2,4 adenohypophyses 1nature  53.5111.5  2,4 adenohypophyses estrous  32.0*9.8  76.0*15.3  147.29  29.55  R*SE (b test/ b standard)  .412 0.590* 0.212  56.5*4.2  455.00  17.45  ESTRUS 9 Q 0 O ± 2 L 2 % OF IMMATURE  DOSE LEVEL  1.603  91  females. The number of antral f o l l i c l e s 1.0-2.0 in diameter at the 2.0 adenohypophyses dose level averages 32.0 in each ovary, significantly less by 40.2% than the same value at 2.0 immature adenohypophyses. Similarly at 4.0 adenohypophyses from estrous females, the follicular average, 56.5 in each ovary, is significantly less by 25.2"L than the same value at 4.0 immature adenohypophyses. The number of antral f o l l i c l e s less than 1.0 mm in diameter, an average 191.1 in each ovary at the dose level of 2.0 adenohypophyses, is significantly less than the same value, an average 349.0 f o l l i c l e s in each ovary at the dose level of 4.0 adenohypophyses; values.  the latter value is significantly greater than control  The percentage of atretic antral f o l l i c l e s in each ovary is  greater at the dose level of 2.0 adenohypophyses, averaging 41.3%, than at the dose level of 4.0 adenohypophyses, averaging 32.07<, in each ovary. The endometrial response to 2.0 adenohypophyses from estrous females is the same as the response to 2.0 adenohypophyses from immature females;  the index of glandular coiling is 92.2, and the columnar  glandular and rugal epithelia are secretory; frequent.  mitotic figures are in-  Similarly, the endometrial response to 4.0 adenohypophyses  from estrous females is the same as the response to 4.0 adenohypophyses from immature females:  the index of glandular coiling is 146.4, the  glandular and rugal epithelia are columnar and secretory, and the rugal epithelia are pseudostratified;  mitotic figures are frequent.  (e) Quantification of F o l l i c l e Stimulating Hormone. The development of antral f o l l i c l e s 1.0-2.0 mm in diameter in the ovaries of recipient pups in response to exogenous gonadotropins was again used as the parameter to quantify FSH potency.  The results of the  92  regression analyses are summarized in Tables 13, 14 and 15. Because of the minimal follicular development in response to 1.0 mg FSH, and the large variances, the indices of precision tend to be close to unity, but do not exceed this permissible limit.  Adenohypophyses from 2- and 3-year-  old immature females, with an average dry weight of 21.2 mg, contain 1.403 mg equivalents of standardizing FSH at a concentration of 0.066 mg per mg adenohypophysial dry weight.  Adenohypophyses from estrous females  about to ovulate, with an average dry weight of 22.16 mg, contain 0.8030 mg equivalents of standardizing FSH at a concentration of 0.036 mg per mg dry adenohypophysial weight.  A regression analysis was used to com-  pare the adenohypophysial FSH potency of immature and estrous females (Table 16):  the potency of adenohypophyses from estrous females is  59.0%, or approximately half that of adenohypophyses from immature females.  The data suggest that at estrus, immediately prior to ovulation,  there is a decrease in adenohypophysial FSH content.  93  3.  Discussion.  There is no direct explanation for the presence of antral f o l l icles in the ovaries of prenatal, neonatal, and infant fur seal pups (Craig 1965). Harrison (in Amoroso et a l , 1965) has suggested that maternal hormones, either FSH or estrogen, may cross the placental barrier to effect a similar precocious ovarian development in the Common seal, Phoca vitulina.  The numbers of antral f o l l i c l e s decrease with each month  in the ovaries of fur seal pups, and by 5 months of age, no f o l l i c l e s are visible (Craig 1965);  this evidence suggests an i n i t i a l maternal in-  fluence, which is either minimally sustained, or not sustained by the endocrine system of the neonate. The present experiments indicate that the ovarian and uterine tissue of fur seal pups 6-8 weeks of age is responsive to exogenous gonadotropic hormones, further suggesting a prenatal maternal influence. In these experiments, HCG acted as LH on the ovaries of the pups to promote estrogen synthesis:  in both series of assays, the adminis-  tration of HCG alone resulted in endometrial estrogenic activation, with no increase in the size or number of antral ovarian f o l l i c l e s over the same parameters among pups given saline.  That estrogen synthesis by the  ovarian f o l l i c l e s is minimal is suggested by the extent of endometrial activation:  the index of glandular coiling is increased over saline  values 21.0% in the f i r s t series and 44.0%  in the second series;  gland-  ular and rugal epithelia were low columnar and slightly secretory, but no mitotic figures were seen. Effectively, in these experiments, HCG, administered concurrently with FSH (either as a purified, standardizing preparation or as unknown  94  quantities in the form of adenohypophysial homogenates), sensitizes the available small antral f o l l i c l e s in the ovaries of the pups to the growth effects of FSH:  with the exception of one ineffective, low dose level,  the ovarian response to HCG and FSH is increased follicular size.  Ster-  oid synthesis is increased by the concurrent administration of HCG and FSH over HCG alone;  the estrogenic endometrial response is increased in  a l l parameters over HCG control values. Given the sensitivity of the recipient pups to exogenous gonadotropins, their use as bioassay animals is limited by 3 factors:  the num-  ber of small antral f o l l i c l e s in the ovaries, and the individual growth capabilities of these f o l l i c l e s ; can respond to estrogen;  the extent to which endometrial tissue  and the effect of contaminating LH in adenohy-  pophysial homogenates. Adenohypophyses from immature 2-year-old females and matured 4and 5-year-old females which had ovulated were chosen to examine these 3 points, as well as to quantify adenohypophysial FSH.  Previous data  (Craig 1966), and the evidence of the differential adenohypophysial c e l l counts described, suggested that adenohypophyses from immature females could be expected to contain large amounts of FSH and LH.  Similarly,  adenohypophyses from recently ovulated females could be expected to contain large amounts of FSH, but relatively l i t t l e LH:  the ovaries con-  tain no antral f o l l i c l e s , suggesting adenohypophysial FSH synthesis and storage;  the recently ovulated f o l l i c l e is actively forming luteal c e l l s ,  suggesting rapid LH synthesis and release from an adenohypophysis recently depleted of LH by ovulation. The development of antral f o l l i c l e s 1.0-2.0 mm in diameter in the  95  ovaries of pups given 3.0 and 4.0 adenohypophyses from 2-year-old immature females is the same as that found in the ovaries of pups receiving 2.0 and 3.0 mg FSH; the associated endometrial development is also the same (Tables 6 and 7). The development of antral f o l l i c l e s 1.0-2.0 mm in diameter of pups given adenohypophyses from from recently ovulated females is greater at a l l dose levels than the same development at corresponding dose levels in the ovaries of pups receiving FSH and adenohypophyses from immature females;  but the measured endometrial parameters  are a l l significantly less (Tables 6, 7 and 8). These data suggest that the assay adapted for use in female fur seal pups is relatively insensitive to contaminating LH in the development of antral ovarian f o l l i c les 1.0-2.0 in diameter:  the ovarian response to adenohypophyses from  immature females closely approximates the response to LH-free, standardizing FSH, while the ovarian response to adenohypophyses from recently ovulated females, containing less LH than adenohypophyses from immature females, is greater than that to standardizing FSH. It is assumed that contaminating LH may reinforce the effects of HCG in promoting steroid synthesis, either directly on ovarian tissue, or by acting to release endogenous adenohypophysial gonadotropins; the endometrial response to adenohypophyses from recently ovulated females, relatively low in LH content, is far less than the endometrial response to LH-containing adenohypophyses from immature females. That the endometrial response to adenohypophyses from immature females was not greater than that to standardizing FSH is probably an expression of the limitation of endometrial tissue to respond to exogenous gonadotropins. Assuming that endometrial development is affected by contaminating  96  LH, and that endometrial tissue is limited in growth response to exogenous gonadotropins, none of the measured endometrial parameters can be considered sufficiently sensitive to FSH alone to act as quantifying endpoints. The obvious limitation of ovarian follicular development is in the growth of no f o l l i c l e s larger than 2.0 mm in diameter.  Neverthe-  less, within the follicular diameter range of 1.0-2.0 mm, dose-response relationships were obtainedand the evidence suggests that this development, while sensitive to FSH, is insensitive to LH.  Consequently,  numbers of ovarian f o l l i c l e s 1.0-2.0 mm in diameter is the endpoint of choice for quantifying FSH. The second series of assays were made to confirm the evidence of the f i r s t series, as well as to quantify adenohypophysial FSH.  Two dose  levels of FSH, 1.0 and 2.0 mg, and 2 dose levels of adenohypophyses, 2.0 and 4.0, from immature females and from estrous females about to ovulate for the f i r s t time, were chosen, based on evidence from the f i r s t series, to obtain the best dose-response regression relationships for FSH quantification.  To confirm the evidence of the f i r s t series by replication,  the responses to 150 IU HCG, to 1.0 and 2.0 mg FSH, and to 2.0 and 4.0 adenohypophyses from immature females should have been not significantly different from those obtained at the same dose levels previously. The measured ovarian and uterine parameters obtained in response to HCG are not significantly different between the 2 series (Tables 6 and 12). Antral follicular development in response to 1.0 adenohypophyses from immature females is significantly greater in the second than in the f i r s t series, but s t i l l f a l l s within the limit of error;  a l l other  measured ovarian and endometrial parameters at 1.0 and 2.0 adenohypophyses  97 are not significantly different between the 2 series (Tables 7 and 12). The ovarian response to 1.0 and 2.0 mg FSH is significantly less in the second than in the f i r s t series:  the number of antral f o l l i c l e s 1.0-2.0  mm in diameter is approximately half those of the f i r s t series at both dose levels (Tables 6 and 12). The endometrial response to 1.0 mg FSH is also approximately half the values of the measured parameters obtained at the same dose level;  but the endometrial responses to 2.0 mg FSH are  not significantly different between the 2 series, possibly an expression of the growth limit of the endometrial tissue. The replication of response to HCG, and to adenohypophyses from immature females in the second series of bioassays tends to confirm the authenticity of the f i r s t series.  There is. no direct explanation for  the response failure to 1.0 and 2.0 mg FSH in the second series, but there are 2 possibilities.  Assuming no technical errors, the only d i f f -  erence between the 2 series was the injection route used, intrathoracic in the f i r s t , intramuscular in the second;  this may have had a reduct-  ion effect. The number of small antral f o l l i c l e s available to the growth effects of FSH was significantly less in the second than in the f i r s t series (Tables 7 and 12), which may also have had a reduction effect in response to FSH, although no such effect was found in response to adenyhypophysial doses in the same circumstances. This argument is reinforced by a similar response failure to adenohypophyses from ovulated females in the f i r s t series:  only at 3.0 adenohypophyses was follicular  development inconsistent with dose;  the number of antral f o l l i c l e s less  than 1.0 mm in diameter was remarkably small in comparison to any other dose level (Table 8), and presumably were too few for a complete dose response.  98  The biological activity of porcine FSH and of FSH from fur seal adenohypophyses appears to be the same; no qualitative differences in response of ovarian and uterine tissue were found. The fact of qualitative equality indicates, however, that the reactive hormone in the adenohypophysial material was FSH. Considered separately, the results of the assays support the original hypotheses and tend to confirm the evidence of the differential adenohypophysial c e l l counts described.  The results of the f i r s t assay  indicate that adenohypophyses from females which have recently ovulated contain 55.87o more FSH than adenohypophyses from immature females (Table 9).  The results of the second assay indicate that adenohypophyses from  estrous females about to ovulate contain 43.5% less FSH than adenohypophyses from immature females (Table 13). of differential c e l l counts:  This data confirms the evidence  adenohypophyses from immature females con-  tained only 8.0% degranulated folliculotrops, with 82.4%. partly granulated, while 44.5% of the folliculotrops in adenohypophyses from estrous, ovulatory females were degranulated, and 53.7% were partly granulated (Table 3).  The evidence suggests a massive release of FSH at or near  ovulation, followed by synthesis and storage of FSH during i n i t i a l corpus luteum formation.  TABLE 17.  RESPONSE OF THE REPRODUCTIVE TRACTS OF FUR SEAL PUPS TO PREGNANT MARE SERUM (PMS). SECOND BIOASSAY SERIES.  Salln« control  8  n  300 ni PMS •ubcutaneoua 8  300 HI PMS Intramuscular  300 BI PMS Intrapleural  8  8  500 UJ PMS subcutaneous 8  500 VI PMS lntralauacular 8  500 Q] PMS Intrapleural 8  700 HJ PMS subcutaneoua 8  700 IU PMS Intra•Macular  8  700 m PMS Intrapleural 8  iKorlan weight avenue gina/ovary  B  0.8810.1  0.91t0.1  1.2t0.1  1.2+0.1  0.9t0.1  1.310.1  0.78+0.1  1.3t0.9  1.4+0.2  1.4+0.1  Ulertne weight xm«  4  2.4*0.2  2.7±0.2  4.8+1.5  4.0+0.9  4.5+1.2  4.1+0.4  3.3+0.5  4.3+0.5  4.3+0.5  5.2+0.5  Antral l o l l I d e a average number/ ,»vary  234.9+46.2  198.8+40.7  281.2*39.9  309.5*66.4  169.1*23.7  592.8*101.9  371.1*31.7  299.1*94.4  97.3*24.7  752.0*187  1.0- 3.0 run diameter  0  3.1*2.1  4.8*2.5  11.4*4.7  11.0*3.0  12.4*1.2  10.6*4.1  9.6*2.5  18.5*4.4  2.6*1.2  Uirgt'St ram diameter  1.0  6.0  2.0  6.0  2.0  11.0  1.0  1.Uramdlametor  B  1.0  5.0  3.0  100  D.  1.  Response of Female Fur Seal Pups and Immature Females to Exogenous Luteinizing Hormone  First year  The attempt to quantify LH in adenohypophyses from 2- and 3-yearold immature females and 4-year-old estrous females about to ovulate for the f i r s t time was not successful.  Pregnant Mare Serum (PMS), an FSH-  like gonadotropin used to induce development of ovulatory f o l l i c l e s in a variety of mammalian species had no effect on the small antral f o l l i c l e s in the ovaries of female fur seal pups at the single dose level used, 150 IU. 2.  Second year  To determine i f higher dose levels of PMS would induce ovulatory f o l l i c u l a r development, a preliminary experiment was performed; sults are summarized in Table 17. PMS were used;  the re-  Dose levels of 300, 500, and 700 IU  within each dose level, 4 pups each received a single  injection subcutaneous, intramuscular, or intrathoracic, and autopsies were made 72 hours later. The results indicate individual response. Although ovarian weight increased at each dose level, for each injection route, these weights were not significantly different from the saline control value or from each other, and ovarian weight at 500 IU PMS intrapleural is less than the control value. Follicular response varies at every dose level and injection route;  the ovaries of at least 2 females in each component de-  veloped antral f o l l i c l e s 1.0 mm in diameter or larger, but the ovaries of at least 1 female in each component contained no f o l l i c l e s larger than  TABLE 18. RESPONSE OF THE REPRODUCTIVE TRACTS OF 2-, 3-, AND 4-YEAR-OLD IMMATURE FEMALES TO PREGNANT MARE SERUM (PMS), LUTEINIZING HORMONE (LH), AND ADENOHYPOPHYSES FROM ESTROUS FEMALES, EARLY PROESTROUS FEMALES, AND 2-AND 3-YEAR-OLD IMMATURE FEMALES. SECOND BIOASSAY SERIES. Saline control  n Age of assay females, yrs. Ovarian weight average gms/ovary Number of antral follicles average/ovary 1.0- 3.0mm diam. Largest, mm diam.  4 3  1000 IU PMS control 6 2 and 3  3.5  2.6  143.5 4.3  97.3 4.2  Total number of ovulatory f o l l i c l e s  4mg LH  8mg LH  6  8  3 5.7  2 and 3 5.8  73.8 6.3 1  1.0 adenohypophyses estrous females 8 3 and 4  3.0 adenohypophyses estrous females  1.0 adenohypophyses proestrous females  3.0 ader hypophys immature females 6  6  6  3 and 4  2 and 3  3  6.4  4.6  4.8  3.6  73.1 7.1  60.6 6.5  93.8 6.8  72.1 8.5  91.5 8.2  12  15  8  10  12  2  3  6  Total number of ovulations Number of uterine glands  115.1  66.8  107.3  107.1  124.9  82.8  153.8  105.8  Endometrial rugal c e l l height, y.  12.5  11.8  22.9  22.3  26.9  25.7  27.8  23.0  Endometrial gland c e l l height, ji  10.8  10.8  17.6  16.2  19.3  19.3  19.8  17.6  102 less than 1.0 ram in diameter.  Only 1 ovulatory f o l l i c l e developed, 11.0  mm in diameter, in the ovary of a female given 700 IU PMS intramuscular. There is a tendency for the number of f o l l i c l e s less than 1.0 mm in diameter to increase with increasing dosage and for each injection route, but this parameter also varied individually. Estrogen secretion by the ovaries is evident.  Uterine weight is  increased significantly over control values at each dose level and injection route.  Histologically, endometrial glandular coiling is in-  creased over control values, and rugal and glandular epithelia are columnar . Because ovarian follicular development in response to PMS was inconsistent and not of ovulatory size, no further attempts were made to induce ovulation in fur seal pups for the purpose of quantifying LH. Immature 3- and 4-year-old females were used in a final effort to develop an assay for LH using the same species as gonadotropin donors and bioassay recipients.  The results are summarized in Table 18. PMS at the  dose level used, 1000 IU, successfully promoted the growth of large antral ovarian f o l l i c l e s ;  further, f o l l i c l e s of ovulatory size and con-  dition were found in the ovaries of a l l females, but the number of ovulatory f o l l i c l e s varied individually.  Endometrial growth is evident, with  glandular coiling, and secretory, columnar rugal and glandular epithelia. The response to 4.0 and 8.0 mg LH, and to 1.0 and 3.0 adenohypophyses from estrous, ovulatory females, and from proestrous, maturing females was inconsistent. Not a l l females in any dose level had ovulated, and the numbers of ovulations in the ovaries of females which had, did not vary with the dose level, so that quantification of LH was not poss-  103  ible.  Many of the ovulatory f o l l i c l e s showed single hemorrhagic spots  on the outer wall, indicative of incipient ovulation. The possibility exists that autopsies made 48 hours following LH administration, rather than 24 hours, might have given more consistent results.  3.  Discussion.  That PMS w i l l act in the female fur seal for the development of ovarian f o l l i c l e s of ovulatory size is indicated by the response of pups to single injections of 300-700 IU PMS, and of immature 3- and 4-yearold females to 1000 IU PMS;  ovulation was achieved among the latter  group following the administration of standardizing LH or adenohypophysi a l LH. Individual variations of ovarian follicular development in response to PMS precluded the use of fur seal pups as bioassay animals. Among the pups, a l l of approximately the same age and weight, with numerous small antral f o l l i c l e s in the ovaries, the follicular response to a given dose of PMS should be the same;  in fact, the only similarity of  response is the secretion of ovarian steroids, resulting in an estrogenic endometrium. PMS may have acted as a complete gonadotropin;  Lamond  (1960) demonstrated in hypophysectomized ewes that PMS has f o l l i c l e stimulating and luteinizing actions in the ratio of 5:1 respectively, at high (800-1500 IU) dosages.  It is suggested that the ovarian f o l l i c l e s of the  pups are preferentially sensitive to the luteinizing component of  PMS;  follicular sensitivity to LH-like HCG, with no increase in f o l l i c l e size, resulting in a similar estrogenic endometrial response, has been demonstrated.  104  Individual variation in numbers of ovulations in response to LH precluded the use of immature 3- and 4-year-old females as bioassay animals;  although a consistent follicular response to PMS was obtained,  the number of f o l l i c l e s ovulated was not consistent with LH dosage. Jainudeen et a l (1965) found similar ovulatory inconsistencies among calves 4-24 weeks of age.  The heifers received a single intramuscular injection  of 2000 IU PMS, followed in 4 days with a single intravenous injection of 5.0, 7.5, or 10.0 mg LH;  ovulatory ovarian f o l l i c l e s developed, but the  number of f o l l i c l e s varies individually, as did the number of ovulations. Lamond (1960) encountered similar inconsistencies with Merino ewes. Jainudeen and Lamond suggest that the exogenous gonadotropins have reinforced endogenous gonadotropins, with some effect on the hypothalamus. Among 3- and 4-year-old females, the action of PMS and exogenous LH is complicated by the active hypothalamic-pituitary-gonad axis, responsible for the annovulatory f o l l i c u l a r cycle;  the effects of exogenous gonado-  tropins on this axis are unknown, but may account for the inconsistent ovulatory response.  105  V.  BIOASSAY OF FOLLICLE STIMULATING HORMONE AND LUTEINIZING HORMONE IN THE ADENOHYPOPHYSES OF FEMALE FUR SEALS, USING IMMATURE RATS AS ASSAY ANIMALS  A.  Introduction  A series of bioassays for FSH and LH in the adenohypophyses of immature and maturing female fur seals are described in the following section;  intact, immature female rats were used as recipient assay  animals. The assays were intended to provide a more c r i t i c a l analysis of gonadotropic factors affecting reproductive maturity in female fur seals than the analyses provided by adenohypophysial c e l l counts and bioassays using fur seal pups and immature females as assay animals. Although the question of quantification of gonadotropic hormones from one species against a standardizing gonadotropin from a second species by means of response parameters in a third species remains, practical considerations dictated the use of a universally used and accepted system.  The white rat, in a variety of inbred strains, is commonly used  as a recipient bioassay animal;  inbreeding reduces response variation  among individuals, an important factor where experimental material is limited, and where results may be expected to vary significantly within a small range of response.  106  B.  1.  Material and Methods  The collection and classification of hypophyses from female  seals. Adenohypophyses for this series of bioassays were collected during the commercial k i l l of female fur seals in land, Alaska.  August, 1965, on St. Paul Is-  Collection, transport, and storage of the glands have been  described for, and did not differ from, the treatment of hypophyses taken for assay in fur seal pups. Adenohypophyses from immature and maturing female fur seals were classified f i r s t by age, then by reproductive condition. The latter classification was made on the basis of 3 ovarian parameters, ovarian weight, numbers of antral f o l l i c l e s , and size of the largest antral f o l licle;  and 3 uterine parameters, the index of endometrial coiling, and  the rugal and glandular epithelium c e l l height.  The results are summar-  ized in Table 19. The reproductive tracts of immature 2-year-old females are characterized by small ovaries, each containing an average 67.9 antral f o l l i c les, the largest 3.8 mm in diameter;  the index of glandular coiling in  the associated endometria averages 41.7, and the glandular and rugal epithelia are cuboidal. The average ovarian weight of immature 3-year-old females is increased 33.47o over that of 2-year-olds, and there are a significantly greater number of antral f o l l i c l e s , an average 88.7 in each ovary, the largest 4.8 mm in diameter;  similarly, the index of endomet-  r i a l coiling in the associated endometria is increased over that of 2year-olds by 34.67o, and the rugal epithelia are low columnar.  TABLE 19. GROSS AND HISTOLOGICAL ANALYSES OF REPRODUCTIVE TRACTS ASSOCIATED WITH HYPOPHYSES COLLECTED ON ST. PAUL ISLAND, ALASKA, FOR BIOASSAY IN INTACT, IMMATURE RATS Immature 2Immature 3Early Late year-old year-old proestrus proestrus females females P<0.0E>* P<0.05 P<0.0 5 Number of females Age  12  56  2  3  Ovarian weight average gms/ovary  2.8±0.18** < ;  4.2±0.10  Average/ovary number of antral follicles 1-2+mm diameter 2.5-3mm diameter largest, mm diameter % atretic  30.3*5.3 2.8*0.9 3.8*0.6 32.0  37.7*2.8 4.9*0.2 4.9*0.2 36.0  <^  33  13  3 and 4  4 and 5  4 and 5  5.0±0.16  <  5.7*0.45  5.5-0.2  36.8*2.9  I>  26.5*3.3 3.8-0.3  21.9*5.7  +  > <I  7.6-0.3 75.0  <^  9.8-0.3 87.0  110.5*14.4  <[  137.1*8.1  <'  187.1*20.8  <  31.76-2.0  +  Endometrial rugal cell height _u  10.8*0.72  12.0*0.27 < '  16.42*0.5  <  25.56-1.3  Endometrial gland cell height >i  8 . 78 +0. 4  8.93*0.3  12.13*0.3  <[  14.79*0.5  <[  <[  +  2.9-0.7  6.8-0.9 5.9*0.2 42.0  41.7*5.4  64.6*3.0  P<C).05  32  Number of uterine glands ( index of glandular coiling)  <[  Estrus  * Significant differences between means.  **.±standard error.  <  +  16.8*1.2  TABLE 20.  24 hours postpartum Postcoitem  GROSS AND HISTOLOGICAL ANALYSES OF REPRODUCTIVE TRACTS FROM MATURE FEMALES TAKEN 24 HOURS POSTPARTUM OR IMMEDIATELY POST-COITEM, ON ST PAUL ISLAND.  Ovarian weight gms  No. Graafi an follicles (mm diamete r) in ovulate ry ovary 1-2+ 2.5-3 1  6.29±0.3*  48.2±9.6  16.8±4.4  8.2±2.2  9.19i0.9  ** 32.5±7.3  ** 14.714.4  ** 2.7+J.0  * **  standard error Significant difference P 0.05  Largest  % atretic Graafian follicles  7.4±1.5  8.7±0.4  74. 2  ** 2.5+1.0  ** 85. 7 11.3+0.2  3  Mucosal epithelia height Rugal Glandular  23.2±1.0  19.8±0.5  ** 37.8±1.1  20.2+1.4  109  The ovaries of 3- and 4-year-old females in early proestrus average 5.0 gms, significantly larger than those of immature females, and each contains an average 91.1 antral f o l l i c l e s , the largest 5.9 mm in diameter, both values significantly greater than the same parameters for immature females. The endometrial index of glandular coiling is increased 41.67o over that of 3-year-old immature females, and the glandular and rugal epithelia are columnar, and significantly greater in height than those of immature 3-year-olds.  It should be noted that this class-  ification is arbitrary, as are the 2 following classifications, to the extent that adenohypophyses from females whose reproductive parameters did not f a l l within the confidence limits set by the analysis were excluded, and either placed in another, fitted reproductive classification or discarded as anamolous. Females in late proestrus and females in estrus were so classified as the result of an investigation made by Craig (1966); are summarized below and in Table 20.  the results  Twelve females were marked at  parturition on St. Paul Island, Alaska, and shot 24 hours later;  9  females were shot immediately post-coitem. The average ovarian weight of females taken postpartum is significantly less than that of females taken post-coitem;  the number of antral f o l l i c l e s is significantly  greater than that of females taken post-coitem, and the size of the ovulatory f o l l i c l e is significantly less than that in the ovaries of postcoitem females. Bartholomew (1953) estimated that mating occured 3-5 days postpartum;  presumably at parturition, females are in late proes-  trus, come into estrus during the next 3-5 days, and ovulate post-coitem. The assumption was made that a female ovulating for the f i r s t time  110 would have the same proestrus-estrus pattern as the mature females described.  Consequently, reproductive tracts with ovaries containing a  f o l l i c l e approaching ovulatory size were carefully examined, and the same parameters that marked the difference between estrus and late proestrus among mature females, did so among females ovulating for the f i r s t time. The ovaries of late proestrous females averaged 5.7 gms, significantly greater than that of early proestrous females, but the same as that of estrous females. The ovaries of late proestrous females contain 66.4 antral f o l l i c l e s each, significantly less than that of early proestrous females, but significantly more than that in the ovaries of estrous females, with an average 51.8 f o l l i c l e s in each ovary. The largest antral f o l l i c l e in the ovaries of late proestrous females averages 7.6 mm in diameter, that in the ovaries of estrous females, 9.8 mm in diameter; in both conditions, the f o l l i c l e is pre-ovulatory.  Atresia of antral  f o l l i c l e s among early proestrous females is 42.07o, among late proestrous females 75.2%, and among estrous females 87.0%. The endometrial index of glandular coiling in the uteri of late proestrous females averages 137.1, and is significantly greater among estrous females, averaging 187.1;  similarly, the height of rugal and glandular epithelia among  estrous females are increased 20.0%o and 12.07. respectively from the late proestrous condition.  It was assumed that the differences in the reprod-  uctive tracts between early proestrus, late proestrus, and estrus, represented a c r i t i c a l difference in the release of adenyhypophysial gonadotropins, which could be measured quantitatively in bioassay.  Ill  2.  Treatment of hypophyses from female fur seals.  Frozen hypophyses were pooled by reproductive condition, then thawed in ice water, and the pars anterior separated from the neural elements of the gland.  Adenohypophyses were homogenized in pairs in 4.0'  cc distilled water, using a variable speed lab motor with a glass-embedded teflon stirring rod in a stainless steel tube sunk in a beaker of ice water. The homogenate was pipetted into an 8 cc capacity flask for lyophilization.  At the end of lyophilization, the flasks were heat-  closed to maintain vacuum, and the homogenates were stored at room temperature. For each assay, the total amount of homogenate to be used in that assay was weighed, then divided by weight into individual daily doses, which were placed into small tubes and kept in a freezer at -32 degrees C until used. Doses were suspended in sterile distilled water, the amount of diluent depending on dose level and assay.  3.  Bioassay for F o l l i c l e Stimulating Hormone.  Wistar-strain intact immature female rats were used in the Steelman-Pohley bioassay for FSH, previously described. The rats were 25-28 days of age at the beginning of each assay, and weighed 30-35 gms. rats were randomized into groups of 6 for treatment;  The  food and water were  given ad libitum, and the light regime was 12 hours of dark and 12 of light. The United States National Institutes of Health, Endocrinology Study Section, donated the standardizing FSH, NIH-FSH-S5 (ovine). Human Chorionic Gonadotropin, "Antuitrin" brand, was supplied by Burrough-  112  Wellcome, Ltd. The dose levels of experimental and standardizing material were determined in preliminary assays to be the optimum dose levels which made'the most conservative use of the experimental material, and gave legitimate dose-response relationships for quantitative analyses. Two dose levels, 0.1 mg and 0.2 mg of standardizing FSH, and 2 dose levels, one-third (0.33) and two-thirds (0.66) of an adenohypophysis, were used. A total dose of 50 IU HCG was administered concurrently with FSH or adenohypophysial homogenate to each rat.  Two control groups of 6 rats  each were given saline or 50 IU HCG. Total doses of standardizing FSH or adenohypophysial homogenates with 50 IU HCG were given as 6 injections, twice a day (9:00 AM and 9:00 PM) for 3 days. Doses of FSH and HCG were made fresh to 0.5 cc with sterile saline for each injection.  Adenohypophysial homogenates and  HCG were made fresh to 0.5 cc with sterile distilled water for each injection.  A l l injections were made subcutaneously over the shoulder  blades, alternating left and right. the last injection;  Autopsies were made 24 hours after  a l l ovaries were removed and weighed individually  to the nearest tenth of a milligram on a torsion balance.  4.  Bioassay for Luteinizing Hormone,  (a) Basis for the assay. The bioassay used for quantification of LH was suggested by Zarrow et al (1959), based on superovulation in the immature, intact rat in response to ovulating hormones, subsequent to "priming" with Pregnant Mare Serum (PMS), an FSH-like gonadotropin which induces ovarian ovulatory  113  follicular development. Zarrow and Wilson (1961) found that superovulation in the immature,  intact rat is age-dependent:  only females 21 days of age and older  respond completely to an i n i t i a l dose of PMS followed by an ovulating dose of HCG;  further, maximum numbers of ova were shed at 23-32 days  of age. The data suggest the necessity of antral ovarian f o l l i c l e s (17 days of age) for response to PMS, and for optimum bioassay procedures, an age of 23 days. Zarrow and Quinn (1963) found that 30-70 IU PMS administered to intact rats 23-32 days of age resulted in individual ovulation of an average 16.7 ova without subsequent administration of ovulating hormones. The evidence suggested endogenous LH release; a l though PMS contains some LH activity the latter is insufficient for the number of ovulations observed. Immature rats hypophysectomized at 25 days, and given 30 IU PMS at 28 days, did not ovulate without the subsequent administration of 10 IU HCG, confirming the suspected release of endogenous LH.  Hypophysectomies performed up to 56 hours after the  administration of PMS to intact rats blocked PMS-induced ovulation; hypophysectomies performed after 56 hours failed to block PMS-induced ovulation, indicating that endogenous LH release occurs within 56 hours after PMS administration, and suggesting a " c r i t i c a l period" in immature rats.  Rennels and O'Steen (1967) confirmed the c r i t i c a l period in  immature rats.  Hagino (1969) demonstrated the neural control of endog-  enous LH release in immature female rats:  PMS administered to 28-day-  old females resulted in ovulation at 31 days;  systemic phenobarbitol  administered at 2-4 PM on day 30 blocked PMS ovulation, and bilateral electrical stimulation of the ventro-medial.-arcuate nuclei overcame the  114  pharmacological blockade, for the release of ova. (b) Method of the assay. The evidence of ovulation in response to PMS alone, among intact, immature rats, as a result of endogenous LH release, was incorporated into the bioassay used to quantify LH in the adenohypophyses of female fur seals.  In preliminary experiments, ovulation occurred in response  to 50 IU PMS in a l l of the Wistar-strain, intact, 26-day-old rats used, with an average 11.6 ova shed by each female. At 26 days of age, 50 IU PMS, followed in 56 hours (to coincide with the c r i t i c a l period) by ovulating hormones, induced individual ovulation significantly greater in number of shed ova than that induced by PMS alone. Further preliminary experiments established that, using intact females 26-30 days of age, weighing 30-36 gms, dose-response relationships could be obtained with exogenous LH and adenohypophysial homogenate administered 56 hours after administration of 50 IU PMS.  The optimum dose-response to LH was  achieved at 5 and 10 micrograms;  the optimum dose of adenohypophysial  homogenate which made the most conservative use of the material available was one-third (0.33) and two-thirds (0.66) of an adenohypophysis. The United States National Institutes of Health, Endocrinology Study Section, donated the standardizing LH, NIH-LH-S5 (ovine). Pregnant Mare Serum, "Wellcome" brand, was supplied by Burroughs Wellcome, Ltd. Wistar-strain intact female rats, 26-30 days of age and weighing 30-35 gms, were randomized and caged in groups of 6 for treatment. Food and water were given ad libitum;  the light regime was 12 hours of light,  12 of dark. Fifty IU PMS in 0.5 cc sterile saline was given subcutan-  115  eously over the shoulder blade to each female, followed in 56 hours by a single subcutaneous injection of LH in 0.5 cc sterile saline at 2 dose levels, 5 micrograms or 10 micrograms, or by a single subcutaneous injection of adenohypophysial homogenate in 1.0 cc sterile d i s t i l l e d water at 2 dose levels, 0.33 adenohypophysis or 0.66 adenohypophysis. Two control groups of 6 females each received saline or 50 IU PMS alone, the ovulatory dose replaced by saline. Autopsies were made 24 hours following administration of ovulatory hormones. Ovaries were removed and weighed to the nearest tenth of a milligram.  A l l ovaries were fixed and stored in 10% formalin for sub-  sequent paraffin serial sectioning. Ovulated f o l l i c l e s were counted from each serially sectioned ovary, and the number of ovulated f o l l i c l e s was used as the response parameter in the quantifying regression analyses. (c)  Statistical analysis.  The regression analysis suggested by Burn (in Burn et a l , 1950), and illustrated by Steelman and Pohley (1959) was used to quantify adenohypophysial FSH and LH.  The number of ovulated f o l l i c l e s in response  to PMS only, in the assays for LH, was incorporated in the regression analysis as a control value, in the same way that ovarian weight from the HCG control level is incorporated in the regression analysis for quantification of FSH.  Comparisons for significant differences between bio-  assays were made with Scheffe's test (in L i 1964).  116  C. The Adenohypophysial Content of F o l l i c l e Stimulating Hormone and Luteinizing Hormone  The results of bioassays for the quantification  of FSH and LH in  the adenohypophyses of female fur seals are summarized in Tables 21 and 32, and Figure 5. Adenohypophysial concentration and content of FSH and LH are given in Tables 26 and 32, and Figures 5 and 6.  1. Two-year-old immature females (Tables 21 and 26; Tables 27 and 32). The adenohypophyses of 2-year-old females contain significantly more FSH than adenohypophyses in any other reproductive group examined. Adenohypophyses from these females, with an average dry weight of 22.4 mg each, contain 0.6773 mg equivalents of standardizing FSH at a concentration of 0.0165 mg FSH per mg dry adenohypophysial weight.  Similarly,  the adenohypophyses of 2-year-old females contain significantly more LH than adenohypophyses in any other reproductive group examined. Adenohypophyses, with an average dry weight of 20.0 mg each, contained 34.64 microgram equivalents of standardizing LH at a concentration of 1.732 micrograms LH per mg dry adenohypophysial weight.  2. Three-year-old immature females (Tables 22 and 26; Tables 28 and 32). The values for FSH concentration and content in the adenohypophyses of these females are significantly less than those for 2-year-old females, and represent a decrease of 54.6% in concentration and 19.7% in content (Figure 5). Adenohypophyses from these females, with an average  117  dry weight of 34.8 mg each, contain 0.5111 mg equivalents of standardizing FSH at a concentration of 0.0147 mg FSH per mg dry adenohypophysial weight. The values for adenohypophysial concentration and content of LH among these females are also significantly less than the same values for 2-year-old females, constituting a decrease of 59.7% in LH concentration and 44.3% in LH content (Figure 6). Adenohypophyses from 3year-old females, with an average dry weight of 27.2 mg, contain 19.32 microgram equivalents of standardizing LH, at a concentration of 0.7103 microgram LH per mg adenohypophysial dry weight.  3.  Females in early proestrus (Tables 23 and 26; Tables 29 and  32). Adenohypophyses from early proestrous females, with an average dry weight of 20.6 mg each, contain 0.4262 mg equivalents of standardizing FSH at a concentration of 0.0207 mg FSH per mg dry adenohypophysial dry weight. The values for concentration and content of adenohypophysi a l FSH among these females do not differ significantly from the same values for 3-year-old immature females.  Although the values are not  significantly different, FSH concentration is 28.2% greater than among 3-year-old immature females, but paradoxically, because of a lesser adenohypophysial weight, the total amount of FSH available to early proestrous females is 16.4% less than that available to 3-year-old immature females (Figure 5). The values for concentration and content of LH among early proestrous females also do not differ significantly from the same values for 3-year-old immature females.  Adenohypophyses from early proestrous  118  TABLE 21. BIOASSAY FOR FSH OF ADENOHYPOPHYSES FROM 2-YEAR-OLD IMMATURE FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT; THE END POINT MEASURED IS OVARIAN WEIGHT.  n  Ovarian weight, mg mean reaponse*SE A  B  Total variance of response  Slope b  Index of precision  Treatment 0.1. 0.2 mg FSH  12  31.2*1.5  47.9*1.6  56.43  158.611  .067  0.33, 0.66 12 adenohypophyses  47.6*2.6  87.4-4.4  224.45  354.805  .069  90-,  A DOSE LEVEL  B  R-SE , (b test b standard) 2.236* 0.2535  119  TABLE 22. BIOASSAY FOR FSH OF A DENOH YPOPH YS ES FROM 3-YEAR-OLD IMMATURE FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT; THE END POINT MEA8URED IS OVARIAN WEIGHT.  Treataent  n  Ovarian w<tight, ag aean response*SE A B  Total varlance of response  Slope b  Index of precision  1) 0.1,0,2 12 mg FSH 0.33,0.66 12 adeno.  31.2+1.5  47.9il.6  57.43  123.8  0.087  43.5*1.5  67.2*3.7  92.34  230.3  0.086  2) 0.1,0.2 mg FSH  12  20.8*0.6  36.9*2.5  77.32  128.1  0.072  0.33,0.66 12 adeno.  34.0*1.1  52.2*1.1  29.47  215.6  0.035  17.6-0.6  27.10±0.9  13.31  108.5  0.046  23.8*0.8  44.013.2  32.85  188.0  0.067  23.2*1.1  37.3±1.8  155.14  129.3  0.122  33.7±1.5  54.4-2.3  167.79  218.8  0.103  3) 0.1,0.2 12 mg FSH 0.33,0.66 12 adeno. 4) Coablned 0.1,0.2 36 mg FSH 0.33,0.66 36 adeno.  D FSH • ADENOHYPOPHYSES 1 A DOSE LEVEL  B  R*SE (b test' b standard) 1.860* 0.285S  1.683^ 0.0989  1.7270.1819  1.6925* 0.1462  120  TABLE 23.  BIOASSAY FOR FSH OF ADENOHYPOPHYSES FROM EARLY PROESTROUS FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT; THE END POINT MEASURED IS OVARIAN WEIGHT. Ovarian w<sight, mg mean respc>nse*SE B A  Total variance of response  Slope b  Index of precision  R*SE (b test b standard) 1.53070.1878  Treatment  n  1) 0.1,0.2 mg FSH  12  17.6*0.6  27.1*0.9  13.31  96.8  0.052  0.33,0.66 12 adeno.  22.5*0.8  37.5*3.6  62.70  148.3  0.102  12  20.8*0.6  36.9*2.4  77.32  116.0  0.081  0.33,0.66 adeno. 12  35.3*0.6  50.9*2.4  70.60  200.7  0.051  24  19.2*0.5  32.0*1.6  71.09  106.4  0.099  0.33.0.66 adeno. 24  28.9*1.4  44.2*2.5  100.71  174.5  0.1110  2) 0.1,0.2 mg FSH  3) Combined 0.1,0.2 mg FSH  a FSH • ADENOHYPOPHYSES  DOSE LEVEL  1.7300* 0.1694  1.6393* 0.3268  121  TABLE 24. BIOASSAY FOR FSH OF ADENOHYPOPHYSES FROM LATE PROESTROUS FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT; THE END POINT MEASURED IS OVARIAN WEIGHT.  n  Ovarian weight, mg mean response-SE B A  Total var- Slope b iance of response  Index of precision  0.1, 0.2 mg FSH  12  31.2*1.5  47.9*1.6  57.43  134.1  0.079  0.33, 0.66 adenohypophyses  12  42.0+1.2  58.6*1.8  _ 56.85  198.5  0.052  Treatment  DOSE LEVEL  R*SE (b test/ b standard) 1.481* 0.1024  122  TABLE 25. BIOASSAY FOR FSH OF ADENOHYPOPHYSES FROM ESTROUS FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT; THE END POINT MEASURED IS OVARIAN WEIGHT.  Treatment  n  0.1, 0.2 mg FSH  12  Ovarian weight, mg mean responselSE B A  Total variance of response  Slope b  Index of precision  R*SE (b test b standard 0.727 • 0.055 ~  27.6*0.8  43.9*0.8  13.65  140.7  .041  0.33, 0.66 adenohy12. 25.8*0.7 pophyses  33.5*0.8  12.81  102.4  .050  6Q_  A DOSE LEVEL  B  TABLE 26.  CONCENTRATION AND CONTENT OF ADENOHYPOPHYSIAL FOLLICLE STIMULATING HORMONE, QUANTIFIED IN BIOASSAY, USING IMMATURE INTACT FEMALE RATS AS ASSAY ANIMALS. ' ' Immature 2year-old females P<0.05*  Immature 3year-old females  Late proestrus  Early proestrus P<0. C )5  P<0.C)5  Estrus  V<$).05  Number of assays  1  3  2  1  1  Number of rats/ dose level  6  6  6  6  6  20.59+3.4  26.14  24.37  Adenohypophysial dry weight, mg-SE  22.43  34.8+2.6  FSH contration mg equivalents NIHFSH- SE 1 2 3 combined  0.0302±0.0034 « >  FSH content mg equivalents NIH-FSH^SE 1 2 3 combined  0.5626+0.0852+ 0.4507±0.0431+ 0.5096i0.0276+ 0.3977^0.0482+ 0.5148+0.0492+ : 0.677310.0767 " > 0.5111+0.0417 = 0.4262+0.0844 =  0.0165+0.0025+ 0.0188+0.0018+ 0.0166l'0.0009+ 0.0231+0.0844+ 0.0104±0.0001+ 0.0147-0.0012 == 0.0207-0.0041 = : 0.0147+0.0007 >> 0.0076+0.0005  * Significant differences between means. + No significant differences between means  0.3842+0.0182 I > 0.1889^0.0124  724> F I G U R E S . THE G R A P H ILLUSTRATES ADENOHYPOPHYSIAL C O N T E N T AND CONCENTRATION  MG EQUIVALENTS  2  B  e  8  J  I  L.  __L_  2-YEAR-OLD  3-YEAR-OLD  8  8-  S  L  I  L  IMMATURE  IMMATURE  EARLY PROESTRUS  LATE PROESTRUS  ESTRUS  2-YEAR-OLD  IMMATURE  3-YEAR-OLD IMMATURE EARLY PROESTRUS  LATE PROESTRUS  ESTRUS  NIH-FSH  8 J  FSH  125  TABLE 27. BIOASSAY FOR LH OF ADENOHYPOPHYSES FROM 2-YEAR-OLD IMMATURE FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT; THE END POINT MEA3URED IS NUMBER OF OVULATIONS.  Treatment 5,10,ng  LH  n 12  Number of ovulations Total vcirMean reaponse*SE lance oi response A B  35.3*1.3  53.5*1.9  62.48  Slope b  11.23  Index of precision  0.982 2. 2863* 0.2567  0,33, 0.66  adenohypophyses  RtSE (b test/ b standard)  12  59.75*1.5  77.6*2.2  83.74  25.68  A DOSE LEVEL  0.457  B  126  TABLK 28.  Treatment  BIQA8SAY FOR LH OF ADBNGHYPOPHYSKS FROM 3-YEAR-OLD IMMATURE FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT; THE END POINT MEASURED IS NUMBER OF OVULATIONS.  n  1) 5,10 ^ig LH 12 0.33,0.66 12 adeno. 2) S.lO^ug 12 LH 0.33,0.66 12 adeno. 3) 5 , 1 0 ^ 12 LH 0.33,0.66 12 adeno. 4) Combined 5.10 jig 36 LH 0.33,0.66 36 adeno.  Number of ovulations mean reap*>nse*SE B A  Total var- Slope b iance of response  Index of precision  35.3*1.3  53.5*1.9  62.544  18.1  0.609  45.2*1.4  72.8*3.3  54.666  27.8  0.594  38.8*1.1  54.0*2.6  94.0222  16.6  0.774  43.1*1.5  67.0*2.9  126.9927  22.6  0.667  28.6*1.9  50.5*1.7  80.72  19.0  0.667  31.5*1.3  46.7*2.3  82.3332  18.1  0.680  34.2*1.1  52.7*1.1  85.8753  17.9  0.732  39.9*1.3  62.2*2.5  L80.9642  22.8  0.981  A DOSE LEVEL  B  R*8E (b teat b • tanda rd) 1.536* 0.1300  1.. 366 0.098  0.95OO* 0.1341  1.2763* 0.0685  127  TABLE 29. BIOASSAY FOR LH OF ADENOHYPOPHYSES FROM EARLY PROESTROUS FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT; THE END POINT MEASURED IS NUMBER OF OVULATIONS. Number of ovulations Total vai mean response*SE lance of response B  Treatment 5.10 /tg LH 0.33,0.66 adenohypophyses  Slope b  Index of precision  12  28.6*1.9  50.5*1.7  80.7196  18.1  0.702  12  32.8*1.1  46.1*2.0  60.4696  17.1  0.613  A DOSE LEVEL  B  128  TABLE 30.  BIOASSAY FOR LH OF ADENOHYPOPHYSES FROM LATE PROESTROfS FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT: THE END POINT MEASURED IS NUMBER OF OVULATIONS. Number of ovulation!9 Total variance of •ean resp<onsel SE response B A  Slope b  Index of precision  Treatment  n  S.lO^ig LH  12  35.2^1.2  50.3^1.2  33.2648  14.0  0.582  12  50.9±1.1  78.2^3.6  70.0500  28.3  0.572  0.33,0.66 Adenohypophyses  80-,  A DOSE LEVEL  B  R ! S E  (b test b standard) 2.0220: 0.2054  129  TABLI 31,  BIOASSAY FOR LH OF ADENOHYPOPHYSES FROM ESTROUS FEMALE FUR SEALS. THE RECIPIENT ANIMAL IS THE IMMATURE FEMALE RAT; THE END POINT MEASURED IS NUMBER OF OVULATIONS. Nuaiber of ovulations Total var•ean response-8K lance of response B  Treatment  Slope b  Index of  1USE  (b test, b standard)  5,10 jig LH  12  38.8-1.2  54.0-2.1  70.3802  14.20  0.800  0.33,0.66 adenohypophyses  12  38.8-1.0  50.711.7  41.1214  13.88  0.622  0.9776^ 0.1396  60-  g 50. O  9776±t3M Of L H  \  P  3 o &  40J  cc  D  LH • ADENOHYPOPHYSES  DOSE LEVEL  TABLE 32. CONCENTRATION AND CONTENT OF ADENOHYPOPHYSIAL LUTEINIZING HORMONE, QUANTIFIED IN BIOASSAY, USING INTACT, IMMATURE FEMALE RATS AS. ASSAY ANIMALS. Immature 2year-old females  Immature 3year-old females  K0.05*  Late proestrus  Early proestrus P<0.05  P<0.05  Estrus  P<0.05  Number of assays  1  3  1  1  1  Number of rats/ dose level  6  6  6  6  6  22.88  30.0  Adenohypophys i a l dry weight, mg+SE LH concentration jig equivalents NIH-LH-SE 1 2 3 combined LH content ^jg equivalents NIH-LH+SE 1 2 3 combined  20.0  27.2*1.47  1.7320+0.1944 > >  34.64+3.888  0.9309*0.0787+ 0.6901*0.499 + 0.5390*0.0760+ 0.7103+0.0381 « > 0.538+0.0331  22.5  < ^ 0.9190*0.0933 >>  23.2725*1.9675 20.7030*1.4970 14.3690*2.0210 I > 19.3201*0.7576 I > 12.2800*0.7576 <C 27.57*2.7990 i  * Significant difference between means. + No significant difference between means  0.4929+0.0705  '> 11.0902*1.5862 •  IB 1  FIGURE 6. THE GRAPH ILLUSTRATES ADENOHYPOPHYSIAL LH CONTENT AND CONCENTRATION  MICROGRAM EQUIVALENTS —  ro  _L  L_  oi  2 YEAR OLD  NIH-LH  o  8  in  s  IMMATURE  3 YEAR OLD IMMATURE O  m  EARLY PROESTRUS LATE PROESTRUS  ESTRUS  S  g  £  g  J_  L_  2-YEAR-OLD 3-YEAR-OLD IMMATURE  3 2  EARLY PROESTRUS LATE PROESTRUS  ESTRUS  IMMATURE  _L_  OB J  132  females, with an average dry weight of 22.9 mg, contain 12.28 microgram equivalents of standardizing LH, at a concentration of 0.538 microgram LH per mg dry adenohypophysial weight. These values are 36.8% less in content and 24.37o less in concentration than the same values for 3-year-old immature females (Figure 6).  4.  Females in late proestrus (Tables 24 and 26; Tables 30 and 32).  Adenohypophyses' from females in late proestrus, with an average dry weight of 26.1 mg each, contain 0.3842 mg equivalents of standardizing FSH, at a concentration of 0.0147 mg FSH per mg dry adenohypophysial weight. These values do not differ significantly from the same values for early proestrous females;  nevertheless, FSH content is 28.27o less,  and concentration 9.9% less among late proestrous than among early proestrous females (Figure 5). Adenohypophyses from late proestrous females, with an average dry weight of 30.0 mg each, contain 27.57 microgram equivalents of standardizing LH, at a concentration of 0.9190 microgram LH per mg dry adenohypophysial weight. Both values are approximately 50% more than the same values for early proestrous females (Figure 6).  5.  Females in estrus (Tables 25 and 26;  Tables 31 and 32).  Adenohypophyses from females in estrus, with an average dry weight of 24.9 mg each, contain 0.1889 mg equivalents of standardizing FSH at a concentration of 0.0076 mg FSH per mg dry adenohypophysial weight. Both values are significantly less than those for late proestrous females, a decrease of approximately 50% for both concentration and content of FSH  133  (Figure 5). Adenohypophyses from females in estrus, with an average dry weight of 22.5 mg, contain 11.0902 microgram equivalents of standardizing LH, at a concentration of 0.4929 microgram LH per mg dry adenohypophysial weight. Both values are significantly less than the same values for late proestrous females, a decrease of 46.47, in concentration and 59.87, in content (Figure 6).  134  D.  Discussion  It should be emphasized that the gonadotropic content of an adenohypophysis represents the difference between adenohypophysial synthesis and release of that gonadotropin at the time the gland is taken.  To  make comparisons between females of different ages and varying reproductive conditions, and to draw inferences from the evidence obtained, as has been done in the present investigation, the females must be taken at times when reproductive conditions resulting from gonadotropic synthesis and release may be considered equivalent in time.  In the present invest-  igation, a l l donor females were taken, for each experiment, at the same time in August during annual commercial k i l l s , and for each reproductive group, at the peak of ovarian and uterine development. A l l the females, regardless of age or reproductive condition, may be considered equal in time;  the assumption is.made that adenohypophysial gonadotropic  activ-  ity is at or near maximum in each reproductive condition examined.  The  difference in synthesis, storage, and release of gonadotropic hormones which mark the difference between reproductive immaturity and the ovulatory capability of reproductive maturity should be more obvious at this culmination than at any other time during the seasonal follicular cycle which extends from May to August. The following inferential analyses of reproductive conditions have been made from the results of bioassays, using rats as assay animals, which quantified LH and FSH in the adenohypophyses of female fur seals.  The results of differential adenohypophys-  i a l c e l l counts, and bioassays using fur seal pups, are used as supportive evidence, and are compared to the bioassays using rats.  135  1. Two-year-old immature females. The concentration of adenohypophysial FSH, 0.0302 mg per mg dry adenohypophysial weight appears to be particularly high among these females;  the content of FSH, 0.6773 mg per adenohypophysis, provides a  more conservative and acceptable estimate of the amount of FSH available to the female (Table 26). FSH has been synthesized and released for ovarian antral follicular development since May, suggesting not storage, but synthesis at a greater rate than release.  The evidence of differ-  ential adenyhypophysial c e l l counts is supportive:  only 9.57, of the  folliculotrops counted were fully granulated storage c e l l s , while 82.47° of the folliculotrops were partly granulated, indicative of synthesis at a greater rate than release (Figure 4).  In general, the evidence sug-  gests a high concentration and content of FSH, primarily due to a rate of synthesis greater than rate of release.  The results of bioassays  using fur seal pups confirms the high concentration and content of adenohypophysial FSH among 2-year-old immature females.  In the f i r s t assay,  FSH content was 0.5655 mg per adenohypophysis, at a concentration of 0.0370 mg FSH per mg dry adenohypophysial weight, both values very close to those obtained in the present assay (Table 9). FSH content and concentration were apparently doubled in the second assay, with an FSH content of 1.4030 mg per adenohypophysis, at a concentration of 0.0660 mg FSH per mg dry adenohypophysial weight.  The apparent disparity of re-  sults between the two assays is the effect of a decreased response to standardizing FSH in the second assay, and a large variance;  neverthe-  less, the lower limits of the confidence interval for the second year reach upper confidence interval limit for the f i r s t year (Table 13).  136  Concentration and content of LH in the adenohypophyses of 2-yearold females is greater than the same values for any other reproductive condition examined, suggesting LH storage (Table 32).  Storage is also  suggested by endometrial quiescence during the follicular cycle:  LH  necessary for.complete f o l l i c u l a r maturation and estrogen synthesis and release, the latter initiating endometrial growth, has not been available to the ovaries.  The relatively high percentage, 24.67,, of fully  granulated interstitiotrops, significantly greater than in any other reproductive condition examined, is supportive evidence for LH storage. Further, 56.67, of the interstitiotrops counted were partly granulated, significantly greater than in any other reproductive condition examined, indicating a greater rate of synthesis than release (Figure 4). A total 78.47, of the interstitiotrops counted would maintain the high levels of LH concentration and content demonstrated in bioassay.  2. Three-year-old immature females. The ovaries of 3-year-old females contain antral f o l l i c l e s no greater in size and number than those in the ovaries of 2-year-old females taken at the same time in August;  but ovarian weight is doubled,  and associated endometria show estrogen activation in increased glandular coiling and height of rugal epithelia over the same parameters in the endometria of 2-year-old females (Table 19).  Similar endometrial  activation is evident during the follicular cycle in June and July. During the cycle, the release of adenohypophysial LH has matured the ovarian f o l l i c l e s , which consequently synthesize and release estrogen, resulting in endometrial growth.  137  Predictably, there is a dramatic decrease in adenohypophysial LH concentration and content, 59.77, and 44.3% respectively, from the same values for 2-year-old females (Table 32). The evidence of cyclic LH i release suggests l i t t l e LH storage, but a continuous release of LH since June.  The adenohypophyses of 3-year-old females show a marked decrease  in FSH concentration, 54.67. of the same value for 2-year-old females (Table 26). The decrease in FSH content, 19.77, less than the same value for 2-year-old females, is a more conservative estimate resulting from a 35.77, increase in adenohypophysial weight, although significantly less than FSH content among 2-year-old females, probably more accurately estimates FSH available to the female.  The size and number of ovarian an-  tral f o l l i c l e s among 3-year-old females is the same as those among 2-year old females, suggesting rates of FSH synthesis and release nearly equal between the two age groups. 3.  Early proestrous females.  The concentration of adenohypophysial FSH among early proestrous females is increased 28.27, from FSH concentration among 3-year-old female but adenohypophysial content, the total amount of FSH available to the female, is decreased 16.47, (Table 26). The apparent contradiction is explained only in part by the 40.97, decrease in adenohypophysial weight of early proestrous females, compared to 3-year-old females. Nevertheless, the release of FSH has been cyclic since June, for the maintenance of ovarian f o l l i c l e s , which, in August, do not differ in size and number from those found in the ovaries of immature 3-year-old females, and presumably rates of FSH synthesis and release would approximate those of  138  3-year-old females.  Consequently, the high concentration of FSH suggests  storage. The evidence of differential c e l l counts of folliculotrops in the adenohypophyses of early proestrous females is confirmatory: 29.7% of the folliculotrops counted were fully granulated storage c e l l s , significantly more than in any other reproductive condition examined, and 56.07, were partly granulated, suggesting a rate of synthesis greater than the rate of release (Figure 4).  A preponderant 85.77o of the f o l l i c u l o -  trops counted would account for the high FSH concentration demonstrated in bioassay. Concentration and content of adenohypophysial LH are decreased 24.37, and 36.57, respectively from the same values for 3-year-old immature females.  Similar to FSH, the release of LH has been cyclic since  June, for f o l l i c u l a r maturation and estrogen synthesis and release, the latter indicated by estrogen activation of the associated endometrium, which, in August, is greater in a l l measured parameters than those of 3year-old females (Table 32).  The evidence suggests a rate of LH release  greater than that of 3-year-old immature females. The suggestion is confirmed by the evidence of differential adenohypophysial c e l l counts: 59.67> of the interstitiotrops counted were degranulated, indicating a rate of release equal to the rate of synthesis, 35.27, were partly granulated, and only 5.17, were fully granulated (Figure 4).  4.  Late proestrous females.  Adenohypophysial FSH concentration and content among late proestrous females are decreased 28.27, and 9.97, respectively from the same values for early proestrous females (Table 26).  Most remarkable is the  139  increase in LH concentration and content, 41.57o and 54.57 greater respectively than.the same values for early proestrous females (Table 32). Numbers of antral f o l l i c l e s are significantly fewer in the ovaries of late proestrous females than in the ovaries of early proestrous females, but 75.07, of the f o l l i c l e s in the ovaries of late proestrous females are atretic, while only 42.07o are atretic in the ovaries of early proestrous females (Table 19). The ovaries of late proestrous females are characterized by a single healthy f o l l i c l e approaching ovulatory size.  The as-  sociated endometria have approached maximum estrogenic development: the measured parameters are a l l significantly greater than those of early preostrous females, but are significantly less than those of estrous females. The evidence suggests a further release of FSH from the early proestrous condition.  The evidence further suggests LH retention, which  undoubtedly contributes to the marked follicular atresia.  The estrogenic  endometrial development., greater than that of early proestrous, must be assumed to be the result of LH release prior to late proestrous;  there  is presently no way of estimating the time elapsed between early and late proestrous, but obviously sufficient time for the characteristic enlargement of a single ovulatory f o l l i c l e , and for endometrial growth.  5.  Estrous females.  The values for adenohypophysial concentration and content of FSH among estrous females are respectively 48.37, and 53.47, less than the same values for late proestrous females (Table 26). The values for adenohypophysial concentration and content of LH are respectively 46.47 and 59.87, less than the same values for late proestrous females (Table 32). The  140  results of the second bioassay using female fur seal pups tends to confirm the decrease in adenohypophysial FSH; although the values for FSH concentration and content are greater than those obtained in the present bioassays, they are nevertheless 45.57, and 42.77. less than the same values for 2-year-old immature females obtained in the same assay (Table 13). The evidence suggests massive release of both gonadotropins. are confirmed by differential adenohypophysial c e l l counts.  The data Of the f o l -  liculotrops counted, 44.57. are degranulated, 68.47 more than the same value for early proestrous females (Figure 4). counted interstitiotrops are degranulated.  Similarly, 57.07. of the  It should be noted that the  values for fully granulated, partly granulated, and degranulated interstitiotrops are not significantly different between early proestrous and estrous females (Table 3, Figure 4); similarly, the values for adenohypophysial LH concentration and content do not differ between early proestrous and estrous females.  This evidence suggests LH release from the  stored condition demonstrated among late proestrous females. Presumably the massive release of gonadotropins is associated primarily with ovulation, which has not yet occured.  The release of LH  and FSH i s , however, reflected in the associated reproductive tracts. The size of the characteristic  ovulatory f o l l i c l e is increased 22.57.  from that of the late proestrous condition, and is significantly larger. The index of endometrial glandular coiling is increased 26.77. from that of the late proestrous condition, and is significantly greater, and both glandular and rugal epithelia are significantly increased in height over the late proestrous condition (Table 19).  141  VI.  DISCUSSION AND CONCLUSIONS  A.  Introduction  The hypothalamic neurohormonal control of adenohypophysial gonadotropic storage, synthesis and release is well established and has been extensively reviewed (Markee et al 1952; McCann and ett 1959; Harris 1964; Sawyer 1964; McCann and  Ramirez 1964; Ever-  Dhariwal 1967; Flerko  1967). For the present discussion, the mechanisms salient to a consideration of the hypothalamic control of reproductive maturity are reviewed. Of necessity, the mechanisms described are primarily those controlling reproduction in the rat, since only in this mammal has the neural control of reproductive capacity been directly and completely examined.  142  B.  Evidence for the Dual Hypothalamic Control of Adenohypophysial Gonadotropins in the Adult Cyclic Rat.  A discrete area of the hypothalamus synthesizes the small polypeptide neurohormones which have been isolated and identified as Luteinizing Hormone Releasing Factor (LH-RF) and F o l l i c l e Stimulating Hormorte Releasing Factor (FSH-RF) (Dhariwal et al 1965; Igarashi and McCann 1964; Igarashi et al 1964; McCann et al 1964; Ramirez et al 1965), and which act as factors for the cellular release of adenohypophysial gonadotropic hormones. This "hypophysiotropic area" (Halasz et al 1962) includes the median eminence and parts of the arcuate and ventro medial nuclei (Halasz et al 1965). The tubero-infundibular neural tract, originating in the hypophysiotropic area and terminating on the portal vascular system at the level of the median eminence, neural stalk, and pars tuberalis, is the transport mechanism for LH-RF and FSH-RF to the portal vascular system (Szentagothai 1964; Daniel 1967; Tejasen and  Everett 1967).  The  portal vascular system carries the gonadotropic releasing factors to discrete areas of the adenohypophysis for cellular release of gonadotropic hormones (Adams et al 1964, 1966). Lesions within the hypophysiotropic area result in a condition of constant diestrus (McCann and Friedmann .1960; Taleisnik and McCann 1961), as do implants of estrogen (Lisk 1960; Palka et al 1966), FSH (Corbin and Daniels 1967), and LH (David et al 1966), suggesting a blockage of gonadotropin release. Areas of the hypothalamus which do not secrete gonadotropic releasing factors are also implicated in the control of reproductive c y c l i c i t y .  Lesions of the preoptic-suprachiasmatic area result in a  condition of constant estrus (Bogdanove 1963), as do implants of estrogen  143  (Palka et al 1966; Lisk 1960);  pharmacological blockade of ovulation is  overcome by electrical stimulation of this area (Taleisnik and McCann 1961; Markee et al 1952). Deafferentation of the hypophysiotropic area which precludes the anterior hypothalamus (preoptic, suprachiasmatic, and anterior hypothalamic nuclei) results in constant diestrus;  inclusion  of the anterior hypothalamus in the surgically isolated area induces constant estrus (Butler and Donovan 1969; Halasz and Pupp 1965; Koves and Halasz 1969; Palka et al 1969). The evidence indicates 2 levels of reproductive control.  The hy-  pophysiotropic area of gonadotropic releasing factor synthesis and release is concerned directly with release of adenohypophysial gonadotropic hormones. The anterior hypothalamus, which does not synthesize gonadotropic releasing factors, but is neurally connected to the hypophysiotropic area, may control the release of ovulatory amounts of gonadotropins.  The 2 levels act together for reproductive c y c l i c i t y .  The feedback of gonadal steroid to the hypothalamus and adenohypophysis for the control of reproductive cyclicity has long been established (Nalbandov 1964). Most recently, the sites of gonadal steroid reception in the hypothalamo-hypophysial  complex have been demonstrated directly.  Estrogen receptor sites have been identified with radioactively labelled estrogens in the median eminence, and in the arcuate and ventro-medial hypothalamic nuclei (hypophysiotropic area); in the anterior hypothalamus; and in the adenohypophysis (Palka et al 1966; McGuire and Lisk 1969; Anderson and Greenwald 1969). has also been demonstrated:  Internal feedback of gonadotropic hormones  LH implanted in the median eminence decreases  plasma LH concentration (Corbin and Story 1967; David et al 1965);  FSH  144  implanted in the median eminence decreased adenohypophysial FSH content and hypothalamic FSH-RF (Corbin and Story 1967; Szontagh and Uhlarik 1964). Ovarian steroids and gonadotropic hormones are assumed to act in a feedback mariner either as depressants or activators, of the synthesis and release of gonadotropic releasing factors from the hypophysiotropic area.  145  C.  Evidence for the Dual Hypothalamic Control of Adenohypophysial Gonadotropins in the Adult Cyclic Rat.  With the identification of the hypothalamic control of adenohypophysial gonadotropin release, and the development of sensitive  assays  for FSH and LH, an integrated explanation of the cyclic release of gonadotropic hormones in the female rat has been possible. The release or "surge" of ovulatory quanta of LH during a 3 hour " c r i t i c a l period" on the afternoon of  proestrus between 2 PM and 3 PM  has been established, as has its neurological control (Everett 1956; Markee et al 1952; Sawyer et al 1949). Adenohypophysial LH content decreases through estrus;  plasma LH is detectable at metestrus, but undetectable  at diestrus, although adenohypophysial LH concentration and content are s t i l l low, relative to peak values on the morning of proestrus (Anderson and McShan 1966; Schwartz and Bartosik 1962; Ramirez and McCann 1964). The highest values of adenohypophysial FSH concentration and content during the estrus cycle are found on the morning of proestrus;  a  sharp decline in FSH concentration occurs between 9 AM and 5 PM on the day of proestrus (Caligaris et al 1967). The proestrus release of FSH is prevented by pharmacological blocking or ovariectomy before, but not after 9 AM on the day of proestrus, establishing neurological control of an FSH "surge" during a " c r i t i c a l period" which precedes the LH surge by 5 hours (Caligaris et al 1967; McClintock and Schwartz 1968). Goldman and Mahesh (1968, 1969) confirmed the proestrus release of FSH in rats, and further established that FSH by itself w i l l induce ovulation, suggesting that the proestrus release of FSH is in some way necessary to normal ovulation.  146  Lawton and Sawyer (1968) examined the timing of gonadotropin and ovarian steroid release at diestrus, necessary for ovarian and uterine changes preceding proestrus. Hypophysectomy at 9 AM to 2 PM on the day of diestrus prevented the uterine weight increase and fluid ballooning characteristic of proestrus; hypophysectomy at 3 PM to 6 PM on the day of diestrus had no such effect.  Pharmacological blockade of gonadotrop-  in release at diestrus was ineffective, indicating that the hypothalamic timing mechanism for diestrus gonadotropin release is not susceptible to neurological suppression. The evidence strongly suggests dual hypothalamic control of cyci  .  lie gonadotropin release in the rat.  •  The release of FSH and LH at dies-  trus, necessary for the ovarian steroid secretion which controls the events of proestrus and estrus is not blocked by pharmacological agents, and is continuous for 6 hours.  This tonic gonadotropin release may be  controlled by negative feedback of estrogen on the hypophysiotropic area: deafferentation, lesions, and estrogen implants in this area result in constant diestrus and eventual gonadal atrophy (Taleisnik and McCann 1961; Palka et al 1966), indicating blockage of gonadotropin release. The "surges" of FSH and LH at proestrus, necessary for ovulation, are prevented by anterior hypothalamic lesions, and by pharmacological blockade which may be overcome with electrical stimulation of the anterior hypothalamus (Everett 1956; McClintock and Schwartz 1968). Deafferentation of this area results in constant estrus, the result of uninterrupted gonadotropin release, probably mediated at the level of the uninterrupted hypophysiotropic area.  Estrogen receptor sites have been identi-  fied in the anterior hypothalamus (Palka et al 1966);  assuming that es-  147  trogen controls the release of proestrus gonadotropin "surges", the feedback must be positive, since both surges occur at proestrus, when ovarian estrogen secretion is maximum. The evidence of Labhsetwar (1970 a and b) strongly supports the concept of positive estrogen feedback on the hypothalamus, and further, suggests that the stimulatory estrogen is secreted at dies.trus.  Anti-estrogen administered to cyclic rats on the day  of diestrus inhibits ovulation;  anti-estrogen administered on the morn-  ing of proestrus does not inhibit ovulation. Adenohypophysial LH- concentration among anti-estrogen blocked rats killed on the day of estrus was greater than control values, indicating blockage of the LH "surge". Exogenous administration of LH-RF, or high dosages of estrogen, at proestrus, overcame the anti-estrogen ovulation blockade, indicating that the anti-estrogen interferes with LH-RF release, which is restored by large (positive feedback) amounts of estrogen.  148  D. Cyclic Adenohypophysial Gonadotropin Release in the Adult Female Sheep, Pig and Cow.  No direct investigation of the hypothalamic control of adenohypophysial gonadotropic synthesis, storage, and release has been made in mammals larger than the rat. Quantification of adenohypophysial and plasma gonadotropins have been made for the cyclic sheep, pig and cow, and an inferential explanation of hypothalamic control may be made on the basis of this evidence. Adenohypophysial LH and FSH concentration and content are highest at proestrus in the ewe (Santolucito 1960; Robertson and Hutchinson 1962; Roche et al 1970), and pig (Parlow et al 1964); neither gonadotropin is detectable in the plasma at this time, and the ovaries are characterized by numerous atretic antral f o l l i c l e s and relatively few healthy, ovulatory f o l l i c l e s , while the associated endometria are maximally estrogenic. Adenohypophysial FSH concentration and content decrease 12 hours prior to the onset of estrus in the ewe and pig, followed at estrus by a further precipitous release of FSH, and an equally precipitous release of LH, resulting in ovulation (Rakha and Robertson 1965; Wheatly and Radford 1969; Roche 1970; Parlow et al 1964). There is a similar "surge" of adenohypophysial FSH and LH release during estrus in the. cow;  pharmacological  blockade administered at proestrus blocks this gonadotropin release and prevents ovulation (Rakha and Robertson 1965). Adenohypophysial FSH and LH concentration and content increase from low estrus values during the luteal phase in ewe and pig;  with luteal regression at diestrus, adeno-  hypophysial FSH and LH again decrease, and are detectable in the plasma, concommitant with increasing ovarian follicular development and estrogenic  149  endometrial development. Despite the disparity in the length of the estrus cycles between the domestic ewe, pig and cow (averaging 20 days) and the rat (4 or 5 days), the pattern of adenohypophysial and ovarian events are equivalent, and suggest a dual hypothalamic control of the cycle for the ewe, pig and cow similar to that described for the rat. As in the rat, the diestrus release of FSH and LH results in ovarian antral follicular development, ovarian estrogen synthesis, and endometrial proliferation in the ewe and pig;  increasing blood titres of estrogen during diestrus and early pro-  estrus may act as a negative feedback at the level of the hypothalamic hypophysiotropic area, resulting in the marked adenohypophysial retention of FSH and LH in ewe and pig at late proestrus.  The diestrus release  of FSH and LH in the rat has been demonstrated to initiate the release of high levels of estrogen which act as a positive feedback on the anterior hypothalamus for the proestrus release of ovulatory quanta of FSH and LH (Labhsetwar 1970 a and b).  Presumably in the ewe, pig and cow, the  high levels of blood estrogen at proestrus, initiated by the diestrus release of gonadotropins, may act as a positive feedback on the anterior hypothalamus for the release of the ovulatory quanta of FSH and LH demonstrated at proestrus and estrus.  150  E. Dual Hypothalamic Control of the Onset of Reproductive Maturity in the Female Rat.  Ovarian maturation of the rat may be described in 3 periods of postnatal development (Critchlow and Bar Sela 1967). During the infantile period, from birth to 10 days, the ovary is f i r s t characterized by cortical primary oocytes, which, at 6 days, are surrounded by granulosa c e l l s , and at 10 days by rudimentary thecal tissue;  this follicular de-  velopment is self-determinative in the absence of detectable plasma gonadotropins or steroids (Donovan and Ten Bosch 1963). Ovarian inters t i t i a l tissue volume increases from 10-20 days of age, the early juveni l e period;  rapid growth of antral f o l l i c l e s is characteristic, with  concommitant synthesis and release of steroids.  By 10 days of age,  ovarian steroids are secreted in amounts sufficient for endometrial stimulation, but circulating steroids do not exert a feedback effect on adenohypophysial gonadotropins until 20 days of age (Baker and Kraght 1969). During the late juvenile period, 20-40 days of age, ovarian follicular atresia results in thecal hypertrophy, and the formation of secondary i n t e r s t i t i a l tissue.  The ovaries differ from adult ovaries only in the  absence of corpora lutea.  The adenohypophyses of 25-26-day-old prepub-  eral female rats contain high concentrations of FSH and LH;  the median  eminence content of LH-RF. and FSH-RF is low at 25-26 days compared to a sudden increase in both releasing factors prior to vaginal opening at 35 days (Ramirez and McCann 1963; Moore 1966; Corbin and Daniels 1967; Goldman and Mahesh 1968). Adenohypophysial FSH and LH decline 24 hours prior to vaginal opening, concommitant with the release of LH-RF and FSH-RF (Corbin and Daniels 1967; Ramirez and Sawyer 1965);  at estrus (38 days)  151  there is a further decline in FSH and LH concentration, but no further decline in median eminence FSH-RF or LH-RF. Ovariectomy of 20-day-old rats results in decreased adenohypophysial LH concentration; the postovariectomy release of LH is inhibited with half the dose of estrogen necessary for the same negative feedback effect in ovariectomized adults, suggesting a greater hypothalamic sensi t i v i t y to estrogen among immature females (Ramirez and McCann 1963). Chronic estrogen administration begun at 5 days of age results in ovarian atrophy, the effect of negative steroid feedback inhibiting gonadotropin release; again, the total dose is half that necessary for the same effect in adult females, confirming the greater hypothalamic sensi t i v i t y to estrogen of immature rats (Ramirez and Sawyer 1965). Conversely, acute estrogen administration begun at 2(3 days of age results in precocious puberty at 30 days, with release of median eminence LH-RF and FSH-RF and adenohypophysial FSH and LH, vaginal opening, and ovulation (Corbin and Daniels 1967);  the onset of precocious puberty is the  result of positive estrogen feedback on the hypothalamus promoting the release of gonadotropic releasing factors and hormones. The evidence for negative and positive feedback of estrogen suggests dual hypothalamic control of the events preceding and resulting in reproductive maturity, similar to the dual hypothalamic control of reproductive cycl i c i t y among adults.  Evidence from hypothalamic steroid implants con-  firms dual hypothalamic control: estrogen implanted in the hypophysiotropic hypothalamic area of 26-day-old females induces constant diestrus and ovarian atrophy, the result of negative steroid feedback block of gonadotropin release; estrogen implanted in the anterior hypothalamus of 26-day-old females induces precocious puberty, the result of positive  152  steroid feedback for gonadotropin release (Smith and Davidson 1968). Annovulatory s t e r i l i t y is induced with the administration of testosterone to neonatal female rats (Barraclough 1966). The syndrome is characterized by polyfollicular ovaries and the release of a l l adenohypophysial LH synthesized (Barraclough 1970). The estrogen binding capacity of the anterior hypothalamus is reduced from control values by neonatal androgen administration (Flerko et al 1969), suggesting that the androgen blocks the development of, or destroys, the estrogen receptor sites in the anterior hypothalamus. The consequent loss of positive feedback from the anterior hypothalamus results in tonic gonadotropin release, polyfollicular ovaries which synthesize estrogen, and ovulation failure.  Barraclough (1970) suggests that the anterior hypothalamus is  undifferentiated from birth to 10 days; i f normally differentiated, i t regulates the release of ovulatory quanta of gonadotropins, but.differentiation in the presence of androgens desensitizes the anterior hypothalamus to the positive effect of steroid feedback, and tonic gonadotropin secretion is controlled at the level of the hypophysiotropic area by negative estrogen feedback.  This concept is supported by evidence  that estrogen retention by the hypophysiotropic area and the anterior hypothalamus is age dependent between birth and puberty in the female rat (Presl et al 1970). Estrogen retention by both areas is high at 5 days of age, but retention decreases between 5 and 10 days of age.  The  hypophysiotropic area retains more estrogen than the anterior hypothalamus between 10 and 20 days, the time period coinciding with the period during which tonic gonadotropin release in response to ovarian estrogen is established and during which chronic estrogen administration results in ovarian atrophy.  The evidence suggests that the hypophysiotropic area  153  differentiates before the anterior hypothalamus for mediation of the release of FSH and LH through negative estrogen feedback.  Anterior hy-  pothalamus retention of estrogen is greater than that of the hypophysiotropic area from 20-25 days, the time period coinciding with the period during which acute estrogen administration (positive feedback) results in precocious puberty.  The evidence suggests that the anterior hypo-  thalamus differentiates after the hypophysiotropic area for the mediation of the release of ovulatory amounts of FSH and LH through positive estrogen feedback. The. evidence discussed may be summarized in the following manner. At birth, the hypothalamus of the female rat is undifferentiated with respect to response to steroids (Barraclough 1970 ), and w i l l not be exposed to endogenous steroids until 5-10 days of age (Critchlow and BarSela 1967). That the hypothalamus w i l l retain androgen as well as estrogen (Flerko et al 1969) from birth to 5 days suggests an indiscriminate acceptance of any steroids;  the androgen s t e r i l i t y syndrome (Bar-  raclough 1966) suggests that the hypothalamus is developmentally influenced by a particular steroid, normally female in the presence of estrogens, normally male in the presence of androgens. The decline in hypothalamic acceptance of estrogen at 10 days (Presl et al 1970) argues for the development of specific estrogen sites, coincident with the secretion of measurable amounts of ovarian estrogens which do not exert a feedback effect (Baker and Kraght 1969). The hypophysiotropic area retains more estrogen than the anterior hypothalamus between 10-20 days (Presl et al 1970), coincident with the rapid ovarian and antral f o l l i c l e growth of the early juvenile period (Critchlow and Bar-Sela 1966);  154  negative feedback of estrogen on the hypothalamus is evident (Ramirez and McCann 1962), and at this time, chronic estrogen administration results in diestrus and gonadal atrophy (Ramirez and Sawyer 1967).  The  coincidence of events suggests maturation of the hypophysiotropic area, which becomes responsive to steroid feedback prior to the maturation of the anterior hypothalamus. Between 20-25 days, the anterior hypothalamus retains more estrogen than the hypophysiotropic area (Presl et al 1970), coincident with the age at which precocious puberty may be induced with acute exogenous estrogen (Corbin and Daniels 1967), suggesting maturation of the anterior hypothalamus, responsive to positive feedback of ovarian estrogen for the adenohypophysial release of ovulatory amounts of gonadotropins, resulting in ovulation and reproductive maturity. The sequence of events described is simplistic in the sense that it makes use only of established phenomena, leaving out of consideration phenomena for which there are presently no reasonable explanation. The greater sensitivity of the immature hypothalamus to estrogen is undeniably established (Ramirez and McCann 1963; Ramirez and Sawyer 1965); its relevance to immaturity has no satisfactory explanation, nor is the decreased sensitivity of maturity easily explained. The position of the synthesis and release of gonadotropic releasing factors in the time sequence of maturation has yet to be investigated.  Internal negative feed-  back of adenohypophysial gonadotropins on the hypothalamus among immature female rats has been established (Ojeda and  Ramirez 1969 and 1970), but  the physiological implications have yet to be explained. Apropos of the questions remaining is the statement of Ojeda and  Ramirez (1969):  "The  mechanism of the onset of puberty is s t i l l beyond reasonable understand-  155  ing.", perhaps unduly pessimistic in the light of the most recent investigations, but s t i l l descriptive of the present state of knowledge.  156  F.  Gonadotropic Factors Affecting the Onset of Reproductive Maturity in the Female Pig, Cow and Sheep.  There has been very l i t t l e direct investigation of the hypothalamic or gonadotropic factors affecting the onset of reproductive maturity in mammalian species other than rodents. The information available is confined to domestic species.  The adult response of the immature ovaries  of the cow (Howe et al 1964; Jainudeen et al 1966) and sheep (Lamond 1962) to exogenous gonadotropins has been discussed. The adenohypophyses of immature female pigs (Hollandbeck et al 1956), cows (Hansel 1959), and sheep (Robinson 1959) have concentrations of gonadotropins greater than those of adults, but the gonadotropin content of immature and adult glands are similar, and the total amount of gonadotropin available to each is equal. Ovarian antral f o l l i c u l a r development prior to ovulatory maturity is a prominent characteristic of these species;  in no case do  f o l l i c l e s reach ovulatory size prior to the onset of puberty. No direct investigation of the hypothalamic or gonadotropic control of the onset of reproductive maturity among these species has been made. As in the immature rat, adult ovarian capability and adult adenohypophysial gonadotropic content are available to prepubertal cow, pig and sheep;  pre-  sumably the ultimate factor controlling the onset of reproductive maturity i s , as in the rat, hypothalamic maturation.  157  G.  Gonadotropic Factors Affecting the Onset of Reproductive Maturity in the Female Fur Seal.  Apparently the present direct examination of gonadotropic factors affecting the onset of reproductive maturity in the female fur seal is the f i r s t of its kind among large wild mammals. It should be noted that the annual availability of large numbers of immature and maturing females at a particular time, coincident with a commercial culling of these females, made the investigation possible;  the circumstances are rarely  duplicated for any other species. To attempt an analysis of the hypothalamic-hypophysial-gonad control of the onset of reproductive maturity in a monestrous, monovulatory, late-maturing female, in the absence of any closely related  precedents  is d i f f i c u l t , and requires the following basic assumption. Critchlow and Bar-Sela (1967) make the point that although the temporal features of ovarian maturation vary between species, they do not differ;  since  ovarian maturation is controlled by the hypothalamic-hypophysial complex, then the development and maturation of this complex must differ only temporally between species.  Given this assumption, with the evidence  for hypothalamic control of the onset of reproductive maturity and subsequent cyclicity in rats, and the evidence for gonadotropic hormone storage and release resulting in estrus and ovulation for the sheep, pig and cow, an attempt to explain the onset of reproductive maturity in the female fur seal may be made. It must be emphasized that the analysis is hypothetical, based on the evidence of bioassays for gonadotropin content, and differential adenohypophysial c e l l counts, and not on any direct evidence of hypothalamic control.  158  1.  Pups.  The ovaries of female fur seal pups 6-8 weeks of age correspond to the infantile ovarian period of the rat (Critchlow and Bar-Sela 1966), and are characterized by numerous antral f o l l i c l e s less than 1.0 mm in diameter.  The f o l l i c l e s develop prenatally (Craig 1966), probably in  response to maternal gonadotropins passing through the placental barrier. The ovarian f o l l i c l e s of the neonate may be self-determinative, maintained without the influence of adenohypophysial gonadotropins:  no ovar-  ian steroid synthesis, necessary for the feedback release of adenohypophysial gonadotropins, is evident;  further, the f o l l i c l e s become atretic  without replacement, and the ovaries of 5-month-old females are devoid of antral f o l l i c l e s , a condition which maintains through the f i r s t year. The ovarian and uterine tissue of 6-8-week-old pups is nevertheless responsive to exogenous gonadotropins.  Human Chorionic Gonadotrop-  in induces ovarian steroid synthesis without follicular  enlargement;  steroid release is evident in endometrial activation (Table 6).  Follicle  Stimulating Hormone and HCG administered to pups concurrently induces ovarian follicular growth and steroid synthesis and release, with concommitant endometrial growth (Table 6). Adenohypophyses from 2-and 3year-old immature females and from estrous females, given to pups concurrently with HCG, similarly induce ovarian and endometrial growth (Table 7). Pregnant Mare Serum induces the development of ovarian f o l licles of ovulatory size, steroid synthesis and release, and endometrial growth (Table 17). The ovarian tissue of the pups is limited in response to exogenous gonadotropins.  No f o l l i c l e s larger than 2.0 mm in diameter  develop in response to HCG and FSH, or in response to HCG and adenohypo-  159  physes from immature, estrous, or ovulated female seals;  despite the  development of ovulatory f o l l i c l e s in response to PMS, no ovulations were obtained with subsequent administrations of LH or of adenohypophyses from immature female seals (Tables 6-8). To summarize, during the ovarian infantile period of the female seal, extending from birth to 1 year, the ovaries i n i t i a l l y contain numerous small antral f o l l i c l e s , which become atretic without replacement, so that the ovaries are devoid of antral f o l l i c l e s by 5 months of age; during this period, no steroid synthesis is evident.  Nevertheless,  ovarian and endometrial tissues respond to exogenous gonadotropins at 6-8 weeks with ovarian antral follicular development, steroid synthesis, and endometrial growth. The evidence indicates that the existing ovarian and endometrial potential for growth is not exploited during the infantile period, suggesting steroid insensitivity of the possibly undifferentiated hypothalamus. Similarly, during the ovarian infantile period of the rat, follicular development is self-determinative (Critchlow and Bar-Sela 1967) in the absence of detectable plasma gonadotropins and steroid (Donovan and Ten Bosch 1963), and the hypothalamus is undifferentiated to the effect of ovarian steroids (Barraclough 1970; Flerko et al 1969).  2.  Two-year-old immature females.  The ovaries of 2-year-old immature female fur seals correspond to the early juvenile ovarian period of the rat (Critchlow and  Bar Sela  1966), and are characterized by antral follicular development which is progressive from May to August (Craig 1966):  the associated endometria  160  show minimal estrogen activation;  no f o l l i c l e s are larger than 5.0 mm  in diameter, and the follicular cycle culminating in August is annovulatory.  Similarly, the ovaries of immature pigs (Hollandbeck et al 1956),  cow (Howe et al 1964) and sheep (Lamond 1962) contain antral f o l l i c l e s prior to ovulatory maturity. The ovaries are capable of ovulation.  The administration of PMS  followed by LH or by adenohypophyses from female fur seals induces ovulation in the ovaries of 2-year-old females (Table 18). Further, the adenohypophysis of a 2-year-old female contains sufficient LH to induce an ovulation in this manner (Table 18). Bioassay has demonstrated that adenohypophyses from 2-year-old females contain both FSH and LH in amounts greater than in adenohypophyses from proestrous females preparing to ovulate for the f i r s t time (Tables 26, 32). The adenohypophyses of immature pigs (Hollandbeck et a l 1956), cows (Hansel 1959), and sheep (Robinson 1959) also contain adult quantities of gonadotropins. The release of FSH and LH from adenohypophyses of 2-year-old females has been demonstrated in bioassay (Tables 26, 32), and is suggested by differential adenohypophysial c e l l counts;  the growth and  maturation effect of these gonadotropins on the ovarian f o l l i c l e s of the female fur seal has been demonstrated in bioassays using female fur seal pups (Table 7). Presumably gonadotropin release for ovarian follicular development in 2-year-old females is mediated by steroid feedback on the hypothalamus, although ovarian steroid synthesis is sufficient to induce only minimal endometrial activation without growth. The evidence suggests that during the early juvenile ovarian period of the 2-year-old female, sufficient adenohypophysial FSH and LH are  161  released, in response to feedback on the hypothalamus of minimal amounts of ovarian steroids, for ovarian follicular development. Although the ovarian capability exists, and sufficient adenohypophysial gonadotropins are available, no ovulation occurs. That the hypothalamus of the rat during the juvenile period is more sensitive to estrogen than at later periods of maturation has been demonstrated (Ramirez and McCann 1963; Ramirez and Sawyer 1965); • during this period, the hypophysiotropic area retains more estrogen than the anterior hypothalamus (Presl et al 1970), and chronic estrogen administration results in gonadal atrophy, the result of negative feedback block of gonadotropin release (Ramirez and Sawyer 1965). The hypophysiotropic area has been demonstrated in adult rats to control the release of gonadotropins for follicular development and maturation, mediated by the negative feedback of ovarian steroid (Koves and Halasz 1969; Palka et al 1969). By analogy, i t is suggested that among 2-year-old immature female fur seals, the hypothalamic hypophysiotropic area has differentiated, and is responsive to the negative feedback of minimal amounts of ovarian estrogen for the ovarian antral follicular development characteristic of this age group.  Ovulat-  ion failure may be due to an undifferentiated anterior hypothalamus, which would be responsive to positive steroid feedback for the release of ovulatory gonadotropins;  alternatively, if. the anterior hypothala-  mus has differentiated, the sensitivity of the hypothalamus to estrogen may be so great that insufficient estrogen is synthesized for a positive feedback effect.  162  3.  Three-year-old immature females.  The ovaries of 3-year-old immature female fur seals correspond to the late juvenile ovarian period of the rat (Critchlow and Bar-Sela 1966). The ovaries are characterized by antral follicular development which is progressive from May to culmination in August, without ovulation (Craig 1966). The ovarian cycle differs from that of the 2-yearold immature female only in increased ovarian weight, and increased ovarian steroidogenesis resulting in endometrial activation and growth (Table 19). The evidence of the bioassays indicates that more adenohypophysial FSH and LH are released than among 2-year-old immature females concentration and content of FSH and LH in adenohypophyses from 3-yearold females are significantly less than in the glands of 2-year-old females (Tables 26, 32). Adenohypophysial content of FSH and LH is nevertheless more than in glands from early proestrous females (Tables 26, 32 Further, the ovaries of 3-year-old females are capable of ovulatory f o l licular development and ovulation in response to exogenous administration of PMS and LH or adenohypophyses from female seals (Table 18). That gonadotropin release among 3-year-old females in response to larger amounts of circulating estrogen than among 2-year-old females, despite the equivalence of f o l l i c u l a r development, suggests decreasing sensitivity of the hypothalamic hypophysiotropic area to estrogen among 3-year-old females. The failure of ovulation despite the availability of ovarian competence and sufficient adenohypophysial gonadotropins suggests that, as among 2-year-old females, the hypothalamic hypophysiotropic area is responsive to the negative feedback of ovarian steroids for the release of gonadotropins sufficient only for antral follicular  163  development and steroidogenesis, and that either the anterior hypothalamus is not responsive to steroids, or insufficient steroicfe are synthesized for a positive feedback effect on the anterior hypothalamus to induce the release of ovulatory amounts of gonadotropins.  4.  Females in early proestrus, late proestrus, and estrus.  The ovarian cycle of the 4-year-old female marks the onset of reproductive maturity.  The cycle differs from those of 2-and 3-year-old  immature females from March to July in increased number and size of ovarian antral f o l l i c l e s , and in increased ovarian steroidogenesis, reflected in greater endometrial proliferation (Craig 1966). The cycle culminates in ovulation during August (Craig 1963, 1966). Among early and late proestrous females, and estrous females, the evidence of the bioassays, and of differential adenohypophysial c e l l counts suggest the following sequence of gonadotropic events leading to ovulation. At early proestrus, adenohypophysial FSH and LH content are less than the same values for immature 3-year-old females (Tables 26, 32), suggesting a significantly greater release of both gonadotropins.  At the same time, FSH concentration is high relative to the  same value among 3-year-old females, suggesting a decreased rate of FSH release or a rate of synthesis greater than rate of release;  the re-  tention of FSH may be necessary for the marked f o l l i c u l a r atresia and the development of a single ovulatory f o l l i c l e characteristic of late proestrus and estrus. That adenohypophysial FSH is released between early and late proestrus is indicated  by decreased adenohypophysial  FSH concentration and content at late proestrus (Table 26).  The rate of  164  LH release is decreased between early and late proestrus, or the rate of LH synthesis is greater than rate of LH release, since adenohypophysial LH concentration and content are doubled at late proestrus from the early proestrus values (Table 32).  At estrus, the adenohypophysial con-  tent and concentration of both FSH and LH are half the values of late proestrus, indicating a massive release of both gonadotropins prior to ovulation (Tables 26, 32). The anterior hypothalamus of the rat, responsive to positive estrogen feedback in the cyclic adult rat (Labhsetwar 1970) selectively retains estrogen at 26 days of age in the prepubertal female (Presl et al 1970), 10 days prior to ovulatory maturity, and 6-10 days later than similar estrogen retention by the hypothalamic hypophysiotropic area. Ovulatory maturity at 36 days in the rat is preceded by the release of FSH and LH at day 35, concommitant with vaginal opening, and uterine fluid ballooning; at estrus, prior to ovulation, there is a further release or "surge" of FSH and LH, the result of positive estrogen feedback (Corbin and Daniels 1967; Ramirez and Sawyer 1965). The evidence suggests that ovulatory maturity is dependent on anterior hypothalamic maturation, responsive to positive ovarian steroid feedback for the release of ovulatory quantities of FSH and LH; and that anterior hypothalamic maturation precedes maturation of the hypothalamic hypophysiotropic area, responsive to negative steroid feedback for the release of gonadotropins which effect ovarian antral follicular development without ovulation.  In the extended temporal sequence of reproductive maturity  among larger mammals, the differential development of the 2 hypothalamic areas would account for the characteristic annovulatory follicular  165  development of immature pigs (Hollandbeck et al 1956), cows (Hansel 1959), sheep (Robinson 1959) and fur seal.  In the prepubertal rat, the gonado-  tropic sequence leading to ovulatory maturity, after anterior hypothalamic maturity, parallels that of the mature cyclic rat: diestrus release of gonadotropins resulting in ovarian follicular development and estrogen synthesis (Lawton and Sawyer 1968), the latter i n i t i a t i n g , by positive feedback on the anterior hypothalamus (Labhsetwar 1970 a and b) the release of ovulatory amounts of FSH and LH.  Similarly, in the adult ewe  (Santolucito et al 1960; Roche et al 1970) and pig (Parlow et al 1964), the release of FSH and LH at diestrus promotes ovarian antral follicular development and steroid synthesis, the latter initiating, at proestrus, the release of ovulatory amounts of FSH and LH, as a result of positive feedback, presumably on the anterior hypothalamus. By analogy, the following events are suggested as responsible for ovulatory maturity in the female fur seal.  During the follicular cycle,  from May to July, complete hypothalamic hypophysiotropic maturation is accomplished among 4-year-old females, with decreased sensitivity to the negative feedback of estrogen;  consequently sufficient ovarian steroid  synthesis may be generated for positive feedback on the anterior hypothalamus. The release of FSH and LH at early proestrus may correspond to the similar release in the prepubertal rat, and to the diestrus gonadotropin release in the adult rat, ewe arid pig, and results in the ovarian antral follicular development and estrogen synthesis, with concommitant endometr i a l proliferation, of late proestrus. The late proestrus release of adenohypophysial FSH among fur seals parallels that of the adult ewe and pig, as does the estrus release of FSH and LH.  The release of adenohypo-  166  pophysial FSH and LH at estrus among the female fur seals, which w i l l result in ovulation, occurs in a condition of maximum steroid  synthesis,  the result of early proestrus adenohypophysial gonadotropin release, and must be effected by positive steroid feedback. The positive steroid feedback suggests the maturation of the anterior hypothalamus, the final and ultimate factor in the reproductive maturity of the female fur seal.  167  VII. SUMMARY  1. The morphology of the fur seal hypophysis is described. The pars anterior and pars intermedia surround the pars nervosa;  the posit-  ion of the residual lumen is marked by colloid deposits. The pars tuberalis encloses the long neural stalk and prominent median eminence, and extends onto the exterior surface of the pars anterior.  The zona tuber-  alis extends from the median eminence to the pars nervosa, along the line of the neural stalk. pars anterior; 2.  The serous cells are zonated laterally within the  the mucoid cells are zonated medially and peripherally.  The cytology of the pars anterior is described, and 5 cells  are identified morphologically and histochemically.  Two are serous c e l l s ,  with granules composed of simple proteins; by comparison and analogy to cells similarly identified in the adenohypophyses of other mammals, these cells are considered to be the somatotrop and the luteotrop. Three are mucoid c e l l s , with granules composed of mucoproteins;  by comparison and  analogy to cells similarly identified in the adenohypophyses of other mammals, these cells are considered to be the thyrotrop, the folliculotrop, and the interstitiotrop. 3.  Differential c e l l counts were made of gonadotrops in the pars  anteriors of immature, early proestrous, and estrous female seals. In the adenohypophyses of immature females, 10% of the folliculotrops were fully granulated, 80% partly granulated, and 8% degranulated;  25% of the  interstitiotrops were fully granulated, 51% were partly granulated, and 20%, were degranulated.  In the adenohypophyses of early proestrous fe-  168  males, 30% of the folliculotrops were fully granulated, 55% were partly granulated, and 15% degranulated;  57, of the interstitiotrops were fully  granulated, 35%. partly granulated, and 557, degranulated.  In the adeno-  hypophyses of estrous females, 20%, of the folliculotrops were fully granulated, 507, partly granulated, and 407. degranulated;  7% of the intersti-  tiotrops were fully granulated, 407. partly granulated, and 54% were degranulated.  The data suggest minimal gonadotropic hormone release among  immature females, extensive release of gonadotropic hormones at early proestrus in preparation for estrus, and the further release of ovulatory amounts of gonadotropic hormones at estrus. 4. Adenohypophyses from 2- and 3-year-old immature females and estrous females were assayed for quantification of F o l l i c l e Stimulating Hormone (FSH) and Luteinizing Hormone (LH), using female fur seal pups 6-8 weeks old as assay animals for FSH, and immature females 2 and 3 years old, as well as pups, as assay animals for LH. The end point measured for FSH quantification was ovarian antral f o l l i c l e s 1.0-2.0 mm in diameter. The concentration and content, of adenohypophysial FSH among immature females is approximately double that of estrous females;  the evidence is  commensurate with the results of the differential adenohypophysial c e l l counts of gonadotrops, and suggest minimal gonadotropin release among immature females, and the release of ovulatory amounts of FSH among estrous females. The bioassays for LH were only partly successful, but demonstrated the ovulatory capability of 2- and 3-year-old immature females, and the presence of ovulatory amounts of LH in the adenohypophyses of 2and 3-year-old immature females.  169  5. Adenohypophyses from 2-year-old and 3-year-old immature females, early proestrous females, late proestrous females, and estrous females were assayed for concentration and content of FSH and LH, using intact, immature female rats as assay animals. Adenohypophysial FSH content decreases from a peak 0.8 mg FSH among 2-year-old females to 0.5 mg FSH among 3-year-old females, 0.4 mg FSH among early proestrous females, 0.35 mg FSH among late proestrous females, and 0,18 mg FSH among estrous females;  FSH concentration similarly decreases, except  among early proestrous females, where a relatively high concentration suggests FSH synthesis at a rate greater than rate of release.  Adenohy-  pophysial LH content is highest among 2-year-old immature females, 34 _ug, and decreases to 19 }xg among 3-year-old immature females, and to 10 yig among early proestrous females;  LH content is increased to 28 jig among  late proestrous females, and decreases to 10 ^ig among estrous females. Adenohypophysial LH concentration follows the same pattern.  The evid-  ence suggests minimal release of FSH and LH among 2- and 3-year-old immature females, sufficient to initiate the annovulatory ovarian f o l l i c ular cycle in March and support i t to its conclusion in September; the evidence of differential adenohypophysial c e l l counts and assay of adenohypophysial FSH in fur seal pups supports this conclusion.  Increased  release of FSH and LH at early proestrus probably initiates the ovarian ovulatory follicular development and estrogen synthesis characteristic of late proestrus and estrus. The release of FSH at late proestrus is similar to proestrus FSH release among pubertal rats, and among cyclic sows and ewes in proestrus.  Similarly, the high concentration and con-  tent of LH at late proestrus is characteristic of pubertal proestrous rats, and of cyclic sows and ewes in proestrus.  The release of FSH and  170  LH at estrus, for ovulation, is also characteristic of rats, and of cows ewes, and pigs, at estrus;  i t should be noted that the release of ovul-  atory quanta.of adenohypophysial gonadotropins occurs at the peak of ovarian estrogen synthesis, and must be the result of positive estrogen feedback. 6. Recent investigation has established that in the female r a t , reproductive maturity is dependent on the differential maturation of 2 areas of the hypothalamus. The "hypophysiotropic area" differentiates f i r s t , and controls, through the synthesis and release of neurohormonal gonadotropic releasing factors, the release of adenohypophysial FSH and LH for ovarian antral development and estrogen synthesis; the hypophysiotropic area is responsive to negative estrogen feedback.  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