Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Role of the thyroid in ovarian maturation in the goldfish, Carassius auratus L Hurlburt, Mary E. 1977

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1977_A6_7 H87.pdf [ 6.5MB ]
Metadata
JSON: 831-1.0094005.json
JSON-LD: 831-1.0094005-ld.json
RDF/XML (Pretty): 831-1.0094005-rdf.xml
RDF/JSON: 831-1.0094005-rdf.json
Turtle: 831-1.0094005-turtle.txt
N-Triples: 831-1.0094005-rdf-ntriples.txt
Original Record: 831-1.0094005-source.json
Full Text
831-1.0094005-fulltext.txt
Citation
831-1.0094005.ris

Full Text

ROLE OF THE THYROID IN OVARIAN MATURATION IN THE GOLDFISH CARASSIUS AURATUS L. by MARY E. HURLBURT B.Sc. (Hons.), Un i v e r s i t y of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1977 (T) Mary E. Hurlburt, 1977. MASTER OF SCIENCE in THE DEPARTMENT OF ZOOLOGY In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date fiffstY /f,/f7r-?-ABSTRACT Thyroxine (T^) administration by c h o l e s t e r o l p e l l e t implantation, by immersion, or by feeding r a i s e d plasma T^ l e v e l s of g o l d f i s h , Carassius  auratus, above values of control f i s h (usually l e s s than 1 ug/100 ml). Values for treated f i s h i n the lower dosage groups generally f e l l between 1 and 4 ug/100 ml, while i n the higher dosage groups plasma T^ l e v e l s ranged from 5 to 16 yg/100 ml. Immersion was the most e f f e c t i v e method of creating sustained p h y s i o l o g i c a l plasma T^ elevations; both implantation and feeding are more sui t a b l e f or flowing water systems where immersion i s not f e a s i b l e . Thyroxine treatment of maturing f i s h r e s u l t e d i n a depression of gonado-t r o p i n c e l l a c t i v i t y , while immersion of immature f i s h i n the goiterogen, p r o p y l t h i o u r a c i l , stimulated gonadotropin c e l l a c t i v i t y s l i g h t l y ; t h i s e f f e c t was reversed by T^ replacement therapy. T^ administration to i n t a c t g o l d f i s h accelerated ovarian development i n immature i n d i v i d u a l s , but had no apparent e f f e c t i n mature f i s h . In hypophysectomized i n d i v i d u a l s , T^ alone f a i l e d to i n i t i a t e v i t e l l o g e n e s i s or maintain yolky oocytes; the hormone did, however, augment the ovarian response to salmon gonadotropin (SG-G100) and ovine l u t e i n i z i n g hormone (LH) following p i t u i t a r y a b l a t i o n . Thyroxine treatment tended to increase r e l a t i v e l i v e r weights i n both i n t a c t and hypophysectomized f i s h , except when administered with LH. In the l a t t e r case, a combination of LH and T^ prevented the usual l i v e r hypertrophy a f t e r p i t u i t a r y removal; e i t h e r hormone alone, however, was i n e f f e c t i v e , suggesting a s y n e r g i s t i c action of the two hormones on l i v e r function. In i n t a c t and hypophysectomized g o l d f i s h , SG-G100 and LH stimulated t h y r o i d function, as measured by h i s t o l o g i c a l c r i t e r i a and plasma T 4 a n a l y s i s . The findings indicate that thyroid hormones act synergistically with gonadotropin to influence ovarian development in goldfish, and suggest that these effects are mediated, directly or indirectly, by a thyroxine-induced increase in ovarian sensitivity to gonadotropic stimulation. Thyroxine appears also to modulate gonadotropin production by a negative feedback on the hypothalamo-hypophysial axis; the physiological significance of this i s , however, uncertain. i v TABLE OF CONTENTS Page ABSTRACT «. i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS • • > * INTRODUCTION • • • 1 L i t e r a t u r e Review 1 F i s h 1 Other Vertebrates 4 Mechanisms of Thyroid Action 5 GENERAL TECHNIQUES , 8 Maintenance of Goldfish , 8 Histology and H i s t o l o g i c a l Techinques 8 Ovary 8 Thyroid 10 P i t u i t a r y 11 Hypophysectomy 12 I. EFFECTS. OF THYROXINE ON PLASMA THYROXINE LEVELS 14 INTRODUCTION 15 MATERIALS AND METHODS 16 a. P e l l e t Implantation 16 b. Immersion 16 c. Feeding 17 Blood Sampling and Plasma T^ Analysis 18 RESULTS 19 DISCUSSION 26 I I . EFFECTS OF THYROID HORMONES ON THE PITUITARY, GONADS, AND LIVER OF INTACT FISH 29 INTRODUCTION 30 V TABLE.OF CONTENTS (Continued) Page MATERIALS AND METHODS 33 a. E f f e c t s of Thyroxine i n Maturing F i s h 33 b. E f f e c t s of P r o p y l t h i o u r a c i l and Thyroxine i n Immature F i s h 33 RESULTS 34 P i t u i t a r y 34 Ovary 40 L i v e r 44 DISCUSSION 44 P i t u i t a r y 44 Ovary 47 L i v e r 49 Thyroid 1 \ . ... 50 I I I . EFFECTS OF THYROXINE AND SALMON GONADOTROPIN ON OVARIES AND LIVER OF LONG-TERM HYPOPHYSECTOMIZED FISH ' 51 INTRODUCTION 5 2 MATERIALS AND METHODS . 52 a. E f f e c t s of Thyroxine, i n Long-Term Hypophysectomized F i s h . 52 b. E f f e c t s of Salmon Gonadotropin and Thyroxine i n Long-Term Hypophysectomized F i s h 53 RESULTS 53 Ovary 53 Thyroid 55 L i v e r 63 DISCUSSION 64 Ovary 6 4 Thyroid . 64 L i v e r 65 IV. EFFECTS OF THYROXINE AND LH ON OVARIES, THYROID, AND LIVER OF SHORT-TERM HYPOPHYSECTOMIZED FISH 66 INTRODUCTION 67 MATERIALS AND METHODS 68 v i TABLE OF CONTENTS (Continued) Page a. E f f e c t s of LH i n Intact F i s h 68 b. E f f e c t s of LH and Thyroxine i n Short-Term Hypophysectomized F i s h 69 RESULTS 70 Ovary 70 Thyroid 70 L i v e r 79 DISCUSSION 82 Ovary 8 2 Thyroid 8 3 L i v e r 8 4 GENERAL DISCUSSION 8 6 P i t u i t a r y 8 7 Ovary 9 0 L i v e r 9 3 Conclusions 9 6 SUMMARY. 9 9 REFERENCES 1 0 0 v i i LIST OF TABLES Table P a 9 e 1. E f f e c t s of thyroxine on gonadotropin c e l l and nuclear s i z e i n maturing f i s h 35 2. E f f e c t s of p r o p y l t h i o u r a c i l and thyroxine on gonado-t r o p i n c e l l and nuclear s i z e i n immature f i s h 37 E f f e c t s of p r o p y l t h i o u r a c i l and thyroxine on thy r o i d e p i t h e l i a l c e l l height and p i t u i t a r y TSH c e l l frequency 41 4. E f f e c t s of thyroxine on ovarian development of maturing f i s h 42 5. E f f e c t s of p r o p y l t h i o u r a c i l and thyroxine on ovarian development of immature f i s h , 43 6. E f f e c t s of thyroxine on ovarian development and l i v e r weight of long-term hypophysectomized f i s h 54 7. E f f e c t s of salmon gonadotropin and thyroxine on ovarian development and thyroid e p i t h e l i a l c e l l height i n long-term hypophysectomized f i s h 56 8. E f f e c t s of ovine LH on ovarian development and thyr o i d e p i t h e l i a l c e l l height of i n t a c t f i s h 71 9. E f f e c t s of ovine LH and thyroxine on ovarian main-tenance and plasma thyroxine l e v e l s i n short-term hypophysectomized f i s h 72 v i i i LIST OF FIGURES Figure Page 1. Plasma T^ concentrations of f i s h implanted with p e l l e t s containing 0, 60, 150, and 750 ug T 4 20 2. Plasma T^ concentrations of f i s h immersed i n T^ solutions of 0, 2, and 10 yg/100 ml 22 3. Plasma T^ concentrations of f i s h fed d i e t s containing T^ i n proportions of 0, 10, and 100 ppm 24 •4. P i t u i t a r y gonadotrophs and thyrotrophs of untreated maturing female f i s h 38 5. P i t u i t a r y gonadotrophs of maturing female f i s h implanted with T^ f o r 80 days.. 38 6. P i t u i t a r y gonadotrophs and thyrotrophs of untreated immature female f i s h 38 7. P i t u i t a r y gonadotrophs of immature female f i s h immersed i n p r o p y l t h i o u r a c i l for 40 days 38 8. P i t u i t a r y gonadotrophs of immature female f i s h treated with p r o p y l t h i o u r a c i l and T^ for 40 days 38 9. P i t u i t a r y thyrotrophs of immature female f i s h immersed i n p r o p y l t h i o u r a c i l for 40 days 38 10. Hepato-somatic indices of i n t a c t immature f i s h treated with thyroxine and p r o p y l t h i o u r a c i l 45 11. E f f e c t s of salmon gonadotropin and thyroxine on oocyte composition of ovaries of long-term hypophysectomized f i s h 57 12. Ovarian section of an untreated long-term hypophysec-tomized f i s h (Exp. I l i a ) 59 13. Ovarian section of a long-term hypophysectomized f i s h following immersion i n T^ f o r 25 days . 59 14. Ovarian section of an untreated long-term hypophysec-tomized f i s h (Exp. I l l b ) 5 9 i x LIST OF FIGURES (Continued) Figure Page 15. Ovarian section of a long-term hypophysectomized f i s h treated with SG-G100 for four weeks 59 16. Ovarian section of a long-term hypophysectomized f i s h treated with SG-G100 and for four weeks 59 17. Thyroid f o l l i c l e s of an untreated long-term hypophysectomized f i s h 61 . 18. Thyroid f o l l i c l e s of a long-term hypophysectomized f i s h treated with SG-G100 for four weeks 61 19. E f f e c t s of ovine LH and thyroxine on oocyte composition of ovaries of short-term hypophysectomized f i s h 73 20. Ovarian section of an i n t a c t immature f i s h (Exp. IVa) 75 21. Ovarian section of an i n t a c t f i s h treated with ovine LH for three weeks 75 22. Thyroid f o l l i c l e s of an i n t a c t c o n t r o l f i s h (Exp. IVa) 75 23. Thyroid f o l l i c l e s of an i n t a c t f i s h treated with LH f o r three weeks 75 24. Ovarian section of an i n t a c t control f i s h (Exp. IVb) 77 25. Ovarian section of an untreated f i s h hypophysectomized fo r four weeks 77 26. Ovarian section of a hypophysectomized f i s h treated with ovine LH for four weeks post-operatively 77 j .27. Ovarian section of a hypophysectomized f i s h immersed i n T^ for four weeks post-operatively 77 28. Ovarian section of a hypophysectomized f i s h treated with LH and T^ f o r four weeks post-operatively 77 29. E f f e c t s of ovine LH and thyroxine on the hepato-somatic index of short-term hypophysectomized female f i s h 80 ACKNOWLEDGEMENTS I would l i k e to thank my supervisor, Dr. W. S. Hoar, for h i s help and guidance throughout the pro j e c t and i n the w r i t i n g of t h i s t h e s i s , and also my committee members, Dr. E. M. Donaldson and Dr. A. M. Perks, f o r t h e i r advice and c a r e f u l c r i t i c i s m s of t h i s manuscript. I am also indebted to W. S. Marshall, Dr. Y. Nagahama, N. E. Stacey, and Dr. T. J . Lam f o r t h e i r generous assistance and support. The help of Dr. G. B e l l and associates of the P a c i f i c B i o l o g i c a l Station, Nanaimo, B. C. i n diagnosing and tr e a t i n g f i s h disease i s g r a t e f u l l y acknowledged. In addition, I would l i k e to thank the s t a f f of the Department of Nuclear Medicine, Shaughnessy Hospital, Vancouver, B. C. f o r the use of t h e i r laboratory f a c i l i t i e s . Ovine l u t e i n i z i n g hormone (NIH-LH-S19) was generously donated by the National I n s t i t u t e of A r t h r i t i s , Metabolism, and Digestive Diseases. This study was supported through National Research Council of Canada grants-in-aid to Dr. W. S. Hoar, and a National Research Council post-graduate scholarship to M. E. Hurlburt. 1 INTRODUCTION I t i s recognized that thyroid hormones influence the reproductive pro-cesses of vertebrates (see reviews by Pickford and Atz, 1957; B a l l , 1960; Matty, 1960; Dodd and Matty, 1964; Reinboth, 1972; Leatham, 1973; Dodd, 1975). In t e l e o s t s , these hormones have been implicated i n spawning ( B a l l , 1960) and the i n i t i a t i o n of spawning migrations (M. Fontaine and Leloupe, 1960). Changes i n t h y r o i d a l a c t i v i t y often coincide with sexual maturation (Hoar, 1955) and a c t i v i t y of the thyroid gland appears to increase during gonadal development ( B a l l , 1960). Experimentally induced hypothyroidism can slow gonadal development i n both t e l e o s t s (Barrington and Matty, 1952; Scott, 1953; P i c k f o r d and Atz, 1957) and mammals (Leatham, 1973), while administra-t i o n of thyroid hormones can stimulate sexual maturation i n some f i s h (Grobstein and Bellamy, 1939; Hurlburt, 1975). There appears to be a complex i n t e r a c t i o n between thyr o i d hormones, sex st e r o i d s , and p i t u i t a r y thyrotrophs and gonadotrophs i n t e l e o s t s (Sage and Bromage, 1970b), with hypothalamic-pituitary control over the th y r o i d and reproductive systems c l o s e l y l i n k e d (Sage, 1973). However, the actual mechanisms by which thyr o i d hormones a f f e c t reproductive processes have only recently been studied; while many fact s have emerged, few generalizations have as yet been made. L i t e r a t u r e Review Fi s h . Cycles of thyro i d a c t i v i t y occur i n many t e l e o s t s . In part, these cycles appear to be a response to seasonal environmental v a r i a t i o n s (Hoar, 1959). They may, however, have a fun c t i o n a l r e l a t i o n s h i p with the reproduc-t i v e cycle as well (Swift, 1960; Matty, 1960; Sage, 1973). I f associated 2 with reproduction, these cycles might serve as an i d e a l mechanism for co-ordi n a t i n g seasonal adaptation to environmental changes by a l t e r i n g s e n s i t i v -i t y of the f i s h to i t s surroundings (Sage, 1973). Annual increases i n t h y r o i d a c t i v i t y have been c o r r e l a t e d with both ova maturation (Hickman, 1962; Takashima et a l . , 1972; Ichikawa et a l . , 1974; Singh et a l . , 1974) and spawning (Matty, 1960; Berg e t a l . , 1959). I t has been demonstrated that t h i s surge i n thyroid a c t i v i t y may be at l e a s t p a r t i a l l y independent of en-vironmental influence. Peak thyro i d a c t i v i t y occurs i n summer i n Pl a t i c h t h y s  s t e l l a t u s and i n l a t e f a l l i n Coregonus clupeaformis; i n neither case was i t p o s s i b l e to c o r r e l a t e a c t i v i t y with any one environmental f a c t o r (Hickman, 1962). A c y c l i c change i n thyr o i d a c t i v i t y has also been observed i n the viviparous f i s h , P o e c i l i a r e t i c u l a t a (Bromage and Sage, 1968); changes were corre l a t e d with the gestation c y c l e , and occurred i n the absence of changes i n temperature and day length. Thus, thyroid cycles may occur p a r t i a l l y or completely independently of changes i n the environment, and may be associated with the reproductive c y c l e . These increases i n t h y r o i d a l a c t i v i t y , as well as a l t e r i n g s e n s i t i v i t y to the environment.in as s o c i a t i o n with reproduction, may serve to stimulate gonadal maturation. Normal gonadal a c t i v i t y i n t e l e o s t s i s dependent on the presence of a functioning t h y r o i d (Novales et a l ^ , 1973), and a seasonal increase i n hormonal iodine has been c o r r e l a t e d with ova maturation i n P l a t i c h t h y s s t e l l a t u s (Hickman, 1962). Thyroidectomy or treatment with a n t i t h y r o i d drugs w i l l i n h i b i t gonadal maturation, whereas t h y r o i d hormone treatment tends to reverse t h i s e f f e c t (Bern and Nandi, 1964). Grobstein and Bellamy (1939) found that feeding thyr o i d powder to sexually immature males of the genus P l a t y p o e c i l i u s r e s u l t e d i n precocious maturation i n the 3 two species tested. Conversely, destruction of the t h y r o i d gland of Xipho- phorous with 1 3 1 i r e s u l t e d i n the retardation of gonadal maturation. Using the a n t i - t h y r o i d drug thiourea, Barrington and Matty (1952) induced hypothy-roidism i n the minnow, Phoxinus phoxinus, and found that spermatogenesis was arrested. Thiourea treatment also retarded gonadal development i n young zebra f i s h , Brachydanio r e r i o , although maturation of the testes i n adults was unaffected (Scott, 1953). Administration of a n t i - t h y r o i d drugs or thy r o i d hormones has sometimes a f f e c t e d ovarian maturation as w e l l . Treatment with thiourea e f f e c t s a profound drop i n ovarian a c t i v i t y (Raizada, 1974) and blocked oocyte maturation (Mukherjee, 1976) i n the c a t f i s h , Heteropneustes  f o s s i l i s ; further, i t r e s u l t e d i n an i n h i b i t i o n of oocyte growth i n P_. phoxinus (Barrington and Matty, 1952) and P o e c i l i a r e t i c u l a t a (Grosso, 1961). Thyroxine (T^) treatment stimulated ovarian maturation i n immature g o l d f i s h (Hurlburt, 1975), and tr i i o d o t h y r o x i n e (T^) restored the a b i l i t y of sturgeon oocytes to respond to hypophysial stimulation a f t e r prolonged c a p t i v i t y or cooling of the f i s h (Detlaf and Davydova, 1974). In addition, the increase i n a c t i v i t y of the t h y r o i d gland during gestation i n P_. r e t i c u l a t a (Bromage and\ Sage, 1968) may function i n the maturation of the next batch of ova, since B a l l (1962) has shown that the p i t u i t a r y i s not necessary f o r mainten-ance of gestation i n P o e c i l i a . I t appears that the thy r o i d i s f u n c t i o n a l l y involved i n reproduction i n elasmobranchs as w e l l . The thyro-somatic index i n the dogfish, Scyliorhinus  c a n i c u l i , fluctuates annually and i s highest i n maturing females, i n d i c a t i n g an increasing demand for thyroid hormones at t h i s time. In addition, thyroidectomy prevented ovarian maturation i n t h i s species (Lewis and Dodd, 1974). 4 Other Vertebrates. An e f f e c t of t h y r o i d hormones on gonadal function i s also evident i n other vertebrates. Complete maturation of oocytes i n amphibia i s dependent on a functioning thyroid (Pickford and Atz, 1957), and i n Rana cyanophyctis both ovulation and spawning are reduced i n hypothyroid i n d i v i d u a l s (Sarkar and Rao, 1971). In r e p t i l e s there i s evidence of a r e l a t i o n s h i p between t h y r o i d function and many aspects of reproduction. Seasonal changes i n thyr o i d function are correlated with spermatogenesis i n Naja naja and Elaphe taeniura (Wong and Chiu, 1974), and i n the l i z a r d s , Sceloporous o c c i d e n t a l i s and Agama agama, v i t e l l o g e n e s i s occurs at the time of greatest thyr o i d a c t i v i t y (Eyeson, 1970; Stebbins and Cohen, 1973); i n addition, hyperthyroidism i n S_. o c c i d e n t a l i s resulted i n increased rates of ova maturation (Stebbins and Cohen, 1973) . Ovarian weight and egg production i n b i r d s are stimulated by thyr o i d hormone administration (Maqsood, 1952; R a i l et a l . , 1964; Hendrich and Turner, 1966), and i n mammals th y r o i d hormones generally enhance both f e r t i l i t y and sexual behaviour (Rail et a l . , 1964) and can induce precocious sexual maturation (Pickford and Atz, 1957; Kanwar and Chaudry, 1976). Thyroidectomy of female rats leads to a reduction i n v o v a r i a n weight, a retardation of uterine and vaginal development, and a decreased ovulatory capacity (Leatham, 1973; Peppier et a l . , 1975; Kovacs and Mess, 1976); neonatal hypothyroidism can r e s u l t i n permanent defects i n the c o n t r o l of estrous cycles (Phelps and Leatham, 1976). In women, myxedema (a form of hypothyroidism) i s frequently associated with menstrual i r r e g u l a r i -t i e s and r e l a t i v e i n f e r t i l i t y , both of which respond p o s i t i v e l y to thy r o i d hormone treatment (Leatham, 1973) . An i n t e r e s t i n g , though rare, example of thyroid-ovarian i n t e r a c t i o n s i n women i s the occurrence of thyroid tumors within the ovary (Fredlund, 1976). 5 Mechanism of Thyroid Action The t h y r o i d i s involved i n gonadal function i n both poikilotherms and homeotherms, but the question remains as to exactly how the t h y r o i d hormones a f f e c t the reproductive system. They may act d i r e c t l y on the gonads, or a f f e c t them i n d i r e c t l y v i a the hypothalamo-hypophysial a x i s . Furthermore, thyro i d hormones have a profound e f f e c t on metabolic rates and may i n d i r e c t l y influence gonadal function i n t h i s way. There i s evidence that thyroxine stimulates the a c t i v i t y of s t e r o i d dehydrogenases i n the r a t ovary, thus increasing the rate of steroidogenesis, but whether t h i s e f f e c t i s d i r e c t or mediated v i a the p i t u i t a r y i s not known (Leatham, 1973). E a r t l y and Leblond (1954), working also with r a t s , observed that thyroxine stimulated t e s t i c u l a r development when the p i t u i t a r y was present, whereas no e f f e c t was evident i n the absence of the p i t u i t a r y . This indicates that thyroxine e i t h e r acts on the hypothalamo-hypophysial axis, as the authors suggest, or that i t a f f e c t s the gonads v i a some other . mechanism but i s dependent on the presence of p i t u i t a r y gonadotropin. No s i m i l a r work on t e l e o s t s has been published. A complex i n t e r p l a y between thyroid hormones, sex s t e r o i d s , gonadotrophs, and thyrotrophs i s evident i n t e l e o s t s . Sage and Bromage (1970b) found thyroxine i n h i b i t e d gonadotropin production i n Xiphophorous spp. and P o e c i l i a  r e t i c u l a t a i n v i t r o , while the thyrotropic c e l l s of P_. r e t i c u l a t a were stimulated by estrogens and i n h i b i t e d by androgens i n vivo. Sex steroids may also a f f e c t t h y r o i d function d i r e c t l y , since i t has been found that methyl testosterone can stimulate the thyroid gland i n Sparisoma (Matty, 1960), P o e c i l i a r e t i c u l a t a (Sage and Bromage, 1970b), and the hypophysectomized c a t f i s h , Mystus v i t t a t u s (Singh, 1969). In the c a t f i s h , estrogens and pro-6 gesterone e l i c i t a s i m i l a r response, though not as strongly as that induced by methyl testosterone (Singh, 1969). Thus, there appears to be a r e c i p r o c a l e f f e c t , the reproductive hormones i n f l u e n c i n g t h y r o i d a c t i v i t y and the thy r o i d hormones i n turn i n f l u e n c i n g the reproductive system. The many i n t e r a c t i o n s may be a r e s u l t of evolutionary associations. Sage (1973) has proposed that the o r i g i n a l r o l e of thyroid hormones was i n some manner associated with reproduction. The thyroid gland has evolved from the protochordate endostyle, a structure which secretes iodinated mucous used to trap food p a r t i c l e s . There i s , however, no evidence that thyroxine evolved f u n c t i o n a l l y as a g a s t r o - i n t e s t i n a l hormone i n vertebrates. I t s e a r l i e s t r o l e may have been an involvement i n gonadal maturation; t h i s perhaps l e d to other morphogenic e f f e c t s on growth, development and metamorphosis. The close r e l a t i o n s h i p of t h y r o i d a l and gonadal c o n t r o l systems i n te l e o s t s supports t h i s concept. Both gonadotropic and thyrotropic hormones (GTH and TSH) are glycoproteins, and TSH may have evolved from a gonadotropin-l i k e molecule by gene d u p l i c a t i o n and subsequent divergence. There i s no evidence f o r a p i t u i t a r y control over thyr o i d function i n cyclostomes. Thyrotropic a c t i v i t y i s evident i n elasmobranchs, and both t h i s and gonadotrop-i c a c t i v i t y are l o c a l i z e d i n the ventral lobe of the p i t u i t a r y . There i s a general p a r a l l e l i s m between thyroid and reproductive function i n t h i s group, and i t remains to be demonstrated whether or not two d i s t i n c t t h y r o t r o p i c and gonadotropic molecules are involved i n t h e i r control (Dodd and Matty, 1964; Lewis and Dodd, 1974). In t e l e o s t s , TSH and GTH have evolved as separate hormones, but factors regulating t h e i r production remain c l o s e l y l i n k e d . The many accounts of thyroid a c t i v i t y p a r a l l e l i n g reproductive a c t i v i t y i n t e l e o s t s are therefore not s u r p r i s i n g . So close are these 7 p a r a l l e l s that Sage finds i t d i f f i c u l t to imagine how the t h y r o i d could have any e f f e c t that i s not c l o s e l y r e l a t e d to reproduction. Thus, a f u n c t i o n a l and perhaps evolutionary r e l a t i o n s h i p e x i s t s between the reproductive and t h y r o i d a l systems. The involvement of the t h y r o i d i n gonadal maturation i s , however, an obviously complex problem, and one that i s poorly understood. Previous studies have demonstrated that thyroxine adminis-t r a t i o n may stimulate ovarian maturation i n immature g o l d f i s h ; t h i s e f f e c t was dose-dependent, with low doses stimulating gonadal development, while at high doses both stimulatory and i n h i b i t o r y influences were apparent (Hurlburt, 1975). However, i t i s s t i l l unknown whether thyroidal-gonadal i n t e r a c t i o n s i n t e l e o s t s are p i t u i t a r y mediated, or whether they are mediated by e f f e c t s of the t h y r o i d on aspects of general or ovarian metabolism. Hence, the present study was designed to elucidate further the mechanisms by which th y r o i d hormones influence ovarian maturation i n the g o l d f i s h , Carassius  auratus. A p h y s i o l o g i c a l dose for t h y r o i d hormone administration was f i r s t established, and the responses of the p i t u i t a r y , ovary, and l i v e r of i n t a c t f i s h to hyper- and hypothyroid states then investigated. In a d d i t i o n , the e f f e c t s of t h y r o i d hormones alone or i n combination with p i s c i n e and mammal-ian gonadotropins on the ovary and l i v e r of hypophysectomized f i s h were studied, i n order to evaluate the nature and mechanisms of the thyroid's involvement i n ovarian development i n t h i s species. 8 GENERAL TECHNIQUES Maintenance of Goldfish s Goldfish (Carassius auratus L.) of the common comet v a r i e t y were obtained from Hartz Mountain Pet Supply Company, Richmond, B r i t i s h Columbia and Grassyforks F i s h e r i e s Company, M a r t i n s v i l l e , Indiana. Stock f i s h were main-tained i n flowing, dechlorinated fresh water under natural photoperiod, and fed r e g u l a r l y on brine shrimp. The f i s h were frequently diseased on a r r i v a l from suppliers; they were r o u t i n e l y treated i n stock tanks. Skin parasites (Gyrodactylus spp. and Trichodianis spp.) were eradicated by immersion i n formalin (1:5000) f o r 30-60 min or methylene blue (2-4 ppm) f o r several days; the former treatment was found to be more successful. Secondary b a c t e r i a l i n f e c t i o n ( p r i n c i p a l l y Aeromonas hydrophilia) was common, and t h i s was treated i n the e a r l y stages of i n f e c t i o n very s u c c e s s f u l l y by immersion of the f i s h i n 10% sea water containing chloramphenicol (10-15 mg/1) for three to f i v e days. The g o l d f i s h was used as an experimental animal since i t i s of a conven-i e n t s i z e and e a s i l y obtained. Moreover, the structure of the ovary and the p i t u i t a r y under varying environmental and hormonal conditions i s well docu-mented. Histology and H i s t o l o g i c a l Techniques Ovary. The normal sequence of events i n the growth of the g o l d f i s h oocyte includes the f i r s t , or p r e - v i t e l l o g e n i c , growth stage and the second, or v i t e l l o g e n i c growth stage. The second growth stage i s marked i n i t i a l l y by the appearance of yolk v e s i c l e s ( c o r t i c a l a l v e o l i ) when the oocyte i s about 150 ym i n diameter, while the next phase of v i t e l l o g e n e s i s , that of yolk granule formation, begins when the oocyte reaches a diameter of 350 to 450 ym (Yamazaki, 1965) (see F i g . 21). Not a l l developing f o l l i c l e s mature to ovulation; many of them become a t r e t i c at some stage i n development. V i t e l l o g e n i c oocytes are r e g u l a r l y resorbed i n the g o l d f i s h (Khoo, 1974),, and Stacey (1977) has described pre-v i t e l l o g e n i c oocyte a t r e s i a i n t h i s species. i H i s t o l o g i c a l changes i n the ovary of the g o l d f i s h following hypophysec-tomy are documented by Yamazaki (1965) and Khoo (1974). A l l yolky oocytes undergo a t r e s i a following p i t u i t a r y removal (Yamazaki, 1965). Those with more yolk become a t r e t i c f a s t e r than those with le s s yolk; thus, four weeks a f t e r the operation a l l yolk granule stage oocytes are i n various stages o f degeneration, while i n t a c t yolk v e s i c l e oocytes may be found i n the ovary up to 6 or 7 weeks post-hypophysectomy (Khoo, 1974). In f i s h hypophysectomized f o r a period o f several months, a reduction i n the number o f p r e - v i t e l l o g e n i c oocytes i s apparent i n the g o l d f i s h (Stacey, 1977), suggesting that these f o l l i c l e s may undergo a t r e s i a as w e l l . In the present study, ovaries were removed and weighed on termination of experiments. They were then f i x e d i n Bouin's s o l u t i o n , embedded i n p a r a f f i n , and sectioned at 7 ym. Yolk laden ovaries are b r i t t l e , and were often d i f f i c u l t to s e c t i o n r o u t i n e l y . This problem was overcome by soaking the trimmed blocks:, i n 60% e t h y l a l c ohol and g l y c e r i n e (9:1) p r i o r to sectioning. A l l ovaries were stained by the Masson's trichrome technique. Ovarian development was analyzed i n several ways. The gono-somatic index (GSI = gonad weight/body weight x 100) was ca l c u l a t e d f or each f i s h , and means f o r each experimental group determined. A l l values were trans-10 formed by the arc s i n square root method for analysis, and the GSI's for each group compared by analysis of variance (ANOVA) and Duncan's multiple range t e s t f or comparison among means. Next, the diameter of the l a r g e s t oocyte i n a randomly chosen h i s t o l o g i -c a l section was measured. As oocyte si z e i s d i r e c t l y r e l a t e d to the stage of maturity and yolk deposition i n g o l d f i s h (Khoo, 1974), maximum oocyte diameter may be used as a s e n s i t i v e c r i t e r i o n of the stage of ovarian maturity. As with GSI values, these data were analyzed by ANOVA and Duncan's multiple range t e s t . In experiments with hypophysectomized f i s h , i n which hormone administra-t i o n often not only a f f e c t e d s i z e but also the number of oocytes, the stage of ten f o l l i c l e s (both developing and a t r e t i c ) i n a section was then measured; these f o l l i c l e s were sampled randomly using the microscope stage coordinates. Data were pooled for each group, and the number of a t r e t i c and developing oocytes c a l c u l a t e d . Data were analyzed by the Kolmogorov-Smirnov t e s t f or cumulative d i s t r i b u t i o n s . This technique allows determination of the proportion of ovaries occupied by developing and v i t e l l o g e n i c oocytes and by degenerated f o l l i c l e s , while avoiding tedious c e l l counts. Thyroid. Lower jaws containing thy r o i d t i s s u e were f i x e d i n Bouin's s o l u t i o n and t r a n s f e r r e d to d e c a l c i f i c a n t (5% formalin:5% formic acid) for one week. They were then embedded i n p a r a f f i n , sectioned s e r i a l l y at 5 ym, and stained by the Masson's trichrome technique. E p i t h e l i a l c e l l height was used as a h i s t o l o g i c a l index of thy r o i d a c t i v i t y . The highest and lowest c e l l heights i n ten f o l l i c l e s were measured, and mean values for each f o l l i c l e determined. The mean e p i t h e l i a l c e l l 11 height f o r i n d i v i d u a l f i s h was then cal c u l a t e d . Results were analyzed by ANOVA and Duncan's multiple range t e s t . Numbers of thyro i d f o l l i c l e s were not determined, since Chavin (1956) found t h i s parameter extremely variable i n the g o l d f i s h , with l i t t l e or no c o r r e l a t i o n between f o l l i c l e number and thyroid a c t i v i t y . P i t u i t a r y . The bony capsule containing the p i t u i t a r y was removed and f i x e d i n Bouin Holland-saturated mercuric chloride (9:1). . Following d e c a l c i f i c a -t i o n (1 week i n 5% formalin:5% formic acid) t i s s u e s were embedded i n p a r a f f i n , sectioned s e r i a l l y at 5 ym, and stained with the a l c i a n blue (pH 2.5)-periodic a c i d S c h i f f (PAS)-orange G method of Herlant (1960). This method not only allows for d i f f e r e n t i a t i o n between b a s o p h i l i c and a c i d o p h i l i c c e l l s , but also for d i s t i n g u i s h i n g between the b a s o p h i l i c c e l l types. Gonadotropic and thyrotropic c e l l s are basophils, the former s t a i n i n g with both a l c i a n blue and PAS while the l a t t e r s t a i n mainly with a l c i a n blue by t h i s technique. Staining with PAS alone produces a p o s i t i v e pink c o l o r a t i o n i n both c e l l types i n g o l d f i s h (Nagahama, 1973). GTH and TSH c e l l s may also be d i s t i n -guished by t h e i r shape and l o c a t i o n i n the p i t u i t a r y ; i n the g o l d f i s h , GTH c e l l s are generally round or e l l i p t i c a l and are si t u a t e d i n the mesoadeno-hypophysis, whereas TSH c e l l s are polygonal and are located i n the proadeno-hypophysis (Nagahama, 1973). The a c t i v i t y of GTH c e l l s was studied according to the c r i t e r i a estab-l i s h e d by Sage and Bromage (1970a), by which reduced c e l l and nuclear s i z e r e f l e c t r e l a t i v e i n a c t i v i t y . C e l l and nuclear diameters of 15 to 20 GTH c e l l s were measured i n a mid-saggital section of each p i t u i t a r y , and mean values of each f i s h c a l c u l a t e d . Results were analyzed by ANOVA and Duncan's multiple range t e s t . In addition, the amount of PAS p o s i t i v e granulation 12 and the frequency of i n t r a c e l l u l a r spaces were examined; a decrease of one or both of these factors may in d i c a t e that a c e l l i s r e l a t i v e l y i n a c t i v e (Nagahama, personal communication). / Hypophysectomy Goldfish were hypophysectomized using a modification of Yamazaki's (1965) technique as demonstrated by N. E. Stacey (personal communication). The f i s h were f i r s t anaesthetized i n ic e water containing 0.01% tric a n e methane sulphonate (MS 222) f o r about 30 minutes. F i s h acclimated previously at 12°C needed a s l i g h t l y longer period f o r anaesthesia than those acclimated at 20°C, but s u r v i v a l rate of the former was much higher, presumably because temperature shock was l e s s severe. F i s h were next placed on the "operating table" (a grooved sponge) with the l e f t side uppermost and covered with crushed i c e . The gular membrane anterior to the l e f t operculum was s l i t with a small s c a l p e l , and the l e f t operculum together with the f i r s t two g i l l arches retr a c t e d . An oblique i n c i s i o n of about 1.0 mm was made i n the underlying dorsal mucosa, exposing the parasphenoid bone. A hole was then d r i l l e d with a round dental burr immediately p o s t e r i o r to the pterygoid bone and l a t e r a l l y to the path of a conspicuous p a i r of nerves (probably the f a c i a l [vii c r a n i a l ] nerves). The p i t u i t a r y was thus exposed, and was then removed with a curved p i p e t t e a s p i r a t o r . Bleeding was often profuse during the operation, but ceased soon afterwards. A f t e r the operation the f i s h were placed i n buckets containing col d (4°C), well oxygenated 25% sea water (about 7°/oo s a l i n i t y ) . Upon recovery, the f i s h were transferred to holding tanks (8°C; 25% sea water); the tanks were then allowed to warm slowly to room temperature. With the exception of 13 Experiment IVb, 10 mg chloramphenicol/litre was added to the holding tanks for the f i r s t week to prevent i n f e c t i o n ; a f t e r t h i s time the wound was healed, and the exposed area of the s k u l l was covered with connective t i s s u e . Treat-ment with chloramphenicol increased s u r v i v a l rates of 70-80% to greater, than 90%. Following hypophysectomy, there i s a gradual l o s s of skin pigmentation which takes several days or weeks to complete; t h i s whitening of the body may be used as an index of operational success. In addition, completeness of hypophysectomy was determined upon termination of experiments i n the following manner. The p i t u i t a r y region was f i r s t examined under a binocular microscope to check for p i t u i t a r y remnants. I f remnants seemed to be present, t h i s region was f i x e d i n Bouin's s o l u t i o n , d e c a l c i f i e d , and sectioned at 10 ym. Upon s t a i n i n g , s e r i a l sections were examined h i s t o l o g i c a l l y f o r adenohypophy-s i a l t i s s u e . The only disease attacking hypophysectomized f i s h resembled "red f i n disease" (Van Duin, 1973). This was characterized i n i t i a l l y by a lowering of the dorsal f i n and a general l i s t l e s s n e s s . In l a t e r stages there was a rupturing of blood vessels close to the surface. I f detected before the disease was well advanced, the f i s h could be cured e f f e c t i v e l y by immersion i n potassium dichromate (1:25,000) for one week. During t h i s period the f i s h would not feed. 14 SECTION I. THE EFFECTS OF THYROXINE ON PLASMA THYROXINE LEVELS 15 INTRODUCTION Hormone dosage i s an important f a c t o r when considering the e f f e c t s of T^ on ovarian maturation, since mild hyperthyroidism may stimulate while extreme hyperthyroidism may i n h i b i t ovarian development i n both g o l d f i s h (Hurlburt, 1975) and mammals (Leatham, 197-3) . Thus, before commencing further studies on the i n t e r a c t i o n s between the gonadal and t h y r o i d a l systems i n g o l d f i s h , a d e t a i l e d study of the e f f e c t s of hormone dosage and of various vehicles f o r T^ administration on plasma T^ l e v e l s was undertaken. Several methods have been employed to increase plasma T^ l e v e l s i n t e l e o s t s , including i n j e c t i o n , immersion, feeding, and p e l l e t implantation. Their effectiveness i n creating chronic p h y s i o l o g i c a l elevations has, however, only recently been studied. Eales (1974) investigated the e f f e c t s of immersion and i n t r a p e r i t o n e a l i n j e c -tions on plasma T^ l e v e l s i n several species of t e l e o s t s . Intraperitoneal i n j e c t i o n was found to be im p r a c t i c a l as major surges of plasma T^ are created a f t e r i n j e c t i o n of large dosages, while small dosages of T^ were cleared r a p i d l y from the blood, so that more frequent i n j e c t i o n s , i n v o l v i n g considerable stress to the f i s h , would be needed. Immersion was found to be highly e f f e c t i v e i n creating sustained and predictable elevations i n plasma T^ i n the species studied (Eales, 1974). In the present study, the effectiveness of immersion and two a d d i t i o n a l vehicles of T^ administration (feeding and T^-cholesterol p e l l e t implantation) were investigated. 16 MATERIALS AND METHODS The following experiments were conducted at 20°C, and with the exception of the t h i r d experiment, f i s h were fed r e g u l a r l y on brine shrimp throughout the experimental period. a. P e l l e t Implantation F i s h were divided into four groups and implanted with T^-cholesterol p e l l e t s . Cholesterol and L-thyroxine (both from Sigma Chemical Company, St. Louis, Missouri) were mixed thoroughly with a metal spatula, and p e l l e t e d i n 10 mg l o t s by means of a hand press (Parr Instruments Company, Moline, I l l i n o i s ) . The f i n a l amount of T^ i n the p e l l e t s of the four groups was n i l (control), 60 yg, 150 yg, and 750 yg r e s p e c t i v e l y . F i s h were l i g h t l y anaes-thetized i n MS 222 (1:5000); a small i n c i s i o n was made i n the skin between the l a t e r a l l i n e and dorsal f i n and a 2 mm s t a i n l e s s s t e e l spatula used to tunnel i n t o the musculature. The p e l l e t was inserted by means of f i n e forceps, and a drop of terramycin (50 mg/ml) applied to the wound which was then sealed with Eastman 910 adhesive. F i s h were sampled before the s t a r t of the experiment, and again on days 5, 12, 21, and 28. b. Immersion F i s h were divided into three groups and held i n 5 0 - l i t r e glass aquaria. The aquaria were cleaned and f i l l e d with fresh water on the day before the s t a r t of the experiment. At noon on day 0, h a l f the water was removed from each tank and replaced with fresh water to which was added T^ dissolved i n 5 ml of 0.1 N NaOH. The amount of hormone dissolved was such that the f i n a l T„ concentrations i n the three tanks were n i l (control) , 2 yg/100 ml, and 17 10 yg/100 ml r e s p e c t i v e l y . Half the water i n each aquarium was again exchanged at noon on days 2, 4, and 6, and the amount of added at these times was h a l f that added o r i g i n a l l y . F i s h were sampled j u s t p r i o r to the i n i t i a l im-mersion i n T^-treated water, and again on day 0 at 1600 hours and at 1200 hours on days 1, 3, 5, and 7. c. Feeding F i s h were divided i n t o three groups, and starved for one week before the -experiment. On day 0, they were fed a meal-gelatin d i e t prepared according to Peterson et a l . (1967) at a l e v e l of 3% body weight. The T^ was f i r s t ground i n a mortar and p e s t l e , and added i n suspension to the aqueous meal mixture. This was blended for several minutes before addition of the g e l a t i n (cooled to 35°C). In l a t e r experiments, when T^ was administered f o r a period of weeks, the meal-gelatin d i e t was frozen and stored i n t h i s manner. The control group received the unmodified d i e t , while T^ was added to the d i e t of the two treatment groups i n proportions of 10 ppm or 100 ppm. The f i s h were observed to consume a l l food within one-half hour a f t e r addition of the d i e t to the tank. Samples were taken p r i o r to treatment, and again at 2, 4, 8, 12, 24, and 48 hours a f t e r feeding. Sample times i n each experiment were chosen according to the nature of the treatment. Hence, T^ l e v e l s i n p e l l e t implanted f i s h were studied over several weeks to determine the duration as well as the pattern of T^ uptake by the plasma. In the immersion experiment, f i s h were sampled for 1 week only, as the method i s presumably e f f e c t i v e as long as fresh T^ i s added to the water. Oral administration d i f f e r s from the other two methods i n that the f i s h i s exposed to' exogenous T^ f o r a r e l a t i v e l y short time a f t e r feeding; 18 thus the pattern of T^ uptake and subsequent excretion a f t e r a single feeding was studied. Blood Sampling and Plasma Analysis Sampled f i s h were anaesthetized i n MS 222 and blood drawn from the caudal artery by heparinized Natelson blood c o l l e c t i n g tubes (Fisher S c i e n t i f -r i c Company). The blood was then centrifuged, and the plasma c o l l e c t e d and stored at -10°C for up to two and one-half weeks. Plasma stable T^ l e v e l s were determined by the competitive protein-binding technique, using Tetralute k i t s obtained from Ames Company D i v i s i o n , Miles Laboratories, Rexdale, Ontario. The standard method was modified according to Ames Technical Services B u l l e -t i n no. 1-75-D, increasing s e n s i t i v i t y of the t e s t to encompass the r e l a t i v e l y low plasma T^ l e v e l s previously encountered i n t e l e o s t s (Higgs and Eales, 1973). Competitive protein-binding analysis of plasma T^ i s known to be both accurate, and highly reproducible (Braverman et a l . , 1971). In addition, Higgs and Eales (1973) found the Ames Company Tetralute k i t to be the most useful f o r studies of f i s h plasma l e v e l s , as i t i s rapid, precise, and lacks the exactly timed stages of other k i t s tested i n t h e i r laboratory. In the present study, duplicate analyses were usually not possible due to the low amounts of plasma obtainable from experimental animals. However, i n the few instances that they were performed, r e s u l t s were found to be reproducible within 0-0.1 yg T./100 ml plasma. 19 RESULTS F i g . 1 shows the mean plasma T^ l e v e l s i n co n t r o l and T^-implanted f i s h . Control values remained below 1 yg/100 ml and usually below 0.5 yg/100 ml plasma for the duration of the experiment, while T^ l e v e l s i n treated f i s h rose r a p i d l y a f t e r p e l l e t implantation. In the highest and medium dosage groups, these rose to 15-30 times the control values f or the f i r s t three weeks. Those of the lowest dosage group peaked at a mean of about 4 yg T^/ 100 ml plasma a f t e r 5 days, and f e l l to within the control range a f t e r 3 weeks. V a r i a b i l i t y within each sample group was greatest at the medium dosage, while at the highest and lowest dosages, values generally f e l l within 2 yg and 1 yg of the mean re s p e c t i v e l y . Mean plasma T^ l e v e l s for f i s h immersed i n T^-treated water are given i n F i g . 2. Except for the sample 4 hours a f t e r the i n i t i a l immersion, a l l v a l -ues represent means of f i s h sampled 24 hours a f t e r addition of fresh T^ s o l u t i o n . T^ l e v e l s i n control f i s h remained below 1.0 yg/100 ml plasma. In the high dosage group, plasma T^ l e v e l s rose s t e a d i l y , reaching a mean of 5.5 yg/100 ml a f t e r 5 days. Plasma l e v e l s i n the low dosage group increased sharply i n the f i r s t few hours, but f e l l to about 1 yg/100 ml a f t e r 24 hours. A f t e r 7 days, plasma T^ l e v e l s i n t h i s group had r i s e n to a mean of 2.5 yg/ 100 ml. Values f o r both dosage groups are s i m i l a r to those observed i n other t e l e o s t s a f t e r immersion (Eales, 1974). V a r i a b i l i t y i n several of the samples of the higher dosage group was considerable, while i n the lower dosage group, values generally f e l l within 1 yg of the mean. F i g . 3 gives the plasma T^ l e v e l s of f i s h fed d i e t s containing T^. Values for c o n t r o l f i s h were l e s s than 1 yg/100 ml plasma, with the exception 20. Figure 1. Plasma concentrations of f i s h implanted with p e l l e t s contain-ing 0, 60, 150, and 750 yg T 4. Means and standard errors of values from 3 to 5 f i s h are repre-sented. Mean body weights (g) and ranges were: 22.7,18.1-35.5 (controls); 16.4,11.1-20.4 (60 yg. T ) ; 24.9,16 .1-30.8 (150 yg T ) ; 24.2,15.8-38.9 (750 yg T^). 21 |w 0 0 l / * l b H 22 Figure 2. Plasma 1^ concentrations of f i s h immersed i n solutions of 0, 2, and 10 ug/100 ml for up to 7 days. Means and standard errors of values from 3 to 6 f i s h are represent-ed. Mean body weights (g) and ranges were: 10.7,7.9-14.9 (con- . t r o l s ) ; 10.4/7.3-14.4 (2 yg/100 ml); 11.4/8.0-17.8 (10 yg/100 ml). 23 I1" O O L / *1 b H 24 Figure 3. Plasma concentrations of f i s h fed die t s containing i n pro-portions of 0, 10, and 100 ppm f o r up to 48 hours a f t e r feeding. Means and standard errors of values from 3 to 6 f i s h are repre-sented. Mean body weights (g) and ranges were: 10.9,9.6-12.5 (controls); 11.1,8.3-17.2 (10 ppm); 11.0/7.8-14.9 (100 ppm). 2& of two f i s h i n which plasma l e v e l s of 1.1 and 1.8 yg/100 ml were recorded. l e v e l s i n the higher dosage group rose dramatically i n the f i r s t few hours, reaching a peak a f t e r 4 hours. V a r i a b i l i t y i n t h i s and the 8- and 12-hour samples was high, but was reduced and T^ l e v e l s s t a b i l i z e d a f t e r 24 and 48 hours. Plasma l e v e l s i n the f i s h fed 10 ppm were considerably lower and l e s s v a r i a b l e than those of the f i s h fed 100 ppm T 4; values were again highest a f t e r 4 hours, with plasma l e v e l s dropping to a mean of 1.3 yg/ ml a f t e r 48 hours. DISCUSSION Eales (1974) defines a p h y s i o l o g i c a l dose of as one which r a i s e s plasma to a l e v e l which the f i s h i t s e l f could maintain by endogenous production. In the g o l d f i s h , plasma concentrations of untreated f i s h were s i m i l a r to those found i n other t e l e o s t species by Higgs and Eales (1973) and Leloup and Hardy (1976) (usually l e s s than 1 yg/100 ml), but were considerably lower than those reported by Refetoff et a l . (1970) i n Salmo i r i d e u s and Catastomus commersoni (4.5 and 4.3 yg/100 ml r e s p e c t i v e l y ) . Bovine TSH administration may r a i s e endogenous plasma T^ l e v e l s to over 3 yg/100 ml and oc c a s i o n a l l y up to 5 yg/100 ml i n the brook trou t (Chan and Eales, 1976), and perhaps also i n the g o l d f i s h . Hence, a l e v e l of 4 to 5 yg T^/100 ml plasma was considered the upper p h y s i o l o g i c a l l i m i t i n the following studies on the g o l d f i s h . A l l methods of T^ administration elevated plasma T^ l e v e l s of treated f i s h . At the highest dosages, plasma l e v e l s were several times the p h y s i o l o g i c a l values, while the lowest dosages generally r a i s e d plasma T^ l e v e l s to the upper p h y s i o l o g i c a l range. 27 The T^-cholesterol p e l l e t implantation method was found to be a conven-ie n t means of elevating plasma T^ l e v e l s e s p e c i a l l y i n flowing water systems where immersion i s not f e a s i b l e . A minimum of handling time and stress i s incurred, since many f i s h may be implanted i n a day and then l e f t undisturbed fo r a period of three to four weeks. Few problems were encountered with i n -f e c t i o n , and a l l f i s h began to feed well within a day. I f , however, i n j e c t i o n with another hormone i n add i t i o n to T^ treatment i s considered, t h i s method i s impractical as frequently the wound may be aggravated during handling and serious i n f e c t i o n s may occur. Immersion of f i s h i n T^-treated water i s also a convenient and e f f e c t i v e method of T^ administration. The f i n d i n g that exchange of only h a l f the water every second day, rather than a complete exchange every day, created chronic plasma T^ elevations i s s i g n i f i c a n t f o r two reasons. F i r s t l y , stress to the f i s h i s reduced as they need not be removed from the tank, and secondly, i t i s advantageous i n long term experiments where complete exchange of the water every day may not be p r a c t i c a l . In f i s h l e s s t o l e r a n t to metabolic waste, however, more frequent water exchange may be necessary. Although samples were taken i n the present experiment only up to 7 days a f t e r immer-sion, Eales (1974) found plasma T^ l e v e l s i s starved brook trou t d i d not change s i g n i f i c a n t l y between 7 and 21 days of immersion i n 10 yg T^/100 ml water. Feeding res u l t e d i n small elevations of plasma T^ l e v e l s i n the 10 ppm group, and at t h i s dosage o r a l administration appears to be an e f f e c t i v e means of creating sustained increases i n c i r c u l a t i n g T . Treatment with the higher dosage (100 ppm), however, produced a major surge i n plasma T^ l e v e l s comparable to that observed by Eales (1974) a f t e r i n t r a p e r i t o n e a l i n j e c t i o n s , 28 and hence feeding of the hormone at t h i s l e v e l was considered unsuitable for creating chronic p h y s i o l o g i c a l plasma elevations. Plasma concentrations at both dosages peaked 4 hours a f t e r feeding; t h i s c o rrelates well with a study by Hayes (1968) which showed that i n t e s t i n a l absorption of i n man was complete 4 hours a f t e r o r a l administration of the hormone. Hayes (1968) concluded that which was not absorbed within t h i s period becomes f i r m l y bound i n the i n t e s t i n e and i s not a v a i l a b l e for l a t e r absorption. In the present study, v a r i a b i l i t y i n both treatment groups was large perhaps as a r e s u l t of v a r i a t i o n s i n the amount of d i e t consumed by i n d i v i d u a l f i s h . The f i n d i n g that feeding of T^ e f f e c t i v e l y elevates plasma T^ l e v e l s i n a t e l e o s t i s of importance, since i t has been suggested that t h i s i s the most p r a c t i c a l procedure f o r T^ administration i n f i s h culture (Higgs et a l . , 1976) . 29 SECTION I I . EFFECTS OF THYROID HORMONES ON THE PITUITARY, GONADS AND LIVER OF INTACT FISH 30 INTRODUCTION While mild hyperthyroidism may stimulate gonadal function i n vertebrates, severe hyperthyroidism appears to i n h i b i t gonadal development i n several groups studied, i n c l u d i n g t e l e o s t s (Hurlburt, 1975) , birds (Chandola et al_., 1974), and mammals (Leatham, 1973). This i n h i b i t o r y influence i s possibly due to a negative feedback of t h y r o i d hormones on p i t u i t a r y gonadotropin production. In mammals, i t i s well established that changes i n t h y r o i d a c t i v i t y a f f e c t plasma gonadotropin l e v e l s . Both d i u r n a l LH peaks and p i t u i -tary response to l u t e i n i z i n g hormone-releasing hormone (LH-RH) administration are s i g n i f i c a n t l y increased i n hypothyroid r a t s , although basal plasma gonadotropin l e v e l s remain unchanged (Dunn et a l . , 1976; Kalland- et a l . , 1976). In addition, plasma l u t e i n i z i n g hormone (LH) and f o l l i c l e stimulating hormone (FSH) concentrations r i s e to higher l e v e l s i n hypothyroid than i n euthyroid rats following gonadectomy; replacement with T^ r e s u l t s i n a reduction of LH and FSH l e v e l s to those normally found i n euthyroid-gonad-ectomized controls (Larochelle and Freeman, 1974) or even euthyroid-intact controls (Kalland et al_., 1976) . Metabolic clearance rates of LH and FSH were unaffected by t h y r o i d state, suggesting that these changes r e s u l t from a d i r e c t a f f e c t of T^ on the hypothalamo-hypophyseal axis (Larochelle and Freeman, 1974). Midcycle peaks i n LH release may also be a f f e c t e d by thyroid hormones. LH surges i n hyperthyroid r a t s were depressed to 25% of control l e v e l s , and the amount of estrogen needed to induce an LH surge i n ovariectomized-thyroidectomized animals was much g r e a t e r ' i f they had received T^ replacement than i f no T. replacement was given (Freeman et a l . , 1976). 31 Evidence of the e f f e c t s of thy r o i d hormones on gonadotropin production i n man c o n f l i c t s with the above fin d i n g s . Two studies (Akande and Anderson, 1975; Clyde et a l . , 1976) report elevated gonadotropin concentrations i n hyperthyroid patients; i n both cases, treatment with a n t i - t h y r o i d drugs r e -stored serum l e v e l s to those found i n euthyroid i n d i v i d u a l s . These r e s u l t s , however, may not be due to a d i r e c t e f f e c t of t h y r o i d hormones on the p i t u i t a r y . ~T^ stimulates the formation of sex hormone binding globulins i n man, decreasing the l e v e l of unbound sex steroids i n the c i r c u l a t i o n . Nega-t i v e feedback of the sex steroids on the p i t u i t a r y i s consequently reduced; hence, gonadotropin production i s increased (Akande and Anderson, 1975). Thyroid hormones appear to i n h i b i t gonadotropin production i n b i r d s as w e l l . Plasma LH l e v e l s i n the Pekin duck were depressed following thyroxine treatment (Jallegeas et a l . , 1974) and, i n the chick, t e s t i c u l a r 3 2 P uptake was elevated following goiterogen treatment, suggesting that endogenous plasma gonadotropin l e v e l s were increased (Lehman and Frye, 1976). In addition, c y t o l o g i c a l studies of the avian p i t u i t a r y i n d i c a t e a suppression of gonadotropin c e l l a c t i v i t y i n hyperthyroid i n d i v i d u a l s (Thapliyal, 1969; Chandola et a l . , 1974). Studies of the e f f e c t s of thy r o i d hormones on gonadotropin production i n t e l e o s t s are l i m i t e d . Sage and Bromage (1970b) found gonadotropic c e l l s of P o e c i l i a r e t i c u l a t a and Xiphophorous spp. to be i n h i b i t e d by T^ i n v i t r o , and the same e f f e c t was evident i n immature g o l d f i s h i n vivo (Hurlburt, 1975). In the l a t t e r experiment, sample si z e s were r e l a t i v e l y small, and the f i s h were sampled only a f t e r 17 weeks of treatment. In addition, the two dosages used (100 and 1000 ppm orally): may both be considered pharmacological. The present study was designed to investigate the e f f e c t s of known p h y s i o l o g i c a l 32 and supra-physiological doses of on gonadotropin c e l l a c t i v i t y i n mature g o l d f i s h . The e f f e c t s of induced hypothyroidism, with or without T^ replace-ment, on the p i t u i t a r y were also investigated, and l i v e r weights, an e a s i l y measurable metabolic parameter, taken i n t h i s experiment. Thyroid t i s s u e i n g o l d f i s h and other t e l e o s t s does not form a d i s c r e t e glandular body as i s the case i n mammals, but rather consists of scattered f o l l i c l e s l y i n g along the ventral aorta and i n the head kidney. Hence, thyroidectomy i s not possible, and hypothyroidism must be induced by the administration of goiterogenic drugs (chemical thyroidectomy) or N a 1 3 1 I (radiothyroidectomy). The l a t t e r method was not considered i n the present study, since N a 1 3 1 I must be administered i n large doses to e f f e c t destruction of the t h y r o i d a l t i s s u e ; extra-thyroidal t i s s u e s may also be affected, as the isotope i s detectable i n s i g n i f i c a n t concentrations i n the v i s c e r a and other "body components following i n j e c t i o n i n the g o l d f i s h (Chavin, 1956).,Instead, the goiterogen, p r o p y l t h i o u r a c i l , was used. This drug was chosen as i t has several times the goiterogenic a c t i v i t y of re l a t e d a n t i - t h y r o i d compounds ( L i b e r t i and Stanbury, 1971), and thus may be used i n r e l a t i v e l y small dosages. Clearance of these substances from the c i r c u l a t i o n and t h e i r con-centration i n t h y r o i d a l t i s s u e (see Tausk, 1975) would thus be more e f f i c i e n t than when large dosages are administered. This i s important, as most goiterogens act as anti-oxidants and may exert pharmacological e f f e c t s on other metabolic functions. 33 MATERIALS AND METHODS. a. E f f e c t s of Thyroxine i n Mature F i s h S i x t y maturing female g o l d f i s h were di v i d e d among three tanks and main-tain e d under long photoperiod (16L:8D) at 12°C. On day one of the experiment, the three groups were implanted with c h o l e s t e r o l p e l l e t s containing T^ i n amounts of n i l ( c o n t r o l s ) , 60, or 150 yg. Control f i s h and those of the 60 yg group were reimplanted on days 20, 40, and 60, while f i s h of the 150 yg T 4 group were reimplanted on days 30 and 60. A l l groups were fed r e g u l a r l y on brine shrimp throughout the experiment. One-half of the f i s h i n each group was k i l l e d by decapitation on day 40 and the r e s t on day 80. The p i t u i t a r y regions and ovaries were f i x e d f o r subsequent h i s t o l o g i c a l examination. Unfortunately, several of the f i s h implanted with 60 yg T^ died from b a c t e r i a l i n f e c t i o n e a r l y i n the experiment, so sample s i z e s f o r t h i s group are r e l a t i v e l y small. b. E f f e c t s of P r o p y l t h i o u r a c i l and Thyroxine i n Immature F i s h S i x t y immature g o l d f i s h were divided among four tanks and maintained under long photoperiod (16L:8D) at 13-14°C f o r three weeks. The groups were immersed i n 50 ppm 6n-propyl-2-thiouracil (PTU-Sigma) or implanted with c h o l e s t e r o l p e l l e t s containing 60 yg T^ as follows: Group 1: Controls Group 2: PTU Group 3: T 4 Group 4: PTU and T. 34 Groups 1 and 2 were implanted with p e l l e t s containing c h o l e s t e r o l only, and a l l groups were reimplanted on day 20. The PTU was p a r t i a l l y dissolved i n 100 ml 0.01 N NaOH before addition to the tanks. Half the water i n a l l tanks was exchanged once a week, and e i t h e r 100 ml 0.01 N NaOH (groups 1 and 3) or 100 ml 0.01 N NaOH + 50 ppm PTU (groups 2 and 4) added with the fresh water. The f i s h were k i l l e d by decapitation 40 days a f t e r the s t a r t of the experiment. The gonads and l i v e r s of a l l f i s h were weighed, and the ovaries f i x e d f o r subsequent h i s t o l o g i c a l examination. P i t u i t a r y regions and thyroid-a l t i s s u e of females were also preserved. RESULTS P i t u i t a r y E f f e c t s of thyroxine. Table 1 summarizes the r e s u l t s of measurements of gonadotropin c e l l and nuclear diameters i n maturing, T^-treated f i s h . Analy-s i s of variance showed a s i g n i f i c a n t e f f e c t of on both c e l l and nuclear s i z e . A f t e r 40 and 80 days of treatment, both values were s i g n i f i c a n t l y smaller i n the 150 yg T^ group than i n the controls, while the 60 yg T^ group d i f f e r e d from controls only a f t e r 80 days. Mean c e l l volumes ( c a l c u l a t -ed from mean diameters) were reduced to 70% and 55% of the control value i n the 60 yg and 150 yg T^ groups r e s p e c t i v e l y a f t e r 80 days of treatment; nuclear volumes of both treatment groups were 70% of con t r o l values at t h i s time. Within each group, changes i n mean c e l l and nuclear diameters between the two sample times were small, and both analysis of variance and comparison among means found no s i g n i f i c a n t d i f f e r e n c e s . TABLE 1. EFFECTS OF THYROXINE ON GONADOTROPIN CELL AND NUCLEAR SIZE IN MATURING FISH. DAYS OF CELL SIZE NUCLEAR SIZE TREATMENT GROUP .h DIAM(p) t S.E. VOLUME(p3) DIAM(p) ± S.E. VOLUME(p3) 40 Control 8 9.0 + 0.4 a 382 4.8 ± o . i a 58 60 pg T 4 5 8.3 + 0.2 299 4.6 ± 0.1 51 150 pg T 4 9 7.5 + 0.2 b 220 4.2 b, c ± 0.1 ' 39 80 Control 9 9.6 + 0.4 463 5.0 ± 0.1 65 60 pg T 4 5 8.5 + 0.3 d 321 4.5 ± o . i e 48 150 pg T 4 9 7.8 + 0.3 6 248 4.5 ± 0.04 6 48 aANOVA p < 0.001 for e f f e c t of T 4 on diameter, NS for e f f e c t of time < con t r o l (40 day) p < 0.01 C< 60 pg T 4 (40 day) p < 0.05 < con t r o l (80. day) p < 0.05 S< cont r o l (80 day) p < 0.01 36 Morphologically, GTH c e l l s of control f i s h appeared quite active (Fig. 4); they contained large vacuoles, and large granules were frequent. In T^-treated f i s h , however, the vacuoles were reduced i n s i z e (Fig. 5), although large granules were s t i l l numerous. E f f e c t s of p r o p y l t h i o u r a c i l and thyroxine replacement. Results of p i t u i t a r y measurements i n immature con t r o l and treated f i s h (Exp. l ib) are recorded i n Table 2. Mean c e l l and nuclear sizes of the controls are le s s than those of controls i n the previously described experiment (Ila); t h i s i s probably due to the differences i n gonadal development i n the two groups, since GTH c e l l a c t i v i t y (hence c e l l and nuclear size) increases as the gonads mature (Naga-hama, personal communication). Values for PTU-treated f i s h are s i g n i f i c a n t l y elevated over those of both the controls and f i s h r e c e i v i n g a combination of PTU and T^, suggesting gonadotropin production i s stimulated i n hypothyroidism., GTH c e l l s of a l l groups i n t h i s experiment were morphically l e s s active than those of f i s h i n Experiment I la and both vacuolation and frequency of large granules were reduced (Fig. 6, 7, and 8). There was l i t t l e d i f f e r -ence i n gonadotroph cytology between con t r o l and treated f i s h , however, and the main di f f e r e n c e among the groups appeared to be the s l i g h t increase i n c e l l and nuclear s i z e i n PTU-treated f i s h . Since plasma samples were not taken for analysis i n experiment l i b , t h y r o i d e p i t h e l i a l c e l l height and TSH c e l l a c t i v i t y were studied to deter-mine whether the PTU was indeed i n h i b i t i n g t h y r o i d hormone production. PTU i s concentrated by the thyr o i d gland i n mammals and acts to suppress both t h y r o i d hormone synthesis, by blocking organic binding of iodine ( L i b e r t i and Stanbury, 1971), and peripheral conversion of T^ to the more p h y s i o l o g i c a l l y a c t i v e T form (Tausk, 1975). As a r e s u l t of reduced plasma thyroid TABLE 2. EFFECTS OF PROPYLTHIOURACIL (PTU) AND THYROXINE ON GONADOTROPIN CELL AND NUCLEAR SIZE IN IMMATURE FISH. CELL SIZE NUCLEAR SIZE GROUP .n DIAM(U) ± S.E. VOLUME(y 3) DIAM(y) ± S.E. VOLUME ( y 3 ) Control 6 7.0 ± 0 . l a 180 3.8 ± 0.1 a 29 PTU 4 7.8 ± 0.1 b , C 248 ' 4.5 ± 0 . 1 b , d 48 PTU + T„ 6 7.2 ± 0.1 195 4.0 ± 0.1 33 aANOVA p < 0.05 b> control p < 0.01 C> PTU + T 4 p < 0.05 < PTU + T„ p < 0.01 38 Figure 4. P i t u i t a r y gonadotrophs (G) and thyrotrophs (T) of untreated maturing female g o l d f i s h . A l c i a n blue-PAS-orange G. X610. Figure 5. P i t u i t a r y gonadotrophs (G) of maturing female g o l d f i s h implanted with T 4 (150 pg) for 80 days. A l c i a n blue-PAS-orange G. X610. Figure 6. P i t u i t a r y gonadotrophs (G) and thyrotrophs (T) of untreated immature g o l d f i s h . A l c i a n blue-PAS-orange G. X610. Figure 7. P i t u i t a r y gonadotrophs (G) of immature g o l d f i s h immersed i n p r o p y l t h i o u r a c i l for 40 days. A l c i a n blue-PAS-orange G. X610. Figure 8. P i t u i t a r y gonadotrophs (G) of immature g o l d f i s h treated with p r o p y l t h i o u r a c i l and for 40 days. A l c i a n blue-PAS-orange G. X610. Figure 9. P i t u i t a r y thyrotrophs (T) of immature g o l d f i s h immersed i n p r o p y l t h i o u r a c i l f o r 40 days. A l c i a n blue-PAS-orange G. X610. 40 hormone l e v e l s , p i t u i t a r y TSH production r i s e s dramatically and t h y r o i d e p i t h e l i a l c e l l s consequently hypertrophy. In the present study, only one of the PTU-treated f i s h had greatly elevated e p i t h e l i a l c e l l s a f t e r 42 days, while i n another, enlarged f o l l i c l e s were evident (active f o l l i c l e s are gener-a l l y small i n the g o l d f i s h (Chavin, 1956)), and the mean e p i t h e l i a l c e l l height in. t h i s group d i d not d i f f e r s i g n i f i c a n t l y from the controls (Table 3). Chavin (1956) also found l i t t l e evidence of h i s t o l o g i c a l change i n the gold-f i s h thyroid following goiterogen treatment, yet t h y r o i d a l a c t i v i t y as measured by 1 2 5 i uptake was s i g n i f i c a n t l y suppressed. In the present study, p i t u i t a r y TSH production was apparently increased following PTU treatment, since c e l l frequency was greater and a f f i n i t y f o r basic stains l e s s than i n cont r o l p i t u i t a r y glands (Table 3; F i g . 9); both these changes are thought to be associated with an increase i n TSH production i n g o l d f i s h and other t e l e o s t s (Nagahama, 1973). In f i s h r e c e i v i n g a replacement dose of T^, both thyroid e p i t h e l i a l c e l l height and TSH c e l l frequency were reduced over con t r o l values. Ovary Thyroxine treatment of maturing females had no e f f e c t on e i t h e r GSI or maximum oocyte diameter (Table 4). In a l l groups, the v a r i a b i l i t y i n both values was large, and no s i g n i f i c a n t differences were evident a f t e r 40 or 80 days. Results of the e f f e c t s of T^ and PTU treatment of immature f i s h are given i n Table 5. Ovaries of con t r o l f i s h showed l i t t l e development over the experimental period, and no s i g n i f i c a n t differences were evident i n the mean GSI values of co n t r o l and treated f i s h . The mean maximum oocyte s i z e of the TABLE 3. EFFECTS OF PROPYLTHIOURACIL AND THYROXINE ON THYROID EPITHELIAL (TH EPITH) CELL HEIGHT AND PITUITARY TSH CELL FREQUENCY GROUP TH EPITH CELL HEIGHT (y) ± S.E. TSH CELL FREQUENCY Control PTU PTU + T, 6 4 6 4.0 ± 0.2 5.4 ± 1.5 3.2 ± 0.1* ++(+) ++++(+) + (+) ANOVA p < 0.01 b.. <control p < 0.01 TABLE 4. EFFECT OF THYROXINE ON OVARIAN DEVELOPMENT OF MATURING FISH. DAYS OF TREATMENT 40 BODY WT 80 GROUP n (g) + S.D, GSI + S.E.. Control 9 11.8 + 2.5 \ 5.70 + 1.78 3 60 yg T 4 5 15.1 + 3.8 5.16 + 1.00 150 yg T 4 9 13.1 + 2.3 6.93 + 2.47 Control 8 12.5 + 2.1 6,93 + 2.47 60 yg T 4 5 13.1 + 1.2 5.02 + 2.14 150 yg T 4 9 13.6 + 1.7 10.10 + 1.90 MAX OOCYTE DIAM(y) ± S. 495 ± 63 J 465 ± 79 565 ± 57 550 ± 51 470 ± 93 603 ± 24 ANOVA NS TABLE 5. EFFECT OF PROPYLTHIOURACIL AND THYROXINE ON OVARIAN - DEVELOPMENT OF IMMATURE FISH. GROUP n BODY WT (g) ± SD GSI ± SE MAX OOCYTE DIAM (y) ± SE I n i t i a l c o n t r o l 10 9.3 ± 2.2 1.70 ± 0.14a 146 ± 6 b F i n a l c o n t r o l 8 11.3 ± 3.2 1.66 ± 0.13 157 ± 4 T4 10 10.3 ± 3.1 2.07 ± 0.18 195 ± 21 C PTU 5 11.7 ± 5.2 1.95 ± 0.26 175 ± 20 PTU + T„ 4 8 11.0 ± 3.2 2.12 ± 0.24 185 ± 12 3ANOVA NS ANOVA p < 0.01 > i n i t i a l control p < 0.05 44 group was s i g n i f i c a n t l y greater than that of the i n i t i a l c ontrols, although not of the f i n a l c o n trols. L i v e r The mean hepato-somatic index (HSI) was calculated f o r each group i n Experiment l i b , and the r e s u l t s are given i n Figure 10. PTU treatment of female f i s h r e s u l t s i n HSI values which were s i g n i f i c a n t l y lower than those of both c o n t r o l and-T^-treated f i s h . In the groups r e c e i v i n g T^ alone or PTU + T , HSI values were s i m i l a r to con t r o l s . In males, however, r e l a t i v e l i v e r weights were s i g n i f i c a n t l y elevated i n the T^-treated f i s h , while values for the PTU and PTU + T^ groups remained unchanged from c o n t r o l s . The mean HSI of male controls i s s i g n i f i c a n t l y l e s s than that of female controls, although treatment values f o r the two sexes are very s i m i l a r . DISCUSSION P i t u i t a r y I t appears that, as i n mammals, thyro i d hormones may modulate gonado-t r o p i n production i n t e l e o s t s . The present study supports the findings of Sage and Bromage (1970b) and Hurlburt (1975) which suggest that t h i s i n f l u -ence i s of an i n h i b i t o r y nature. T^ treatment of maturing g o l d f i s h r e s u l t e d i n a dose-dependent suppression of gonadotropic c e l l a c t i v i t y , while t r e a t -ment of immature f i s h with the goiterogen, p r o p y l t h i o u r a c i l , r e s u l t e d i n a s l i g h t stimulation of c e l l a c t i v i t y ; the l a t t e r e f f e c t was reversed by T^ replacement therapy. 45 Figure 10. Hepato-somatic indic e s of i n t a c t immature f i s h treated with thyroxine and p r o p y l t h i o u r a c i l . P l a i n bars represent mean values of female f i s h , and s t i p p l e d bars represent mean values of male f i s h . V e r t i c a l l i n e s i n d i c a t e standard errors, and numbers adjacent to each standard histogram are sample s i z e s . S i g n i f i c a n t differences are evident between the HSI values of: a. male controls and female controls (p < 0.05). b. PTU-treated females and control females (p< 0.05). c. PTU-treated females and T^-treated females (p < 0.01). d. male controls and T^-treated males (p < 0.01). e. T 4 ~ t r e a t e d males and both PTU-treated and PTU + T^-treated males (p < 0.05). 46 J L o o co q CN I S H The mechanisms whereby T^ may a f f e c t gonadotropin production are uncer-t a i n , but i t i s most l i k e l y v i a an action on the hypothalamo-pituitary a x i s . In t e l e o s t s a d i r e c t e f f e c t on the p i t u i t a r y was demonstrated by Sage and Bromage (1970b), who found that addition of to the medium of cultured p i t u i t a r i e s r e s u l t e d i n gonadotropin c e l l i n h i b i t i o n . Mammalian work suggests e f f e c t s on both the p i t u i t a r y and hypothalamus. The responsiveness of the p i t u i t a r y to LH-RH was enhanced i n hypothyroid r a t s (Kalland e_t al_. , 1976), and T^ treatment of t h i s species r e s u l t e d i n an increase i n the estrogen receptor content of the ant e r i o r p i t u i t a r y (Cidlowski and Muldoon, 1975). Since 173-estradiol a l t e r s s e n s i t i v i t y of the gonadotrophs to LH-RH (Cidlowski and Muldoon, 1975), a T4~stimulated increase i n estrogen binding would presumably decrease the gonadotropin c e l l response to endogenous LH-RH, as suggested by Larochelle and Freeman (1974) . This may r e s u l t from a decreased rate of estrogen receptor catabolism, rather than a stimulation of receptor anabolism, as the metabolic rate of the p i t u i t a r y i n mammals i s , i n contrast to most other t i s s u e s , diminished under the influence of thyroi d hormones (Tonoue and Yamamoto, 1967; Lee et a l . , 1968; Cidlowski and Muldoon, 1975) . Freeman et al_. (1976) , however, found the LH response to LH-RH unaltered i n hyperthyroid r a t s while LH production following hypothalamic stimulation was s i g n i f i c a n t l y reduced, i n d i c a t i n g the hypothalamus must also be consider-ed as a s i t e of negative feedback of th y r o i d hormones on gonadotropin production. Ovary Thyroxine had no detectable e f f e c t on ovarian development of mature f i s h a f t e r 40 or 80 days of treatment. V a r i a b i l i t y i n both GSI and maximum 48 oocyte diameter i n a l l groups was very large and, although both values were greatest i n the high dose group, s t a t i s t i c a l differences were not evident. T^ treatment of immature f i s h , however, resu l t e d i n a s l i g h t but s t a t i s t i c a l -l y s i g n i f i c a n t increase i n ovarian maturation a f t e r 40 days. This i s consistent with the r e s u l t s of Hurlburt (1975) i n g o l d f i s h and Higgs et a l . (1976) i n coho salmon; T^ i n both cases stimulated ovarian development of immature f i s h . An i n h i b i t o r y e f f e c t of high doses of T^ on ovarian maturation was described by Hurlburt (1975), and t h i s was a t t r i b u t e d to a thyroxine-induced decrease i n gonadotropin production. Retardation of gonadal development i n T^-treated f i s h was not apparent i n the present study. The dosages used, however, were considerably le s s than the high dose of Hurlburt (1975) (1000 ppm administered o r a l l y ) , hence t h i s i s perhaps not s u r p r i s i n g . Although gonadotropin c e l l a c t i v i t y was apparently i n h i b i t e d i n mature treated f i s h (Exp. I l a ) , c e l l and nuclear diameters were s t i l l greater than those values of immature controls i n Exp. l i b . Any decrease i n gonadotropin production i n the former case may have been compensated for by an increase i n plasma T^ l e v e l s . Previous studies have reported a retardation of oocyte maturation following goiterogen treatment i n t e l e o s t s (e.g. Barrington and Matty, 1952; Grosso, 1961; Mukherjee, 1976). In the present study immersion i n propyl-t h i o u r a c i l d i d not a f f e c t gonadal development; however, ovarian development of c o n t r o l f i s h was disappointingly s l i g h t over the experimental period, and any i n h i b i t o r y e f f e c t s of PTU would be d i f f i c u l t to demonstrate. I t i s also doubtful that th y r o i d hormone l e v e l s were depressed f o r the f u l l 40 days of treatment, since only synthesis and not release of stored thyroxine 49 i s a f f e c t e d by goiterogens (Tausk, 1975). A l t e r n a t e l y , the r e s u l t s of previous studies may have been due to a pharmacological action of the a n t i -t h y r o i d drug d i r e c t l y on the ovary; dosages used i n the present study were comparatively low, and MacKay (1973) found that, i n the f i r e t a i l gudgeon, a small dose of thiourea was e f f e c t i v e i n suppressing t h y r o i d a c t i v i t y but had no e f f e c t on ovarian development while a higher dose i n h i b i t e d both thyroid function and ovarian maturation. L i v e r The mean HSI of female f i s h was greater than that of male f i s h i n Exp. l i b , suggesting a sexual d i f f e r e n c e i n l i v e r weights. This may be a r e s u l t of estrogen-induced l i v e r hyperplasia as has been described by B a i l e y (1957) i n i n t a c t g o l d f i s h , since Khoo (1974) found p r e v i t e l l o g e n i c and e a r l y yolk v e s i c l e oocytes capable of s t e r o i d production i n t h i s species; however, Schreck and Hopwood (1974) report estrogen l e v e l s to be s i m i l a r i n immature g o l d f i s h of both sexes. A l t e r n a t e l y , the d i f f e r e n c e s i n l i v e r weights may represent a d i f f e r e n c e i n endogenous T^ s e c r e t i o n rates i n males and females, since administration of exogenous T^ increased the HSI of males to a value s i m i l a r to that of female c o n t r o l s , while PTU treatment reduced the HSI of females to a value s i m i l a r to that of male c o n t r o l s . Thyroid hormones have also been reported to increase l i v e r weight i n the trout, Salmo g a i r d n e r i i (Takashima et a l . , 1972) and i n thyroidectomized water snakes, Natrix  p i s c a t o r (Gupta et. a l . , 1975). The mechanisms by which t h y r o i d hormones may a f f e c t l i v e r metabolism i n t e l e o s t s are uncertain, and w i l l be discussed i n a l a t e r s e c t i o n . 50 Thyroid The absence of f o l l i c u l a r hypertrophy i n most of the p r o p y l t h i o u r a c i l -treated f i s h i s perhaps not s u r p r i s i n g , since s i m i l a r findings have previous-l y been reported i n the g o l d f i s h (Chavin, 1956). In addition, Subhedar and Rao (1975) found that immersion i n t h i o u r a c i l produced stimulatory changes i n the thyroid f o l l i c l e s of the c a t f i s h , Heteropneustes f o s s i l i s , up to 30 days only; a f t e r t h i s time, values for t h y r o i d e p i t h e l i a l c e l l heights i n treated f i s h dropped to within the c o n t r o l range. This suggests that the goiterogen, as well as i n h i b i t i n g hormonal synthesis by the thyroid t i s s u e , may perhaps reduce the s e n s i t i v i t y of the f o l l i c u l a r c e l l s to TSH stimulation a f t e r prolonged treatment. Since the goiterogen dosage used i n the present study was rather low (50 ppm), i t might also be argued that t h y r o i d hormone synthesis was not i n h i b i t e d s i g n i f i c a n t l y . However, t h i o u r a c i l , which has one-tenth the goiterogenic a c t i v i t y of p r o p y l t h i o u r a c i l ( L i b e r t i and Stanbury, 1971), severely reduced 1 2 5 i uptake by the g o l d f i s h t h y r o i d a t a dosage of 500 ppm i n the absence of f o l l i c u l a r hypertrophy (Chavin, 1956). In addition, morphological studies of TSH c e l l a c t i v i t y i n the present experiment indic a t e that TSH production was increased somewhat i n goiterogen treated f i s h , suggesting that thyroid hormone synthesis was indeed reduced. 51 SECTION I I I . EFFECTS OF THYROXINE AND SALMON GONADOTROPIN ON OVARIES AND LIVERS OF LONG TERM HYPOPHYSECTOMIZED FISH 52 INTRODUCTION Hurlburt (1975) found that low doses of exogenous T4.stimulated ovarian maturation i n immature g o l d f i s h , while at high doses both stimulatory and i n h i b i t o r y e f f e c t s were evident. Preliminary studies of GTH c e l l a c t i v i t y i n t h i s study ind i c a t e d that T^ may i n h i b i t GTH production, since at high (pharmacological) dosages GTH c e l l and nuclear size were considerably reduced and gonadal maturation retarded. These r e s u l t s suggested that thyroid hormones act s y n e r g i s t i c a l l y with gonadotropin i n t h e i r e f f e c t s on ovarian development, and that i n the absence of c r i t i c a l l e v e l s of gonadotropin, T^ i s i n e f f e c t i v e i n stimulating gonadal maturation. To t e s t t h i s hypothesis, the e f f e c t s of T^ alone and i n combination with salmon gonadotropin were studied i n long term hypophysectomized f i s h . t MATERIALS AND METHODS a. E f f e c t s of Thyroxine i n Long Term Hypophysectomized F i s h Goldfish hypophysectomized f o r three months were divided among three tanks (20°C; 16L:8D). A f t e r a two-week acclimation period, the groups were immersed i n T^ at concentrations of n i l (controls), 2, or 10 ug/100 ml. Half the water was exchanged three times a week and fresh T^ added. The experiment was terminated 25 days a f t e r the i n i t i a l immersion. F i s h were k i l l e d by decapitation, and the l i v e r s and ovaries removed and weighed; the ovaries were f i x e d f o r subsequent h i s t o l o g i c a l examination. 53 b. E f f e c t s of Salmon Gonadotropin and Thyroxine i n Long Term  Hypophysectomized F i s h G o l d f i s h hypophysectomized three to f i v e months were divided i n t o three groups and maintained at 20°C and 16L:8D f or two weeks. They were then injec t e d i n t r a p e r i t o n e a l l y with s a l i n e (controls), or 3-4 ug/g salmon gonadotropin (SG-G100 - N u t r i t i o n and Applied Endocrinology Program, F i s h e r i e s and Marine Service, Research and Resource Services Directorate, West Vancouver, B. C , batch no. 4, May 1972). In addition to salmon gonadotropin, one group was fed T^ (25 ppm) throughout the experiment; the cont r o l group and the group r e c e i v i n g SG-G100 alone were fed the unmodified meal-gelatin d i e t (see Section I ) . The SG-G100 was dissolved i n f i s h s a l i n e (Wiebe, 1969) and 0.1 ml of the s o l u t i o n i n j e c t e d with a 1.0 ml tuberculin syringe and 30 gauge needle. F i s h were i n j e c t e d three times a week for i four weeks. The experiment was terminated 24 hours a f t e r the l a s t i n j e c t i o n . A l l f i s h were k i l l e d by decapitation, and both ovaries and lower jaws, contain-ing t h y r o i d t i s s u e s , removed and f i x e d f o r subsequent h i s t o l o g i c a l examination. RESULTS Ovary Thyroxine alone had no apparent e f f e c t on ovarian development of long term hypophysectomized f i s h . Ovaries of both control and treated f i s h were h i s t o l o g i c a l l y regressed, containing only p r e - v i t e l l o g e n i c oocytes and, since the f i s h were hypophysectomized when immature, few o l d a t r e t i c f o l l i c l e s (Fig. 12 and 13). The GSI and maximum oocyte s i z e values of a l l three groups were s i m i l a r (Table 6). TABLE 6. EFFECTS OF THYROXINE ON OVARIAN DEVELOPMENT AND LIVER WEIGHT IN LONG-TERM HYPOPHYSECTOMIZED FISH. GROUP BODY WT (g) ± SD GSI ± SE MAX OOCYTE DIAM(p) ± SE HSI ± SE Control 2 pg T 4 10 pg T 9 8 6 14.1 ± 3.0 12.5 ± 1.5 12.1 ± 2.3 0.92 ± 0.14 1.15 ± 0.19 1.23 ± 0.16 129 ± 8 134 ± 7 137 ± 5 7.65 ± 0.78 11.69 ± 0,72 9.60 ± 0.59 a,b > control p < 0.01 b> 10 pg T 4 p < 0.05 °> control p < 0.05 55 Salmon gonadotropin, with or without thyroxine supplement, stimulated a s i g n i f i c a n t increase i n ovarian weight over hypophysectomized controls (Table 7). While the GSI values of the SG-G100 and the SG-G100 + T 4 groups d i f f e r e d only s l i g h t l y , the mean maximum oocyte diameter of the l a t t e r group was s i g n i f i c a n t l y greater than those of both the SG-G100 group and the cont r o l s . S i g n i f i c a n t d i f f e r e n c e s i n the frequency of developing oocytes and a t r e t i c f o l l i c l e s are also evident i n t h i s experiment (Fig. 11). As with the previous experiment, co n t r o l ovaries were h i s t o l o g i c a l l y completely regressed. Most of each ovary i n t h i s group consisted of o l d a t r e t i c f o l l i c l e s , with a r e l a t i v e l y small number of i n t a c t oocytes (Fig. 14). Those oocytes which were present were p r e - v i t e l l o g e n i c , and few exceeded 100 ym i n diameter. Ovaries of f i s h r e c e i v i n g SG-G100 alone had a la r g e r proportion of developing ova (Fig. 15) and 2 out of 6 f i s h had oocytes i n the early yolk v e s i c l e stage. In those f i s h r e c e i v i n g both SG-G100 and T^, the number of a t r e t i c f o l l i c l e s was greatly reduced and v i t e l l o g e n i c oocytes were more frequent than i n controls or i n SG-G100-treated f i s h . Yolk v e s i c l e formation was evident i n f i v e of the eight f i s h , and only i n t h i s group were developing f o l l i c l e s over 200 ym encountered (Fig. 16). Thyroid Thyroid e p i t h e l i a l c e l l heights were measured i n Experiment I l l b to determine whether SG-G100 stimulated t h y r o i d a l function i n treated f i s h (Table 7; F i g . 17 and 18). As i s evident, f o l l i c u l a r c e l l heights of the two treatment groups are s i g n i f i c a n t l y greater than those of the controls, suggesting that t h y r o i d a c t i v i t y and perhaps also thyroid hormone production are indeed stimulated by the SG-G100 preparation. 7. EFFECTS OF SALMON GONADOTROPIN AND THYROXINE ON OVARIAN DEVELOPMENT AND THYROID EPITHELIAL CELL HEIGHT IN LONG-TERM HYPOPHYSECTOMIZED FISH. BODY WT MAX OOCYTE THYROID CELL GROUP n .(g) ± SD GSI ± SE DIAM(u) ± SE HT(y) ± SE Control 4 24.8 ± 11.3 0.56 ± 0.10 70 ± 13 2.2 ± 0.16 a h a f SG-G100 6 27.1 ± 11.8 1.57 ± 0.34 165 ± 9 4.8 ± 0.20 SG-G100 a ± 2 Q a , c 4 _ 9 ± a + T„ a> control p < 0.01 k> control p < 0.05 C> SG-G100 p < 0.05 57 Figure 11. E f f e c t s of salmon gonadotropin and thyroxine on oocyte composi-t i o n of ovaries of long-term hypophysectomized f i s h . The r e l a t i v e frequencies of a t r e t i c (A), p r e - v i t e l l o g e n i c (PV), and v i t e l l o g e n i c (V) f o l l i c l e s are given f o r each group. Both treatment groups d i f f e r from the control group at p < 0.01, and the SG-G100 + T 4 group d i f f e r s from the SG-G100 group at p < 0.05. % of O V A R Y CO o T 4*. O T Cn O T f> N O O T—r 00 o T o T o o 1 8S 5 9 Figure 12. Ovarian section of an untreated long-term hypophysectomized f i s h (Exp. I l i a ) showing only p r e - v i t e l l o g e n i c oocytes. Masson's trichrome. X140. Figure 13. Ovarian section of long-term hypophysectomized f i s h immersed i n T 4 (10 yg/100 ml) for 25 days. Only p r e - v i t e l l o g e n i c oocytes are evident. Masson's trichrome. X140. Figure 14. Ovarian section of an untreated long-term hypophysectomized f i s h (Exp. I l l b ) . Only small p r e - v i t e l l o g e n i c (PV) and a t r e t i c (A) f o l l i c l e s are evident. Masson's trichrome. X140. Figure 15. Ovarian section of a long-term hypophysectomized f i s h treated with SG-G100 for four weeks. Note ea r l y yolk v e s i c l e stage oocyte (YV). Masson's trichrome. X140. Figure 16. Ovarian section of a long-term hypophysectomized f i s h treated with SG-G100 and for four weeks. Note oocyte with well developed yolk v e s i c l e s (YV). Masson's trichrome. X140. 61 Figure 17. Thyroid f o l l i c l e s (F) from a con t r o l long-term hypophysectomized f i s h showing low e p i t h e l i a l c e l l s . Masson's trichrome. X450. Figure 18. Thyroid f o l l i c l e s (F) from a long-term hypophysectomized f i s h treated with SG-G100 for four weeks. Note hypertrophied e p i t h e l i a l c e l l s . Masson's trichrome. X450. -(ol: 63 L i v e r Thyroxine treatment, e s p e c i a l l y at the lower dosage, res u l t e d i n an increase i n r e l a t i v e l i v e r weights i n long-term hypophysectomized f i s h (Table 6). Since HSI values r i s e following p i t u i t a r y a b l a t i o n i n untreated g o l d f i s h and other t e l e o s t s as a r e s u l t of increased l i v e r glycogen stores (Walker and Johansen, 1975; B a l l and Hawkins, 1976), the e f f e c t of T^ i n the present study was to augment the l i v e r response to hypophysectomy. Whether t h i s was due to a further stimulation of glycogen accumulation could not be assessed; l i v e r samples were taken for hepatic glycogen deter-mination, but l a b e l s were l o s t during processing. 64 DISCUSSION Ovary The r e s u l t s of the present study demonstrate that T^ alone has no e f f e c t on ovarian development i n long-term hypophysectomized g o l d f i s h , but the hormone does augment the ovarian response to p i s c i n e gonadotropin stimula-t i o n . This suggests that the e f f e c t s of T^ are dependent on the p i t u i t a r y , since the presence of gonadotropin i s necessary for thyroxine to influence ovarian development. Thus, as suggested by Hurlburt (1975), t h y r o i d a l -gonadal i n t e r a c t i o n s appear to be regulated by gonadotropin l e v e l s , and i n the absence of the l a t t e r , thyroxine i s i n e f f e c t i v e . A stimulatory e f f e c t of salmon gonadotropin on the ovary of g o l d f i s h hypophysectomized 6 weeks has been described previously by Yamazaki and Donaldson (1968). In the present study, SG-G100 was e f f e c t i v e i n re-i n i t i a t i n g ovarian development 3 to 5 months post-operatively, i n d i c a t i n g that the ovary does not lose i t s a b i l i t y to respond to gonadotropin even a f t e r several months of p i t u i t a r y deprivation. Thyroid Salmon gonadotropin stimulated t h y r o i d a l a c t i v i t y , as measured by h i s t o l o g i c a l c r i t e r i a , i n hypophysectomized f i s h . This l i k e l y resulted i n an el e v a t i o n i n plasma T^ l e v e l s i n treated f i s h since, i n a study on i n t a c t g o l d f i s h , plasma T^ concentrations rose from a mean of 0.3-0.5 ug/100 ml to 0.9 ug/100 ml following SG-G100 treatment (unpublished r e s u l t s ) . Donaldson and McBride (1974) a t t r i b u t e t h i s s i t u a t i o n to TSH contamination i n the SG— G100 preparation, rather than to an inherent thyrotropic a c t i v i t y of the 65 gonadotropin molecule i t s e l f . L i v e r Thyroxine treatment s i g n i f i c a n t l y elevated l i v e r weights of hypophysec-tomized f i s h . This e f f e c t was greatest at the lower dosage, while at the higher dosage HSI values were intermediate between those of the control and low dose groups. Thyroxine i n small concentrations may promote the accumulation of n u t r i t i o n a l stores i n the l i v e r , perhaps v i a a stimulation of general metabolic processes i n the animal, while administration of the hormone at high concentrations may r e s u l t i n a depletion of l i v e r glycogen as i s apparent i n P o e c i l i a l a t i p i n n a ( B a l l and Hawkins, 1976) and mammals (White et a l . , 1973). Whether these e f f e c t s r e f l e c t d i r e c t or i n d i r e c t actions of thyroxine on the l i v e r i n the g o l d f i s h i s uncertain. 66 SECTION IV. EFFECTS OF THYROXINE AND LH ON OVARIES, THYROID, AND LIVER OF SHORT-TERM HYPOPHYSECTOMIZED FISH \ 67 INTRODUCTION The e f f e c t s of mammalian gonadotropins on gonadal development have been studied extensively i n f i s h (see reviews by P i c k f o r d and Atz, 1957; Hoar, 1966; de Vlaming, 1974; M. Fontaine, 1976). LH and HCG (human chorionic gonadotropin) have been more successful i n . a c c e l e r a t i n g gonadal maturation than FSH i n t e l e o s t s (de Vlaming, 1974)/ although a l l are considerably l e s s e f f e c t i v e than any of t h e i r p i s c i n e equivalents (M. Fontaine, 1976). LH and HCG have usually stimulated v i t e l l o g e n e s i s i n i n t a c t f i s h (e.g. / stickleback, Ahsan and Hoar, 1963; G i l l i c h t h y s . m i r a b i l i s , de Vlaming, 1972; Cytomagaster aggregata, Wiebe, 1969). Treatment of hypophysectomized females, however, has produced c o n f l i c t i n g r e s u l t s . LH was i n e f f e c t i v e i n the c a t f i s h and HCG i n e f f e c t i v e i n the g o l d f i s h i n i n i t i a t i n g v i t e l l o g e n e s i s following p i t u i t a r y a b l a t i o n (Yamazaki, 1965; Sundararaj and Anand, 1972). LH did, however, restore steroidogenic enzyme a c t i v i t y i n hypophysectomized g o l d f i s h (Khoo, 1974), and HCG prolongs i n v i t r o s u r v i v a l of v i t e l l o g e n i c oocytes i n t h i s species (Remacle et al_. , 1976) . Mammalian gonadotropins are more co n s i s t e n t l y e f f e c t i v e i n stimulating gonadal development i n male than i n female t e l e o s t s (M. Fontaine, 1976). LH stimulated t e s t i c u l a r development i n Couesius plumbeus (Ahsan, 1966), Fundulus h e t e r o c l i t u s (Pickford et a l . , 1972), and Heteropneustes f o s s i l i s (Sundararaj and Mayyar, 1967) following p i t u i t a r y a b l a t i o n . Mammalian gonadotropins were also successful i n ac c e l e r a t i n g t e s t i c u l a r maturation i n several species of i n t a c t f i s h studied (see reviews by de Vlaming, 1974; M. Fontaine, 1976). This, coupled with the f a c t that these hormones 68 appear to be more e f f e c t i v e i n i n t a c t than i n hypophysectomized females, suggests that a d d i t i o n a l p i t u i t a r y factors are necessary f o r mammalian gonadotropins to influence oocyte development. Hence the e f f e c t s of LH and T^ .,. alone or i n combination, on the mainten-ance of v i t e l l o g e n i c oocytes following hypophysectomy were investigated. In addition, since mammalian LH i s known to have an i n t r i n s i c capacity to stimulate the t e l e o s t thyroid (Wallis, 1975), plasma samples of treated f i s h were analyzed f o r T 4 content. A preliminary experiment, designed to t e s t the a b i l i t y of LH alone to stimulate ovarian maturation i n i n t a c t g o l d f i s h i s included. MATERIALS AND METHODS a. E f f e c t s of LH i n Intact F i s h In July, 1976, g o l d f i s h were divided into three groups, and placed i n tanks supplied with flowing dechlorinated water at 12°C under natural photo-period. Gonads of these f i s h had not matured as they do normally i n t h i s species through the l a t e winter and spring months (Yamazaki, 1965), presumably as a r e s u l t of poor environmental and n u t r i t i o n a l conditions at the s u p p l i e r s . Ten females were sampled at the s t a r t of the experiment to check the h i s t o l o g i c a l condition of the ovary ( i n i t i a l c o n t r o l s ) . These f i s h had very l i t t l e or no f a t , i n d i c a t i n g that though the ovaries were immature they were probably not r e f r a c t o r y to exogenous stimulation (Nagahama, personal communication). The three experimental groups were i n j e c t e d three times a week for 21 days with ei t h e r s a l i n e , 1 yg/g, or 10 yg/g NIH-LH-S19. The LH was dissolved 69 i n f i s h s a l i n e (Wiebe, 1969), and 0.1 ml of t h i s s o l u t i o n was i n j e c t e d i n t r a p e r i t o n e a l l y with a 1.0 ml tuberculin syringe and 30 gauge needle. The experiment was terminated 24 hours a f t e r the l a s t i n j e c t i o n . F i s h were k i l l e d by decapitation„after weighing, and portions of each ovary and of the lower jaw were f i x e d f o r subsequent h i s t o l o g i c a l examination. b. E f f e c t s of LH and Thyroxine i n Short-Term Hypophysectomized F i s h In September, 1976, 48 maturing g o l d f i s h were hypophysectomized. These f i s h were of the same stock as those used i n Exp. IVa, and had been maintain-ed at 12°C and long photoperiod (16L:8D) to stimulate gonadal development. Two of the f i s h died s h o r t l y a f t e r the operation, and the remainder were divided i n t o four groups. The groups were treated with e i t h e r ovine LH (NIH-LH-S19; 5 yg/g i n t r a p e r i t o n e a l l y ) , T^ (immersion; 10 yg/100 ml), a combination of LH and T^, or s a l i n e i n j e c t i o n (controls). T^ treatment was begun immediately following hypophysectomy, and h a l f the water was exchanged three times a week and fresh T^ added. LH i n j e c t i o n s were begun two days a f t e r the operation, and repeated three times a week for a t o t a l of 12 i n j e c t i o n s . The experiment was terminated 24 hours a f t e r the l a s t i n j e c t i o n and water change. F i s h were anaesthetized i n MS 222 and blood samples taken from the caudal artery. The f i s h were then k i l l e d by decapitation, and t h e i r ovaries and l i v e r s removed and weighed; the ovaries were f i x e d f o r subsequent h i s t o l o g i c a l examination. Plasma samples were analyzed f o r T^ l e v e l s using the Ames Tetralute k i t as described i n Section I. 70 RESULTS Ovary Mean values for GSI and maximum oocyte si z e i n i n t a c t LH-treated f i s h are given i n Table 8. Control ovaries matured l i t t l e over the experimental period, while LH administration apparently stimulated ovarian development (Fig. 20 and 21). Differences i n GSI and maximum oocyte s i z e are, however, only s i g n i f i c a n t when comparing the 10 yg/g LH group with the controls. Hypophysectomy resu l t e d i n a decrease i n both GSI and maximum oocyte s i z e values (Table 9). Treatment with LH or T^ alone had no e f f e c t on post-operative ovarian regression, while a combination of the two hormones appears to have retarded t h i s process s l i g h t l y but s i g n i f i c a n t l y (Table 9). Ovaries of hypophysectomized control and T^-treated f i s h contained mainly p r e - v i t e l l o g e n i c and a t r e t i c oocytes (Fig. 19, 25, and 27), although a few oocytes with small yolk v e s i c l e s (less than 8 ym i n diameter) were evident i n some of the f i s h studied (Table 9). V i t e l l o g e n i c oocytes were found i n ovaries of most f i s h treated with LH or LH + T^, and yolk v e s i c l e s i n these groups ranged up to 12 and 18 ym i n diameter r e s p e c t i v e l y (Table 9; F i g . 26 and 28). Only i n the LH + T^ group, however, di d values for maximum oocyte s i z e , GSI, and frequency of v i t e l l o g e n i c oocytes d i f f e r ' s i g n i f i c a n t l y from c o n t r o l s . Thyroid LH treatment of i n t a c t f i s h r e s u l t e d i n a s i g n i f i c a n t increase i n thyroi d e p i t h e l i a l c e l l heights (Table 9; F i g . 22 and 23). This response was greatest at the highest dosage (10 yg/g), and values for t h i s group TABLE 8. EFFECTS OF OVINE LH ON OVARIAN DEVELOPMENT AND THYROID EPITHELIAL HEIGHT IN INTACT FISH. BODY WT MAX OOCYTE THYROID EPITH GROUP n (g) ± SD GSI ± SE DIAM(p) ± SE CELL HT (y), ± SE I n i t i a l c o ntrol 10 9.3+2.2 1.54 ± 0.21 142 ± 6 F i n a l control 15 10.8 ± 2.2 1.85 ± 0.12 188 ± 18 3.4 ± 0.1 LH (1 yg/g) 12 10.3 ± 2.4 , 2.13 ± 0.29 266 ± 47 4.0 ± 0.06° LH (10 yg/g) 11 11.1 ± 1.7 2.40 ± 0.42 320 ± 5 9 a , b 4.4 ± 0.16°' d a > i c p < 0.01 b >FC p < 0.05 C>FC p < 0.01 >LH (1 yg/g) p < 0.05 TABLE 9. EFFECTS OF OVINE LH AND THYROXINE ON OVARIAN MAINTENANCE AND PLASMA THYROXINE LEVELS IN SHORT-TERM HYPOPHYSECTOMIZED FISH. *No. BODY WT * MAX OOCYTE DIAM PLASMA T. ± 4 SE GROUP n YV . (g) ± SD •: GSI ± SE (y) ± SE (yg/100 ml) n Intact c o n t r o l 8 8 15.6 ± 2.77 3.20 ± 0.04 a 475 ± 49 a 0.60 ± 0.14 (4) Sham 2 2 9.5, 14.4 2.31, 5.14 310, 670 -HPX-control 5 3 13.6 ± 3.3 1.21 ± 0.14b 134 ± 12° 0.48 ± 0.16 (5) HPX-LH 9 7 13.7 ± 1/8 1.30 ± 0.13 151 ± 6 - 0.83 + 0.14 (6) HPX-T4 8 3 12.7 ± 2.4 1.17 ± 0.10 140 ± 11 2.35 ± 0.23 ( 5 ) g ' h HPX-LH + T 4 9 8 13.6 ± 2.5 1.70 ± 0.13 d' 6' f 177 ± 8 f' g 2.92 + 0.24 ( 6 ) g ' h *Number v i t e l l o g e n i c ; HPX-C + HPX-T4 < HPX-LH + HYP-LH+T4 p < 0.05 (x 2 a n a l y s i s ) . a>HPX groups p < 0.01 e>HPX-LH p < 0.05 bANOVA (HPX groups) p < 0.025 f>HPX-T4 p < 0.05 °ANOVA (HPX groups) p < 0.01 g>HPX-control p < 0.01 d>HPX-control p < 0.05 h>HPX-LH p < 0.01 -73 Figure 19. E f f e c t s of ovine LH and thyroxine on oocyte composition of ovaries of short-term hypophysectomized f i s h . The r e l a t i v e frequencies of a t r e t i c (A), p r e - v i t e l l o g e n i c (PV), and v i t e l l o g e n i c (V) f o l l i c l e s are given for each group. IC = i n t a c t c o n t r o l ; SH = sham operated; HPX = hypophysec-tomized. A l l hypophysectomized groups d i f f e r s i g n i f i c a n t l y from i n t a c t f i s h at p < 0.01, and the HPX-LH+T4 group d i f f e r s s i g n i f i c a n t l y from HPX-controls at p < 0.01. 74 < > O l O O i 80 6 0 40 20 IC IOO S O 60 4 0 2 0 TOO 80| 60 4 0 2 0 r ~ HPX C HPX T4 SH HPX LH HPX LH +T4 A PV V A PV V 75 Figure 20. Ovarian section of i n t a c t c o n t r o l f i s h (FC) i n Exp. IVa. Only p r e - v i t e l l o g e n i c oocytes are evident. Masson's trichrome. X140. Figure 21. Ovarian section of i n t a c t f i s h treated with LH (10 yg/g) for three weeks showing yolk v e s i c l e (YV) and yolk granule (YG) stage oocytes. Masson's trichrome. X140. Figure 22. Thyroid f o l l i c l e s (F) from i n t a c t control f i s h (Exp. IVa). Masson's trichrome. X450. Figure 23. Thyroid f o l l i c l e s (F) from an i n t a c t f i s h treated with LH (10 pg/g) for three weeks. Note hypertrophied f o l l i c u l a r epithelium. Masson's trichrome. X450. 77 Figure 24. Section of i n t a c t c o n t r o l ovary showing yolk granule (YG) stage oocyte. Masson's trichrome. X140. Figure 25. Ovarian section of c o n t r o l f i s h hypophysectomized f o r four weeks. Only p r e - v i t e l l o g e n i c (PV) and a t r e t i c (A) f o l l i c l e s are present. Masson's trichrome. X140. Figure 26. Ovarian section of hypophysectomized f i s h treated with ovine LH for four weeks post-operatively. Note early yolk v e s i c l e (YV) stage oocytes. Masson's trichrome. X610. Figure 27. Ovarian section of hypophysectomized f i s h immersed i n thyroxine fo r four weeks post-operatively. Masson's trichrome. X140. Figure 28. Ovarian section of hypophysectomized f i s h treated with both ovine LH and thyroxine for four weeks post-operatively. Note mid yolk v e s i c l e (YV) stage oocyte. Masson's trichrome. X140. / 79 were s i g n i f i c a n t l y greater than those of both the controls and the low (1 yg/g) dosage group. Thus i t appears that th y r o i d a c t i v i t y i s stimulated by mammalian LH treatment. However, no conclusions can be drawn as to whether plasma l e v e l s of thy r o i d hormones are i n f a c t elevated i n treated f i s h , since th y r o i d f o l l i c u l a r c e l l height i s s t r i c t l y a morphological index that does not necess a r i l y r e f l e c t the rate of hormone production (Eales, 1964). Hence, i t was decided to measure plasma T^ l e v e l s i n experiment IVb to determine whether c i r c u l a t i n g T^ l e v e l s are indeed elevated by LH treatment. T^ i s detectable i n the plasma of hypophysectomized controls at a l e v e l not s i g n i f i c a n t l y d i f f e r e n t from i n t a c t controls, i n d i c a t i n g that hormonal release from the t h y r o i d i s not completely abolished by p i t u i t a r y removal. Administration of LH alone and i n combination with T^ elevated plasma l e v e l s over those values of co n t r o l and T^-immersed f i s h r e s p e c t i v e l y ; however, these r e s u l t s are not s t a t i s t i c a l l y s i g n i f i c a n t , perhaps as a r e s u l t of the small sample s i z e s . L i v e r HSI values for i n t a c t and hypophysectomized f i s h are given i n Figure 29. In hypophysectomized controls, the mean HSI i s elevated over that of untreated i n t a c t animals, and treatment with LH or T^ alone had no e f f e c t on t h i s post-operative increase. In those f i s h r e c e i v i n g a combination of LH and T^, however, the increase i n l i v e r weight following hypophysectomy appears to have been prevented, and HSI values i n t h i s group are s i m i l a r to those of i n t a c t c o n t r o l s . 80 Figure 29. E f f e c t s of ovine LH (5 yg/g) and thyroxine (10 yg/100 ml) on the hepato-somatic index of short-term hypophysectomized female f i s h . Bars represent mean HSI values, and v e r t i c a l l i n e s repre-sent standard er r o r s . Numbers adjacent to each histogram represent sample s i z e s . IC = i n t a c t controls; HPX = hypo-physectomized. Values for the IC and HPX-LH+T^ groups are s i g n i f i c a n t l y l e s s than those of a l l other groups at p <0.01. ANOVA i s s i g n i f i c a n t at p. < 0.001. 81 Q- + X X X 1= CL. U X 1 1 I I I 1 o K Q o Cl'C 0 b O q co o CN o 6 o o CO I S H 82 DISCUSSION Ovary The r e s u l t s of Experiment IVa indic a t e that mammalian LH i s capable of stimulating ovarian maturation i n i n t a c t g o l d f i s h . Control ovaries matured only s l i g h t l y over the experimental period, while ovarian development was accelerated s i g n i f i c a n t l y i n f i s h treated with 10 yg/g LH. LH alone had no e f f e c t on preventing ovarian regression following hypo-physectomy. This i s probably due to the absence of endogenous p i t u i t a r y f a c t o rs (gonadotropins), and indicates that LH has only a p a r t i a l gonado-t r o p i c potency i n g o l d f i s h . The 3 protein subunits of mammalian LH and p i s c i n e gonadotropins d i f f e r g r eatly (Pierce et a l . , 1976)/ and, since the 3 subunit of the glycoprotein hormones confers hormonal s p e c i f i c i t y i n mammals at l e a s t (Wallis, 1975), the lack of effectiveness of LH alone i n the present study may be explained by phylogenetic differences i n structure between the ovine LH preparation and g o l d f i s h gonadotropin(s); gonadotropin receptors i n the g o l d f i s h ovary are presumably more responsive to endogenous hormones. Studies of the g o l d f i s h with another mammalian gonadotropin, HCG, show s i m i l a r r e s u l t s ; HCG was unable to i n i t i a t e v i t e l l o g e n e s i s i n long-term hypophysectomized f i s h (Yamazaki, 1965), and was only p a r t i a l l y e f f e c t i v e i n maintaining yolky oocytes In v i t r o '(Remade e_t a l . , 1976) . The i n a b i l i t y of T^ alone to a f f e c t ovarian regression i n hypophysec-tomized g o l d f i s h supports the hypothesis that thyroid hormones act i n synergy with endogenous gonadotropins i n t h e i r e f f e c t s on ovarian development i n i n t a c t f i s h . A combination of both LH and TA apparently did retard post-operative 83 a t r e s i a . These r e s u l t s are somewhat s u r p r i s i n g i n l i g h t of the f a c t that T^ was present i n s i g n i f i c a n t amounts i n hypophysectomized, LH-treated f i s h . However, since ovarian development i n the l a t t e r group was s l i g h t l y greater than i n untreated f i s h , ovarian regression may indeed have been retarded but the sampling time was perhaps too l a t e to detect t h i s phenomenon. In support of t h i s , Anand and Sundararaj (1972) found treatment with 5 ug/g NIH-LH-S16 d a i l y f or 20 days p a r t i a l l y e f f e c t i v e i n maintaining yolky oocytes i n the c a t f i s h , Heteropneustes f o s s i l i s . In the g o l d f i s h , the high plasma T^ l e v e l s present i n the LH+T^ group may have augmented the ovarian response to LH, r e s u l t i n g i n s i g n i f i c a n t l y greater ovarian development i n t h i s group than i n the controls a f t e r 28 days. Thyroid The a b i l i t y of mammalian gonadotropins to stimulate t h y r o i d function i n t e l e o s t s i s well documented (Wallis, 1975) . These "heterothyrotropic f a c t o r s " , as they have been c a l l e d , have been shown to induce thy r o i d f o l l i c u l a r c e l l hypertrophy and increase 1 3 1 i uptake by the gland i n f i s h (Pickford and Grant, 1968; Y. Fontaine, 1969a,b), but the author knows• of no studies measuring the actual changes i n plasma T^ i n response to mammalian gonadotropins. In the present study, c i r c u l a t i n g T^ l e v e l s were s l i g h t l y but not s i g n i f i c a n t l y elevated i n hypophysectomized f i s h following ovine LH treatment. This d i f f e r e n c e may have been greater e a r l i e r i n the experimental period, since the t h y r o i d a l response to mammalian TSH, as measured by plasma T^ analysis, drops a f t e r repeated i n j e c t i o n i n the brook trout (Chan and Eales, 1976); t h i s may be due to a depletion of thyroid hormone stores i f hormone secretion exceeds hormone production (Chan and Eales, 1976). The actual thyroid-stimulating capacity of the LH dosage 84 used i n the present study was equivalent to about 3 mlU TSH/g body weight (this includes both TSH contamination of the LH preparation and the hetero-thyrotropic a b i l i t y of the LH molecule i t s e l f as c a l c u l a t e d from Y. Fontaine, 1969b). In a 15 g brook trout, a dose of 3 mlU/g would r a i s e plasma l e v e l s by 0.5 yg/100 ml or l e s s (Chan and Eales, 1976), and, since c i r c u l a t -ing T^ l e v e l s i n untreated g o l d f i s h and brook trout are very s i m i l a r (see Section I ) , the LH dosage used i n the present study might be expected to r a i s e plasma T^ l e v e l s by a s i m i l a r amount i n a 15 g g o l d f i s h . As the r e s u l t s i n d i c a t e , t h i s was indeed the case. The f a c t that plasma T^ l e v e l s i n untreated f i s h hypophysectomized for four weeks f a l l within the range of values observed i n i n t a c t controls i s s u r p r i s i n g , since Chavin (1956) found a severe reduction i n t h y r o i d a l 1 3 1 i uptake 24 days post-hypophysectomy i n the g o l d f i s h ; the l a t t e r would indi c a t e that t h y r o i d hormone synthesis was also greatly reduced. However, hormonal stores may have been released i n the absence of p i t u i t a r y stimulation or de novo synthesis i n the present study. In addition, the metabolic clearance rate of t h y r o i d hormones from the plasma i s perhaps les s i n hypophysectomized than i n i n t a c t f i s h , r e s u l t i n g i n an accumulation of plasma T^. L i v e r As i n P o e c i l i a l a t i p i n n a ( B a l l and Hawkins, 1976), l i v e r weights of untreated g o l d f i s h increased following hypophysectomy. This i s due to an accumulation of hepatic glycogen and water i n the g o l d f i s h , with the absolute content of p r o t e i n and l i p i d remaining unchanged a f t e r p i t u i t a r y a b l a t i o n (Walker and Johansen, 1975). Treatment with LH alone had no apparent e f f e c t , while T - administration augmented s l i g h t l y the post-operative l i v e r hyper-85 trophy. A combination of LH and T^, however, prevented t h i s increase i n hepatic weight, suggesting a s y n e r g i s t i c action of the two hormones on l i v e r function. I t might be argued that t h i s e f f e c t i s a r e s u l t of elevated l e v e l s i n the LH+T^ treated f i s h , since administration of high doses of can lead to a depletion of l i v e r glycogen i n te l e o s t s ( B a l l and Hawkins, 1976); t h i s i s , however, u n l i k e l y , as plasma values i n the LH+T^ group were only s l i g h t l y and not s i g n i f i c a n t l y greater than i n those treated with T„ alone. 86 GENERAL DISCUSSION The involvement of the thyr o i d i n gonadal maturation of t e l e o s t s has been l a r g e l y i n f e r r e d from experiments i n v o l v i n g chemical thyroidectomy (Barrington and Matty, 1952; Pickford and Atz, 1957; Matty, 1960; Raizada, 1974), from observations of increased t h y r o i d a l a c t i v i t y at the time of gonadal maturation (Berg et a l . , 1959; Hickman, 1962; Takashima et a l . , 1972; Singh et al_., 1974), and from the stimulation of precocious development of secondary sexual development by thyr o i d hormones (Grobstein and Bellamy, 1939). In the early part of t h i s study (Hurlburt, 1975), i t was found that thyroxine administration i n low dosages stimulated ovarian maturation i n immature goldfish> while at high dosages a secondary i n h i b i t o r y influence was evident. Preliminary data on p i t u i t a r y gonadotropin c e l l a c t i v i t y i n treated f i s h suggested that the thyr o i d may stimulate gonadal development through an action on general metabolism or, more s p e c i f i c a l l y , ovarian metabolism, and also may modulate p i t u i t a r y gonadotropin production v i a a negative feedback on the hypothalamo-hypophyseal axis (Hurlburt, 1975) . The present study confirms the above hypothesis, and also demonstrates that thyroxine, i n the absence of the p i t u i t a r y , has no e f f e c t on the i n i t i a t i o n of v i t e l l o g e n e s i s or on the maintenance of yolky oocytes. The hormone does, however, augment the ovarian response to gonadotropin stimula-t i o n , suggesting that th y r o i d hormones act s y n e r g i s t i c a l l y with endogenous gonadotropin i n t h e i r e f f e c t s on gonadal development. No conclusions can be drawn as to the a c t i v i t y of gonadotropin i n the absence of thyroxine, as both gonadotropin preparations used i n t h i s study have apparent thyroid-87 stimulating a c t i v i t y . Thus, thyroid hormones appear to influence gonadal maturation i n t e l e -osts through e f f e c t s at both the p i t u i t a r y and target organ l e v e l s . The following discussion w i l l consider the evolutionary associations of the t h y r o i d a l and gonadal systems, and also several of the s p e c i f i c mechanisms by which the t h y r o i d may regulate reproductive processes i n t e l e o s t s . These include an i n h i b i t o r y influence of thyroid hormones on p i t u i t a r y gonadotropin production, and stimulatory e f f e c t s of thyroxine on aspects of general or ovarian metabolism. In addition, the involvement of the thyroid i n l i v e r function w i l l be considered,, since l i v e r metabolism i s a f f e c t e d both by general metabolic processes (e.g. Walker and Johansen, 1975; Birnbaum et a l . , 1976; Lewander et a l . , 1976) and ovarian a c t i v i t y i n t e l e o s t s . P i t u i t a r y In the present study, hyperthyroidism re s u l t e d i n a decrease i n gonado-t r o p i n c e l l a c t i v i t y as measured by h i s t o l o g i c a l c r i t e r i a , while goiterogen-induced hypothyroidism stimulated gonadotropin c e l l function s l i g h t l y . This supports the findings of Sage and Bromage (1970b) and Hurlburt. (1975) and suggests that, as i n mammals, thyro i d hormones exert a negative feedback on p i t u i t a r y gonadotropin production i n t e l e o s t s (see Section II for discussion of possible mechanisms). These e f f e c t s may be a r e s u l t of evolutionary associations of the t h y r o i d a l and gonadal co n t r o l systems, and, although a thyroxine-induced suppression of gonadotropin production may not be important p h y s i o l o g i c a l l y i n t e l e o s t s , i t serves to emphasize the close involvement of the t h y r o i d with reproductive processes i n t h i s group. An i n h i b i t o r y influence of t h y r o i d hormones on p i t u i t a r y gonadotropin production has been reported i n several vertebrate classes i n c l u d i n g 88 mammals (e.g. Larochelle and Freeman, 1974; Freeman et_ a l . , 1976), b i r d s (Chandola et a l . , 1974), and te l e o s t s (Sage and Bromage, 1970b; Hurlburt, 1975). Thyroxine a l t e r s TSH production by negative feedback on the p i t u i t a r y r i n several t e l e o s t s studied (Baker, 1965; Baker, 1969; Sage and Bromage, 1970b), inc l u d i n g the g o l d f i s h (Peter, 1971), hence i t i s perhaps not sur-p r i s i n g that GTH production i s af f e c t e d i n the same way. Evidence points to both a close evolutionary l i n k and a complex, fu n c t i o n a l i n t e r a c t i o n between the t h y r o i d a l and the gonadal systems. GTH and TSH are s t r u c t u r a l l y r e l a t e d glycoprotein molecules and i n mammals contain common a protein subunits, while v a r i a t i o n s i n the 8 subunits confer hormonal s p e c i f i c i t y (Wallis, 1975). They may have ar i s e n from a sing l e molecule which performed gametogenic, steroidogenic, and thyrotropic functions by i n t e r a c t i o n s with s p e c i f i c target organs (Hoar, 1975). Divergence of a TSH molecule d i s t i n c t from GTH has not occurred i n cyclostomes, and pos s i b l y not i n elasmobranchs (see account of Sage [1973] summarized i n the introduction to t h i s paper). Although separate TSH and GTH molecules are evident i n te l e o s t s and higher vertebrates, t h e i r c o n t r o l mechanisms remain c l o s e l y l i n k e d (Sage and Bern, 1971). Hormones regulating both GTH and TSH production o r i g i n a t e from s i m i l a r regions of the hypothalamus i n g o l d f i s h (Peter, 1970) and, : i n mammals at l e a s t , the structure of hypothalamic thyrotropin-releasing hormone (TRH) i s s i m i l a r to part of the LH-RH molecule (Sage, 1973). A factor s i m i l a r to mammalian TRH has been i d e n t i f i e d i n the hypothalamus of t e l e o s t s (Jackson and R e i c h l i n , 1974), although no substances immunoreactive with mammalian LH-RH has been i d e n t i f i e d i n t h i s group (Crim et a l . , 1976). Mammalian LH-RH does, however, stimulate gonadotropin production i n g o l d f i s h , and a gonadotropin-releasing 89 substance has been i d e n t i f i e d i n the hypothalamus of t h i s species (Crim ejt a l . , 1976). Thyroid hormones and sex steroids influence both GTH and TSH c e l l s i n t e l e o s t s v i a common pathways within the brain (Sage and Bromage, 1970b); a s i m i l a r response occurs i n b i r d s (Chandola et a l . , 1974). In addition, evidence indicates that gonadotropin production i s under i n h i b i t o r y c o n t r o l of t h y r o i d hormones i n several avian species, t h i s mechanism perhaps prevent-ing unseasonal gonadal growth i n t r o p i c a l finches (Thapliyal, 1969; Weisel-t h i e r and Van Tienhoven, 1971; Chandola et a l . , 1974) and promoting t e s t i c u -l a r regression following the spring breeding season i n the domestic duck (Jallegeas and Assenmacher, 1974). Thus, a close r e l a t i o n s h i p between gonadal and t h y r o i d a l systems i s found i n t e l e o s t s , and t h i s r e l a t i o n s h i p i s further supported by evidence from other vertebrate groups. These r e s u l t s are s i g n i f i c a n t i n that they indicate that thyroxine stimulates gonadal maturation by some mechanism other than v i a a stimulation of p i t u i t a r y gonadotropin production. Other p i t u i t a r y e f f e c t s of thyroxine cannot, however, be excluded. Production of growth hormone, which stimulated gonadal development i n the female toad ( B i l l e t e r and Jorgensen, 1976) and male k i l l i f i s h (Pickford et a l . , 1972), may be induced by thyroxine i n some te l e o s t s (Sage> 1967; Higgs et a l . , 1976) and mammals (Ishikawa et a l . , 1976). In addition, TRH appears to regulate p r o l a c t i n and i n some cases growth hormone production i n mammals (Chihara et a l . , 1976; Dannies et a l . , 1976; Hirvonen et a l . , 1976; Jeppson et a l . , 1976). Whether t h i s i s true i n t e l e o s t s as well i s unknown; i n t h i s group, however, the hypothalamic t h y r o t r o p i n - c o n t r o l l i n g factor appears to i n h i b i t rather than stimulate p i t u i t a r y TSH production (Peter, 1972; Bromage, 1975; Bromage et a l . , 1976). 90 Ovary Thyroxine was e f f e c t i v e i n stimulating gonadal maturation i n i n t a c t g o l d f i s h but was i n e f f e c t i v e i n hypophysectomized i n d i v i d u a l s , i n d i c a t i n g that the actions of t h i s hormone on gonadal development are pituitary-depend-ent. This i s most l i k e l y explained by a s y n e r g i s t i c a c t i v i t y of thyroxine with gonadotropin, rather than a stimulatory action of the former on p i t u i -tary function, since thyroxine d i d augment the ovarian response to SG-G100 i n hypophysectomized f i s h , and i t s e f f e c t s on the p i t u i t a r y appear to be i n h i b i t o r y i n nature. Thyroid hormones and gonadotropin perhaps act syner-g i s t i c a l l y i n the s t e l l a t e sturgeon as well; i n t h i s species T^ administra-t i o n restored the ovarian response to exogenous hypophyseal stimulation following prolonged c a p t i v i t y or cooling of the f i s h (Detlaf and Davydova, 1974) .' A s y n e r g i s t i c e f f e c t of t h y r o i d hormones and p i t u i t a r y gonadotropins on ovarian development i s well established i n mammals. E a r t l y and Leblond (1954) found thyroxine e f f e c t i v e i n stimulating t e s t i c u l a r function i n i n t a c t r a t s but i n e f f e c t i v e i n hypophysectomized i n d i v i d u a l s . Thyroidectomy of r a t s r e s u l t s i n a reduction i n ovarian weight (Leatham, 1973; Kovacs and Mess, 1976), and the c h a r a c t e r i s t i c weight increase of the ovary i n response to gonadotropin stimulation i s abolished (Johnson and Mietes, 1950; Janes, 1954). H i s t o l o g i c a l l y , ovaries of thyroidectomized r a t s treated with gonadotropin several weeks post-operatively contain c y s t i c , a t r e t i c , and l u t e i n i z e d f o l l i c l e s with few normally developing oocytes (Janes, 1954). In addition, T^ or T^ added to the medium of porcine granulosa c e l l s stimulated' an increase i n progesterone production i n response to FSH and LH (Channing et a l . , 1976). Thyroxine treatment of euthyroid r a t s , however, di d l i t t l e to 91 a l t e r the ovulatory capacity of the ovary i n response to gonadotropins (Dubin, 1974). The above suggests both that a normally functioning t h y r o i d i s e s s e n t i a l for gonadotropins to stimulate ovarian development, and that > gonadotropins i n turn are necessary f o r thyroid hormones to influence gonadal function i n mammals. The nature of t h i s s y n e r g i s t i c e f f e c t i s uncertain, but both i n d i r e c t and d i r e c t actions of thyroxine on the ovary must be considered. Thyroid hormones play an important r o l e i n the regulation of general metabolism i n mammals; i f t h i s i s true i n t e l e o s t s , they might serve to increase the a v a i l a b i l i t y of nutrients, metabolites, and yolk precursors necessary for ovarian development. In addition, thyroid a c t i v i t y may a f f e c t s t e r o i d b i o -synthesis within the ovary or the extra-ovarian metabolism of these hormones. Since steroids i n the g o l d f i s h a f f e c t both yolk v e s i c l e and yolk granule formation (Khoo, 1974), and appear to regulate l i v e r yolk precursor produc-t i o n i n t h i s and some other t e l e o s t s (Bailey, 1957; I s h i i and Yamamoto, 1970; Plack et a l . , 1971; Campbell and I d l e r , 1976), and involvement of the thyroid i n steroidogenesis might a f f e c t yolk formation. The influence of t h y r o i d hormones on metabolism i n t e l e o s t s i s s t i l l poorly understood. I t i s known that t h y r o i d hormones a f f e c t both growth and development (Novales et a l . , 1973) and influence l i v e r metabolism (Hochachka, 1962; B a l l and Hawkins, 1976; Ray et a l . , 1976). Regulatory e f f e c t s of and T^ on nitrogen excretion (Ray and Medda, 1976), erythropoiesis (Slicher, 1961; Srivastava, 1976), and plasma l i p i d components (Takashima et a l . , 1972) have also been reported i n t e l e o s t s . However, metabolic responses as measured by changes i n oxygen consumption following administration of t h y r o i d hormones, TSH, or a n t i - t h y r o i d drugs, have been inconsistent i n f i s h 92 (Higgs and Eales, 1971). Hochachka (1962) suggests that whether or not th y r o i d a l stimulation of metabolic a c t i v i t y i s r e f l e c t e d i n oxygen uptake could depend on the major metabolic pathway influenced by thyroxine, and upon the predominant oxidation scheme operating at the time. Higgs and Eales (1971) conclude that, although the evidence i s s t i l l f a r from conclu-sive, an involvement of the thyroid i n the regulation of metabolic rates i n te l e o s t s i s l i k e l y . The energy needs of the t e l e o s t ovary are great since the ovary may increase from le s s that 1% to over 20% of the body weight during maturation; hence, the importance of a t h y r o i d a l stimulation of general metabolism on ovarian development cannot be overestimated. In mammals, by contrast, t h i s r o l e might be less important, since there i s no yolk associated with the ova and the energy requirements of the ovary are minor when compared with t e l e o s t s and other lower vertebrate groups. A d i r e c t e f f e c t of thyro i d hormones on general or s p e c i f i c aspects of ovarian metabolism i s a l i k e l y p o s s i b i l i t y i n mammals, and perhaps also i n t e l e o s t s . From the r e s u l t s of the present study i t appears that, as suggest-ed by Myant (1964), thyroxine increases gonadal s e n s i t i v i t y to gonadotropic stimulation. S t e r o i d production i n mammals i s influenced by thyro i d hormones; t h i s i s po s s i b l y a r e s u l t of increased l e v e l s of steroidogenic enzymes, since thyroxine i s known to stimulate the formation of many enzymes (Hardy et. a l . , 1960; Snedecor et a l . , 1972; Goodrich and Adelman, 1976) including those i n the steroidogenic pathway (White et a l . , 1973). In hypothyroid r a t s , 3g-hydroxysteroid dehydrogenase a c t i v i t y and u t i l i z a t i o n of ovarian c h o l e s t e r o l f o r s t e r o i d biosynthesis are decreased (Leatham, 1973), and the ovulatory response to gonadotropin i s i n h i b i t e d , leading to the formation of 93 c y s t i c (anovulatory) f o l l i c l e s (Janes, 1954; C a l l a r d and Leatham, 1965). Studies on the steroidogenic capacity of p o l y c y s t i c ovaries induced by t r e a t i n g hypothyroid rats with gonadotropin reveal a dramatic decrease i n estrogen production, possibly due to a decreased conversion from androgen intermediates (Callard and Leatham, 1965) . Hyperthyroidism, on the other hand, r e s u l t s i n an increase i n plasma estrogen l e v e l s (Southren et a l . , 1974; Akande, 1975; Olivo et a l . , 1975). Southren et a l . (1974) suggest that th y r o i d hormones may stimulate ovarian androstenedione production and also increase the a c t i v i t y of 173-hydroxysteroid dehydrogenase, which converts androstenedione to testosterone and estrone to e s t r a d i o l . Whether , these r e s u l t s are due to a s p e c i f i c action of thyroid hormones on steroido-genic enzyme a c t i v i t y , or to a more generalized stimulation of ovarian c e l l J metabolism as suggested by Channing et al_. (1976) remains to be elucidated. L i v e r Thyroxine treatment of both i n t a c t and hypophysectomized g o l d f i s h tended to increase r e l a t i v e l i v e r weights except i n Experiment IVb, while administra-t i o n of a combination of LH and T^ prevented post-hypophysectomy l i v e r hypertrophy. The mechanisms mediating these responses i n t e l e o s t s are uncertain, but may involve e f f e c t s of thyroxine on hepatic glycogen, protein, and l i p i d metabolism (Baker-Cohen, 1962; Hochachka, 1962; Takashima et a l . , 1972; B a l l and Hawkins, 1976). L i v e r glycogen concentrations i n hypophysectomized P o e c i l i a l a t i p i n n a were unaffected by presumed p h y s i o l o g i c a l l e v e l s of thyroid hormones (induced by TSH administration) but decreased dramatically by a pharmacological dose of T„ (50 yg/100 ml immersion - B a l l and Hawkins, 1976). This i s consistent 94 with mammalian studies, which demonstrate a depletion of l i v e r glycogen only when excessive dosages of t h y r o i d hormones are administered (White et a l . , 1973). The l a t t e r e f f e c t may r e s u l t from a d i r e c t action of t h y r o i d hormones on c e l l u l a r metabolism, or i n d i r e c t l y by, for example, increasing t i s s u e s e n s i t i v i t y to glucagon (Stouffer and Dunaway, 1976) or adrenalin (Guttler et a l . , 1975). However, p h y s i o l o g i c a l l e v e l s of thyroxine appeared to increase rather than decrease l i v e r weights i n the present study. This may r e s u l t from increased general metabolic rates stimulating a build-up of n u t r i t i o n a l stores i n the l i v e r . Takashima et al_. (1972) found that l i v e r weights increased but r e l a t i v e proportions of hepatic l i p i d and protein were unchanged following thyroid hormone treatment i n rainbow trout, i n d i c a t i n g that these constituents may increase i n absolute content under thyr o i d stimulation. In addition, hyperthyroidism i n mammals leads to increased ACTH and C o r t i s o l secretion, the l a t t e r promoting l i v e r glycogen deposition (White et a l . , 1973). Whether t h i s i s true i n t e l e o s t s i s uncertain. B a l l and Hawkins (1976) found C o r t i s o l production stimulated by mammalian TSH i n hypophysectomized P_. l a t i p i n r i a , but t h i s was a t t r i b u t e d to a d i r e c t e f f e c t of TSH on the i n t e r r e n a l rather than an i n d i r e c t e f f e c t mediated by the thyroid, since thyroxine administration d i d not stimulate i n t e r r e n a l function h i s t o l o g i c a l l y ; C o r t i s o l l e v e l s were not, however, measured following thyroxine treatment. Treatment of hypophysectomized g o l d f i s h with a combination of LH and T^ prevented post-operative l i v e r hypertrophy, suggesting a s y n e r g i s t i c action of the two hormones, ei t h e r d i r e c t l y or i n d i r e c t l y , on l i v e r function. High p h y s i o l o g i c a l doses of T 4 are apparently necessary to e l i c i t t h i s s y n e r g i s t i c 95 e f f e c t , since was detectable i n the plasma of f i s h treated with LH alone but l i v e r weight was unaffected. Mammalian LH may a f f e c t l i v e r metabolism i n t e l e o s t s through a stimula-t i o n of ovarian s t e r o i d production. Androgen secretion i n male f i s h i s augmented by LH or HCG therapy (see review by de Vlaming, 1974), and s t e r o i d o -genic enzyme a c t i v i t y i n ovaries of hypophysectomized g o l d f i s h i s stimulated by LH (Khoo, 1974) i n d i c a t i n g estrogens are being produced. In t e l e o s t s , i n c l u d i n g the g o l d f i s h , estrogens appear to act d i r e c t l y on the l i v e r to stimulate production of yolk granule precursors or " v i t e l l i n " , which consist of phospholipoproteins; these are released i n t o the c i r c u l a t i o n f o r transport to the developing oocytes (Bailey, 1957; I s h i i and Yamamoto, 1970; Plack et a l . , 1971; Aida, 1973; Campbell and I d l e r , 1976). Takashima et a l . (1972) suggest that the thy r o i d i s also involved i n regulation of c i r c u l a t i n g v i t e l l i n l e v e l s , since plasma l i p i d and l i p o p r o t e i n components were increased following estrogen treatment and decreased following t h y r o i d hormone t r e a t -ment i n rainbow trout. Hence LH, through a proposed stimulation of estrogen production, and may have acted s y n e r g i s t i c a l l y i n regulating v i t e l l i n metabolism i n the present study. The f a i l u r e of the two hormones to do more than p a r t i a l l y maintain v i t e l l o g e n i c oocytes following hypophysectomy i s perhaps explained by the absence of endogenous p i t u i t a r y f a c t o r s . In amphibia, v i t e l l i n uptake by developing oocytes i s d i r e c t l y c o n t r o l l e d by gonadotropins ( F o l l e t and Redshaw, 1974). While studies on yolk uptake i n t e l e o s t s are l i m i t e d , Campbell and I d l e r (1976) demonstrated that a non-glycoprotein p i t u i t a r y f a c t o r i s involved i n stimulating ovarian yolk accumulation i n the winter flounder, suggesting that some substance other than gonadotropins regulate t h i s process i n t e l e o s t s . 96 Conclusions The e f f e c t s of the thyroid on gonadal maturation are complex, and i n -volve actions at both the p i t u i t a r y and target organ l e v e l . Thyroxine appears to both i n h i b i t p i t u i t a r y gonadotropin production by a negative feedback on the hypothalamo-hypophyseal axis, and stimulate ovarian develop-ment i n synergy with gonadotropin. The l a t t e r e f f e c t may be mediated by a th y r o i d a l stimulation of general metabolism, increasing the a v a i l a b i l i t y of nutrients, yolk and s t e r o i d precursors u t i l i z e d i n oocyte development, and perhaps through a regulation of plasma v i t e l l i n l e v e l s . A l t e r n a t e l y , t h y r o i d hormones may act d i r e c t l y on the ovary, increasing f o l l i c u l a r s e n s i -t i v i t y to gonadotropin stimulation or regulating c e r t a i n aspects of steroidogenesis. Thyroxine, i n high p h y s i o l o g i c a l and pharmacological dosages at l e a s t , appears to i n h i b i t p i t u i t a r y gonadotropin c e l l a c t i v i t y . While t h i s phenom-enon may be an important mechanism regulating gonadal development i n some avian species, i t s p h y s i o l o g i c a l importance i n te l e o s t s i s uncertain. Since thy r o i d a c t i v i t y increases at the same time as gonadotropin c e l l a c t i v i t y i n many t e l e o s t species, i t i s doubtful that the thyroid normally plays a s i g n i f i c a n t r o l e i n the regulation of gonadotropin i n t h i s group. Ovarian maturation appears to be unaffected except when high pharmacological dosages are administered (see Hurlburt, 1975). At lower thyroxine dosages, gonadotropin production i s perhaps not severely reduced, or a l t e r n a t e l y , thyroxine may increase gonadal s e n s i t i v i t y to gonadotropic stimulation thus compensating f o r reduced plasma gonadotropin l e v e l s . A s y n e r g i s t i c action of thyroxine and gonadotropin on ovarian develop-ment i s evident i n the g o l d f i s h . While ovarian maturation was stimulated 97 i n i n t a c t f i s h by thryoxine treatment, the hormone was i n e f f e c t i v e i n i n i t i -a t i n g v i t e l l o g e n e s i s or maintaining yolky oocytes following p i t u i t a r y a b l a t i o n . Ovarian response to exogenous gonadotropins was, however, augment-ed by thyroxine i n hypophysectomized f i s h , i n d i c a t i n g that the thy r o i d acts to stimulate aspects of gonadotropin-induced gonadal development. This may be v i a an a c t i o n of the thyroid on general metabolism, increasing the a v a i l a b i l i t y of nutrients and metabolites necessary i n oocyte growth, while a d i r e c t stimulation by thyroxine of steroidogenic processes within the ovary might enhance yolk v e s i c l e formation and yolk granule precursor production i n the l i v e r . Thyroid hormones may also regulate plasma v i t e l l i n l e v e l s . The effectiveness of gonadotropin on ovarian development i n the absence of thyroxine could hot be elucidated i n t h i s study. P i s c i n e gonadotropin preparations, as a r e s u l t of TSH contamination, and mammalian gonadotropins, due to inherent heterothyrotropic a c t i v i t y , both stimulate t h y r o i d a c t i v i t y i n t e l e o s t s . S u r g i c a l thyroidectomy i s not possible i n most t e l e o s t s , since the t h y r o i d t i s s u e i s scattered along the v e n t r a l aorta and functional heteroptic (extra-pharyngeal) f o l l i c l e s are frequently found. In addition, complete chemical or radiothyroidectomy may have various pharmacological e f f e c t s on e x t r a - t h y r o i d a l t i s s u e . Hence, the question of whether thyro i d hormones are e s s e n t i a l for gonadotropin to stimulate normal ovarian matura-t i o n i s d i f f i c u l t to assess. In conclusion, i t i s evident that the thy r o i d plays an important r o l e i n the reproductive processes i n the g o l d f i s h and other t e l e o s t s . In view of the many i n t e r a c t i o n s and probable<evolutionary l i n k s between the thyroid and gonadal systems, however, the problem of the mechanisms mediating these 98 e f f e c t s i s obviously complex, and the d i v e r s i t y of p h y s i o l o g i c a l functions influenced by t h y r o i d hormones leads to the p o s s i b i l i t i e s of both d i r e c t and i n d i r e c t involvement i n reproductive processes. 99 SUMMARY 1. Thyroxine administration by c h o l e s t e r o l p e l l e t implantation, immersion, or feeding r a i s e d plasma thyroxine l e v e l s i n the g o l d f i s h , Carassius  auratus. Immersion was found to be the most e f f e c t i v e means of creating sustained p h y s i o l o g i c a l thyroxine elevations, while both c h o l e s t e r o l p e l l e t implantation and feeding are p r a c t i c a l for flowing water systems where immersion i s not f e a s i b l e . 2. Thyroxine administration to maturing f i s h r e s u l t e d i n a decrease i n gonadotropin c e l l a c t i v i t y , while goiterogen treatment of immature f i s h stimulated gonadotropin c e l l a c t i v i t y s l i g h t l y . 3. Ovarian maturation of i n t a c t mature f i s h was not a f f e c t e d by thyroxine treatment. In immature f i s h , thyroxine stimulated oocyte development. 4. Thyroxine alone was i n e f f e c t i v e i n i n i t i a t i n g v i t e l l o g e n e s i s or maintain-ing yolky oocytes i n hypophysectomized i n d i v i d u a l s . The hormone did, however, augment the ovarian response to exogenous p i s c i n e or mammalian gonadotropins i n both short- and long-term hypophysectomized f i s h . ' 5. Thyroxine treatment of both i n t a c t and hypophysectomized f i s h tended to increase r e l a t i v e l i v e r weights. When administered with mammalian LH, however, post-hypophysectomy l i v e r hypertrophy was prevented, i n d i c a t i n g a s y n e r g i s t i c action of the two hormones on l i v e r function. 6. P i s c i n e and mammalian gonadotropins stimulated thyr o i d function, as measured by both h i s t o l o g i c a l c r i t e r i a and plasma T^ analysis, i n i n t a c t and hypophysectomized g o l d f i s h . . 100 REFERENCES Ahsan, S. A. 1966. E f f e c t s of gonadotropic hormones on male hypophysectom-ize d lake chub, Couesius plumbeus. Can. J . Zool. 44: 703-717. Ahsan, S. and W. S. Hoar. 1963. Some e f f e c t s of gonadotropic hormones on the threespine stickleback, Gasterosteus aculeatus. Can. J . Zool. 41: 1045-1053. Akande, E. 1975. Plasma estrogens i n euthyroid and thyrotoxic) women. Am. J. Obstet. Gynecol. 122: 880-886. Akande, E. and D. Anderson. 1975. Role of sex hormone-binding gl o b u l i n i n hormonal changes and amenorrhoea i n thyrotoxic women. B r i t . J . Obstet. Gynec. 821: 557-561. Anand, T. and B. Sundararaj. 1974. Ovarian maintenance i n the hypophysec-tomized c a t f i s h , Heteropneustes f o s s i l i s (Bloch), with mammalian hypophyseal and pla c e n t a l hormones, and gonadal and adrenocorti-c a l hormones. Gen. Comp. Endocrinol. 22_: 154-168. Bailey, R. E. 1957. The e f f e c t of e s t r a d i o l on serum calcium, phosphorous, and protein of g o l d f i s h . J . Exp. Zool. 136: 455-469. Baker, B. 1965. Dir e c t action of thyroxine on the trout p i t u i t a r y i n v i t r o . Nature London 208: 1234-1235. Baker, B. 1969. The response of t e l e o s t p i t u i t a r y thyrotrophs to thyroxine' i n v i t r o - a h i s t o l o g i c a l study. Gen. Comp. Endocrinol. 12: 227-231. Baker-Cohen, K. 1962. The ro l e of the thyro i d i n the development of p l a t y -f i s h . Zoologica 46: 181-222. B a l l , J. N. 1960. Reproduction i n female boney f i s h e s . Symp. Zool. Soc. London 1_: 105-135. B a l l , J . N. 1962. Brood production a f t e r hypophysectomy i n the viviparous t e l e o s t , M o l l i e n e s i a l a t i p i n n a . Nature 194: 787. B a l l , J . N. and E. Hawkins. 1976. Adrenocortical (interrenal) responses to hypophysectomy and adenohypophysial hormones i n the t e l e o s t P o e c i l i a l a t i p i n n a . Gen. Comp. Endocrinol. 2£3: 59-70. Barrington, E. J . and A. J . Matty. 1952. Influence of thiourea on reproduc-t i o n i n the minnow. Nature London 170: 105-106. Berg, O., A. Gorbman, and H. Kobayashi. 1959. The thyroid hormones i n invertebrates and lower vertebrates. In "Comparative Endocrin-ology", pp. 302-319 (A. Gorbman, Ed.). 1 John Wiley and Sons, New York. 101 Bern, H. and J . Nandi. 1964. Endocrinology of poikilotherm vertebrates. In "The Hormones", V o l . 4, pp. 199-298 (G. Pincus, K. Thimann, and E. Astwood, Eds.). Academic Press. B i l l e t e r , E. and C. Jorgensen. 1976. Ovarian development i n young toads, Bufo bufo bufo (L): E f f e c t s of u n i l a t e r a l ovariectomy, hypo-physectomy, treatment with gonadotropin (HCG), growth hormone, and p r o l a c t i n , and importance of body growth. Gen. Comp. Endocrinol. 29i 531-544. Birnbaum, M. J . , J . Schultz, and J . Fain. 1976. Hormone-stimulated glyco-genolysis i n i s o l a t e d g o l d f i s h hepatocytes. Amer. J . Physiol. 231; 191-197. Braverman, L. E., A. Vagenakis, A. Foster, and S. Ingbar. 1971. Evaluation of a s i m p l i f i e d technique f o r the s p e c i f i c measurement of serum thyroxine concentrations. J . C l i n . Endocrinol. .32: 497-502. Bromage, N. R. 1975. The e f f e c t s of thyrotropin-releasing hormone on the p i t u i t a r y - t h y r o i d axis of t e l e o s t f i s h . Gen. Comp. Endocrinol. 25: 292-297. Bromage, N. R. and M. Sage. 1968. The a c t i v i t y of the thyroid gland of P o e c i l i a during the gestation c y c l e . J . Endocrinol. 41: 303-311. Bromage, N. R., C. Whitehead, and T. Brown. 1976. Thyroxine secretion i n te l e o s t s and the e f f e c t s of TSH, TRH, and other peptides. Gen. Comp. Endocrinol. 29i 246. C a l l a r d , G. V. and J . H. Leatham. 1965. In v i t r o synthesis of steroids by experimentally induced c y s t i c ovaries. Proc. Soc. Exp. B i o l . Med. 118: 996-999. Campbell, C. and D. R. I d l e r . 1976. Hormonal con t r o l of v i t e l l o g e n e s i s i n hypophysectomized winter flounder (Pseudopleuronectes americanus Walbaum) . Gen. Comp. Endocrinol. 28_: 143-150. Chan, H. and J . G. Eales. 1976. Influence of bovine TSH on plasma thyroxine l e v e l s and thyro i d function i n brook trout, Salvelinus f o n t i n a l - i s . Gen. Comp. Endocrinol. 28_: 461-472. Chandola, A., J . T h a p l i y a l , and J . Pavnaskar. 1974. E f f e c t s of t h y r o i d a l hormones on the ovarian response to photoperiod i n a t r o p i c a l f i n c h (Ploceus p h i l i p p i n u s ) . Gen. Comp. Endocrinol. 24: 437-441. Channing, C , V. T s a i , and D. Sachs. 1976. Role of i n s u l i n , thyroxine, and C o r t i s o l i n l u t e i n i z a t i o n of procine granulosa c e l l s grown i n chemically defined media. B i o l . Reprod. 15_: 235-247. 102 Chavin, W. 1956. Thyroid d i s t r i b u t i o n and function i n the g o l d f i s h , Carassius auratus L. J . Exp. Zool. 133: 259-279. Chihara, K.,Y. Kato, S. Ohgo, Y. Iwasaki, K. Maeda, Y. Miyamoto, and H. Imura. 1976. E f f e c t s of hyperthyroidism on r a t growth hormone release induced by thyrotropin r e l e a s i n g hormone. Endocrinol. 98: 1396-1400. Clyde, H.,P. Walsh, and T. Eng l i s h . 1976. Elevated plasma testosterone and gonadotropin l e v e l s i n i n f e r t i l e males with hyperthyroidism. F e r t . S t e r i l . TTh. 662-666. Crim, L. W., R. E. Peter, and R. B i l l a r d . 1976. Stimulation of gonadotropin secretion by i n t r a v e n t r i c u l a r i n j e c t i o n of hypothalamic extracts i n the g o l d f i s h , Carassius auratus. Gen. Comp. Endocrinol. 30: 77-82. Dannies, P., K. Gautvik, and A. Tashjian. 1976. A possible r o l e of c y c l i c AMP i n mediating the e f f e c t s of thyrotropin-releasing hormone on p r o l a c t i n release and on p r o l a c t i n and growth hormone synthe-s i s i n p i t u i t a r y c e l l s i n cult u r e . Endocrinol. 98_: 1147-1159. Detlaf, T. A. and S. Davydova. 1974. E f f e c t of triiodothyronine on ripen-ing of oocytes i n s t e l l a t e sturgeon a f t e r the action of low temperatures and reservation of females. Ontogenet. 5_: 454-462. de Vlaming, V. 1972. The ro l e of the endocrine system i n temperature-c o n t r o l l e d reproductive c y c l i n g i n the estuarian gobid f i s h , G i l l i c h t h y s m i r a b i l i s . Comp. Biochem. Ph y s i o l . 41A: 697-713. de Vlaming, V. 1974. Environmental and endocrine control of t e l e o s t repro-duction. I r i "Control of Sex i n Fishes" (C. Schreck, Ed.). V i r g i n i a Polytechnic I n s t i t u t e , Publ. Dodd, J . M. 1945. The hormones of sex and reproduction and t h e i r e f f e c t s i n f i s h and lower chordates: twenty years on. Amer. Zool. 15 (Suppl. 1.): 137-171. Dodd, J . M. and A. J . Matty. 1964. Comparative aspects of thyro i d function. In "The Thyroid Gland", pp. 303-356 (R. P i t t - R i v e r s and W. Tr o t t e r s , Eds.). Butterworth. Donaldson, E. M. and J . R. McBride. 1974. E f f e c t of ACTH and salmon gonado-tr o p i n on i n t e r r e n a l a c t i v i t y of gonadectomized adult sockeye salmon (Oncorhynchus nerka). J . F i s h . Res. Bd. Canada 31: 1211-1214. 103 Dubin, N. 1974. Rat ovarian and uterine response to gonadotropin as modified by trii o d o t h y r o n i n e . B i o l . Reprod. JL1: 529-533. Dunn, J . , M. Hess, and D. Johnson. 1976. E f f e c t of thyroidectomy on rhythmic gonadotropin release. Proc. Soc. Exp. B i o l . Med. 151: 22-27. Eales, J . 1964. The influence of temperature on thyroid histology and radioiodine metabolism of y e a r l i n g steelhead trout, Salmo  g a i r d n e r i . Can. J . Zool. 42_: 829-841. Eales, J . 1974. Creation of chronic p h y s i o l o g i c a l elevations of plasma thyroxine i n brook trout, Salvelinus f o n t i n a l i s ( M i t c b i l l ) and other t e l e o s t s . Gen. Comp. Endocrinol. ^22: 209—217-E a r t l y , H. and C. Leblond. 1954. I d e n t i f i c a t i o n of the e f f e c t s of thyrox-ine mediated by the hypophysis. Endocrinol. 54_: 249-271. Eyeson, K. 1970. The ro l e of the thyroid i n reproduction of the west A f r i c a n l i z a r d , Agama agama. Gen. Comp. Endocrinol. 1_5_: 1-5. F o l l e t , B. K. and J . Redshaw. 1974. The physiology of v i t e l l o g e n e s i s . In: "Physiology of the Amphibia", Vol. 2, pp. 219-298 (B. Lof t s , Ed.). Academic Press, New York. Fontaine, M. 1976. Hormones and the cont r o l of reproduction i n aquaculture. J . F i s h . Res. Bd. Canada 33_: 922-939. Fontaine, M. and J . Leloup. 1962. Le fonctionement thyroidien du saumon adulte (Salmo sa l a r L.) a quelques etapes de son cycle migratoire. Gen. Comp. Endocrinol. 2_: 317-322. Fontaine, Y. A. 1969a. La s p e c i f i c i t e zoologique des proteines hypophys-ai r e s capables de stimuler l a thyroide. Acta Endocrinol. Supp. 138. Fontaine, Y. A. 1969b. Studies on the heterothyrotropic a c t i v i t y of prep-arations of mammalian gonadotropins on i n t a c t f i s h . Gen. Comp. Endocrinol. Supp. 2_: 417-424. Fredlund, P. 1976. The c l i n i c a l spectrum of hyperthyroidism. B u l l . Mason C l i n i c 30_: 3-15. Freeman, M. E., F. LaRochelle, and R. Moore. 1976. E f f e c t of thyro i d status on spontaneous and induced surges of l u t e i n i z i n g hormone. Endocrinol. 99: 713-719. 104 Goodrich, G. and T. Adelman. 1976. Regulation of malic enzyme synthesis by i n s u l i n , t r i iodothyronine, and glucagon i n l i v e r c e l l s i n cul t u r e . J . B i o l . Chem. 251: 3027-3032. Grobstein, C. and A. Bellamy. 1939. Some e f f e c t s of feeding t h y r o i d to immature fishes (Platypoecilus). Proc. Soc. Exp. B i o l . Med. 41: 363-365. Grosso, L. 1961. The e f f e c t of thiourea, administered by immersion of the maternal organism, on the embryos of Lebistes r e t i c u l a t u s , with notes on the adult gonadal changes. Bio. B u l l . 121: 481-496. Gupta, S., J . T h a p l i y a l , and R. Garg. 1975. E f f e c t s of thyro i d hormones on the chemical constituents of d i f f e r e n t t i s s u e s of the chequered water snake, Natrix p i s c a t o r . Gen. Comp. Endocrinol. 27: 223-229. Guttler, R., C. O t i s , J . Shaw, D. Warren, and J . N i c o l o f f . 1973. The e f f e c t of t h y r o i d hormone on adenyl cyclase - a p o t e n t i a l s i t e f o r thy r o i d hormone a c t i o n . In: "Thyroid Hormone Metabolism", pp. 201-211 (W. A. Harland and J . S. Ou, Eds.). Academic Press. Hardy, H., Y. Lee, and A. Takemori. 1960. Enzyme responses to thyroid hormones. Ann. N. Y. Acad. S c i . 8(5: 506-511. Hayes, M. T. 1968. Absorption of o r a l thyroxine i n man. J . C l i n . Endocrin-o l . _28: 749-756. Hendrich, C. E. and C. W. Turner. 1966. E f f e c t s of radiothyroidectomy and various replacement l e v e l s of thryoxine on growth, organ and gland weights of cornish cross chickens. Gen. Comp. Endocrinol. 7_: 411-419. Herlant, M. 1960. Etude c r i t i q u e de deux techniques nouvelles destinees a mettre en evidence l e s d i f f e r e n t e s categories c e l l u l a i r e s presentes dans l a glande p i t u i t a i r e . B u l l . Microsc. Appl. 10: 37-44. Hickman, C. P. 1962. Influence of environment on the metabolism of iodine i n f i s h . Gen. Comp. Endocrinol. Supp. 1_: 48-62. Higgs, D. A., E. M. Donaldson, H. Dye, and J . McBride. 1976. Influence of bovine growth hormone and L-thyroxine on growth, muscle composi-t i o n and h i s t o l o g i c a l structure of the gonads, thy r o i d , pancreas, and p i t u i t a r y of coho salmon (Oncorhynchus k i s u t c h ) . J . F i s h . Res. Bd. Canada 33.= 1585-1603. Higgs, D. A. and J . G. Eales. 1971. Iodide and Thyroxine metabolism i n the brook trout, Salvelinus f o n t i n a l i s , during sustained exercise. Can. J . Zool. 49: 1255-1269. i 105 Higgs, D. A. and J . G. Eales. 1973. Measurement of c i r c u l a t i n g thyroxine i n several fresh water t e l e o s t s by competitive binding analysis. Can. J . Zool. 51: 49-53. Hirvonen, E., T. Ranta, and M. Seppala. 1976. P r o l a c t i n and thyrotropin responses to thyrotropin-releasing hormone i n patients with secondary amenorrhea: the e f f e c t of bromocriptine. J . C l i n . Endocrinol. Metab. 42: 1024-1030. Hoar, W. S. 1955. Reproduction i n t e l e o s t f i s h e s . Mem. Soc. Endocrinol. 4: 5-22. Hoar, W. S. 1959. Endocrine factors i n the e c o l o g i c a l adaptation of f i s h e s . I n i "Comparative Endocrinology", pp. 1-23 (A. Gorbman, Ed.). • John Wiley and Sons, New York. Hoar, W. S. 1965. Comparative physiology: hormones and reproduction i n f i s h . Ann. Rev. P h y s i o l . 27: 51-70. Hoar, W. S. 1966. Hormonal a c t i v i t y of the pars d i s t a l i s i n cyclostomes, f i s h , and amphibia. In "The P i t u i t a r y Gland", Vol. 1, pp. 242-294 (G. Harris and B. Donovan, Eds.). Butterworth, Washington, D. C. Hoar, W. S. 1975. "General and Comparative Physiology." Prentice H a l l . Hochachka, P. W. 1962. Thyroidal e f f e c t s on pathways for carbohydrate metabolism i n a t e l e o s t . Gen. Comp. Endocrinol. 2: 499-505. Hurlburt, M. E. 1975. The e f f e c t s of thyroxine on ovarian maturation i n the g o l d f i s h , Carassius auratus L. Bachelor's Thesis, Univ. of B r i t i s h Columbia. Ichikawa, M., T. Mori, S. Kawashima, K. Ueda, and S. Shirahata. 1974. H i s t o l o g i c a l changes i n the thyroid and i n t e r r e n a l t i s s u e of the Kokanee (Oncorhynchus nerka) during sexual maturation and spawning. J . Fac. S c i . Univ. Tokyo Sec. IV 13: 175-182. I s h i i , D. and K. Yamamoto. 1970. Sexual differences i n the l i v e r c e l l s i n the g o l d f i s h , Carassius auratus L. B u l l . Fac. F i s h . Hokkaido Univ. 21: 161-167. Ishikawa, H., T. Nagayama, C. Kato, and M. Takahashi. 1976. The e f f e c t s of growth hormone-releasing hormone (GH-RH) and thyroxine on the synthesis and release of GH i n a c l o n a l s t r a i n of r a t p i t u i t a r y tumor c e l l s . Bioch. Biophy. Rsch. Comm. 70: 241-247. 106 Jackson, I. and S. Reichl in . 1974. Thyrotropin.-releasing hormone (TRH) : dis tr ibution i n hypothalamic arid extrahypothalamic brain tissues of, mammalian and sub mammalian chordates. Endocrinol. 95_: 854-862. Jallageas, M. and I. Assenmacher. 1974. Thyroidal gonadal interactions in the male domestic duck i n relat ion with the sexual cycle. Gen. Comp. Endocrinol. 22_: 13-20. Jallageas, M . , I. Assenmacher, and B. F o l l e t . 1974. Testosterone secretion and plasma lute iniz ing hormone during a sexual cycle in the Pekin duck, and after Thyroxine treatment. Gen. Comp. Endocrinol. 23: 472-475. Janes, R. G. 1954. Effect of a p i tu i tary gonadotropin on the ovaries of hypothyroid rats . Endocrinol. _3_4: 464-470. Jeppsson, S . , K. Nilsson, G. Rannevik, and L . Wide. 1976. • Influence of suckling and of suckling followed by TRH of LH-RH on plasma prolact in , TSH, GH, and FSH. Acta Endocrinol. 82_: 246-263. Johnson, T . , and J . Meites. 1950. Effects of hypo- and hyperthyroidism in rats and mice on ovarian response to equine gonadotropin. Proc. Soc. Exp. B i o l . Med. 75: 155-157. Kalland, G. A . , A. Vera, and R. Swerdloff. 1976. Reproductive hormonal axis in experimental hypothyroidism. 58th Ann. Meet. Endocrine S o c , San Francisco. Kanwar, K. and V. Chaudry. 1976. Thyroid tes t is interrelationships i n Indian palm s q u i r r e l , Funambulus pennanti Wroughton. Endocrinologie j37_: 307-314. Khoo, K. 1974. Steroidogenesis and the role of steroids in the endocrine control of oogenesis and vitellogenesis i n the goldfish, Carassius auratus. Ph.D. Thesis, Univ. of B r i t i s h Columbia. Kovacs, L . and B. Mess. 1976. Investigation on luteinizing-inducing effect of pinealectomy or thyroidectomy•in different types of anovulatory syndromes in rats . Endocrinol. Exper. 10_: 83-90. LaRochelle, F. T. and M. Freeman. 1974. Superimposition of thyroid hormone regulation on gonadotropin secretion. Endocrinol. 95_: 379-387. Leatham, J . H. 1973. Role of the thyroid. In. "Reproductive Biology", pp. 857-876 (H. Balin and S. Glasser, Eds . ) . Excerpta Medica, i Amsterdam. 107 Lee, K., E. Bowers, and O. M i l l e r . 1968. Some studies of the e f f e c t of thyroi d hormones on the RNA synthesis of the an t e r i o r p i t u i -tary gland of r a t s . Endocrinol. 83: 763-768. Lehman, G., and B. Frye. 1976. The e f f e c t s of changes i n thyro i d function on t e s t i s 3 2 P uptake and the response to gonadotropin i n the chick. Gen. Comp. Endocrinol. 28^ 446-453. Leloup, J . and A. Hardy. 1976. Hormones thyroidiennes c i r c u l a n t e s chez , un cyclostome et des poissons. Gen. Comp. Endocrinol. 29_: 258. Lewander, K., G. Dave, M. Johansson-Sjobeck, A. Larsson, and U. Lidman. 1976. Metabolic e f f e c t s of i n s u l i n i n the European e e l , A n g u i l l a a n g u i l l a L. Gen. Comp. Endocrinol. 29: 455-467. Lewis, M. and J . M. Dodd. 1974. Thyroid function and the ovary i n the spotted dogfish, Scyliorhinus canicula. J . Endocrinol. 63_: 63. L i b e r t i , P. and J . Stanbury. 1971. The pharmacology of substances a f f e c t i n g the t h y r o i d gland. Ann. Rev. Pharmac. 2_: 113-142, MacKay, N. J . 1973. The e f f e c t s of methallibure (I. C. I. 33, 828) and thiourea on gametogenesis i n the f i r e t a i l gudgeon, Hypseleotris  r g a l i i . Gen. Comp. Endocrinol. _20_: 221-235. Maqsood, M. 1952. Thyroid functions i n r e l a t i o n to reproduction of mammals and b i r d s . B i o l . Rev. _2J7: 281-319. Matty, A. 1960. Thyroid cycles i n f i s h . Symp. Zool. Soc. London 2i 1-15. Mukherjee, A. 1975. E f f e c t s of thiourea treatment on thyro i d and ovary of the c a t f i s h , Heteropneustes f o s s i l i s (Bloch). Indian J . Exp. B i o l . 1_3: 327-332. Myant, N. B. 1964. The thyro i d and reproduction i n mammals. I_n "The Thyroid Gland", pp. 283-302 (R. P i t t - R i v e r s and W. Tr o t t e r , Eds.). Butterworths, London. Nagahama, Y. 1973. Hi s t o p h y s i o l o g i c a l studies bn the p i t u i t a r y gland of some t e l e o s t f i s h e s , with s p e c i a l reference to the c l a s s i f i c a -t i o n of hormone-producing c e l l s i n the adenohypophysis. Mem. Fac. F i s h . Hokkaido Univ. 21: 1-63. Novales, R., L. G i l b e r t , and F. Brown. 1973. Endocrine mechanisms. In "Comparative Animal Physiology", pp. 857-908 (C. L. Prosser, Ed.). W. B. Saunders. Ol i v o , J . , G. Gordon, F. R a f i i , and A. Southren. 1975. Estrogen metabolism i n hyperthyroidism and i n c i r r h o s i s of the l i v e r . Steroids 26: 47-56. 108 Peppier, R., M. Hess, and J . Dunn. 1975. Compensatory ovulation a f t e r u n i l a t e r a l ovariectomy i n thyroidectomized r a t s . J . Endocrinol. 66: 137-138. Peter, R. E. 1970. Hypothalamic c o n t r o l of th y r o i d gland a c t i v i t y and gonadal a c t i v i t y i n the g o l d f i s h , Carassius auratus. Gen. Comp. Endocrinol. 14: 334-356. Peter, R. E. 1971. Feedback e f f e c t s of thyroxine on the hypothalamus and p i t u i t a r y of g o l d f i s h , Carassius auratus. J . Endocrinol. 51: 31-39. Peter, R. E. 1972. Feedback e f f e c t s of thyroxine i n g o l d f i s h Carassius auratus with an auto-transplanted p i t u i t a r y . Neuroendocrinol. 17; 273-281. Peterson, E. J . , R. Robinson, and H. Willoughby. 1967. A meal-gelatin d i e t for aquarium f i s h e s . Prog. F i s h C u l t . 29_: 170-171. Phelps, C. and J . Leatham. 1976. E f f e c t s of postnatal thyroxine administra-t i o n on brain development, response to postnatal androgen and thyroid regulation i n female r a t s . J . Endocrinol. 69_: 175-182. Pickford, G. E. and J . Atz. 1957. "Physiology of the P i t u i t a r y Gland of Fishes". New York Zoological Society. Pickford, G. E. and F. B. Grant. 1968. The response of hypophysectomized male k i l l i f i s h (Fundulus heteroclistus) to thyrotropin prepara-tions and to the bovine heterothyrotropic f a c t o r . Gen. Comp. Endocrinol. _10: 1-7. Pickford, G. E., B. L o f t s , G. Bara, and J . Atz. 1972. Testes stimulation i n hypophysectomized male k i l l i f i s h , Fundulus h e t e r o c l i s t u s , treated with mammalian growth hormone and/or l u t e i n i z i n g hormone. B i o l . Reprod. l_l 370-386. Pierce, J . G., M. F a i t h , and E. M. Donaldson. 1976. Antibodies to reduced S-carboxymethylated alpha subunit of bovine l u t e i n i z i n g hormone and t h e i r a p p l i c a t i o n to study of the p u r i f i c a t i o n of gonado-t r o p i n from salmon (Oncorhynchus tschawytscha) p i t u i t a r y gland. Gen. Comp. Endocrinol. 30_: 47-60. Plack, P., D. Prit c h a r d , and N. Fraser. 1971. Egg proteins i n cod serum. Natural occurrence and induction by estrogen i n j e c t i o n s . Biochem. J . 121; 847-858. Raizada, R. B. 1974. Summary of Ph.D. Thesis. Banaras Hindu University, India. 109 R a i l , J . E., J . Robbins,-and C. G. Lewallen. 1964. The Thyroid. In "The Hormones", V o l . 5, pp. 159-440 (G. Pincus, K. V. Thimann, and E. Astwood, Eds.). Academic Press. Ray, A., S. Bhattacharjee, and A. Medda. 1976. Histochemical studies on the e f f e c t of thyroid hormone on a l k a l i n e and acid phosphatase a c t i v i t i e s i n the l i v e r of f i s h and amphibia;.: Endokrinologie 68: 80-85. Ray, A. and A. Medda. 1976. E f f e c t of thyroid hormones and analogues on ammonia and urea excretion i n l a t a f i s h (Ophicephalus punctatus). Gen. Comp. Endocrinol. 29_: 190-197. Refetoff, S., N. Robbin, and V. Fang. 1970. Parameters of thyroid function i n serum of 16 selected vertebrate species: a study of PBI, serum T4, free T4 and the pattern of T4 and T3 binding to serum proteins. Endocrinol. 86_: 793-805. Reinboth, R. 1972. Hormonal control of the t e l e o s t ovary. Amer. Zool. 12: 307-324. Remacle, C , P. Delare, and P. Jacquet. 1976. Actions hormonales sur l e s c e l l u l e s germinales femelles de Carassius auratus L., en culture organotypique. Renversement sexuel et ovogenese i n v i t r o . Gen. Comp. Endocrinol. 29_: 212-224. Sage, M. 1967. Responses of p i t u i t a r y c e l l s of P o e c i l i a to changes i n growth induced by thyroxine and thiourea. Gen. Comp. Endocrinol. 8: 314-319. Sage, M. 1973. The evolution of t h y r o i d a l function i n f i s h e s . Amer. Zool. 13: 899-905. Sage, M. and H. Bern. 1971. Cytophysiology of the t e l e o s t p i t u i t a r y . Int. Rev. Cy t o l . 31: 339-376. Sage, M. and N. Bromage. 1970a. A c t i v i t y of the p i t u i t a r y c e l l s of the t e l e o s t P o e c i l i a during gestation cycle and the control of gonadotropic c e l l s . Gen. Comp. Endocrinol. 14_: 127-136. Sage, M. and N. Bromage. 1970b. Interactions of the TSH and thyro i d c e l l s with gonadotropic c e l l s and gonads i n p o e c i l i d f i s h e s . Gen. Comp. Endocrinol. 14: 137-140. Sarkar, H. and M. Rao. 1971. E f f e c t of thyroidectomy and administration of thyroxine on ovulation and spawning i n vivo, i n v i t r o and i n transplantation i n the skipper frog, Rana cyanophlyctes (Schn). Gen. Comp. Endocrinol. 16: 594-598. 110 Schreck, C. and M. Hopwood. 1974. Seasonal androgen and estrogen patterns i n the goldfish,'Carassius auratus. Trans. Amer. F i s h . Soc. 2: 375-378, Scott, J . L. 1953. The e f f e c t s of thiourea treatment upon the thyroid, p i t u i t a r y , and gonads of the zebra f i s h , Brachydanio r e r i o . Zoologica 38.: 53-62. Singh, B. R., R. Thakur, and B. Yadav. 1974. The r e l a t i o n s h i p between the changes i n the i n t e r r e n a l , gonadal, and t h y r o i d a l t i s s u e of the a i r breathing f i s h , Heteropneustes f o s s i l i s (Bloch) at d i f f e r e n t periods of the breeding c y c l e . J . Endocrinol. _61: 309-316. Singh, T. P. 1969. Observations on the e f f e c t of gonadal and-adrenal c o r t i -c a l s t e r o i d s upon t h y r o i d gland i n hypophysectomized c a t f i s h , Mystus v i t t a t u s (Bloch). Gen. Comp. Endocrinol. 1_2: 556-560. S l i c h e r , A. 1961. En d o c r i n o l o g i c a l and hematological studies i n Fundulus  h e t e r o c l i t u s (Linn.). B u l l . Binham Ocean. C o l l . 17: 1-55. Snedecor, J . G., L. Krichan, and R. Freedland. 1972. E f f e c t of a sing l e i n j e c t i o n of L-thyroxine on glycogen, and on g l y c o l y t i c and other enzymes i n p r o p y l t h i o u r a c i l - f e d cockerels. Gen. Comp. Endocrinol. 18: 199-209. Southren, A., J . Ol i y o , G. Gordon, J . V i t t e k , J . Brener, and F. R a f i i . 1974. The conversion of androgens to estrogens i n hyperthyroidism. J. C l i n . Endocrinol. Metab. 38: 207-214. Srivastava, A. 1976. E f f e c t of thyroxine on erythropoiesis i n i n t a c t and hypophysectomized male k i l l i f i s h , Fundulus h e t e r o c l i t u s . Gen. Comp. Endocrinol. 29_: 259. Stacey, N. E. 1977. Regulation of spawning behaviour i n the female g o l d f i s h , Carassius auratus. Ph.D Thesis, Univ. of B r i t i s h Columbia. Stebbins, R. C. and N. Cohen. 1973. The e f f e c t of parietalectomy on the thyroid and gonads i n free l i v i n g western fence l i z a r d s , Sceloporus o c c i d e n t a l i s . Copeia: 662-672. Sto u f f e r , J . and D. Dunaway. 1975. Thyroid hormone modulation of gluconeo-genesis i n i s o l a t e d l i v e r c e l l s . Endocrine Soc. 57th Annual Meeting, New York, p. 119. Subhedar, N. and P. Rao. 1975. E f f e c t of some a n t i - t h y r o i d drugs on the corpuscles of stannius and th y r o i d gland of the c a t f i s h , Heteropneustes f o s s i l i s (Bloch). Gen. Comp. Endocrinol. 26: 327-335. Sundararaj, B. and T. Anand. 1972. E f f e c t s of p i s c i n e and mammalian gonado-tropins on gametogenesis i n the c a t f i s h . Gen. Comp. Endocrinol. Supp. 3^: 688-702. I l l Sundararaj, B. and S. Nayyar. 1967. E f f e c t s of exogenous gonadotropins and gonadal hormones on the testes and seminal v e s i c l e s of hypo-physectomized catfish,' Heteropneustes f o s s i l i s (Bloch) . Gen. Comp. Endocrinol'. 8: 403-416. Swift, D. 1960. C y c l i c a l a c t i v i t y of the th y r o i d gland of f i s h i n r e l a t i o n to environmental changes. Symp. Zool. Soc. London _2: 17-27. Takashima, F., T. Hibiya, P. Ngan, and.K. Aida. "1972. Endocrinological studies on l i p i d metabolism i n rainbow t r o u t . I I . E f f e c t s of sex s t e r o i d s , t h y r o i d powder, and adrenocb&ticotropin on plasma l i p i d content. B u l l . Jap. Soc. A c i . F i s h . _38: 43-49. Tausk, M. 1975. "Pharmacology of Hormones". Geog Thieme, Stuttgart (Publ.). T h a p l i y a l , J . P. 1969. Thyroid i n avian reproduction. Gen. Comp. Endocrin-o l . Supp. _2: 111-122. Tonoue, T. and K. Yamamoto. 1967. Stimulation by thyroidectomy and suppres-sion by thyroxine administration of amino a c i d uptake by the ra t p i t u i t a r y gland. Endocrinol. 81_: 101-104. Van Duin, C. 1973. "Diseases of F i s h . " Butterworth. Walker, R. and P. Johansen. 1973. Changes i n major l i v e r constituents following hypophysectomy i n g o l d f i s h (Carassius auratus). Experimentia 31/ . 1252-1253. W a l l i s , M. 1975. The molecular evolution of p i t u i t a r y hormones. B i o l . Rev. 50: 35-98. White, W., P. Handler, and E. Smith. 1973. " P r i n c i p l e s of Biochemistry". McGraw-Hill. Weibe, J . 1969. Endocrine controls of spermatogenesis and oogenesis i n the viviparous seaperch Cymatogaster aggregata Gibbons. Gen. Comp. Endocrinol. 12_: 267-275. Weiselthier, A. and A. Van. Tienhoven. 1971. The e f f e c t of thyroidectomy on t e s t i c u l a r s i z e and on the photorefractory period i n the s t a r l i n g (Sturnus v u l g a r i s ) . J . Exp. Zool. 179: 331-338. Wong, K. and K. Chiu. 1974. The snake t h y r o i d gland. I. Seasonal v a r i a -t i o n of t h y r o i d a l and serum iodoamino acids. Gen. Comp. Endocrinol. 2_3: 63-70. Yamazaki, F. 1965. Endo c r i n o l o g i c a l studies on the reproduction of the female g o l d f i s h , Carassius auratus L., with s p e c i a l reference to the function of the p i t u i t a r y gland. Mem. Fac. F i s h . Hokkaido Univ. 13_: 1-64. Yamazaki, F. and E. M. Donaldson. 1968. The e f f e c t s of p a r t i a l l y p u r i f i e d salmon p i t u i t a r y gonadotropin on spermatogenesis, v i t e l l o g e n e s i s , and o v u a l t i o n i n the hypophysectomized g o l d f i s h (Carassius aura- tus) . Gen. Comp. Endocrinol. 11: 292-299. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0094005/manifest

Comment

Related Items