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A study into the regulation of ovarian follicular dynamics by progestins in the bovine Taylor, Christopher C. 1994

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A STUDY INTO THE REGULATION OF OVARIAN FOLLICULAR DYNAMICS BY PROGESTINS IN THE BOVINE by CHRISTOPHER C. TAYLOR B.Sc., University of British Columbia, 1987 M.Sc., University of British Columbia, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANIMAL SCIENCE We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January, 1994  0  Christopher C. Taylor, 1994  In presenting this thesis  in  partial fulfilment of the requirements  for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  /  c1z/  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  /C  /9,9V  ABSTRACT  Ovarian follicles grow and regress in a wave-like pattern during the luteal phase of the estrous cycle in the bovine. Each wave of follicular growth gives rise to a dominant follicle. What causes non ovulatory dominant follicles to stop growing, become atretic, and regress is not understood. The synthetic progestin, norgestomet, has been shown to induce the maintenance of dominant follicles in the absence of a corpus luteum in the bovine. This provides an excellent model for studying the regulation of dominant follicle growth, maintenance and regression. A series of in vivo experiments using norgestomet and progesterone 4 (P were undertaken to determine how progestins modulate dominant follicle ) growth, maintenance, and regression. In addition, in vitro experiments were conducted to determine the effects of P 4 on steroidogenesis at the granulosa cell level. The results indicate that in the absence of a corpus luteum norgestomet induces the maintenance of dominant follicles and new follicular growth is arrested. This is the result of persistent high-frequency luteinizing hormone (LH) pulses from the pituitary gland. When the circulating concentration of progestin is increased, either with norgestomet or P , circulating LH decreases, the 4 maintained dominant follicle regresses and new follicular growth is restored. Administration of exogenous P 4 early in the estrous cycle, a period normally characterized by low plasma 4 concentrations and high-frequency LH pulses, induces premature regression of the first P wave dominant follicle. Thus, high circulating P 4 and low circulating LII is permissive to normal follicle turnover. The in vitro results suggest that P 4 may enhance bovine granulosa cell estradiol-17f3 and P 4 production. Progesterone had no suppressive effects on granulosa  ii  Abstract cell steroidogenesis, even at very high concentrations (10.6 M). Taken as a whole, the results suggest that progestins modulate dominant follicle growth, maintenance and regression by regulating pituitary LH release. High-frequency LH pulses induce the maintenance of dominant follicles. A normal pattern of follicular growth and regression is restored with a decrease in LH pulse frequency and decreased circulating LH. These results may have important implications for improving estrus synchronization and superovulatory protocols and lead to a better understanding of some reproductive disorders in cattle such as cystic ovary condition. The norgestomet induced maintained dominant follicle model may also provide a valuable tool for conducting research into the establishment, maintenance and loss of follicular dominance.  iii  TABLE OF CONTENTS  Abstract  ii  Table of Contents  iv  List of Tables  v  List of Figures  vii  Acknowledgements  ix  Dedication  x  Preface  xi  Literature Review  1  Ovarian Follicular Dynamics and Plasma Luteinizing Hormone in Norgestomet Treated Heifers  40  The Effect of Increasing Progestin Concentration on Norgestomet Maintained Dominant Follicles in Cattle  61  Effect of Exogenous Progesterone on the Ovulatory Capacity of the First Wave Dominant Follicle in Cattle  81  Effect of Exogenous GnRH Administration on LH Pulsatility and Follicular Dynamics During the Luteal Phase in Cattle  97  A Serum Free Culture System for Bovine Granulosa Cells: The Effects of FSH and Forskolin  109  Effect of Progesterone on Steroid Production by Bovine Granulosa Cells Collected From Day 7 Dominant Follicles and Cultured In Vitro  126  Effect of Progesterone on Dominant Follicle Growth and Regression in the Bovine: General Discussion  137  iv  LIST OF TABLES  2.1  2.2  3.1  3.2  3.3  3.4  4.1  5.1  5.2  5.3  Growth and regression of dominant follicles and corpus luteum in heifers implanted with a 9 day norgestomet ear implant on day 6, 12 or 18 of the estrous cycle  51  Mean LH pulse frequency, pulse amplitude and mean LH plasma concentration in heifers treated with a nine day norgestomet ear implant starting on day 6, 12 or 18 of the estrous cycle  52  Mean diameter of dominant follicles in cows treated with i) single norgestomet (iN), ii) norgestomet plus a PRID (NP), and iii) double norgestomet (2N)  68  Mean ovulatory follicle size and mean interval to standing estrus and ovulation in cows treated with a single norgestomet (iN) or norgestomet plus a PRID (NP)  69  Mean plasma progesterone in cows treated with a single norgestomet (lN), norgestomet plus a PR1D (NP) or double norgestomet (2N)  70  Mean plasma LH concentration, LH pulse frequency and pulse amplitude on day 6 of treatment in cows treated with a single norgestomet (iN) norgestomet plus a PRID (NP) or double norgestomet (2N)  71  Mean interval in days from PGF , 4 a administration to basal P 2 estrus and ovulation in heifers treated with P 4 on days 3, 4 and 5 and PGF a on day 7 2  89  Maximum diameter, onset of first dominant follicle regression and emergence of the second follicular wave in heifers treated with GnRH or saline  102  Maximum luteal diameter, onset of luteal regression and return to estrus in heifers treated with GnRH or saline  102  Mean plasma LH and LH pulse frequency and pulse amplitude in heifers treated with GnRH or saline  103  V  Tables continued  6.1  6.2  7.1  Estradiol, progesterone and testosterone concentrations in follicular fluid from representative estrogen active and estrogen inactive follicles  114  Viable granulosa cells at termination of a 72 hour culture period when plated directly on plastic (Pla) or on an extracellular matrix (ECL) expressed as a percent of the initial plating density  120  Viable granulosa cells at termination of culture expressed as a percent of the initial plating density  131  vi  LIST OF FIGURES  2.1  2.2  2.3  3.1  3.2  4.1  4.2  4.3  5.1  6.1  6.2  Mean corpus luteum diameter and plasma progesterone concentration of heifers receiving a 9 day norgestomet ear implant on day 6, 12 or 18 of the estrous cycle  49  Mean dominant follicle diameter and number of subordinate follicles in heifers implanted with a 9 day norgestomet ear implant on day 6, 12 or 18  50  Plasma LH concentrations in representative heifers before and after receiving a norgestomet implant on day 8 or on day 18 of the estrous cycle  53  Mean dominant follicle diameter and number of subordinate follicles in heifers treated with a single 9 day norgestomet implant, norgestomet + PRID or 2 norgestomet  67  Secretory profile of LH from representative animals treated with a single norgestomet, norgestomet + PRID or 2 norgestomet  72  Mean plasma P 4 and CL diameter in control and P 4 treated heifers. Progesterone injections were given on days 3, 4 and 5 and PGF a on day 7 2  87  Growth, regression/ovulation of dominant follicles in control and P 4 treated heifers  88  Plasma P 4 and CL diameter in heifers treated with P 4 on days 3 to 5 of the estrous cycle and administered , on day 7 20 PGF  90  Luteinizing hormone profiles in a representative control heifer and a GnRH treated heifer  104  Mean estradiol, progesterone and testosterone accumulation in media from bovine granulosa cell cultures in response to FSH  116  Mean fold change in estradiol, progesterone and testosterone production by bovine granulosa cells in response to FSH  117  vii  Figures continued 6.3  6.4  7.1  7.2  Mean estradiol and progesterone accumulation in media from bovine granulosa cell cultures cells in response to forskolin  118  Mean estradiol, progesterone and testosterone accumulation in media from bovine granulosa cell cultures plated either on plastic or extracellular matrix  119  Mean estradiol and progesterone accumulation in media from bovine granulosa cell cultures in response to progesterone  132  Mean fold change in estradiol and progesterone accumulation in media from bovine granulosa cell cultures in response to progesterone  133  viii  4 hwkdriwr ia  a  a,m  cu1c  cvmc c  tcdm /Q4rv.  wJG  I,I  J4/1Q4  1I  wd/  cvrttnl1  4maI  y / cL-Ldc1 1i4 UJU  &  Q&?TQJTh1  c1&ct  UQJ7b  i/Q  V1Th  &w ?/ac1  u/ a/mc/  Jc  mc1kc4  .  .  ac/ mu t uJlJmc/irta &  & fzj7z. X  sjn  c1 i i/ainL  .  &  c1 cwc1  fzic2aI. Y wuJc1 at& ti/c&,  wn  344tc/  w&cJrt a41  .  .  .  i&  ka,c1 aL  h+Th m  X. cvmcl  t& a1&w m,  Xim&Jrt,  .  5.  1/  .  acJ uda . 4 mc4 t  n, 1/La/, a4l 1& c1a ct i/,  cddu1.  Lcdthu  k &6 1/  &WTt  cv  WII  ct2/a/l  1ad am, Jjrm4  mcta,, &U2,  cvnc/ o’m  ix  &/1,Jt  J&  c/cv,  /U%mcvT4 amc1 /mG&Tt cb&c1I/r, 1&J1.  x  PREFACE  The following work describes research conducted into the regulation of ovarian follicular growth and regression in the cow. Much of the work is either published, in press, or submitted for publication. As such each chapter, save the literature review and general discussion (Chapters 1 and 8), constitutes an independent research paper. The common goal in all of the research was to determine the role of the steroid hormone progesterone in the regulation of dominant follicle growth, maintenance and regression. Chapters 2 and  3  describe the effects of the synthetic progestin norgestomet on follicular dynamics, plasma LH profiles and the use of norgestomet in an in vivo model of dominant follicle maintenance. This model, which we were among the first to describe, provides an excellent means for studying follicular maintenance, as seen in cystic ovary condition in cows. It also provides an in vivo model for studying factors which may induce atresia in non-ovulatory dominant follicles. Chapter 4 provides evidence to show that progesterone can induce premature atresia of non-ovulatory dominant follicles. Chapter 5 describes an attempt to induce highfrequency LH pulses during the mid luteal phase to determine if dominant follicles can be maintained in a high progesterone environment. In Chapter 6 a completely serum free in vitro culture system for bovine ovarian granulosa cells is described. This system can be used to maintain granulosa cell estrogen production and can therefore be used for in vitro research into the regulation of estrogen  production by bovine granulosa cells. Granulosa cells  collected from day 7 dominant follicles were challenged with progesterone in Chapter 7.  xi  Preface Chapter 8 attempts to bring the entire volume together in a general discussion with a final conclusion that progesterone modulates dominant follicle growth, maintenance and regression indirectly, via regulation of luteinizing hormone release from the pituitary gland.  xii  CHAPTER 1  LITERATURE REVIEW  It could be argued that the most important structure in the female reproductive axis is the ovarian follicle. Follicles not only house, nourish and release ova for fertilization but they also produce and secrete steroid hormones which are intricately involved in regulating reproductive cycles. This review will attempt to integrate some of the knowledge on the regulation of fofficular growth, steroidogenesis and atresia. It will refer to research done in several species, especially the rat and sheep where the most information is available, but will focus on the bovine species where possible.  1.1 The Ovarian Follicle The ovarian follicle is composed of an oocyte surrounded by granulosa cells which are attached loosely to a basal lamina. With the initiation of follicular growth the interstitial cells immediately adjacent to the basal lamina differentiate into thecal cells. Each of these layers play important roles in steroidogenesis and the development of ovulatory follicles. Ovarian follicles form early in embryonic development. Extragonadally derived primordial germ cells migrate to the gonadal ridge (Everett, 1943) and subsequently enter meiosis (Peters, 1970). In some species such as sheep, cows, pigs and rabbits, meiosis is  1  LITERATURE REVIEW  delayed while the germ cells become enclosed in germ cell cords. It has been suggested that a transient increase in sex steroids may inhibit the onset of meiosis in these species. At approximately half term, the germ cords degenerate and the primordial germ cells enter meiosis (Mauléon and Mariana, 1969). Meiosis is arrested in the last phase of the meiotic prophase, the diplotene stage, when the oocyte becomes surrounded by a single layer of granulosa cells and a basal lamina to form a primordial follicle (Peters, 1978). Follicle formation always starts in the innermost portion of the ovary immediately after the first oocytes reach the diplotene stage (Mossman and Duke, 1973). Granulosa cells arise from mesonephric derived cells (Byskov and Lintem—Moore, 1973) and are initially connected to the rete cords. Primordial follicles become independent units with the formation of the basal lamina and the breaking of the connection of the granulosa cells and rete cords (Hashimoto and Eguchi, 1955). From birth until the onset of reproductive cycles there is a massive loss of follicles due to a degenerative process termed atresia. From puberty onwards, poois of primordial follicles enter a growing phase, at which time interstitial cells differentiate to form the thecal cell layers (see Paton and Collins, 1992). Growing follicles may progress through preantral, antral and ovulatory follicle stages, however, follicles at any stage may stop growing and degenerate through the process of atresia.  2  LiTERATURE REVIEW 1.2 Gonadotrophic Regulation of Follicular Growth  The first signs of follicular growth are the enlargement of the primary oocyte, granulosa cell proliferation and differentiation of theca cells adjacent to the basal lamina. The initial stimulus for these events is unknown, however it would appear to be gonadotrophin independent. Granulosa cell proliferation occurs in vitro without gonadotrophin stimulation (Baker and Neal, 1973; Peters et aL, 1973; Challoner, 1975). In addition, treatment of adult mice with anti-gonadotrophins does not interrupt early follicular growth (Nakano et aL, 1975), although gonadotrophic stimulation can accelerate the rate of growth (Hansel and Convey, 1983). It has been suggested that an oocyte derived growth factor may stimulate granulosa cell proliferation in primordial follicles (Satoh et al., 1985). Destruction of primordial germ cells in fetal rats results in development of sterile gonads incapable of steroid synthesis (Merchant, 1975). As with the initial stimulus for the growth of primordial follicles, the stimulus for the differentiation of interstitial cells to the thecal layers is not clear. It appears that the signal may arise from the primordial follicle itself, co-cultures of dispersed porcine granulosa and thecal cells tend to re-aggregate with the thecal cells at the periphery (Stoklosawa et aL, 1982). With the acquisition of luteinizing hormone (LH) receptors thecal cells become steroidogenically competent. Further growth of follicles from the preantral to the antral stage and beyond has long been held to require gonadotrophic stimulation, namely that of follicle stimulating hormone  3  LITERATURE REVIEW (FSH). Hypophysectomy results in the arrest of antral follicular growth (Gulyas et aL, 1977) while treatment of rats with antibodies to FSH blocks the development of the ovulatory pool of follicles (Welschen and Dullaart, 1976). With FSH stimulation granulosa cells acquire the enzymes required for the conversion of androgens to estrogens, there is an increase in the mitotic activity, and finally, under the influence of FSH and estradiol-1713 ) the granulosa cell layer acquires LH receptors (Richards, 1980). 2 (E Under the influence of both LII and FSH, the thecal cell layer and the granulosa cell layer of growing antral follicles interact synergistically to synthesize and secrete sex steroid hormones. The thecal layer primarily produces progestins and androgens while the granulosa layer produces estrogens and to a lesser degree progestins.  1.3 Steroid Feedback Sex steroid hormones produced by growing follicles and the resultant corpus luteum (CL) from ovulatory follicles, enter the general circulation and find their way to the hypothalamic-pituitary axis where they can regulate the synthesis and secretion of pituitary gonadotrophins. It has long been known that LH and FSH secretion is modulated by negative feedback ioops from factors secreted by the gonads. Gonadectomy leads to a rapid increase in circulating gonadotrophin levels (Gay and Midgley, 1969). Sex steroid hormone replacement leads to a return of gonadotrophins to pregonadectomy levels.  4  LiTERATURE REVIEW  Progesterone feedback Progesterone (P ) exerts a negative effect on LH secretion by decreasing the LH 4 pulse frequency. This has been demonstrated in various species including sheep (Goodman and Karsch, 1980) and cows (Ireland and Roche, 1982; Roberson et al., 1989). The negative effect of P 4 is probably primarily at the level of the hypothalamus. The opiate antagonist naloxone causes an increase in LH secretion during the luteal phase in sheep (Brooks et al., 1986) by overriding the P 4 negative feedback and inducing a gonadotrophin-releasing hormone (GnRH) pulse. The negative effect of P 4 does not appear to extend to FSH. Treatment of ovariectomized ewes with P 4 does not decrease circulating FSH (Nett et al., 1981). Additionally, treatment with P 4 has no effect on gonadotrophin subunit (a, FSHI3 and LHj3) mRNA content in the pituitary (Nett et al., 1990). In rats, P 4 acts in synergy with F 2 to decrease pituitary LHi3 mRNA but only when pituitary P 4 receptors have been induced by pretreatment with E. 2 (Jutisz et al., 1990). There is some evidence to suggest that P 4 may also act at the level of the pituitary. Ovine pituitary cell cultures treated with P 4 have a decrease in a and FSH(3 subunit mRNA’s and a decrease in responsiveness to GnRH (Miller et al., 1990).  Estrogen feedback Feedback of estrogens on the hypothalamic-pituitary axis is more complicated than that of P . Estrogens have been shown to have both negative and positive feedback effects. 4 A single injection of F 2 in the ewe (Nett et al., 1974) and cow (Beck and Convey, 1977) 5  LJTER4TURE REVIEW leads to an immediate decrease in circulating FSH and LH. This is followed several hours later with an ovulatory-like surge of both gonadotrophins. In ewes, chronic treatment or elevation of E 2 as seen during pregnancy, leads to a decrease in secretion and pituitary content of both LH and FSH (Moss et al., 1981). This is probably the result of a decrease in a, FSH(3 and LHI3 subunit mRNAs in the anterior pituitary (Nilson et al., 1983). Chronic administration of E 2 in the ewe shows a triphasic response. Initially there is a decrease in circulating LH and FSH followed by an ovulatory-like surge between 12 and 20 hours after the onset of treatment. Following the induced gonadotrophin surge there is again a decrease in circulating gonadotrophins and chronic inhibition for the remainder of the treatment (Nett et al., 1990). The a, FSH3 and LH(3 subunit mRNAs follow a similar pattern in response to E , however there is approximately a 24 hour lag period behind the 2 gonadotrophin secretion pattern (Nett et al., 1990). The initial inhibition by E 2 is probably due to a direct effect at the pituitary level. Injection of an E 2 bolus in ovariectomized ewes inhibits LH secretion but has no effect on GnRH secretion from the hypothalamus (Nett et al., 1984; Clarke and Cummins, 1985; Schillo et al., 1985). The positive feedback effect of E , which generates the ovulatory gonadotrophin 2 surges, appears to be the result of 2 E ’ s effect on the hypothalamus. The LH surge is associated with an increase in GnRH secretion in rats (Sarkar et al., 1976), monkeys (Neill et al., 1977) and women (Miyake et al., 1980). In ewes, after the first 12 hours of chronic administration of E , there is an increase in the GnRH pulse frequency in hypophyseal portal 2 blood (Clarke and Cummins, 1985). Recently, surges of GnRH immediately preceding E 2 6  LITERATURE REVIEW  induced LH surges (Moenter et aL, 1990), as well as preceding natural LH surges (Moenter et aL, 1991), have been described in the ewe. The increase in GnRH leads to the secretion of large amounts of both FSH and LH, producing the ovulatory gonadotrophin surges and resulting in depletion of the pituitary gonadotrophin stores (Roche et at, 1970; Nett et at, 1990). This is followed by an increase in the pituitary content of gonadotrophin subunit mRNAs, presumably to replenish the pituitary (Nett et al., 1990). The positive feedback of E 2 may be enhanced by its actions directly on the pituitary. One of the initial effects of E 2 appears to be the induction of an increase in pituitary GnRH receptors (Gregg and Nett, 1989). This would therefore increase the sensitivity of the pituitary to increases in GnRH. Other evidence suggests that E 2 may act directly at the level of the pituitary. The entire menstrual cycle in hypothalamic-lesioned monkeys can be mimicked by treatment with E 2 (Knobil, 1981). If serum F. concentrations remain elevated following the gonadotrophin surges the effect of E 2 again switches from a positive feedback to a negative one. Most evidence suggests that the long term effect of E 2 is due to its actions on the hypothalamus. GnRH secretion is decreased (Karsch et al., 1987) leading to a decrease in gonadotrophin secretion. Additionally, the chronic decrease in GnRH results in a decrease in pituitary mRNA for the gonadotrophin subunits, a corresponding decrease in gonadotrophin synthesis (Nilson et at, 1983) and eventually depletion of pituitary LH and FSH (Moss et aL, 1981). Thus it would appear that E 2 has three relatively distinct effects on pituitary gonadotrophin synthesis and secretion. Firstly, the acute negative feedback of F 2 is probably 7  LiTERATURE REVIEW due to a decrease in LH and FSH secretion with little or no effect on GnRH secretion from the hypothalamus. The positive feedback effect of E 2 that occurs during gonadotrophin surges is probably regulated at both the hypothalamic and pituitary levels. Immediately preceding the gonadotrophin surge, E 2 increases GnRH secretion from the hypothalamus by increasing both GnRH pulse frequency and amplitude, resulting in a GnRH surge which in turn stimulates the gonadotrophin surge. Additionally, E 2 acts at the level of the pituitary to increase the number of GnRH receptors, thus malcing the pituitary more sensitive to GnRH. Finally, the long term negative effect of E 2 would appear to be due to a decrease in GnRH secretion from the hypothalamus leading to a chronic decrease in gonadotrophin synthesis and secretion by the pituitary.  1.4 Intraovarian Regulators of Follicular Growth The pattern of pituitary gonadotrophin secretion, largely regulated by ovarian steroids as outlined above, has long been held to regulate follicular growth, selection and maturation of the ovulatory follicle. In the bovine, the time required for follicles to grow from 0.4 to 10 mm has been estimated at 20 to 22 days. This time frame corresponds to the inter-estrus interval. With this in mind it has been suggested that the post-estrus FSH surge stimulates the growth of a pool of follicles which will ultimately give rise to the follicle which will ovulate 20 to 22 days later (Scaramuzzi et al., 1980). Recent advances in the understanding of the dynamics of follicular growth have lead to the suggestion that the pattern of follicular growth in several species cannot be fully 8  LITERATURE REVIEW  explained by the pattern of gonadotrophin secretion (Goodman et al., 1979; Hansel and Convey, 1983). This has lead to the investigation of intraovarian regulators of follicular growth. These factors may act in an endocrine, paracrine or autocrine manner to modulate gonadotrophin secretion or follicular reponsiveness to circulating gonadotrophins.  Inhibin Inhibin is perhaps the most important of these ovarian factors. The first evidence for a non steroidal gonadotrophin inhibitor came from studies using aqueous extracts from the testes to inhibit FSH secretion in males (McCullagh, 1932). Purification of porcine (Ling et al., 1985; Rivier et al., 1985; Miyamoto et al., 1985) and bovine (Robertson et al., 1985) inhibin has revealed the primary form to be a heterodimer composed of a single 18 kilodalton a-chain,  linked by disulfide bonds, to one of either  (A  or 1 B 14 kilodalton 3  subunits. Inhibin has since been characterized in several different species including the pig, cow, hamster, human, sheep, monkey and rat (sce Tonetta and diZerega, 1989 for review). Inhibin biosynthesis has been localized to granulosa cells using immunoblot and immunocytochemistry techniques (Erickson and Hsueh, 1978). Secretion by granulosa cells is stimulated by FSH, as well as by LII and hCG in cells pretreated with FSH to induce LH receptors (Bicsak et al., 1986). In contrast, GnRH acts to block FSH stimulated inhibin release (LaPolt et al., 1990a). The major action of inhibin is a feedback to the pituitary to inhibit the release of FSH. Circulating inhibin and FSH concentrations are generally inversely related in sows and 9  LITERATURE REVIEW  cows (Hasegawa, 1988). When antisera to inhibin is administered to female rats in either proestrus or estrus there is an increase in circulating FSH but not LH (Rivier et at, 1986). Immunoneutralization of inhibin also appears to increase responsiveness of the pituitary to GnRH (Culler and Negro-Vilar, 1989). Inhibin may exert its effect by suppressing GnRH’s up regulation of its own receptor (Wang et al., 1989). There is also evidence to suggest that inhibin suppresses FSH/3 subunit synthesis, probably at a pretranslational level (Mercer et al., 1987; Carroll et al., 1989; Attardi et aL, 1989). This would help to explain inhibin’s differential effect in suppressing FSH but not LH. Inhibin may also have paracrine and autocrine activities. Inhibin suppresses FSH stimulated aromatase activity in granulosa cells (Hutchinson et al., 1987; Ying et al., 1986; Zhang et al., 1987) and amplifies the action of LH on thecal cells (Findlay, 1993; Hsueh etal., 1987).  Activin  During the efforts to isolate and purify inhibin from follicular fluid it was found that some fractions were capable of stimulating pituitary FSH release. This FSH stimulating factor was found to be a dimer composed of two inhibin 3 subunits; either activin-A, or  13A13B,  3 9 A / ,  to yield  to yield activin-B (Ling et al., 1986; Vale et al., 1986). Dimers of 1 33  have not been identified. Immunostaining would suggest that the cellular source for activin is the granulosa cell layer (Ogawa, et al., 1991; Robinovici et al., 1992).  10  LITERATURE REVIEW  Both activin-A and activin-B increase basal secretion of pituitary FSH with equal potency, but have no effect on LH release (Ying, 1988). The differential stimulation of FSH secretion is probably due to an increase in FSHI3 subunit biosynthesis (Carroll et al., 1989). As with inhibin, activins would appear to have paracrine and autocrine activities. Activin increases FSH stimulated granulosa cell aromatase activity, P 4 production and increases FSH and LH receptor content (Sugino et al., 1988). Activin-A has also been shown to increase FSH-stimulated inhibin secretion (LaPolt et al., 1989).  Insulin-like growth factors The insulin-like growth factors (IGF-I and IGF-II) comprise another class of probable intraovarian regulators. These low molecular weight proteins are structurally related to proinsulin. They are composed of a single polypeptide chain with three intrachain disuiphide bonds (Rinderkneckt and Humbel, 1978a; Rinderkneckt and Humbel, 1978b). Both IGF-I and IGF-II bind to specific cell surface receptors; IGF-I preferentially binds to the type-I receptor while IGF-Il preferentially binds to the type-IT receptor. However, IGF-I, IGF-II and insulin have very similar three dimensional structures, leading to cross reactivity between the IGFs, insulin and their respective receptors (see Giudice, 1992 for review). The first suggestion that IGFs were produced in the ovary came from studies showing that the concentration of immunoreactive IGF-I was far greater in the follicular fluid than in serum (Hammond, 1981). It was then demonstrated that porcine granulosa cells cultured in vitro secreted immunoreactive IGF-I into the culture media (Hammond et al., 1985). 11  LITERATURE REViEW Secretion of IGF-I is stimulated by FSH. In addition, E 2 acts synergistically with FSH to increase IGF-I secretion (Hsu and Hammond, 1987). In situ hybridization studies using cells collected from rat ovaries suggest that IGF-I  is synthesized in granulosa cells of growing follicles but not atretic follicles or the corpus luteum (Oliver et aL, 1989). IGF-II appears to be synthesized exclusively in the theca (Hernandez et al., 1990). The IGFs have several effects on ovarian cells. One of the more important is the mitogenic effect of IGF-I, which stimulates DNA synthesis and cell proliferation in granulosa cells (Baranao and Hammond, 1984; May et al., 1988; Olsson et al., 1990). IGF-I also increases progestin production in porcine granulosa cells cultured in vitro (Veldhuis et al., 1985), probably by acting in synergy with FSH to increase 4P 50 side-chain cleavage activity (Veidhuis and Rodgers, 1987), gene expression (Urban et al., 1990) and increased lipoprotein metabolism (Veldhuis and Gwynne, 1989). In rat granulosa cells IGF-I increases FSH-stimulated, but not basal, P 4 and E 2 production (Adashi et al., 1985a). It also increases the FSH stimulated induction of granulosa cell LH receptors (Adashi et aL, 1985b), FSH stimulated and basal inhibin synthesis (Bicsak et al., 1986), as well as basal and FSH stimulated proteoglycan synthesis (Adashi et al., 1986), suggesting IGF-I plays an important role in follicle selection, maturation and atresia. Less appears to be known of the actions of IGF-II, although it would appear that IGF-II does stimulate P 4 production by granulosa cells collected from mature pigs (Veidhuis etal., 1985).  12  LITERATURE REVIEW  Complicating the IGF story are the IGF binding proteins (IGFBPs). At least six IGFBPs have been cloned and characterized (see Giudice, 1992 for review). These binding proteins are involved in both the transport of the IGFs and their presentation to cell surface receptors where they may act to augment or inhibit the actions of the IGFs. IGFBP-2 has been found to be synthesized by both granulosa cells and theca cells while IGFBP-3 is produced primarily in the theca (Giudice, 1992). Both these binding proteins have been found to inhibit DNA synthesis and FSH stimulated steroid production by cultured rat granulosa cells (Ui et at, 1989; Bicsak et at, 1990; Shimasaki et at, 1990). IGFBP-1 has also been found to inhibit the synergistic effect of IGF-1 and FSH in stimulating rat granulosa cell P 4 production in vitro (Adashi et al., 1992). It has therefore been suggested that the binding proteins act by sequestering IGFs and limiting their availability to the cell (Shimasaki et at, 1990; Adashi et at, 1992). However, IGFBPs have also been found to inhibit forskolin and cholera-toxin stimulated increases in granulosa cell steroidogenesis, suggesting a possible direct action of the binding proteins (Bicsak et al., 1990).  Other intraovarian factors  In recent years a plethora of other factors synthesized in the ovary and affecting follicular growth and steroidogenesis have been identified (see Tonetta and DiZerega, 1989; Paton and Collins, 1992; Urban and Veldhuis, 1992 for reviews).  13  LITERATURE REVIEW  Transforming growth factor beta (TGFj9) is a protein of the inhibin family produced by granulosa cells under FSH stimulation (Hemandez et al., 1987; Mulheron and Schomberg, 1990). The response of granulosa cells to TGFI3 is dependent upon other hormonal influences. TGF13 acts in concert with IGF-I to increase cell proliferation in the absence of FSH. However, when FSH is present TGF13 promotes granulosa cell proliferation and steroidogenesis (Knecht et aL, 1989; Bendell and Dorrington, 1990; LaPolt et al., 1990b). Granulosa cell response is also dependent upon the concentration of FSH. At low FSH concentrations TGFI3 enhances LH receptor acquisition but at high FSH concentrations TGFI3 acts to inhibit receptor induction (Knecht et at, 1989). Transforming growth factor-alpha (TGFa) is a single chain polypeptide unrelated to TGF3. TGFa is thought to be an embryonic form of epidermal growth factor (EGF) to which it is structurally related (May and Schomberg, 1989). Both TGFa and EGF are thought to be produced by theca cells (Rail et aL, 1985; Skinner et al., 1987) under gonadotrophin stimulation (Kudlow et at, 1987; Roy and Greenwald, 1990). Both of these growth factors bind to the same cell surface receptor (Urban and Veidhuis, 1992) and therefore probably produce similar effects, although there appears to be more information in the literature on the effects of EGF. Epidermal growth factor inhibits rat granulosa cell 2 production and LH receptor induction in vitro (Mondschein and Schomberg, 1981; Hsueh E et al., 1984) and promotes mitogenesis in rat, pig and human granulosa cell cultures (Gospodarowicz and Bialecki, 1979).  14  LITERATURE REVIEW  Other intraovarian factors include fibroblast growth factor (FGF) which also promotes granulosa cell mitogenesis (Gospodarowicz and Bialecki, 1978); GnR}l, which can stimulate 4 production (Echstein et al., 1986) or inhibit gonadotrophin stimulated steroido basal P genesis (Jones and Hsueh, 1982a; Wickings et aL, 1990), and may be involved in inducing atresia by decreasing IGF-I receptors (Szende et aL, 1990); and the cytokines, tumour necrosis factor-alpha (TNFr) and interleukin-I (IL-I), both of which appear to suppress granulosa cell steroidogenesis (Gottschall et al., 1989; Emoto and Baird, 1988; Adashi et al., 1989; Veldhuis et al., 1991).  That the above factors can influence granulosa cell proliferation, differentiation and steroidogenesis in vitro seems without question, but their effects in vivo are much less clear. It seems probable that the gonadotrophic hormones FSH and LH are the gross regulators of follicular growth and steroidogenesis, while the intraovarian factors play a fine tuning role in follicle selection, maturation and initiation of atresia.  1.5 Steroidogenesis Growing follicles and the corpus luteum synthesize and secrete most of the body’s sex steroids. Ovarian theca cells and granulosa cells work in concert to produce progestins, androgens and estrogens by what is known as the two cell theory of steroidogenesis (see Hsueh et al., 1984; Gore-Langton and Armstrong, 1988 for reviews). Under stimulation from FSH and LH both theca and granulosa cells synthesize progestins, namely pregnenolone 15  LITERATURE REViEW  and progesterone. These are then converted to androgens in the thecal compartment. Androgens, such as androstenedione, dehydroepiandrosterone and testosterone, diffuse from theca cells, across the basement membrane and into granulosa cells where they can be aromatized to estrogens. All three types of steroids are secreted and enter the circulation to be delivered to target tissues, including cells in the hypothalamic-pituitary axis where they modulate gonadotrophin biosynthesis and secretion.  Progestin biosynthesis The steroidogenic pathway begins with cholesterol, derived from either de novo synthesis from acetyl CoA by the enzyme HMG CoA reductase, delivery of circulating cholesterol by lipoproteins, or from intracellular stores of cholesteryl esters (Rosenbium et aL, 1981; Colbran et al., 1986; Lange et al., 1988). Cholesterol is metabolized by cytochrome 4P 50 cholesterol side chain cleavage (P45Oscc) to yield pregnenolone and isocaproic acid. Cytochrome P45Oscc catalyses this reaction by a two step process; hydroxylation at C20 and C22 and then cleavage between these two positions (Strauss et al., 1981). This would appear to be the rate limiting step in ovarian steroidogenesis (Waterman and Simpson, 1985). Immunoflourescent staining has been used to localize cytochrome P45Oscc to inner mitochondrial membranes in theca and granulosa cells (Farkash et al., 1986; Zlotkin et al., 1986). FSH stimulates small increases in cytochrome P45Oscc mRNA in rat granulosa cells, and this effect is potentiated by E 2 (Goidring et aL, 1987). Stimulation of granulosa and 16  LITERATURE REVIEW  theca cells by LH leads to dramatic increases in cytochrome P45Oscc transcripts (Goidring et aL, 1987). Induction of cytochrome P45Oscc by gonadotrophins is via the cAMP-adenylate cyclase signal transduction system. Forskolin and other agonists which increase intracellular cAMP, stimulate increases in cytochrome P45Oscc mRNA and increase progestin production (Hsueh et al., 1984; Richards and Hedin, 1988). In addition, several intraovarian factors, such as inhibin and the insulin-like growth factors, stimulate progestin production by increasing cytochrome P45Oscc as outlined in the previous section. Pregnenolone produced as a result of P45Oscc action can then be acted upon by the 3$ hydroxysteroid dehydrogenase (3$ HSD)  -  delta-4, delta-5 isomerase enzyme complex  to produce P . This reaction can take place in both granulosa and theca cells (Hsueh et al., 4 1984) and is stimulated by both FSH and LH (Jones and Hsueh, 1982b; Martel et al., 1990) but is inhibited by prolactin (Martel et al., 1990).  Androgen biosynthesis Progestins produced in theca and granulosa cells can be further metabolized in theca cells to yield androgens. Pregnenolone can be converted directly to the androgen, dehydroepiandrosterone via the delta-5 pathway. Similarly, progesterone that has been produced from pregnenolone is converted to the androgen androstenedione via the delta-4 pathway. Both of these reactions are catalyzed by the enzyme cytochrome P 450 l a 7 hydroxylase (P450 a) which has been localized in theca but not granulosa cells (Carson et 17 al., 1981a; Erickson et al., 1985; Richards et al., 1986). This single chain polypeptide has 17  LiTERATURE REVIEW  both hydroxylase and 17-20 lyase activity to convert C-21 progestins to C-19 androgens (Nakajin and Hall, 1981). In rats, small amounts of LH or human chorionic gonadotrophin (hCG) stimulate androgen biosynthesis by cultured theca cells (Bogovich et aL, 1981; Carson et aL, 1981a). The increase in androgen is associated with an increase in theca cell cytochrome P450 a 17 mRNA and protein (Hedin et al., 1987). Conversely, with large amounts of LH, as seen during the ovulatory surge, there is a decrease in theca cell androgen biosynthesis, P450 a 17 activity (Eckstein and Tsafriri, 1986), protein, and mRNA (Hedin et al., 1987). Dehydroepiandrosterone can be acted upon by 3i3 HSD to produce androstenedione. Androstenedione can then be converted to testosterone by 1713 hydroxysteroid dehydrogenase (1713 HSD). The conversion of androstenedione to testosterone occurs in both theca and granulosa cells, although the majority of the activity appears to be associated with granulosa cells (Bogovich and Richards, 1984). Studies in rat granulosa and theca cells suggest that 1713 HSD is present constitutively. It is present in all types of follicles, including those following hypophysectomy (Bogovich and Richards, 1984).  Estrogen biosynthesis The conversion of androgens to estrogens in the ovary occurs primarily, if not exclusively, in granulosa cells. The reaction is catalyzed by the microsomal enzyme aromatase cytochrome P . 4 50 Aromatase has been shown to be a single polypeptide capable of catalyzing both the aromatization of the A ring and the NADPH-cytochrome reductase 18  LITERATURE REVIEW reaction (Mendelson et al., 1990). Aromatase is capable of converting androstenedione to estrone and testosterone directly to estradio1-173. Estrone is converted to estradiol-1713 by 17f3 HSD, although there is evidence to suggest this is a separate isozyme from the 17 HSD that converts androstenedione to testosterone (Tremblay et al., 1989). Aromatase is induced by FSH stimulation, as seen by dramatic increases in both mRNA and protein (Chan and Tan, 1987). Luteinizing hormone has been shown to be capable of maintaining the FSH induced aromatase activity (Urban and Veidhuis, 1992), as has E , testosterone and dihydrotestosterone (Richards et al., 1987). In addition, a number 2 of growth factors synergize with FSH to further increase aromatase activity, including IGF-I (Adashi et al., 1985a) and TGF/3 (Knecht et al., 1989; Bendell and Dorrington, 1990). Conversely, EGF (}Isueh et al., 1981; Mondschein and Schomberg, 1981) and TNFc (Emoto and Baird, 1988; Darbon et aL, 1989) inhibit FSH stimulated aromatase activity.  1.6 Follicular Atresia One of the remarkable aspects of ovarian physiology is the apparent waste of ovarian follicles. The vast majority of follicles never even reach the antral stage but undergo a degenerative process termed atresia. In the human ovary perhaps as many as 99% of follicles become atretic, while in the mouse the number is somewhat lower, at approximately 75% (Byskov, 1978). Although the majority of follicular loss due to atresia occurs in primordial and preantral follicles prior to puberty, follicles can become atretic at any stage of growth.  19  LITERATURE REVIEW  Growing antral and dominant follicles will become atretic if the hormonal milieu is not appropriate for further growth or ovulation. Atretic follicles are marked by pyknotic granulosa cells (Byskov, 1974) and a decrease in the mitotic index, as indicated by a decrease in 3 [ H ]-thymidine incorporation (Pederson, 1970). As atresia progresses, there is leukocyte invasion, breakdown of the basement membrane, invasion of cytotoxic T cells (Bukovsky et al., 1979; Bukovsky et al., 1984) and loss of gap junctions between granulosa cells (Merk et al., 1973). In cows, sheep and pigs the theca undergoes complete regression (Priedkalns et al., 1968; O’Shea et al., 1978; Centola,  1982). In sheep, this process is marked by condensation of cells and  phagocytosis by still healthy theca cells (O’Shea et al., 1978). With atresia there is an alteration in steroidogenesis. In theca cells collected from atretic human follicles, there is an increase in P 4 and testosterone production (McNatty et al., 1979). In cells collected from atretic porcine follicles, there is an increase in androstenedione, testosterone and dihydrotestosterone but a decrease in B 2 production (Maxson et al., 1985). There is an increase in androgens and a decrease in B 2 in follicular fluid from atretic sheep follicles (Carson et al., 1981b; Tsonis et al., 1984), thus it would seem that androgen availability is not the limiting factor in the decrease in estrogen production by atretic follicles. Recent studies suggest that the decrease in estrogen production in atretic follicles is due to a decrease in aromatase activity, and this in turn is due to a decrease in aromatase mRNA (Tilly et al., 1992).  20  LiTERATURE REVIEW  There is a growing school of thought that follicular atresia represents an excellent example of programmed cell death, or apoptosis (see Hurwitz and Adashi, 1992 for review). Atretic follicles exhibit internucleosomal DNA fragmentation, which is characteristic of apoptosis (Thy, et aL, 1992). In addition, Ca /Mg sensitive endonucleases, thought to 2 cause DNA fragmentation, are present in granulosa and luteal cells in rats primed with PMSG to induce atresia (Zeleznick et aL, 1989). Perhaps one of the most dramatic examples of follicular atresia is the regression of large dominant follicles during the luteal phase in the bovine. The estrous cycle in cows is characterized by a wave-like pattern of follicular growth and regression (see Lucy et at, 1992 for review). During the luteal phase there is a regular pattern of growth and regression of dominant follicles. It has been shown that the number of follicular waves, and hence the number of dominant follicles, is related to the duration of the luteal phase and is not associated with the length of the follicular phase (Taylor and Rajamahendran, 1991a). In addition, waves of follicular growth appear to continue through at least the first sixty days of pregnancy (Ginther, et at, 1989; Taylor and Rajamahendran, 1991b) and resume shortly after parturition (Rajamahendran and Taylor, 1990; Savio et at, 1990a). Waves of follicular growth have also been described in prepubertal heifers (Hooper et al., 1993). Thus, the wave-like pattern of follicular growth would appear to be a fundamental characteristic of the bovine ovary. Despite all of the above information, the trigger for the induction of atresia is poorly understood. What causes some follicles to become atretic while others continue to grow? 21  LITERATURE REVIEW  This question is the focus of a great deal of research at the present time. Recently it has been demonstrated in the bovine that dominant follicles can be maintained with synthetic progestins administered in the absence of a corpus luteum (Rajamahendran et aL, 1989; Savio et aL, 1990b; Rajamahendran and Taylor, 1991) or with low levels of P 4 (Sirois and Fortune, 1990). The ability to manipulate the life-span of dominant follicles provides an excellent model for studying the regulation of dominant follicle growth and regression/atresia. The following studies were conducted in an effort to further our understanding of the regulation of follicular dynamics in the bovine. The specific aims were to determine the role of P , or 4 progestins, in: 1) the regulation of dominant follicle maintenance, 2) regulation of dominant follicle regression, and 3) regulation of F 2 production by granulosa cells harvested from dominant follicles.  REFERENCES Adashi, E.Y., Resnick, C.E., D’Ercole, A.J., Svaboda, M.E. and Van Wyk, J.J. 1985a. Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocrine Revs. 6:400-420. Adashi, E.Y., Resnick, C.E., Brodie, A.M.H., Svaboda, M.E. and Van Wyk, J.J. 1985b. Somatomedin-C enhances induction of luteinizing hormone receptors by follicle stimulating hormone in cultured rat granulosa cells. Endocrinology 116:2369-2374. Adashi, E.Y., Resnick, C.E., Svaboda, M.E., Van Wyk, J.J., Hascall, C.C. and Yanagishita, M. 1986. Independent and synergistic actions of somatomedin-C in the stimulation of proteoglycan biosynthesis by cultured rat granulosa cells. 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Developmental expression of / Ca 2 dependent Mg endonuclease activity in rat granulosa and luteal cells Endocrinology 125:2218-2220. Zhiwen, Z., Carson, R.S., Herrington, A.C., Lee, V.W.K. and Burger, H.G. 1987. Follicle-stimulating hormone and somatomedin-C stimulate inhibin production by rat granulosa cells in vitro. Endocrinology 120:1633-1638. Zlotkin, T., Farkash, Y. and Orly, J. 1986. Cell specific expression of immunoreactive cholesterol side chain cleavage cytochrome P450 during follicular development in the rat ovary. Endocrinology 119:2809-2820.  39  CHAPTER 2  OVARIAN FOLLICULAR DYNAMICS AND PLASMA LUTEINIZING HORMONE IN NORGESTOMET TREATED HEIFERS  ABSTRACT  Two experiments were conducted to determine the effects of a synthetic progestin, norgestomet, on ovarian follicular dynamics and plasma luteinizing hormone (LH) concentrations in cycling heifers. In the first experiment, a norgestomet implant was inserted for 9 days starting on day 6 (day 0  =  day of estrus), day 12 or days 18 to 20 (n  =  8 heifers  per group). Follicular dynamics and progesterone profiles were compared among treatment groups. In the second experiment, heifers received the implant either between days 8 to 10 (n  =  3) or days 18 to 20 (n  =  3) and left in place for 9 days. To determine plasma LH  concentrations, serial blood samples (every 20 minutes for 12 hours) were collected on the day before and 3 days after implant insertion. Follicular dynamics and corpus luteum growth and function were unaffected when norgestomet was implanted at either day 6 or day 12 (experiment 1) and between days 8 and 10 (experiment 2). When implanted on days 18 to 20, norgestomet caused the maintenance of the ovulatory follicle, while recruitment of new antral follicles was absent. No change in plasma LH concentrations was observed before and during norgestomet treatment when the implant was inserted between days 8 and 10 of the cycle. Increased LH pulse frequency (P < 0.05), decreased pulse amplitude (P < 0.05) and  40  Follicular dynamics and plasma LH with norgestomet increased mean plasma concentrations were observed following norgestomet insertion between days 18 to 20. It is concluded that maintenance of an ovulatory follicle in the absence of a functional corpus luteum may be due to high-frequency, low-amplitude LH pulses.  INTRODUCTION The ovary is a dynamic organ with the growth and regression of follicles and the corpus luteum (CL). The dynamics and regulation of follicular growth and regression (atresia) have received a great deal of attention. In recent years ultrasound imaging has been extensively used to study the pattern of follicular growth in cattle. Several studies (Pierson and Ginther, 1987a; Rajamahendran and Walton, 1988; Savio et al., 1988; Sirois and Fortune, 1988) confirm the hypothesis first proposed by Rajakoski (1960) that follicular growth in cattle occurs in waves. A wave of follicular growth is characterized by the appearance of a pooi of small follicles (2 to 3 mm in diameter) and the emergence and differential growth of a single dominant follicle while the remainder of the cohort regress. Estrous cycles with two (Ginther et al., 1989) and three (Savio et al., 1988; Sirois and Fortune, 1988) follicular waves have been reported for heifers. In our laboratory the majority of cycles monitored in postpartum cows were of two waves (Rajamahendran and Taylor, 1990), while heifers had three waves (Rajamahendran and Taylor, 1991). The dominant follicle of the first wave in both two and three wave cycles was identifiable by day 4 (day 0  day of estrus), reached its maximum diameter between days 6 and 7, went 41  Follicular dynamics and plasma LH with norgestomet through a static phase of no growth between days 8 and 12 and was no longer identifiable by day 15. The dominant follicle of the second wave was identifiable by day 14 and was the ovulatory follicle in two wave cycles while the third dominant follicle was the ovulatory follicle in three wave cycles. The number of waves of follicular growth during the estrous cycle in mature cows appears to be related to the length of the luteal phase of the cycle and not the length of the follicular phase (Taylor and Rajamahendran, 1991a). The synthetic progestin, norgestomet (l7o  ,  1113  ,  19 nor preg-4-ene 3, 20 dione) has  been used in combination with estradiol valerate to control the estrous cycle in cattle (Wiltbank and Gonzalez-Padilla, 1975; Short et al., 1976; Humphrey et al., 1977). This treatment is now commercially available as Syncro-Mate B (Ceva Labs, Overland Park, Kansas, USA) for synchronization of estrus in dairy and beef heifers and postpartum beef cows. Numerous studies have reported lowered pregnancy rates following norgestomet treatment (Rentfrow et al., 1987; Brown et aL, 1988; Mikeska and Williams, 1988; Brink and Kiracofe, 1988). In a recent study we have reported that: a) norgestomet treatment is an effective method of estrus synchronization with no effect on the relationship among the onset of standing estrus, the luteinizing hormone (LH) surge and ovulation following implant removal; b) follicular dynamics, CL growth and regression and plasma progesterone (P ) 4 concentration were unaffected by norgestomet treatment when implanted between days 9 and 11 (static phase of the first dominant follicle) and left in place for 9 days, and c) if a 9 day treatment was started between days 18 and 20 the dominant follicle present was maintained for the entire treatment period and ovulated following implant removal (Rajamahendran and 42  Follicular dynamics and plasma LH with norgestomet Taylor, 1991). It is not clear if norgestomet will induce the maintenance of the first dominant follicle if treatment is started during the growth phase of the follicle, prior to day 8. The aims of the present experiments were: 1) to determine the effect of initiation of norgestomet treatment on day 6 of the estrous cycle on subsequent follicular dynamics and CL growth and function and 2) to characterize LH pulsatility during a 9 day norgestomet treatment started at different stages of the estrous cycle.  MATERIALS AND METHODS  Experiment 1. Twenty four Holstein heifers between 12 and 14 months of age and having displayed at least one normal estrous cycle (length, 19 to 23 days) were selected. Estrus was synchronized with two injections of prostaglandin F a (25mg PGF; Lutalyse, The Upjohn 2 Co., Kalamazoo, MI) administered 12 days apart. Heifers were then chosen at random to receive a norgestomet ear implant for 9 days (6 mg norgestomet; Sanofi Canada Inc., Victoriaville, Quebec) during one of three stages of the estrous cycle (day 0  =  day of  estrus): early luteal phase (day 6, 8 heifers), mid luteal phase (day 12, 8 heifers) and the follicular phase, once the CL had begun to regress and the ovulatory follicle was identifiable according to ultrasound imaging (days 18 to 20, 8 heifers).  43  Follicular dynamics and plasma LH with norgestomet Ultrasound examination: Heifers were examined daily by ultrasound imaging from the onset of the PGF a 2 synchronized estrus until estrus and ovulation following norgestomet withdrawal. Ultrasound examinations were conducted as described by Rajamahendran and Taylor (1990) using a linear array ultrasound scanner (Tokyo Keiki LS 300 Tokyo Keiki Co. Ltd., Tokyo, Japan) equipped with a 5 MHz rectal transducer. The ovaries were scanned in several planes to identify all visible follicles and the CL. Appropriate images were arrested and relevant structures measured using a built-in caliper system. Permanent records were made using a video processing unit (Mitsubishi Electronics Co. Ltd., Tokyo, Japan).  Blood sampling and radioimmunnoassays: A blood sample (10 ml) was collected from a coccygeal blood vessel into a heparinized tube (Vacutainer, Canlab, Toronto, Ontario) prior to each ultrasound examination. The samples were centrifuged immediately and plasma collected for the subsequent analysis of P . Plasma P 4 4 concentrations were determined using a commercially available radioimmunoassay kit (Coat-A-Count, Diagnostics Products Corp., Los Angeles, CA). This kit was previously validated in our laboratory for measurement of P 4 in bovine milk and plasma (Rajamahendran et aL, 1989). Briefly, 100 LL of unextracted plasma from each sample was added to appropriately labelled polypropylene test tubes coated with antibodies to P . Standards contained 0, 0.1, 0.5, 2, 10 and 40 ng/mL P 4 4 in processed human serum. One mL of 1-labelled 25 P ‘ 4 (0.05 1 LCi/mL) was added to each sample and standard 44  Follicular dynamics and plasma LH with norgestomet tube. Tubes were left to incubate for three hours after which the liquid phase was gently aspirated. Radioactivity was determined using an LKB gamma counter. All plasma samples from individual heifers were analyzed within the same assay, with heifers from each treatment group represented within each assay. The inter- and intra-assay coefficients of variation were 9% and 7%, respectively, and the sensitivity was 0.05 ng/ml. Cross reactivity with 20o-dihydroprogesterone and 17c-hydroxyprogesterone was approximately 2 and 0.5%, respectively.  Statistical analysis: Differences among treatment groups with regard to follicular and CL dynamics were analyzed by comparing the means of individual characteristics of the estrous cycle defined below, using a General Linear Model analysis of variance (SAS Inc., 1985). Characteristic days of each cycle were determined retrospectively and defined as follows: day of emergence of a wave of follicles was defined as the day on which a pool of individually identifiable follicles 2 to 5 mm in diameter was first observed. The day of emergence of the dominant follicle was defined as the day on which a single follicle from the original pool achieved a diameter 2 standard deviations greater than the mean of its cohorts. Day of onset of regression of dominant follicles and the CL was defined as the first of two consecutive days of decrease with a continued decrease in diameter thereafter. Profiles of P 4 were compared using an analysis of variance for repeated measures. Heifers receiving the norgestomet implant during the follicular phase (implanted between days 18 and 20) served as controls 45  Follicular dynamics and plasma LH with norgestomet for all parameters tested against heifers receiving implants during the early (day 6) and mid (day 12) luteal phase.  Experiment 2. Six heifers, none of which was used in the first experiment, were selected and synchronized as in experiment 1. Heifers were chosen randomly to receive a norgestomet ear implant at one of two stages of the estrous cycle: between days 8 and 10 (luteal phase, 3 heifers) or between days 18 and 20 (follicular phase, 3 heifers). The norgestomet implant was removed after 9 days.  Ultrasound examinations and blood sampling: Heifers were bled and scanned daily as in experiment 1. In addition, blood samples were collected into heparinized tubes at 20 minute intervals for 12 hours via a jugular catheter 1 day before and 3 days after norgestomet implant insertion. Blood samples were centrifuged immediately and plasma stored at -20° C until assayed for LH. Plasma LH concentrations were determined in the laboratory of Dr. John Walton, Guelph University by radioimmunoassay procedures previously described (Vostermans and Walton, 1985). Breifly, 1-labelled 2 ‘ 5 bovine LH was used as tracer, the assay standard was NAIMMD-bLH-4 (2.2 X NIH-LH-B1). All results were corrected to NIH-LH-B1. The assay utilized anti-ovine LH antiserum at a final dilution of 1:400 000 in a double antibody separation system. Cross reactivity with highly purified bovine FSH and TSH were negligable (< 0.5%; Vostermans 46  Follicular dynamics and plasma LH with norgestomet and Walton, 1985). The inter- and intra-assay coefficients of variation were 11% and 8%, respectively, and the sensitivity was 0.1 ng/ml. All samples from individual heifers were analyzed within the same assay with heifers from each treatment group included in each assay.  Statistical analysis: Mean LII concentrations were calculated for each 12 hour sampling period. An LH pulse was defined as a sample meeting two of the following criteria (Cook et al., 1991): 1) An increase equal to or greater than 1 standard deviation from the mean or previous reading; 2) Peak reading 1 standard deviation or greater than the mean or subsequent reading; 3) two consecutive decreasing readings after the peak. Pulse frequencies (number of pulses per 12 hour sampling period) and pulse amplitudes (ng/ml greater than the previous nadir) were compared within groups before and during norgestomet treatment by a nested analysis of variance for repeated measures (heifer nested within treatment). The model tested the main effects of day and treatment and the day by treatment interaction. Mean plasma LH concentration for all heifers within a treatment were compared between treatments by an analysis of variance for repeated measures.  Breeding and pregnancy diagnosis: All heifers in both experiments were artificially inseminated with frozen  -  thawed  semen approximately 12 hours after the onset of standing estrus following norgestomet 47  Follicular dynamics and plasma LH with norgestomet withdrawal. Pregnancy was diagnosed by the visualization of an embryo with a heartbeat (Taylor and Rajamahendran, 1991b) approximately 28 days after breeding in those heifers that had not returned to estrus.  RESULTS Experiment 1 In the first experiment, 20 of 24 heifers had cycles consisting of three waves of follicular growth, the remaining four heifers had two waves. Only heifers with three waves were considered in the statistical analysis. There were no significant differences (P > 0.05) among groups for day of emergence of first and second dominant follicles (Fl and F2), day of onset of regression of either Fl or F2, or onset of regression of the CL (Table 2.1).  Experiment 2 Corpus luteum diameter and plasma P 4 concentrations were similar for all three groups (Fig. 2. la and b). The dominant follicle at the time of luteolysis (F2 or F3) became the ovulatory follicle in all heifers treated with norgestomet from days 6 to 15 and days 12 to 21. In heifers treated with norgestomet between days 18-20 and 27-29 the dominant follicle was maintained for the entire 9 day treatment period. There was an absence of recruitment of new antral follicles during the period of dominant follicle maintenance. Upon implant removal the maintained follicle ovulated in all cases (Fig. 2.2a and b).  48  Follicular dynamics and plasma LH with norgestomet a  40  30 E E  0 18-20 to 27-29 Implant 0 12 to 21 implant 0 6 to 15 implant  5  0  10  15 Day of cycle  20  25  30  b 8 D 18-20 to 27-29 implant 4  D 12 to 21 implant 6  0 6 to 15 implant  -J  4  E  0  a, (0 a, 0) 0  a-  2  0 0  5  10  15 Day of cycle  20  25  30  Fig. 2.1. Mean ( S.E.M.) corpus luteum diameter (a) and plasma progesterone concentration (b) of heifers receiving a nine day norgestomet ear implant on day 6 (o), 12 (G) or 18 (—) of the estrous cycle (day 0 • day of estrus).  49  Follicular dynamics and plasma LH with norgestomet  a  Norgestomet treatment  I  Norgestomet treatnn  Ovulation T  T.2  +-h 11  4)  T 1’! 1”!  TA Q  ‘1  C  I  1/1  i’t  I  If,  I  Ti’ ,j  0  I  I IJ 9 ] F1 [i F1 [ F1 ri  C  0 O  b  I  2  4  6  8  10  12  14  16  18  20  22  Norgestomet treatment Ovulation  25  _>_  E  -  9-a-a --  20  E  TI 0  15  C  10  T’  I  ‘cF  5 0 C  0  o  2  4  6  8  10  12  14  16  18  20  Day of estrous cycle (day 0 # sf  —  1st df  =  22  24  26  28  estrus)  2nd df  —  3rd df  Fig. 2.2. Mean (± SEM) dominant follicle (df) diameter and number of subordinant follicles (sf; 3-5mm) in heifers implanted with a 9 day norgestomet ear implant on day 6, 12 (a; n = 7 & n = 6, respectively) or on day 18 (b; n = 7) 50  Follicular dynamics and plasma LH with norgestomet  Table 2.1.  Growth and regression of dominant follicles’ and corpus luteum (CL) in  heifers implanted with a nine day norgestomet ear imlant on day 6, 12 or 18 of the estrous cycle.  Staae of estrous cycle 2 Day 6 (n=7)  Day 12 (n=6)  Day 18 (n-7)  Day of emergence Fl F2 F3  2.5 ± 0.3 12.4 ± 0.8 18.6 ± 0.3  2.4 ± 0.4 12.3 ± 0.9 18.3 ± 0.5  2.3 ± 0.3 12.5 ± 0.9 18.3 ± 0.2  Day of regression Fl F2 CL  13.7 ± 0.2 18.8 ± 0.1 18.0 ± 0.6  14.2 ± 0.3 18.3 ± 0.2 18.6 ± 0.4  13.5 ± 0.3 17.9 ± 0.3 18.3 ± 0.6  Day of ovulation  21.3 ± 1.6  23.1 ± 1.2  29.6 ± 0.8  ‘Fl, F2 and F3 are dominant follicles of the 1st, 2nd and 3rd wave, respectively. 2 D ay of estrus is designated as Day 0.  Follicular dynamics, CL diameter changes and plasma P 4 concentrations in the second experiment were consistent with those in experiment 1. Plasma LH results are summarized in Table 2.2  51  Follicular dynamics and plasma LH with norgestomet  Table 2.2.  Mean (± SEM) LH pulse frequency (pulses/12 hours), pulse amplitude (ng/mL) and mean LH plasma concentration (ng/mL) in heifers treated with a nine day norgestomet ear implant starting on day 6, 12 or 18 of the estrous cycle.  Implant treatment period Days 8-17 Pre implant n=3 Freq. Amp. Mean  1.7 ± 0.6a 0.6 ± o.i’° 0.7 ± 0.02a  Days 18-27  Post implant n=3 4.8 ± l.7ab  0.6 ± 0.lb 0.8 ± 0.04°  Pre implant n=3 3.5 ± 0.5a 0.9 ± 0.2° 0.6 ± 0.03”  Post implant nt=3 7.3 ± 0.6b 0.4 ± o.la 0.8 ± 0.03°  Values with different superscripts within rows differ (P < 0.05).  Briefly, LH pulse frequency and amplitude were not significantly (P > 0.05) altered by norgestomet when implanted during the luteal phase (Fig. 2.3a). However mean plasma LH concentration did increase (P < 0.05) during treatment. Following norgestomet implant during the follicular phase a significant (P < 0.05) increase in LH pulse frequency, decrease in pulse amplitude and increase in mean plasma LH concentrations were observed (Fig 2.3b). Combined for both experiments, five of eleven heifers in the follicular phase treated group were diagnosed pregnant by ultrasound imaging at day 28 post breeding. This did not differ from the pregnancy ratees in the mid luteal phase treated heifers (6/11 pregnant) and early luteal phase treated group (4/8 pregnant).  52  Follicular dynamics and plasma LH with norgestomet  a  2  1 -J  E  C  I -J  2  1  0:00  2:00  4:00  6:00  8:00  10:00  12:00  8:00  10:00  12:00  Hours  b  2  -J  E C  =  -J  2  0:00  2:00  4:00  6:00  Hours  Fig. 2.3. Plasma LH concentrations in representative heifers before and after recieving a norgestomet implant on day 8 (a) or on day 18 (b) of the estrous cycle (day 0 - day of estrus).  53  Follicular dynamics and plasma LH with norgestomet DISCUSSION Results from the first experiment show that norgestomet will induce the maintenance of a dominant follicle only when administered in the absence of a functional CL. However norgestomet had no effect on growth and regression of dominant fofficles when administered in the presence of a functional CL. This confirms an earlier report from our laboratory (Rajamahendran and Taylor, 1991), in which norgestomet was implanted for 9 days either between days 9 to 11 or days 18 to 20. However, the first dominant follicle normally begins to decrease in size by approximately day 11 to 12 of the cycle (Pierson and Ginther, 1987a; Savio et al., 1988; Taylor and Rajamahendran, 1991a), therefore the possibility exists that the first wave dominant follicle had already become atretic in some heifers when norgestomet was implanted. The results from the first experiment in the present study demonstrate that norgestomet does not alter follicular dynamics even when implanted on day 6, during the growth phase of the first dominant follicle. These observations suggest the CL may be involved, either directly or via the hypothalamic-pituitary axis, in the regulation or initiation of atresia of large dominant follicles during the luteal phase. The second experiment was designed to monitor LH pulsatulity during norgestomet treatment at different stages of the estrous cycle. The pulsatile pattern of LH varies throughout the estrous cycle and has been well characterized in cattle (Rahe et al., 1980; Walters et al., 1984). During the luteal phase when plasma P 4 concentration is relatively high, (> 2 ng/ml) LH is secreted in low-frequency, high-amplitude pulses (1 to 3 ng/ml every 3 to 5 hours). During the follicular phase prior to the LH surge, when plasma P 4 54  Follicular dynamics and plasma LH with norgestomet concentration is low, LH is characterized by high-frequency, low-amplitude pulses (0.5 to 1.5 ng/ml every 1 to 2 hours). Norgestomet had no effect on LH pulsatility when implanted during the luteal phase (day 12); Low-frequency, high-amplitude LH pulses were observed both before and during luteal phase norgestomet treatment. The pulsatile pattern of LH observed in the present experiment prior to the follicular phase-norgestomet implant was similar to the pattern observed in the luteal phase. This is probably the result of continuing negative feedback from P , as the norgestomet was 4 implanted prior to full regression of the CL. Thus the increase in LH pulse frequency and decrease in amplitude observed after 3 days of norgestomet treatment during the follicular phase could not be attributable to norgestomet but more probably reflects the normal highfrequency, low-amplitude LH pulses observed between luteolysis and the LH surge (Rahe etal., 1980). Although there are no bled control heifers for the follicular phase we are confident that the results demonstrate that norgestomet does not prevent the LH pulse pattern normally associated with the follicular phase. However, norgestomet does appear to block the very rapid, high-amplitude pulses observed during the ascending stage of the LH surge (Rahe et al., 1980). In a study using one or two P 4 releasing intravaginal devices (PRID) to provide different concentrations of P 4 to heifers administered PGF a to induce luteolysis, Roberson 2 et al. (1989) found that when one PRID (providing 1 to 2 ng/ml circuculating P ) was used, 4 LH pulse frequency was greater than when 2 PRIDs were used (providing > 5 ng/ml 4 P ) . 55  Follicular dynamics and plasma LH with norgestomet In a similar experiment, Sirois and Fortune (1990) found that the use of one controlled intravaginal drug releasing (CIDR) device induced the maintenance of dominant follicles in the absence of a CL while two CIDR devices restored the wave-like pattern of follicular growth. Recently, Savio et al. (1990) reported that in non-lactating Holstein cows administered PGF a on day 8, the first wave dominant follicle could be maintained with 2 norgestomet treatment. They also reported a higher LH pulse frequency in treated cows compared to controls. These observations, coupled with the present work suggest that a single norgestomet implant in the follicular phase provides a low level of progestin which allows for a high-frequency, low-amplitude LH pulse pattern, and this pattern may induce the persistence of dominant follicles. Alternatively, progestins may have a direct effect on follicular growth and atresia. Pierson and Ginther (1987b) found a greater number of follicles on the CL bearing ovary than on the contralateral ovary and have suggested a positive effect of the CL on follicular growth. Rexroad and Casida (1977) found that P 4 hastened follicle growth when it was injected into the ovary of sheep and that the effect was independent of ovarian estradiol-17j3 content. Conversely, studies of in vitro cultured granulosa cells suggest that high concentrations P 4 (106 M) inhibit granulosa cell aromatase activity (Schreiber et al., 1981; Kharbanda et al., 1990). Perhaps 4 P is acting at both levels. At low circulating P 4 concentrations, high-frequency, low-amplitude LH pulses induce maintenance of the  56  Follicular dynamics and plasma LH with norgestomet dominant follicle whereas high levels of P 4 result in low-frequency, high-amplitude LH pulses and possibly inhibit granulosa cell aromatase activity leading to atresia. It is unclear how prolonged high-frequency, low-amplitude LH pulses affect maintained dominant follicles. Based on both the ultrasound images of the sustained dominant follicles and the plasma P 4 values, there was no evidence of luteinization. In addition, all maintained follicles went on to ovulate after norgestomet withdrawal. Combined for both experiments five of the eleven heifers treated with norgestomet during the follicular phase were diagnosed pregnant at 28 days post insemination. This indicates that at least some oocytes remained viable and capable of being fertilized in spite of these abnormally long follicular waves. In summary, a single norgestomet implant induces the maintenance of dominant follicles in the absence of a functional CL. This is likely the result of a maintained pattern of high-frequency, low-amplitude LH pulses normally seen for only a short period during the follicular phase in the bovine. Norgestomet would appear to delay ovulation of the dominant follicle by blocking the increase in pulse amplitude associated with the ascending  stage of the preovulatory LH surge.  REFERENCES Brink, J.T. and Kiracofe, G.H. 1988. Effects of estrous cycle stage at Syncro-Mate B treatment on conception and time to estrus in cattle. Theriogenology, 29:513-518.  57  Follicular dynamics and plasma LII with norgestomet Brown, L.N., Odde, K.G., King, M.E., Lefever, D.G. and Newbauer, C.J. 1988 Comparison of MGAPGF a and Syncro-Mate B estrous synchronization in beef 2 heifers. Theriogenology, 30:1-12. Cook, D.L., Parfet, J.R., Smith, C.A., Moss, G.E., Youngquist, R.S. and Garverick, H.A. 1991. Secretory patterns of LH and FSH during development and hypothalamic and hypophyseal characteristics following development of steroid induced ovarian follicular cysts in dairy cattle. J. Reprod. Fertil. 91:19-28. Ginther, O.J., Knopf, L. and Kastelic, P., 1989. Temporal associations among ovarian events in cattle during estrous cycles with two and three follicular waves. J. Reprod. Fertil. 87:223-230. Humphrey, N.D., Hopper, L.D., Clemente, P., Dunn, T.G. and Kaltenbach, C.C. 1977. Estrus and conception in heifers treated with norgestomet and estradiol valerate vs norgestomet alone. J. Anim. Sci., (Suppl 1) 45:357. Kharbanda, S.M., Band, V., Murugesan, K. and Farooq, A. 1990. Modulation of steroid production in goat ovarian cells: Effect of progestins and anti progestins. Endocrine Res. 16:293-309. Mikeska, J.C. and Williams, G.L. 1988. Timing of preovulatory endocrine events, estrus and ovulation in Brahman X Hereford females synchronized with norgestomet and estradiol valerate. J. Anim. Sci. 66:939-943. Pierson, R.A. and Ginther, O.J. 1987a. Follicular populations during the estrous cycle in heifers I: Influence of day. Anim. Reprod. Sci. 14:165-176. Pierson, R.A. and Ginther, O.J. 1987b. Follicular populations during the estrous cycle in heifers II: Influence of right and left sides and intraovarian effect of the corpus luteum. Anim. Reprod. Sci. 14:177-186. Rahe, C.H., Owens, R.E., Fleeger, J.L., Newton, H.J. and Harms, P.G. 1980. Pattern of plasma luteinizing hormone in the cyclic cow: dependence upon the period of the cycle. Endocrinology 107:498-503. Rajakoski, E. 1960. The ovarian follicular system in mature heifers with special reference to seasonal, cyclical and left-right variations. Acta Endocronol., (SuppL) 52:7-68.  58  Follicular dynamics and plasma LH with norgestomet Rajamahendran, R. and Walton, J.S. 1988. Follicular development and corpus luteum formation in postpartum dairy cattle. 11th Tnt. Cong. Anim. Repro. and Artif. Insem., 2:60-62. Rajamahendran, R., Robinson, 3., Desbottes, S. and Walton, J.S. 1989. Temporal relation ships among estrus, body temperature, milk yield, progesterone and luteinizing hormone levels and ovulation in dairy cows. Theriogenology 31:1173-1182. Rajamahendran, R. and Taylor, C. 1990. Characterization of ovarian activity in postpartum dairy cows using ultrasound imaging and progesterone profiles. Anim. Reprod. Sci. 22:171-180. Rajamahendran, R. and Taylor, C. 1991. Follicular dynamics and temporal relationships among body temperature, oestrus, the surge of luteinizing hormone and ovulation in Holstein heifers treated with norgestomet. I. Reprod. Fertil. 92:46 1-467.  Rentfrow, L.R., Randel, R.D. and Neuendorff, D.A. 1987. Effect of estrous synchronization with Syncro-Mate B on serum luteinizing hormone, progesterone and conception in Brahman heifers. Theriogenology 28:355-362. Rexroad, C.E. and Casida, L.E. 1977. Effect of injection of progesterone into one ovary of PMSG-treated anestrous ewes on follicle growth and ovarian estradiol-17J3. J. Anim. Sci. 44:84-88. Roberson, M.S., Wolfe, M.W., Stumpf, T.T., Kittok, R.J. and Kinder, J.E. 1989. Luteinizing hormone secretion and corpus luteum function in cows receiving two levels of progesterone. Biol. Reprod. 41:997-1003. Savio, J.D., Keenan, L. Boland, M.P. and Roche, J.F. 1988. Pattern of growth of dominant follicles during the oestrous cycle in heifers. 3. Reprod. Fertil. 83:663-671. Savio, J.D., Thatcher, W.W., Badinga, L. and de la Sota, R.L. 1990. Turnover of dominant ovarian follicles is regulated by progestins and dynamics of LH secretion in cattle. J. Reprod. Fertil. Abstract Series #6:23. Schreiber , J.R., Nakamura, K. and Erickson, G.F. 1981. Progestins inhibit FSH-stimulated granulosa estrogen production at a post-cAMP site. Mol. and Cell. Endocrinology 21:161-170.  59  Follicular dynamics and plasma LH with norgestomet Short, R.E., Bellows, R.A., Carr, J.B., Staigmiller, R.B. and Randel, R.D. 1976. Induced or synchronized puberty in heifers. J. Anim. Sci. 43:1254-1263. Sirois, I. and Fortune, I.E. 1988. Ovarian follicular dynamics during the estrous cycle in heifers monitored by ultrasonography. Biol. Reprod. 39:308-3 17. Sirois, I. and Fortune, I.E. 1990. Lengthening of the bovine estrous cycle with low levels of exogenous progesterone: A model for studying ovarian follicular dominance. Endocrinology 127:916-925. Statistical Analysis Institute, Inc., 1985. User’s Guide: Statistics, Version 5. SAS Institute Inc. Cary, N.C. Taylor, C. and Rajamahendran, R. 1991a. Follicular dynamics and corpus luteum growth and regression in lactating dairy cattle. Can. J. Anim. Sci. 71:61-68. Taylor, C. and Rajamahendran, R. 199 lb. Follicular dynamics and corpus luteum growth and function in pregnant vs non-pregnant dairy cows. I. Dairy. Sci. 74:115-123. Vostermans, J.N. and Walton, J.S. 1985. Effect of intermittent injections of gonadotropin releasing hormone and ovulation in dairy cows. Anim. Reprod. Sci. 8:335-347. Walters, D.L., Schams, D. and Schallenberger, E. 1984. Pulsatile secretion of gonado tropins, ovarian steroids and ovarian oxytocin during the luteal phase of the estrous cycle of the cow. J. Reprod. Fertil. 71:479-491. Wiltbank, J.N. and Gonzalez-Padilla, E. 1975. Synchronization and induction of estrus in heifers with a progestagen and estrogen. Ann. Biol. Anim. Biochem. Biophys. 15:255-262.  60  CHAPTER 3  THE EFFECT OF INCREASING PROGESTIN CONCENTRATION ON NORGESTOMET MAINTAINED DOMINANT FOLLICLES IN CATTLE  ABSTRACT Previous research has shown that a single norgestomet ear implant will maintain the dominant follicle present in cattle when implanted during the follicular phase. The aim of the present study was to determine if the addition of a progesterone releasing intravaginal device (PRID), or a second norgestomet ear implant would restore follicular turnover. Four cows were allocated in a crossover design to three different treatments administered during the follicular phase during three different cycles, each treatment cycle separated by two normal cycles. Treatments consisted of: 1) A single norgestomet ear implant (iN) for 9 days (day 0  =  day of implant, 2) addition of a PRID 3 days after a single norgestomet implant (NP),  and 3) addition of a second N implant 3 days after the first (2N). Treatments were terminated on day 9. Ultrasound examinations were carried out before, during and after treatments to monitor follicular dynamics. Serial blood samples were obtained at different times during treatment to characterize LH pulsatility. The dominant follicle was maintained for 9 days in all iN cows and ovulated following implant removal. In all NP and 2N treated cows, the dominant follicle regressed and a new pool of follicles appeared before the end of treatment. The dominant follicle from this new pool ovulated in all NP treated cows. The  61  Effect of increasing progestin on dominant follicles new dominant follicle in the 2N treated cows became cystic in three of four animals. A significant decrease (P < 0.05) in LH pulse frequency, amplitude and mean plasma LH concentration was observed in NP treated cows when compared to iN treated cows. Pulse amplitude and mean plasma LH concentration were also significantly lower in 2N treated cows compared to iN. The results demonstrate that higher concentrations of progestin cause regression of the dominant follicle in cattle and this is associated with a decrease in LH pulse frequency and amplitude.  INTRODUCTION  The wave-like pattern of follicular growth is well established in cattle and has been outlined in the previous chapter. Each wave of follicular growth gives rise to a single dominant follicle which may either regress or ovulate depending on the hormonal milieu. What causes non-ovulatory dominant follicles to become atretic and regress is unclear. Recent research has shown that the synthetic progestin norgestomet, when implanted for 9 days during the follicular phase, will induce the maintenance of the dominant follicle present for the entire 9 day treatment period (Rajamahendran et al., i989a; Rajamahendran and Taylor, 1991; Taylor et at., 1993). Maintenance of the dominant follicle would appear to be due to high-frequency, low-amplitude luteinizing hormone (LH) pulses (Taylor and Rajamahendran, 1991; Savio et al., 1992; Taylor et at., 1993). Maintenance of the first dominant follicle with low levels of progesterone (Pj, following induced luteolysis, or the dominant follicle present during the natural follicular phase, and regression of these follicles 62  Effect of increasing progestin on dominant follicles with high levels of P 4 have been reported by other researchers (Sirois and Fortune, 1990; Savio et aL, 1990). The above observations suggest that the increases in circulating P 4 may in some way be involved in the induction of atresia and regression of non-ovulatory dominant follicles. The objective of the present study was to use the norgestomet model of maintained dominant follicles to test the hypothesis that regression of dominant follicles is associated with increasing amounts of progestin.  MATERIALS AND METHODS  Animals and treatment Four regularly cycling dry cows from the University of British Columbia dairy herd were used in the present study. Cows received three different treatments, in a crossover design, over three estrous cycles separated by two normal cycles between each treatment. Treatments consisted of: 1) implantation of a single norgestomet (Sanofi Canada Inc., Victoriaville, Quebec, Canada) 9 day ear implant (iN) during the follicular phase (day 0  =  day of implant insertion); 2) implantation of a single norgestomet implant during the follicular phase followed by insertion of a single progesterone releasing intravaginal device (PRID; CEVA, Paris, France) 3 days following norgestomet implantation (NP); 3) implantation of a second norgestomet implant 3 days after receiving a single norgestomet implant during the follicular phase (2N).  63  Effect of increasing progestin on dominant follicles Ultrasound examination of the ovaries Ultrasound examinations of the ovaries were conducted as described in the previous chapter. Cows were examined daily from the day prior to the insertion of the first norgestomet implant until estrus and ovulation, or 10 days post-treatment, whichever came first. The ovaries were scanned in several planes to identify the dominant follicle and all other visible follicles. Appropriate images were arrested, structures measured and hard copies were made using a video processor (Mitsubishi Electronics Co. Ltd., Tokyo, Japan).  Blood sampling and hormone measurements Prior to each ultrasound examination on days 0, 3, 6, 9, 12 and 18 a 10 mL blood sample was collected via a coccygeal vein or artery in heparinized tubes (Vacutainer, Bectin-Dickinson, Rutherford, NJ, USA). All samples were centrifuged (400 X g) within 1 hour of collection and plasma was separated and frozen at -20° C until assayed for P . In 4 addition, jugular catheters were put in place for serial blood sampling on days 3 and 6 in all treated cows and on day 10 in two iN treated cows. During serial bleeding blood samples were collected into heparinized tubes at 15 minute intervals for 8 hours. Samples were immediately centrifuged and plasma stored at -20°C until assayed for LH. Plasma P 4 concentrations were determined using a commercially available solid-phase radioimmunoassay kit (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA, USA). The kit had previously been validated in our laboratory for measurement of P 4 in bovine milk and plasma (Rajamahendran et al., 1989b). Plasma LH concentrations were determined by 64  Effect of increasing progestin on dominant follicles radioimmunoassay as previously described (Vostermans and Walton, 1985). The assay standard for LH determination was NAIMMD-bLH-4 (2.2 X NIH- -LH-B1). All results were corrected to NIH-LH-B1. The inter- and intra-assay coefficients of variation were 11% and 8%, respectively, and the sensitivity was 0.1 ng/mL. Samples from cows in each treatment were analyzed within a single assay.  Detection of estrus and ovulation Cows were observed for signs of estrus four times daily following the end of treatments; at the morning and afternoon milkings (3:00 A.M. to 6:00 A.M. and 2:00 P.M. to 4:00 P.M.), during the mid morning while ultrasound examinations were being conducted (9:00 A.M. to 10:00 A.M.), and for 1/2 hour in the evening by a night watchperson (10:00 P.M. to 10:30 P.M.). Once a cow was observed in standing estrus ultrasound examinations were performed every 4 hours until the acute disappearance of the ovulatory follicle, which marked ovulation.  Statistical analyses Mean LH concentrations were calculated for each cow for each 8 hour sampling period. Luteinizing hormone pulses, pulse frequency and pulse amplitude were defined as in the previous chapter and as published (Taylor et al., 1993), and were compared between treatments by an analysis of variance. Follicle diameter and plasma P 4 concentrations were compared by an analysis of variance using General Linear Model procedures (SAS, 1985). 65  Effect of increasing progestin on dominant follicles The model tested the main effects of treatment and day, and the treatment by day interactions. Additionally, intervals between the termination of treatment and estrus and ovulation were compared between treatments by an analysis of variance.  RESULTS Follicular dynamics The mean diameter  (± SEM) of dominant follicle and number of subordinate follicles  (3 to 5mm in diameter) in iN, NP and 2N treated cows during and after treatment are shown in Figure 3.1. The dominant follicle was maintained for the entire 9 day treatment in all iN treated cows and ovulated upon implant removal. Uterine turgidity was noted upon rectal palpation during the ultrasound examinations throughout the treatment period. In all NP and 2N treated cows the dominant follicle was maintained until following insertion of either the PRID or the second norgestomet, after which the dominant follicle eventually regressed. Uterine turgidity gradually decreased during the treatment period. Coincident with the regression of the dominant follicle a new pool of follicles became apparent, giving rise to a new dominant follicle. This follicle ovulated after the termination of treatment in all NP cows. However, in the 2N treated cows, this new dominant follicle ovulated in only one cow. In the remaining three animals this new dominant follicle became cystic and persisted beyond 10 days after the termination of treatment. Mean (± SEM) diameter (mm) of dominant follicles on different days of treatment are shown in Table 3.1. 66  Effect of increasing progestin on dominant follicles a  Ovulation -,  20 1  Norgestomet  .4-  10  .4-  C  0 0  1  2  3  5  4  --  -=-  7  8  6  9  b.  10 11 12 Ovulation  _I—  :ç  ‘S  IS_ 4) 4)  •:  Norgestomet  C .44,)  4)  10  4) C  .4-  5  .4-  C  0 0 C 4)  •0 4) 4)  4) C) C  1  2  3  4  5  6  7  8  9  10 11 12 13  25 20 15  Cystic (n 3)  Norgestomet  10  5  .4.4-  o  0 0 1 2 3 4 5 6 7 8 9 1011121314151617 Day of treatment # sf  ——  df  —-  Newdf  Fig. 3.1. Mean (± SEM) dominant follicle diameter (dO and number of subordinate follicles (sf; 3-5mm) in heifers treated with a single 9 day norgestomet implant (a), norgestomet plus a progesterone releasing intravaginal device (b) or 2 norgestomet implants (c) following natural luteolysis. 67  Effect of increasing progestin on dominant follicles A significant decrease (P < 0.01) in dominant follicle diameter was observed in both NP and 2N treated cows on day 12, three days after implant removal.  Table 3.1.  Mean (± SEM) diameter of dominant follicles in cows treated with a single norgestomet (1W), ii) norgestomet plus a progesterone releasing intravaginal device (NP), and iii) double norgestomet (2N).  Day  iN (n=4)  NP (n=4)  2N (n=4)  0  19 5 + 0 5  19 8 + 0 28  19 3 + 1 38  3  6  20.0 ± 0.58 20.2 ± 0.48  21.3 ± 0.28 20.5 ± 0.38  21.3 ± 0.98 21.5 ± 1.3k  9  20.4 ± 0.3k  19.6 ± 0.7k  17.8 ± 1.3k  12  21.5 ± 0.8k  13.0 ± 1.1”  12.3 ± 1.2’  Treatment means with different superscripts within rows and columns differ (P < 0.01).  Estrus, ovulation and progesterone profiles There was no difference in the diameter of the ovulatory follicle in iN (maintained follicles) versus NP treated (new follicles) cows (Table 3.2). However, the mean interval to estrus and ovulation following the termination of treatment was significantly longer (P <  68  Effect of increasing progestin on dominant follicles 0.05) for NP treated cows (Table 3.2). As only one cow in the 2N treated group ovulated within 10 days of termination of treatment the 2N group was not considered in this analysis.  Table 3.2.  Mean (± SEM) ovulatory follicle size (mm) and mean (± SEM) interval to standing estrus and ovulation in cows treated with a single norgestomet (iN) or norgestomet plus a progesterone releasing intravaginal device (NP).  Parameter Ovulatory follicle diameter (mm) on day of ovulation  N (n=4)  NP (n=4)  20.5 ± 0.8a  20.3 ± 0.5k  Interval from termination of treatment to standing estrus (h)  75.2 ± 3•9a  90.0 ± 3.6b  Interval from termination of treatment to ovulation (h)  94.0 ± 9.0k  113.5 ± 7•3b  Treatment means with different superscripts within row a differ (P < 0.05).  Mean (± SEM) concentration of plasma P 4 was significantly higher (P < 0.05) on days 6 and 9 in NP treated cows compared to cows in the iN and 2N treatment groups (Table 3.3). All cows across all treatments had basal (< 1 ng/mL) concentrations of P 4 on day 12. Cows belonging to the iN group had the highest P 4 concentration on day 18 (Table  3.3).  69  Effect of increasing progestin on dominant follicles  Table 3.3.  Mean (± SEM) plasma progesterone (ng/mL) in cows treated with a single norgestomet (iN), norgestomet plus a progesterone releasing intravaginal device (NP) or double norgestomet (2N).  Day  iN (n = 4)  NP (n = 4)  2N (n = 4)  0  0.28 ± 0.05k 0.18 ± 0.02k  0.26 ± 0.04k 0.15 ± 0.02k  0.23 ± 0.06k 0.20 ± 0.03k  0.17 ± 0.Ola 0.21 ± 0.03k  2.13 ± 0.15” 1.11 ± 0.04l  0.20 ± 0.03k 0.19 ± 0.03k  0.18 ± 0.Ola 3.01 ± 0.23a  0.10 ±  3 6 9 12 18  o.oia  2.15 ± 0.11”  0.25 ± 0.01” 0.74 ± 0.40c  Treatment means with different superscripts within a row differ (P < 0.01).  Luteinizing hormone profiles Profiles of plasma LII over the 8 hour sampling period, on day 6 of treatment, for individual representative animals from each treatment group are shown in Figure 3.2. Mean concentration of LH, the LH pulse frequency and pulse amplitude on day 6 of treatment are shown in Table 3.4. Mean plasma LII concentration and pulse amplitude were greater (P <  0.05) in N treated cows than in the other two treatments. Mean plasma concentration of LH and amplitude of LH pulses did not differ (P > 0.05) between the NP and 2N groups. Pulse  70  Effect of increasing progestin on dominant follicles frequency was lower (P < 0.05) in NP treated cows than in N and 2N treated cows. Mean plasma LH concentration, pulse frequency and amplitude in iN, NP and 2N treated cows on day 3 of treatment, ie. before the addition of a PRID or second norgestomet, were similar to the values observed for iN treated cows on day 6. In two iN treated cows, where serial blood samples were collected 16 hours after termination of treatment, the LH pulse frequency increased to 7.0 ± 1.2 pulses per 8 hours. However, there were no changes in the mean LH concentration or the LII pulse amplitude when compared to day 6.  Table 3.4.  Mean (± SEM) plasma LH concentration, LH pulse frequency and pulse amplitude on day 6 of treatment in cows treated with a single norgestomet (iN), norgestomet plus a progesterone releasing intravaginal device (NP) or double norgestomet (2N).  iN (n=4)  NP (n=4)  2N (n=4)  Mean LH ng/mL  0.77 ± 0.29a  0.40 ± 0.09b  0.34 ± 0.16’  Frequency pulses/8 h  4.3 ± i.2  2.3 ± 0.6b  4.3 ± 0.6a  Amplitude ng/mL  0.71 ± 0.35a  0.16 ± 0.12b  0.39 ± 0.18’  Treatment means with different superscripts within a row differ (P < 0.01).  71  Effect of increasing progestin on dominant follicles  iN  I -J  0.5  0 0:00  1:00  2:00  3:00  4:00  5:00  8:00  7:00  8:00  7:00  8:00  Time (Hrs) 2  NP 1.5 1  E ‘S  C  I -J  0:00  1:00  2:00  3:00  4:00  5:00  6:00  Time (Hrs) 2  2N 1.5 J  E  ‘S C  I J  0.5  0  111111  0,00  1:00  I  2,00  1:111111111::  3,00  4,00  5:00  1111:11111  8:00  7:00  8:00  Time (Hrs) Fig. 3.2. Secretory profile of LH from representative animals treated with a single norgestomet (iN), norgestomet + PRID (NP), or 2 norgestomet (2N). 72  Effect of increasing progestin on dominant follicles DISCUSSiON Treatment of cows with a single norgestomet implant during the follicular phase resulted in the maintenance of the dominant follicle for the duration of treatment. All follicles ovulated following implant removal. This observation confirms earlier reports from our lab (Rajamahendran et aL, 1989a; Taylor and Rajamahendran, 1990; Rajamahendran and Taylor, 1991) as well as observations made by others following treatment resulting in low concentrations (1 to 2 ng/mL) of circulating P 4 (Sirois and Fortune, 1990). A recent study has demonstrated that a low P / progestin environment could maintain the first dominant 4 follicle, following induced luteolysis, beyond day 18 of the estrous cycle (Savio et al., 1992). In the present study the addition of a second norgestomet or PRID 3 days after the first norgestomet implant caused regression of the dominant follicle in all treated cows, confirming our hypothesis that increasing the concentration of circulating progestin would restore follicular turnover. Others (Sirois and Fortune, 1990; Bergfelt et aL, 1991) have also reported that follicular turnover is restored when high concentrations (4 to 6 ng/mL) of circulating P 4 are maintained during the follicular phase. These findings suggest that high circulating concentrations of progestins may initiate a cascade of events leading to the regression of dominant follicles, either by acting directly at the follicular level and/or indirectly, via regulation of LH release from the pituitary. The plasma LH profiles demonstrate that a single norgestomet implant maintains the high-frequency, low-amplitude LII pulse pattern normally associated with the follicular phase and blocks the very rapid high amplitude pulses normally seen during the ascending stage 73  Effect of increasing progestin on domini2nt follicles of the LII surge (Rahe et aL, 1980). Recent reports suggest that maintenance of dominant follicles is associated with high-frequency LII pulses (Savio et aL, 1990; Taylor and Rajamahendran, 1991; Taylor et aL, 1993). In the present study, addition of a PRID lead to a decrease in basal LH, decreased LH pulse frequency and pulse amplitude. Addition of a second norgestomet implant lead to a decrease in basal LH and a decrease in LH pulse amplitude. This is in agreement with previous work suggesting that increasing circulating P , 4 as seen during the luteal phase of the estrous cycle (Rahe et al., 1980), or with administration of exogenous progesterone (Ireland and Roche, 1982; Roberson et al., 1989), leads to a decrease in circulating LH and a decrease in the LH pulse frequency. Thus, it would appear that high-frequency, low-amplitude LII pulses will induce the maintenance of dominant follicles while a decrease in circulating LH is at least permissive to a normal pattern of follicular turnover. Alternatively, high circulating concentrations of progestin may have a direct effect on dominant follicle regression. Progestin binding sites have been reported in the ovary of the cow (Jacobs and Smith,  1980). Progestins have been shown to suppress estrogen  production by rat (Schreiber et al., 1981) and goat (Kharbanda et al. ,1990) granulosa cells, cultured in vitro, by inhibiting FSH induction of aromatase activity. Perhaps high concentrations of P 4 inhibit granulosa cell aromatase leading to the loss of estrogenic capacity and regression of the dominant follicle. The short interval between the termination of treatment and estrus and ovulation in cows treated with a  single norgestomet implant is likely explained by the advanced 74  Effect of increasing progestin on dominant follicles development of the dominant follicle. Short intervals between P 4 withdrawal and the LH surge have been reported following treatment with subnormal levels of P 4 (Roberson et aL, 1989) or with norgestomet implanted during the follicular phase  (Rajamahendran and  Taylor, 1991). The delay in ovulation in NP treated cows is probably due to the time required for recruitment and growth of a new dominant follicle. The tonicity of the uterus observed in 1N treated cows could be attributed to the normal follicular phase estrogen secretion from the dominant follicle (Badinga et at, 1992). The loss of tonicity following insertion of either a PRID or a second  norgestomet is suggestive of regression of the  dominant follicle and its inability to produce estrogen. It has been previously reported that the dominant follicle arising from a pool of follicles suppresses the growth of its cohorts and possibly causes atresia (Ireland and Roche, 1987; Ko et at, 1991). The presence of a dominant follicle at the time of initiation of superovulatory treatments has also been shown to decrease the ovulatory response (Guilbault et al., 1991; Huhtinen et al., 1992). In the present study, during the single norgestomet treatment there was no recruitment of new antral follicles during the time of dominant follicle maintenance, suggesting the suppressive activity of the dominant follicle. Addition of a PRID or second norgestomet lead to recruitment and growth of new antral follicles and regression of the former dominant follicle. Both estrogen and inhibin produced by the dominant follicle have been implicated in the suppressive effect (Kastelic et al., 1990; Goulding et al., 1991). Furthermore, it has been concluded that these inhibitory factors  75  Effect of increasing progestin on dominant follicles produced by the dominant follicle suppressed other follicular growth through systemic endocrine channels rather than through intraovarian paracrine effects (Ginther et aL, 1989). A single norgestomet implant in combination with estradiol valerate has been used for estrus synchronization in cattle (Wiltbank and Gonzalez-Padilla, 1975; Short et aL, 1976; Humphrey et al., 1977). The synchrony of estrus following the above treatment is high (Miksch et aL, 1978; Spitzer et al., 1978) however numerous studies have reported lower pregnancy rates (Rentfrow et al., 1987; Mikeska and Williams, 1988). Recently we (Rajamahendran and Taylor, 1991) have shown that there is synchrony between estrus, the LH surge and ovulation following norgestomet treatment. We suggested that decreased fertility with norgestomet may be due to the prolonged maintenance of the dominant follicle. Lowered pregnancy rates following maintenance of the first wave dominant follicle with low level progesterone has been reported (Savio et aL, 1992). This implies that high levels of progesterone must be maintained in a synchronization regime to insure follicular turnover and to obtain acceptable pregnancy rates. However, in the present study three of four 2N treated cows became cystic, suggesting that non synthetic P 4 may be more appropriate. In conclusion the present study has shown that treatment of cows during the fofficular phase with a single norgestomet implant induces the maintenance of dominant follicles, most likely due to persistent high frequency LH pulses. 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Turnover of dominant ovarian follicles is regulated by progestin and dynamics of LH secretion in cattle. J. Reprod. Fertil. Abstr. Series 6:23 Savio, J.D., Thatcher, W.W., Morris, G.D., Entwistle, K. and Drost, M. 1992. Terminal follicular development and fertility in cattle is regulated by concentration of plasma progesterone. In: 12th Tnt. Cong. Anim. Reprod. and Art. Insem. 2:999-1001. Savio, J.D., Thatcher, W.W., Badinga, L., de la Sota, R.L., and Wolfenson, D., 1993. Regulation of dominant follicle turnover during the oestrus cycle in cows. J. Reprod. Fertil. 97:197-203. Schreiber, J.R., Nakamura, K. and Erickson, G.F. 1981. Progestins inhibit FSH-stimulated granulosa estrogen production at a post cAMP site. Mol. Cell. Endocrinol. 21:161-170. Short, J.R.E., Bellow, R.A., Carr, J.B., Staigmiller, R.B. and Randall, R.D. 1976. Induced or synchronized puberty in heifers. J. Anim. Sci. 43:1254-1263. Sirois, J. and Fortune, J.E. 1990. Lengthening of the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance. Endo crinology 127:916-925. Spitzer, J.C., Jones, D.L., Miksch, E.D. and Wiltbank, J.N. 1978. Synchronization of estrus in beef cattle. III. Field trial in heifers using a norgestomet implant and injection of norgestomet and estradiol valerate. Theriogenology 10:223-229. Statistical Analysis Institute Inc. SAS 1985. User’s Guide Statistics, Version 5. SAS Institute Inc., Cary, NC. Taylor, C. and Rajamahendran, R. 1990. The effect of norgestomet on follicular dynamics in cycling heifers. Biol. Reprod. 42 (Suppi. 1):244. Taylor, C. and Rajamahendran, R. 1991. The effect of norgestomet and progesterone supple mentation on LH release and follicular dynamics in dairy cattle. Biol. Reprod. 44 (Suppl. 1):56.  79  Effect of increasing progestin on dominant follicles Taylor, C., Rajamahendran, R. and Walton, J.S. 1993. Ovarian follicular dynamics and plasma luteinizing hormone concentrations in norgestomet-treated heifers. Anim. Reprod. Sci. 32: 173-184. Vorstermans, J.N. and Walton, J.S. 1985. Effect of intermittent injections of gonadotrophin releasing hormone and ovulation in dairy cows. Anim. Reprod. Sci. 8:335-347. Wiltbank, J.N. and Gonzalez-Padilla, E. 1975. Synchronization and induction of estrus in heifers with a progestagen and estrogen. Annal. Biol. Anim. Biochem. Biophys. 15:225-262.  80  CHAPTER 4  EFFECT OF EXOGENOUS PROGESTERONE ON THE OVULATORY CAPACITY OF THE FIRST WAVE DOMINANT FOLLICLE IN CATTLE  ABSTRA CT  A wave-like pattern of follicular growth and regression has been described during the luteal phase in the bovine. The factors responsible for inducing the onset of regression of non ovulatory dominant follicles are unknown. The present study was designed to examine the effect of progesterone (P ) administration early in the estrous cycle on the first wave 4 dominant follicle. Nine heifers were administered P 4 on day 3 (200 mg), day 4 (100 mg) and day 5 (50 mg) of the estrous cycle, in addition seven heifers received vehicle to serve as controls. All heifers received a luteolytic dose of prostaglandin F j on day 7. 2 a (PGF 2 Follicular dynamics were monitored by daily ultrasonography. All 7 control heifers ovulated the first wave dominant follicle. Four of nine P 4 treated heifers ovulated a second wave dominant follicle. In these heifers regression of the first wave dominant follicle began prior to PGF a administration on day 7. The remaining five P 2 4 treated heifers ovulated the first wave dominant follicle following 2 4 treated heifers that ovulated the first wave PGF a . In the P dominant follicle, growth of the first dominant follicle was slowed (0.65 ± 0.13 mm/day between days 3 and 7 for treated vs 1.46 ± 0.23 mm/day for control; P < 0.05) and estrus  81  Effect of exogenous P 4 in the early estrous cycle and ovulation were delayed compared to controls (3.8 ± 0.3 vs 2.4 ± 0.2 and 5.2 ± 0.4 vs 3.9 ± 0.2 days after 2 PGF a , respectively; P < 0.05). The results indicate that P 4 administered early in the estrous cycle to mimic the mid luteal phase alters follicular dynamics and is capable of inducing premature regression of the first wave dominant follicle.  INTRODUCTION The wave-like pattern of antral follicular growth in the bovine has been well established (see Lucy et al., 1992 for review). A pool of small follicles becomes apparent around the time of estrus, there is a period of growth, at which time a single follicle becomes dominant and continues growing while the remainder become atretic and regress. This first wave dominant follicle normally enters a static phase of no growth at about day 7 to 8 of the cycle and then itself becomes atretic and begins to regress between day 11 and 13 to be replaced by a second wave of follicular growth. The majority of estrous cycles are characterized by two or three waves of follicular growth, with the dominant follicle at the time of luteolysis becoming the ovulatory follicle. What regulates this pattern and initiates the regression of non-ovulatory dominant follicles is not clear. The ability to manipulate and alter the normal pattern of follicular growth may lead to a better understanding of its regulation. The synthetic progestin norgestomet has been shown to induce maintenance of dominant follicles in the absence of a corpus luteum (CL) (Rajamahendran et al., 1989; Savio et al., 1990a; Rajamahendran and Taylor, 1991). The maintenance of dominant follicles in this model would appear to be due to a persistent 82  Effect of exogenous P 4 in the early estrous cycle high-frequency, low-amplitude luteinizing hormone (LH) pulse pattern (Savio et at, 1990a; Taylor and Rajamahendran, 1991a; Taylor et at, 1993). This has been supported by studies using various doses of exogenous progesterone (P ) administered during the follicular phase 4 (Sirois and Fortune, 1990; Savio et at, 1992) to either maintain the dominant follicle (circulating doses of P 4 < 2 ng/mL) or initiate growth of subsequent follicular waves (circulating doses of 3 to 5 ng/mL of P ). 4 The dominant follicle of the first wave of follicular growth has been shown to be capable of ovulation when prostaglandin F a2 2 (PGF a ) is administered on day 7 of the estrous cycle (Kastelic et at, 1990; Savio et al., 1990b). In addition, administration of hCG on day 7 induces ovulation of the first dominant follicle in the presence of a CL (Rajamahendran and Sianangama, 1992). The aim of the present experiment was to attempt to use ovulation of the first dominant follicle after PGF2a administration on day 7 as a functional end point to determine if P 4 administered early in the estrous cycle could induce the premature regression of the first wave dominant follicle, resulting in ovulation of a second wave dominant follicle.  MATERIALS AND METHODS  Animals Sixteen heifers between 12 and 14 months of age and having exhibited at least one normal length estrous cycle (18 to 22 days) were selected from the University of British Columbia dairy herd. Heifers were then selected at random to serve either as control (n 83  =  Effect of exogenous P 4 in the early estrous cycle 7) or treated (n  =  9) animals. Both control and treated heifers were then synchronized with  a (25mg Lutalyse, Upjohn Co., Kalamazoo, MI). Heifers were observed for signs of 2 PGF estrus during the two milking periods (3:00 A.M. to 6:00 A.M. and 2:30 P.M. to 5:30 P.M.) and for one hour during the mid morning (9:00 A.M. to 10:00 A.M.) while the ovaries of heifers were examined by ultrasonography.  Progesterone treatment Heifers in the treated group were injected intramuscularly with 4 P (Sigma, St. Louis, MO) dissolved in corn oil (20 mg/mL) on day 3 (200 mg), day 4 (100 mg) and day 5 (50 mg) of the treatment cycle (day 0  =  estrus). Control heifers received vehicle only. All  heifers were administered 25 mg PGF2a intramuscularly on day 7 of the treatment cycle.  Ultrasound examination The ovaries of all heifers were examined daily by ultrasound imaging from the onset of the PGF a synchronized estrus until ovulation of the treatment cycle. Ultrasound 2 examinations were conducted as described by Rajamahendran and Taylor (1990) using a linear array ultrasound scanner (Tokyo Keiki LS 300, Tokyo Keiki Co. Ltd., Tokyo, Japan) equipped with a five MHz rectal transducer. The ovaries were scanned in several planes to identify all visible follicles and the CL. The largest and second largest follicles and the CL were measured using built-in calipers. Permanent records were made using a video processor (Mitsubishi Electronics Co. Ltd., Tokyo, Japan). 84  Effect of exogenous P 4 in the early estrous cycle Blood collection and steroid assays Blood samples were collected from a tail vein prior to each ultrasound examination. Samples were centrifuged and the plasma was collected and stored at -20°C until analysis for P . Plasma P 4 4 concentrations were determined using a solid phase radioimmunoassay (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA). This system was previously validated in our laboratory for measurement of P 4 in bovine milk and plasma (Rajamahendran et al., 1989). All plasma samples from individual heifers were analysed within the same assay, with heifers from each treatment group represented within each assay. The inter- and intra-assay coefficients of variation were 7% and 5%, respectively, and the sensitivity was 0.05 ng/mL.  Statistical analyses Comparisons between control and treated heifers with regard to diameter of the largest follicle present on the ovaries and profiles of plasma P 4 were made by an analysis of variance for repeated measures (SAS Inc., 1985). The model tested the main effects of day, treatment and the day by treatment interaction. The difference between dominant follicle diameter on day 3 and day 7 devided by the number of days (ie. 4) was used to determine the rate of growth of the dominant follicle. Mean dominant follicle growth rate and the interval in days from PGF a administration to basal P 2 4 (< 1 nglmL), estrus, ovulation, and the interval between the return of 4 P to basal concentrations and day of ovulation were compared using an analysis of variance. 85  Effect of exogenous P 4 in the early estrous cycle RESULTS Plasma P 4 concentration increased in treated heifers starting on day 4 of the treatment cycle, attained mid luteal phase concentrations of 5 to 6 ng/mL by day 5, plateaued between day 5 and 7 and sharply declined following PGF a administration on day 7 (Fig. 4.1). In 2 control heifers plasma P 4 increased gradually between days 3 and 7, reached maximum concentrations of 2 to 3 ng/mL and declined rapidly following PGF a administration. There 2 was no difference between control and treated heifers (P > 0.05) in the interval between 4 returning to basal concentrations. Additionally, treatment had a administration and P 2 PGF no effect on CL growth or regression (Fig. 4.1). All control heifers ovulated the first wave dominant follicle following PGF 2 administration on day 7. In four of nine P 4 treated heifers a follicle from a second wave of follicular growth ovulated. In these four heifers the first wave dominant follicle reached its maximum diameter on days 4 to 5 and began to regress by day 6. A new wave of follicular growth was apparent by day 6, and this second wave gave rise to the ovulatory follicle. The remaining five P 4 treated heifers ovulated the first wave dominant follicle (Fig. 4.2). Even among P 4 treated heifers that ovulated the first wave dominant follicle there were some significant differences from control heifers; The first dominant follicle grew at a slower rate in treated heifers (0.65 ± 0.13 mm/day for treated vs 1.46 ± 0.23 mm/day for control). Also the interval from PGF a administration to both estrus and ovulation was longer (P < 2 0.05) in treated heifers (Table 4.1; Fig. 4.2). Most interestingly, when data for treated animals was analysed on the basis of the number of follicular waves, it became apparent that 86  Effect of exogenous P 4 in the early estrous cycle  10  30  CL  8  S E  LU  6  E ..-  E  0 1) cl,j  4.  i1I  0  /  2  /  \ \  _I_  10  P4  0  U .  0 0  r,)  I  I  I  I  1  2  3  4  5  I  I  I  6  7  8  Days After Estrus (0  =  0 9  10  estrus)  Fig. 4.1. Mean (± SEM) plasma P 4 and CL diameter in control (_ ) and P 4 treated (___) heifers. Progesterone injections were given on days 3, 4 and 5 and PGF 2 on day 7. —  87  11  Effect of exogenous P 4 in the early estrous cycle  1  . 2 PGF  ovulation  n=7  I  ovulation  =s  ovulation  —  I  n  E  C)  0  5  0 0  1  2  3  4  5  6  7  Days After Estrus (0  8 =  9  10  estrus)  Fig. 4.2. Growth, regression/ovulation of dominant follicles in control (_) and P 4 treated (___ first wave dominant follicle ovulated; second wave dominant follicle ovulated) heifers. PGF a given on day 7. 2  88  11  12  13  Effect of exogenous P 4 in the early estrous cycle heifers with two waves of growth had significantly higher circulating P 4 on days 4, 5, 6 and 7 than treated heifers with a single wave of follicular growth (Fig. 4.3).  Table 4.1.  Mean interval (± SEM) in days from PGF 4 (< 1 a administration to basal P 2 ng/mL), estrus and ovulation in heifers treated with P 4 on days 3, 4 and 5 and a on day 7 (day 0 = estrus). 2 PGF  N  Basal P 4  Control  7  1.0 ± 0.2k  Treated 1 wave  5  Treated 2 waves  4  Estrus  Ovulation  2.4 ± 0.2’  3.9 ± 0.3a  1.4 ± 0.4a  3.8 ± 0.3”  5.2 ± 0.4b  1.8 ± 0.3k  4.8 ± 0.3c  5.8 ± 0.3b  Different superscripts within columns denote differences (P < 0.05).  89  Effect of exogenous P 4 in the early estrous cycle  10  30  8  T  CL  20  .1  6  E E  0  LI)  4 •10  4 P  0  2  :1  0 0  I  I  1  2  I  I  I  I  4 3 5 6 Days After Estrus (0  =  I  I  7  8  0  9  10  estrus)  Fig. 4.3. Plasma P 4 and CL diameter (± SEM) in heifers treated with P 4 on days 3 to 5 of the estrous cycle and administered PGF a on day 7 2 (——- treated-one wave of follicular growth; treated-two waves of follicular growth).  90  11  Effect of exogenous P 4 in the early estrous cycle DiSCUSSION In the present study exogenous P 4 administered during the early luteal phase in an attempt to mimic the mid luteal phase with regard to circulating P 4 concentrations. Administration of P 4 was initiated on day 3 to insure that a dominant follicle had been selected. Treatment was terminated on day 5 to insure that all exogenous P 4 had been cleared from the circulaion by the time of luteolysis following PGF 2 administration on day 7. The pooied plasma P 4 profiles from all treated heifers indicate that treatment resulted in elevation of P 4 concentrations to mid luteal phase levels of 5 to 6 ng/mL by day 5 of the treatment cycle, leveled off until PGF a administration on day 7 and then declined rapidly. Plasma P 2 4 concentrations do not normally reach their maximum before day 10, and then plateau between 4 and 6 ng/mL (Taylor and Rajamahendran, 1991b). The decreasing dose of P 4 employed in the treatment would therefore appear to have been effective in mimicking the mid luteal phase. The dominant follicle of the first wave of follicular growth in the bovine has been shown to be capable of ovulation at a very high frequency when luteolysis is initiated on day 7 of the estrous cycle (Kastelic et al., 1990; Savio et al., 1990b). The present study attempted to use this as an end point to determine if P 4 administered early in the cycle could initiate the early regression or atresia of the first wave dominant follicle. The result of premature atresia would be the loss of the ovulatory capacity of the first wave dominant follicle and subsequent growth and ovulation of a second wave dominant follicle.  91  Effect of exogenous P 4 in the early estrous cycle Four of nine heifers treated with P 4 ovulated a second wave dominant follicle. We are confident that this is an effect of treatment. The first wave dominant follicle had begun to decrease in diameter in these heifers prior to PGF a admicnistration on day 7 and a 2 second wave of follicular growth was already apparent. Typically, the first wave dominant follicle attains its maximum diameter between day 7 to 8, enters a static phase until approximately day 11 and then begins to regress (Ginther et. al, 1989; Taylor and Rajamahendran, 1991c). This premature regression of the first wave dominant follicle in four of the treated heifers is in agreement with a recent report showing 4 P administered on day  o through 5  of the estrous cycle had a suppressive effect on the growth of the first wave  dominant follicle (Adams et al., 1992). The effects of P 4 on dominant follicles could be at two levels; via the hypothalamic-pituitary axis or directly on the follicle itself. Evidence to suggest that P 4 is acting via the hypothalamic-pituitary axis comes from studies with exogenous progestins administered during the follicular phase. Dominant follicles have been maintained in the absence of a CL using synthetic progestins (Rajamahendran et al., 1989; Savio et al., 1990a; Rajamahendran and Taylor, 1991a) and low doses of circulating P 4 (Sirois and Fortune, 1990; Savio et al., 1992). It has been established that treatments which induce the maintenance of dominant follicles result in a persistent high-frequency, low-amplitude LH pulse pattern (Roberson et al., 1989; Savio et al., 1990a; Taylor and Rajamahendran, 1991a; Taylor et al., 1993). Mid luteal phase levels of circulating P 4 reestablish the wave-like growth of antral follicles (Sirois and Fortune, 1990; Savio et al., 1992) and result in low 92  Effect of exogenous P 4 in the early estrous cycle frequency LH pulses (Roberson et aL, 1989; Savio et aL, 1992). This has led Savio et al. (1993) to hypothesize that low-frequency LH pulses characteristic of the mid luteal phase fail to support thecal androgen production, thus impairing granulosa cell function and leading to atresia of the dominant follicle and a new wave of follicular growth. It has not been conclusively demonstrated that P 4 has any effect on FSH secretion in cattle (Ireland and Roche, 1982; Bolt et aL, 1990; Adams et aL, 1992). However, Adams et al. (1992) have recently shown that P 4 administered early in the estrous cycle hastens the second FSH surge, presumably due to the removal of inhibin suppression on FSH with the early regression of the first wave dominant follicle. Alternatively, or coincidently, P 4 may be acting directly at the level of the follicle. Progesterone receptors have been isolated in the bovine ovary (Jacobs and Smith, 1980) and may be induced by increasing estrogen (Nardulli et aL, 1988) during follicular growth. Progesterone has been shown to suppress estradiol-173 synthesis by granulosa cells (Saidapur and Greenwald, 1979; Schreiber et. al, 1981; Fortune and Vincent, 1983), therefore high concentrations of 4 P during the mid luteal phase may suppress granulosa cell estradiol synthesis leading to atresia of the dominant follicle and a new wave of follicular growth. Five treated heifers displayed a single wave of follicular growth. A within treatment analysis of plasma P 4 demonstrated that heifers with a single wave of growth had significantly lower circulating P 4 than treated heifers that displayed two waves of growth. This may suggest a threshold at which 4 P is capable of inducing follicular turnover. Treated heifers that ovulated the first dominant follicle showed other significant effects of treatment; 93  Effect of exogenous P 4 in the early estrous cycle the dominant follicle grew at a slower rate and both estrus and ovulation were delayed in comparison to controls. There was no difference between treated and control heifers with regard to the day on which P 4 fell below 1 ng/mL. This suggests that the delay in ovulation was not due to high circulating P 4 following 2 PGF , , on day 7 but more likely due to a delay in follicular maturation. In summary, the present study suggests that high circulating concentrations of P early in the estrous cycle can alter follicular dynamics, in some instances inducing the premature regression of the first wave dominant follicle.  REFERENCES Adams, G.P., Matteri, R.L. and Ginther, O.J., 1992. Effect of progesterone on ovarian follicles, emergence of follicular waves and circulating follicle-stimulating hormone in heifers. J. Reprod. Fert. 95:667-640. Bolt, D.J., Scott, V. and Kiracofe, G.H., 1990. Plasma LH and FSH after estradiol, norgestomet and Gn-RH treatment in ovariectomized beef heifers. Anim. Reprod. Sci. 23:263-271. Fortune, J.E. and Vincent, S.E. 1983. Progesterone inhibits the production of aromatase activity in rat granulosa cells in vitro. Biol. Reprod. 28:1078-1089. Ginther, O.J., Knopf, L. and Kastelic, P. 1989. Temporal associations among ovarian events in cattle during estrous cycles with two and three follicular waves. J. Reprod. Fertil. 87:223-230. Ireland, J.J. and Roche, J.F., 1982. Effect of progesterone on basal LH and episodic LH and FSH secretion in heifers. J. Reprod. Fertil. 64:295-302. Jacobs, B.R. and Smith, R.G., 1980. Evidence for a receptor like protein for progesterone in bovine ovarian cytosol. Endocrinology 106:1226-1230.  94  Effect of exogenous P 4 in the early estrous cycle Kastelic, J.D., Knopf, L. and Ginther, O.J., 1990. Effect of day of prostaglandin F2 alpha treatment on selection and development of the ovulatory follicle in heifers. Anim. Reprod. Sci. 23:169-180. Lucy, M.C., Savio, J.D., Badinga, L., delaSota, R.L. and Thatcher, W.W., 1992. Factors that affect follicular dynamics in cattle. 3. Anim. Sci. 70:3615-3626. Nardulli, A.M., Greene, G.L., O’Malley, B.W. and Katzenellenbogen, B.S., 1988. Regulation of progesterone receptor messenger ribonucleic acid and protein levels in MCF-7 cells by estradiol: analysis of estrogens effect on progesterone receptor synthesis and degradation. Endocrinology 122:935-944. Rajamahendran, R., Robinson, J., Desbottes, S. and Walton, J.S., 1989. Temporal relation ships among estrus, body temperature, milk yield, progesterone and luteinizing hormone levels and ovulation in dairy cows. Theriogenology, 31:1173-1182. Rajamahendran, R., Eide, A., Robinson, J., Taylor, C. and Walton, J.S., 1989. Effect of norgestomet on follicular dynamics, corpus luteum growth , progesterone, LH, oestrus and ovulation in cycling heifers. J. Anim. Sci. 67 (Suppl. 1):383. Rajamahendran, R. and Taylor, C., 1990. Characterization of ovarian activity in postpartum dairy cows using ultrasound imaging and progesterone profiles. Anim. Reprod. Sci. 22: 171-180. Rajamahendran, R. and Taylor, C., 1991. Follicular dynamics and temporal relationships among body temperature, oestms, the surge of luteinizing hormone and ovulation in Holstein heifers treated with norgestomet. J. Reprod. Fertil. 92:46 1-467. Rajamahendran, R. and Sianangama, P.C., 1992. Effect of human chorionic gonadotrophin on dominant follicles in cows: formation of accessory corpora lutea, progesterone production and pregnancy rates. 3. Reprod. Fertil. 95: 577-584. Roberson, M.S., Wolfe, M.W., Stumpf, T.T., Kittok, R.J. and Kinder, J.E., 1989. Luteinizing hormone secretion and corpus luteum function in cows receiving two levels of progesterone. Riol. Reprod. 4 1:997-1003. Saidapur, S.K. and Greenwald, J.S. 1979. Regulation of 17/3-estradiol synthesis in the proestrus hamster: Role of progesterone and luteinizing hormone. Endocrinology 105:1432-1439.  95  Effect of exogenous 4 P in the early estrous cycle Savio, J.D., Thatcher, W.W., Badinga, L. and de la Sota, R.L., 1990a. Turnover of dominant ovarian follicles as regulated by progestins and dynamics of LH secretion in cattle. I. Reprod. Fertil. Abstr. Ser. 6:23. Savio, J.D., Boland, M.P., Hynes, N., Mattiacci, M.R. and Roche, J.F., 1990b. Will the first dominant follicle of the estrous cycle ovulate following luteolysis on day 7? Theriogenology 33:677-688. Savio, J.D., Thatcher, W.W., Morris, G.D., Entwistle, K. and Drost, M., 1992. Terminal follicular development and fertility in cattle is regulated by plasma progesterone. In: 12th International Congress of Animal Reproduction and Artificial Insemination 2:999-1001. Savio, J.D., Thatcher, W.W., Badinga, L., de la Sota, R.L. and Wolfenson, D., 1993. Regulation of dominant follicle turnover during the oestrous cycle in cows. I. Reprod. Fertil. 97:197-203. Schreiber, J.R., Nakanura, K., and Erikson, G.G. 1981. Progestins inhibit FSH-stimulated granulosa estrogen production at a post cAMP site. Mol. Cell. Endocrinology 21:161170. Sirois, J. and Fortune, J.E. 1990. Lengthening of the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance. Endocrinology 127:916-925. Statistical Analysis System Institute, Inc. 1985. User’s Guide: Statistics, Version 5. SAS Institute Inc. Cary, NC. Taylor, C. and Rajamahendran, R., 1991a. The effect of norgestomet and progesterone supplementation on LH release and follicular dynamics in dairy cattle. Biol. Reprod. 44 (Suppl. #1): Abstract #56. Taylor, C. and Rajamahendran, R., 199 lb. Follicular dynamics and corpus luteum growth and function in pregnant vs non pregnant dairy cows. J. Dairy. Sci., 74:115-123. Taylor, C. and Rajamahendran, R., 1991c. Follicular dynamics, corpus luteum growth and regression in lactating dairy cattle. Can. J. Anim. Sci. 71:61-68. Taylor, C., Rajamahendran, R. and Walton, J.S. 1993. Ovarian follicular dynamics and plasma luteinizing hormone concentrations in norgestomet treated heifers. Anim. Reprod. Sci. 32:173-184. 96  CHAPTER 5  EFFECT OF EXOGENOUS GnRH ON LH PULSATILITY AND FOLLICULAR DYNAMICS DURING THE LUTEAL PHASE IN CATTLE  ABSTRACT  In an effort to determine the effect of high-frequency LH pulses on follicular dynamics in an environment of high circulating progesterone, three heifers were treated with high-frequency GnRH pulses between days 7 and 17 of the estrous cycle (day 0  =  day of  estrus). Three other heifers received saline pulses to serve as controls. Serial blood samples were collected on day 12 to determine LH pulsatility and ovaries were examined daily by ultrasonography to determine follicular dynamics. Treatment had no effect on any of the parameters examined. Day of onset of regression of the first wave dominant follicle, emergence of the second wave of follicular growth and onset of regression of the CL were similar in control and treated heifers. Mean plasma LH during the sampling period, LH pulse frequency and pulse amplitude were also not different between treated and control heifers. The failure of treatment to alter LH pulsatility render the remainder of the results inconclusive at best. Future trials with either a different delivery system for GnRH or the use of LH may lead to better success.  97  Exogenous GnRH and follicular dynamics INTRODUCTION Previous research in our lab  (Rajamahendran et aL,  1989;  Taylor and  Rajamahendran, 1991a; Taylor et aL, 1993) and others (Savio et al., 1990; Sirois and Fortune, 1990; Savio et at, 1992) has shown that low concentrations of circulating progestins will induce the maintenance of dominant follicles in the bovine. The evidence suggests that dominant follicle maintenance is the result of persistent high-frequency, low-amplitude luteinizing hormone (LII) pulses (Savio et at,  1990; Taylor and  Rajamahendran, 1991a; Savio et at, 1992; Savio et al., 1993; Taylor et at, 1993). With increasing concentrations of progestin, LH pulsatility decreases and follicular turnover is restored. Thus it would appear that high-frequency LH pulses induce the maintenance of dominant follicles, whereas low-frequency LH pulses, or low circulating LH due to high circulating progesterone 4 (P ) , is at least permissive for normal follicular turnover. However, one cannot determine from the above results whether dominant follicle regression is due to the decrease in circulating LH or due to a possible direct effect of high concentrations of P 4 acting at the level of the ovary. Progestin binding sites have been isolated in the ovary (Jacobs and Smith, 1990) and P 4 has been shown to suppress FSH induction of aromatase activity in cultured granulosa cells (Schreiber et at, 1981; Kharbanda et al., 1990). The objective of the present study was to attempt to induce high-frequency LH pulses, with exogenous gonadotrophin-releasing hormone (GnRH), during the mid luteal phase and determine the effects on follicular dynamics.  98  Exogenous GnRH and follicular dynamics MATERIALS AND METHODS  Animals Six heifers between 14 and 18 months of age and having exhibited at least two normal length estrous cycles were used for this study.  GnRR treatment GnRH pulses were delivered to three heifers by loading a length of PE 60 tubing with intermittent sequences of air and human GnRH (Sigma, St. Louis, MO) designed to deliver 5 g pulses over 15 minutes every one to two hours for 10 days. Catheters were attached to mini-osmotic pumps (Aizet osmotic pumps, 2ML4, Aiza, Palo Alto, CA) and the entire unit was implanted in a subcutaneous pouch made in the neck of each heifer. The free end of the catheter was inserted into the jugular vein for delivery of GnRH directly into the blood stream. Pumps and catheters were implanted on day 6 or 7 of the estrous cycle (day 0  =  estrus) and removed between 15 and 20 days later. The remaining three heifers were implanted with pumps and catheters loaded with saline to serve as controls. A pump and catheter unit was tested in physiological saline in vitro.  Ultrasound imaging Ovaries were examined daily by ultrasound imaging, as previously described (Taylor and Rajamahendran, 1991b), in all heifers from the time of estrus at the commencement of the treatment cycle until the subsequent estrus. 99  Exogenous GnRFJ and follicular dynamics  Blood sampling On day 12 heifers were fitted with a jugular catheter on the contralateral side to the osmotic pump. A 7 mL blood sample was collected into heparinized tubes (Vacutainer, Becton-Dickinson, Missisauga, Ont.) every 15 minutes for 8 hours. Samples were centrifuged (400 X g) immediately, and plasma separated and stored at 200 C until analysis for LH. In addition, blood samples were collected from a coccygeal artery or vein prior to each ultrasound examination for determination of plasma P . 4 Plasma LH was determined by a double antibody radioimmunoassay in the laboratory of Dr. Lee Sanford (Sanford, 1987). All samples from all heifers were analysed in a single assay. The intra-assay coefficient of variation was less than 13%. The minimum detectable concentration was  0.20  ng/mL.  Plasma P 4 was  determined by a solid phase  radioimmunoassay as described in previous chapters. All samples were analysed within a single assay. The Intra-assay coefficient of variation was 6% and the limit of sensitivity was 0.05 ng/mL.  Statistical analyses Differences between control and treated groups with regard to follicular and CL dynamics were analysed by comparing the means of individual characteristics of the estrous cycle defined below, using a General Linear Model analysis of variance (SAS Inc., 1985). Characteristic days of each cycle were determined retrospectively and defined as follows: day 100  Exogenous GnRH and follicular dynamics of emergence of a second wave of follicles was defined as the day on which a pooi of individually identifiable follicles 2 to 5 mm in diameter was first observed. The day of emergence of the dominant follicle was defined as the day on which a single follicle from the original pooi achieved a diameter 2 standard deviations greater than the mean of its cohorts. Day of onset of regression of dominant follicles and the CL was defined as the first of two consecutive days of decrease with a continued decrease in diameter thereafter. Plasma 4 profiles were compared using an analysis of variance for repeated measures. P Mean plasma LH concentrations were calculated for each cow for each 8 hour sampling period. Luteinizing hormone pulses, pulse frequency and pulse amplitude were defined as in Chapter 2 and as published (Taylor et aL, 1993), and were compared between treatments by an analysis of variance.  RESULTS Follicular dynamics Follicular dynamics were similar for both control and treated heifers (Table 5.1). There was no difference with regard to the maximum diameter or the onset of regression of the first wave dominant follicle. Emergence of the second wave of follicular growth was also similar for both control and treated heifers. In addition, there was no difference in the diameter of the corpus luteum, the day of onset of luteal regression, or return to estrus (Table 5.2).  101  Exogenous GnRR and follicular dynamics  Table 5.1.  Maximum diameter, onset of first dominant follicle regression and emergence of the second follicular wave in heifers treated with GnRH or saline (day 0 estrus).  Control (n = 3) Treated (n = 3)  Maximum diameter (m)  Onset of regression (day)  Day of emergence (day)  16.0 ± 0.& 16.6 ± 0.9a  12.0 ± 0.6a  11.3 ± 0.3k 11.6 ± 0.6  12.3 ± 0.Y  Means (± SEM) with similar superscripts were not different (P > 0.05).  Table 5.2.  Maximum luteal diameter, onset of luteal regression and return to estrus in heifers treated with GnRH or saline (day 0 = day of estrus at beginning of treatment cycle).  Maximum diameter (mm)  Control (n = 3) Treated (n = 3)  Onset of regression (day)  27.3 ± 0.3k 27.6 ± 0.7a  Return to estrus (day)  16.6 ± 0.7 16.3 ± 0.3  Means (± SEM) with similar superscripts were not different (P > 0.05).  102  19.3 ± 0.3k 19.6 ± 0.6  Exogenous GnRH and follicular dynamics  Hormone profiles Treatment with high frequency GnRH pulses had no effect on P 4 or LH pulsatility (Table 5.3). LH pulse frequency, pulse amplitude and mean plasma LH were similar for both control and treated heifers. One treated heifer did have 4 LH pulses over the 8 hour sampling period (Fig. 5.1).  Table 5.3.  Mean plasma LH and LH pulse frequency and pulse amplitude in heifers treated with GnRH or saline.  Mean LH (nglmL)  Pulse frequency (pulses/8h)  Pulse amplitude (ng/mL)  2.7 ± 0.88 1.7 ± 0.3k  0.72 ± 0.078 0.42 ± 0.148  Control (n  =  3)  0.45 ± O.198  Treated (n  =  3)  0.40 ± 0.l6  Means (± SEM) with similar superscripts were not different (P > 0.05).  DISCUSSION Previous research has demonstrated that in a low progestin environment, high frequency LH pulses will induce the maintenance of dominant follicles (Savio et al., 1990; Taylor and Rajamahendran, 1991a; Savio et al., 1992; Savio et al., 1993; Taylor et al., 1993). In the present study an attempt was made to treat heifers with high-frequency GnRH pulses during the luteal phase in an effort to generate high-frequency LH pulses. The aim 103  Exogenous GnRR and follicular dynamics  a 1.6  1.2  :  0.8  55 0.4  th  ‘  -D  ‘El  0  i  I  0:00  I  I  2:00  1:00  I  I  I  I  4:00  3:00  5:00  6:00  7:00  8:00  6:00  7:00  8:00  Time (hrs)  b 1.6  1.2 -J  E 0, C  0.8  u  0.4  1 o 0:00  I  I  1:00  I  I  I  2:00  I  I  I  3:00  I  I  I  I  I  4:00  5:00  Time (hrs)  Fig. 5.1.  Luteinizing hormone profiles in a representative control heifer (a) and a GnRH treated heifer (b).  104  Exogenous GnRH and follicular dynamics was to determine if high-frequency LII pulses could maintain dominant follicles in an environment of high circulating P . 4 Treatment of heifers with GnRH had no effect on any of the responses examined.  Most importantly treatment did not appear to affect LH pulse frequency or pulse amplitude. There could be several explanations for this apparent failure of response to GnRH treatment. There may have been desensitization of pituitary gonadotrophs due to prolonged GnRH exposure (deKoning et aL, 1978; Smith and Vale, 1981; Smith and Conn, 1983). In addition, 4 has been shown to decrease GnRH receptor number in cultured ovine pituitary cells (Laws P et al., 1990). Finally there is the possibility that GnRH was not delivered as designed, unfortunately plasma GnRH was not determined. Examination of catheters at the termination of the trial did not provide information as to the delivery of the GnRH. Several catheters broke or became kinked and damaged during withdrawl. Examination of the pumps revealed that approximately half of the reservoir had been expelled suggesting that the pumps had been functional. Despite this, one heifer did have four LH pulses during the 8 hour sampling period and had a normal pattern of follicular turnover. This approaches the pulse frequency that has been reported to induce the maintenance of dominant follicles (Savio et al., 1992; Taylor et al., 1993). If follicular turnover can be demonstrated in a high progesterone, high LH pulse frequency environment this would demonstrate a possible direct P 4 effect on follicular dynamics. However, in a similar study (Glencross, 1987) GnRH pulses were infused every hour during the luteal phase and a period of artificially elevated 4 P Estradiol-1713 levels . 105  Exogenous GnRH and follicular dynamics approached preovulatory concentrations and were maintained for the duration of treatment. This data suggests that high frequency LH pulses can overcome a high P 4 environment and induce the maintenance of dominant follicles. To overcome possible desensitizing effects of prolonged GnRH treatment on pituitary gonadotrophs, future studies should possibly utilize LH, although the cost may be prohibitive. Also a different delivery system may yield more certain results. The advantage of the mini osmotic pumps is that animals do not have to be restrained for the entire treatment, thus decreasing animal stress. In conclusion, the attempt to treat heifers during the luteal phase with high frequency GnRH pulses had no effect on LH or follicular dynamics. The lack of response to GnRH with regard to LH calls into question whether GnRH was delivered as designed and render the results on follicular dynamics inconclusive.  REFERENCES deKoning, J.A., van Dietan, M.J. and van Rees, G.P. 1978. Refractoriness of the pituitary gland after continuous exposure to luteinizing hormone releasing hormone. J. Endocrinol. 79:311-318. Glencross, R.G. 1987. Effect of pulsatile infusion of gonadotrophin-releasing hormone on plasma estradiol-17f3 concentrations and follicular development during naturally and artificially maintained high levels of plasma progesterone in heifers. J. Endocrinol. 112:77-85. Jacobs, B.R. and Smith, R. G. 1990. Evidence for a receptor-like protein for progesterone in bovine ovarian cytosol. Endocrinology 106:1226-1230.  106  Exogenous GnRH and follicular dynamics Kharbanda, S.M. Band, V., Murugesan, K. and Farooq, A. 1990. Modulation of steroid production in goat ovarian cells. Effects of progestins and anti progestins. Endocrine Res. 16:293-309. Laws, S.C., Beggs, M.J., Webster, J.C. and Miller, W.L. 1990. Inhibin increases and progesterone decreases receptors for gonadotropin-releasing hormone in ovine pituitary culture. Endocrinology 127:373-380. Rajamahendran , R., Eide, A., Robinson, J., Taylor, C. and Walton, J.S. 1989. Effect of norgestomet on follicular dynamics, corpus luteum growth, progesterone, LH, estrus and ovulation in cycling heifers. J. Anim. Sci. 67 (Suppl. 1):388. Sanford, L.M. 1987. Luteinizing hormone release in intact and castrate rams is altered with immunoneutralization of endogenous estradiol. Can. 3. Physiol. Pharmacol. 65: 1442-1447. Savio, J.D., Thatcher, W.W., Badinga, L. and de la Sota, R.L. 1990. Turnover of dominant ovarian follicles is regulated by progestin and dynamics of LH secretion in cattle. J. Reprod. Fertil. Abstr. Series 6:23 Savio, J.D., Thatcher, W.W., Morris, G.D., Entwistle, K. and Drost, M. 1992. Terminal follicular development and fertility in cattle is regulated by concentration of plasma progesterone. In: 12th International Congress of Animal Reproduction and Artificial Insemination. 2:999-1001. Savio, J.D., Thatcher, W.W., Badinga, L., de la Sota, R.L. and Wolfenson, D., 1993. Regulation of dominant follicle turnover during the oestrous cycle in cows. 3. Reprod. Fertil. 97:197-203. Schreiber, 3. R., Nakamura, K. and Erickson, G. F. 1981. Progestins inhibit FSH-stimulated granulosa estrogen production at a post cAMP site. Mol. Cell. Endocrinol. 21:161-170. Sirois, 3. and Fortune, J.E. 1990. Lengthening of the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance. Endocrinology 127:916-925. Smith, M.A. and Vale, W. 1981. Desensitization to gonadotropin-releasing hormone in superfused pituitary cells on cytodex beads. Endocrinology 108:752-759.  107  Exogenous GnRH and follicular dynamics Smith, W.A. and Conn, P.M. 1983. GnRH-mediated desensitization of the pituitary gonado trope is not calcium dependent. Endocrinology 112:408-410. Statistical Analysis System Institute Inc., 1985. SAS User’s Guide Statistics, Version 5. SAS Institute Inc. Cary, NC. Taylor, C. and Rajamahendran, R. 1991a. The effect of norgestomet and progesterone supplementation on LH release and follicular dynamics in dairy cattle. Biol. Reprod. 44 (Suppi. 1):56. Taylor, C. and Rajamahendran, R. 199 lb. Follicular dynamics, corpus luteum growth and regression in lactating dairy cattle. Can. J. Anim. Sci. 71:61-68. Taylor, C., Rajamahendran, R. and Walton, J.S. 1993. Ovarian follicular dynamics and plasma luteinizing hormone concentrations in norgestomet-treated heifers. Anim. Reprod. Sci. 32: 173-184.  108  CHAPTER 6  SERUM FREE CULTURE SYSTEM FOR BOVINE GRANULOSA CELLS: THE EFFECTS OF FSH AND FORSKOLIN  ABSTRACT  A serum free culture system for bovine granulosa cells was developed in order to examine the effects of FSH on steroid production. Granulosa cells were harvested from large (15 to 20 mm) antral follicles and plated either directly on plastic or on an entactin collagen-IV-laminin extracellular matrix. Media were supplemented with insulin, transferrin and sodium selenite. Addition of FSH (0.5 to 100 ng/mL) caused a dose dependent decrease in estradiol-17f3 (E ) secretion after 24 hours in culture and a dose dependent increase in 2 progesterone (P ) and testosterone (T) secretion. Production of F 4 2 was maintained or increased over time in controls and cultures supplemented with low concentrations (0.5 to 5 ng/mL) of FSH but decreased dramatically in cultures treated with high concentrations (20 and 100 ng/mL) of FSH. Treatment with forskolin mimicked the effects of FSH. Cell attachment was greater when cells were cultured on matrix compared to cells plated directly on plastic. Production of E 2 and P 4 was greater on the 3rd and 4th day of culture when cells were plated on matrix. However T production was greater in cultures plated directly on plastic. The results suggest that high concentrations of FSH induce functional luteinization of bovine granulosa cells. Therefore, to maintain F 2 secretion over a long period of time for  109  Granulosa cell culture study of the regulation of F 2 production, only low concentrations of FSH (< 5.0 ng/mL) should be used.  iNTRODUCTION The previous four chapters examined the role of progesterone (P ) and luteinizing 4 hormone (LH) in regulating fofficular dynamics. While most of the evidence suggests that 4 acts via its regulation of LH to modulate follicular turnover, a direct effect at the level P of the ovary cannot be discounted. To this end, in vitro studies to examine regulation of steroidogenesis by ovarian cells become necessary. An insight into the regulation of estrogen production by the ovary is important for understanding the process of atresia of antral fofficles, differentiation of growing follicles and luteinization following ovulation. The conversion of androgens to estrogens is accomplished by the enzyme complex cytochrome P450 aromatase (see Richards and Hedin, 1988 for review). In the ovary this conversion takes place primarily, if not exclusively, in the granulosa cells. The regulation of aromatase and estrogen production is not well understood. The bovine ovary may provide an excellent model for studying steroidogenesis; it is a single ovulating species and follicles grow to a size (20 to 30mm) large enough to provide sufficient cells from a single follicle for culture studies. Most in vitro studies involving the culture of bovine granulosa cells have used P 4 production as an end point for the regulation of steroidogenesis. However, P 4 and estradiol-l7( (F ) production are probably regulated 2 differentially and appear to be inversely related (Skinner and Osteen, 1988). The study of 110  Granulosa cell culture 2 production by granulosa cells is made difficult by the apparent spontaneous loss of F estrogenic capacity of the cells when cultured in vitro in serum free systems (Fortune and Hansel, 1979; Henderson and Moon, 1979; Skinner and Osteen, 1988; Langhout et aL, 1990). There are few reports of maintained F 2 production in long term (>12 hours) granulosa cell cultures. One such report showed that high levels of follicle stimulating hormone (FSH) decreased E 2 production (Saumande, 1991) and that granulosa cells cultured on a matrix with insulin and low concentrations of FSH (< 5 ng/mL) could remain estrogenic for several days. However, only E 2 production on the last day of culture and not changes in production over time were reported. The aim of the present study was to determine the effect of FSH on cultured bovine granulosa cells in a serum free culture system and to examine changes in steroidogenesis over time.  MATERiALS AND METHODS  Media Medium for all cultures consisted of DMEM/Ham’ s F 12 (1:1; Terry Fox Laboratory, Vancouver, B.C.) supplemented with penicillin (100 U/mL), streptomycin (100 gImL), insulin-transferrin-sodium selenite (5 g/mL, 5 g/mL and 5 ng/mL, respectively; Sigma, St. Louis, MO) and androstenedione (106 M; Sigma, St. Louis, MO) as an aromatizable  111  Granulosa cell culture substrate. Porcine FSH (Sigma, St. Louis, MO) or forskolin (Sigma, St. Louis, MO) were added to cultures as described below.  Granulosa cell harvest and culture Bovine ovaries were collected from a local slaughter-house and transported to the lab in physiologic saline (39°C). Granulosa cells were harvested from visually healthy (well vascularized follicle wall, translucent follicular fluid) 15 to 20 mm follicles. The follicular fluid was aspirated and stored at -20°C until analyzed for steroid content. A small incision was made in the follicle wall with a scalpel blade. Media was flushed in and out of the follicle repeatedly and the interior of the walls were scraped gently with an inoculating loop. Collected cells were washed two times in fresh media, centrifuged (250 x g) and resuspended in media for seeding. Granulosa cells were seeded at 2 x 10 cells per mL either directly on plastic (24 well Falcon culture plates, Becton Dickinson, Mississauga, Ont.) or in wells coated with an entactin-collagen IV-laminin attachment matrix (ECL; approximately 6 pg/well, Promega, Madison, WI). Cultures were treated with FSH (0.5 to 100 ng/mL) or forskolin (0.5 to 10 .LM). Controls received vehicle only. Cultures were maintained for 72 to 96 hours at 39°C 1 in a water saturated atmosphere of 5 % CO 2 and 95 % air. Media was replaced every 24 hours with fresh media and cultures were visually inspected using an incubated phase contrast inverted microscope (Nikon, Diaphot II, Nikon Co. Ltd., Tokyo, Japan). Conditioned medium was stored at -20°C until analyzed for steroid content. 112  Granulosa cell culture Numbers of viable granulosa cells were determined at the end of the culture period by a trypan blue exclusion test. Cells were detached from culture wells with 0.5 mL of media containing 0.25% trypsin (wt:vol) and incubated at 30°C with gentle agitation for 30 minutes. After collecting, cells were washed in fresh media and resuspended in media containing 0.2% trypan blue. Cells were counted on a haemocytometer under light microscopy.  Steroid assays Media recovered from cultures was analyzed for E P and testosterone (T) content , 4 2 by commercially available solid phase radioimmunnoassay kits (Coat-A-Count, Diagnostics Products Corp., Los Angeles, CA). Aliquots for the E 2 determination were diluted 10 times. Fresh media spiked with known concentrations of steroids showed approximately 96% recovery. Serial dilutions showed parallelism. All samples for a given experiment were analyzed within a single assay. The intra-assay coefficients of variation were 6% for F, 7% 4 and 7% for T. The sensitivities were 10 pg/mL for E for P , 0.05 ng/mL for P 2 4 and 0.05 ng/mL for T.  Statistical analyses Only results from cultures obtained from estrogen active follicles (follicular fluid ] > 4 2 [F [P ] ) were analyzed (see Table 6.1). Steroid concentrations were transformed to log concentrations to obtain homogeneity of variance (Steel and Torrie, 1960). The experimental 113  Granulosa cell culture data are presented as the mean ± SEM log steroid concentration from quadruplicate wells within an experiment consisting of cultures derived from a single follicle. Each experiment was repeated three times. In addition, the fold change in steroid production compared to the first 24 hours of culture was calculated using the non transformed real number steroid concentrations for each individual well. Mean changes were then calculated for the four replicate wells within treatment. Treatment effects were analyzed by a two way analysis of variance with specific differences between treatments determined using Fisher’s least significant difference test.  RESULTS Follicular fluid E 2 and P 4 concentrations were determined to identify estrogen active ] > 4 2 ([E [P ] ) and estrogen inactive follicles ([EJ < [Pj; Table 6.1). Steroid accumulation in media of cultures derived from estrogen active follicles are presented  Table 6.1.  Estradiol (E ), progesterone (P 2 ) and testosterone (T) concentrations in 4 follicular fluid of representative estrogen active and estrogen inactive follicles.  Follicle  2 (nM) E  4 (nM) P  T (nM)  Fl F2 F3 F4  0.4 78.7 67.7 4.7  3 494.6 16.2 22.3 140.5  12.5 79.7 11.5 10.3  114  Granulosa cell culture Over the first 24 hours of culture there was no difference in steroid accumulation in culture media between control cultures and cultures receiving FSH. After the first 24 hours there was a dose dependent decrease in E 2 accumulation in response to FSH (Fig. 6. la). Conversely, there was a dose dependent increase in P 4 and T accumulation (Fig. 6. lb and c). A marked increase in accumulation of E 2 over time was observed in controls and cultures supplemented with low concentrations ( 5.0 ng/mL) of FSH (Fig. 6.2a). High concentrations of FSH (> 5.0 ng/mL) lead to a decrease in 2 E accumulation over time. Accumulation of P 4 in culture media increased over time in all cultures, however, there was a much greater fold increase in P 4 accumulation in cultures supplemented with high concentrations of FSH (Fig. 6.2b). A similar but much less dramatic pattern was observed for T accumulation (Fig 6.2c). Granulosa cells treated with forskolin displayed a similar response to cells treated with FSH. Low concentrations of forskolin (0., 0.1 and 0.5 M) had no effect on E 2 production over time, whereas higher concentrations (2.0 and 10 M) lead to a significant decrease in F 2 after the first 24 hours of culture and large increases in P 4 accumulation (Fig. 6.3a and b). Granulosa cells cultured directly on plastic and cells cultured on ECL showed no significant difference in steroid accumulation in culture media for the first 2 days of culture. By the third day cells cultured on ECL produced significantly more F 2 and P 4 than cells cultured on plastic while T was greater in cultures plated directly on plastic (Fig. 6.4a to c).  115  Granulosa cell culture  5 a F-’  \  ‘S \ 03  b.  —  4  ‘-I r  -E--I  -  3  I  I  I  I  I  I  I  I  20 100 C  .5  I  I  4 C  2  i  C 0.5  I  1  5  I  I  20 100 C  .5  1  5  I  I  I  I  1  5  20 100  FSH (ngmL) — —  1st 24 hrs  —  —  2nd 24 hrs  —  -  3rd 24 hrs  Fig 6.1. Mean (+ SEM) estradiol (a), progesterone (b) and testosterone (c) accumulation in media from bovine granulosa cell cultures in response to FSH. 116  Granulosa cell culture  a  Jc Jc  b  I  a  C  a  a  b  6 C  Q  ab 3  ab ab  I..  0 0  C  2  e I  S 0  btà  C C  ‘0  0 0  2nd 24 Hrs  C  3rd 24 Hrs Culture period  0.5  5  20  100  FSH (ng/mL)  Fig. 6.2. Mean (+ SEM) fold change in estradiol (a), pro gesterone (b) and testosterone (C) production by bovine granulosa cells in response to FSH.  117  Granulosa cell culture  a 5.  — -  -  —  —--  —.  -.—-  ..— -  —  I..  o  3  2  I  C  .1  .5  2  I  10  C  .1  .5  I  I  I  I  2  10  C  .1  .5  2  b r  / /7 3-  2  I  C  .1.5  2  10  C  I  I  .1.5  I  2  10  C  I  .1.5  210  Forskolin (uM) -  -  1st 24 hrs  2nd 24 hrs  -  3rd 24 hrs  Fig. 6.3. Mean (± SEM) estradiol (a) and progesterone (b) accumulation in media from bovine granulosa cell cultures in response to forskolin. 118  10  Granulosa cell culture  a 4.50  4.00  8 DO  I_t. I  3.50  ‘r  i  I:  Gd  N DO 0  3.00  2.50  ‘1  2.00  b  4.50  t. .r  4.00  T’  a  E 013  .1 3.50  ,r  t  1.  ±.  ‘  :  I-  Gd Gd 00 0  I  I.  3.00  2.50  2.00  C 4.50  4.00  I  -  E  3.50  x.  C.  F. 013 0  3.00  2.50  lst24hrs  3rd24brs  4th24hzs  2.00  C  .5  1  5  20100C  .5  1  520100 C 0.5  1  520100  FSH (ng/mL) —  —  ECL  Plastic  Fig. 6.4. Mean (i- SEM) estradiol (a), progesterone (b) and testosterone (c) accumulation in media from bovine granulosa cell cultures plated either on plastic or matrix (ECL). 119  Granulosa cell culture Cell viability was unaffected by treatment (Table 6.2). At the termination of the culture period trypan blue exclusion suggested that approximately 65 % of the original plated cells remained viable across all treatments. Attachment, as assessed by daily inspection under phase contrast microscopy, appeared greater in cultures plated on ECL compared to cells plated directly on plastic, this did not appear to have an effect on viability.  Table 6.2.  Viable granulosa cells at termination of a 72 hour culture period when plated directly on plastic or on an extracellular matrix (ECL) expressed as a percent of the initial plating density (2 X i0 cells/mL)’.  FSH treatment (ng/mL) 0.00 Plastic ECL  65 ± 3 66±4  0.5 67 ± 4 65±6  1.0  5.0  20  100  64 ± 2 71±3  64 ± 3 68±5  66 ± 4 69±5  67 ± 3 70±4  1 M ean ± SEM of four replicate wells in a single representative experiment  DiSCUSSION The aim of the present study was to use a serum free culture system to determine the effects of FSH on bovine granulosa cell steroidogenesis over a long term culture period. Most previous research using bovine granulosa cells have used either P 4 production as an end point or have involved short term cultures. These conditions are inadequate for studying the  120  Granulosa cell culture regulation of E 2 production. Agonists or antagonists which induce a chain of events leading to an alteration in steroid production may not show their effect in 2 or 3 hour cultures. The results of the present study show that granulosa cells cultured in serum free medium supplemented with insulin are capable of remaining estrogenic with little or no FSH. This confirms a previous study by Saumande (1991) using highly purified porcine FSH. However, that study reported steroid production only for the final 24 hours of a 4 day culture period. Our results demonstrate a change in cell response over time. During the first 24 hours of culture, increasing concentrations of FSH had no significant effect on steroidogenesis. However, after the first 24 hours of culture FSH had a dose dependent negative effect on F 2 and a positive effect on P 4 and T accumulation in the culture media. The delay in the negative response of granulosa cells to FSH with regard to 2 F production may reflect the relatively long half-life of the aromatase enzyme complex. It has been demonstrated in rat granulosa cells that a decline in aromatase mRNA was not associated with an immediate loss of aromatase activity (Fitzpatrick and Richards, 1991) or enzyme protein (Wong et al., 1989). The binding of FSH to its receptor leads to increased cAMP levels in granulosa cells (Erickson and Ryan, 1975; Richards et al., 1979). In the present study, bovine granulosa cells treated with forskolin, an activator of the adenylate cyclase catalytic subunit, responded in a similar manner to those treated with FSH, increased concentrations of forskolin lead to a decrease in accumulation of E 2 in the culture media and an increase in P . This suggests 4  121  Granulosa cell culture that even at very high concentrations, the adenylate cyclase-cAMP second messenger system remains the main signal transduction pathway for FSH. Granulosa cells cultured directly on plastic and cells cultured on an extracellular matrix showed no difference in steroidogenesis during the first 48 hours of culture. Following the first 48 hours, concentrations of E 2 and 4 P were generally greater from cultures of cells plated on ECL. This is in agreement with Morley et al. (1987) using rat granulosa cells but at odds with other reports that rat (Orly et aL, 1980) and bovine (Saumande, 1991) granulosa cells plated on fibronectin as an attachment matrix have increased E 2 but decreased P 4 production over cells plated on plastic. Interestingly, levels of T were greater in cultures plated directly on plastic. This argues against a difference in cell number as the explanation for the differences. Although cell attachment was greater in cultures plated on ECL there appeared to be little or no loss of cells during media changes as assessed by visual inspection in cultures plated on plastic, or at the end of the culture period as assessed by trypan blue exclusion and cell counting. The increased T may reflect an increase inapoptosis in cultures plated on plastic. The 17-ketosteroid reductase enzyme that converts androstenedione to testosterone has been shown to be constitutive in rat granulosa cells (Bogovich and Richards, 1984). Although neither protein nor DNA assays to quantify cell number at the end of the culture period were attempted, cell quantification and viability by trypan blue exclusion suggest that treatment had no differential effect on cell viability. We are confident that the results cannot be attributed to changes in viable cell number with increasing concentrations 122  Granulosa cell culture of either FSH or forskolin. While E 2 accumulation decreased, P 4 increased dramatically with increasing concentrations of FSH. Similarly, the increases in the rate of steroid accumulation in the culture media cannot be explained by increasing cell number. The fold increases in 2 and P E 4 production over the culture period are of too great a magnitude to be explained by cell doubling. Both Skinner and Osteen (1988) and Saumande (1991) reported 25 to 30 percent loss in viable cells over their culture periods, which is in line with the present results. Previous research has shown an additive effect of insulin and FSH (Skinner and Osteen, 1988; Saumande, 1991). Although in the present study, cells were not cultured without insulin supplementation, addition of FSH had no stimulatory effect over cultures with insulin alone until the fourth day of culture (Fig 6.4a). Saumande (1991) reported a stimulatory effect of low concentrations of FSH over cultures supplemented with insulin alone after 4 days of culture. Skinner and Osteen (1988) reported an additive effect of 100 ng/mL FSH and insulin. However they were unable to maintain E 2 production beyond the first 24 hours of culture. Why low concentrations of FSH (1 ng/mL) stimulate granulosa cells greater than insulin alone only after several days in culture may be due to a slight increase in viability with FSH. Although we were unable to demonstrate any significant change in viability with treatment the magnitude of the increase in E 2 accumulation with 1 ng/mL FSH was relatively small. Saumande (1991) did demonstrate increased viability with FSH over controls, thus a  123  Granulosa cell culture small undetected change in viability may explain the apparent FSH stimulation by day 4 of culture. In summary, a serum free culture system for bovine granulosa cells that will maintain 2 production in the absence of FSH over time has been developed. Addition of high E concentrations (> 5 ng/mL) of FSH inhibits F production and stimulates production of P . 4  REFERENCES Bogovich, K. and Richards, J.S., 1984. Androgen synthesis during ovarian follicular development: evidence that rat granulosa 17-ketosteroid reductase is independent of hormonal regulation. Biol. Reprod. 31:122-131. Erickson, G.F. and Ryan, K.J. 1975. The effect of LH/FSH, dibutyryl cyclic AMP, and prostaglandins on the production of estrogens by rabbit granulosa cells in vitro. Endocrinology 97:108-113. Fitzpatrick, S. L. and Richards, J. S. 1991. Regulation of cytochrome P450 aromatase mRNA and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452-1462. Fortune, J.E. and Hansel, W. 1979. The effects of 17f3-estradiol on progesterone secretion by bovine theca and granulosa cells. Endocrinology 104:1834-1838. Henderson, K.M. and Moon, Y.S. 1979. Luteinization of bovine granulosa cells and corpus luteum formation associated with loss of androgen aromatizing ability. 3. Reprod. Fertil. 56:89-95. Langhout, D.J., Spicer, L.J. and Geisert, R.D. 1990. Development of a culture system for bovine granulosa cells: effects of growth hormone, estradiol, and gonadotropins on cell proliferation, steroidogenesis, and protein synthesis. 3. Anim. Sci. 69:3321-3334. Orley, J., Sato, G. and Erickson, G.F. 1980. Serum suppresses the expression of hormonally induced functions in cultured granulosa cells. Cell 20:817-827.  124  Granulosa cell culture Morley, P., Armstrong, D.T. and Gore-Langton, R.E. 1987. Fibronecton stimulates growth but not follicle-stimulating hormone-dependent differentiation of rat granulosa cells cultured in vitro. J. Cell. Physiol. 132:226-236. Richards, J.S., Jonassen, J.A., Rolfes, A.I., Kersey, K., Reichert Jr., L.E. 1979. Adenosine 3’,5’-monophosphate, luteinizing hormone receptor, and progesterone during granulosa cell differentiation: effects of estradiol and follicle-stimulating hormone. Endocrinology 104:765-774. Richards, J.S. and Hedin, L. 1988. Molecular aspects of hormone action in ovarian follicular development, ovulation and luteinization. Ann. Rev. Physiol. 50:44 1-463. Saumande, 1. 1991. Culture of bovine granulosa cells in a chemically defined serum-free medium: the effect of insulin and fibronectin on the response to FSH. J. Steroid. Biochem. Molec. Bio. 38:189-196. Skinner, M.K. and Osteen, K.G. 1988. Developmental and hormonal regulation of bovine granulosa cell function in the preovulatory follicle. Endocrinology 1668-1675. Steel, R.G.D. and Torrie, J.H. 1960. Principles and Procedures of Statistics. McGraw Hill, New York. Wong, W.Y.L., DeWitt, D.L., Smith, W.L. and Richards, J.S. 1989. Rapid induction of prostaglandin endoperoxide synthase in rat preovulatory follicles by luteinizing hormone and cAMP is blocked by inhibitors of transcription and translation. Mol. Endocrinol. 3:1714-1723.  125  CHAPTER 7  EFFECT OF PROGESTERONE ON STEROID PRODUCTION BY BOVINE GRANULOSA CELLS COLLECTED FROM DAY 7 DOMINANT FOLLICLES AND CULTURED IN VITRO  ABSTRA CT Researchers have demonstrated the ability of progesterone to modulate the turnover of dominant follicles in the bovine. This study was conducted to determine whether there is a direct effect of progesterone at the level of the granulosa cell in suppressing estrogen production. Granulosa cells harvested from day 7 (day 0  =  estrus) first wave dominant  follicles were cultured without (control) or with (10 to iO M) progesterone for 48 hours. Treatment with progesterone failed to suppress accumulation of estradiol-173 in the culture media at any of the tested concentrations. In fact, treatment with iO M to iO M progesterone enhanced estradiol-1713 accumulation compared to controls. In the final 24 hours of culture more estradiol was accumulated in the culture media than in the initial 24 hours (1.27 fold increase for controls) in cultures treated with progesterone (lOs to iO M) showing a greater increase over time (up to 1.87 fold increase). A similar pattern was observed for progesterone accumulation. The results demonstrate that progesterone does not suppress estradiol secretion by bovine granulosa cells from estrogen active dominant follicles and may in fact stimulate steroid secretion in general.  126  Effect ofprogesterone on granulosa cell estrogen production IIVTROD UCTION The previous chapter describes the development of a serum free culture system capable of maintaining estrogen production by bovine granulosa cells. With this system, it becomes possible to challenge granulosa cells with various factors and determine the effect of those factors on steroid production. There is some suggestion in the literature that progesterone acts at the level of the follicle to suppress the follicle-stimulating hormone (FSH) induction of aromatase activity in rat (Schreiber et al., 1981) and goat (Kharbanda et aL, 1990) granulosa cells. In vivo research suggests a role for progesterone 4 (P in regulating the turnover of non-ovulatory ) dominant follicles in the bovine (see chapters 2 to 5). Although the research suggests that the mechanism is probably via regulation of luteinizing hormone (LH), a direct action at the level of the ovary has not been ruled out. The first wave dominant follicle achieves its maximum diameter on day 7 of the estrous cycle (Taylor and Rajamahendran, 1991). Day 7 is also approximately the time when the dominant follicle is secreting its highest levels of estrogens. Thus it was decided to use granulosa cells collected from day 7 dominant follicles to determine the effect of P 4 on steroid production by bovine granulosa cells cultured in vitro.  127  Effect ofprogesterone on granulosa cell estrogen production MATERIALS AND METHODS Animals The ovaries of cows scheduled to be culled were examined by ultrasound imaging to determine the presence of a corpus luteum. Once a corpus luteum was confirmed cows were administered two injections of prostaglandin F 2 (15 mg PGF , Lutalyse, Upjohn Co., 2 Kalamazoo, MI), intramuscularly twelve hours apart. Cows were examined daily thereafter until ovulation was confirmed. Cows were sent for slaughter at a local abattoir on day 7 of the induced estrous cycle (day 0  =  estrus). The ovaries were collected and transported to the  laboratory in physiologic saline at 37°C.  Cell cultures Granulosa cells were harvested and cultured as described in the previous chapter. Briefly, granulosa cells were harvested from the day 7 dominant follicles, washed and cultured in 1 mL (2 X lO cells/mL) DMEM/Ham’s F12 (1:1; Terry Fox Laboratory, Vancouver, B.C.). Media was supplemented with penicillin (100 U/mL), streptomycin (100 g/mL), 1 ng/mL porcine FSH (Sigma, St. Louis, MO), insulin-transferrin-sodium selenite (5 ig/mL, 5 1 Lg/mL and 5 ng/mL, respectively; Sigma, St. Louis, MO) and androstenedione (106  M; Sigma, St. Louis, MO) as an aromatizable substrate. Cultures were maintained at  39°C in a water saturated 5% CO 2  -  95% air atmosphere.  128  Effect ofprogesterone on granulosa cell estrogen production Treatments Cells were cultured for an initial 24 hours with no treatment. After the first 24 hours, media was replaced and P 4 added to cultures (10 to i0 M in triplicate wells) for 48 hours. The range of treatment concentrations was based upon in vivo circulating P 4 concentrations (!0 M) and the highest concentrations seen in follicular fluid (10.6 M). Three wells never exposed to P 4 served as controls. Finally, after 48 hours of exposure to P , cells were washed 4 two times and cultured for a final 24 hours without any P . In addition, a group of wells were 4 left unseeded to serve as blanks. The blanks were exposed to P 4 treatments to determine the residual P 4 from the treatments (ie. P 4 derived from the treatments, not secreted by granulosa cells). Numbers of viable granulosa cells were determined at the end of the culture period by a trypan blue exclusion test. Cells were detached from wells with 0.5 mL of media containing 0.25% trypsin (wt:vol) and incubated at 30°C with gentle agitation for 30 minutes. Cells were collected, washed in fresh media and resuspended in media containing 0.2% trypan blue. Cells were counted on a haemocytometer under light microscopy.  Steroid assays Media from the first 24 hours and the final 24 hours was collected, stored at -20°C and analyzed for estradiol- l73 (E ) and P 2 4 by commercially available solid phase radioimmunnoassay kits (Coat-A-Count, Diagnostics Products Corp., Los Angeles, CA). Aliquots for the E 2 determination were diluted 10 times. All samples for a given experiment 129  Effect ofprogesterone on granulosa cell estrogen production were analyzed within a single assay. The intra-assay coefficients of variation were 8% for 2 and 6% for P E . The sensitivities were 10 pg/mL and 0.05 ng/mL for F 4 2 and P , 4 respectively.  Statistical analyses Steroid concentrations were transformed to log concentrations to obtain homogeneity of variance. The experimental data are presented as the mean ± SEM log steroid concentration from triplicate wells within an experiment. Each experiment was repeated three times using cells harvested from 3 different follicles. Mean F 2 and P 4 production for each 24 hour culture period was calculated for each treatment and compared across treatments by a two way analysis of variance with specific differences between treatments determined using Fisher’s least significant difference test. In addition, the fold change in steroid production compared to the first 24 hours of culture was calculated using the non-transformed real number steroid concentrations for each individual well. Mean changes were then calculated for the three replicate wells within treatment. Treatment effects were analyzed by a two way analysis of variance with specific differences between treatments determined using Fisher’s least significant difference test.  RESULTS The effect of P 4 on B 2 accumulation in culture media by granulosa cells from a representative experiment is shown in Fig 7. la. Progesterone treatment failed to suppress B 2 130  Effect ofprogesterone on granulosa cell estrogen production accumulation, even at the most extreme concentrations (10 M). Progesterone treatment stimulated E secretion at concentrations ranging from lO M to 10 M. Concentrations greater than 1O M returned E 2 accumulation to control levels. Granulosa cells treated with P 4 (l0 M to iO M) had a greater fold increase in F 2 when comparing accumulation from the first 24 hours of culture to the final 24 hours (Fig. 7.2a). Treatment did not affect cell viability (Table 7.1). Progesterone treatment also stimulated the accumulation of P 4 (Fig 7. ib). When comparing the first 24 hours of P 4 accumulation with the final 24 hours, cells treated with 4 had a greater fold increase (P < 0.05) than controls (Fig. 7.2b). This was true across all P concentrations of P 4 treatment. Analysis of P 4 from the blank wells (wells not seeded with cells) showed residual P 4 from the treatment was undetectable.  Table 7.1.  Viable granulosa cells at termination of culture expressed as a percent of the initial plating density (2 X 10 cells/mL)’.  Progesterone treatment (tM)  1  0.00  0.001  68±4  65±3  Mean  0.01  0.1  1.0  10.0  66±2  65±4  66±4  64±5  (± SEM) of three replicate wells from a single representative experiment  131  Effect ofprogesterone on granulosa cell estrogen production 3.50  1  3.00  2.50 C  .001  .01  .1  16  10  C  I  I  .001  .01  I  .1  1  10  Progesterone concentration (uM) 4.00-  350-  3.00  I  C  .001  .01  .1  16  I  I  I  10  C  001  I  .01  .1  16  10  Progesterone concentration (uM) 1st 24 Hrs  Final 24 Hrs  Fig. 7.1. Mean (± SEM) estradiol (a) and progesterone (b) accumulation in media from bovine granulosa cell cultures in response to progesterone.  132  Effect ofprogesterone on granulosa cell estrogen production  a Cd)  C’)  b  0  ab a C.)  0 I.4  1  Final 24 Hrs  b C’)  C’)  4  3.  0  C.)  0 1  Final 24 Hrs Culture period c  001  .01  ::::)  .1  1  10  P4 (uM) Fig. 7.2. Mean (± SEM) fold change in estradiol (a) and progesterone (b) accumulation in media from bovine granulosa cell cultures in response to progesterone (different superscipts signify significant differences; P < 0.05).  133  Effect ofprogesterone on granulosa cell estrogen production DISCUSSION The results of the present study do not support the findings of previous research demonstrating that P , or progestins, suppress FSH stimulated E 4 2 production by rat (Schreiber et al., 1981) and goat (Kharbanda et al., 1990) granulosa cells. In the present study, P 4 enhanced E 2 accumulation in the culture media over a wide range of concentrations (10-s to iO M). Higher concentrations (> i0’ M) returned E 2 to control levels but had no suppressive effect.  A more recent study examining the effects of P 4 on FSH stimulated  induction of aromatase mRNA in rat granulosa cells also failed to find any suppressive effect (Fitzpatrick and Richards, 1991). In fact, at the highest doses (1 jIM), 4 P enhanced FSH stimulation of cAMP, aromatase mRNA and aromatase activity. Treatment with P 4 also enhanced granulosa cell P 4 production. This is in agreement with recent studies in rat (Ruiz de Galarreta et at, 1985; Pridjian et al., 1987) and human (Parinaud et at, 1990) granulosa cells. There could be several explanations for the differences in results from the different studies. It would appear that species differences is the least likely, the contradictory results of Schreiber et al. (1981), Ruiz de Galarreta et al. (1985) and Fitzpatrick and Richards (1991) were produced using rat granulosa cells. More plausible explanations include differences in P 4 metabolism, stage of differentiation of granulosa cells or abundance of P 4 receptors. In addition, differences in culture conditions could lead to differing results. The use of serum (Kharbanda et al., 1990) and high concentrations of FSH (Schreiber et al., 1981; Kharbanda et al., 1990) may have had some effects. Results outlined in the previous 134  Effect ofprogesterone on granulosa cell estrogen production chapter demonstrated that high concentrations of FSH (> 5.0 ng/mL) lead to luteinization of cultured bovine granulosa cells. Similarly, high concentrations of FSH lead to a rapid loss of P450 aromatase mRNA in rat granulosa cells both in vivo (Hickey et aL, 1988) and in vitro (Fitzpatrick and Richards, 1991). The present study utilized granulosa cells harvested from day 7 first wave dominant follicles. The dominant follicle achieves its maximum diameter at this time and has its highest estrogenic capacity. Results in Chapter 4 demonstrated that exogenous P 4 administered in vivo could induce the premature atresia of the first wave dominant follicle. This was accomplished prior to day 7. The present results suggest that the action of P 4 is probably not at the level of the ovary. The concentration of P 4 in the plasma when P 4 was administered would stimulate E 2 production according to the present results. Although P 4 levels in follicular fluid do reach the M concentrations (see Table 6.1 in previous chapter) which have been demonstrated to have a suppressive effect in other species, it is not clear whether these concentrations are present in healthy, estrogenic follicles or are the result of follicular atresia. To summarize, the present study suggests that P 4 may enhance F 2 and P 4 production by granulosa cells from bovine dominant follicles and that there appears to be no suppressive effect as seen in previous studies in other species.  135  Effect ofprogesterone on granulosa cell estrogen production REFERENCES Fitzpatrick, S.L. and Richards, J.S. 1991. Regulation of cytochrome P450 aromatase mRNA and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452-1462. Hickey, G.J., Chen, S., Besman, M.J., Shively, J.E., Hall, P.F., Gaddey-Kurten, D. and Richards, J.S. 1988. Hormonal regulation, tissue distribution and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis. Endocrinology 122: 1426-1436. Kharbanda, S.M., Band, V., Murugesan, K. and Farooq, A. 1990. Modulation of steroid production in goat ovarian cells: Effect of progestins and anti progestins. Endocrine Res. 16:293-309. Parinaud, 3., Perret, B., Ribbes, H., Vieitez, G. and Baulieu, E.E. 1990. Effects of RU486 on progesterone secretion by human preovulatory granulosa cells in culture. J. Clin. Endocrinol. Metab. 70: 1534-1537. Pridjian, G., Schmit, V. and Schreiber, J.R. 1987. Medroxy-pogesterone acetate: receptor binding and correlated effects onsteroidogenesis in rat granulosa cells. J. Steroid Biochem. 26:313-319. Ruiz de Galarreta, C.M., Fanjul, L.F. and Hsueh, A.J.W. 1985. Progestin regulation of progesterone biosynthetic enzymes in cultured rat granulosa cells. Steroids 46:987-1002. Schreiber, J.R., Nakamura, K. and Erickson, G.F. 1981. Progestins inhibit FSH-stimulated granulosa estrogen production at a post-cAMP site. Mol. and Cell. Endocrinology 21:161-170. Taylor, C. and Rajamahendran, R., 1991. Follicular dynamics, corpus luteum growth and regression in lactating dairy cattle. Can. J. Anim. Sci. 71:61-68.  136  CHAPTER 8  EFFECT OF PROGESTERONE ON REGULATION OF DOMINANT FOLLICLE GROWTH AND REGRESSION IN THE BOVINE: GENERAL DISCUSSION  It has long been held that pituitary gonadotrophins regulate antral follicular growth in mammalian species. However, with an increased understanding of the dynamics of follicle growth and regression/atresia it has become increasingly apparent that the regulation of this process is more complex than initially thought. Many intraovarian factors which may be involved in growth, selection and maturation have been identified (see Tonnetta and diZerega, 1989; Paton and Collins, 1992; Urban and Veidhuis, 1992 for reviews). It is now widely acknowledged that inhibin from dominant follicles suppresses pituitary FSH secretion, and this in turn leads to atresia of subordinate antral follicles. What is not clear is, why nonovulatory dominant follicles stop growing, become atretic, and regress to give way to new follicular growth. The work described in this thesis has examined the role of progesterone in regulating follicular growth in the bovine. The emphasis has been on the regulation of dominant follicles, specifically, regulation of their maintenance and onset of regression. A better understanding of the regulation of dominant follicle turnover may lead to a better understanding of the  137  GENERAL DISCUSSION  etiology of conditions such as cystic ovary condition in cows. Both in vivo and in vitro approaches were used in an effort to elucidate progesterone’s effects. Research in several laboratories has demonstrated that progesterone, or progestins, can influence the lifespan of large dominant follicles in the bovine. Sub mid-luteal phase circulating concentrations of progesterone have been shown to induce the maintenance of dominant follicles (Sirois and Fortune, 1990; Savio et aL, 1992). This phenomena can be mimicked with the synthetic progestin, norgestomet, when treatment is applied during the follicular phase of the estrous cycle (Rajamahendran et al., 1989; Savio et al., 1990; Rajamahendran and Taylor, 1991; Savio et al., 1993; Taylor and Rajamahendran, 1993). Results from Chapter 2 demonstrate that the maintenance of dominant follicles is associated with high-frequency LH pulses. This confirms published reports from our laboratory and others (Savio et al., 1990; Taylor and Rajamahendran, 1991; Taylor et al., 1993). One cannot conclude with certainty that high-frequency LH pulses induce the maintenance of dominant follicles. To demonstrate this one must show that in the absence of high-frequency LH pulses follicular turnover will be restored. This was demonstrated in Chapter 3. Addition of a progesterone releasing intra-vaginal device abolished LH pulsatility and restored follicular turnover. Savio et al. (1990) reported that implantation of a new norgestomet implant after a period of prolonged high-frequency LH pulses and follicular maintenance lead to a decrease in LH pulsatility, a decrease in dominant follicle size and an increase in new follicular growth. In our study (Chapter 3) addition of a second norgestomet did not decrease pulse frequency but did cause a decrease in LH pulse amplitude and mean circulating LH. The dominant follicle 138  GENERAL DISCUSSION  present regressed and a new dominant follicle developed. Interestingly, in three of the four heifers treated with two norgestomet implants, the new dominant follicle persisted and became cystic after the termination of treatment. Thus low-frequency, high-amplitude LH pulses would appear to be required for a normal pattern of follicular growth and regression. The results in Chapter 4 demonstrate that administration of exogenous progesterone early in the luteal phase of the estrous cycle, a stage characterized by high-frequency LH pulses (Rahe et at, 1980), will induce the premature regression or atresia of the first wave dominant follicle. In a recent report, progesterone administered early in the estrous cycle (days 0 to 5) was found to have a suppressive effect on dominant follicle growth (Adams et al., 1992). This was accompanied by an acceleration in the timing of the second FSH surge, presumably due to a decrease in inhibin. This lends further support to the idea that high circulating progesterone concentrations may start a chain of events leading to the onset of atresia in dominant follicles.  The evidence outlined above demonstrates that progestins are capable of modulating dominant follicle growth and regression. Maintenance of dominant follicles would certainly appear to be due to high frequency LH pulses. However, the results do not definitively show that a decrease in LH pulse frequency, due to high progesterone, induces regression of dominant follicles. The possibility also exists that progesterone acts directly at the level of the ovary to cause atresia. Unfortunately, the experiment designed to increase LH pulse frequency in a high progesterone environment failed to yield conclusive results (Chapter 5). The attempt to deliver high-frequency GnRH pulses failed to stimulate high-frequency LH 139  GENERAL DISCUSSION  pulses. It is not clear whether this due to a desensitization of the pituitary or a failure in the delivery of GnRH. To further test the effect of progesterone at the level of the ovary, a serum free bovine granulosa cell culture system was developed (Chapter 6). Challenging bovine granulosa cells with progesterone failed to suppress estradiol- 1713 production (Chapter 7). In fact, progesterone may have a stimulatory effect. This is in contradiction with previous studies in other species (Schreiber et al., 1981; Kharbanda et al., 1990) which demonstrated a suppressive effect of progesterone on estradiol- 1713 production at high concentrations. However, a recent study using molecular techniques found that progesterone did not inhibit cytochrome P450 aromatase mRNA or activity (Fitzpatrick and Richards., 1991). Taken together the evidence would suggest that high-frequency LH pulses induce the maintenance of dominant follicles in the bovine. Increases in progesterone, as seen during the mid luteal phase, feed back on the hypothalamic-pituitary axis and decrease LH pulsatility. The decrease in available LH probably leads to a decrease in the estrogenic capacity of the dominant follicle. This may be due to decreased availability of androgen substrate, although there is little change in follicular fluid androgen content between estrogen active and estrogen inactive follicles (McNatty et al., 1884). The loss of ability to produce estrogens may cause a chain of events, including a decrease in granulosa cell insulin-like growth factor-I and transforming growth factor-13 synthesis (see Chapter 1), leading to a decrease in granulosa cell proliferation, steroidogenesis and atresia.  140  GENERAL DISCUSSION  The observation that norgestomet and low circulating concentrations of P 4 induce maintenance of dominant follicles may have important implications in estrous synchronization schemes. A regular turnover of follicles is probably essential for optimum fertility rates, and therefore estrous synchronization regimens must take this into account. Understanding the mechanisms involved in the maintenance of follicles may lead to a better understanding of the etiology of cystic ovary condition in cows. Insights into the establishment and maintenance of follicular dominance may also be gained by the norgestomet maintained dominant follicle model. By using the model one can test other factors to determine if they will induce regression. Yet, questions remain. How is it that new dominant follicles can grow and secrete estrogens in a low LH environment? Is androgen availability a limiting factor in estrogen biosynthesis in the bovine? What is the role of FSH in this scheme? Clearly more research is required. The Regulation of follicular growth and atresia is still largely a mystery.  REFERENCES Adams, G.P., Matteri, R.L. and Ginther, 0.1., 1992. Effect of progesterone on ovarian follicles, emergence of follicular waves and circulating follicle-stimulating hormone in heifers.J. Reprod. Fert. 95:667-640. Fitzpatrick, S.L. and Richards, J.S. 1991. Regulation of cytochrome P450 aromatase mRNA and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452-1462. Kharbanda, S.M. Band, V., Murugesan, K. and Farooq, A. 1990. Modulation of steroid production in goat ovarian cells. Effects of progestins and anti progestins. Endocrine Res. 16:293-309. 141  GENERAL DISCUSSION  McNatty, K.P., Heath, D.A., Henderson, K.M., Lun, S., Hurst, P.R., Ellis, L.M., Montgomery, G.W., Morrison, L. and Thurley, D.C. 1984. Some aspects of thecal and granulosa cell function during follicular development in the bovine ovary. 3. Reprod. Fertil. 72:39-53. Paton, A.C. and Collins, W.P. 1992 Differentiation process of granulosa cells. Oxford Revs. Reprod. Biol. 14:169- 223. Rahe, C.H., Owens, R.E., Fleeger, J.L., Newton, H.J. and Hear, P.G. 1980. Pattern of plasma luteinizing hormone in the cyclic cow: Dependence upon the period of the Endocrinology 107:498-503. Rajamahendran, R., Eide, A., Robinson, 3., Taylor, C. and Walton, J.S., 1989. Effect of norgestomet on follicular dynamics, corpus luteum growth , progesterone, LH, oestrus and ovulation in cycling heifers. 3. Anim. Sci. 67 (Suppi. 1):383. Rajamahendran, R. and Taylor, C., 1991. Follicular dynamics and temporal relationships among body temperature, oestrus, the surge of luteinizing hormone and ovulationh Holstein heifers treated with norgestomet. J. Reprod. FertiL 92:46 1-467. Savio, J.D., Thatcher, W.W., Badinga, L. and de la Sota, R.L., 1990a. Turnover of dominant ovarian follicles as regulated by progestins and dynamics of LII secretion in cattle. 3. Reprod. Fertil. Abstr. Ser. 6:23. Savio, J.D., Boland, M.P., Hynes, N., Mattiacci, M.R. and Roche, J.F., 1990b. Will the first dominant follicle of the estrus cycle ovulate following luteolysis on day 7? Theriogenology 33:677-688. Savio, J.D., Thatcher, W.W., Morris, G.D., Entwistle, K. and Drost, M., 1992. Terminal follicular development and fertility in cattle is regulated by plasma progesterone. In: 12th International Congress of Animal Reproduction and Artificial Insemination 2:999-1001. Savio, J.D., Thatcher, W.W., Badinga, L., de la Sota, R.L and Wolfenson, D., 1993. Regulation of dominant follicle turnover during the oestrous cycle in cows. J. Reprod. Fertil. 97:197-203. Schreiber, J.R., Nakamura, K. and Erikson, G.G. 1981. Progestins inhibit FSH-stimulated granulosa estrogen pro duction at a post cAMP site. Mol. Cell. Endocrinol. 21: 61-170.  142  GENERAL DISCUSSION  Sirois, J. and Fortune, J.E., 1990. Lengthening the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance. Endocrinology 127:916-925. Taylor, C. and Rajamahendran, R., 1991. The effect of norgestomet and progesterone supplementation on LH release and follicular dynamics in dairy cattle. Biol. Reprod. 44 (Suppl. #1): Abstract #56. Taylor, C. and Rajamahendran, R., 1993. Ovarian follicular dynamics and plasma luteinizing hormone concentrations in norgestomet treated heifers. Anim. Reprod. Sci. 32: 173-184. Tonetta, S.A. and diZerega, G.S. 1989. Intragonadal regulation of follicular maturation. Endocrine Rev. 10:205- 229. Urban, R.J. and Veidhuis, J.D. 1992. Endocrine control of steroidogenesis in granulosa cells. Oxford Revs. Reprod. Biol. 14:225-262.  143  

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