Relaxin increases the release of plasminogen activator, collagenase, and proteoglycanase from rat granulosa cells in vitro

Relaxin (Rlx) is shown in vitro to increase the release of plasminogen activator (PA) activity from granulosa cells obtained from 28-day-old rats after priming 48 h before with PMSG. Priming with PMSG was essential for the subsequent marked increase in PA by the addition of Rlx to these cells in vitro. Under the same conditions Rlx also increased the release of both total collagenase and total proteoglycanase activities but not of beta-glucuronidase activity. The total collagenase and proteoglycanase activities of control cells are made up of essentially equal amounts of their respective active and latent enzymes. Rlx stimulation increases the amounts of the respective active enzymes while the latent collagenase and proteoglycanase activities are unchanged or decreased, respectively. The enzyme beta-glucuronidase was not stimulated by Rlx and appears not to be involved in follicular proteoglycan degradation. Granulosa cells harvested from preantral follicles responded most to FSH by PA production whereas cells from antral follicles responded more to LH, reflecting the known changes in concentration of FSH and LH receptors on these cells. The release of PA is maximal by all four hormones studied (FSH, LH, prostaglandin E1, and Rlx) on granulosa cells harvested from rats 48 h after PMSG treatment and this suggests that the follicles at this time are a mixture of both preantral and antral stages. The PA response to FSH is lost by 60 h after PMSG at the same time that the response to prostaglandin E1 is maintained at the same level, whereas that to Rlx and LH, although still significantly higher than controls, were decreased. By 70 h after PMSG, postovulatory, the responses to all hormones studied were lost. Thus, the involvement of PA in ovarian connective tissue alterations appears to be greatest in the period of follicular antrum formation rather than just before ovulation. Rlx is one of a number of hormones involved in the sequence of events culminating in follicle connective tissue remodeling as shown by its action on the release of three intrafollicular enzymes.     

Suppression of ovulation rate by antibodies to tissue-type plasminogen activator and alpha 2-antiplasmin

Indirect evidence has suggested a role for plasminogen activator (PA) in ovulation. Our recent studies demonstrated that 1) tissue-type PA (tPA) is the predominant PA produced by preovulatory rat follicles in response to gonadotropins or GnRH; and 2) several inhibitors of the serine proteases, to which PA and plasmin belong, block ovulation. Here, the role of tPA and plasmin in ovulation was examined directly by the use of specific antibodies to tPA and alpha 2-antiplasmin (alpha 2AP). Immature female rats at 25-26 days of age were treated (sc) with 15 IU PMSG to induce multiple preovulatory follicles. Fifty-four hours later, tPA antibodies and alpha 2AP were injected into one of the ovarian bursae to check their ability to block ovulation, which was initiated with an ovulatory dose (4 IU) of hCG. The data are expressed as percent inhibition of ovulation in the treated vs. the untreated ovaries. A significant decrease in the ovulation rate was obtained by administration of 500 micrograms antibodies to tPA (39.6%) or 1-50 micrograms alpha 2AP (36-44%), whereas minimal inhibition (12%) was found at lower doses of anti-tPA (10 micrograms) or alpha 2AP (0.1 micrograms). Furthermore, nonimmune immunoglobulin G (500 micrograms) and heat-inactivated alpha 2AP were not effective. Anti-tPA and alpha 2AP suppressed ovulation only when injected at the time of hCG administration; later injections (4-h delay) were ineffective, suggesting that PA and plasmin are involved in the early follicular responses to the ovulatory stimulus. Histological observation of the ovaries did not reveal any pathological changes associated with the anti-tPA and alpha 2AP treatment. Suppression of ovulation, as evidenced by decreased number of tubal ova, was frequently accompanied with intraovarian release of the eggs into the follicular thecal compartment. Thus, these results provide direct evidence for an essential role of tPA and plasmin in ovulation.     

Studies on the hormonal regulation of plasminogen activator production in the rat ovary

When granulosa cells are prepared from rats primed with PMSG, it is possible to induce plasminogen activator (PA) production in vitro with a variety of hormones. We have compared the effects of the gonadotropins FSH and LH on granulosa cells in culture. Although both hormones can induce enzyme secretion, the time courses of their effects are different. Whereas FSH yields a linear increase in PA production starting 2 h after the time of hormone addition, the effect of LH shows a longer lag phase, but it eventually reaches the same degree of stimulation as FSH. Experiments with an inhibitor of prostaglandin (PG) synthesis, indomethacin, and with PGs suggest the following interpretation. Granulosa cells that can produce PA can be directly stimulated by FSH, but only a small fraction of these cells can be induced by LH. However, the population that is responsive to LH begins to produce PGs after hormone treatment, and the PGs can then stimulate the entire population. Cumulus cells, which have few LH receptors, can be induced by FSH and PGs, but not LH. Experiments involving treatment of rats with indomethacin in vivo or treatment of follicles in vitro demonstrate that this compound can suppress PA secretion in the ovary, which may partially explain its ability to inhibit ovulation.     

Blockade of the final phase of ovulation has no effect on the post-ovulatory surge of serum FSH in the rabbit

Mating induces a surge of both LH and FSH in the blood of female rabbits, followed 10-12 h later by a surge of FSH only, which begins at the time of ovulation. We have studied the effect of suppression of ovulation on the post-ovulatory surge of FSH. In the first experiment, follicular fluid and oocytes were withdrawn from the largest follicles 8 h after coitus. In the second experiment, ovulation was inhibited by injecting the rabbits with 25 mg indomethacin/kg body weight 7.5 h after mating. Levels of serum FSH and LH were measured for 24-48 h after mating. Control rabbits ovulated normally in both experiments. The treatments did not significantly affect the levels of serum FSH in either experiment, although the second surge of FSH was slightly higher after fluid had been aspirated from the preovulatory follicles. These observations show that the post-ovulatory surge of serum FSH is not dependent upon the completion of ovulation and that it is programmed before 7.5-8 h post coitum.     

Follicle-stimulating hormone-induced prostaglandin E formation in isolated rat ovarian theca

The aim of this study was to search for direct biochemical effects of highly purified FSH on isolated ovarian follicular theca in vitro. Granulosa cells (GC; approximately 1 x 10(5) cells per follicle) were flushed from isolated follicles of pro-oestrous rats. The remaining theca layer and the isolated GC were incubated with highly purified ovine FSH. Prostaglandin E (PGE) accumulation was measured by radioimmunoassay. Follicle-stimulating hormone induced a 15-fold increase in PGE accumulation over the basal level in the follicular theca, the stimulated rate exceeding threefold that observed in the GC fraction derived from the same follicle. Follicle-stimulating hormone caused no significant increase in cyclic AMP level or steroidogenesis in the theca layer, but was active on these parameters in the GC. In contrast, LH increased the accumulation of cyclic AMP, progesterone and testosterone, as well as of PGE, in follicular theca. Exogenous 8-bromo cyclic AMP or cyclic GMP also stimulated PGE production in follicular theca or GC, but FSH was without any effect on the level of endogenous cyclic GMP in GC or follicular theca. Antibodies to FSH prevented the effect of FSH (but not that of LH) on PGE formation by follicular theca and GC, while antibodies to the beta-subunit of LH blocked the effect of LH but not of FSH. We conclude that highly purified FSH has a stimulatory effect on PGE formation by the follicular theca.     

The involvement of collagenolysis in ovulation in the rat

Collagenolytic activity in ovarian follicles was previously demonstrated by using synthetic peptides and reconstituted collagen fibers. However, attempts to demonstrate degradation of ovarian collagen and to correlate collagenase activity with ovulation were not successful. By administration of L-(5-3H) proline, we have labeled ovarian and follicular collagen and followed collagenolytic activity by separation of 3H-hydroxyproline (3H-Hyp) from acid hydrolyzates of ovarian tissue by HPLC. The level of ovarian and follicular 3H-Hyp decreased by about 40% on the afternoon of proestrus or after exogenous stimulation of ovulation by human CG (hCG), and this decrease was abolished by blocking the surge of gonadotropins with Nembutal. To verify that the observed reduction in 3H-Hyp was due to the action of a typical collagenase, the collagenous fraction was prepared from ovarian tissue and from preovulatory follicles before and after the ovulatory stimulus. The extracts were treated with trypsin (25 min, 25 C, 0.01 mg/ml) plasmin and p-amino-phenyl-mercuric acetate to fully activate the collagenase extracted along with collagen. Both, enzymatic and chemical activation of collagenase in vitro resulted in degradation of collagen. This degradation could be inhibited by cysteine and EDTA; both are classic inhibitors of mammalian collagenases. The activity of ovarian collagenase increased within 3 h after hCG-stimulation, peaked at 5-fold 6 h after hCG, and declined afterwards. Administration of cysteine (0.001-0.01 mmol) into the bursal cavity of proestrous rats blocked ovulation and breakdown of ovarian collagen in a dose-dependent manner. Cysteine effectively inhibited ovulation even when injected 7 h after the hCG stimulus. Inhibitors of arachidonic acid metabolism prevent ovulation. Indomethacin (inhibitor of cyclooxygenase) and nordihydroguaiaretic acid (inhibitor of lipoxygenase) blocked ovulation and inhibited hCG-induced ovarian collagenolysis. Collectively, these results corroborate the essential role of collagenolysis in follicular rupture in mammals.     

Changes in the plasma concentrations of luteinizing hormone, progesterone, oestradiol and testosterone and in the binding of follicle-stimulating hormone to the theca of follicles during the ovulation cycle of the hen (Gallus domesticus)

The changes in the binding of FSH during follicular maturation were examined in the hen using 125I-labelled bovine FSH (bFSH) and unlabelled bFSH. The binding of 125I-labelled bFSH was not inhibited by bovine LH or chicken LH but was inhibited by extracts of chicken pituitary glands. The ovarian stroma, which contained both interstitial tissue and small follicles, bound the greatest amount of FSH. As the follicles progressed through the yolk-filled hierarchy of maturation, they bound decreasing amounts of FSH. In the two largest follicles of the hierarchy, there was a significant increase in the binding of FSH 12-16h before ovulation. There were two peaks in the concentrations of LH; a preovulatory peak occurred 4-6h before ovulation and a second peak occurred 14-16h before ovulation. Plasma concentrations of testosterone, oestradiol and progesterone began to rise 9, 8 and 6h, respectively, before ovulation. These data are consistent with the hypothesis that changes in the gonadotrophin concentration and binding regulate the order of the follicular hierarchy and the onset of preovulatory steroidogenesis in the hen.     

Oxytocin inhibits LH-stimulated production of androstenedione by bovine theca cells

Oxytocin secretion by bovine granulosa cells increases dramatically after the LH/FSH surge. We have shown that oxytocin stimulates progesterone secretion and inhibits FSH-stimulated estradiol secretion in vitro by granulosa cells from bovine preovulatory follicles obtained before the LH/FSH surge. To determine if oxytocin regulates LH-stimulated steroid production by bovine theca interna cells, theca cells were isolated from preovulatory follicles obtained before the LH surge and were cultured for 4 days in the presence or absence of LH (2 or 4 ng/ml), without or with graded doses of oxytocin (125-1000 ng/ml). LH increased accumulation of androstenedione and progesterone. Oxytocin inhibited LH-stimulated androstenedione production, but had no effect on LH-stimulated progesterone production by cultured theca interna. The next objective was to determine if oxytocin regulates LH-stimulated steroidogenesis by modulating the levels of mRNA for steroidogenic enzymes and/or Steroidogenic Acute Regulatory protein (StAR). Low doses of LH alone increased the levels of mRNA for P450 17 alpha-hydroxylase (17 alpha-OH), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450 side-chain cleavage, but not for StAR. In contrast, the effects of oxytocin on LH-stimulated androstenedione production were not associated with changes in the levels of mRNA for steroidogenic enzymes or StAR. These results suggest that oxytocin may play a paracrine role in regulating the follicular/luteal phase shift in steroidogenesis by decreasing androstenedione secretion by theca cells of the ovulatory follicle and that this effect is not mediated by changes in the levels of mRNA for steroidogenic enzymes and StAR.     

Ovulation in plasminogen-deficient mice.

Many different studies suggest that plasmin generated from plasminogen plays a crucial role in the degradation of the follicular wall at the time of ovulation. We have assessed the physiological relevance of plasmin on ovulation by studying plasminogen-deficient mice. Ovulation efficiency (mean number of ova released per mouse) was determined both in a standardized ovulation model in which 25-day-old immature mice were injected with finite amounts of gonadotropins to induce ovulation and during physiological ovulation using adult normally cycling mice. Our results revealed that the temporal onset of follicular wall rupture (first ova observed in bursa or oviduct) was not delayed in plasminogen-deficient mice during gonadotropin-induced ovulation. However, there was a trend toward slightly reduced ovulation efficiency in the plasminogen-deficient mice. This reduction was only 13% and not statistically significant (P = 0.084) and may be connected to a delayed maturation of these mice manifested in reduced body and ovary weights. During physiological ovulation adult plasminogen-deficient mice had normal ovulation efficiency compared with plasminogen wild-type mice. Taken together our results indicate that under the conditions used in this study plasmin is not required for efficient follicular rupture or for activation of other proteases involved in this process. Alternatively, the role of plasmin may be effectively compensated for by other mechanisms in the absence of plasmin.     

Initiation of follicular maturation and ovulation after removal of the litter from the lactating rat

In order to elucidate the mechanism of the resumption of follicular activity and ovulation in rats, levels of FSH, LH and prolactin in plasma and pituitary gland and ovarian follicular development were quantified after removal of the litter on day 3 of lactation (day of parturition = day 0 of lactation). Such removal resulted in ovulation of 13 oocytes 4 days later, a number comparable with that found in normal cyclic rats. Plasma levels of prolactin were high during lactation but markedly decreased after removal of the litter. Although plasma concentrations of FSH and LH did not change during days 3-7 of lactation, there was an FSH surge between 24 and 30 h after removal of the litter. Plasma concentrations of LH also increased slightly but significantly by 24 h after removal of the litter and this value persisted during the following 2 days. Surges of FSH, LH and prolactin occurred at 17.00 h 3 days after pups were removed. Removal of the litter did not increase pituitary contents of FSH, LH and prolactin and a marked reduction in pituitary levels of FSH and LH, but not of prolactin, occurred at 17.00 h 3 days after removal of the litter. A quantitative study of follicular development indicated that follicles larger than 401 micrometers in diameter were absent during days 3-7 of lactation. However, the number and size of antral follicles increased by 30 h after removal of the litter, probably due to the increases in plasma levels of FSH and LH, and follicles larger than 601 micrometers in diameter appeared 3 days after the young were removed. Although ovulation could not be induced by human chorionic gonadotrophin from days 3 to 5 of lactation, its administration 30 h after removal of the litter produced ovulation in all rats by the following morning. These results indicated that a moderate increase in FSH, although below the amounts released at the preovulatory surge, together with basal levels of LH which were within the range observed on the day of dioestrus during the normal cycle were responsible for the initiation of follicular maturation after removal of the litter.     

Effect of gonadotrophin-releasing hormone agonist-induced suppression of LH and FSH on follicle growth and corpus luteum function in the ewe

Continuous infusion of a gonadotrophin-releasing hormone (GnRH) agonist (buserelin) by osmotic minipump from day 1 of the luteal phase in five Welsh ewes resulted in a sustained suppression of plasma concentrations of FSH which increased three- to eightfold within 2 days after the end of infusion 29 days later. Plasma concentrations of LH increased three- to eightfold over the first 5 days of infusion and then became basal and non-pulsatile until 1 day after the end of infusion. Duration of the luteal phase and plasma concentrations of progesterone were not significantly different in control and treated ewes. Pulses of LH in control ewes were followed by increases in concentrations of progesterone in samples collected at 10-min intervals for 7 h on days 10 and 14 of the luteal phase. However, progesterone was also released in a pulsatile manner in the absence of LH pulses in both control and GnRH agonist-treated ewes. After natural luteolysis, no ovulation or corpus luteum function occurred in treated ewes up to 15 days after the end of treatment on day 29, even though oestrus, indicating follicular development and oestrogen secretion, had occurred 8-11 days after treatment ended. After 30 days of infusion the ovaries of GnRH agonist-treated ewes contained no follicles greater than 2.5 mm in diameter. In follicles of 1-2 mm in diameter the basal and LH-stimulated production of oestradiol and testosterone in vitro were similar in both control and GnRH agonist-treated ewes, and a similar proportion of these follicles was oestrogenic (greater than 370 mol oestradiol per follicle) in GnRH agonist-treated and control ewes. These results show (1) that progesterone secretion by the corpus luteum of the ewe can be sustained in the presence of basal concentrations but absence of pulsatile secretion of LH, and progesterone is released in a pulsatile manner whether or not LH pulses are present, (2) that follicular development beyond 2.5 mm in diameter in the ewe is dependent upon adequate stimulation by both LH and FSH and (3) that the continuous infusion of GnRH agonist is a simple method for providing reproducible suppression of LH and FSH and follicular development in the ewe to allow the study of gonadotrophin action on the ovary in vivo.     

Clinical review 126: Roles and novel regimens of luteinizing hormone and follicle-stimulating hormone in ovulation induction

Although FSH is universally recognized as the key driver of ovarian follicle growth and maturation, the role of LH in these processes is more controversial. LH acts on theca cells to induce androgen substrate for estrogen conversion by the aromatase system; furthermore, LH can affect granulosa cell function starting in the mid- follicular phase, when these cells express LH receptors. The capacity of LH to stimulate granulosa cells in larger follicles (>10 mm diameter) may be the critical mechanism involved in the selection of the dominant follicle in the normal menstrual cycle. Furthermore, the addition of LH activity can shorten and optimize FSH ovulation induction and reduce the development of smaller preovulatory ovarian follicles that are associated with the severe complications of this procedure. Novel mixed gonadotropin administration regimens that incorporate graded amounts of LH and FSH activity may improve efficacy, safety, and cost of ovulation induction, particularly in the area of assisted reproduction.     

Laminin-alpha6beta1 integrin interaction enhances survival and proliferation and modulates steroidogenesis of ovine granulosa cells.

This study aimed to determine the physiological role of laminin (LN) and its receptor, alpha(6)beta(1) integrin, in controlling the functions of granulosa cells (GC) during follicular development in sheep ovary. Immunohistochemistry experiments showed the presence of increasing levels of LN (P<0.0001), and high levels of mature alpha(6)beta(1) integrin in GC layers of healthy antral follicles during the follicular and the preovulatory phases of the estrous cycle. In vitro, the addition of a function-blocking antibody raised against alpha(6) subunit (anti-alpha(6) IgG) to the medium of ovine GC cultured on LN impaired cell spreading (P<0.0001), decreased the proliferation rate (P<0.05) and increased the apoptosis rate (P<0.05). Furthermore, addition of anti-alpha(6) IgG enhanced estradiol (E2) secretion by GC in the presence or absence of follicle-stimulating hormone (FSH), luteinizing hormone or insulin-like growth factor-I in culture medium (P<0.0001), and inhibited progesterone (P4) secretion in basal conditions or in the presence of low (0.5 ng/ml) FSH concentrations only (P<0.0001). The anti-alpha(6) IgG effect was specific to an interaction of LN with alpha(6)beta(1) integrin since it was ineffective on GC cultured on heat-denatured LN, RGD (arginine-glycine-aspartic acid) peptides and non-coated substratum. Hence, this study established that alpha(6)beta(1) integrin 1) was expressed in GC of antral follicles, 2) mediated the actions of LN on survival, proliferation and steroidogenesis of GC, and 3) was able to dramatically modulate P4 and E2 secretion by GC in vitro. It is suggested that during the follicular and the preovulatory phases of the estrous cycle, the increasing levels of LN in GC of large antral follicles might support their final development to ovulation.     

Selective release of FSH in lactating rats during the period of follicular atresia induced by the administration of antiserum to LH-releasing hormone

Passive immunoneutralization of LHRH by injecting a caprine antiserum to LHRH (LHRH-AS) in lactating rats nursing two pups on day 5 of lactation resulted in an immediate decline in concentrations of LH in the plasma during the 24-h study period, followed by a gradual increase to control levels 30 h later. Concentrations of oestradiol-17 beta and inhibin activity in ovarian venous plasma also decreased abruptly in LHRH-AS treated animals and recovered to control levels 36 h later. These changes were correlated with changes in concentrations of LH in the plasma. On the other hand, plasma concentrations of FSH increased abruptly in the LHRH-AS treated animals within 3 h after injection, but the concentrations declined gradually to control levels 48 h later. The ability of follicles to ovulate in response to human chorionic gonadotrophin (hCG) began to decrease within 6 h after treatment with LHRH-AS, and further decreased until 18 h after injection of LHRH-AS, when hCG induced ovulation (with two oocytes) in only one of five animals. A gradual increase in ovulation rate to control levels was noted by 36 h after injection of LHRH-AS. These results indicate that Graafian follicles present at the time of LHRH-AS injection had become atretic and that a new set of follicles had then begun to mature. Selective release of FSH could not be induced by injection of LHRH-AS in ovariectomized animals. Treatment with inhibin (porcine follicular fluid) suppressed the selective release of FSH, whereas treatment with oestradiol-17 beta had no inhibitory effect on the selective release of FSH. These findings indicate (1) that tonic secretion of LH is an important factor in normal follicular maturation and maintenance in lactating rats, (2) that selective release of FSH after injection of LHRH-AS is attributed to the removal of a negative influence of inhibin from antral follicles during the period of follicular atresia and (3) that a selective surge of FSH is responsible for initiation of new follicular maturation.     

Luteinizing hormone increases the number of ova shed in the cyclic hamster and guinea-pig

Osmotic minipumps containing 400 micrograms ovine LH installed subcutaneously on day 1 (oestrus) of the cycle in the hamster induced superovulation of 30.0 +/- 2.1 ova (n = 5) at the next expected oestrus. Controls ovulated 12.0 +/- 0.8 ova (n = 6). Bovine LH, human LH, porcine LH, human chorionic gonadotrophin and pregnant mare serum gonadotrophin were effective in approximately doubling the number of ova spontaneously shed in the hamster. Ovine FSH (200 micrograms/pump) was most effective in increasing the number of ova spontaneously shed (55 +/- 6, n = 5) in the hamster. Infusion of ovine LH on days 1-4 prevented the reduction of the number of antral follicles that occurs normally between days 3 and 4 of the 4-day cycle. Since this reduction in follicular numbers in control cyclic hamsters is due to atresia, the exogenous LH might prevent atresia of the developing follicles. In the hamster, exogenous ovine LH significantly increased the serum concentrations of androstenedione, oestradiol and LH but not of FSH. Hamsters were hypophysectomized on the day of oestrus, given immediate LH (400 micrograms) or FSH (200 micrograms) replacement therapy and autopsied on day 4. Ovarian histology revealed that immediate LH treatment after hypophysectomy sustained development of histologically normal preovulatory follicles but had no effect on the number of smaller sizes of follicles. Immediate FSH treatment after hypophysectomy increased only the number of smaller sized follicles. Since LH did not increase the smaller sized follicles, no 'FSH-like' effect on follicular development was observed. In the hamster, the ability of various preparations of LH to induce superovulation did not correlate with their ability to displace 125I-labelled ovine FSH from its ovarian binding sites. The superovulatory action of LH required the presence of the pituitary gland, indicating that LH might synergize with FSH and/or prolactin (or hamster LH) for spontaneous superovulation and it appears that exogenous LH might induce superovulation by prevention of atresia. Infusion of LH into the guinea-pig beginning on day 12 of the cycle (day 1 is the day of ovulation) doubled the ovulation rate whereas in the cyclic rat and mouse LH treatment throughout the cycle was ineffective in increasing the number of ova shed.     

Stimulation of ovarian follicular maturation with pure follicle-stimulating hormone in women with gonadotropin deficiency

According to the 2-cell theory, ovarian steroidogenesis requires the coordinate action of both FSH and LH. To evaluate the relative importance of these hormones in follicular maturation, a randomized cross-over study was performed in 10 women with complete gonadotropin deficiency (absence of pulsatile LH secretion and no LH response to LHRH). Five women were treated with highly purified FSH (LH bioactivity, 0.09%) and 3 months later with human menopausal gonadotropin (hMG; LH bioactivity, 65%), each given for 10 days at a daily dose of 225 IU FSH, im. The sequence was reversed in the other 5 women. hCG (5000 IU) was administered im 24 h after the last injection of FSH or hMG. Plasma estradiol (E2), estrone (E1), androstenedione (A), testosterone, LH, and FSH concentrations and urinary LH and FSH were measured daily by RIA. Ultrasonography was performed during each treatment and 2 days after each hCG injection. After FSH treatment, mean plasma and urinary FSH levels increased, mean plasma LH did not change, and urinary LH increased slightly but not significantly from 91 +/- 32 (SE) to 164 +/- 55 mIU/24 h (10(-3) IU/24 h). After hMG treatment, mean plasma and urinary LH and FSH levels increased accordingly. The mean basal plasma E2 [11 +/- 1 pg/mL (40 +/- 4 pmol/L)] and E1 [14 +/- 4 pg/mL (52 +/- 15 pmol/L)] levels increased after FSH treatment to 207 +/- 69 pg/mL (760 +/- 253 pmol/L) and 82 +/- 21 pg/mL (303 +/- 78 pmol/L), respectively (P less than 0.01), but plasma A did not change. In response to hMG, the mean plasma E2, E1, A, and testosterone levels increased more than during FSH treatment. Ultrasonography revealed multiple preovulatory follicles (greater than or equal to 16 mm) in 2 women after hMG and 1 woman after FSH treatment; therefore, hCG was not administered. In 3 women given FSH, hCG did not induce ovulation. hCG induced ovulation in 8 women given hMG and in 6 women given FSH, based on ultrasonography and plasma progesterone levels. Thus, in the presence of profound gonadotropin deficiency pharmacological doses of FSH, with minute LH contamination, are capable of stimulating ovarian follicular maturation, underlining the key role of FSH in folliculogenesis.     

Norepinephrine in Graafian follicles is depleted by follicle-stimulating hormone

The purpose of the present study was to measure norepinephrine (NE) in Graafian follicles and correlate changes in its concentration with circulating gonadotropins secreted endogenously or administered exogenously. Graafian follicles were removed from the ovaries of adult cycling rats. The follicles were pooled in groups of 10-13, and NE was determined by high performance liquid chromatography. Follicular NE(picograms per micrograms protein) did not change between 0900 h (3.61 +/- 0.34) and 1300 h (3.12 +/- 0.25) on proestrus, but was reduced significantly to 1.45 +/- 0.16 at 2100 h, which is 4 h after the peak of the gonadotropin surge. There was a further reduction to 0.83 +/- 0.08 in fresh corpora lutea taken on estrus at 0900 h. The decrease in follicular NE was prevented in estrous rats which were either hypophysectomized 24 h previously or treated with sodium pentobarbital at 1330 h on proestrus. To determine which pituitary hormone was responsible for follicular NE depletion, rats were injected at 0900 h on proestrus with LH (5 micrograms), FSH (20 micrograms), LH plus FSH (5 and 20 micrograms, respectively), or PRL (20 micrograms), and follicular NE was determined 4 h later. FSH reduced follicular NE significantly to 1.86 +/- 0.16 compared to both the control (3.12 +/- 0.25) and the PRL-injected group (2.92 +/- 0.32), whereas LH caused a small but nonsignificant decrease (2.49 +/- 0.2). Both LH and FSH doses used resulted in ovulation, as determined by counting tubal ova 12 h after hormonal treatment. We conclude that 1) NE in Graafian follicles is markedly reduced within 4 h after the preovulatory gonadotropin surge in the normal cycling rat; this reduction is prevented when the surge is abolished; 2) the hormone responsible for follicular NE depletion is FSH rather than LH or PRL; and 3) finally, it is suggested that follicular NE may be involved with the formation and/or functioning of the corpus luteum.     

Evidence that changes in tonic luteinizing hormone secretion determine the growth of preovulatory follicles in the rat

Physiological concentrations of progesterone (20-100 ng/ml), maintained by the insertion of implants into 30-day-old rats, delayed first ovulation, and withdrawal of progesterone on day 47 of age synchronized first ovulation in rats. Inhibition of ovulation involved negative feedback regulation of tonic LH and FSH secretion, blockage of gonadotropin surges, and suppression of preovulatory, but not antral, follicular growth. Removal of implants resulted in a rapid decline in serum progestrone from 100 to 5 ng/ml within 0-12 h. Between 0-36 h there were progressive increases in serum concentrations of LH and FSH, enhanced accumulation of estradiol by individual follicles incubated in vitro with or without exogenous substrate, and marked progressive increases in the content of LH (but not FSH) receptors in both thecal and granulosa cells. These events were followed by gonadotropin surges at 48 h (1800 h on day 49), ovulation, and morphological and biochemical signs of luteinization, including decreases in follicular gonadotropin receptor content and estradiol accumulation, evident by 60 h. With the exception of changes in basal LH, this sequence of events is remarkably similar in time and pattern to that after the decline of progesterone on diestrous day 2 and ovulation on proestrus of a 5-day cycle. Although a direct effect of progesterone on ovarian follicular cell function cannot be excluded, the data suggest that subtle but sustained increases in LH (and possibly FSH) are required for the enhanced follicular accumulation of estradiol and LH-binding activity occurring between diestrus and proestrus of the rat estrous cycle. Thus, perhaps some of the mystery surrounding the endocrine events between diestrus and proestrus can be ascribed to changes in serum LH that have been too small and/or variable for current nonserial sampling methods and RIAs to detect reliably.     

Gonadotropins enhance caspase-3 and -7 activity and apoptosis in the theca-interstitial cells of rat preovulatory follicles in culture

Apoptosis causes the elimination of ovarian germ cells and the atretic degeneration of ovarian follicles. Here we have used cultured rat preovulatory follicles to examine the regulation of effector caspase-3 and -7 in follicles undergoing apoptosis in the presence or absence of gonadotropins or IGF-I. Culturing follicles in the presence or absence of serum resulted in the induction of apoptosis of granulosa cells (GC), which was accompanied by effector caspase activation. Surprisingly, the addition of the survival factors LH or FSH, but not IGF-I, further increased caspase-3 and -7 activity. Immunohistochemistry studies of the LH- and FSH-treated follicles indicated that cleaved caspase-3 was predominantly localized to the peripheral theca-interstitial cells (TIC). Western blot analysis and caspase-3 and -7 activity assays of the separated follicular compartments confirmed that both LH and FSH treatments significantly enhance caspase-3 and -7 activity in TIC. The elevation in caspase-3 and -7 activity in TIC was accompanied by an increase in apoptosis. Interestingly, LH and FSH also induced an increase in caspase-3 and -7 activity in GC; however, this increase was accompanied by a decrease in apoptosis. Finally, we demonstrate that in freshly isolated preovulatory follicles and in antral follicles in intact ovaries, the expression level of procaspase-3 is significantly higher in TIC than in GC. Thus, LH and FSH have a dual effect on the cultured rat preovulatory follicle: an antiapoptotic effect on GC and a proapoptotic effect on TIC.     

Dynamics of ovarian inhibin secretion during the oestrous cycle of the rat

Plasma and ovarian concentrations of inhibin were determined at 3-h intervals throughout the 4-day oestrous cycle of rats by a radioimmunoassay (RIA) based on a bovine RIA. Plasma concentrations of LH, FSH, progesterone, testosterone, oestradiol-17 beta, and pituitary contents of FSH and LH were also determined during the cycle. Plasma levels of inhibin showed a marked increase on the morning of oestrus and the afternoon of metoestrus, and a further increase was noted on the morning of pro-oestrus. These increases in plasma levels of inhibin were probably due to the following three events in the ovary, follicular recruitment on the morning of oestrus, selection of follicles on the day of metoestrus, and final maturation of follicles for ovulation on the morning of pro-oestrus with an increase in oestradiol-17 beta secretion. A striking decrease in inhibin secretion occurred during the process of ovulation after the preovulatory gonadotrophin surge on the afternoon of pro-oestrus. Basal levels of plasma FSH gradually decreased from metoestrus to pro-oestrus as plasma levels of inhibin increased. There was a significant inverse relationship between plasma levels of FSH and inhibin throughout the oestrous cycle (r = -0.51). The present findings suggest that changes in the plasma levels of inhibin during the oestrous cycle provide a precise indicator for follicular recruitment, selection and ovulation, and that changes in concentrations of oestradiol-17 beta in the plasma are associated with follicular maturation.