Prolonged stimulation by follicle-stimulating hormone is required for the induction of ovarian follicular cysts by human chorionic gonadotropin in hypophysectomized rats

Combined stimulation by follicule-stimulating hormone (FSH) and subovulatory doses of human chorionic gonadotropin (hCG, luteinizing hormone [LH]-like activity) produces large ovarian follicular cysts in hypophysectomized (HYPOXD) immature rats. To obtain a better understanding of the extent to which stimulation by FSH is required in order for hCG to induce these ovarian cysts, immature HYPOXD rats were given subcutaneous (sc) injections of 1 IU hCG twice daily for 9 d, either alone or with daily injections of 2 μg of highly purified ovine FSH on 1. Day one of hCG treatment; 2. Days one and two of hCG treatment; 3. Days one through five of hCG treatment; or 4. All 9 d of hCG treatment. Ovaries and serum samples were collected on the morning of d 10 of treatment. Animals that were treated for 9 d with hCG, but that received either no FSH or only 1 or 2 d of FSH treatment, did not display antral follicles on day 10 of treatment. The largest cross-sectional areas for the ovaries from animals that received 1 or 2 d of FSH treatments ranged between 6.84±0.51 mm(2) and 8.94±0.89 mm(2). The diameters of the largest preantral follicles in the ovaries of these two groups ranged between 0.278±0.011 and 0.320±0.028 mm, respectively. In contrast, ovaries from hCG-treated HYPOXD rats that received FSH treatments for either 5 or 9 d displayed follicular cysts by the morning of day 10. The largest cross-sectional areas for the ovaries from these two treatment groups were similar (15.68±1.61 and 18.7±5.13 mm(2), respectively), as were the mean diameters of the cystic follicles in these two groups (0.929±0.096 and 0.830±0.063 mm, respectively). Although serum androstenedione and testosterone concentrations were greater for HYPOXD rats that received combined FSH+hCG treatments than for animals that received hCG treatments alone, these concentrations did not increase with increasing numbers of days of FSH treatment. As with serum androstenedione and testosterone concentrations, serum estradiol and estrone concentrations for HYPOXD rats treated with hCG alone were limited (0.002±0.001 and 0.004±0.002 ng/mL, respectively), but had increased by day 10 after a single injection of FSH on day one of treatment. In contrast to serum androgen concentrations, serum estradiol and estrone concentrations continued to increase as the number of days of combined FSH+hCG treatment increased. These observations indicate that, in the rat, a significant period of exposure to tonic stimulation by both FSH and LH-like activity is required for the development of large ovarian cysts. Further, this period of exposure to FSH appears to be linked to increased peripheral serum estrogen concentrations, rather than to increased androgen concentrations. Therefore, the data provide indirect support for the concept that estrogens play a direct role, at the level of the ovary, in the induction of large ovarian cysts in the rat.     

Differential action of decidual luteotropin on luteal and follicular production of testosterone and estradiol

Decidual tissue of the rat produces a hormone with physiological and biochemical characteristics similar to those of PRL. Because PRL affects both follicular and luteal production of testosterone and estradiol, it was of interest to determine whether decidual luteotropin affects basal and/or LH-stimulated ovarian secretion of steroids and whether it differentially affects follicular and luteal synthesis of testosterone and estradiol. The uteri of pseudopregnant adult rats were scratched on day 5 to induce decidual tissue formation. Pseudopregnant animals without decidua were used as controls. Rats were either hypophysectomized on day 8 or left intact. They were treated with 1.5 IU hCG/day or with vehicle between days 8-9. On day 9, blood was obtained from the ovarian vein, and both corpora lutea and large antral follicles were isolated and incubated in vitro. The presence of the decidua significantly suppressed both basal and hCG-stimulated ovarian secretion of estradiol, yet enhanced progesterone production. A similar inhibitory effect of decidual tissue on hCG stimulation of testosterone and estradiol was observed in the hypophysectomized rats. When the effect of decidua on follicles and corpora lutea was studied separately, it was found that follicles of rats with decidua produced significantly less testosterone and estradiol than follicles of rats without decidua. hCG administration to either intact or hypophysectomized rats markedly enhanced the follicular capacity to produce these two steroids. However, the degree of hCG stimulation of follicular steroidogenesis was significantly reduced by the presence of decidual tissue. In contrast, the decidua did not inhibit the in vitro steroidogenic capacity of corpora lutea. Luteal tissue of intact rats with or without decidua produced similar basal amounts of testosterone and estradiol and responded to a hCG challenge with comparable increases in the production of both steroids. After hypophysectomy, however, the responsiveness of corpora lutea to hCG stimulation differed in rats with or without decidual tissue. Whereas luteal cells of rats without decidual tissue gradually lost their responsiveness to hCG stimulation, luteal cells of rats with decidua remained highly responsive to hCG and produced high levels of testosterone and estradiol. In summary, the present investigation demonstrates that decidual luteotropin impairs ovarian secretion of estradiol and significantly inhibits the stimulatory effect of hCG on ovarian secretion of testosterone and estradiol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective stimulation of luteal androgen biosynthesis by luteinizing hormone: comparison of hormonal regulation of P45017 alpha activity in corpora lutea and follicles

Recent investigations have indicated that luteal cells of pregnant rats become capable of synthesizing androgen and estradiol when stimulated by sustained low levels of LH/hCG. In this investigation we sought 1) to determine whether hCG causes the induction/activation of the luteal enzymes responsible for the conversion of progesterone to estradiol, 2) to investigate the time course of hCG action, and 3) to compare the effect of hCG on luteal and follicular P45017 alpha activities. To determine first the minimum stimulatory dose of hCG, pregnant rats were treated with 0, 1.5, 3, 9, or 30 IU hCG twice on days 12 and 13 and once on day 14. Two hours after the last injection, rats were bled from the ovarian vein, and corpora lutea were isolated and incubated for the determination of in vitro steroid production. Exposure of rats to twice daily injections of 1.5 IU hCG caused a dramatic increase in the ovarian secretion and luteal production of both testosterone and estradiol. However, an inverse dose-related response was observed with higher doses of hCG. To determine the time course of hCG action, pregnant rats were injected with a single dose of 3 IU hCG, and steroid production was determined at different times thereafter. There was no increase in either in vivo or in vitro testosterone and estradiol production until 12 h after hCG administration, at which time a dramatic increase in the secretion of both steroids was observed. hCG administration did not affect the capacity of luteal cells to secrete progesterone, nor did it enhance aromatase activity. It did, however, increase P45017 alpha activities; lyase and hydroxylase activities were stimulated 5- and 1.7-fold, respectively. To compare the effects of hCG on luteal and follicular P45017 alpha, both corpora lutea and follicles were isolated from ovaries of pregnant rats treated with either 1.5 IU hCG or vehicle between days 12-14. In vivo hCG administration caused at least a 1000-fold increase in the specific activity of follicular 17 alpha-hydroxylase and 17,20-lyase. The hCG-induced increase in the specific activities of both hydroxylase and lyase in the follicle far exceeded that in the corpora lutea. However, total lyase and hydroxylase activities in each follicle were only 3- and 5.5-fold higher, respectively, than those in the corpus luteum. In summary, the results of the present investigation have revealed that hCG stimulation of luteal estradiol production is due to a selective effect of this gonadotropin on P45017 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

The impact of unabated stimulation by human chorionic gonadotropin on the steroid hormone environment of pregnant rats and the spontaneous expression of ovarian cysts in female progeny

Unabated stimulation by low doses of human chorionic gonadotropin (hCG) induces ovarian cysts in pregnant rats. In order to determine the impact of these in vivo treatments on the hormonal milieu of pregnancy, and the potential impact of an aberrant cystic-ovary state during pregnancy on the resulting female offspring, pregnant rats were treated with either 0 (control), 1, or 3 IU hCG twice daily for at least 9 days, beginning on day 13 of pregnancy. Serum was harvested from control and hCG treated animals on days 15, 17, 19, and 22 of pregnancy. Control pregnant rats and animals treated with 1 IU hCG shared similar serum profiles for progesterone (P4), androstenedione (A4), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), androsterone (A5), and estrone (E1) between days 15 and 22 of pregnancy. Testosterone serum concentrations were similar for control and 1 IU hGG-treated pregnant rats between days 15 and 19 of pregnancy; whereas, on day 22, 1 IU hGG-treated pregnant rats displayed lower serum testosterone than control pregnant rats (P < or = 0.05). In contrast, serum estradiol (E2) concentrations for 1 IU hCG- treated pregnant rats were greater than E2 values observed for control rats on days 15-19 of pregnancy (P < or = 0.05). Serum testosterone and 3alpha-diol values for 3 IU hCG-treated pregnant rats differed from those of control pregnant rats only on day 19 when these values were transiently greater for these hCG-treated animals compared with serum values for control pregnant rats (P < or = 0.05). Serum A4 values for 3 IU hCG-treated pregnant rats were elevated compared to values for control pregnant rats only on days 15 and 17 (P < or = 0.05). In contrast, serum E1, A5, and E2 were elevated on days 19-22, 17-22, and 15-22, respectively, in 3 IU hCG-treated pregnant rats compared to control pregnant rats (P < or = 0.05). No pups from control pregnant rats displayed ovarian cysts during the time they were observed postnatally. In contrast, 6 of 25 pups from 3 IU hCG-treated pregnant rats displayed cystic ovaries, without corpora lutea, on day 55 of age. Serum steroid concentrations for these cyst-bearing progeny were similar to those of female progeny from control pregnant rats, whereas female progeny without ovarian cysts from 3 IU hCG-treated pregnant rats displayed differences in serum steroid values from those of progeny from control pregnant rats (P < or = 0.05). The data support the concept that an aberrant, yet physiologic hormonal environment associated with the induction of ovarian cysts during pregnancy in rats, can lead to the spontaneous establishment of an ovarian cystic state in at least a subset of the female progeny. Further, the date suggest that tonically increased ovarian estrogen production during pregnancy, reflected by tonically elevated peripheral serum estrogen concentrations, may play a pivotal role in the etiology of an ovarian cystic state in this subset of daughters from hCG-induced, cyst-bearing pregnant rats.     

Exposing cultured mouse ovarian follicles under increased gonadotropin tonus to aromatizable androgens influences the steroid balance and reduces oocyte meiotic capacity

Acquisition of oocyte developmental competence relies on the well-controlled events accompanying antral follicular development. Elevated basal androgen levels, as in PCOS, potentially affect oocyte quality. Current experiments in an in vitro follicle bioassay studied dose-effects of androstenedione and testosterone on FSH and hCG stimulated antral follicle growth and meiotic maturation. The addition of either androgens altered follicle's endogenous production of androstenedione, testosterone, estradiol, and progesterone and affected the oocyte's capacity to resume meiosis. Exposure to 200?nM androstenedione induced an increased production of testosterone and estradiol. Exposure to a concentration of ≥200?nM testosterone induced elevated levels of estradiol and progesterone. Significant dose-dependent negative effects on polar body extrusion were seen at concentrations of ≥200?nM of either androgen. In addition, chromosome displacement on the metaphase plate was observed in oocytes obtained from androstenedione-treated follicles. Follicles exposed to a combination of 25?mIU/ml FSH and 3?mIU/ml hCG and elevated aromatizable androgens altered the steroid production profile, affected the follicular development and impaired oocyte meiotic competence.     

The hormonal activity of the postmenopausal ovary.

The origin of plasma sex hormones in postmenopausal women was studied by determining plasma levels under basal conditions, after ACTH stimulation, and after dexamethasone suppression, as well as after hCG stimulation. Values obtained in postmenopausal women were compared with values observed during the follicular phase of the cycle in young women on the one hand, and with values in ovariectomized women of postmenopausal age on the other hand. All sex steroid levels studied with the exception of estrone, were significantly lower in postmenopausal women than in young women during the early follicular phase of the cycle. In ovariectomized women only androgen levels (testosterone, androstenedione, dihydrotestosterone, and to a lesser extent dehydroepiandrosterone,) were lower than in normal postmenopausal women, estrogen, 17 hydroxyprogesterone, and progesterone levels being similar. ACTH increased all plasma steroid levels except estradiol, whereas after dexamethasone, all sex hormone levels were significantly decreased. hCG stimulation finally caused an increase of borderline statistical significance in testosterone, dehydroepiandrosterone, and 17-hydroxyprogesterone levels. We have concluded from this study that the adrenal cortex is almost the exclusive source of plasma estradiol, estrone, progesterone, and 17OH progesterone and the most important source of plasma dehydroepiandrosterone; that the postmenopausal ovary appears to be responsible for about 50% of plasma testosterone and 30% of androstenedione levels; and that hCG stimulation with 5000 IU daily for 3 days, hardly influences steroid secretion by postmenopausal ovaries.     

Hyperinsulinemia and human chorionic gonadotropin synergistically promote the growth of ovarian follicular cysts in rats.

Tonically elevated serum luteinizing hormone (LH) and hyperinsulinemia are prominent features of polycystic ovary syndrome (PCO) in women, but the relative roles of LH and insulin in the pathogenesis of PCO is still unknown. The present study was undertaken to determine the effect(s) hyperinsulinemia might have on the induction of follicular cysts by LH/human chorionic gonadotropin (hCG) in the rat. Beginning on day 85 of age, adult female rats were given one of the following in vivo treatments: (1) vehicle alone; (2) a high-fat diet to control for the effects of weight-gain; (3) up to 6 U insulin per day; (4) 50 micrograms gonadotropin-releasing hormone (GnRH) antagonist (GnRHant) per day; (5) 1.5 IU hCG twice daily; (6) insulin + hCG; (7) insulin + GnRHant; (8) hCG + GnRHant; or (9) hCG + insulin + GnRHant. After 22 days of treatment, animals were killed on day 23, trunk blood was collected, and ovaries were excised for histological study. Regular cycles, assessed by vaginal smears, ceased after 10 days for most animals in treatment groups receiving hCG, but continued in all other treatment groups. All the animals in each hCG-treated group developed either unilateral or bilateral cystic ovaries, while no animals in the groups not receiving hCG developed follicular cysts. More animals from each group treated with both hCG and insulin possessed bilateral ovarian cysts than did rats treated with hCG alone: 80% and 60%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Obligatory roles for follicle-stimulating hormone (FSH), estradiol and androgens in the induction of small polyfollicular ovarian cysts in hypophysectomized immature rats

Immature hypophysectomized (HYPOXD) rats develop large, polyfollicular ovarian cysts in response to unabated, combined stimulation by subovulatory doses of human chorionic gonadotropin (hCG) and highly purified ovine follicle-stimulating hormone (FSH). Further, circulating amounts of androstenedione (A4) and estradiol (E2), but not testosterone or dihydrotestosterone (DHT), change in parallel with the development of these cysts. To determine the potential roles of either A4 or E2 at the level of the ovary in the induction of ovarian cysts, pellets containing either (1) cholesterol (placebo; controls); (2) A4; or (3) E2 were administered subcutaneously (sc) to immature HYPOXD rats. Some of these animals also received either twice-daily sc injections of 1 IU hCG, or daily sc injections of 2 μg FSH, for 13 days. Ovaries and sera were harvested from all treatment groups on the morning of day 14 of the combined-hormone treatment schedule. As expected, ovaries from HYPOXD rats treated with placebo, A4, or E2 pellets (with or without hCG) failed to display antral follicles. Ovaries from HYPOXD rats treated with FSH and a placebo pellet displayed polyfollicular, atretic, small antral follicles with unstimulated thecal shells. In addition, the ovarian stromal-interstitial tissue had an unstimulated appearance. In contrast, ovaries from HYPOXD rats treated with FSH plus either A4 or E2 implants displayed stimulated stromal-interstitial tissue as well as small follicular cysts and precysts with stimulated thecal shells. The number of cysts and precysts observed in the largest ovarian cross-sections for animals treated with FSH + A4 (17.0 ± 3.0) was less than that observed in the largest ovarian cross-sections for HYPOXD rats treated with FSH + E2 (40.2 ± 10.1; p < 0.05). To determine if the development of ovarian cysts in response to FSH + A4 was due, at least in part, to the metabolism of A4 to E2, HYPOXD rats were treated with either (1) placebo pellets; (2) pellets containing dihydrotestosterone (DHT) which cannot be metabolized to estrogen; (3) E2 pellets plus DHT pellets (E2 + DHT); (4) FSH + DHT; or (5) FSH + E2 + DHT. The largest ovarian cross-sections from FSH + DHT-treated HYPOXD rats displayed 18.3 ± 4.1 small follicles with a mean diameter of ∼0.437 mm which possessed few granulosa cells. The thecal and stromal-interstitial tissues in these ovaries were unstimulated, which indicates that these small degenerating follicles were atretic rather than cystic. In contrast, the largest ovarian cross-sections from FSH + E2 + DHT-treated HYPOXD rats displayed 51.6 ± 2.4 cysts with stimulated thecal shells and a mean diameter of ∼0.634 mm. Further, these cysts were arranged in a “string of pearls” pattern and the ovarian stromal-interstitial tissue possessed a stimulated appearance. These data demonstrate a direct, unambiguous role at the level of the ovary for unabated tonic stimulation by FSH plus estrogen in the development of small polyfollicular cysts in HYPOXD rats. Further, the data also indicate that, at least in HYPOXD rats, combined, tonic stimulation by FSH plus estrogen and androgen is sufficient for the development of small, polyfollicular ovarian cysts in a “string of pearls” pattern. These observations are in distinct contrast to our previous observations that tonic stimulation by FSH + hCG results in the induction of large ovarian cysts in HYPOXD rats and provide tantalizing new insights regarding the potential importance of specific hormones at the level of the ovary in the induction of specific types of cystic follicles.     

Alterations in follicle development, steroidogenesis, and gonadotropin receptor binding in a model of ovulatory blockade

Immature female rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) before gonadotropin-induced follicle development and ovulation ovulate significantly fewer ova compared with controls. This study was designed to investigate potential ovarian-specific mechanisms of TCDD-mediated inhibition of ovulation. Immature hypophysectomized rats were treated with TCDD (32 microg/kg) or corn oil vehicle. Follicle development was initiated by injection of 10 IU PMSG 24 h after TCDD, and ovulation was induced 52 h after PMSG by injection of 10 IU hCG. The number of ova flushed from the oviduct was assessed the morning after hCG injection, and ovaries were collected at multiple times throughout the treatment schedule for histological analysis and analysis of FSH and hCG receptor binding and ovarian cAMP levels. In addition, serum levels of estradiol and progesterone were determined. Control rats ovulated 9.3 +/- 1.5 ova, whereas TCDD-treated rats ovulated 0.6 +/- 0.3. Gonadotropin receptor binding was evaluated 52 h after PMSG at the usual time of hCG injection to induce ovulation. Both FSH binding and hCG binding were significantly reduced in animals treated with TCDD. Serum estradiol levels in control animals were increased by 52 h after PMSG administration. In contrast, serum levels of estradiol in TCDD-treated animals remained low. In response to the ovulatory dose of hCG, serum levels of both estradiol and progesterone increased in control animals. Steroid levels also increased in TCDD-treated animals, but did not reach the peak levels observed in controls. TCDD treatment further resulted in lower ovarian cAMP levels at 52 h post-PMSG and at 5 h post-hCG. Together the data indicate that TCDD treatment altered the ability of the ovary to respond to PMSG, resulting in the development of follicles not comparable to controls (lower gonadotropin binding, lower estradiol production, lower levels of cAMP). It appears that critical steps in the development and maturation of follicles are disrupted by TCDD.     

Steroidogenesis by equine preovulatory follicles: relative roles of theca interna and granulosa cells

Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17 beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of estrus; n = 4) and during late estrus (fourth or fifth day of estrus; n = 4). Preparations of theca interna and granulosa cells were cultured for 3 days in medium with or without equine LH, FSH, LH plus FSH, or CG (100 ng/ml) in the presence or absence of 0.5 microM testosterone, and culture media were assayed for progesterone, androstenedione, and 17 beta-estradiol. Progesterone was the predominant steroid secreted by granulosa cells in the absence of exogenous testosterone. Its accumulation was significantly higher in cultures of granulosa cells from late vs. early estrus (P less than 0.05), and all gonadotropins stimulated progesterone secretion at both stages of follicular development (P less than 0.05). In contrast, granulosa cells secreted very low amounts of androstenedione in vitro, and only very small amounts of 17 beta-estradiol were produced when cells were cultured in medium without testosterone. However, the addition of testosterone caused a 170-fold increase over control values in estradiol accumulation over 3 days of culture (P less than 0.0001), clearly indicating the presence of a very active aromatase enzyme system in equine granulosa cells. Steroid secretion by theca interna differed in several respects from secretion by granulosa cells. Theca interna from early and late estrous follicles secreted negligible amounts of progesterone in vitro, and equine gonadotropins had no effect on its secretion. Also, theca interna secreted only small amounts of estradiol in vitro, and its accumulation was not increased by the addition of exogenous testosterone. Also, in contrast to granulosa cell cultures, androstenedione was the predominant steroid secreted by theca interna from early and late estrous follicles. In conclusion, this study does not support the current model of equine follicular steroidogenesis, which holds that 17 beta-estradiol biosynthesis derives primarily from the theca interna layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estrogen regulation of thecal cell steroidogenesis and differentiation: thecal cell-granulosa cell interactions

Estrogen regulation of thecal cell steroidogenesis and differentiation was investigated with cells from ovarian antral follicles. Bovine theca interna cells were isolated and cultured in serum-free conditions to evaluate the effects of estradiol on thecal cell production of androstenedione, testosterone, and progesterone. Estradiol increased thecal cell androgen production throughout a 6-day culture period; however, the basal and stimulated levels of androgen production diminished after day 3 of culture. Androstenedione accumulation was approximately 10-fold greater than that of testosterone. In contrast to the stimulatory effects that estradiol had on androgen production, estradiol suppressed progesterone production throughout the 6-day culture period. Comparison of the effects of estradiol and hCG on thecal cells from small (less than 5 mm), medium (5-10 mm), and large (greater than 10 mm) antral follicles demonstrated that estradiol stimulated androgen production to a greater extent than hCG with cells from all of these stages of follicle development. Estradiol stimulation of androstenedione was greater in theca from small follicles than in theca from medium or large follicles. In contrast, suppressive effects of estradiol on progesterone were most apparent on thecal cells from medium and large follicles and less apparent on theca from small follicles. Estradiol stimulated androstenedione production in a dose-dependent fashion, with a minimum effective concentration of 10(-9) M and a maximum effective concentration of 10(-7)-10(-6) M. Concentrations greater than 10(-6) M estradiol resulted in a decline in the stimulatory response and may be important in the preovulatory follicle to suppress thecal cell androgen production and initiate the process of luteinization. Progesterone production was slightly stimulated by 10(-9) M estradiol, whereas higher concentrations (10(-7)-5 x 10(-6) M) resulted in a dose-dependent suppression of progesterone production. Interestingly, combined treatment of thecal cells with estradiol and hCG resulted in a greater than additive stimulation of androstenedione production, and estradiol decreased the ability of hCG to stimulate progesterone production. Observations demonstrate that estradiol can dramatically alter thecal cell production of steroids and support a hypothesis that steroid-mediated interactions between granulosa and thecal cells play an important role in regulating cellular function within follicles. The data provide evidence that a local feedback loop may exist in ovarian follicles, where androgens produced by thecal cells are used as a substrate for granulosa cell aromatization into estrogens, which, in turn, may feed back to stimulate thecal cell production of androgens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ovulation induction with human menopausal gonadotropin compared to human urinary follicle-stimulating hormone results in a significant shift in follicular fluid androgen levels without discernible differences in granulosa-luteal cell function

Follicular fluid estradiol, progesterone, testosterone, and androstenedione levels were compared in 2 groups of spontaneously ovulatory women undergoing ovulation induction with human menopausal gonadotropin (hMG; which contains equal amounts of LH and FSH) or human urinary FSH (huFSH). The results were correlated with the ratios of embryo cleavage and pregnancy. Although significantly more FSH [1268 +/- 38 (+/- SEM) vs. 953 +/- 38 IU; P less than 0.05] was required for equivalent hyperstimulation in hMG compared to huFSH cycles, the number of oocytes retrieved and fertilized and the number of embryos transferred were similar for the 2 ovulation induction protocols. Forty-two follicles from 21 women stimulated with hMG and 38 follicles from 15 women stimulated with huFSH were examined and found to be representative of the total cohort of aspirated follicles. Follicular fluid estradiol and progesterone levels were similar, but hMG-stimulated follicles contained significantly more testosterone [7.83 +/- 0.52 (+/- SEM) vs. 6.30 +/- 0.42 ng/ml; P less than 0.03] and less androstenedione (24.4 +/- 3.6 vs. 37.8 +/- 5.0 ng/ml; P less than 0.03) than did huFSH-stimulated follicles. Embryonic cleavage rates were similar for all fertilized oocytes from both hMG- and huFSH-stimulated cycles, although pregnancy rates were significantly higher in huFSH cycles (40% vs. 9.5%; P less than 0.05). In addition, aromatase activity, progesterone production, and [125I]hCG-binding activity were compared in granulosa-luteal cells isolated from some of these women. Cells from 21 follicles from 9 women stimulated with hMG and 24 follicles from 9 women stimulated with huFSH were studied. There were no significant differences in aromatase activity, progesterone production, or [125I]hCG binding. Thus, the presence or absence of exogenous LH during ovulation induction with FSH has little direct effect on granulosaluteal cell function. However, the presence of LH during ovulation induction with FSH does appear to alter thecal androgen metabolism, resulting in higher testosterone and lower androstenedione levels in follicular fluid. Such a shift in androgen milieu may impair oocyte development and successful implantation.     

Contrasting effects of prolactin on luteal and follicular steroidogenesis

To determine whether prolactin affects both luteal and follicular production of testosterone and oestradiol, pseudopregnant rats, either intact or hypophysectomized on day 8, were injected daily between days 8 and 9 with 1.5 i.u. human chorionic gonadotrophin (hCG), 250 micrograms prolactin or a combination of both. Control rats were given vehicle. On day 9, blood was obtained from the ovarian vein and corpora lutea and follicles were isolated and incubated in vitro for 2 h. Administration of hCG to intact rats increased ovarian secretion of testosterone and oestradiol dramatically, but did not affect progesterone secretion. Hypophysectomy on day 8 of pseudopregnancy was followed by a drop in ovarian steroid secretion. Prolactin treatment of hypophysectomized rats markedly enhanced progesterone production but had no stimulatory effect on either testosterone or oestradiol. In contrast, hCG dramatically enhanced ovarian secretion of both testosterone and oestradiol without affecting progesterone secretion. Prolactin administered together with hCG antagonized the stimulation of both testosterone and oestradiol secretion by hCG, yet increased progesterone production. When the specific effects of hCG and prolactin administration on follicles and corpora lutea were studied separately, it was found that hCG treatment in vivo greatly stimulated testosterone and oestradiol production by both tissues in vitro. Since hCG only marginally affected aromatase activity in the follicle, had no effect on aromatase activity in luteal cells and did not increase progesterone synthesis, it appears that hCG acts to increase the formation of androgen substrate for oestradiol biosynthesis. Prolactin, administered with or without hCG, inhibited both basal and hCG-stimulated testosterone and oestradiol synthesis by the follicle. In sharp contrast to its inhibitory effect on follicular production of steroids, prolactin appears to be essential for LH stimulation of testosterone and oestradiol by the corpus luteum. In the absence of prolactin, luteal cells gradually ceased to respond to LH and decreased their output of testosterone and oestradiol. Prolactin administration to hypophysectomized rats did not affect luteal cell production of either steroid. However, corpora lutea of rats treated with prolactin responded to the hCG challenge with an increase in testosterone and oestradiol synthesis. In summary, results of this investigation demonstrate that prolactin affects follicular and luteal production of testosterone and oestradiol in opposite ways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interference with the gonadotropin-suppressing actions of estradiol in macaques overrides the selection of a single preovulatory follicle

We examined the role of the gonadotropin-suppressing effects of estradiol on the maturation of a single ovulatory follicle in cynomolgus monkeys (Macaca fascicularis) by administering ovine antiestradiol antibodies during the mid through late follicular phase of the menstrual cycle. In each of three control animals, when the ovary containing the maturing follicle was removed during the late follicular phase, histological examination of the remaining ovary 10 days later revealed the presence of a single large maturing follicle. In contrast, in three experimental animals, when estradiol antibodies were infused from days 5 through 10 after unilateral ovariectomy, serum FSH and LH concentrations were elevated above those of control animals, and histological examination of ovaries 10 days after unilateral ovariectomy revealed the presence of two large maturing follicles in the remaining ovary of two animals and four large maturing follicles in the remaining ovary of the third animal. The ability of follicles recruited during passive immunization with estradiol antibodies to respond to exogenous gonadotropin was studied. In three control animals, the maturing follicle was destroyed on day 10 of the follicular phase, and 3 days later, each animal received an ovulatory dose of human CG. None of these control animals produced progesterone. In three experimental animals a continuous infusion of estradiol antibodies was initiated on day 5 of the follicular phase, and the largest antral follicle was destroyed on day 10. Three days thereafter the antibody infusions were terminated and each animal received an ovulatory dose of human CG. Each of these animals produced progesterone despite the destruction of the largest follicle 3 days earlier. These observations demonstrate that estradiol is the principal ovarian modulator of gonadotropin secretion during the follicular phase of the cycle and that interference with the gonadotropin-suppressing actions of estradiol results in continued recruitment and maturation of secondary follicles in the presence of a dominant follicle.     

Regulation of steroidogenesis in cultured porcine theca cells by growth factors

Growth factors [insulin-like growth factors (IGF-I, IGF-II), transforming growth factor-beta (TGF beta), epidermal growth factors (EGF)], found in the ovary and known to alter granulosal function, were assessed for their ability to modulate porcine thecal steroidogenesis. Theca cells from large porcine follicles (8-10 mm) were plated (5 x 10(5) cells/ml.well) in serum-free M199, treated with increasing doses of growth factors: IGF-1 (0.1-50 ng/ml), IGF-II (0.5-200 ng/ml), EGF (0.021-100 ng/ml), TGF beta (0.001-40 ng/ml), or insulin (0.01-50 micrograms/ml), with or without human CG [(hCG); 20 ng/ml], and incubated for 72 h. Levels of steroids in media were determined by RIA. Insulin increased (P less than 0.05) basal and gonadotropin-induced secretion of androstenedione, progesterone, estradiol, and testosterone. IGF-I increased (P less than 0.05) the basal and hCG-induced secretion of progesterone and androstenedione at the highest doses, but did not affect basal secretion of estradiol or testosterone. IGF-II, at the highest doses, increased (P less than 0.05) thecal steroidogenesis, but only after administration of hCG. In contrast, TGF beta increased (P less than 0.05) basal and gonadotrophin-induced secretion of estradiol but inhibited thecal secretion of progesterone, androstenedione, and testosterone. EGF did not alter thecal secretion of progesterone, androstenedione, or testosterone but significantly (P less than 0.05) inhibited basal and hCG-stimulated secretion of estradiol. In conclusion, insulin IGF-I, IGF-II, EGF, and TGF beta can modulate steroidogenesis in porcine theca cells.     

Intraovarian excess of nerve growth factor increases androgen secretion and disrupts estrous cyclicity in the rat

A single injection of estradiol valerate induces a form of cystic ovary resembling some aspects of the human polycystic ovarian syndrome. Preceding the development of follicular cysts, there is an increase in intraovarian synthesis of nerve growth factor (NGF) and the low affinity NGF receptor (p75 NGFR). Selective blockade of NGF actions and p75 NGFR synthesis in the ovary restored estrous cyclicity and ovulatory capacity in estradiol valerate-treated rats, suggesting that an increase in NGF-dependent, p75 NGFR-mediated actions within the ovary contributes to the development of cystic ovarian disease. We have tested this hypothesis by grafting NGF-producing neural progenitor cells into the ovary of juvenile rats that have been induced to ovulate precociously by a single injection of PMSG. The NGF-producing cells, detected by their content of immunoreactive p75 NGFR material, were found scattered throughout the ovary with some of them infiltrating the granulosa cell compartment of large, precystic follicles. Ovarian NGF content was 2-fold higher than in the ovary of rats receiving control cells. Estrous cyclicity was disrupted, with the animals showing prolonged periods of persistent estrus, and an almost continuous background of vaginal cornified cells at other phases of the estrous cycle. Morphometric analysis revealed that the presence of NGF-producing cells neither reduced the total number of corpora lutea per ovary nor significantly increased the formation of follicular cysts. However, the ovaries receiving these cells showed an increased incidence of precystic, type III follicles, accompanied by a reduced number of healthy antral follicles, and an increased size of both healthy and atretic follicles. These changes in follicular dynamics were accompanied by a selective increase in serum androstenedione levels. The results show that an abnormally elevated production of NGF within the ovary suffices to initiate several of the structural and functional alterations associated with the development of follicular cysts in the rat ovary.     

Ovarian responses of pregnant mare serum gonadotropin- and human chorionic gondotropin-primed rats: desensitizing, luteolytic, and ovulatory effects of a single dose of human chorionic gonadotropin.

We conducted a study to determine the morphological appearance and functional responsiveness of ovarian tissues after administration of hCG to 28-day-old rats primed 65 h earlier with PMS gonadotropin (PMSG) and after administration of a second dose of hCG 5 days later, i.e. to 33-day-old rats containing heavily luteinized ovaries. Sixty-five hours after the administration of 50 IU PMSG sc to 25-day-old rats, ovaries already contained an abundance of luteinized follicles and an adenylyl cyclase (AC) system that was responsive to LH, epinephrine, and NaF. The administration of 50 IU hCG sc at this time initially resulted in a loss of LH-responsive ovarian AC. Within 4 days of the hCG injection, the ovaries of the now 32-day-old rats were heavily luteinized, and ovarian AC was highly responsive to LH, epinephrine, and NaF. The administration of a single sc dose of 200 IU hCG to 33-day-old PMSC- and hCG-primed rats with luteinized ovaries resulted in a rapid desensitization of the ovarian AC to LH and a drop in serum progesterone levels, During the subsequent 7 days, serum progesterone levels continued to decline, while total ovarian AC reacquired responsiveness to LH by days 4--5 after the densensitizing dose of hCG. Dissection of ovarian components revealed, however, that the AC system of the corpora lutea originally present at the time of the second hCG injection remained permanently refractory to LH and that the AC in corpora lutea newly formed from freshly ovulated follicles exhibited a significant responsiveness to LH, epinephrine, and NaF. However, these new corpora lutea were not fully active, since serum progesterone never rose. Subcutaneous administration of 50 IU hCG to 33-day-old PMSG- and hCG-primed rats also promoted a rapid loss of AC responsiveness to LH. This lower concentration of hCG was not sufficient to promote follicular development or ovulation, and the ovarian AC remained refractory to LH for at least 7 days. Intravenous administration of 75 IU hCG to 33-day-old PMSG- and hCG-primed rats similarly promoted a rapid and permanent loss of luteal AC responsiveness to LH; again, follicles did not mature to a preovulatory state and, in fact, appeared to undergo atresia rather than ovulation. These results indicate that in heavily luteinized ovaries 1) hCG promotes desensitization of rat luteal AC to LH, 2) Desensitization of AC to LH stimulation in corpora lutea is permanent and irreversible, and 3) only under conditions where follicles mature and ovulate and new corpora lutea are formed does total ovarian AC reacqure responsiveness during the subsequent week.     

Regulation of 17 beta-hydroxysteroid oxydoreductase of the Xenopus laevis follicle by gonadotropin and prolactin

Ovarian follicles of Xenopus laevis, incubated in vitro actively metabolized radioactive 4-androstene-3, 17-dione to testosterone, indicating the presence of a 17 beta-hydroxysteroid-oxydoreductase (17 beta-HSOR). This enzymatic activity was found to be solely localized in the follicular envelopes. In vitro treatment of follicles with hCG for 2 hr induced a 40% decrease of the 17 beta-HSOR activity. The gonadotropin-induced inhibition was dose-dependent and only observed with gonadotropic LH-like hormones. Concomitant treatment with prolactin completely prevented the hCG effect.     

Synergistic effects of insulin-like growth factor-I and human chorionic gonadotropin in the rat ovary

Insulin and low doses of lutenizing hormone (LH) activity (human chorionic gonadotropin [hCG]) act synergistically in the rat to produce anovulation, large ovarian cysts, and elevated plasma androstenedione levels. Further, both insulin and insulin-like growth factor-I (IGF-I) affect the ability of gonadotropins to enhance both ovarian theca and granulosa cell function in vitro. The present series of experiments were performed to determine if recombinant human IGF-I (rhIGF-I) can act in a manner similar to insulin when combined with subovulatory doses of hCG in adult normally cycling rats. Fifty-four female Sprague-Dawley rats were randomly assigned to the following treatment groups at the age of 64 days: (A) vehicle alone (controls, phosphate-buffered saline containing 0.09% pig gelatin), (B) twice-daily subcutaneous injections of 0.5 to 3.0 U insulin, (C) twice-daily subcutaneous injections of 1.5 U hCG, (D) both insulin and hCG, (E) twice-daily subcutaneous injections of rhIGF-I (2.5 mg/kg/d), and (F) both hCG and rhIGF-I. After 22 days of treatment, the animals were killed on day 23, trunk blood was collected, and the ovaries were excised for histological study. Eight of 9 control rats and 5 or 6 of 9 rats treated with insulin, hCG, or rhIGF-I alone displayed normal estrus cycles throughout the in vivo treatment period as assessed by daily vaginal smears. In marked contrast, only 1 animal treated with hCG + insulin and 2 animals treated with hCG + rhIGF-I continued to display vaginal smears indicative of normal cycling. Multiple large ovarian follicular cysts were found only in these latter 2 groups (3 of 9 animals in each group). Mean serum testosterone levels were significantly elevated in animals receiving insulin + hCG (0.72 +/- 0.28 v 0.17 +/- 0.03 ng/mL in controls, P = .05). Mean serum androstenedione levels were significantly elevated in animals receiving hCG and animals receiving rhIGF-I + hCG (5.57 +/- 0.99 and 2.39 +/- 0.68 ng/mL, respectively, v0.14 +/- 0.14 ng/mL in controls, P< .01 and P< .05, respectively). We conclude that rhIGF-I and insulin act synergistically with subovulatory doses of hCG to disrupt normal reproductive cycling, elevate serum androgen concentrations, and induce large ovarian cysts in intact adult rats.     

Use of gonadotropin-releasing hormone agonist to trigger follicular maturation for in vitro fertilization

In spontaneous cycles both LH and FSH are secreted in a surge at midcycle. In in vitro fertilization (IVF) cycles, hCG administration results in elevation of LH-like activity only. The objective of this study was to compare the effectiveness of a single midcycle dose of GnRH agonist with hCG on follicular maturation. Eighteen IVF cycles in 14 women were randomized to receive either 0.5 mg leuprolide acetate or 5000 IU hCG at midcycle. Both groups underwent identical ovarian stimulation and cycle monitoring. On the day of GnRH agonist or hCG administration, estradiol concentrations and the number of follicles 1.5 cm or larger were the same in both groups. Mean serum LH and FSH levels were elevated for 34 h after GnRH agonist administration. In contrast, mean serum hCG levels were elevated for approximately 6 days after the administration of hCG, and serum FSH levels did not change. Mean luteal phase serum estradiol concentrations were lower in the GnRH agonist group than in the hCG group (P less than 0.02). No differences were observed in mean serum progesterone or PRL during the luteal phase or in the length of the luteal phase in the two groups. The mean number of oocytes retrieved and embryo number and quality did not differ between the two groups. Three of nine GnRH agonist cycles and none of nine hCG cycles resulted in clinical pregnancy (P = 0.1). The results of this study indicate that GnRH agonist is able to simulate a midcycle surge of gonadotropins, leading to follicular maturation and pregnancy. Further work is needed to determine whether there is any clinical advantage of GnRH agonist over hCG administration with regard to pregnancy rates.