Role of the peripubertal pattern of FSH, LH and prolactin secretion in regulating in-vitro steroidogenesis and follicular growth within juvenile rat ovaries

Juvenile rat ovaries were placed in perifusion culture and exposed to (1) tonic FSH (200 ng PR-1 equiv./ml), (2) LH pulses (2/h, amplitude = 80 ng RP-1 equiv./ml), (3) tonic FSH and LH pulses, (4) tonic FSH with LH mini-surges, or (5) tonic FSH with LH and prolactin mini-surges. The LH mini-surge consisted of a series of 80 ng/ml pulses (2/h) with LH increasing to 180 ng/ml for 2 h then returning to the 80 ng/ml pulses. The prolactin mini-surge consisted of a series of 15 ng/ml pulses (2/h) with prolactin increasing to 40 ng/ml for 2 h before returning to the 15 ng/ml pulses. The LH mini-surge occurred at 14:00 h daily while a prolactin mini-surge occurred at 14:00 h and 06:00 h daily. Ovaries were perifused for 0 (in-vivo control), 24 or 48 h, incubated for 1 h in hormone-free medium to assess steroid secretion and subsequently prepared for histological analysis. After a 24 h exposure to FSH, oestradiol secretion was increased, while exposure to LH pulses enhanced progesterone secretion. Treatment with FSH, LH pulses or FSH plus LH pulses decreased the number of small antral follicles by 24 h of perifusion compared to control (P less than 0.05). The LH mini-surge maintained the small and medium-sized antral follicles after 24 h and increased the number of preovulatory-sized follicles over controls by 48 h (P less than 0.05). Prolactin/LH mini-surges increased the number of preovulatory-sized follicles within 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose-related effects of luteinizing hormone on the pattern of steroidogenesis and cyclic adenosine monophosphate release in superfused preovulatory rat follicles

The secretion of steroids and the release of cAMP in response to repeated luteinizing hormone (LH) stimulation were examined during superfusion of isolated preovulatory rat follicles. A high dose of ovine LH (1 microgram/ml for 20 min) caused a prolonged increase in the secretion of progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OHP) and a transient increase in the secretion of testosterone (T) and estradiol-17 beta (E2), and was accompanied by a peak of cAMP release. A single pulse of LH at a low dose level (10 mg/ml for 20 min) gave a limited increase in T secretion, but no clear change in P, 20 alpha-OHP and E2 secretion or cAMP release. When the follicles were challenged with a second pulse of LH (at 1 microgram/ml), the response varied according to the dose of LH delivered in the preceding pulse. Following exposure to the high dose of LH, the follicles were partially refractory to the second LH challenge in terms of cAMP and P and the secretion of T and E2 remained low. The low dose of LH, however, had a conditioning effect on the follicles since the response to the second LH challenge was amplified in terms of P, 20 alpha-OHP and cAMP. In this case a secondary increase in T and E2 secretion was found. The differential response to varying doses of LH are likely to reflect the physiological control of steroidogenesis during final follicular maturation.     

Role of gonadotropins and insulin in controlling steroidogenesis and growth of antral bovine follicles in perifusion culture

Bovine follicles (2 to 4 mm in diameter) were isolated from the ovaries of 4-to 6-mo-old Holstein calves and placed in perifusion culture. Groups of 6 to 8 follicles/flask were cultured for 4 or 21 h with 1) no hormones; 2) tonic follicle-stimulating hormone (FSH) (10 ng/ml) and luteinizing hormone (LH) pulses (4 ng/ml) once every 4 h; 3) insulin (200 I.U./l); or 4) tonic FSH, LH pulses and insulin. After 0, 4 and 21 h of perifusion culture, each follicle was incubated in 1 ml of medium containing 3(H)-thymidine for 1 h. The 3(H)-thymidine incorporated into DNA of the follicle as well as the amount of estradiol-17 (E2) and testosterone (T) secreted into the medium were determined. Follicles treated with or without gonadotropins secreted higher levels of E2 and T after 4 h of perifusion compared to the 0 h controls. This elevated secretion rate was not maintained and 3(H)-thymidine incorporation was not increased over 0 h control values after 21 h of culture. Insulin suppressed the T secretion after 4 h in culture and increased 3(H)-thymidine incorporation at both 4 and 21 h of culture. After 21 h of culture, the gonadotropin and insulin treatment also enhanced 3(H)-thymidine incorporation. These results demonstrate that insulin is more mitogenic than the gonadotropin treatment tested, suggesting that insulin or insulin-like factors may play a physiological role in the growth of bovine follicles in vivo.     

Inhibitory action of leptin on early follicular growth differs in immature and adult female mice.

In order to investigate the action of leptin on early follicular growth, preantral follicles, 95-115 microm in diameter were mechanically isolated from the ovaries of BDF1 hybrid immature (11-day-old) and adult (8-wk-old) mice, and cultured for 4 days in vitro. Follicular growth was assessed by daily changes in follicular diameter and by the amount of estradiol and immunoreactive (IR)-inhibin released into the culture medium at Day 4. Preantral follicles from immature mice showed a significant development in follicular growth as a result of stimulation by GH (1 mIU/ml), insulin-like growth factor (IGF)-I (100 ng/ml) + FSH (100 mIU/ml), and GH (1 mIU/ml) + FSH (100 mIU/ml). Although leptin at concentrations of 1-1000 ng/ml did not have any significant effect on follicular growth stimulated by IGF-I or GH, it significantly inhibited follicular growth in a dose-related manner when follicles were stimulated by IGF-I + FSH and GH + FSH, respectively, suggesting that leptin attenuated the additive effect of FSH. On the other hand, preantral follicles from adult mice were cultured in the presence of FSH, and FSH-dependent follicular growth was inhibited by leptin in a dose-related manner. Because FSH stimulates cAMP production, we investigated the involvement of cAMP in the inhibitory mechanisms of leptin. Preantral follicles from immature and adult mice were cultured in the presence of either 8-Br-cAMP or forskolin. Both 8-Br-cAMP and forskolin significantly increased follicular diameter and hormone secretion in both immature and adult mice. However, 8-Br-cAMP and forskolin-stimulated follicle growth and hormone secretion were significantly inhibited in immature mice by coadministration of leptin, whereas growth of preantral follicles from adult mice was not inhibited by addition of leptin to cultures. These results indicate that leptin causes an inhibitory effect on the early follicular development of both immature and adult mice, but the inhibitory mechanisms of leptin are different.     

Effect of the peripubertal pattern of LH and FSH secretion on in-vitro oestradiol-17 beta secretion and follicular growth within juvenile rat ovaries

Juvenile rat ovaries were placed in perfusion culture and exposed to (1) no gonadotrophin, (2) tonic NIH-FSH (200 ng RP-1 equiv./ml) or (3) NIH-FSH + NIH-LH pulses (2/h, amplitude = 80 ng RP-1 equiv./ml). After 3 h of perifusion, the ovaries were prepared for histological analysis and the perifusate assayed for oestradiol-17 beta. Since the NIH-FSH preparation is contaminated with LH, a second experiment was conducted using recombinant bovine LH and FSH. Ovaries were perifused for 3 h with (1) no hormones, (2) recombinant FSH (200 ng/ml) or (3) recombinant FSH plus 25 ng recombinant LH/ml. NIH-FSH alone increased the number of mid-size antral follicles (P less than 0.05) and decreased the number of small antral follicles (P less than 0.05). Pulsatile LH in the presence of FSH increased the number of mid-size antral follicles without reducing the number of small antral follicles. Studies with recombinant FSH and LH demonstrated that both FSH and LH are necessary to stimulate follicles to grow, indicating that the growth-promoting property of the NIH-FSH is due to the presence of both FSH and LH. Regardless of whether NIH or recombinant gonadotrophins were used, follicular growth was induced without increasing oestradiol secretion. These results demonstrate that enhanced oestradiol secretion is not essential for the induction of follicular growth, while both LH and FSH are necessary to stimulate small antral follicles to grow into mid-size antral follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

正常月经周期妇女卵泡液中激素水平的变化

本文测定了24例正常月经妇女在不同时相、不同大小卵泡的卵泡液中雌二醇(E_2)、孕酮(P_0)、雄烯二酮(A)、睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)和催乳素(PRL)的含量,并分析其与外周血中相应激素浓度的关系。测定结果显示:小卵泡的卵泡液中E_2、Po,FSH,LH水平低于大卵泡中水平,而A和T水平则相反。排卵前大卵泡中E_2(9815nmol/L),P_0(3316nmol/L),FSH(1.34IU/L)和LH(3.9lIU/L)达最高值。A(280nmol/L)和T(137nmol/L)却较小卵泡中水平低(相应为692nmol/L和176nmol/L)。PRL水平在大小卵泡中无显著性差异。卵泡液中甾体激素水平高于外周血7—20.000倍,FSH、LH水平为外周血的10—80％,PRL水平为60％—3倍。     

Preovulatory biosynthesis and granulosa cell secretion of immunoreactive oxytocin by goat ovaries

Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (less than 0.1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1-2 mm), medium (3-5 mm) and large (greater than 5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6-12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4-12 ng/mg protein). Addition of PGE-2 (1 microgram/ml) caused a further significant (P less than 0.05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2 alpha, FSH and LH were ineffective when added to culture media. Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0.4 pg/ml to 2.4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0.1 ng/mg protein) but rose to 1.4 and 3.5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. (ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of estrogen deprivation on the reproductive physiology of male and female primates

The availability of CGS 16949A, CGS 20267 and CGP 47645, a series of aromatase inhibitors (AIs) having high specific activity and specificity, made possible this study wherein the need for estrogen (E) for regulating (a) follicular maturation/ovulation, luteal function and pregnancy establishment, and (b) testicular function of the bonnet monkey (Macaca radiata) has been examined. Generally these compounds, used in the range of 500 μg to 2.5 mg/day did not inhibit follicular maturation although they did reduce E levels. Although low doses had no effect on ovulation it appears that relatively high doses of CGS 20267 and CGP 47645 could be inhibiting it. Three oral doses of letrozole (CGS 20267, each dose of 2 mg) during the follicular phase resulted in the formation of multiple follicles in cycling females, and these could be ovulated by exogenous hCG (1000 IU) treatment. Although administration of AI during the early luteal phase had no effect on progesterone (P) production, it prevented pregnancy establishment. Whereas AI administration in the female had no significant effect on luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels (except at high drug dosages), it significantly increased serum testosterone (T) levels in the male. Sustained high levels of T (30–50 ng/ml) could be maintained for 100 days by administering 2.5 mg of CGP 47465 orally once in 5 days. Blockade of E synthesis in the male led to the disruption of testicular germ cell transformation, which in turn resulted in a significant reduction in sperm production. These studies with aromatase inhibitors in the monkey suggest that these compounds have a potential for use as fertility regulating agents in both the male and female primate.     

Steroid production in vitro by granulosa, theca, and luteal cells from goat ovaries

Basal progesterone (P4) production by isolated goat ovarian cells in vitro was in the order corpus luteum (CL) greater than granulosa (G) greater than theca (TH), while estradiol (E2) production was in the order TH greater than G greater than CL. In G cells, various concentrations (0.01 to 100 micrograms/ml) of luteinizing hormone (LH), human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) increased P4 and E2 secretion. Testosterone (T, 10(-9) to 10(-5) M) produced dose-dependent increases in P4 and E2 secretion. Testosterone and LH together had an additive effect on E2 secretion. The combined effect of the lower (less than 10(-6) M) concentrations of T and LH on P4 production was marginally higher than either agent alone, but the increase was statistically insignificant; at higher concentrations of T (10(-6) and 10(-5) M) in combination with LH, P4 secretion was similar to that with LH alone, but was significantly (p less than 0.01 and less than 0.001, respectively) less compared to that with T alone. Follicle-stimulating hormone and T together produced a synergistic effect on E2 and an additive effect on P4 production. In TH cells, a dose-dependent increase in P4 and E2 production was observed with LH and hCG, but the effect of FSH was not significant. Testosterone produced a dose-dependent increase in P4 and E2 secretion. Testosterone and LH together induced higher steroid production than either agent alone. However, the increase was not statistically significant compared to T alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation

The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.     

The ovarian follicle and fertility

The precise roles of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in the control of preovulatory follicle growth has been re-examined. Suppression of both pulsatile LH secretion and FSH or specific suppression of FSH results in an inhibition of preovulatory follicle growth beyond 2.5 mm dia. Infusion of sheep FSH alone in physiological amounts in the presence of basal, non-pulsatile LH results in the growth of preovulatory follicles. Co-infusion of large amplitude pulses of LH reduced or abolished this effect of FSH. It is suggested that: (1) FSH controls the number of follicles which develop; (2) selection of the large follicle destined to ovulate is directly related to the decline in the plasma concentration of FSH occurring during the period of follicle selection--thus, only the follicle(s) which can withstand this withdrawal of FSH will continue to develop; and (3) pulses of LH may directly affect the action of FSH on the follicle and play an important, hitherto unrecognized role in the selection of the ovulatory follicle by actively inducing atresia.     

An in vitro method to determine the selective inhibition of estrogen biosynthesis by aromatase inhibitors

Potency and selectivity of aromatase inhibition are parameters which ultimately influence the therapeutic efficacy of aromatase inhibitors. This report describes an in vitro model which allows an assessment of the selectivity with which aromatase inhibitors inhibit estrogen biosynthesis. Estrogen production was stimulated by incubating adult female hamster ovarian tissue with ovine LH. The production rates of estrogens (E), testosterone (T) and progesterone (P) were determined using radioimmunoassays to measure the amount of these steroids released into the incubation medium over a 4-hour incubation period. The selectivity of aromatase inhibition was assessed by determining the IC50S with which each inhibitor inhibited the production of E (end product), T (immediate precursor of E) and P (early precursor of E). Selectivity was studied for each of the 4 aromatase inhibitors, CGS 16949A (a new non-steroidal compound), 4-OH-androstenedione, aminoglutethimide and testolactone. CGS 16949A was the most potent of the four, followed by 4-OH-androstenedione, aminoglutethimide and testolactone. As far as selectivity was concerned, both CGS 16949A and 4-OH-androstenedione selectively inhibited aromatase judging from the IC50s for E and P production (CGS 16949A: IC50 for E & P = 0.03 & 160 microM, resp.; 4-OH-androstenedione: IC50 for E & P = 0.88 & greater than or equal to 330 microM, resp.). Aminoglutethimide was the least selective inhibitor of aromatase (IC50 for E & P = 13 & 60 microM, resp.). For testolactone, the least potent of the four (IC50 for E = 130 microM), no conclusive data were obtained concerning the selectivity of aromatase inhibition. Thus a simple, effective and reproducible method is described for assessing the selectivity with which aromatase inhibitors inhibit aromatase.     

Role of increased estradiol on altering the follicle diameters and gonadotropin concentrations that have been reported for double-ovulating heifers

During the preovulatory period in heifers that ovulate from two compared to one follicle, circulating concentrations of estradiol-17β (E2) are greater, diameter of follicles and concentration of FSH are reduced, and the LH surge occurs sooner. The effect of increased E2 on the reported characteristics of double ovulation was studied by treating heifers with 0.07 mg E2, 0.09 mg E2, or vehicle in four treatments at 6-h intervals (n=6 heifers/group), beginning at the time of expected follicle deviation (largest follicle, 8.5mm). There were no significant differences on follicle diameters or hormone concentrations between the 0.07 and 0.09 mg E2 groups, and heifers were combined into one E2 group (n=12). The E2 treatments induced concomitant preovulatory surges in LH and FSH at 34.0 ± 2.6h after first treatment, compared to 57.6 ± 4.5h in the vehicle group (P<0.0002). The E2 treatments did not affect FSH concentrations during the preovulatory gonadotropin surge. The diameter of the preovulatory follicle at the LH peak was smaller (P<0.0001) in the E2-treated group (10.2 ± 0.2mm) than in the vehicle group (13.1 ± 0.6mm). The hypothesis was not supported that the previously reported increase in circulating E2 in heifers with double preovulatory follicles accounts for the reported lesser concentrations in the preovulatory FSH surge in heifers with double ovulations. Hypotheses were supported that the reported earlier occurrence of the preovulatory LH surge and smaller preovulatory follicles in heifers with double ovulations are attributable to the reported increase in E2 from the double preovulatory follicles.     

Some endocrine traits of transgenic rabbits. II. Changes in hormone secretion and response of isolated ovarian tissue to FSH and ghrelin

In the present in vitro experiments we examined FSH- and ghrelin-induced changes in ovarian hormone secretion by transgenic rabbits. Fragments of ovaries isolated from adult transgenic (carrying mammary gland-specific mWAP-hFVIII gene) and non-transgenic rabbits from the same litter were cultured with and without FSH or ghrelin (both at 0, 1, 10 or 100 ng/ml medium). The secretion of progesterone (P4), estradiol (E2) and insulin-like growth factor I (IGF-I) was assessed by RIA. It was observed that ovaries isolated from transgenic rabbits secreted much less P4, E2 and IGF-I than the ovaries of non-transgenic animals. In control animals FSH reduced E2 (at doses 1-100 ng/ml medium) and IGF-I (at 1-100 ng/ml), but not P4 secretion, whereas ghrelin promoted P4 (at 1 ng/ml) and IGF-I (at 100 ng/ml), but not E2 output. In transgenic animals, the effects were reversed: FSH had a stimulatory effect on E2 (at 100 ng/ml) and ghrelin had an inhibitory effect on P4 (at 10 ng/ml). No differences in the pattern of influence of FSH on P4 and IGF-I and of ghrelin on E2 and IGF-I were found between control and transgenic animals. The present observations suggest that 1) both FSH and ghrelin are involved in rabbit ovarian hormone secretion, 2) transgenesis in rabbits is associated with a reduction in ovarian secretory activity, and 3) transgenesis can affect the response of ovarian cells to hormonal regulators.     

In vitro steroidogenesis by primary to antral follicles in the hamster during the periovulatory period: effects of follicle-stimulating hormone, luteinizing hormone, and prolactin

Hamster ovarian follicles at Stages 1 to 10 (Stages 1-4: follicles with 1-4 layers of granulosa cells (GC); Stages 5-7: 5-10 layers GC plus theca; Stages 8-10: antral follicles) were isolated on the morning of proestrus or estrus and incubated for 2 h in the absence or presence of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (Prl), progesterone (P4), 17 alpha-hydroxyprogesterone (17OHP), or androstenedione (A). Steroid accumulations in the media were measured by radioimmunoassay (RIA). On proestrus, without any hormonal stimulus, consistent accumulation of P4 through estradiol-17 beta (E2) occurred in low amounts only from Stage 6 and on; both FSH (5-25 ng) and LH (1-25 ng) significantly stimulated steroidogenesis by Stage 6-10 follicles, and the effects of FSH, except for Stage 10, were largely attributable to LH contamination. However, 25 ng FSH significantly stimulated A production by Stages 1-4, whereas 1-25 ng LH was ineffective. On estrus, follicles at all stages, especially 1-6, showed significant and dose-dependent increases in P4 production in response to FSH; both FSH and LH significantly stimulated P4 and 17OHP accumulation from Stage 5 onwards; however, there was no increase in A and E2 compared to controls. Even the smallest estrous follicles showed a shift to predominance of P4 accumulation. On proestrus, Prl had a negative influence on LH-induced accumulation of P4 and 17OHP by Stages 7-9 and 6-8, respectively, without affecting A or E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of GnRH antagonists treatment on gonadotrophin secretion, follicular development and inhibin A secretion in goats

The aim of this study was to determine, for goats, the effects of daily doses of GnRH antagonist on ovarian endocrine and follicular function. Ten does were given 45 mg FGA intravaginal sponges and then five were treated with daily injections of 0.5mg of the GnRH antagonist Teverelix for 11 days from 2 days after the day of sponge insertion, while five does acted as controls. Pituitary activity was monitored by measuring plasma FSH and LH daily from 2 days before the first GnRH injection to Day 12. Follicular activity was determined by ultrasonographic monitoring and by assessing plasma inhibin A levels during the same period. In treated does, the FSH levels decreased linearly (0.8 +/- 0.1 ng/ml to 0.5 +/- 0.1 ng/ml, P < 0.01) and remained lower than the mean concentration in control goats (0.8 +/- 0.1 ng/ml, P < 0.005). LH levels were also lower during the period of antagonist treatment (0.6 +/- 0.2 ng/ml versus 0.4 +/- 0.1 ng/ml, P < 0.0005). During GnRH antagonist treatment, there was a significant decrease in the number of large follicles (> or = 6 mm) from Day 3 of treatment (1.2 +/- 0.6, P < 0.0001), with no large follicles from Day 9. The number of medium follicles (4-5 mm in size) also decrease during the period of treatment (4.2 +/- 0.7 to 1.0 +/- 0.6, P < 0.0001), leading to a significant decrease in inhibin A levels when compared to the control (143.7 +/- 31.3 pg/ml versus 65.2 +/- 19.1 pg/ml, P < 0.00005). In contrast, the number of small follicles (2-3 mm) increased in treated goats from Day 4 of treatment (9.6 +/- 2.9 to 20.2 +/- 6.3, P < 0.005). Such data indicate that GnRH antagonist reduced plasma levels of FSH and LH with suppression of the growth of large dominant ovarian follicles and a two-fold increase in number of smaller follicles. The results confirm that GnRH antagonist treatment can be used in goats to control gonadotrophin secretion and ovarian follicle growth in superovulatory regimes.     

Follicular development and reproductive endocrinology during and after superovulation in heifers and mature cows displaying contrasting superovulatory responses

To understand the causes for poor response to superovulation in mature cows of high genetic potential, endocrine and follicular events during and after superovulation were compared in heifers (<2 yr old) yielding large numbers of embryos and cows (9 to 13 yr old) known to be poor embryo donors. Follicular development was monitored by daily ultrasonography. Blood samples were taken 2 to 3 times a day for the measurements of P4, E2, FSH and LH by RIA. Intensive blood collections at 15-min intervals for 6 h were also performed during preovulatory and luteal phases. The number of embryos produced in the heifers (15.2 +/- 2; mean +/- SEM) and the cows (0.6 +/- 0.4), was similar to the number of ovulatory follicles derived from ultrasonographic observations in the heifers (16.2 +/- 3.7), but not in the cows (7.8 +/- 2.8). Contrary to that observations in heifers, there was no increase in the number of 4- to 5-mm follicles in cows during superovulation. The number of larger follicles (>5 mm) increased during superovulation in both cattle groups, but it was significantly lower in cows than in heifers. During superovulation, the maximal E2 concentration was greater (P < 0.0001) in heifers than in cows. One cow showed delayed luteolysis during superovulation, while another had abnormally high FSH (>10 ng/ml) and LH (>3 ng/ml) concentrations following superovulation. All the cows had a postovulatory FSH rise which was not detected in the heifers. The results showed that attempts to improve superovulatory response in mature genetically valuable cows are hampered by a number of reproductive disorders that are not predictable from the study of the unstimulated cycle.     

Modulatory action of FSH on LH-induced follicular growth in rats

Three to four ovaries from rats on the day of di-oestrus I were placed in perifusion culture at 10:30 h and exposed to (1) no gonadotrophin (in-vitro controls); (2) tonic FSH (200 ng RP-1/ml); (3) tonic LH (30 ng RP-1/ml); (4) tonic FSH + tonic LH; or (5) tonic FSH plus hourly pulses of LH (amplitude = 50 ng/ml). The total amount of LH administered was 3060 ng RP-1 regardless of mode of delivery. After culture for 3 h, the ovaries were prepared for histological analysis. Compared to in-vitro controls, tonic LH stimulation increased the number of follicles with greater than 1.6 X 10(5) granulosa cells (P less than 0.05); it was estimated that each follicle in the larger size class increased by 5.5 +/- 2.7 X 10(4) cells. Tonic FSH or tonic FSH + tonic LH treatment did not promote growth into the 1.6 X 10(5) cell class. In the presence of tonic FSH, hourly LH pulses induced follicular growth similar to that observed after tonic LH treatment. The data demonstrate that LH promotes the growth of follicles in vitro. FSH modulates this stimulatory action of LH, allowing it to be expressed when LH is administered in hourly pulses.     

Differential steroid and gonadotropin response by individual tertiary porcine follicles in vitro. Possible physiologic role of atretic follicles

Since atretic follicles contain significant amounts of androgen and/or progesterone in their follicular fluid, we examined whether they also contribute to ovarian steroid secretion. Steroid secretion by atretic porcine follicles and their responsiveness to FSH was assessed by a perifusion system that allows for separate dynamic incubation of whole follicles in vitro. Identically treated nonatretic follicles of comparable size served as a reference group. The extent of granulosal pyknosis, on which the staging of atresia was based, was inversely related to follicular estradiol (E2) secretion and its responsiveness to FSH. Both basal and FSH-stimulated secretion of testosterone (T), androstenedione (A), and progesterone (P) were maintained by follicles in all stages of atresia. Secretion of A by late atretic follicles was greater than that in earlier stages or by nonatretic follicles. Atretic follicles may therefore release comparable or larger amounts of androgen and P into their intraovarian environment than do nonatretic follicles. We examined whether steroids secreted by atretic follicles in vitro could be utilized by nonatretic follicles. A static incubation system was used that allows for simultaneous incubation of a number of individual follicles. When nonatretic follicles were exposed to A, T, or P in physiologic concentrations (10(-7)-10(-5) M), their secretion of E2 increased 2-8-fold. Doses of FSH or LH that stimulated follicular steroid in vitro had no additional stimulatory effect when combined with A or P treatment, respectively. In conclusion, atretic follicles may contribute significantly to intraovarian levels of androgen and P.(ABSTRACT TRUNCATED AT 250 WORDS)