Infusion Of FSH Research Articles

Reproductive and metabolic determinants of granulosa cell dysfunction in normal-weight women with polycystic ovary syndrome

To determine the degree to which E2 hyperresponsiveness to FSH and antimüllerian hormone (AMH) overproduction in normal-weight women with polycystic ovary syndrome (PCOS) correlate with increased antral follicle number (AFN), hyperandrogenism, and/or metabolic dysfunction. Prospective cohort study. Academic medical center. Seven normal-weight women with PCOS (1990 National Institutes of Health criteria) ages 20-34years and 13 age- and body mass index- (BMI-; 18.5-25kg/m2) matched normoandrogenic ovulatory women were studied. All women underwent basal serum hormone and metabolic measurements, FSH stimulation testing with transvaginal ovarian sonography, frequently sampled IV glucose tolerance testing, and whole-body dual-energy x-ray absorptiometry. Serum hormone/metabolite levels, 24-hour serum E2 response to 150 IU recombinant human (rh) FSH infusion, AFN, insulin sensitivity, and body mass measurements. Serum E2 responsiveness to rhFSH and AMH levels were greater in women with PCOS than in BMI- and age-matched control women, as were serum androgen levels, AFN, and abdominal fat mass. In all women combined, serum E2 responsiveness to rhFSH was associated with AFN. Serum AMH levels, however, positively correlated with AFN but remained positively correlated with serum LH and free T levels and negatively correlated with total body fat and percent body fat, adjusting for AFN. In normal-weight women with PCOS, serum E2 hyperresponsiveness to rhFSH represents increased AFN, while elevated serum AMH levels reflect opposing effects of stimulatory reproductive (hyperandrogenism and increased AFN) versus inhibitory metabolic (body fat) factors. Given the small number of subjects reported, additional follow-up studies are required to confirm these data.

Effects of the Rate and Duration of Physiological Increases in Serum FSH Concentrations on Emergence of Follicular Waves in Cyclic Ewes1

Large antral follicles grow in waves in the ewe, with each wave triggered by a peak in serum FSH concentrations. In this study, our objectives were to determine if the slope of the rise in the FSH peak affects the ability of the peak to trigger wave emergence (experiment 1), and whether increasing serum FSH concentrations and holding them at peak concentrations would provide a stimulus for constant emergence of large antral follicles (experiment 2). In experiment 1, cyclic ewes received ovine FSH (n = 6; 0.1 μg/kg, s.c.) or vehicle (n = 6; control) every 6 h for 42 h. This treatment created a peak in serum FSH concentrations (P < 0.05) during the early growth phase of the first follicular wave of the interovulatory interval and enhanced the growth of follicles in that wave (P < 0.05), but did not trigger emergence of a follicular wave. In experiment 2, cyclic ewes were infused constantly with oFSH (1.98 μg/h; n = 6) or vehicle (control; n = 6) for 60 h starting at the time of the second endogenously driven FSH peak of the interovulatory interval. Infusion of oFSH resulted in a super-stimulatory effect, with a peak in the mean number of large follicles (≥5 mm) on Day 2 after the start of FSH infusion (13 ± 1.2 large follicles per ewe, 1.8 ± 0.2 in control ewes; P < 0.001). In conclusion, exposing early growing antral follicles in a wave to a gradual increase in serum concentrations of FSH enhanced their growth, but did not trigger the expected new follicular wave, and infusion of a dose of oFSH within the physiological range caused a super-ovulatory response in cyclic ewes.

Effects of gonadotrophin treatments on meiotic and developmental competence of oocytes in porcine primordial follicles following xenografting to nude mice

Our objective was to improve the developmental ability of oocytes in porcine primordial follicles xenografted to nude mice, by treating the host mice with gonadotrophins to accelerate follicular growth. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Gonadotrophin treatments were commenced around 60 days after vaginal cornification in the mice. Ovarian grafts were obtained 2 or 3 days after treatment with equine chorionic gonadotrophin (eCG-2 and eCG-3 groups), after porcine FSH infusion for 7 or 14 days, or after infusion of porcine FSH for 14 days with a single injection of estradiol antiserum (FSH-7, FSH-14 and FSH-14EA groups, respectively). Gonadotrophin treatments accelerated follicular growth within the xenografts compared with that in control mice given no gonadotrophins, consistent with higher (P < 0.05) circulating inhibin levels in the gonadotrophin-treated mice. In contrast, circulating mouse FSH levels were significantly (P < 0.05) depressed. We recovered large numbers of full-sized oocytes with meiotic competence to the mature stage from the eCG-3, FSH-7, and FSH-14EA, unlike in the control group. Moreover, 56% of matured oocytes with the first polar body (n = 39) were fertilized in vitro in the FSH-14EA group. After in vitro fertilization and subsequent culture for 7 days, one blastocyst was obtained from each of the eCG-3, FSH-7 and, FSH-14EA groups, whereas no blastocysts appeared in the other groups. Exogenous gonadotrophins--not mouse FSH--stimulated the growing follicles that had developed from the primordial follicles in the xenografts: the effects were incomplete but improved to some extent the meiotic and developmental abilities of the oocytes.

Gonadotropin-Independent Proliferation of the Pale Type A Spermatogonia in the Adult Rhesus Monkey (Macaca mulatta)1

The goal of the present study was to examine the relative roles of testosterone (T) and FSH in the proliferation and differentiation of pale type A (Ap) spermatogonia in the rhesus monkey (Macaca mulatta). Twenty adult male monkeys were treated with daily injections of a GnRH-receptor antagonist, acyline, to suppress endogenous gonadotropin secretion during an experiment comprising three phases. Phase 1 established a chronic hypogonadotropic state marked by a profound decrease in testicular size. During phase 2, half the monkeys were implanted with T-filled capsules, and the other half received control implants. Treatment with T produced circulating T levels of approximately 15 ng/ml and normal testicular T content. At the end of phase 2, monkeys were fitted with indwelling i.v. catheters and housed in remote sampling cages for the final phase. During phase 3, five monkeys from the T- and non-T-treated groups were stimulated with recombinant human FSH. The remaining five monkeys from each group received an infusion of vehicle. On the last day of FSH or vehicle infusion, monkeys were bilaterally castrated after receiving an i.v. bolus of bromodeoxyuridine (BrdU). The BrdU labeling of Ap spermatogonia was robust in the hypogonadotropic group and was uninfluenced by treatment with T and FSH, either alone or in combination. In contrast, both T and FSH stimulated spermatogonial differentiation, and this effect was amplified by combined treatment. We conclude that marked Ap spermatogonial proliferation occurs constitutively and in a gonadotropin-independent manner and that differentiation of Ap into B spermatogonia is absolutely gonadotropin dependent and may be driven by either T or FSH.

In the adult male rhesus monkey (Macaca mulatta), unilateral orchidectomy in the face of unchanging gonadotropin stimulation results in partial compensation of testosterone secretion by the remaining testis.

This study examined, in adult monkeys, the role that gonadotropin-independent mechanisms play in compensation of testosterone (T) secretion by the testis that remains after unilateral orchidectomy (UO). We employed a model (testicular clamp), in which endogenous gonadotropin secretion was abolished with a GnRH receptor antagonist, and the gonadotropin drive to the testes was concomitantly replaced with an invariant iv pulsatile infusion of recombinant human LH and FSH (1-min pulse every 2.5 h: LH, 0.08-0.12 IU/kg.pulse; FSH, 0.12-0.32 IU/kg.pulse) that provided the Leydig cells with a physiological stimulus. Within 5 h of UO (n = 5), circulating T concentrations had declined to 43% of pre-UO levels. By d 4, however, loss of the first testis was partially compensated, as reflected by the finding that circulating T had reached a plateau of 67% of the pre-UO level, where it remained for the duration of the study (39 d). That the recovery in circulating T was the result of increased T secretion by the remaining testis was suggested by the finding that the pulsatile pattern and decay of T during the intergonadotropin pulse interval before and after UO were indistinguishable. Interestingly, inhibin B production by the remaining testis also showed a delayed, albeit, minor, compensation (13% on d 10-11; P > 0.05) after loss of the first testis. These results suggest that compensation in T production by the remaining testis after UO in adult monkeys may be achieved in part by a gonadotropin-independent mechanism that probably involves direct neural inputs to the primate testis.

Gonadotropin requirements for dominant follicle selection in GnRH agonist-treated cows.

A study was conducted to examine the effects of gonadotropins on ovarian follicular development and differentiation in GnRH agonist (GnRHa)-treated cattle. Holstein cows were allotted into two pre-treatment groups: controls (n = 5) and GnRHa-treated (n = 9). Ovaries were removed from control cows on day 5 following a synchronized estrus. Treatment with GnRHa resulted in follicular arrest at <5 mm. Following follicular arrest, GnRHa-treated cows received a constant infusion of FSH for 96 h (GnRHa/FSH), with a randomly selected subset receiving hourly pulses of LH in addition to FSH during the last 48 h of infusion (GnRHa/FSH + LH). At the end of infusion, ovaries were removed, follicles were counted and measured, and follicular fluid samples were collected from large follicles (>10 mm). Differences in expression of mRNA for LH receptor, FSH receptor, cytochrome P450 side-chain cleavage, 3beta-hydroxysteroid dehydrogenase, cytochrome P450 17alpha-hydroxylase (P450c17) and cytochrome P450 aromatase were determined in large follicles using in situ hybridization. The number of large follicles did not differ between GnRHa/FSH-treated and GnRHa/FSH + LH-treated cows (P = 0.64), but was greater than control animals (P < or = 0.004). Follicular fluid concentrations of estradiol-17beta and androstenedione were highest in GnRHa/FSH + LH-treated cows (P < or = 0.04), intermediate in control cows, and lowest in GnRHa/FSH-treated cows. Hybridization intensity of P450c17 was greater in GnRHa/FSH + LH-treated versus control or GnRHa/FSH-treated cows (P < or = 0.03). These results indicate that while FSH can support bovine follicular growth >10 mm, LH increases androgen production and expression of P450c17.

Administration of dihydrotestosterone to rhesus monkeys inhibits gonadotropin-stimulated ovarian steroidogenesis.

Androgens, in addition to serving as a substrate for estrogen biosynthesis, exert autocrine/paracrine actions on ovarian function. However, much of the information regarding the actions of androgens on the ovary has been obtained using rodents, and the extent to which these results can be extrapolated to higher primates is uncertain. The current study was initiated to determine the effects of dihydrotestosterone (DHT) and testosterone (T) on the responsiveness of the rhesus monkey ovary to exogenous FSH and LH in vivo. Rhesus monkeys whose spontaneous gonadotropin secretion was interrupted with a GnRH antagonist received s. . implants of either DHT or T for 5 d before and continuing throughout a 15-d i.v. infusion of human FSH and LH. Neither T nor DHT treatment synergized with FSH/LH to stimulate estrogen production or increases in ovarian weight. Rather, administration of DHT significantly reduced estrogen secretion and the augmentation of ovarian weight in response to exogenously administered FSH and LH. These results indicate that high concentrations of DHT are antagonistic to gonadotropin-stimulated ovarian function in primates.

The FecB (Booroola) gene acts at the ovary: in vivo evidence.

The aim of this study was to differentiate between pituitary and ovarian actions of the FecB gene by measuring the ovarian response to a standardized treatment with gonadotrophins designed to mimic the changes in FSH and LH that occur in the follicular phase of the ovarian cycle in ewes, with (Fec(B/-), n=6) and without (Fec(+/+), n=9) the gene, that were rendered hypogonadotrophic by pretreatment with a potent antagonist of GnRH. Ewes with ovarian autotransplants were used to facilitate the assessment of follicular function by the collection of ovarian venous blood and ultrasonography. The gonadotrophin regimen resulted in concentrations of FSH and LH that were similar to concentrations found in a normal cycle and did not differ between genotypes. Follicular development and ovulation occurred in all animals, and patterns of secretion of oestradiol, androstenedione and inhibin A were normal. Despite these endocrine similarities, the antral follicle population stimulated by FSH infusion retained the characteristic genotypic difference with the ovaries of Fec(+/+) animals containing a range of follicle sizes with decreasing proportions of small (<3.5 mm in diameter) and medium (3.5-4.5 mm in diameter) follicles as well as large follicles (> or =4.5 mm in diameter), whereas the ovaries of Fec(B/-) ewes contained no follicles of >4.5 mm in diameter. This genotypic difference was retained after ovulation with gene carriers having more preovulatory follicles/corpora lutea (3.8+/-0.3) of a smaller diameter (5.3+/-0.3 mm) than did non-gene carriers (1.7+/-0.3; 11.4+/-0.9 mm; P<0.05). As ewes carrying the FecB gene mutation were able to ovulate more follicles than non-gene carriers, despite identical concentrations and patterns of FSH and LH stimulation, the results of this study support the hypothesis that the FecB gene acts at the ovary to enhance ovarian sensitivity to gonadotrophic stimulation.

Ovarian Follicular Responses to High Doses of Pulsatile Luteinizing Hormone in Lactating Dairy Cattle

Two experiments in lactating dairy cows examined ovarian follicular responses to high, frequent doses of exogenous LH pulses at levels associated with follicular cysts. In Experiment 1, estrus was synchronized in 12 cyclic lactating cows >40 d postpartum. Emergence of the second follicular wave (d 0) was determined by ultrasonography. Starting on d 1, cows received LH (40μg/h; n=7) or saline (2mL/h; n=5) in hourly pulses for up to 5 (n=5) or 7 (n=7) d. On d 2, all cows received two injections of PGF2α, 12h apart. In experiment 2, 14 lactating cows (7 to 12 d postpartum) received LH (40μg/h; n=7) or saline (1mL/h; n=7) in hourly pulses for 7 d, beginning 24h after start of the first follicular wave. Daily samples were used to determine serum concentrations of progesterone (P4), estradiol-17β(E2), LH, and FSH. Profiles of LH were determined from blood samples collected at 12-min intervals for 8h on d 3. During infusion of LH, serum P4 and FSH were similar across treatments in both experiments. Serum E2 concentrations were similar in experiment 1, but serum E2 was greater on d 2, 3, and 5 in LH-treated cows in experiment 2. Infusion increased LH pulse frequency and amplitude in both experiments. Formation of cysts did not differ between LH- and saline-treated cows in either experiment (1 of 7 vs. 0 of 5 and 1 of 6 vs. 0 of 7, respectively). Cows that ovulated had similar intervals to ovulation in experiment 1 [6.0±0.1 d (LH) vs. 6.4±0.2 d (saline)], but in experiment 2, ovulation was 14 d earlier in LH-treated cows (5.6±1.8 d vs 19.9±1.5 d). In conclusion, high concentrations of LH are not solely responsible for formation of cysts in lactating dairy cows. Pulsatile infusion of LH stimulated follicular growth and steroidogenesis and decreased time to first ovulation in anestrous postpartum cows.

Regulation of expression of ovarian mRNA encoding steroidogenic enzymes and gonadotrophin receptors by FSH and GH in hypogonadotrophic cattle

A study was conducted to determine the effects of FSH and bovine somatotrophin on the expression of mRNA encoding the gonadotrophin receptors and steroidogenic enzymes in ovarian follicles of cattle rendered hypogonadotrophic by treatment with a GnRH agonist. Hereford x Friesian heifers were allotted into two pretreatment groups: controls (n = 10) and GnRH agonist-treated (n = 20). Ovaries of control cows were removed on day 2 of the first follicular wave after synchronized oestrus. GnRH agonist-treated heifers were given either FSH or no FSH. FSH was infused at 50 microg h(-1) for 48 h. Ovaries in GnRH agonist-treated heifers were removed at the end of exogenous hormone treatment. The control, GnRH agonist and GnRH agonist plus FSH treatment groups were divided further into bovine somatotrophin or no bovine somatotrophin treatments (n = 5 per treatment). Bovine somatotrophin (25 mg day(-1) by s.c. injection) was administered for 3 days. Ovaries were scanned once a day by ultrasonography. Blood samples for hormone measurements were collected three times a day from oestrus until the time of removal of ovaries. Expression of mRNAs for the FSH and LH receptors and cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom) enzymes was localized by in situ hybridization and quantified by image analysis. Ovarian follicular growth was arrested at < or = 4.5 mm in diameter in GnRH agonist-treated heifers. There was no effect of bovine somatotrophin on follicular dynamics, gonadotrophin secretion or expression of mRNA for either the gonadotrophin receptors or steroidogenic enzymes. Infusion of FSH to GnRH agonist-treated heifers increased FSH concentrations in serum to the physiological concentrations observed in controls and stimulated growth of follicles to a size similar (5.5-8.0 mm in diameter) to recruited follicles in control cows. FSH induced mRNA expression of P450scc and P450arom in granulosa cells of follicles at a smaller size (< or = 4.5 mm in diameter) than in controls and increased (P < 0.001) expression in larger (> 4.5 mm in diameter) follicles. Expression of mRNAs for P450scc and P450c17 increased (P < 0.001) with increasing follicle size and was higher (P < 0.01) in theca cells of GnRH agonist plus FSH-treated heifers than in the other groups. There were no treatment differences in expression of FSH receptor in granulosa cells or LH receptor in theca cells, but expression of both receptors increased with follicle size. There was no expression of LH receptor in the granulosa cells of cows from any treatment group. In conclusion, FSH treatment in GnRH agonist-treated heifers induced similar changes in follicular growth to those observed during the first follicular wave, but despite similar peak concentrations, prolonged exposure to high FSH induced precocious expression of mRNAs for P450scc and P450arom in granulosa cells from small follicles and markedly upregulated expression of these enzymes in granulosa cells from recruited follicles. The results of this study demonstrate the key role that FSH plays in the induction of follicular growth and differentiation.

Regulation of expression of ovarian mRNA encoding steroidogenic enzymes and gonadotrophin receptors by FSH and GH in hypogonadotrophic cattle

A study was conducted to determine the effects of FSH and bovine somatotrophin on the expression of mRNA encoding the gonadotrophin receptors and steroidogenic enzymes in ovarian follicles of cattle rendered hypogonadotrophic by treatment with a GnRH agonist. Hereford x Friesian heifers were allotted into two pretreatment groups: controls (n = 10) and GnRH agonist-treated (n = 20). Ovaries of control cows were removed on day 2 of the first follicular wave after synchronized oestrus. GnRH agonist-treated heifers were given either FSH or no FSH. FSH was infused at 50 microg h(-1) for 48 h. Ovaries in GnRH agonist-treated heifers were removed at the end of exogenous hormone treatment. The control, GnRH agonist and GnRH agonist plus FSH treatment groups were divided further into bovine somatotrophin or no bovine somatotrophin treatments (n = 5 per treatment). Bovine somatotrophin (25 mg day(-1) by s.c. injection) was administered for 3 days. Ovaries were scanned once a day by ultrasonography. Blood samples for hormone measurements were collected three times a day from oestrus until the time of removal of ovaries. Expression of mRNAs for the FSH and LH receptors and cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom) enzymes was localized by in situ hybridization and quantified by image analysis. Ovarian follicular growth was arrested at < or = 4.5 mm in diameter in GnRH agonist-treated heifers. There was no effect of bovine somatotrophin on follicular dynamics, gonadotrophin secretion or expression of mRNA for either the gonadotrophin receptors or steroidogenic enzymes. Infusion of FSH to GnRH agonist-treated heifers increased FSH concentrations in serum to the physiological concentrations observed in controls and stimulated growth of follicles to a size similar (5.5-8.0 mm in diameter) to recruited follicles in control cows. FSH induced mRNA expression of P450scc and P450arom in granulosa cells of follicles at a smaller size (< or = 4.5 mm in diameter) than in controls and increased (P < 0.001) expression in larger (> 4.5 mm in diameter) follicles. Expression of mRNAs for P450scc and P450c17 increased (P < 0.001) with increasing follicle size and was higher (P < 0.01) in theca cells of GnRH agonist plus FSH-treated heifers than in the other groups. There were no treatment differences in expression of FSH receptor in granulosa cells or LH receptor in theca cells, but expression of both receptors increased with follicle size. There was no expression of LH receptor in the granulosa cells of cows from any treatment group. In conclusion, FSH treatment in GnRH agonist-treated heifers induced similar changes in follicular growth to those observed during the first follicular wave, but despite similar peak concentrations, prolonged exposure to high FSH induced precocious expression of mRNAs for P450scc and P450arom in granulosa cells from small follicles and markedly upregulated expression of these enzymes in granulosa cells from recruited follicles. The results of this study demonstrate the key role that FSH plays in the induction of follicular growth and differentiation.

Effect of FSH infusion on follicle development in GnRH agonist-treated gilts

The aim was to investigate the effect of infusion of purified FSH alone on follicle development in hypogonadotrophic GnRH agonist-treated gilts. Large-White hybrid gilts (n = 12) were treated during the mid-luteal phase and again after 28 days (day 0) with a potent slow releasing GnRH agonist. On day 3, seven gilts were infused for 168 h with 1.5 S1 units oFSH h-1 (equivalent to 1.5 units of bioactivity of NIH-FSH-S1 standard) and blood samples were collected. Ovaries were then recovered and all follicles > or = 1 mm in diameter were dissected and incubated for 2 h in 1 ml Eagle's minimum essential medium. The ovaries were recovered from the remaining five GnRH agonist-treated gilts on day 10 and also from five cyclic gilts during the late follicular phase (controls). Plasma FSH concentrations in GnRH agonist-treated gilts were lower (P < 0.01) than in follicular phase controls, increased (P < 0.001) after 1 h of FSH infusion and reached a plateau similar (P > 0.1) to that of controls after 8 h. Basal LH concentrations were similar (P > 0.1) between GnRH agonist-treated and control gilts and remained unchanged (P > 0.1) throughout the infusion period. GnRH agonist treatment reduced (P < 0.01) basal oestradiol concentrations compared with control gilts. Infusion with FSH alone increased (P < 0.001) plasma oestradiol concentrations after 96 h compared with those before infusion; when the animals were killed oestradiol concentrations were higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH than in controls. This was also apparent by vulval swelling and behavioural oestrus. There were more follicles > or 1 mm in diameter in the GnRH agonist-treated groups than in the controls (184, 153 and 86 per animal; P < 0.01). Infusion with FSH increased the maximum follicle diameter (GnRH agonist: < 4 mm; FSH infused: < 12 mm; controls: < 10 mm) and tended to increase (P < 0.07) the mean number of follicles > or = 6 mm diameter per animal (FSH infused: 53; controls: 21). Total oestradiol production in vitro by follicles > or = 1 mm was higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH and in follicular phase controls than in animals treated with GnRH agonist alone. However, oestradiol and testosterone secretion in vitro per follicle > or = 6 mm in diameter was lower (P < 0.05) in FSH-infused animals than in controls. In summary, although infusion of FSH alone stimulated the growth of multiple follicles of preovulatory size in GnRH agonist-treated gilts, steroidogenic output by individual follicles was impaired.

Ovarian function in ewes made hypogonadal with GnRH antagonist and stimulated with FSH in the presence or absence of low amplitude LH pulses.

This study examined the effect of LH pulses, of similar amplitude and frequency to those found in the luteal phase, on the pattern of hormone secretion and follicle development in GnRH antagonist-suppressed ewes stimulated with exogenous FSH. This experiment was conducted on ewes with ovarian autotransplants in a continuous study. Follicle development was suppressed in 18 ewes by 3 weeks of GnRH antagonist treatment (50 micrograms/kg per 4 days s.c.), and was then stimulated by infusion of ovine (o)FSH (5 micrograms NIADDK-oFSH-16/h i.v.) for 3 days. In addition to FSH, 10 animals received pulses of LH (2.5 micrograms NIADDK-oLH-26 i.v.) every 4 h for the entire period of the FSH infusion. The follicle population was determined by daily ultrasound. Samples of ovarian and jugular venous blood were collected at 4-h intervals over the period of the FSH infusion and there were three periods of intensive blood sampling (15-min intervals for 2.5 h at 24, 48 and 72 h after the start of the FSH infusion) when the steroidogenic capacity of the follicles in all 18 ewes was tested around an LH challenge (2.5 micrograms i.v.). GnRH antagonist treatment resulted in a 57% decrease in FSH concentrations and prevented ovarian follicle development beyond 3 mm in diameter. Infusion of FSH resulted in a 60% increase in FSH concentrations and stimulated the development of large antral follicles and a coincident increase in ovarian androstenedione, inhibin and oestradiol secretion in both experimental groups. In the absence of 4-hourly LH pulses basal steroid secretion was negligible (< 1 ng/min; P < 0.001). Daily LH challenges, however, revealed no difference in the steroidogenic capacity of the follicle population in either experimental group. Similarly, LH pulses had no effect on the growth rate and number of antral follicles stimulated by FSH infusion, or the pattern of ovarian inhibin secretion. In conclusion, these results show that while FSH alone can stimulate the development of ovulatory sized follicles in ewes made hypogonadal with GnRH antagonist, physiological patterns of LH stimulation have no deleterious effects on FSH-stimulated follicle development and are essential for normal steroidogenesis.

Follicle-stimulating hormone amplifies the population of differentiated spermatogonia in the hypophysectomized testosterone-replaced adult rhesus monkey (Macaca mulatta).

Although testosterone supports all phases of spermatogenesis in primates, FSH is obligatory for quantitatively normal spermatogenesis. To further investigate the mechanism of action of FSH on spermatogenesis, eight adult male rhesus macaques were hypophysectomized and supplemented daily with cortisone acetate (5 mg/kg BW, sc) and T4 (50 mg/animal, orally). Complete pituitary ablation was established by 1) a decline in mean testicular volume to 8% of the prehypophysectomy value; 2) a failure to secrete gonadotropins in response to 50 micrograms GnRH, iv; and 3) an absence of pituitary in the sella turcica on postmortem examination. Testosterone-filled SILASTIC brand capsules (Dow Corning) were implanted sc to restore testicular testosterone to normal levels. Once the testes had achieved maximum growth under testosterone stimulation alone, the animals were implanted with indwelling venous catheters. In four animals, a pulsatile infusion of human FSH (one pulse of 4 IU/kg BW every 3 h) was administered for 12 days, and the other four monkeys received vehicle. Testosterone replacement continued throughout the experiment. At the termination of the 12 days of FSH stimulation or vehicle administration, the right testes were removed. The left testes were removed 22 days later to investigate whether testosterone was able to maintain the effects, if any, of FSH stimulation. Portions of each testis were fixed in Bouin's solution and subsequently prepared for histological examination, whereas other portions were frozen in liquid nitrogen for determination of testicular testosterone content. Five hundred cross-sections of seminiferous tubules in periodic acid-Schiff-hematoxylin-stained histological sections were randomly selected from each testis. The stage of the seminiferous epithelial cycle in these sections was identified, and the germ cells and Sertoli cells were counted in each. All cell counts were corrected by the Abercrombie method and expressed per cross-section of seminiferous tubule. Treatment with FSH for 12 days failed to influence the numbers of either Sertoli cells or Ad and Ap stem spermatogonia. In striking contrast, the number of all four generations of differentiated (B1, B2, B3, and B4) spermatogonia were significantly amplified by stimulation with human FSH for 12 days. As reflected by the analysis of the left testes collected 22 days after termination of the gonadotropin treatment, the progeny of these B spermatogonia were not maintained in the absence of FSH. In conclusion, the results of the present study indicate that the action of FSH to quantitatively maintain spermatogenesis in the rhesus monkey is mediated by a selective amplification of B spermatogonia.

Follicle-stimulating hormone amplifies the population of differentiated spermatogonia in the hypophysectomized testosterone-replaced adult rhesus monkey (Macaca mulatta)

Although testosterone supports all phases of spermatogenesis in primates, FSH is obligatory for quantitatively normal spermatogenesis. To further investigate the mechanism of action of FSH on spermatogenesis, eight adult male rhesus macaques were hypophysectomized and supplemented daily with cortisone acetate (5 mg/kg BW, sc) and T4 (50 mg/animal, orally). Complete pituitary ablation was established by 1) a decline in mean testicular volume to 8% of the prehypophysectomy value; 2) a failure to secrete gonadotropins in response to 50 micrograms GnRH, iv; and 3) an absence of pituitary in the sella turcica on postmortem examination. Testosterone-filled SILASTIC brand capsules (Dow Corning) were implanted sc to restore testicular testosterone to normal levels. Once the testes had achieved maximum growth under testosterone stimulation alone, the animals were implanted with indwelling venous catheters. In four animals, a pulsatile infusion of human FSH (one pulse of 4 IU/kg BW every 3 h) was administered for 12 days, and the other four monkeys received vehicle. Testosterone replacement continued throughout the experiment. At the termination of the 12 days of FSH stimulation or vehicle administration, the right testes were removed. The left testes were removed 22 days later to investigate whether testosterone was able to maintain the effects, if any, of FSH stimulation. Portions of each testis were fixed in Bouin's solution and subsequently prepared for histological examination, whereas other portions were frozen in liquid nitrogen for determination of testicular testosterone content. Five hundred cross-sections of seminiferous tubules in periodic acid-Schiff-hematoxylin-stained histological sections were randomly selected from each testis. The stage of the seminiferous epithelial cycle in these sections was identified, and the germ cells and Sertoli cells were counted in each. All cell counts were corrected by the Abercrombie method and expressed per cross-section of seminiferous tubule. Treatment with FSH for 12 days failed to influence the numbers of either Sertoli cells or Ad and Ap stem spermatogonia. In striking contrast, the number of all four generations of differentiated (B1, B2, B3, and B4) spermatogonia were significantly amplified by stimulation with human FSH for 12 days. As reflected by the analysis of the left testes collected 22 days after termination of the gonadotropin treatment, the progeny of these B spermatogonia were not maintained in the absence of FSH. In conclusion, the results of the present study indicate that the action of FSH to quantitatively maintain spermatogenesis in the rhesus monkey is mediated by a selective amplification of B spermatogonia.

FSH-stimulated follicle development in ewes in high and low body condition and chronically treated with gonadotrophin-releasing hormone agonist

Ovarian follicle development in response to FSH infusion was investigated in Scottish Blackface ewes with high and low body condition scores in which endogenous gonadotrophin secretion and follicle development to > or = 2.5 mm diameter was suppressed using subcutaneous implants containing a GnRH agonist. In two experiments conducted during the normal breeding season, groups of 20 (Expt 1) and 15 (Expt 2) ewes were fed to achieve body condition scores > or = 2.75 (high; H) or < or = 1.75 (low; L). In both experiments GnRH agonist implants were inserted four weeks before FSH was infused for 72 h at 7 micrograms h-1 to group H animals or at 5 micrograms h-1 to group L animals; the infusion rates were designed to ensure similar circulating FSH concentrations in animals of both groups. In Expt 2, additional subcutaneous implants containing oestradiol were inserted 21 days after insertion of GnRH agonist implants and 7 days before the FSH infusion began. In both experiments, FSH infusion was associated with an increase in circulatory concentrations of LH (P < 0.01) and FSH (P < 0.001), but there was no difference with body condition in mean circulating gonadotrophin concentrations, the numbers of ovarian follicles > or = 2.5 mm diameter, the proportion of these follicles that were oestrogenic or the mean rate of oestradiol secretion in vitro. It is concluded that differences in body condition of ewes do not affect the responsiveness of the ovary to FSH, in the presence or absence of oestradiol, as measured by the number, size and steroidogenic capacity of ovarian follicles present following FSH infusion.

Continuous luteinizing hormone infusion prevents atretic changes of the follicles during superovulation in hamsters.

Superovulation with exogenous gonadotropins has been used widely to study the reproductive events in animals and in vitro fertilization and embryo transfer in human beings. However, details of the mechanism of superovulation are not yet clearly understood. The present study was conducted to study the mechanism of superovulation with purified human FSH and LH preparations. Cyclic hamsters received continuous infusion of purified FSH and LH for 4 days, from estrus to proestrus. The number of ova shed was significantly increased by 5 iu FSH/minipump infusion, and further increased by 5 iu FSH/minipump plus 5 iu LH/minipump group. On the other hand, the number of atretic follicles was decreased significantly (P < 0.01) by the combined infusion of FSH and LH, but not by FSH alone. Among these experimental groups, there was no significant difference in embryonic development which was assessed by the counting of the number of 4-cells and more developed embryos on day 3 of pregnancy. These data indicated the different roles of FSH and LH in the superovulation process, because FSH stimulated some small follicles to grow and LH, in the presence of FSH, rescued the follicles to undergo atresia.

Effect of follicular fluid and inhibin immunoneutralization on FSH-induced preovulatory follicle growth in the ewe

The potential direct ovarian effects of immunoneutralization of inhibin, which increases, and follicular fluid treatment, which inhibits, follicle development in normal ewes was investigated in a sheep model in which endogenous FSH and LH secretion was suppressed thus removing any potential effects of treatment-induced alterations in endogenous FSH or LH secretion. Eighteen Welsh Mountain ewes were treated with two agonist implants containing 3.3 mg buserelin giving a total of 6.6 mg buserelin per animal. During week 5 of treatment all ewes were given a 72-h continuous infusion of ovine FSH (5 micrograms/h) starting at 09.00 h. Six ewes were treated with antiserum to the 1-26 alpha peptide fragment of porcine inhibin 0 h and 24 h after the start of the FSH infusion, and a further six ewes were treated with charcoal-stripped ovine follicular fluid (oFF) as a source of inhibin, at 09.00 and 17.00 h throughout the 72 h of FSH infusion. The plasma concentrations of both FSH and LH were significantly reduced in all ewes after 5 weeks of treatment with buserelin, and no large follicles greater than 2.5 mm in diameter were present. Treatment with inhibin antiserum or oFF had no effect, compared with control ewes, on the plasma concentrations of either FSH or LH during the FSH infusion period. After 72 h of FSH infusion there was no difference in the number of small follicles (less than 2.5 mm in diameter) or large follicles (greater than 2.5 mm in diameter) or the size of the largest follicles between control ewes and ewes treated with either inhibin antiserum or oFF.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of basal and pulsatile LH release on FSH-stimulated follicle growth in ewes chronically treated with gonadotrophin-releasing hormone agonist

Ewes chronically treated with gonadotrophin-releasing hormone (GnRH) agonist were used to investigate the importance of the peripheral concentration of LH in FSH-stimulated follicular development. Twenty-four Welsh Mountain ewes were treated with two agonist implants containing 3.3 mg buserelin. During week 6 of treatment all the ewes were given a 72-h continuous infusion of ovine FSH alone (3 micrograms/h) or FSH with large (7.5 micrograms)- or small (2.5 micrograms) amplitude pulses of ovine LH delivered at 4-hourly intervals. The importance of baseline LH throughout the FSH infusion was evaluated in six animals which were treated with a specific antiserum against bovine LH (LH-AS) 15-20 h before the start of FSH treatment. In the absence of LH-AS, infusion of FSH alone or with large or small pulses of LH stimulated the development of a normal number of small follicles (less than or equal to 2.5 mm in diameter) and large follicles (greater than 2.5 mm in diameter). These follicles had normal diameter and steroid secretion compared with control ewes on day 8 of the luteal phase. In contrast, the animals pretreated with LH-AS developed no follicles greater than 2.0 mm in diameter but the number of small follicles per ewe was significantly (P less than 0.05) increased. These results support the hypothesis that FSH in the absence of pulsatile LH release stimulates preovulatory follicular development in ewes treated with GnRH agonist. The follicular response to LH pulses of different amplitude is dependent on both the stage of development of the follicle and the peripheral concentration of FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Preovulatory LH profiles of superovulated cows and progesterone concentrations at embryo recovery

Forty-two Holstein cows were randomly assigned to three superovulatory treatment groups of 14 cows each. Cows in Group I received follicle stimulating hormone (FSH; 50 mg i.m.); those in Group II received FSH (50. mg i.m.) along with GnRH (250 ug in 2 % carboxymethylcellulose s.c.) on the day of estrus; and cows in Group III were infused FSH (49 mg) via osmotic pump implants. FSH was administered over a 5-d period for cows in Groups I and II (twice daily in declining doses). Cows in Group III received FSH over a 7-d period (constantly at a rate of 7 mg/day). All cows received 25 mg PGF 2α (prostaglandin F 2α) 48 hours after initiation of the FSH treatment. Blood samples were collected from seven cows from each group at 2 hour intervals on the fifth day of superovulation for serum luteinizing hormone (LH) concentration analysis by radioimmunoassay, and blood samples were collected from all cows on the day of embryo recovery for plasma progesterone determination. The LH profile was not altered (P>0.05) by either GnRH administration or by the constant infusion of FSH as compared to FSH treatment alone. Plasma progesterone concentrations were highly correlated with the number of corpora lutea (CL) palpated (r=0.92; P<0.01) and with the number of ova and/or embryos recovered (r=0.88; P<0.01). The accuracy of predicting the number of recoverable ova and/or embryos by the concentration of plasma progesterone was 86%.