Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels

Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSH beta, as well as LH beta and alpha-subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using alpha, LH beta, and FSH beta cDNA and genomic fragments. Treatment with inhibin for 72 h significantly suppressed alpha and FSH beta mRNA levels with parallel changes in FSH secretion. No change in LH beta mRNA levels was observed. A decrease in FSH beta mRNA to undetectable levels was seen 4 h after inhibin administration. Recombinant human Activin A caused dose-dependent and parallel increases in FSH beta mRNA levels and FSH secretion. This increase was evident at 4 h after activin administration and maintained at longer times. alpha and LH beta mRNA levels remained unchanged. Follistatin addition to cultures for 72 h significantly reduced FSH beta mRNA levels. In a time-course experiment, a reduction in FSH beta mRNA to undetectable levels was observed 24 h after follistatin administration. There were no changes in alpha or LH beta mRNA levels. These data demonstrate that the actions of these gonadal peptides on FSH secretion may be accounted for, at least in part at the level of biosynthesis, by reductions in FSH beta mRNA levels directly at the level of the anterior pituitary gland.     

Estrogen and leptin have differential effects on FSH and LH release in female rats

Prior experiments have shown that the adipocyte hormone leptin can advance puberty in mice. We hypothesized that it would also stimulate gonadotrophin secretion in adults. Since the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) is drastically affected by estrogen, we hypothesized that leptin might have different actions dependent on the dose of estrogen. Consequently in these experiments, we tested the effect of injection of leptin into the third cerebral ventricle of ovariectomized animals injected with either the oil diluent, 10 microg or 50 microg of estradiol benzoate 72 hr prior to the experiment. The animals were ovariectomized 3-4 weeks prior to implantation of a cannula into the third ventricle 1 week before the experiments. The day after implantation of an external jugular catheter, blood samples (0. 3 ml) were collected just before and every 10 min for 2 hr after 3V injection of 5 microl of diluent or 10 microg of leptin. Both doses of estradiol benzoate equally decreased plasma LH concentrations and pulse amplitude, but there was a graded decrease in pulse frequency. In contrast, only the 50-microg dose of estradiol benzoate significantly decreased mean plasma FSH concentrations without significantly changing other parameters of FSH release. The number of LH pulses alone and pulses of both hormones together decreased as the dose of estrogen was increased, whereas the number of pulses of FSH alone significantly increased with the higher dose of estradiol benzoate, demonstrating differential control of LH and FSH secretion by estrogen, consistent with alterations in release of luteinizing hormone releasing hormone (LHRH) and the putative FSH-releasing factor (FSHRF), respectively. The effects of intraventricularly injected leptin were drastically altered by increasing doses of estradiol benzoate. There was no significant effect of intraventricular injection of leptin (10 microg) on the various parameters of either FSH or LH secretion in ovariectomized, oil-injected rats, whereas in those injected with 10 microg of estradiol benzoate there was an increase in the first hr in mean plasma concentration, area under the curve, pulse amplitude, and maximum increase of LH above the starting value (Deltamax) on comparison with the results in the diluent-injected animals in which there was no alteration of these parameters during the 2 hr following injection. The pattern of FSH release was opposite to that of LH and had a different time-course. In the diluent-injected animals, probably because of the stress of injection and frequent blood sampling, there was an initial significant decline in plasma FSH at 20 min after injection, followed by a progressive increase with a significant elevation above the control values at 110 and 120 min. In the leptin-injected animals, mean plasma FSH was nearly constant during the entire experiment, coupled with a significant decrease below values in diluent-injected rats, beginning at 30 min after injection and progressing to a maximal difference at 120 min. Area under the curve, pulse amplitude, and Deltamax of FSH was also decreased in the second hour compared to values in diluent-injected rats. In contrast to the stimulatory effects of intraventricular injection of leptin on pulsatile LH release manifest during the first hour after injection, there was a diametrically opposite, delayed significant decrease in pulsatile FSH release. This differential effect of leptin on FSH and LH release was consistent with differential effects of leptin on LHRH and FSHRF release. Finally, the higher dose of E2 (50 microg) suppressed release of both FSH and LH, but there was little effect of leptin under these conditions, the only effect being a slight (P < 0.04) increase in pulse amplitude of LH in this group of rats. The results indicate that the central effects of leptin on gonadotropin release are strongly dependent on plasma estradiol levels. These effects are consistent w     

Evidence that testosterone and follicular fluid do not interact in the control of FSH secretion in rams

The hypothesis that testosterone and inhibin interact in the control of FSH secretion in rams was tested. Adult rams were castrated and were simultaneously given testosterone implants and 3-times daily sc injections of 0, 0.4, 0.8 or 1.6 ml charcoal-treated bovine follicular fluid (bFF). After 1 wk, the implants were removed, and the bFF injections continued as before. Blood samples were taken daily for mean LH, FSH and testosterone concentrations, and every 10 min for 12 h in the presence and in the absence of testosterone for assessment of pulsatile LH release. The bFF specifically inhibited FSH secretion from rat pituitary cells in culture. In the presence of testosterone, there were no main effects of bFF on mean plasma FSH or LH concentrations, nor were these values different from their pre-treatment means (P>0.05). Treatment with bFF did not affect LH pulse frequency or amplitude, but the number of rams showing LH pulses was reduced in the 0.8 and 1.6-ml dose groups (P<0.05). Removal of testosterone increased (P<0.05) both gonadotropins. In the absence of testosterone, no main effect of bFF on mean LH or FSH concentrations was observed, although the 1.6-ml dose suppressed the postcastration rise of both LH and FSH. These data suggest that inhibin does not interact with testosterone and that a physiological level of testosterone is sufficient for the regulation of FSH secretion in adult rams.     

A Mathematical Model for the Actions of Activin,Inhibin, and Follistatin on Pituitary Gonadotrophs

The timed secretion of the luteinizing hormone (LH) and follicle stimulating hormone (FSH) from pituitary gonadotrophs during the estrous cycle is crucial for normal reproductive functioning. The release of LH and FSH is stimulated by gonadotropin releasing hormone (GnRH) secreted by hypothalamic GnRH neurons. It is controlled by the frequency of the GnRH signal that varies during the estrous cycle. Curiously, the secretion of LH and FSH is differentially regulated by the frequency of GnRH pulses. LH secretion increases as the frequency increases within a physiological range, and FSH secretion shows a biphasic response, with a peak at a lower frequency. There is considerable experimental evidence that one key factor in these differential responses is the autocrine/paracrine actions of the pituitary polypeptides activin and follistatin. Based on these data, we develop a mathematical model that incorporates the dynamics of these polypeptides. We show that a model that incorporates the actions of activin and follistatin is sufficient to generate the differential responses of LH and FSH secretion to changes in the frequency of GnRH pulses. In addition, it shows that the actions of these polypeptides, along with the ovarian polypeptide inhibin and the estrogen-mediated variations in the frequency of GnRH pulses, are sufficient to account for the time courses of LH and FSH plasma levels during the rat estrous cycle. That is, a single peak of LH on the afternoon of proestrus and a double peak of FSH on proestrus and early estrus. We also use the model to identify which regulation pathways are indispensable for the differential regulation of LH and FSH and their time courses during the estrous cycle. We conclude that the actions of activin, inhibin, and follistatin are consistent with LH/FSH secretion patterns, and likely complement other factors in the production of the characteristic secretion patterns in female rats.     

Circadian and pulsatile variations in plasma levels of inhibin, FSH, LH and testosterone in adult Murrah buffalo bulls

The present study investigated pulsatile and circadian variations in the circulatory levels of inhibin, gonadotrophins and testosterone. Six adult buffalo bulls (6 to 7 yr of age) were fitted with indwelling jugular vein catheters, and blood samples were collected at 2-h intervals for a period of 24 h and then at 15-min interval for 5 h. Plasma concentrations of inhibin, FSH, LH and testosterone were determined by specific radioimmunoassays. Plasma inhibin levels in Murrah buffalo bulls ranged between 0.201 to 0.429 ng/mL, with a mean of 0.278 +/- 0.023 ng/mL. No inhibin pulses could be detected during the 15-min sampling interval. Plasma FSH levels ranged between 0.95 to 3.61 ng/mL, the mean concentration of FSH over 24 h was 1.66 +/- 0.25 ng/mL. A single FSH pulse was detected in 2 of 6 bulls. The LH levels in peripheral circulation ranged between 0.92 to 9.91 ng/mL, with a mean concentration of 3.33 +/- 1.02 ng/mL. Pulsatility was detected in LH secretion with an average of 0.6 pulses/h. Plasma testosterone levels in 4 buffalo bulls ranged from 0.19 to 2.99 ng/mL, the mean level over 24 h were 1.34 +/- 0.52 ng/mL. Testosterone levels in peripheral circulation followed the LH secretory pattern, with an average of 0.32 pulses/h. The results indicate parallelism in inhibin, FSH and LH, and testosterone secretory pattern. Divergence in LH and FSH secretory patterns in adult buffalo bulls might be due to the presence of appreciable amounts of peripheral inhibin.     

Neuroendocrine control of follicle-stimulating hormone (FSH) secretion: II. Is follistatin-induced suppression of FSH secretion mediated via changes in activin availability and does it involve changes in gonadotropin-releasing hormone secretion?

The objective of the present study was to determine to what extent activin participates in setting the level of FSH secretion and if this regulation includes mediation via changes in GnRH secretion. We administered follistatin, the high-affinity binding protein for activin, to five ovariectomized sheep; we reasoned that the resultant binding of follistatin to activin should lower activin bioavailability and FSH secretion. Hypophyseal portal and peripheral blood samples were collected simultaneously at 10-min intervals for 18 h to measure GnRH, LH, FSH, and both activin-free and total follistatin. Six hours into collection, each ewe received 150 microg/kg i.v. of recombinant human follistatin-288. A week later, the same ewes were subjected to a second series of blood collections of similar length (time control). The FSH levels in pituitary portal blood were approximately 8-fold higher than those in the peripheral circulation. The FSH secretory patterns changed minimally during the time-control period. In contrast, follistatin had profound suppressive effects on FSH secretion. Maximal FSH suppression after FS-288 administration occurred at 5-6 h in the pituitary portal (65% suppression) and 9-10 h in the peripheral (48% suppression) circulation. Follistatin had no effect on GnRH or LH secretory patterns. Disappearance of total follistatin (i.e., free follistatin plus activin-bound follistatin) from the circulation was slower (P < 0.05) than that of free follistatin alone, suggesting that some of the follistatin was complexed with circulating activin, thus reducing the bioavailability of activin. The slower clearance of total follistatin and the lack of follistatin effects on GnRH secretion suggest that changes in activin bioavailability dictate the level of pituitary FSH secretion and that this is a pituitary-specific effect.     

Effect of alterations in pulsatile luteinizing hormone release on ovarian follicular atresia and steroid secretion on diestrus 1 in the rat estrous cycle

This study examined the importance of pulsatile luteinizing hormone (LH) release on diestrus 1 (D1; metestrus) in the rat estrous cycle to ovarian follicular development and estradiol (E2) secretion. Single injections of a luteinizing hormone-releasing hormone (LHRH) antagonist given at -7.5 h prior to the onset of a 3-h blood sampling period on D1 reduced mean blood LH levels by decreasing LH pulse amplitude, while frequency was not altered. Sequential injections at -7.5 and -3.5 h completely eliminated pulsatile LH secretion. Neither treatment altered the total number of follicles/ovary greater than 150 mu in diameter, the number of follicles in any size group between 150 and 551 mu, or plasma E2, progesterone, or follicle-stimulating hormone (FSH) levels. However, both treatments with LHRH antagonist significantly increased the percentage of atretic follicles in the ovary. These data indicate that: 1) pulsatile LH release is an important factor in determining the rate at which follicles undergo atresia on D1; 2) reductions in LH pulse amplitude alone are sufficient to increase the rate of follicular atresia on D1; 3) an absence of pulsatile LH release for a period of up to 10 h on D1 is not sufficient to produce a decline in ovarian E2 secretion, most likely because the atretic process was in its early stages and had not yet affected a sufficient number of E2-secreting granulosa cells to reduce the follicle's capacity to secrete E2; and 4) suppression or elimination of pulsatile LH release on D1 is not associated with diminished FSH secretion.     

The inhibitory action of corticotropin-releasing hormone on gonadotropin secretion in the ovariectomized rhesus monkey is not mediated by adrenocorticotropic hormone

Earlier observations in our laboratory indicated that i.v. infusion of human/rat corticotropin-releasing hormone (hCRH) suppresses pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in ovariectomized rhesus monkeys. Since cortisol secretion increased significantly as well, it was not possible to exclude the possibility that this inhibitory effect of hCRH on gonadotropins was related to the activation of the pituitary/adrenal axis. The purpose of the present study was to determine the role of pituitary/adrenal activation in the effect of hCRH on LH and FSH secretion. We compared the effects of 5-h i.v. infusions of hCRH (100 micrograms/h, n = 7) and of human adrenocorticotropic hormone (ACTH) (1-24) (5 micrograms/h, n = 3; 10 micrograms/h, n = 3, 20 micrograms/h, n = 3) to ovariectomized monkeys on LH, FSH, and cortisol secretion. As expected, during the 5-h ACTH infusions, cortisol levels increased by 176-215% of baseline control, an increase similar to that observed after CRH infusion (184%). However, in contrast to the inhibitory effect observed during the CRH infusion, LH and FSH continued to be released in a pulsatile fashion during the ACTH infusions, and no decreases in gonadotropin secretion were observed. The results indicated that increases in ACTH and cortisol did not affect LH and FSH secretion and allowed us to conclude that the rapid inhibitory effect of CRH on LH and FSH pulsatile release was not mediated by activation of the pituitary/adrenal axis.     

Rapid recovery of estrogen-induced pituitary gonadotropin surges after luteectomy in rhesus monkeys

In the presence of a functional corpus luteum, positive estrogen feedback on the surge modes of gonadotropin secretion is blocked in rhesus monkeys. We investigated the effects of luteectomy (Lx) on the time required for recovery of pituitary responsiveness (LH/FSH surges) to positive estrogen feedback. Estradiol-17 beta-3- benzoate (EB, 50 microgram/kg sc) was given: 1) 24th prior to, 2) the day of, or 3) 24 h after luteal ablation. Daily measurements of serum follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol-17 beta (e2) and progesterone (P) were made on each monkey for 5 days. Serum P fell to undetectable levels within 24 h after Lx, whereas E2 levels in circulation peaked within 24h after injection of EB. Among early follicular phase monkeys, this EB treatment results in typical midcycle type LH/FSH surges within 48h. Lx alone was not soon followed by significant changes in pituitary gonadotropin secretion. When circulating P levels were undetectable the pituitary responded fully to EB; that is, typical midcycle type FSH/LH surges occurred. When serum P was in the midst of declining after Lx, gonadotropin surges were present, but attenuated. However, when P levels remained elevated for more than 24 h after EB injection, the surge modes of FSH/LH secretion remained fully blocked. These results demonstrate that the suppressive influence of luteal secretions (principally progesterone) on positive estrogen feedback regulation of the surge modes of pituitary gonadotropin secretion is quite transient in these primates.     

The pituitary-gonadal axis before puberty: Effects of various estrogenic steroids in the ovariectomized rat

A series of experiments were conducted to determine the effects of several estrogens upon FSH and LH secretion in immature ovariectomized rats. Groups of animals were castrated at 26 days of age and treated for 5 days post-operatively with various dosages of one of the following steroids: estrad1ol-17β(E₂), estradiol benzoate (EB), estrone (E₁), equilenin (EQ), 17α-ethinyl estradiol (EE), or mestranol (ME). Uterine weights were recorded and blood taken for radioimmunoassay.Estradiol was able to suppress both FSH and LH within a “physiologic dosage range” (PDR), wherein both gonadotropins were suppressed to intact control levels by a dose which did not stimulate uterine weight higher than intact control weight. EB and ME suppressed LH but not FSH at the PDR; the other steroids suppressed at higher than PDR doses. LH was preferentially suppressed, as compared to FSH, by all 6 steroids. By biological potency the order of activity was E₂ = EE ? EB ? ME ? E₁ ? EQ. For relative ability to suppress FSH (compared to LH), the order was E₁ or E₂, ? ME ?EB ? EE ? EQ. At higher doses (near maximum uterine stimulation), e₁, E₂ and EE produced higher FSH and LH than suppressed levels seen at lower doses; a pharmacologie dosage of E₂ caused re-suppression of both gonadotropins.Results indicate that a feedback system is present before puberty and this system is sensitive to very low levels of estrogens. Likewise, there is a potential for positive feedback present for higher estrogen levels, and a complete suppression occurs at pharmacologie levels. There appears to be a significant discrepancy between the biologic activity (by uterine weight) of estrogens and their ability to affect gonadotropin     

Sex differences in acute luteinizing hormone responses to gonadectomy remain after progesterone antagonist and dopamine agonist treatment.

In male rats, LH pulse frequency and amplitude increase dramatically by 24 h after gonadectomy; in females they increase only slightly by this time. Mean FSH levels increase significantly in both sexes by 24 h after gonadectomy. The objectives of the present studies were to compare pulsatile LH, FSH, and prolactin (PRL) secretion in intact versus gonadectomized and in male versus female rats, and to determine whether the acute postovariectomy lag in LH rise is due to a lingering effect of the higher PRL and/or progesterone (P) levels seen in intact females. LH pulse amplitude, frequency, and mean levels increased significantly by 24 h after gonadectomy in both sexes, but the increases were greater in the males. FSH mean levels, but not pulse amplitude or frequency, increased similarly in both sexes by 24 h after gonadectomy. PRL did not change with gonadectomy. Treatment with CB-154 (a dopamine agonist), with or without RU486 (a P antagonist), 1 h before gonadectomy significantly suppressed pulsatile PRL secretion 1 day later in both sexes. There was no effect of either treatment on LH secretion. We have demonstrated that there is a sex difference in LH, but not FSH or PRL, pulsatility at 24 h after gonadectomy, and that female rats' higher PRL and P levels do not account for their slow rate of LH rise after ovariectomy.     

Plasma LH, FSH and testosterone concentrations in adult rams which were homozygous carriers or non-carriers of the Booroola fecundity gene

No gene-specific differences were found with respect to LH or testosterone pulsatile secretion (over 12 h), or in 12 hourly mean FSH concentrations in adult Booroola FF and ++ rams. Also, no differences between genotypes in the LH response to an injection of testosterone propionate, the FSH response to an infusion of bovine follicular fluid, or the testosterone response to injections of PMSG were noted. However, during the phase of seasonal testicular development, mean testosterone pulse amplitude (over 12 h) and the FSH response to 25 micrograms GnRH were higher in FF than in ++ rams (P less than 0.05); there were also significant effects of sire (P less than 0.05 in FF genotype only) and litter size (P less than 0.05) on testosterone pulse amplitude and GnRH-stimulated FSH release, respectively. During the breeding season, mean LH, but not FSH, concentrations were higher in FF than in ++ rams, after an injection of 0.5 micrograms GnRH; LH release was not affected by sire or litter size (P greater than 0.05). Long-term studies revealed that the FF rams were born in significantly larger litters, they weighed significantly less than ++ rams (P less than 0.05), and that bodyweight was significantly correlated (P less than 0.05) with litter size. There were no differences in testis size, and testis size was not significantly correlated with bodyweight. There was a strong tendency (P = 0.056) for overall mean FSH concentrations, measured weekly for 9 months, to be highest more often in FF than in ++ rams.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of gonadotropin-releasing hormone pulse-frequency modulation on luteinizing hormone, follicle-stimulating hormone and testosterone secretion in hypothalamo/pituitary-disconnected rams

The effects of changes in pulse frequency of exogenously infused gonadotropin-releasing hormone (GnRH) were investigated in 6 adult surgically hypothalamo/pituitary-disconnected (HPD) gonadal-intact rams. Ten-minute sampling in 16 normal animals prior to HPD showed endogenous luteinizing hormone (LH) pulses occurring every 2.3 h with a mean pulse amplitude of 1.11 +/- 0.06 (SEM) ng/ml. Mean testosterone and follicle-stimulating hormone (FSH) concentrations were 3.0 +/- 0.14 ng/ml and 0.85 +/- 0.10 ng/ml, respectively. Before HPD, increasing single doses of GnRH (50-500 ng) elicited a dose-dependent rise of LH, 50 ng producing a response of similar amplitude to those of spontaneous LH pulses. The effects of varying the pulse frequency of a 100-ng GnRH dose weekly was investigated in 6 HPD animals; the pulse intervals explored were those at 1, 2, and 4 h. The pulsatile GnRH treatment was commenced 2-6 days after HPD when plasma testosterone concentrations were in the castrate range (less than 0.5 ng/ml) in all animals. Pulsatile LH and testosterone secretion was reestablished in all animals in the first 7 days by 2-h GnRH pulses, but the maximal pulse amplitudes of both hormones were only 50 and 62%, respectively, of endogenous pulses in the pre-HPD state. The plasma FSH pattern was nonpulsatile and FSH concentrations gradually increased in the first 7 days, although not to the pre-HPD range. Increasing GnRH pulse frequency from 2- to 1-hour immediately increased the LH baseline and pulse amplitude. As testosterone concentrations increased, the LH responses declined in a reciprocal fashion between Days 2 and 7. FSH concentration decreased gradually over the 7 days at the 1-h pulse frequency. Slowing the GnRH pulse to a 4-h frequency produced a progressive fall in testosterone concentrations, even though LH baselines were unchanged and LH pulse amplitudes increased transiently. FSH concentrations were unaltered during the 4-h regime. These results show that 1) the pulsatile pattern of LH and testosterone secretion in HPD rams can be reestablished by exogenous GnRH, 2) the magnitude of LH, FSH, and testosterone secretion were not fully restored to pre-HPD levels by the GnRH dose of 100 ng per pulse, and 3) changes in GnRH pulse frequency alone can influence both gonadotropin and testosterone secretion in the HPD model.     

Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone beta by inhibin from primate Sertoli cells

We showed previously that inhibin, partially purified from cynomolgus monkey Sertoli cell culture medium (primate Sertoli cell inhibin referred to as pSCI), selectively suppressed basal FSH secretion from dispersed rat pituitary cells and decreased total cellular FSH, but not LH content, suggesting a decrease in FSH biosynthesis. In order to investigate the mechanism of action of inhibin at the molecular level, we have now examined the effects of pSCI on steady state levels of the subunit mRNAs encoding LH and FSH and correlated these with release and intracellular content of LH, FSH, and glycoprotein alpha-subunit. Dispersed pituitary cells from 7- to 8-week-old adult male rats were cultured in the presence of pSCI or control medium for 2-72 h. FSH secretion was reduced significantly by 6 h (P less than 0.05) and reached a nadir (38% of control) by 48 h. LH secretion was unchanged, while release of the alpha-subunit was decreased to 89% of control at 72 h (P less than 0.05). Also by 72 h, cell content of both FSH (73% of control) and alpha-subunit (81% of control) were significantly suppressed (P less than 0.001, P less than 0.01), while LH was slightly affected. Total RNA was extracted from the pituitary cell cultures, electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for the rat alpha-, LH beta-, and FSH beta-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroendocrine control of FSH secretion: IV. Hypothalamic control of pituitary FSH-regulatory proteins and their relationship to changes in FSH synthesis and secretion

The current dogma is that the differential regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and secretion is modulated by gonadotropin-releasing hormone (GnRH) pulse frequency and by changes in inhibins, activins, and follistatins both at the pituitary and at the peripheral level. To date no studies have looked at the overlapping function of these regulators in a combined setting. We tested the hypothesis that changes in GnRH pulse frequency alter the relative abundance of these regulators at the pituitary and peripheral levels in a manner consistent with changes in pituitary and circulating concentrations of FSH; that is, an increase in FSH will be accompanied by increased stimulatory input (activin) and/or reduced follistatin and inhibin. Ovariectomized ewes were subjected to a combination hypothalamic pituitary disconnection (HPD)-hypophyseal portal blood collection procedure. Hypophyseal portal and jugular blood samples were collected for a 6-h period from non-HPD ewes, HPD ewes, or HPD ewes administered GnRH hourly or every 3 h for 4 days. In the absence of endogenous hypothalamic and ovarian hormones that regulate gonadotropin secretion, 3-hourly pulses of GnRH increased pituitary content of FSH more than hourly GnRH, although these differences were not evident in the peripheral circulation. The results failed to support the hypothesis in that the preferential increase of pituitary content of FSH by the lower GnRH pulse frequency could be explained by changes in the pituitary content of inhibin A, follistatin, or activin B. Perhaps the effects of GnRH pulse frequency on FSH is due to changes in the balance of free versus bound amounts of these FSH regulatory proteins or to the involvement of other regulators not monitored in this study.     

Hypothalamic site of progesterone action on gonadotropin release

Tonic gonadotropin secretion was monitored at 20 min intervals for a total of 9 hours in 3 female volunteers during the mid-luteal phase of an ovulatory cycle. This control period was followed by repeated LH-RH stimulation (12 micrograms LH-RH as i.v. bolus once every hour for another 5 hours). During the control period spontaneous albeit low-frequent pulsatile secretion was observed for LH (a pulse occurring once every 3-8 hours) but not for FSH. While intermittent exogenous LH-RH stimulation was being performed at circhoral LH-RH pulse frequency pulsatile gonadotropin release was established at synchronous episodicity and systemic gonadotropin levels consecutively increased. These data provide indirect evidence that the pituitary gland is not rendered refractory to LH-RH by luteal progesterone secretion but readily responds to LH-RH stimuli even when these simulate a follicular phase LH-RH pulse frequency. Thus, it is concluded that spontaneous pulsatile LH release at low frequency during the luteal phase of the cycle reflects low frequent LH-RH discharges from the hypothalamus. Underlying mechanisms are discussed.     

Studies of the effectiveness of gonadotrophin-releasing hormone, steroids and follicular fluid in modulating ovine gonadotrophin output in vivo and in vitro

Gonadotrophin-releasing hormone (GnRH) readily stimulated LH output by sheep pituitary cells in vitro, and raised plasma LH concentrations in vivo in sheep, in a dose-dependent fashion. However, increases in FSH levels were only marginal by comparison. Dose-dependent decreases in sheep pituitary cell FSH output and in plasma FSH concentrations were caused by sheep follicular fluid and oestradiol-17 beta in vitro, and by bovine follicular fluid and oestradiol benzoate in vivo. In contrast, LH concentrations were only reduced slightly at the higher doses of these reagents. Cumulative suppressive effects of follicular fluid and oestradiol-17 beta (oestradiol benzoate) on FSH levels were observed both in vitro and in vivo. The transient positive feedback effect of oestradiol benzoate on FSH output negated the suppressive effect of bovine follicular fluid on plasma FSH concentrations. Progestagens, androgens and catechol oestrogens also suppressed mean FSH output in vitro, though not as effectively as oestradiol-17 beta. While only 1-5 pg/ml of oestradiol-17 beta was needed to suppress significantly mean FSH output in vitro, greater than 500 pg/ml of the other steroids was required. Seminal plasma inhibin-like peptide failed to suppress mean FSH output by cultured sheep pituitary cells at doses from 1 pg/ml to 500 ng/ml. At higher doses, both FSH and LH output was suppressed and this was accompanied by morphological deterioration of the cells. It is suggested that, to raise plasma FSH concentrations with a view to increasing ovulation rates in sheep, the development of means to reduce the negative feedback effects of steroids, notably oestradiol-17 beta, and inhibin on FSH secretion may be a more appropriate pharmacological strategy than increasing pituitary exposure to GnRH.     

Opioidergic control of gonadotropin secretion in the female rabbit: divergent effects of morphine on secretion of follicle-stimulating hormone and luteinizing hormone

The nature of secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was followed in female rabbits on a daily basis from age 36 to 60 days by sequential 5-min blood sampling over 1- to 2-h periods each day. Both LH and FSH were found to be secreted in a pulsatile manner. The mean LH pulse amplitude over the 25 days was 0.95 +/- 0.32 ng/mL and for FSH it was 10.15 +/- 1.11 ng/mL. Mean plasma LH levels were significantly increased from 1.46 +/- 0.08 ng/mL in 36 to 42-day-old rabbits to 1.89 +/- 0.12 ng/mL in 43 to 50-day-old rabbits and remained elevated from 50 to 60 days. FSH levels during the same periods also rose significantly from 14.93 +/- 0.79 to 19.57 +/- 2.05 ng/mL. To examine the influence of endogenous opioid peptides on the release of LH and FSH in 36 to 60-day-old female rabbits, morphine sulfate at 0.2, 0.5, 2.0, and 5.0 mg/kg was administered subcutaneously after 30 min baseline sampling, and blood was taken for another 60-120 min. Morphine at all doses and at all ages inhibited the amplitude and frequency of LH pulses but had no effect on FSH secretion. To determine whether the effects of morphine on LH secretion could be reversed with naloxone, females aged 82-114 days were used. Naloxone administered 1 h after morphine reversed the inhibitory effects of morphine, whereas the simultaneous administration of naloxone with morphine had variable effects but seemed to delay the LH increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of mouse epidermal growth factor on plasma concentrations of LH, FSH and testosterone in rams

Two experiments were conducted to examine the effects of mouse epidermal growth factor (EGF) on the concentrations of testosterone, LH and FSH in jugular blood plasma and on the pituitary responsiveness to LHRH. In 20 rams treated with subcutaneous doses of EGF at rates of 85, 98 or 113 micrograms/kg fleece-free body weight, mean plasma LH and testosterone concentrations were significantly reduced (P less than 0.05) at 6 h after treatment but not at 24 h. EGF treatment at 130 micrograms/kg fleece-free body weight suppressed the plasma content of these hormones for up to 48 h. Mean plasma FSH concentrations decreased significantly (P less than 0.05) for up to 48 h after EGF treatment, the effect being most pronounced in rams with mean pretreatment FSH values greater than or equal to 0.5 ng/ml. Intravenous injections of 1.0 micrograms LHRH given to each of 5 rams before and at 6 h, 24 h and 72 h after EGF treatment produced LH and testosterone release patterns which paralleled those obtained in 5 control rams similarly treated with LHRH. These results suggest that, in rams, depilatory doses of mouse EGF temporarily impair gonadotrophin and androgen secretion by inhibiting LHRH release from the hypothalamus. Such treatment appears to have no effect on the responsiveness of the pituitary to LHRH.