Effects of neonatal administration of monosodium glutamate and castration on neurokinin A levels in the hypothalamus and anterior pituitary of rats.

The effects of neonatal administration of monosodium glutamate (MSG) and castration on hypothalamic and anterior pituitary levels of neurokinin A (NKA) were studied in male and female rats killed at 46 days of age. In male rats treated neonatally with MSG, body, anterior pituitary, testis, ventral prostate, and seminal vesicle weights and serum testosterone levels were significantly lower than in saline-injected controls. Hypothalamic NKA was significantly lower in MSG-treated male rats as compared with the controls, and no apparent changes were recorded in anterior pituitary NKA. Orchidectomy was followed by a significant decrease in hypothalamic NKA in saline controls, but not in MSG-treated rats. In female rats treated with MSG, there was a significant decrease in body, anterior pituitary, and ovarian weights, as compared with saline-injected controls, but no significant differences were observed in uterine weights and serum estradiol levels. Hypothalamic NKA was lower, although not significantly, in MSG-treated rats as compared with the respective controls, and no differences were recorded in anterior pituitary NKA levels. Ovariectomy was followed by a significant decrease in hypothalamic NKA in both MSG-treated and control rats, but NKA in the anterior pituitary was significantly increased after ovariectomy only in saline-treated controls, whereas MSG-treated females failed to show this response. It is concluded that neonatal MSG treatment resulted in a decrease of hypothalamic NKA, which was particularly pronounced in male rats without any significant change in anterior pituitary NKA levels. The response of hypothalamic NKA to castration and the response of anterior pituitary NKA to ovariectomy were also altered in MSG-treated rats; this may reflect a functional block of some neuroendocrine functions of the hypothalamus that resulted from the neuronal lesions induced by MSG.     

Circulating radioimmunoassayable inhibin during periods of transient follicle-stimulating hormone rise: secondary surge and unilateral ovariectomy

We have measured changes in circulating immunoreactive (ir-) inhibin in male and female rats using an RIA with an antiserum raised against porcine inhibin alpha (1-26)-Gly-Tyr. The same synthetic peptide was used for standards and for the preparation of tracer. Serum ir-inhibin levels were significantly higher in intact female than in intact male rats (p less than 0.001). Immunoreactive inhibin was significantly reduced in both sexes 24 h after bilateral gonadectomy (p less than 0.0001). Unilateral ovariectomy (ULO) of female rats on metestrus caused a transient decrease in serum inhibin 8 h after surgery, but levels were not significantly different from those of sham-operated controls at later times after surgery. Increases in serum FSH and LH were observed for 8-18 h after ULO. Serum ir-inhibin levels were also measured on the early morning of estrus during the secondary FSH surge. At this time, ir-inhibin levels were low, while FSH levels were high and LH levels were low. These results show that serum ir-inhibin levels in rats are decreased at times when serum FSH levels are high.     

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.     

A role for inhibin in the control of follicle-stimulating hormone secretion in male rats

We examined the relationship of testosterone (T) and porcine follicular fluid (pFF) in the negative feedback control of FSH and LH secretion in adult male rats. Either at the time of castration (acute) or at least 30 days after castration (chronic), we implanted T-filled Silastic capsules, which were 2 mm, 10 mm, or 30 mm long; empty capsules (30 mm) served as controls. Seven days later, we injected either 0.15 ml of pFF or saline (i.v.), decapitated the rats 6 hours later, and collected trunk blood for subsequent serum analysis of FSH, LH, and T by RIA. In the acute groups, T implants suppressed the postcastration rises in plasma FSH and LH levels in a dose-dependent manner, with only the largest implant, 30 mm, able to return them to intact levels. PFF injection significantly suppressed FSH levels in intact and acute rats but had no effect on serum LH. In chronic rats, T therapy for 7 days suppressed plasma LH levels in a dose-dependent relationship, yet did not do so to plasma FSH levels. FSH levels were significantly higher in rats with the 30 mm T implants than in intact rats, but were significantly suppressed as compared to chronic controls. PFF significantly suppressed serum FSH levels in all chronic groups with the chronic controls showing the greatest amount of suppression. We conclude that the role for inhibin in the normal control of FSH secretion is that of a secondary modulator which is superimposed on, yet independent of, the steroid feedback mechanism. At any given moment this modulation is dependent upon the secretory activity of the FSH gonadotrope.     

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.     

Inhibin activity and secretion of gonadotropin during the period of follicular maturation

To evaluate the relative contributions of the ovarian inhibin and estradiol-17 beta (E) on the regulation of FSH secretion, inhibin and E in ovarian venous plasma (OVP) and FSH and LH in peripheral plasma were simultaneously measured using superovulating rats with special reference to follicular maturation. By the transplantation of a pituitary gland from adult male rats under the kidney capsule between 1100 and 1200 hr on diestrus-1 in cyclic rats, superovulation was successfully induced on the morning of the next estrus without any additional treatment with human chorionic gonadotropin (hCG). The number of maturing follicles capable of ovulating in response to hCG significantly increased at 12 hours after the grafting as compared with sham-operated controls and further increases occurred until the afternoon of proestrus. In the superovulating rat, first and second surges of FSH were completely blocked and an LH surge was also partially suppressed during the periovulatory period when surges of FSH and LH were normally observed in controls. Contents of FSH as well as LH in the animal's own pituitary gland were suppressed significantly after the grafting as compared with controls. A marked increase in inhibin activity in OVP of rats with a pituitary transplant occurred concomitantly with an increase in the number of follicles capable of ovulating whereas E levels in OVP did not so. Inhibin activity in OVP at each point was much higher in the pituitary grafted rats than in controls but this was not true for E levels. These results suggest that ovarian inhibin derived from the maturing follicles rather than E may be a primary factor for regulation of FSH secretion, and high levels of endogenous inhibin can suppress synthesis of LH as well as FSH in the pituitary gland of the female rat.     

Blockade of the selective increase in serum follicle-stimulating hormone concentration in immature female rats and its effects on ovarian follicular development

We investigated whether administration of monosodium L-glutamate (MSG) to neonatal female rats would block the selective increase in serum follicle-stimulating hormone (FSH) concentration in immature rats in an attempt to provide a model in which to study the importance of the selective FSH rise on ovarian follicular development. In two separate experiments, s.c. injections of MSG (4 mg/g BW) on Days 1, 3, 5, 7 and 9 after birth blocked the selective increase in serum FSH concentration observed on Days 7 and 15 without blocking basal FSH secretion. Serum luteinizing hormone (LH) levels were unaffected in the first experiment and changed little in the second. MSG-treated rats had smaller ovaries on Days 15 and 23. The ovaries of MSG-treated rats on Day 15 showed decreased follicular growth as evidenced by a decrease in the number and percentage of follicles with diameters greater than 50 microns, in the number of follicles with greater than 1 layer of granulosa cells, and in the number of follicles beyond the primary stage of follicular development. These differences between MSG-treated rats and controls all but disappeared by Day 23. The results demonstrate that neonatal administration of MSG blocks the selective increase in serum FSH concentration in immature female rats and suggest that this selective increase in serum FSH levels plays a role in the normal acceleration of ovarian follicular development but is not needed for the development of preovulatory follicles by the sixth week after birth.     

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)     

Evidence of an epididymis factor inhibiting the hypothalamic synthesis of FSH-RH in the rat]

We have measured, by radioimmunoassay, FSH and LH in the blood plasma and in the hypophysis of castrated male rats, injected with epididymal inhibin; we have also evaluated the FSH and LH releasing activities of their hypothalamus by measuring plasma FSH and LH levels of spayed female rats, treated by hypothalamic extracts of the previous rats. The FSH and LH pituitary levels do not change compared with controls, and it is impossible to know if inhibin acts directly on hypophysis; it is likely that, directly or indirectly, inhibin restrains at the same time the synthesis and the release of FSH. On the contrary, the hypothalamic extracts lose their FSH-RH, but not their LH-RH, activities; then, inhibin operates on hypothalamus by suppressing of the synthesis of FSH-RH.     

Regulation of gonadotropin secretion and number of gonadotropin-releasing hormone receptors by inhibin, activin-A, and estradiol.

Primary cultures of ovine pituitary cells were used to characterize the effects of inhibin and activin on the secretion of gonadotropins and on the regulation of number of GnRH receptors in the presence or absence of estradiol. Number of GnRH receptors was determined by the specific binding of a saturating dose of [125I]des-Gly10-D-Trp6-GnRH-ethylamide (GnRH-A). Recombinant human inhibin-A (rh-inhibin-A) or inhibin in porcine and bovine follicular fluid (pFF and bFF, respectively) decreased secretion of FSH in a dose-dependent manner, with maximum inhibition at an inhibin concentration of approximately 0.1 nM. Neither pFF or bFF affected secretion of LH, although rh-inhibin-A caused a modest decrease (p less than 0.05) in secretion of LH. Treatment of cells with rh-inhibin-A, bFF, or pFF approximately doubled the number of GnRH receptors. Scatchard analysis indicated that increases in GnRH-A binding were due to an increase in receptor number rather than a change in affinity. Additionally, rh-inhibin-A, at a dose that doubled numbers of GnRH receptors, increased GnRH-induced LH release above that caused by GnRH alone, indicating that the increase in receptor number leads to increased responsiveness to GnRH. Recombinant human activin-A (rh-activin-A) increased secretion of FSH but did not affect secretion of LH. Number of GnRH receptors was not affected by lower concentrations of rh-activin-A but was decreased (p less than 0.05) by 3.0 nM activin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of testicular feedback in male rats using artificial cryptorchidism as a model.

Plasma LH, FSH and testosterone were measured in testosterone-treated and untreated cryptorchid and castrated male rats. Exogenous testosterone prevented the increase in basal LH but not FSH levels seen in the untreated cryptorchids. Increases in plasma LH and FSH in response to LH-RH were greater in the cryptorchid as compared to the control group and this could not be reversed by exogenous testosterone, suggesting that spermatogenesis-related feedback factors regulate LH as well as FSH at the pituitary level in the intact rat. The results were consistent with a reduced but nevertheless significant secretion of inhibin by the cryptorchid testis. Basal plasma testosterone levels and ventral prostate weights were not significantly different from intact animals.     

In vivo suppression of pituitary and circulatory follicle stimulating hormone by human seminal plasma inhibin

Human seminal plasma inhibin (HSPI), of prostatic origin, has been shown to bring about a dose dependent suppression in pituitary and circulatory FSH concentrations in intact rats. No significant changes in LH levels either in pituitary or in circulation were observed at the doses used. This has further been substantiated by an immunocytochemical staining. A marked reduction in staining intensity for FSH was observed in the pituitary of inhibin treated rats as compared to the controls. None of the purified inhibin peptides from other sources have so far been reported to act on pituitary FSH in vivo. This study thus, for the first time demonstrates an in vivo effect of inhibin (HSPI) on pituitary FSH concentration and secretion.     

Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures

The actions of two inhibin preparations and cycloheximide on gonadotropin release were investigated in superfused pituitary cell cultures. Pituitary cells isolated from 18-day-old male rats were grown in Matrigel-coated superfusion chambers in chemically defined medium. After stationary culture for 4 days, the cell monolayers were superfused at a constant speed (0.25 ml/min) and were intermittently stimulated (6 min/h) with 10 nM gonadotropin-releasing hormone (GnRH). Groups of cultures were exposed to the test substances for varying time periods during stationary culture and/or during superfusion. Inhibitory effects of both inhibin preparations on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in response to GnRH pulses were observed after 2 h of exposure and became maximal after about 6 h. Basal secretion of FSH between GnRH pulses was also suppressed, whereas the basal interpulse secretion of LH was not changed. When exposure to inhibin was discontinued, the secretion of both FSH and LH progressively increased and returned to control values by approximately 6 h. Cycloheximide (500 ng/ml) affected gonadotropin release with dynamics similar to those observed for the inhibin preparation. These data support the hypothesis that inhibition of gonadotropin synthesis may be an important step in the molecular mechanism of action by which inhibin regulates gonadotropin release.     

Inhibin secretion during the rat estrous cycle: relationships to FSH secretion and FSH beta subunit mRNA concentrations

Serum inhibin and FSH and FSH beta subunit mRNA levels were measured at 3h intervals throughout the 4 day estrous cycle in female rats and hourly between 1000 and 2400 h of proestrus. On proestrus, serum inhibin concentrations fell during the late morning-early afternoon, then increased transiently during the late afternoon gonadotropin surges. Inhibin levels decreased during the late evening of proestrus, coincident with the FSH surge-related rise in FSH beta mRNA levels. Serum inhibin remained relatively stable during estrus and early metestrus, but rose during the late evening of metestrus and remained elevated until early diestrus. FSH beta mRNA levels were elevated on late estrus and early metestrus and declined during the evening of metestrus as serum inhibin levels increased. These data show that concentrations of serum inhibin change during the estrous cycle and that a general inverse relationship exists between serum inhibin and FSH levels and FSH beta mRNA concentrations in the pituitary. This suggests that inhibin may inhibit FSH beta gene expression and FSH secretion during the 4 day cycle in female rats.     

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.     

Immunological study of ovarian inhibin

Antisera to purified porcine follicular fluid inhibin of 32 K protein (pFF 32 K inhibin) were raised in rabbits. Increasing doses of an antiserum with high titer could neutralize the maximal suppression of FSH secretion caused by 10 ng of pFF 32 K inhibin from rat anterior pituitary cells in culture. A radioimmunoassay was developed using the antiserum and 125I-labelled pFF 32 K inhibin. Specificity of the antiserum was examined by comparing immunological and biological potencies of various inhibin preparations. Cross-reactivity tests revealed that the antiserum almost recognizes rat ovarian inhibin preparations. The antiserum also recognizes purified bovine follicular fluid inhibin of 32 K protein (bFF 32 K inhibin), but with a cross-reactivity of approximately 20%. Cross-reactivity of human follicular fluid to the antiserum was less than 10%. The antiserum also recognizes inhibin forms of higher molecular weights, 100 K, 80 K, and 55 K proteins, which have previously been identified by gel filtration or SDS-PAGE of crude pFF inhibin preparations under protein-dissociation conditions, indicating that these inhibin forms have common or closely related immunodetermining sites.     

Effects of discrete medial preoptic hypothalamic lesions on the androgen gonadotropin feedback system in the male rat

The effect of discrete lesions of the Anterior Medial Preoptic Area of the Hypothalamus (MPOA) in the control of pituitary gonadotropins in the adult male Wistar rat has been studied. Electrolytic lesions were made by passing an anodal current through tungsten electrodes. Electrodes were oriented stereotaxically into the MPOA and lesion placement was histologically checked. In sham controls, electrodes were lowered into the MPOA but no current was applied. Serum LH and FSH were measured by RIA. MPOA lesioned animals showed significantly lower plasma LH (p less than 0.01) in comparison to sham lesioned group. Plasma FSH remained unaltered. To test whether these results were related to an alteration in the negative feedback system, the response to administration of Testosterone Propionate (TP) and the secretion patterns of LH and FSH after castration were analyzed. Administration of TP revealed similar LH and FSH 24 and 48 h decrements and the pattern of LH and FSH secretion after gonadectomy was not significantly different in lesioned and sham lesioned animals. Responsiveness to exogenous LHRH was not impaired by MPOA lesions. The results suggest that neural elements within the MPOA are functionally related to pituitary LH secretion in the male rat. The LH control alteration in lesioned animals is not associated with modifications in negative feedback of androgens and pituitary sensitivity to LHRH remains unaltered.     

Dexamethasone increases inhibin and estradiol secretion mediated by endogenous FSH in equine chorionic gonadotropin (eCG)-primed immature female rats.

In the present study, we have examined whether the effects of dexamethasone on follicle stimulating hormone (FSH) secretion were mediated by hypophysiotropic factors, and whether the increased levels of FSH induced by dexamethasone can stimulate ovarian functions in equine chorionic gonadotropin (eCG)-primed immature female rats. Dexamethasone (500 microg) significantly increased serum concentrations of FSH in hypophysectomized rats implanted with pituitary under the kidney capsule, as well as in intact rats. Serum concentrations of inhibin and estradiol in eCG (2.5, 5 i.u.)-primed rats were significantly increased by simultaneous treatment with dexamethasone (500 microg) and eCG. These simultaneous effects were not confirmed in hypophysectomized rats. The results had shown that hypophysiotropic factors do not mediate the selective increase of FSH secretion caused by dexamethasone. Dexamethasone induces the excess amount of FSH secretion from anterior pituitary and this FSH can stimulate inhibin and estradiol secretion in eCG-primed immature female rat.     

Effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion in rats

The effect of uni- and bilateral cryptorchidism on testicular inhibin and testosterone secretion and their relationships to gonadotropins were studied in rats. Mature Wistar male rats weighing approximately 300 g were made either uni- or bilaterally cryptorchid. Testicular inhibin and testosterone content and plasma levels of LH and FSH were examined 2 weeks later. A similar remarkable decrease in testicular inhibin content was found in uni- and bilaterally cryptorchid testes. On the other hand, the testicular testosterone content was significantly decreased only in unilaterally cryptorchid testis with an inverse increase in the contralateral testis. Plasma testosterone levels were normal and plasma LH and FSH increased significantly in both of the cryptorchid groups. These results showed that cryptorchidism impairs both Sertoli and Leydig cell functions. While testosterone production was compensated by increased LH for 2 weeks, neither inhibin secretion nor storage changed in cryptorchid or contralateral testes during the same period.