FSH-releasing Factor Research Articles

Growth hormone, luteinizing hormone, and follicle-stimulating hormone regulation by neuropeptide Y in both sexes of the cichlid fish, Cichlasoma dimerus.

Neuropeptide Y (NPY) is considered the most potent orexigenic peptide, increasing before meal time and during fasting. In teleost, most studies on NPY action upon growth hormone (GH) and luteinizing hormone (LH) were conducted in females or group of animals without sex discrimination. The aim of this study was to evaluate whether NPY modulates the expression and release of GH and gonadotropins in both sexes of Cichlasoma dimerus. By double-label immunofluorescence, we first determined the association between NPY fibers and pituitary cells. In addition, we performed in vitro studies to evaluate the effect of NPY on GH and gonadotropins expression by real-time PCR, and release by Western blot, in males and females separately. Contacts between NPY fibers and GH and follicle-stimulating hormone (FSH)-producing cells were detected, indicating possible functional relationships. We observed an increase in GH release in the culture medium at 2 nM for males (p = 0.043) and 20 nM for females (p = 0.028). Pituitary FSH release was stimulated at 20 nM (p = 0.026) and 200 nM (p = 0.033) for males and females, respectively. Finally, NPY only increased β-LH mRNA expression at 20 nM in females (p = 0.028) and its release at 2 nM (p = 0.049) and 200 nM for males (p = 0.005) and 200 nM for females (p = 0.018). In conclusion, NPY acts as a GH-, LH- and FSH-releasing factor, in a dose- and sex-dependent way.

Lamprey GnRH-III acts on its putative receptor via nitric oxide to release follicle-stimulating hormone specifically.

Lamprey gonadotropin-releasing hormone-III (l-GnRH-III), the putative follicle-stimulating hormone (FSH)-releasing factor (FSHRF), exerts a preferential FSH-releasing activity in rats both in vitro and in vivo. To test the hypothesis that l-GnRH-III acts on its own receptors to stimulate gonadotropin release, the functional activity of this peptide at mammalian (m) leutinizing hormone (LH)RH receptors transfected to COS cells was tested. l-GnRH-III activated m-LHRH receptors only at a minimal effective concentration (MEC) of 10(-6) M, whereas m-LHRH was active at a MEC of 10(-9) M, at least 1,000 times less than that required for l-GnRH-III. In 4-day monolayer cultured cells, l-GnRH-III was similarly extremely weak in releasing either LH or FSH, and, in fact, it released LH at a lower concentration (10(-7) M) than that required for FSH release (10(-6) M). In this assay, m-LHRH released both FSH and LH significantly at the lowest concentration tested (10(-10) M). On the other hand, l-GnRH-III had a high potency to selectively release FSH and not LH from hemipituitaries of male rats. The results suggest that the cultured cells were devoid of FSHRF receptors, thereby resulting in a pattern of FSH and LH release caused by the LHRH receptor. On the other hand, the putative FSH-releasing factor receptor accounts for the selective FSH release by l-GnRH-III when tested on hemipituitaries. Removal of calcium from the medium plus the addition of EGTA, a calcium chelator, suppressed the release of gonadotropins induced by either l-GnRH-III or LHRH, indicating that calcium is required for the action of either peptide. Previous results showed that sodium nitroprusside, a releaser of nitric oxide (NO), causes the release of both FSH and LH from hemipituitaries incubated in vitro. In the present experiments, a competitive inhibitor of NO synthase, L-NG-monomethyl-L-arginine (300 micro M) blocked the action of l-GnRH-III or partially purified FSHRF. The results indicate that l-GnRH-III and FSHRF act on putative FSHRF receptors by a calcium-dependent NO pathway.

Lamprey Gonadotropin Hormone‐Releasing Hormone‐III has No Selective Follicle‐Stimulating Hormone‐Releasing Effect In Rats

Lamprey gonadotropin releasing-hormone (LGnRH)-III, a hypothalamic neurohormone recently isolated from sea lamprey, was reported to have a selective stimulatory effect on follicle-stimulating hormone (FSH) release in rats and suggested to be the mammalian FSH-releasing factor. In this study, we determined the relative luteinizing hormone (LH)- and FSH-releasing potency of LGnRH-III compared to mammalian gonadotropin-releasing hormone (LHRH) in normal female rats, ovariectomized (OVX) and oestrogen/progesterone substituted rats and the superfused rat-pituitary cell system. The specificity of LGnRH-III for the mammalian LHRH receptor was investigated by blocking the receptor with an LHRH antagonist, MI-1544. In vitro, LGnRH-III dose-dependently stimulated both LH and FSH secretion from rat pituitary cells at 10(-7) to 10(-5) M concentrations, while LHRH stimulated gonadotropin secretion at a 1000-fold lower doses (10(-10) to 10(-8) M). The difference between its LH- and FSH-releasing potency was similar to that of LHRH. LGnRH-III bound to high affinity binding sites on rat pituitary cells with a Kd of 6.7 nM, B(max)=113 +/- 27 fmol/mg protein. In vivo, LGnRH-III also stimulated both LH and FSH secretion in a dose-dependent manner and, similar to LHRH, induced a greater rise in the serum LH than the FSH level. In normal cycling rats, it showed 180-650-fold weaker potency than LHRH in stimulating LH secretion and 70-80-fold weaker effect in stimulating FSH secretion. In OVX rats, LGnRH-III demonstrated a similarly weak effect on both gonadotropins. It was found to be 40-210-fold less potent than LHRH regarding LH release and 50-160-fold weaker regarding FSH release. LHRH-receptor antagonist MI-1544 prevented both the LH- and the FSH-releasing effect of LGnRH-III both in vitro and in vivo. These results do not support the hypothesis that LGnRH-III might be the mammalian FSH-releasing factor but demonstrate that it is a weak agonist for the pituitary LHRH receptor and stimulates both gonadotropins in a dose-dependent fashion.

Is there an FSH-releasing factor?

A thorough understanding of the factors that regulate the secretion of FSH is critical for the development of efficient methods for fertility regulation. The purpose of this review is to evaluate what is currently known about the existence of FSH-releasing factor(s). It considers the obstacles encountered in understanding the control of FSH secretion, current knowledge of the nature of FSH secretion and the mechanisms involved in controlling FSH secretion, the arguments that have been posed against the need for an FSH-releasing factor and finally the evidence available to date supporting the existence of FSH-releasing factor(s).

Is there an FSH-releasing factor?

Is there an FSH-releasing factor?

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

Median eminence lesions reveal separate hypothalamic control of pulsatile follicle-stimulating hormone and luteinizing hormone release.

The effects of hypothalamic lesions designed to destroy either the anterior median eminence (ME) or the posterior and mid-ME on pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined in castrated male rats. In sham-operated animals, mean plasma FSH concentrations rose to peak at 10 min after the onset of sampling, whereas LH declined to a nadir during this time. In the final sample at 120 min, the mean FSH concentrations peaked as LH decreased to its minimal value. In rats with anterior ME lesions, there was suppression of LH pulses with continuing FSH pulses in 12 of 21 rats. On the other hand, in animals with posterior to mid-ME lesions, 3 out of 21 rats had elimination of FSH pulses, whereas LH pulses were maintained. Fifteen of 42 operated rats had complete ME lesions, and pulses of both hormones were abolished. The remaining 12 rats had partial ME lesions that produced a partial block of the release of both hormones. The results support the concept of separate hypothalamic control of FSH and LH release with the axons of the putative FSH-releasing factor (FSHRF) neuronal system terminating primarily in the mid- to caudal ME, whereas those of the LHRH neuronal system terminate in the anterior and mid-median eminence. We hypothesize that pulses of FSH alone are mediated by release of the FSHRF into the hypophyseal portal vessels, whereas those of LH alone are mediated by LHRH. Pulses of both gonadotropins simultaneously may be mediated by pulses of both releasing hormones simultaneously. Alternatively, relatively large pulses of LHRH alone may account for simultaneous pulses of both gonadotropins since LHRH has intrinsic FSH-releasing activity.

A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat.

Previous studies indicated that there is a separate hypothalamic control of follicle-stimulating hormone (FSH) release distinct from that of luteinizing hormone (LH). An FSH-releasing factor (FSHRF) was purified from rat and sheep hypothalami, but has not been isolated. We hypothesized that FSHRF might be an analogue of mammalian luteinizing hormone-releasing hormone (m-LHRH) and evaluated the activity of many analogues of m-LHRH and of the known LHRHs found in lower forms. Here we demonstrate that lamprey (l) LHRH-III has a potent, dose-related FSH- but not LH-releasing action on incubated hemipituitaries of male rats. l-LHRH-I on the other hand, had little activity to release either FSH or LH. m-LHRH was equipotent to l-LHRH-III to release FSH, but also had a high potency to release LH in contrast to l-LHRH-III that selectively released FSH. Chicken LHRH-II had considerable potency to release both LH and FSH, but no selectivity in its action. Salmon LHRH had much less potency than the others tested, except for l-LHRH-I, and no selectivity in its action. Because ovariectomized, estrogen, progesterone-treated rats are a sensitive in vivo assay for FSH- and LH-releasing activity, we evaluated l-LHRH-III in this assay and found that it had a completely selective stimulatory effect on FSH release at the two doses tested (10 and 100 pmols). Therefore, l-LHRH-III is a highly potent and specific FSH-releasing peptide that may enhance fertility in animals and humans. It may be the long sought after m-FSHRF.

Control of follicle-stimulating hormone and luteinizing hormone release by hypothalamic peptides.

Lesion, stimulation, and pharmacological studies point to separate hypothalamic control of pulsatile FSH and LH secretion. LH release is controlled by a region extending from the preoptic area to the anterior and mid-median eminence, whereas FSH release is controlled by a region extending from the dorsal anterior hypothalamic area to the caudal median eminence. We have separated an FSH-releasing factor from LHRH by gel-filtration on Sephadex G-25, confirming results obtained over 25 years ago; and we are attempting its isolation in collaboration with Vale and River. In the meantime, reasoning that FSH-releasing factor might be related to LHRH, we tested many analogs of LHRH and found one that has selective FSH-releasing activity over a 50-fold dose range; however, it is relatively weak. This led us to the possibility that the GAP might be FSH-RF. Indeed, GAP1-13 has FSH but no LH-releasing activity over a 100-fold dose range; however, it is less potent than we would expect of the natural product. Substituting D-Trp-9 into the molecule to inhibit enzymatic degradation yielded a more potent and completely selective FSH-releasing peptide,24 which could be clinically useful. Alpha-inhibin-92 of Li et al. has been shown to have a highly selective dose-related suppressive action on FSH release in castrate male rats.25 Smaller fragments (35-65 and 66-92) of this molecule also possess the activity, albeit at higher doses. That this molecule may be physiologically significant is indicated by elevations in plasma FSH in immature rats obtained following intravenous injection of antisera raised against the peptide. Because of its much smaller size than that of 32-kDa alpha, beta inhibins and the lack of carbohydrate in the molecule, this can be relatively easily synthesized and might have clinical utility as an FSH release-inhibiting peptide.

Differential gonadotropin responses to N-methyl-D,L-aspartate in intact and castrated male rats.

Peripheral administration of N-methyl-D,L-aspartate (NMA), a neuroexcitatory amino acid agonist, probably stimulates LH release through an increase in endogenous LHRH secretion. In the present study, NMA and a potent LHRH antagonist were used to determine the degree to which release of FSH is similarly dependent upon the acute secretion of LHRH. A second aim was to compare responsiveness of LHRH neurons to NMA in castrated and intact male rats. Adult male rats were castrated (n = 10) or sham castrated (n = 11) on the morning of Day 0. After 8 days, rats were fitted with atrial catheters between 0900 and 1200 h; at 2100 h they received s.c. either oil vehicle or 100 micrograms of an LHRH antagonist. Starting at 0900 h on Day 9, 0.5-ml blood samples were collected every 10 min for 3 h. After 1 h of sampling each animal received i.v. 5 mg of NMA in 0.5 ml 0.9% saline. An hour later each rat received i.v. 500 ng of LHRH in 0.5 ml saline. Plasma LH, FSH, and prolactin (PRL) levels were determined by RIA. In the oil-treated sham castrates, mean plasma LH levels were increased by 110% (p < 0.01) within 10 min and remained elevated for 30 min after the injection of NMA. The profile of this LH secretory response was similar to or slightly more robust than endogenous LH pulses observed previously. The NMA-induced LH release was completely blocked by pretreatment with LHRH antagonist. In both oil- and antagonist-treated sham-castrated rats, NMA administration failed to elicit a concomitant increase in plasma FSH levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Feedback of Follicle-Stimulating Hormone to Inhibit Luteinizing Hormone and Stimulate Follicle-Stimulating Hormone Release in Ovariectomized Rats

Injection of ovine follicle-stimulating hormone (o-FSH) into the third cerebral ventricle significantly suppressed plasma luteinizing hormone (LH) release in conscious, unrestrained ovariectomized rats. Intraventricular injection of o-FSH (0.5 microgram) significantly lowered the plasma levels of LH by 70 min and decreased pulse frequency, whereas the pulsatile release of FSH was unchanged during the 180-min duration of the experiment. In a 12-hour experiment, the suppression of LH release reached the maximum within 3 h (p less than 0.001), followed by a return of plasma LH level to initial values 6-8 h after intraventricular injection of o-FSH at doses of 0.5, 4 and 8 micrograms. Plasma levels of FSH were not significantly changed by the low dose of o-FSH (0.5 microgram), but there was a delayed elevation of plasma FSH during the 8-12 h after intraventricular injection of o-FSH at higher doses (4 and 8 micrograms). On the other hand, plasma prolactin was unchanged after intraventricular injection of o-FSH. Since the pituitary responsiveness to synthetic LH-releasing hormone (LHRH) was unimpaired at the end of the sampling and incubation with o-FSH suppressed the release of LHRH from median eminence terminals, these data indicate that FSH acts intrahypothalamically to suppress LHRH release. The late elevation of plasma FSH may be the result of enhanced release of FSH-releasing factor or of a delayed, direct effect of the hormone on the gonadotropes to increase release of FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered gonadotropin and prolactin release induced by median eminence (ME) lesions and pharmacological manipulation of prolactin release: Further evidence for separate hypothalamic control of FSH and LH release

To delineate more clearly the regions of the median eminence involved in the control of gonadotropin secretion, electrolytic lesions were made in the median eminence of adult male rats. Also the role of prolactin in the maintenance of gonadotropin secretion and sex organs was further evaluated by the administration of α-bromo-ergocryptine (CB154) or its diluent SC to lower prolactin via its dopamine agonist action in these animals with lesions. The effects of elevation of prolactin by the administration in the drinking water of the dopamine antagonist, sulpiride, were also evaluated in intact rats. At the conclusion of the experiments the localization of lesions was carefully evaluated by microscopic examination of serial sections through the hypothalamic region. The results indicate that lesions which completely destroyed the median eminence led to elevation in plasma prolactin and profound decreases in plasma FSH and LH associated with atrophy of testes and accessory sex organs. This result was not affected if CB 154 was given to lower the elevated prolactin levels. Lesions which destroyed the rostral 2/3 of the median eminence were frequently associated with declines in plasma LH and relatively normal plasma FSH, whereas lesions which extended caudally to destroy the posterior median eminence were associated uniformly with a profound lowering of FSH. If these lesions did not include the anterior portions of the median eminence, LH levels remained normal. The results support the concept of separate control of FSH and LH release. LH release appears to be controlled particularly by the rostral 2 3 of the median eminence which is the presumed site of termination of the terminals of LHRH-secreting neurons, whereas FSH release is predominantly controlled by the posterior 2 3 of the median eminence, the presumed termination of the axons of the putative FSH-releasing factor neurons. The development of testicular atrophy and target gland atrophy was more closely associated with low plasma and pituitary FSH than LH which led to the suggestion that the release of FSH may be more important for maintenance of gonadal and target gland weight than that of LH. Elevation of plasma prolactin by administration of sulpiride failed to alter gonadotropin levels, testes weight or accessory organ weight. Since profound elevation or lowering of plasma prolactin did not alter the weights of testes or sex accessory organs, it appears that prolactin has little effect on these organs in adult in contrast to young rats.

Simultaneous Measurement of Luteinizing Hormone (LH)-Releasing Hormone, LH, and Follicle-Stimulating Hormone Release in Intact and Short-Term Castrate Rats*

The temporal relationship between LHRH release and gonadotropin secretion as well as the effects of castration on LHRH release were investigated in conscious, freely moving male rats. LHRH release was measured in hypothalamic/median eminence perfusates, while levels of pituitary gonadotropins (LH, FSH) were determined in sequential blood samples obtained via atrial catheters. Twenty-four to 26 h before experiments, rats underwent sham surgery or castration. LHRH release in push-pull perfusates from both groups was pulsatile, and nearly all identified LH pulses (83.3%) were temporally associated with LHRH pulses. Of the fewer irregular FSH pulses that were observed, only 43.7% were temporally associated with LHRH pulses. Mean LHRH pulse amplitude and mean LHRH levels were not different in intact and castrate animals. The frequency of LHRH pulses was moderately increased in castrate rats (1.30 pulses/h) compared to that in intact animals (0.83 pulses/h), and this acceleration was accompanied by a significant increase in LH pulse frequency, pulse amplitude, and mean level. It was also noted that the number of silent LHRH pulses (those not associated with LH pulses) was dramatically reduced in castrate animals. Characteristics of gonadotropin release (pulse frequency, pulse amplitude, and mean level) were not significantly different in animals undergoing push-pull perfusion/bleeding procedures from those in rats not receiving push-pull cannula implants. We conclude from these studies that 1) LH pulses show a high concordance with LHRH pulses, providing evidence that the LHRH pulse generator operates as the neural determinant of LH pulses in male rats, 2) FSH secretion is not associated with LHRH release in an obvious and consistent manner, suggesting that LHRH/FSH relationships are not easily discerned in these animals or that a FSH-releasing factor distinct from the LHRH decapeptide may regulate FSH secretion, 3) a modest increase in LHRH pulse frequency occurs 24-30 h after castration, and 4) silent LHRH pulses occur with much greater regularity in intact than in castrate rats. The latter two observations suggest that both hypothalamic and intrapituitary sequelae of castration may be critically important in the development of postcastration increases in LH secretion and the negative feedback of gonadal steroids.

5Inhibin—a non-steroidal regulator of pituitary follicle stimulating hormone

Inhibin is a gonadal glycoprotein produced by the granulosa and Sertoli cell under the influence of FSH and acts to specifically suppress pituitary FSH secretion. Recently, ovarian inhibin has been purified from several species and its amino acid sequence deduced using cloning techniques. Inhibin consists of two disulphide-linked heterologous subunits of which the smaller may exist in two different forms accounting for two different forms of inhibin in humans and pigs. Heterogeneity of inhibin also exists as a result of proteolytic processing of the molecule during its passage into the circulation. Significant homology exists between the subunits of inhibin and the dimeric peptides TGF-beta and Mullerian inhibitory substance (MIS), suggesting they are all derived from a common ancestral gene. Furthermore, dimers of the smaller subunit of inhibin (FSH-releasing protein (FRP) or activin) have now been found in follicular fluid (FF) and, along with TGF-beta, shown to be potent and specific stimulators of FSH secretion. These proteins may be involved in controlling FSH by another as yet unknown pathway and may prove to be the FSH-releasing factor, analogous to LHRH, which has been postulated to exist for some years. Inhibin can no longer be simply considered as an isolated FSH-suppressing protein. The physiological significance and relationship between inhibin and its related proteins represent one of the most challenging and interesting areas in reproductive endocrinology. Further studies, particularly with the development and use of sensitive assays for both the FSH releasing hormone and inhibin will clarify their role in reproduction and their usefulness in monitoring or treating fertility.

Purification of FSH-releasing factor: Its dissimilarity from LHRH of mammalian, avian, and piscian origin

Sheep stalk median eminence fragments were lyophylized, extracted and filtered through a column of Sephadex G-25. The fractions were then assayed for the presence of LHRH by radioimmunoassay (RIA) and bioassayed for FSH and LH-releasing activity following their IV injection into ovariectomized, estrogen progesterone-blocked rats. The radioimmunoassayable LHRH emerged from the column at the same position from which it emerged many years before when LH was measured by bioassay. This same region also contained the LH-releasing activity as measured by bioassay. FSH-releasing activity was present in two tubes just preceding the emergence of the bio- and immunoassayable LHRH. The activity was highly significant and there was no LH- releasing activity in the fractions. They contained much less LHRH as determined by RIA than is sufficient to evoke LH release in this assay. The FSH-releasing activity was recovered in the same fractions in which it was found many years ago with this same assay but with measurement of plasma FSH by bio-rather than immunoassay as employed here. A dose-related release of LH was obtained by injection of LHRH in this assay but there was no significant FSH release even with a dose of 27 ng of LHRH per rat. To determine if one of the LHRHs of lower forms might be FSH-RF, Chicken I and II LHRH and Salmon LHRH were also assayed for FSH- and LH-releasing activity. Each of these peptide possessed LH-releasing activity, albeit much less than that of the mammalian peptide but had no FSH-releasing activity whatsoever. It is concluded that an FSH-RF probably mediates the selective hypothalamic control of FSH release. The structure of the presumed peptide remains to be determined.

Effect of destruction of the dorsal anterior hypothalamus on follicle-stimulating hormone secretion in the rat.

The role of the paraventricular nucleus-dorsal anterior hypothalamus (PVN-DAHA) in the control of anterior pituitary gland secretion of FSH and LH in castrated male and female rats was examined. Bilateral radiofrequency lesions of the PVN-DAHA in chronically ovariectomized (OVX) rats lowered plasma FSH levels by 33% (P less than 0.005) compared to values in unoperated and sham-operated control rats; plasma LH concentrations were unaltered. RIA of median eminence (ME) LHRH concentrations in these animals revealed no differences among the three experimental groups. Other categories of diencephalic destruction did not result in this pattern of selectively reduced FSH release. Bilateral radiofrequency destruction of the PVN-DAHA also attenuated by 50% (P less than 0.025 to P less than 0.005) the progesterone-induced surge of FSH in estrogen-primed OVX rats. Progesterone-induced LH release was unaffected by PVN-DAHA lesions. Other lesion categories failed to show the same result. Bilateral ablation of the PVN-DAHA in male rats resulted in a selective diminution of the postcastration rise of plasma FSH beginning 48 h postcastration (P less than 0.05 to P less than 0.005) and persisting for 14 days (P less than 0.005) after orchidectomy, thus revealing the time course and permanence of this procedure on plasma FSH levels. The postcastration rise of plasma LH levels was not affected by PVN-DAHA lesions. The concentration of ME LHRH was the same among orchidectomized male rats whether they bore PVN-DAHA lesions, sham lesions, or no lesions. In summary, destruction of the PVN-DAHA was found to reduce significantly the elevation of plasma FSH, but not LH, in the OVX rat and the estrogen-progesterone-stimulated OVX rat. PVN-DAHA lesions also attenuated the postcastration rise of FSH, but not that of LH, in the male. The failure of lesions of the PVN-DAHA to alter ME LHRH concentrations in the face of decreased FSH release does not prove that LHRH release is totally unaffected by this procedure. This finding is, however, consistent with the concept that diminished FSH secretion could be the result of a deficiency of a hypothalamic releasing factor (FSH-releasing factor?) other than that of the LHRH decapeptide.

Recent studies on the role of brain peptides in control of anterior pituitary hormone secretion

Recent work in our laboratory on the role of peptides to influence release of pituitary hormones by direct action on the gland and also some of the interactions of these peptides at the hypothalamic level to alter release of pituitary hormones will be reviewed. Considerable evidence from hypothalamic stimulation and lesion studies suggests the existence of a separate FSH-releasing factor (FSHRF). We have been able to purify a bioactive FSHRF which appears to be distinct from LHRH. Consequently, we believe that a distinct FSHRF will ultimately be isolated. With regard to prolactin, it is now clear that it is under dual control by both prolactin-inhibiting (PI) and prolactin-releasing factors (PRF). Although dopamine acts as a PIF, our recent fractionation studies indicate the existence of a peptidic PIF in hypothalamic extracts which can be separated from dopamine and GABA. The peptidic PIF is eluted from Sephadex in the same position originally described by us a number of years ago. Thus, inhibitory control is probably mediated by a combination of factors which would include dopamine, possibly GABA and a peptidic PIF. A number of peptides have been shown to have PRF activity which include TRF and also VIP. In recent studies, we have shown a prolactin-releasing action of oxytocin on male hemipituitaries or dispersed pituitary cells. Furthermore, high doses of oxytocin given intravenously released prolactin in male rats. There is a correlation between estrogen-induced prolactin release and an increase in plasma oxytocin and a correlation between suckling-induced oxytocin and prolactin release. These results suggest that oxytocin may be an important PRF. Control of growth hormone (GH) secretion is similarly under dual control via panhibin (somatostatin) and GRF. We have recently shown that motilin can stimulate release of GH from hemipituitaries or dispersed pituitary cells in vitro and that large doses are also effective in vivo. Furthermore, antisera raised against motilin were capable of lowering plasma GH. Motilin has been found in the median eminence and also in somatotrophs. Therefore, these results suggest that motilin may be a physiologically important GRF. In the meantime, a tumor (t) GRF has been synthesized by two laboratories and is extremely potent to release GH in vivo and in vitro. This peptide is much more potent than motilin and has an additive effect with motilin to increase GH release in vitro. We also have evidence for an important interplay amongst the various releasing and inhibiting factors at the hypothalamic level. For example, somatostatin injected into the ventricle will elevate GH instead of lowering it. This action is probably due to an ultrashort loop feedback to suppress somatostatin release and/or also stimulate GRF release. Similarly, peptides which stimulate prolactin release via direct action on the pituitary tend to inhibit it when given intraventricularly. Motilin, which stimulates GH release when given systemically, inhibits it when given intraventricularly and we have found similar results with synthetic GRF, low doses of which given intraventricularly suppress instead of stimulating GH release.

Evidence for an FSH-releasing factor in the posterior portion of the rat median eminence

To test the hypothesis that an FSH-releasing factor might be contained within the posterior portion of the median eminence (ME), the anterior half of the ME (aME) and the posterior half of the ME (pME) were removed separately from the brains of adult male rats and extracted in 0.2 N acetic acid. LH and FSH-releasing activities of the extracts were measured in vitro by incubating 8 hemipituitaries from adult male rats for 6 h at a dose of 5 tissue equivalents and determining the radioimmunoassayable LH and FSH released into the medium. LH release induced by the aME extracts was significantly greater than that induced by the pME in both experiments, whereas there were no differences in FSH release between aME and pME extracts. A significant dose-related increase in FSH release was noted in this system when 1 and 2 ng of synthetic LHRH were tested which indicates that the assay was sensitive to different amounts of LHRH with regard to FSH-releasing action. The content of immunoreactive LHRH in the extracts was almost twice as high in the aME as in the pME. Therefore, the results indicate that the pME has greater FSH-releasing activity than can be accounted for by its content of LHRH. The additional FSH-releasing activity is presumably due to an FSH-releasing factor distinct from LHRH.

Purification of a bioactive FSH-releasing factor (FSHRF)

Before the advent of radioimmunoassay (RIA), FSH-releasing factor (FSHRF) appeared to be separable from LH-releasing hormone (LHRH) by chromatography followed by bioassay for FSH. In this study, we re-examined hypothalamic extracts for the existence of an FSHRF distinct from LHRH, utilizing the Steelman-Pohley bioassay as well as RIA for identification of PSH. Acid extracts of rat hypothalamic fragments were chromatographed on Sephadex G-25. LH- and FSH-releasing activities of each fraction were assessed by bio- and immunoassay of FSH and immunoassay of LH released after incubation with hemipituitaries from adult male rats. The immunoreactive LHRH(IR-LHRH) concentration of each fraction was also measured by RIA. In order to evaluate the FSH-releasing activity of LHRH, three doses of synthetic LHRH were tested and FSH-releasing activity determined by bio- and immunoassay. By RIA, the FSH-releasing activity of each column fraction could be accounted for by IR-LHRH contamination. However, greater FSH-releasing activity than could be predicted by IR-LRH contamination was detected by Steelman-Pohley assay in fractions eluted prior to the LHRH peak in 2 separate fractionations. These fractions from the second fractionation were pooled and eluted from a CMC column with ammonium acetate buffers. Again greater FSH-releasing activity than could be accounted for by IR-LHRH was detected prior to the IR-LHRH peak by Steelman-Pohley assay. These results agree with early work from our laboratory and suggest the presence of a bioactive FSHRF in hypothalamic extracts.

Differential hypothalamic control of FSH secretion: A review

There are many circumstances in which the release of FSH and LH is dissociated; however, many of these are now thought to be brought about by interactions of LH-releasing hormone (LHRH), which stimulates not only LH but also FSH release, and the gonadal peptide, inhibin, which acts at the pituitary to suppress FSH release selectively. There are also many examples which can only be explained by postulating separate hypothalamic control of FSH and LH release. For example, electrochemical stimulation of the medial preoptic area elicited only LH release, whereas stimulation further caudally elicited equivalent LH release but FSH release as well. Points of stimulation particularly in the dorsal anterior hypothalamic area (DAHA) evoked only FSH release. Furthermore, implantation of prostaglandin E2 in various hypothalamic loci in a region extending from the DAHA caudally and ventrally to the caudal median eminence (ME) selectively elicited FSH release. Lesions of the DAHA resulted in a decrease of plasma FSH but not LH in castrated male and female rats and also suppressed the post-castration rise in FSH in males. In ovariectomized estrogen-primed rats with DAHA lesions, injection of progesterone provoked a normal LH surge but a significantly depressed FSH surge. Anterior ME lesions in castrates lowered LH levels more than FSH levels. Extracts of the DAHA evoked greater FSH and LH release in vitro than could be accounted for by the content of LHRH in the extracts, but there was no preferential release of FSH. On the other hand, extracts of the organum vasculosum lamina terminalis (OVLT) evoked dramatically increased FSH release above that which could be accounted for by the content of LHRH. Lastly, posterior ME extracts had more FSH-releasing activity than could be accounted for by their content of LHRH. All these results suggest the existence of an FSH-releasing factor (FSHRF) and lead to the speculation that the cell bodies of FSHRF neurons are located in the DAHA, with axons projecting to the OVLT and to the posterior ME. In other experiments, attempts were made to purify rat and sheep hypothalamic extracts by gel filtration on Sephadex G-25 and to assay the FSH-releasing activity by both bio- and immunoassay. Using this approach, we obtained evidence for the early emergence of a bioactive FSHRF prior to the emergence of LHRH from the column. Although much more work remains to be done, the accumulated evidence strongly supports the concept of a distinct FSHRF.