Basal Follicle-stimulating Hormone Release Research Articles

In vitro pituitary responsiveness to LHRH in young and old female rats. Influence of melatonin

The effect of aging and melatonin on in vitro pituitary responsiveness to luteinizing hormone-releasing hormone (LHRH) was studied. Young cyclic (3-months-old) control (cyclic-control, N=15), and melatonin (MEL) treated for 2 months (150 μg/100 g BW) (cyclic-MEL, N=15), old acyclic (23-months-old) control (acyclic-control, N=13), and MEL-treated (acyclic-MEL, N=18) rats were used. The hormones analyzed were luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL). The results showed a different influence of the reproductive status as well as of melatonin on the basal secretion rate of both gonadotropins, i.e. LH and FSH. Only the basal FSH release was significantly reduced in cyclic-MEL and acyclic-controls compared to cyclic-controls. The hemipitutary FSH content raised to values similar to those observed for FSH secretion and only the cyclic-MEL group showed significantly higher FSH pituitary content than for release. LHRH addition to the incubation medium resulted in increased LH release for both cyclic and acyclic rats, but FSH release was only stimulated in acyclic rats. Melatonin treatment blunted this response in both cases. In addition, melatonin treatment inhibited prolactin release in acyclic-MEL group after LHRH stimulation but not the basal levels. Pituitary LH and prolactin contents, were significantly higher than the pituitary LH and prolactin levels released from all groups studied, and were not affected by reproductive senescence nor by exogenous melatonin. These data indicate that aging influences more the secretory than the biosynthetic processes. Melatonin influences is endocrine status-dependent, being inhibitory when pituitary hormones reach their higher values.

Effect of somatostatin on the release of gonadotropins in male rats.

Since somatostatin, the growth hormone release-inhibiting hormone, has inhibitory actions in many cell types and is delivered to the anterior pituitary gland via the hypophysial portal vessels, as well as being synthesized by cells within the gland, we tested the hypothesis that it might inhibit the release of gonadotropins from anterior pituitaries in vitro. Consequently, the effect of somatostatin on gonadotropin release from incubated anterior pituitaries of male rats with and without the stimulatory action of luteinizing hormone-releasing hormone (LHRH) was studied. After a preincubation period of 1 hr, hemipituitaries from adult male rats were incubated in fresh Krebs-Ringer bicarbonate (KRB) buffer in a Dubnoff incubator with an atmosphere of 95% O(2)-5% CO2 at 37 degrees C for 3 hr. Incubation with somatostatin (10(-6), 10(-7), and 10(-8) M) had no effect on basal release of either LH or follicle-stimulating hormone (FSH). However, somatostatin (10(-6)-10(-8) M) suppressed LHRH (1.7 x 10(-8) M)-induced release of LH (P < 0.01 to P < 0.0001), but not FSH. Furthermore, somatostatin antiserum (1:1000) had no significant effect on basal LH or FSH release, whereas incubation with the antiserum plus LHRH (1.7 x 10(-9) or 1.7 x 10(-8) M) increased LH (P = 0.015 and P=.005, respectively), but not FSH release. In summary, our results suggest that somatostatin exerts a physiologically significant inhibitory effect on LH but not FSH release in the presence of LHRH in vitro. Presumably, somatostatin is secreted in vitro by pituitary cells since not only have anterior pituitaries of rats been shown to contain somatostatin, but also somatostatin mRNA. Somatostatin then diffuses to the LH gonadotropes, where it exerts its inhibitory action. However, the release of somatostatin is insufficient to alter basal in vitro release. On the other hand, at least at the concentrations employed, there was no significant effect either of somatostatin or the antiserum to alter basal or stimulated FSH release.

The identification of gonadotrophin surge inhibiting factor and its role in the regulation of pituitary gonadotrophin secretion

Evidence from several laboratories suggests that the ovaries of rats, pigs, monkeys and women produce a non-steroidal factor which inhibits or attenuates the mid-cycle gonadotrophin surge. This substance is present in follicular fluid and we have called it gonadotrophin surge inhibiting factor (GnSIF). Utilizing a rat pituitary cell bioassay, we have monitored GnSIF activity during purification from porcine follicular fluid (PFF). Rat pituitary cells in short-term culture were incubated with GnSIF for 48 h, washed and then exposed to gonadotrophin releasing hormone (GnRH) plus GnSIF for 4 h. GnSIF activity was defined as suppression of GnRH-stimulated LH secretion over 4 h, and inhibin activity was expressed as suppression of basal follicle stimulating hormone (FSH) secretion over 48 h. Approximately 5 l of PFF was fractionated through heparin-Sepharose, Q-Sepharose, Mono-S, hydroxylapatite, and gel permeation chromatography steps. This fractionation removes all detectable inhibin and follistatin immunoactivity and bioactivity. The purified GnSIF inhibits GnRH-stimulated LH secretion with little or no effect on basal FSH release. In summary, we have obtained a purified preparation of GnSIF which contains undetectable inhibin and follistatin. As such, GnSIF appears to be distinct from other known gonadal proteins regulating reproductive function, and may participate along with the inhibins, activins and follistatins in the gonadal regulation of pituitary gonadotrophin secretion.

Influence of dopamine on the altered release of prolactin, luteinizing hormone, and follicle-stimulating hormone induced by interleukin-2 in vitro.

Interleukin-2 (IL-2) alters the release of anterior pituitary hormones at femtomolar concentrations from hemipituitaries incubated in vitro. This cytokine significantly lowered luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and stimulated prolactin (PRL) release, thus demonstrating a reciprocal action of the lymphokine on lactotrophs and gonadotrophs. Since dopamine (DA) is a powerful inhibitor of PRL release, in the present experiments were evaluated possible dose dependent effects of DA on IL-2-induced alterations of the release of PRL, LH, and FSH. Hemipituitaries were incubated with varying concentrations of DA, a combination of IL-2 plus DA, or a combination of haloperidol (1 x 10(-5) M) with DA for 1 h, followed subsequently by incubation with medium containing only high potassium (K+) to study the effects on depolarization-induced hormone release. DA induced a dose-related, significant lowering of the basal PRL release with a minimal effective dose (MED) of less than 19 nM. The depolarization-induced PRL release was also inhibited, but the MED was 100-fold higher than the MED to inhibit basal PRL release. DA at much higher concentrations (30, 60, and 90 microM) significantly reduced pituitary PRL content. The addition of 0.187, 3.75, 15, or 60 microM DA to IL-2-induced PRL release. IL-2 (10(-15) M) produced a significant decrease in LH and FSH release. The combination of 3.75 or 15 microM DA plus IL-2 failed to alter the IL-2 suppressed LH release, whereas the addition of 0.187 microM DA to IL-2 blocked its suppressive influence, and 60 microM DA added to Il-2 produced an additive inhibitory effect. Thus, the interaction of IL-2 and DA is biphasic on LH release. The significant reduction of FSH release induced by IL-2 was blocked in the presence of 0.187, 3.75, 15, or 60 microM DA. DA alone at relatively high concentrations of 30, 60, and 90 microM suppressed basal LH and FSH release. The effects of DA on PRL, LH, and FSH at all doses tested were blocked by the DA receptor blocker, haloperidol which by itself at the concentration tested (1 x 10(-5) M) had no effect. Thus, the actions of DA at all concentrations tested appear to be mediated via DA receptors. In conclusion, DA was capable of blocking the stimulatory action of IL-2 on PRL release and its inhibitory action on FSH release by a DA receptor mediated action.(ABSTRACT TRUNCATED AT 400 WORDS)

Basal Luteinizing Hormone and Follicle-Stimulating Hormone Release Rates as a Function of Time after Castration in Female and Male Rats

We investigated the potential importance of the basal follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release rates in causing the acute and chronic elevations in serum FSH and LH concentrations which occur after ovariectomy (OVX) and orchidectomy (ORCH) of rats. Diestrous day 1 female and male rats were decapitated or castrated and killed 2, 4 or 8 h or 1, 2, 7, 21 or 35 days later. In females, the weight of the anterior pituitary gland (APG) did not change. Serum FSH rose within 4 h and then progressively higher until 35 days after OVX. These increases were paralleled nearly perfectly with increases in APG FSH concentration which was first elevated at 1 day after OVX and in the basal FSH release rate (measured in vitro) which was first elevated at 4 h after OVX. Serum LH levels rose by 7 days after OVX and then more dramatically thereafter. These increases were associated with increased APG LH concentrations. The pronounced increases in serum LH levels between 7 and 35 days after OVX were associated with marked increases in the basal LH release rate. In males, APG weight was increased at 21 and 35 days after ORCH. Serum FSH levels were elevated at 1 day after ORCH and continued to rise until 21 days after ORCH. APG FSH concentration was decreased at 2 and 7 days and increased at 35 days after ORCH. The basal FSH release rate per milligram APG did not change significantly after ORCH. Serum LH levels were elevated at 8 h after ORCH. They rose further by 1 day and then further between 7 and 21 days after ORCH. APG LH concentration and the basal LH release rate per milligram APG were elevated at 21 and 35 days after ORCH. The results suggest that changes in basal FSH and LH release are (1) involved to a major extent in causing the post-OVX rise in serum FSH concentration during the first 5 weeks after OVX and in serum LH concentration between 7 and 35 days after OVX, (2) not involved in causing the post-ORCH rises in serum FSH and LH concentrations during the 1 week after ORCH, and (3) involved to some extent in causing the elevations in serum FSH and LH concentrations observed at 3 and 5 weeks after ORCH. The results also indicate that (1) increases in the basal gonadotropin release rates per milligram APG after castration may be coupled in some way with increased synthesis of gonadotropin, and (2) increases in the basal LH release rate per milligram APG can occur independently of an increase in the basal FSH release rate per milligram APG.

Basal gonadotropin hormone release rates during the period of selective follicle-stimulating hormone release in the juvenile female rat

There are situations in which adult female rats release increased amounts of follicle-stimulating hormone (FSH) independent of increased luteinizing hormone (LH) release. This results from, at least in part, a selective increase in the basal FSH release rate. We investigated whether an increase in the basal FSH release rate is contributory to the rise in serum FSH levels which occurs independent of a rise in serum LH levels in the immature female rat. Rats had high serum FSH concentrations on days 7 and 15 after birth, low serum FSH levels on day 23, and low serum LH levels on all three days. In contrast, anterior pituitary gland (APG) FSH and LH concentrations and contents increased from day 7 to day 15 and the contents increased further from day 15 to day 23. Similarly, basal FSH and LH release rates per mg APG or per APG, as assessed by measurement of FSH and LH released into culture medium containing APG(s) from different aged rats, increased from day 7 to day 15 but did not increase further between days 15 and 23. The results indicate that unlike situations observed to date in adult female rats, a mechanism(s) other than an increase in the basal FSH release rate is involved in selective FSH release in the immature female rat.

Actions of 17β-Estradiol on Gonadotropin Release Induced by Drugs That Activate Intracellular Signal Transduction Mechanisms in Rat Anterior Pituitary Cells1

We studied the effects of 17 beta-estradiol (E2) on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release induced by drugs that activate different intracellular signal transduction mechanisms in rat anterior pituitary cells. Cells were pretreated with E2 (6 x 10(-10) M) or diluent for 24 h. Then, both E2- and diluent-pretreated cells were incubated for 4 h with E2 or diluent, respectively, with or without drugs, and in the presence or absence of gonadotropin-releasing hormone (GnRH). Media were assayed for LH and FSH by radioimmunoassays. E2 treatment had no effect on basal FSH release, but occasionally stimulated basal LH release. Phospholipase C (PLC), L-alpha-1,2-dioctanoyl glycerol (C8), veratridine, 8-bromo-cyclic adenosine 3',5'-monophosphate (8-Br-cAMP), melittin (a phospholipase A2 [PLA2] activator), arachidonic acid, PLA2, and GnRH all stimulated LH and FSH release in both E2- and diluent-treated cells. E2 treatment increased both LH and FSH release induced by GnRH, PLC, C8, veratridine, and 8-Br-cAMP, but not by melittin, arachidonic acid, and PLA2. Neither C8, PLA2, nor arachidonic acid in combination with a maximal dose of GnRH had additive effects on either LH or FSH release, whereas melittin increased the maximal response to GnRH in both E2- and diluent-treated cells. The effects of veratridine and 8-Br-cAMP depended on dose of GnRH and presence or absence of E2. These results suggest that E2 augments stimulus-coupled gonadotropin release by interacting with the Ca2+-, and/or diacylglycerol-, and cAMP-activated pathways, but not with the arachidonic acid-activated pathway.

Participation of voltage-sensitive calcium channels in pituitary hormone release.

The role of extracellular Ca2+ in pituitary hormone release was studied in primary cultures of rat anterior pituitary cells. The basal levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyrotropin (TSH), and adrenocorticotropin (ACTH) secretion were independent of extracellular Ca2+ concentration ([Ca2+]e). In contrast, the basal levels of growth hormone (GH) and prolactin (PRL) release showed dose-dependent increases with elevation of [Ca2+]e, and were abolished by Ca2+-channel antagonists. Under Ca2+-deficient conditions, BaCl2 mimicked the effects of calcium on PRL and GH release but with a marked increase in potency, and also increased basal LH and FSH release in a dose-dependent manner. In the presence of normal [Ca2+]e, depolarization with K+ maximally increased cytosolic [Ca2+] ([Ca2+]i) from 100 to 185 nM and elevated LH, FSH, TSH, ACTH, PRL, and GH release by 7-, 5-, 4-, 3-, 2-, and 1.5-fold, respectively. These effects of KCl were abolished in Ca2+-deficient medium or in the presence of the Ca2+-channel antagonist, Co2+, and were diminished by the dihydropyridine Ca2+-channel antagonist, nifedipine. The Ca2+-channel agonist BK 8644 (100 nM) enhanced the hormone-releasing actions of 25 mM KCl upon PRL, LH, FSH, GH, TSH, and ACTH by 2.3-, 2.0-, 1.8-, 1.7-, 1.6-, and 1.4-fold, respectively. The dose- and voltage-dependent actions of BK 8644 were specific for individual cell types; BK 8644 enhanced GH, PRL, TSH, LH, and ACTH secretion in the absence of any depolarizing stimulus, with ED50 values of 8, 10, 150, 200, and 400 nM, respectively. However, in the presence of 50 mM KCl, the ED50 values for BK 8644 were 1.5, 2, 3, 5, and 7 nM for GH, PRL, ACTH, TSH, and LH, respectively. [3H]BK 8644 bound specifically to pituitary membranes with Kd values of 0.8 nM and concentrations of about 900 channels per cell. These observations provide evidence for the presence and participation of voltage-sensitive calcium channels in the secretion of all five populations of anterior pituitary cells.

Cyclic nucleotides and suppression of follicle-stimulating hormone release by inhibin.

The relative roles of the cyclic nucleotide messengers cAMP and cGMP in the suppression of follicle-stimulating hormone (FSH) secretion in response to inhibin (IBN) were assessed employing rat gonadotropes in monolayer culture. While exposure of cells to gonadotropin-releasing hormone (GnRH) induced a significant increase in the amounts of both FSH and luteinizing hormone (LH) released into the culture medium, these responses were dampened by the administration of IBN (in porcine follicular fluid). Addition of cGMP to the system failed to restore FSH release, while cAMP restored basal FSH release. Modulation of nucleotide metabolism with theophylline, sodium nitroprusside, and a protein kinase inhibitor failed to overcome the IBN-induced suppression of FSH release. The cellular content of calmodulin increased in response to GnRH, a response antagonized by IBN. Cellular levels of cGMP were also increased by GnRH, but this response was unaltered by IBN. The administered drugs all failed to reverse these effects of IBN. These data indicate that the IBN-induced suppression of FSH release is not dependent upon the cyclic nucleotides cAMP and/or cGMP. However, a role in the maintenance of basal FSH synthesis and release for cAMP is indicated.

Follicle-stimulating Hormone Within and Secreted From Anterior Pituitaries of Female Golden Hamsters during the Estrous Cycle and after Ovariectomy1

Anterior pituitary (AP) glands were removed from groups of female golden hamsters at 0900 h on estrus (E), diestrus I (DI), and diestrus II (DII) and at 1200 h and 1500 h on proestrus (P12 and P15), as well as at 0900 h from ovariectomized hamsters (OVX). Hemipituitaries were incubated in culture medium with or without 10(-8) M luteinizing hormone-releasing hormone (LHRH) for 3 h at 37 degrees C to determine the magnitude of basal and LHRH-stimulated follicle-stimulating hormone (FSH) release. All samples were assessed for FSH activity by radioimmunoassay and radioreceptor assay. In a second set of experiments, AP were removed from E, DII, and OVX hamsters and bisected. One hemipituitary was homogenized in 10 mM Tris-HCl and the other half was incubated for 3 h. Follicle-stimulating hormone forms present within pituitary extracts or secreted into medium were separated by an isoelectric focusing technique, chromatofocusing. Basal FSH release was lowest in AP collected on DII and P12, higher in AP collected on E and DI, and highest in AP from OVX. Luteinizing hormone-releasing hormone-stimulated release of FSH was highest in AP obtained on DII and P12, lower in AP collected on E and DI, and lowest in AP from OVX. Radio-receptor-to-radioimmunoassay ratio of secreted FSH was greatest when basal FSH secretion was low and LHRH sensitivity was high (DII and P12) and least when basal FSH secretion was high and LHRH sensitivity low (E and after OVX).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat.

Changes at the anterior pituitary gland level which result in follicle-stimulating hormone (FSH) release after ovariectomy in metestrous rats were investigated. Experimental rats were ovariectomized at 0900 h of metestrus and decapitated at 1000, 1100, 1300, 1500, 1700 or 1900 h of metestrus. Controls consisted of untreated rats killed at 0900 or 1700 h and rats sham ovariectomized at 0900 h and killed at 1700 h. Trunk blood was collected and the serum assayed for FSH and luteinizing hormone (LH) concentrations. The anterior pituitary gland was bisected. One-half was used to assay for FSH concentration. The other half was placed in culture medium for a 30-min preincubation and then placed in fresh medium for a 2-h incubation (basal FSH and LH release rates). The basal FSH release rate and the serum FSH concentration rose significantly by 4 h postovariectomy and remained high for an additional 6 h. The basal FSH release rate and the serum FSH concentration correlated positively (r=0.71 with 72 degrees of freedom) and did not change between 0900 and 1700 h in untreated or sham-ovariectomized rats. In contrast, the serum LH concentration and the basal LH release rate did not increase after ovariectomy. Ovariectomy had no significant effect on anterior pituitary gland FSH concentration. The results suggest that the postovariectomy rise in serum FSH concentration is the result, at least in part, of changes which cause an increase in the basal FSH secretion rate (secretion independent of the immediate presence of any hormones of nonanterior pituitary gland origin). The similarities between the selective rises in the basal FSH release rate and the serum FSH concentration in the ovariectomized metestrous rat and in the cyclic rat during late proestrus and estrus raise the possibility that an increase in the basal FSH release rate may be involved in many or all situations in which serum FSH concentration rises independently of LH.

Interaction of progesterone with testosterone and dihydrotestosterone on follicle-stimulating hormone release by cultures of rat anterior pituitary cells.

Cultures of rat anterior pituitary cells were utilized to examine the effects of testosterone, dihydrotestosterone and progesterone alone as well as the interaction of progesterone with these androgens on follicle-stimulating hormone (FSH) release. Four-day cultures were incubated with the steroids for 48 h to examine their effects on basal FSH release and were then challenged for 6 h with 5 × 10−10 M luteinizing hormone releasing hormone (LHRH), the approximate concentration required to induce half-maximal FSH release (EC50), to assess the effects of these steroids on LHRH-induced FSH release. Incubation of cultures with either testosterone, dihydrotestosterone or progesterone stimulated 48-h basal FSH release with EC50 ’s of 8.6 × 10−10 M, 2.2 × 10−10 M and 6.4 × 10−10 M, respectively. Progesterone (10−6 M) was not synergistic nor antagonistic with either testosterone or dihydrotestosterone in stimulating 48-h basal FSH release. Neither testosterone nor dihydrotestosterone altered LHRH-induced FSH release but progesterone (≥10−7 M) had a stimulatory effect. The stimulatory effect of progesterone on LHRH-induced FSH release was partially reversed by high concentrations (≥10−8 M) of either testosterone or dihydrotestosterone. Each of these 3 steroids increased the total FSH content of culture dishes, suggesting stimulation of FSH synthesis. Progesterone was slightly synergistic with dihydrostestosterone, but not with testosterone, in stimulating total FSH. Because dihydrotestosterone was approximately 4-fold more potent than testosterone in stimulating basal FSH release, we examined the effect of testosterone in the presence of heparin, a substance which may be used to inhibit steroid 5α-reductase activity. Unexpectedly, the addition of 1000 U/ml heparin increased 48-h basal, testosterone- and LHRH-induced FSH release.

Seasonal reproduction in the mongoose, Herpestes auropunctatus: III. Regulation of gonadotropin secretion in the male

The seasonal regulation of gonadotropin secretion in the male mongoose was investigated by evaluating in vitro responsiveness of anterior pituitaries to gonadotropin releasing hormone (GnRH) and by evaluating postcastration gonadotropin responses in vitro and in vivo during the breeding and inactive seasons. The relative increase of luteinizing hormone (LH) from in vitro GnRH-stimulated anterior pituitaries was significantly greater during the active season than during the inactive season. The reduced responsiveness during the inactive season was related to an increase in the negative feedback actions of the testes at the hypophyseal level. In vitro follicle stimulating hormone (FSH) secretion from GnRH-treated anterior pituitaries was not significantly affected by season; however, a more potent negative feedback effect of the testes on basal FSH release was apparent during the inactive season. The postcastration serum LH response was more rapid during the active season than during the inactive season, whereas the postcastration serum FSH response was not affected by season.

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour in vitro incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.