Estradiol differentially modulates the exocytotic proteins SNAP-25 and munc-18 in pituitary gonadotrophs

Long-term treatment with estradiol increases LH secretion from female gonadotrophs. The mechanisms are not fully clarified yet. Our previous data indicated that sexual steroids might affect late steps in GnRH signal transduction such as exocytosis. The secretion of hormones from neuroendocrine cells requires the merger of secretory vesicles with the plasma membrane. This regulated exocytosis is mediated by specific proteins, which are present in the pituitary gland. Here, we examined whether two of these crucial exocytotic proteins, SNAP-25 and munc-18, are affected by estradiol in female gonadotrophs. Female rat anterior pituitary cells and alphaT3-1 cells, derived from a murine immortalized gonadotroph cell line, were treated with 100 pM estradiol for 48 h. LH secretion of anterior pituitary cells, additionally stimulated with eight consecutive pulses of 1 nM GnRH for 15 min at an interval of 1 h, was determined by RIA. Gene expression was measured by quantitative RT-PCR and protein expression by immunoblotting. Additionally, quantitative RT-PCR was performed in single rat gonadotrophs to ascribe effects exclusively to intact gonadotrophs. Pulsatile GnRH enhanced the mRNA expression of SNAP-25 and munc-18 in accordance with the LH secretory response with the greatest increase at the third pulse of GnRH. Estradiol treatment further increased GnRH-induced LH secretion at all GnRH pulses. SNAP-25 gene expression was significantly decreased at the fifth GnRH pulse and unaffected at basal after 48 h of estradiol treatment. In contrast, munc-18 mRNA levels were not significantly affected by estradiol at different GnRH-pulses in mixed anterior pituitary cells, whereas munc-18 gene expression was significantly increased at basal. In alphaT3-1 cells and single gonadotrophs, long-term estradiol treatment significantly reduced SNAP-25 protein and gene expression. In contrast, the protein and gene expression of munc-18 was significantly enhanced in both alphaT3-1 cells and single gonadotrophs. In conclusion, munc-18 and SNAP-25 were oppositionally influenced by estradiol. The results suggest that estradiol modulates the expression of exocytotic proteins in gonadotrophs and thus affects LH secretion.     

Androgen receptors in gonadotrophs in pituitary cultures from adult male monkeys and rats

There is substantial evidence demonstrating that the principal feedback action of androgens to decrease LH secretion in male primates, including man, is to slow the GnRH pulse generator, whereas in male rats androgens not only decrease GnRH but also suppress LH synthesis and secretion through a direct pituitary effect. Previous experiments in our laboratory revealed that testosterone (T) suppresses LH secretion and decreases alpha-subunit mRNA levels in male rat pituitary cell cultures perifused with pulses of GnRH but not in pituitary cells from adult male monkeys. In the present study, we sought to determine whether the lack of responsiveness of gonadotrophs to androgens in the primate is androgen receptor (AR) related. Primary cultures were prepared from the anterior pituitary glands of adult male monkeys and rats. Cells were identified as gonadotrophs if they were immunoreactive for LH-beta or FSH-beta. Of these cells in the monkey, 80% contained both gonadotropins, 17% contained only LH-beta, and 3% contained only FSH-beta. AR immunoreactivity (IR) was nuclear in 22% and 15%, respectively, of monkey and rat FSH-beta-positive cells in the absence of T. Following T treatment, nuclear AR IR was identified in 79% of monkey and 81% of rat gonadotrophs. T treatment similarly intensified AR IR in mouse gonadotroph alphaT3-1 and LbetaT2 cells and in monkey and rat fibroblasts. Single-cell RT-PCR confirmed coexpression of LH-beta and AR mRNA as well as LH-beta and GH mRNA in monkey gonadotrophs. Our data reveal that most monkey, as well as rat, gonadotrophs are AR-positive with nuclear localization in the presence of T. GH expression is not required for AR expression in gonadotrophs. We conclude that the failure of T to inhibit LH secretion and decrease alpha-subunit mRNA expression in the male primate is not due a disturbance in AR nuclear shuttling.     

Melatonin inhibits gonadotropin-releasing hormone-induced elevation of intracellular Ca2+ in neonatal rat pituitary cells.

GnRH stimulates LH release by increasing intracellular Ca2+ ([Ca2+]i). Melatonin is known to inhibit GnRH-stimulated LH release from neonatal rat pituitary cells. In the present report, the issue of whether melatonin acts through [Ca2+]i was addressed. [Ca2+]i was studied in cells in suspension, using Fluo-3 as a fluorescent indicator. In neonatal rat pituitary cells, melatonin inhibited the GnRH-induced [Ca2+]i increase in a dose-dependent manner; the GnRH-induced increase in [Ca2+]i was inhibited 40% by 100 nM melatonin. The relative potencies of several indoles as inhibitors of the GnRH stimulation of [Ca2+]i in neonatal pituitary cells (2-iodo-melatonin greater than melatonin greater than 6-hydroxymelatonin) correlate with their known potencies to inhibit LH release and with their binding affinity to high affinity melatonin receptors, which indicates that these receptors probably mediate the effects of melatonin. Further support for this interpretation comes from the observation that melatonin does not inhibit the GnRH effect on [Ca2+]i in cells obtained from adolescent rat pituitary glands, which lack melatonin receptors and are insensitive to melatonin as an inhibitor of GnRH-stimulated LH release. The possible involvement of an inhibitory G-protein was also investigated by studying the effects of pertussis toxin. Pretreatment with pertussis toxin antagonized the effects of melatonin on [Ca2+]i and LH release. This indicates that melatonin may inhibit the GnRH-induced increase in [Ca2+]i through a mechanism involving a pertussis toxin-sensitive G-protein. To examine the role of extracellular Ca2+ in this effect, the effects of melatonin were examined in a low Ca2+ medium. Under these conditions, the effect of melatonin was markedly reduced, which indicates that melatonin may act by inhibiting Ca2+ influx. These observations indicate that melatonin inhibits GnRH stimulation of [Ca2+]i in neonatal rat gonadotrophs, and this probably explains the inhibitory action of melatonin on GnRH stimulation of LH release.     

Modulation of the estradiol-induced luteinizing hormone surge by progesterone or antiestrogens: effects on pituitary gonadotropin-releasing hormone receptors

The present study examined the question of whether modulation of estradiol-induced LH surges by progesterone or antiestrogens in the immature rat might be related to changes in the concentration of pituitary GnRH receptors (GnRH-R). Rats (28 days old) that received estradiol implants at 0900 h had LH surges approximately 32 h later. Administration of progesterone or nafoxidine (U-11,100 A; 1-(2-[P-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]pyrrolidine hydrochloride) concomitantly with estradiol led to blockade of these LH surges (progesterone or nafoxidine inhibition), while progesterone treatment 24 h after estradiol brought about premature and enhanced LH release (progesterone facilitation). GnRH-R-binding capacity was determined by saturation analysis in homogenates of single pituitaries from immature rats treated with estradiol and progesterone or nafoxidine and controls treated only with estradiol using [125I]iodo-(D-Ala6,Des-Gly10)GnRh ethylamide. The affinity of GnRH-R for this analog ranged from 8.2-15.1 X 10(9) M-1 and was not affected by in vivo steroid or antiestrogen treatment. The number of GnRH-R in gonadotrophs from untreated 28-day-old rats (57.2 +/- 2.6 fmol/pituitary or 177 +/- 11 fmol/mg protein) was comparable to values previously reported for 30 day-old females. GnRH-R levels were first measured 1, 8, 24, 32, and 48 h after estradiol treatment. The pituitary content of GnRH-R paralleled changes in total pituitary protein (nadir at 24 h, rebound at 32 h, continued increase at 48 h), while their concentration (femtomoles per mg protein) was highest at 8 h. Next, GnRH-R levels were examined at 1200 h and at hourly intervals (1400-1800 h) on the afternoon of the LH surge. While GnRH-R concentrations were significantly lower at 1400 and 1700 h than at 1200 or 1800 h in animals treated with estradiol in the progesterone facilitation model, they did not change over time in the other two paradigms. There was no significant difference in pituitary content or concentration of GnRH-R at any time between immature rats treated with estradiol and progesterone or nafoxidine and their respective estradiol-treated controls. These results suggest that changes in GnRH-R levels in pituitary gonadotrophs do not play a major role in enhancement of LH surges by progesterone or in their suppression by progesterone or nafoxidine in the immature rat; therefore, these compounds may affect the pituitary at a site distal to the GnRH receptor.     

Gonadotropin-releasing hormone receptor gene expression in rat anterior pituitary

Gonadotropin-releasing hormone (GnRH) effects on the lactotroph function have been widely studied, but they probably result from paracrine interactions. No visual data about GnRH receptor in the pituitary are available. In order to identify the GnRH target cells in the pituitary of adult rats, the cellular distribution of rat GnRH receptor mRNA was investigated by electron microscopy, usingin situ hybridization on ultrathin pituitary frozen sections.In situ hybridization was performed using a digoxigenin-labeled oligonucleotide probe revealed by an indirect immunogold reaction. Gonadotropin-releasing hormone receptor mRNA was found in the cytoplasmic matrix, apposed to the endoplasmic reticulum and the nucleus of the gonadotrophs, which were identified by their ultrastructural characteristics, and by the presence of luteinizing hormone (LH) immunoreactivity. It was also found in the lactotrophs, which were revealed by the immunocytological detection of prolactin. No GnRH receptor mRNA was detected in corticotrophs, somatotrophs, thyrotrophs or hepatocytes. This result, without excluding paracrine effects, clearly showed that in addition to the gonadotrophs, the lactototrophs are likely to be direct target cells for the hypothalamic GnRH.     

Ontogeny of hypothalamic luteinizing hormone-releasing hormone (GnRH) and pituitary GnRH receptors in fetal and neonatal rats

Although it is known that LH secretion starts at 17 days of gestation in the fetal rat and that this first LH release is most likely driven by hypothalamic GnRH, an earlier role for GnRH during fetal life has been postulated with the observation that presence of GnRH is important before day 13 of gestation for the differentiation of the pituitary anlage. In order to clarify the different roles of GnRH during fetal life, we have studied the first appearance of GnRH in the fetal brain, the expression of GnRH receptors in the fetal pituitary gland, and the presence of GnRH immunoreactivity within the fetal gonadotrophs. GnRH was present in the earliest brain tissue examined (12 days of gestation). From 12-17 days, GnRH content of fetal brain remained low and then increased markedly by the end of gestation. No immunoreactive GnRH-like material could be detected in rat placental tissue throughout gestation. Binding sites for GnRH were detected as early as 12 days of gestation in fetal pituitary glands. However, binding was very low until 16 days. At 17 days, Scatchard analysis indicated the presence of high affinity, low capacity binding sites [affinity constant (Ka) = 10(10) M-1]. Intracellular presence of GnRH as seen by immunocytochemistry using ultrathin sections prepared by cryoultramicrotomy was first visible at 14 days and started to increase at 16 days. LH was first detectable in the fetal pituitary by RIA at 17 days; FSH was first detectable at 21 days, and PRL at 1 day of postnatal life. Thereafter, neonatal pituitary contents of LH, FSH, and PRL increased linearly with-time, as did the number of pituitary GnRH receptors. At 10 days of postnatal life, pituitary contents of LH and FSH were significantly higher in females than in males. In summary, hypothalamic GnRH appears early in fetal life and potentially can induce differentiation of the pituitary anlage. Conversely, the presence at 15 days of gestation of specific binding sites for GnRH and of intracellular GnRH immunoreactivity in gonadotrophs indicates that the hypophysiotropic action of GnRH clearly precedes the start of LH biosynthesis.     

Effects of leptin on gonadotropin secretion in juvenile female rat pituitary cells

OBJECTIVE: Leptin is an adipocyte-derived hormone, which is the product of the obese gene and it is thought to play important roles in pubertal development and maintenance of reproductive function in the female. In a study using adult male or female rats, it was found that leptin stimulated the secretion of gonadotropin directly from the pituitary in a dose-related manner. However, there is no study in juvenile female rats before puberty. METHODS: In this study, we cultured pituitary cells from 4-, 6- and 8-week-old female Wistar rats with leptin (0-10(-7)mol/l) and gonadotropin-releasing hormone (GnRH) (0 or 10(-8) mol/l). Basal or GnRH-stimulated secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and their synthesis within cells were determined by radioimmunoassay (RIA). RESULTS: Leptin induced bell-shaped dose--response curves of basal LH and FSH secretion from cultured cells of every age-group of rats studied. The most effective concentration of leptin on the basal secretion of LH and FSH from 6- and 8-week-old cultured pituitary cells was 10(-10) mol/l. This leptin concentration was consistent with circulating physiological serum leptin levels at each age. As for juvenile 4-week-old pituitary cells, the most effective concentration was 10(-11) mol/l which was lower than that of 6- and 8-week-old rats. It was consistent with the circulating serum leptin levels of 4-week-old rats. Also, the synthesis and the GnRH-stimulated secretion of LH and FSH were effectively controlled by leptin at concentrations similar to the serum leptin levels of given ages. CONCLUSIONS: Leptin induced pituitary cells to synthesize and secrete both LH and FSH regardless of the presence or absence of GnRH. The concentration of leptin that induced the greatest synthesis and secretion of gonadotropins from pituitary cells changed around the pubertal period. The most effective leptin concentrations in each experiment were similar to the physiological serum leptin level at each animal age. These results indicate that leptin stimulates gonadotrophs not only in the pubertal and the mature period but also in the juvenile period before puberty. It is also conceivable that leptin may modulate the sensitivity of gonadotrophs until the appearance of GnRH stimulation, and may be the factor that brings about puberty onset.     

Stimulation of luteinizing hormone release and cyclic nucleotide production by arachidonic acid in cultured pituitary gonadotrophs

Gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone (LH) release and cyclic guanosine 3',5-cyclic monophosphate (cGMP) production in rat anterior pituitary cells through a calcium-dependent activation mechanism that involves increased phospholipid turnover and liberation of arachidonic acid. In enriched pituitary gonadotrophs, LH release was stimulated by arachidonic acid and its oxygenated metabolite, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), in a dose-dependent manner. The prominent LH responses of purified gonadotrophs to arachidonic acid suggest that the secretory actions of arachidonate are exerted primarily on the gonadotroph and do not involve the participation of other pituitary cell types. Preincubation of pituitary cells with stimulatory concentrations of arachidonic acid for up to 120 min did not alter the subsequent LH responses elicited by GnRH, indicating that the secretory mechanism was unimpaired by arachidonate treatment and that no cross-desensitization occurs during sequential exposure of gonadotrophs to the two stimuli of LH release. Cyclic adenosine 3',5-monophosphate (cAMP) production was stimulated by 10 microM arachidonic acid to the same degree (about 2-fold) as by GnRH, but did not parallel the progressive LH response to higher arachidonate concentrations. cGMP production was initially stimulated by addition of arachidonic acid but returned to the control value after 5 min, whereas GnRH typically elicited a prolonged cGMP response. In contrast to the calcium-independent action of arachidonic acid, the stimulatory effect of 5-HETE on LH release required the presence of extracellular Ca2+, as previously observed for GnRH. These findings demonstrate that arachidonic acid and its metabolite, 5-HETE, partially reproduce the actions of GnRH upon LH release and cyclic nucleotide production.(ABSTRACT TRUNCATED AT 250 WORDS)     

A quantitative immunocytochemical study of the luteinizing hormone and follicle-stimulating hormone cells in the adenohypophysis of adult male rats and adult female rats throughout the estrous cycle

We investigated whether 1) the absolute or the relative numbers of LH and FSH cells change during the rat estrous cycle, 2) the percentages of gonadotrophs that contain LH and/or FSH change during the estrous cycle, and 3) gonadotrophs change in size during the rat estrous cycle. Groups of four female rats were decapitated at one of five different times during the estrous cycle. Four male rats were also decapitated. Serum concentrations of LH and FSH were determined by RIA. Paired horizontal flip-flopped or nonflipped paraffin sections were mounted from the dorsal, middle, and ventral portions of each pituitary gland. In each pair of sections, one was stained with a-rat LH-S4 and the other with a-rat FSH-S7 by the unlabeled antibody peroxidase-antiperoxidase method. All immunoreactive cells were counted. Photographs were taken from randomly chosen corresponding areas, and the cells were individually matched to determine the percentage that contained one or both hormones. Correction factors had to be used because in paired flip-flopped or nonflipped sections stained with the same antibody (a-rat LH-S4), not all of the stained cells found in one section were found in the other section. The absolute numbers of LH and FSH cells did not change throughout the estrous cycle. The ratio of LH cells to FSH cells in the pars distalis of female rats was also constant throughout the estrous cycle. In female rats, 75.2% of LH cells also contained FSH, while 99.4% of FSH cells also contained LH. In the male rats, 88.6% of LH cells also contained FSH, while 98.6% of FSH cells also contained LH. Similar results were obtained in paired flip-flopped sections stained with a-rat LH beta and a-rat FSH beta. Sequential staining of additional individual tissue sections with a-rat LH-S4 and then a-rat FHS-S7 or vice versa revealed the following. Staining of LH-stained tissue for FSH revealed less than 1% new cells, but staining of FSH-stained tissue for LH revealed a 8.7% increase in gonadotrophs in males and a 25.4% increase in females. The gonadotrophs in female rats did not change in size during the estrous cycle and were significantly smaller than the gonadotrophs in male rats. The results suggest that in normal adult rats: 1) virtually all FSH-containing cells contain LH, 2) about 25% of the gonadotrophs in females and about 11% of the gonadotrophs in males contain LH but not FSH, 3) the number of cells containing LH or those containing LH and FSH does not change during the estrous cycle, 4) gonadotrophs in female rats do not change in size during the estrous cycle and are smaller than the gonadotrophs in male rats, and 5) FSH release during the early morning of estrus, when the serum FSH concentration is elevated and the serum LH concentration is low, occurs from cells that contain both LH and FSH.     

Ontogenic and sexual differences in pituitary GnRH receptors and intracellular Ca2+ mobilization induced by GnRH

The present experiments were designed in order to elucidate the participation of the developing hypophysis in determining the changing sensitivity of gonadotrophins to gonadotropin-releasing hormone (GnRH) during ontogeny in the rat. To that end, we chose two well defined developmental ages that differ markedly in sexual and ontogenic characteristics of hypophyseal sensitivity to GnRH, 15 and 30 d. In order to study sex differences and the role of early sexual organization of the hypothalamus, experiments were carried out in males, females, and neonatally androgenized females (TP females). We evaluated (1) the characteristics of pituitary GnRH receptors, and (2) associated changes in GnRH-induced mobilization of intracellular Ca²⁺ (a second messenger involved in gonadotropins exocytosis). We measured binding characteristics of the GnRH analog d-Ser(TBu)6-des-Gly10-GnRH ethylamide in pituitary homogenates. We found that K _ds did not vary among the different sex groups. Total number and concentration of receptors decreased in the female rat from 15–30 d of age, whereas in the male and TP female, receptors/pituitary increased, and the concentration/mg tissue did not change. Also, at 30 days of age, males presented higher content and concentration of receptors than females, and higher content than TP females. In order to evaluate if developmental and sexual differences in pituitary sensitivity to GnRH might be expressed through variations in the intracellular Ca²⁺ signal, we studied the mobilization of intracellular Ca²⁺ induced by GnRH (1 × 10⁻⁸ to 1 × 10⁻¹¹ M) in a suspension of dispersed pituitary cells in the six groups. In cells from 15-d-old females, Ca²⁺ response was greater than in 30-d-old females at the doses of 10⁻⁸ to 10⁻¹⁰ M, indicating that in the infantile female rat activation of highly concentrated GnRH receptors is reflected in an increase in signal transduction mediated by Ca²⁺. In males and in female rats androgenized at birth, there was also a decrease in the magnitude of intracellular Ca²⁺ mobilization induced by GnRH (10⁻⁸ to 10⁻¹⁰ M) from 15–30 d of age, even though the concentration of GnRH receptors did not change in the same period. In conclusion, the present results suggest that high sensitivity to GnRH, which has been described in the female infantile rat, may be related to elevated concentration of hypophyseal receptors coupled to an increase of intracellular calcium response to GnRH, both parameters decreasing as the rat matures. In males, the greater sensitivity that has been described for GnRH at 30 d in comparison to 15 d is paralleled by an increase in the total number of GnRH receptors per pituitary (and not in their concentration), but not in an increase in the magnitude of Ca²⁺ mobilization induced by GnRH. On the other hand, neonatal sexual organization of the hypothalamus is involved in the differential expression of GnRH receptors, but does not modulate mobilization of intracellular Ca²⁺ induced by the decapeptide.     

GnRH and oxytocin have nonidentical effects on the cellular LH response by gonadotrophs at pro-oestrus

For full fertility in the female ovulation is necessary, which is dependent on the production of a surge of LH. An understanding of the processes which result in the high levels of LH requires delineation of the activities of individual component cells. In this study the responses of gonadotrophs to two signalling hypothalamic peptides, GnRH and oxytocin, were investigated. A cell immunoblot method was used to identify and distinguish between cells which secrete LH and those which contain LH but do not secrete the glycohormone. Rats were killed on the morning of pro-oestrus, the pituitary collected and the cells dispersed onto a protein-binding membrane for study. Cells were then incubated with GnRH and oxytocin, after which the membranes including the attached cells were stained by immunocytochemistry for LH. GnRH increased the total number of immunopositive cells which were present in a concentration-dependent manner. The most prominent change after 2 h incubation was in the number of secreting cells, whereas after 4 h there was also a marked increase in numbers of nonsecreting cells. Oxytocin also increased the total number of immunopositive cells in a concentration-responsive manner, however the profile of action of oxytocin was different from that observed for GnRH. Oxytocin had a relatively greater effect on numbers of immunopositive nonsecreting cells. Thus, the results reveal the potential for gonadotrophs to be flexibly and appropriately modulated by selected hypothalamic peptides. When cells were preincubated with oxytocin prior to GnRH there was not an additive increase in the numbers of immunopositive cells, suggesting that the two agonists act, in a nonidentical manner, on similar cells. The increase in the total number of immunopositive cells implies that there was a production of LH or post-translational processing, induced by exposure to GnRH or oxytocin. The results confirmed the heterogeneity of gonadotrophs and the existence of functionally distinguishable subpopulations, and revealed a difference between the effects of GnRH and oxytocin on expression and secretion of LH.     

Gonadotropin-releasing hormone-stimulated luteinizing hormone secretion by perifused pituitary cells from normal, gonadectomized, and testicular feminized rats

To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King x Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P less than 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P less than 0.01) than that by cells from intact animals, but lower (P less than 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-dependent LH release. Pituitary cells from intact female rats showed an overall greater (P less than 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P less than 0.0001) or intact female (P less than 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRH-stimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P less than 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact females rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens.     

Characterization of angiotensin II receptors in the anterior pituitary gland

Specific and high affinity binding sites for angiotensin II were demonstrated in the anterior pituitary gland by binding studies with [¹²⁵I]iodoangiotensin II. The binding properties of the pituitary receptors were similar to those of angiotensin II receptors present in the adrenal gland. The concentration of binding sites in rat anterior pituitary (293 ± 50 fmoles/mg protein) was less than in the adrenal gland, but was much greater than in smooth muscle. Angiotensin II receptors were identified in the anterior pituitary tissue of mature and immature animals of both sexes, and in species including rat, rabbit and dog. No binding of angiotensin II was detected in posterior pituitary homogenates, or in GH₃ pituitary tumor cells. Collagenase-dispersed anterior pituitary cells also contained specific binding sites for angiotensin II, with equilibrium binding constant (K_a) of 3.6 × 10⁹ M^?1. The presence of specific high-affinity angiotensin II receptor in the anterior pituitary gland provides a mechanism by which angiotensin-like peptides could modulate the process of pituitary hormone secretion.     

Calcium-dependent actions of gonadotropin-releasing hormone on pituitary guanosine 3',5'-monophosphate production and gonadotropin release

The effects of gonadotropin-releasing hormone (GnRH) on cGMP production and LH release in cultured rat pituitary cells are markedly dependent upon the extracellular calcium concentration. The absence of calcium from incubation media caused almost complete loss of the GnRH effects on cGMP production and LH release but did not change the stimulation of cAMP accumulation by GnRH in the pituitary of the adult male rat. In female rat pituitary cells, reduction of the extracellular calcium concentration increased the concentration of GnRH required to produce half-maximal LH release and decreased the maximal gonadotropin output but had no significant effect on basal LH release. The divalent cation ionophore A23187 stimulated LH release, and this action was dependent on extracellular calcium. Both GnRH and A23187 were found to have maximal effects when the calcium concentration was 0.6 mM, and their actions were not additive. The calcium antagonists, verapamil and lanthanum, caused concentration-dependent inhibition of the actions of GnRH, with half-maximal blockade values of 10(-5) and 3 X 10(-6) M, respectively, and had no effect on basal LH release. The binding of a radioiodinated GnRH analog, [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide, to pituitary GnRH receptors was unchanged in the absence of extracellular calcium. These observations demonstrate that stimulation of pituitary cGMP production and LH release by GnRH is dependent on extracellular calcium. The site at which calcium is required during GnRH action is at a postreceptor locus before cGMP formation.     

Mechanisms of age-related changes in gonadotropin-releasing hormone receptor messenger ribonucleic acid content in the anterior pituitary of male rats.

To determine the mechanism(s) of age-related changes in gonadotropin release from pituitary gonadotrophs in male rats, we measured the number of GnRH (gonadotropin-releasing hormone) receptor containing cells and expression of GnRH receptor mRNA per cell in the anterior pituitary. An in situ hybridization procedure was performed using young (six months) and old (24-25 months) Wistar rats. An image analysis system was employed for the autoradiographic analysis. The number of pituitary cells increased during aging (approximately 45%, p < 0.01). On the other hand, the number of GnRH receptor mRNA-containing cells decreased (approximately 25%, p < 0.05). The percentage of these cells in old rats decreased to less than a half of that in young animals (p < 0.01). GnRH receptor mRNA per cell in old rats was only 7% lower than in young (p < 0.01). These results suggest that loss of pituitary gonadotroph GnRH receptors and response is primarily due to the loss of gonadotrophs, and that the death mechanism(s) are responsible for decreased stimulation of Gn release during aging.     

Modulation of cytoplasmic calcium signaling in rat pituitary gonadotrophs by estradiol and progesterone.

The stimulatory action of GnRH on gonadotropin secretion from cultured rat pituitary cells is modulated by estradiol (E) and progesterone (P). Since secretory responses to GnRH are initiated by phosphoinositide hydrolysis and Ca2+ mobilization, the effects of gonadal steroids on the pattern of Ca2+ signaling were analyzed in single pituitary gonadotrophs. Increasing concentrations of GnRH elicited a spectrum of [Ca2+]i signals in single gonadotrophs, ranging from subthreshold to threshold-oscillatory and biphasic (spike & plateau) responses. In E-treated gonadotrophs, short-term P treatment shifted subthreshold [Ca2+]i responses to oscillatory and oscillatory to biphasic responses, whereas long-term P treatment shifted oscillatory to subthreshold [Ca2+]i response profiles. These changes parallel the effects of P on GnRH-induced LH release, and indicate that the modulatory effects of ovarian steroids on gonadotropin secretion include a significant action on the Ca2+ signaling pathway.     

Molecular characterization and expression of three GnRH forms mRNA during gonad sex-change process, and effect of GnRHa on GTH subunits mRNA in the protandrous black porgy (Acanthopagrus schlegeli)

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in control of reproduction and gonadal maturation in teleost fish. To investigate the action GnRH in black porgy (Acanthopagrus schlegeli), we examined the mRNA expression of GTH subunits (GTHα, FSHβ, and LHβ) in the pituitary as well as plasma estradiol-17β (E₂) level following treatment with a GnRH analog (GnRHa) in immature fish. The expression levels of GTH subunits mRNA and plasma E₂ level were increased after GnRHa injection. We were also able to identify three GnRH forms: salmon GnRH (sGnRH), seabream GnRH (sbGnRH) and chicken GnRH-II (cGnRH-II) by cDNA cloning in the ovary of the black porgy. Black porgy gonadal development is divided into seven stages, involving sex change from male to female (immature testis, mature testis, testicular portion of mostly testis, ovarian portion of mostly testis, testicular portion of mostly ovary, ovarian portion of mostly ovary, and mature ovary). In the present study, we investigated the expression pattern of three GnRH molecular forms in the black porgy gonads at different stages of gonadal development by quantitative polymerase chain reaction (QPCR). The mRNA expressions of sGnRH, sbGnRH and cGnRH-II were found to be higher in mature testis and ovary, compared to gonads at different stages of maturity. The findings support the hypothesis that the three forms of GnRH play important roles in the regulation of hypothalamic-pituitary-gonadal axis, and are likely involved also in gonadal development and sex change in black porgy.     

The localization of gonadotrophs in normal adult male and female rats

We investigated the localization of LH and FSH cells within the pituitary glands of normal adult rats. Groups of four female rats were decapitated at one of five different times during the estrous cycle. Four male rats were also decapitated. Paired horizontal flip-flopped serial paraplast sections from the dorsal, middle, and ventral portions of each pituitary gland were stained. For each pair, one section was stained with antirat LH-S4 and the other section with antirat FSH-S7, by the unlabeled antibody peroxidase-antiperoxidase method. All immunoreactive cells were counted, and the area of pars distalis in each section was determined. We studied the spatial distribution of gonadotrophs within the sections and determined if a polarization along the antero-posterior axis existed. In the "sex zone" of the pars distalis, the cross-sectional area of LH cells and the percentages of LH cells that also contained FSH and vice versa were determined and compared with those obtained from the entire pars distalis. Additional sections were stained for TSH, ACTH, GH, or PRL, and the distribution of stained cells was compared with that of those that stained for LH or LH/FSH, particularly in the sex zone and in the pars intermedia. The results indicate that 1) gonadotrophs are more evenly distributed dorsoventrally within the pars distalis of male rats than in that of female rats; 2) an antero-posterior polarity in gonadotropic distribution is more pronounced in male rats than in female rats; 3) gonadotrophs containing only LH are less numerous in male than female rats, and in the female tend to be centrally located within the pars distalis; 4) the sex zone contains PRL cells and gonadotrophs, and the percentages of gonadotrophs that contain LH or LH and FSH are not different from those of the entire pars distalis; 5) LH, and occasionally LH/FSH cells, are present between lobules of immunoreactive ACTH cells in the pars intermedia; and 6) LH cells in the pars intermedia are smaller than those in the sex zone or entire pars distalis.