A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.     

Human pituitary null cell adenomas and oncocytomas in vitro: effects of adenohypophysiotropic hormones and gonadal steroids on hormone secretion and tumor cell morphology.

Human pituitary null cell adenomas and oncocytomas are not associated with evidence of excess hormone secretion in vivo; their cellular derivation has not been clarified by morphologic investigation. In this study we examined 41 null cell adenomas and 58 oncocytomas in vitro to determine hormone release and its response to several adenohypophysiotropic hormones and gonadal steroids. In vitro, 96/99 tumors released LH, FSH, and/or alpha-subunit of glycoprotein hormones. TSH was released by 11 tumors. GH, PRL, and ACTH were found in small quantities in 11, 8, and 5 tumors, respectively. Only 3 tumors released no detectable hormones. Incubations with test substances were examined at 2- and 24-h periods for up to 72 h. All but 3 of 53 tumors showed marked and persistent increases in the release of LH, FSH, and/or alpha-subunit in response to GnRH in short and long duration experiments. Secretion of LH, FSH, or alpha-subunit was stimulated to more than 150% of control by TRH in 37/48 tumors, by CRH in 10/20, by GRH in 7/20. Estradiol, progesterone, and testosterone increased release of FSH, LH, and/or alpha-subunit in 23/32, 3/12, and 3/12 tumors, respectively, and reduced their release in 6/32, 5/12, and 7/12, respectively. In tumors which showed no response to gonadal steroids, GnRH in combination with estradiol, progesterone, or testosterone yielded the same result as GnRH alone; in tumors inhibited by gonadal steroids, GnRH in combination with one of those substances reduced the response to GnRH. No secretion of GH, PRL, ACTH, or TSH was detected after incubation with GRH, estradiol, CRH, or TRH except in the tumors which initially released GH, PRL, or TSH. Ultrastructural examination of cultured cells from 15 cases revealed morphologic alterations that correlated with changes in hormone release and could be quantified by morphometry. This study represents the largest analysis of hormone production and release in vitro and morphologic correlation of clinically nonfunctioning pituitary adenomas. The responsiveness of gonadotropin secretion by null cell adenomas and oncocytomas to GnRH and gonadal steroids resembles that of gonadotroph adenomas. However, the unexpected increases in gonadotropin release attributable to several other adenohypophysiotropic hormones and the release of multiple hormones suggests that null cell adenomas and oncocytomas may represent neoplasms derived from uncommitted or committed precursor cells that can undergo differentiation towards several cell lines.     

Bihormonal cells producing gonadotropins and prolactin in a rat pituitary tumor cell line (RC-4B/C)

Using single hormone-staining immunocytochemical methods, we have recently characterized a novel cell line, RC-4B/C, established from an aged male rat pituitary adenoma. This cell line contained all known anterior pituitary cell types including gonadotropes. Gonadotropin-releasing hormone receptors were also present. A recent re-examination of the cell types using single hormone stains showed that the cell line underwent an alteration in the percentage of different cell types as compared to the data obtained 2 years ago. The proportion of follicle-stimulating hormone-beta (FSH beta), luteinizing hormone-beta (LH beta), prolactin (PRL), and adrenocorticotropic hormone cells increased significantly (p less than or equal to 0.001), while the proportion of growth hormone (GH), and thyrotropin-beta cells did not change. Dual staining of the monolayers showed that the cell line contained many bihormonal cells producing FSH beta + LH beta, FSH beta + PRL and LH beta + PRL. The proportion of bihormonal FSH beta + LH beta and FSH beta + PRL cells was preponderant over monohormonal cells, while the proportion of bihormonal LH beta + PRL cells was in the same range as that of monohormonal cells. Preliminary data with dual labeling also revealed the presence of GH and PRL in the same cell, but complete absence of a combination such as FSH beta + GH. No search for the presence of other bihormonal or multihormonal cells was performed. In short, our data show that the majority of the cells in the cell line RC-4B/C contain FSH beta, LH beta and PRL and that among these cells many bihormonal cells are present.     

Gonadotropin-releasing hormone agonists are unsuccessful in reducing tumoral gonadotropin secretion in two patients with gonadotropin-secreting pituitary adenomas

Whether GnRH agonist treatment leads to reduced gonadotropin secretion and tumor volume in patients with gonadotropin-secreting pituitary adenomas is controversial. We studied the effect of GnRH analog treatment in two such patients, one with a recurrent FSH- and LH-secreting pituitary adenoma (patient 1) and one with a recurrent FSH- and alpha-subunit-secreting pituitary adenoma (patient 2). Patient 1 was treated with 200 micrograms Buserelin daily for 65 days, and patient 2 received three injections of 3 mg [D-Trp6]-LHRH formulated in microcapsules at 21-day intervals. In both patients, plasma FSH, LH (RIA), and alpha-subunit concentrations increased initially and remained above the pretreatment values throughout the treatment period. Plasma LH, measured by immunoradiometric assay, remained well above the detection limit. Plasma bioactive LH and testosterone became undetectable in patient 2, but did not change in patient 1. In neither patient did pituitary tumor size (determined by computed tomographic scan) change during treatment. We conclude that 1) the overall effect of GnRH analogs in patients with gonadotroph cell adenomas is stimulation of gonadotropin release by the tumor, although LH release varies according to how plasma LH is measured, possibly related to the origin of the hormone (normal or tumor gonadotroph cells), and 2) GnRH analog treatment does not reduce tumor size.     

Pituitary gonadotropin-releasing hormone (GnRH) receptor responses to GnRH in hypothalamus-lesioned rats: inhibition of responses by hyperprolactinemia and evidence that testosterone and estradiol modulate gonadotropin secretion at postreceptor sites

Increased hypothalamic GnRH secretion appears to influence positively the number of pituitary GnRH receptors (GnRH-R). GnRH-R increase after castration in male rats, and this rise can be prevented by testosterone (T), anti-GnRH sera, or hypothalamic lesions. GnRH also increases serum LH and GnRH-R in hypothalamus-lesioned rats, and these animals injected with exogenous GnRH are, therefore, a good model in which to study the site of steroid feedback at the pituitary level. Adult male and female rats were gonadectomized, and radiofrequency lesions were placed in the hypothalamus. Males received T implants, and females received estradiol implants at the time of surgery. Empty capsules were placed in the control animals. Beginning 3-5 days later, animals in each group were injected every 8 h with vehicle (BSA) or GnRH (0.002-200 micrograms/day) for 2 days. After these GnRH injections, all rats received 6.6 micrograms GnRH, sc, 1 h before decapitation to determine acute LH and FSH responses. GnRH-R were determined by saturation analysis using 125I-D-Ala6-GnRH ethylamide as ligand. In males, GnRH injections increased GnRH-R. T inhibited acute LH and FSH responses to GnRH in all groups, but had little effect on GnRH-R, indicating that T inhibits gonadotropin secretion at a post-GnRH receptor site. In females, the GnRH-R response to GnRH was less marked, and only the 200 micrograms/day dose of GnRH increased GnRH-R, indicating that the positive feedback effects of estradiol at the pituitary level are also exerted at a site distal to the GnRH receptor. There was no positive correlation between the number of GnRH-R and GnRH-stimulated gonadotropin release in males or females. Female rats with hypothalamic lesions had markedly elevated serum PRL levels (greater than 300 ng/ml). Suppression of PRL secretion by bromocryptine resulted in augmented GnRH-R responses to GnRH, and GnRH-R concentrations rose to the same values induced in males. This suggests that hyperprolactinemia inhibits GnRH-R responses to GnRH in females by a direct action on the pituitary gonadotroph.     

Hormones in synergy: Regulation of the pituitary gonadotropin genes

The precise interplay of hormonal influences that governs gonadotropin hormone production by the pituitary includes endocrine, paracrine and autocrine actions of hypothalamic gonadotropin-releasing hormone (GnRH), activin and steroids. However, most studies of hormonal regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the pituitary gonadotrope have been limited to analyses of the isolated actions of individual hormones. LHβ and FSHβ subunits have distinct patterns of expression during the menstrual/estrous cycle as a result of the integration of activin, GnRH, and steroid hormone action. In this review, we focus on studies that delineate the interplay among these hormones in the regulation of LHβ and FSHβ gene expression in gonadotrope cells and discuss how signaling cross-talk contributes to differential expression. We also discuss how recent technological advances will help identify additional factors involved in the differential hormonal regulation of LH and FSH.     

Characterization of gonadotropic cells in a new pituitary tumor cell line

The pituitary tumor cell line RC-4B/C was established in The Jackson Laboratory from an aged rat pituitary adenoma. Immunocytochemical studies of this cell line showed that all pituitary cell types were present. Approximately 20% reacted with antisera (AS) to ovine (o) LH beta, 8.6% with AS to oFSH beta, 15% with AS to rat PRL, 12% with AS to equine GH, 9% with AS to porcine TSH beta and 8.6% with AS to ACTH1-24. Using NIDDK rat kits, RIA showed about 0.38, 0.08 and 607.50 ng per 10(6) cells of LH, FSH and PRL, respectively, vs 33.9, 75.6 and 573 ng in freshly dispersed rat pituitary cells. The GnRH receptor content of the cell line was about a half that of normal rat pituitary cells but the receptor affinity was the same. A chronic treatment of the cells for about 5 months with a "sub-physiological" concentration (3.7 pM) of a GnRH agonist had 3 major effects: 1) as compared to the controls, a 3-fold increase in the cell number in the log phase; 2) an increase of the percentage of FSH beta cells from 8.6 to 21.9% whereas LH beta cells and the cell content of LH and FSH remained stationary; 3) a decrease of the percentage of PRL cells from 15 to 6.5% and an almost 250-fold decrease of PRL cell content. Incorporation studies with [35S] Met demonstrated that the alpha subunit in the cell line was only partly glycosylated. Pretreatment of the cells with 5 nM estradiol restored, at least partly, glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selectivity of melatonin pituitary inhibition for luteinizing hormone-releasing hormone

The pineal indole melatonin suppresses the neonatal rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responses to LH-releasing hormone (LHRH), as shown in previous studies from this laboratory. We show in this study that the melatonin inhibition is a selective effect and is not due to general inhibition of pituitary function. The effects of the indole on the responses to thyrotropin-releasing hormone (TRH) and somatostatin (SRIF) and on basal pituitary hormone secretion were examined with cells in culture. Neonatal rat anterior pituitary cells dissociated with collagenase and hyaluronidase were cultured overnight and distributed to 35-mm dishes at the time of use. For examination of melatonin effects on the response to releasing hormones, the cells were incubated for 3 h in control medium or medium containing LHRH (10-9-10-6 M), TRH (10-10-10-6 M), or SRIF (10-9-10-6 M), either alone or in the presence of melatonin (10-8 or 10-6 M). For examination of basal hormone secretion, the cells were incubated for 1.5, 3, 6, 15, or 24 h in either medium alone or medium containing melatonin (10-6 M). Medium and cell lysate concentrations of LH, FSH, thyroid-stimulating hormone (TSh), prolactin (PRL) and growth hormone (GH) were determined by double antibody RIA. As previously, melatonin (10-8 M) significantly suppressed LH and FSH release by all concentrations of LHRH. This concentration of the indole produced maximal suppression of both LH and FSH responses to LHRH. By contrast, melatonin at a 100-fold greater concentration (10-6 M) had no effect on TRH stimulation of TSH or PRL release or on SRIF inhibition of GH release. Similarly, melatonin had no effect on basal release of TSH, PRL, or GH at the times examined. These findings show that melatonin inhibition of the gonadotroph response to LHRH is a selective effect.     

Effects of a novel gonadotropin-releasing hormone antagonist (ORG 30850) on gonadotropin and prolactin secretion by rat pituitary cells in culture

The effect of a new GnRH antagonist (ORG 30850 ANT) on FSH, LH, and PRL secretion was studied using male rat pituitary cells in monolayer cell culture. In the absence of GnRH, ORG 30850 ANT did not alter spontaneous FSH and LH secretion into culture medium or the cell content of these hormones. In the presence of GnRH (10(-8) mol/l), ORG 30850 ANT significantly and dose-dependently inhibited FSH and LH secretion into culture medium while increasing their cell content. Conversely, in the presence of a single dose of ORG 30850 ANT, FSH and LH secretion rose significantly when subjected to increasing amounts of GnRH, whereas the hormonal cell content diminished. Furthermore, inhibition of GnRH-induced FSH and LH release by ORG 30850 ANT was not changed by pre-incubation with the GnRH antagonist regardless of the pre-incubation time. The inhibitory effect of the GnRH antagonist was observed early, with its peak occurring within 6 h of culture. These short-term studies indicate that ORG 30850 ANT specifically inhibits GnRH-induced gonadotropin release into culture medium, exerts no effect on the rate of gonadotropin production in the presence or absence of GnRH, competitively and reversibly inhibits the binding of natural GnRH to its receptors, and does not lead to any modifications in PRL secretion.     

Gonadotropin-releasing hormone and its agonist inhibit testicular luteinizing hormone receptor and steroidogenesis in immature and adult hypophysectomized rats

The direct effect of gonadotropin-releasing hormone (GnRH) and its agonist on testicular LH receptor and steroidogenesis was studied in hypophysectomized immature and adult rats. Hypophysectomized rats were treated daily with varying doses of GnRH or [des-Gly10,D-Leu6(N alpha Me)Leu7, Pro9-NHEt]GnRH(a potent agonist). Some animals were also treated concomitantly with FSH, PRL, GH and/or LH to prevent the hypophysectomy-induced loss of testicular LH receptor and steroidogenic capacity. At the end of 5 days of treatment, testicular LH/hCG receptor concentration was measured by a [125I]-hCG-binding assay and steroidogenic responsiveness was determinded by in vitro incubations. GnRH and the GnRH agonist reduced testicular LH receptor in control and FSH-treated hypophysectomized immature rats. As little as 0.5 microgram agonist/day induced a greater than 40% decrease in the LH receptor content, whereas GnRH was less potent, with 50 micrograms/day inducing about a 50% decrease. The inhibitory effect of GnRH was shown to be the result of decreases in the concentration of LH receptor rather than changes in the receptor affinity (Kd = 1.1 X 10(-10)M). GnRH did not interfere with the [125I]hCG receptor assay. Treatment with PRL, GH, and FSH, alone or in various combinations, increased the testicular LH receptor content. The stimulatory effect of these pituitary hormones was depressed by concomitant treatment with the GnRH agonist. Similar inhibitory effects of GnRH and the agonist on testicular LH receptor were demonstrated in adult hypophysectomized rats. In vitro studies demonstrated that treatment with the GnRH agonist in vivo inhibited both basal and hCG-stimulated androgen production in FSH-primed immature hypophysectomized rats. Associated with decreases in androgens (testosterone and androstenedione) and reduced androgens (dihydrotestosterone, androstanediol, and androsterone), there was marked suppression of 17 alpha-hydroxylated precursors and C-21 steroid intermediates in animals treated with the GnRH agonist, thus suggesting that the inhibitory effect of the GnRH agonist was associated with possible defects in 17 alpha-hydroxylase and side-chain cleavage enzymes. Likewise, treatment with the GnRH agonist inhibited in vitro testicular steroidogenic responses in adult hypopysectomized rats. These results demonstrate the extrapituitary inhibitory effect of GnRH on testicular LH receptor content and Leydig cell steroidogenesis in immature and adult hypophysectomized rats.     

Direct effects of prolactin and dopamine on the gonadotroph response to GnRH

The intrapituitary mechanisms underlying the inhibitory actions of hyperprolactinaemia on the reproductive axis remain unclear. Previous work on primary pituitary cultures revealed combined suppressive effects of prolactin (PRL) and dopamine on the gonadotrophin response to GnRH. However, whether these effects occur directly at the level of the gonadotroph and are accompanied by changes in gene expression is still unresolved. Here, alphaT(3)-1 and LbetaT2 cells were used to investigate the effects of PRL and dopamine on gonadotrophin synthesis and release in gonadotroph monocultures under basal and GnRH-stimulated conditions. PRL receptor and dopamine receptor mRNA expressions were first determined by RT-PCR in both cell lines. Then, PRL and the dopamine agonist bromocriptine (Br), alone or in combination, were shown to block the maximal alpha-subunit and LHbeta-subunit mRNA responses to a dose-range of GnRH. The LH secretory response was differentially affected by treatments. GnRH dose-dependently stimulated LH release, with a 4-5 fold increase at 10(-8) M GnRH. Unexpectedly, PRL or Br stimulated basal LH release, with PRL, but not Br, enhancing the LH secretory response to GnRH. This effect was, however, completely blocked by Br. These results reveal direct effects of PRL and dopamine at the level of the gonadotroph cell, and interactions between these two hormones in the regulation of gonadotrophin secretion. Moreover, uncoupling between LH synthesis and release in both the basal and the GnRH-stimulated responses to PRL and dopamine was clearly apparent.     

Neuroendocrine function in adult female transgenic mice expressing the human growth hormone gene.

Adult female transgenic mice expressing the human GH (hGH) gene with mouse metallothionein-I promoter are sterile. To evaluate the hypothalamic-pituitary function in these animals, adult female transgenic mice and nontransgenic normal littermates were ovariectomized. On days 7 and 8 after ovariectomy, mice were injected with either oil or primed with 0.5 micrograms estradiol benzoate (EB) in oil, 24 h later treated with 10 micrograms EB/100 g body wt and a day later bled for measurements of FSH, LH, and PRL levels. Plasma gonadotropin and PRL levels were also measured in ovary-intact transgenic and normal siblings at estrus. Additional ovariectomized EB-treated transgenic mice and normal siblings were injected with either saline or GnRH in saline (1 ng/g body wt) and were bled 15 min later for determination of circulating hormone levels. At estrus, in transgenic mice, circulating FSH and PRL levels were significantly lower (FSH:P less than 0.001; PRL:P less than 0.025), but plasma LH concentrations were higher (P less than 0.001) than those in nontransgenic mice. As expected, ovariectomy significantly increased (P less than 0.001) circulating FSH and LH levels in both groups of mice relative to ovary-intact animals, but the increase in plasma LH levels was attenuated in transgenic mice. The suppressive effect of estrogen on circulating FSH and LH levels were similar in transgenic and nontransgenic mice. Treatment with GnRH significantly increased plasma FSH and LH levels in both transgenic and normal mice. However, the plasma FSH and LH responses to GnRH administration were significantly reduced (P less than 0.001) in transgenic mice. The results of these studies indicate that adult female transgenic mice expressing the hGH gene are hypoprolactinemic. Yet due to PRL-like activity of hGH, the gonadotropin secretion is altered. Thus, endogenously secreted hGH modulates the hypothalamic-pituitary function of adult female transgenic mice bearing the hGH gene.     

Targeted pituitary overexpression of pituitary adenylate-cyclase activating polypeptide alters postnatal sexual maturation in male mice

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is present in high concentrations within the hypothalamus, suggesting that it may be a hypophysiotropic factor, whereas pituitary expression suggests a paracrine function. PACAP stimulates gonadotropin secretion and enhances GnRH responsiveness. PACAP increases gonadotropin α-subunit (αGSU), lengthens LHβ, but reduces FSHβ mRNA levels in adult pituitary cell cultures in part by increasing follistatin. PACAP stimulates LH secretion in rats; however, acceptance of PACAP as a regulator of reproduction has been limited by a paucity of in vivo studies. We created a transgenic mouse model of pituitary PACAP overexpression using the αGSU subunit promoter. Real-time PCR was used to evaluate PACAP, follistatin, GnRH receptor, and the gonadotropin subunit mRNA in male transgenic and wild-type mice of various ages. Transgenic mice had greater than 1000-fold higher levels of pituitary PACAP mRNA; and immunocytochemistry, Western blot, and ELISA analyses confirmed high peptide levels. FSH, LH, and testosterone levels were significantly suppressed, and the timing of puberty was substantially delayed in PACAP transgenic mice in which gonadotropin subunit and GnRH receptor mRNA levels were reduced and pituitary follistatin expression was increased. Microarray analyses revealed 1229 of 45102 probes were significantly (P < 0.01) different in pituitaries from PACAP transgenic mice, of which 83 genes were at least 2-fold different. Genes involved in small molecule biochemistry, cancer, and reproductive system diseases were the top associated networks. The GnRH signaling pathway was the top canonical pathway affected by pituitary PACAP excess. These experiments provide the first evidence that PACAP affects gonadotropin expression and sexual maturation in vivo.     

Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters

Despite important advances in human therapeutics, no specific treatment for both non-functioning gonadotroph and resistant somatotroph adenomas is available. Gene transfer by viral vectors can be considered as a promising way to achieve a specific and efficient treatment. Here we show the possibility of efficient gene transfer in human pituitary adenoma cells in vitro using a human immunodeficiency virus (HIV)-type 1-derived vector. Using enhanced green fluorescent protein (eGFP) gene as a marker placed under the phosphoglycerate kinase (PGK) promoter, gonadotroph and somatotroph adenomas were transduced even with moderate viral loads. The expression started at day 2, reached a peak at day 5, and it was still present at day 90. For targeting somatotroph and gonadotroph adenomas, human growth hormone (GH) promoter (GH -481, +54 bp) and two fragments of the human glycoprotein hormone alpha-subunit promoter (alpha-subunit 1 -520, +33 bp, and alpha-subunit 2 -907, +33 bp) were tested. In gonadotroph adenomas, the percentage of identified fluorescent cells and the fluorescence intensity analyzed by fluorescence-activated cell sorting indicated that the strength of the alpha-subunit 1 and alpha-subunit 2 promoters were comparable to that of the PGK promoter. Primary cultures of rat pituitary cells showed that alpha-subunit 1 is more selective to thyreotroph and gonadotroph phenotypes than alpha-subunit 2. GH promoter activity appeared weak in somatotroph adenomas. The human GH enhancer did not increase the GH promoter activity at all but the human prolactin promoter (-250 bp) allowed 4-fold more fluorescent cells to be obtained than the GH promoter. Several cell lines appeared too permissive to test cell-specificity of pituitary promoters. However, on human non-pituitary cell cultures, the tested pituitary promoters seemed clearly selective to target endocrine pituitary phenotypes. This study gives a starting point for a gene-therapy program using lentiviral vectors to transfer therapeutic genes in human pituitary adenomas.     

Growth hormone cells as co-gonadotropes: partners in the regulation of the reproductive system.

Through unique receptors, growth hormone (GH) stimulates ovarian follicles and Leydig cells, working alone or synergistically with luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The source of GH might include a unique cell type that expresses mRNA encoding gonadotropin and GH and the antigens themselves, together with gonadotropin-releasing hormone (GnRH) and GH-releasing hormone (GHRH) receptors. This multifunctional cell might provide a cocktail of hormones needed to effect optimal gonadotropic activity.     

Release of dynorphin-like immunoreactivity from rat adenohypophysis in vitro during inhibition of anterior pituitary hormone secretion from individual cell types

LHRH has previously been found to be the only known hypothalamic releasing factor which can specifically stimulate the release of the opioid dynorphin and other proenkephalin B-derived peptides from the rat adenohypophysis in vitro. In the present study the mechanisms that regulate dynorphin release were further characterized. It was examined whether or not dynorphin release from the adenohypophysis in vitro is altered during inhibition of the secretion of various anterior pituitary hormones. Rat anterior pituitary quarters were incubated in vitro and hormone release into the incubation medium was measured by RIAs. Somatostatin, dopamine, T3, dexamethasone, and 5 alpha-dihydrotestosterone were used to inhibit the secretion of GH, PRL, TSH, ACTH/beta-endorphin, or LH/FSH, respectively. GH, PRL, or beta-endorphin release was inhibited without affecting the simultaneous release of dynorphin A-(1-13)-like immunoreactivity (Dyn A1-13-IR). Concentrations of T3, somatostatin, or dopamine which were effective in suppressing the evoked and/or basal release of TSH, GH, or PRL, respectively, produced no effect on Dyn A1-13-IR release caused by high potassium concentration (40 mM) or LHRH (500 pM). The LHRH-induced release of LH and FSH was inhibited by the glucocorticoid dexamethasone or the androgen 5 alpha-dihydrotestosterone. Under these conditions, Dyn A1-13-IR release was also reduced. However, whereas LH release was completely blocked by 5 alpha-dihydrotestosterone, FSH and Dyn A1-13-IR release was reduced only by 50%. The release of FSH and Dyn A1-13-IR in vitro from anterior pituitary glands taken from rats, castrated 3 weeks before, was enhanced to a similar extent (about 2.5-fold); the simultaneous enhancement of LH release was significantly (P less than 0.005) greater (about 5-fold). We conclude that the mechanisms which regulate the release and/or biosynthesis of dynorphin and other proenkephalin B-derived peptides of the adenohypophysis are similar to those of the gonadotropins but different from those of any other anterior pituitary hormone, and may be more closely related with FSH release than LH release. These data support the view that dynorphin of the normal rat adenohypophysis may be localized in at least a subpopulation of gonadotrophs.     

Gonadotropin-releasing hormone increases the amount of messenger ribonucleic acid for gonadotropins in ovariectomized ewes after hypothalamic-pituitary disconnection

To investigate the role of GnRH in regulating the synthesis and secretion of gonadotropins, GnRH (250 ng/6 min every other hour for 7 days) or saline was administered to ovariectomized (OVX) ewes after hypothalamic-pituitary disconnection (HPD). Blood samples were collected from all HPD ewes on the day before and the day after HPD and on days 1 and 7 of GnRH or saline. At the end of day 7, anterior pituitary glands were removed for analysis of hormone, receptor, and mRNA content. The amount of mRNA for gonadotropins was lower (P less than 0.05) in saline-treated HPD ewes than in GnRH-treated HPD or OVX ewes. Administration of GnRH restored the amount of mRNA for FSH beta and alpha-subunits to levels similar (P greater than 0.05) to those measured in OVX ewes. The amount of mRNA for LH beta was higher (P less than 0.05) in GnRH-treated HPD ewes than in saline-treated HPD ewes, but lower (P less than 0.05) than that in OVX ewes. The pituitary content of LH and FSH was lower (P less than 0.05) in saline-treated HPD ewes than in OVX ewes. Administration of GnRH to HPD ewes maintained the ewes. Administration of GnRH to HPD ewes maintained the pituitary content of LH, but not FSH, compared to the pituitary gonadotropin content in OVX ewes. There were no differences (P greater than 0.05) in the amount of mRNA for GH or PRL or the pituitary content of these hormones among treatments. The number of hypophyseal receptors for GnRH was reduced in saline-treated HPD ewes (P less than 0.05) compared to that in OVX ewes and GnRH-treated HPD ewes. The number of hypophyseal receptors for 17 beta-estradiol was lower (P less than 0.05) in GnRH- and saline-treated HPD ewes than in OVX ewes. Serum LH concentrations were lower (P less than 0.05) after HPD than before HPD, but were restored to normal (P greater than 0.05) by GnRH replacement. Serum concentrations of FSH were lower (P less than 0.05) after HPD and were not affected by GnRH replacement. Serum PRL concentrations in all ewes were higher (P less than 0.05) after HPD than before HPD. Serum GH concentrations in all ewes were similar (P greater than 0.05) before and after HPD. Since synthesis and secretion of GH and PRL were not diminished after HPD, it was considered that the pituitary gland remained viable and functioned independently of hypothalamic input in OVX ewes after HPD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel Pituitary Ligands: Peroxisome Proliferator Activating Receptor-γ

Pituitary tumors cause considerable morbidity due to local invasion, hypopituitarism, or hormone hypersecretion. In many cases, no suitable drug therapies are available, and surgical excision is currently the only effective treatment. We have recently demonstrated abundant expression of nuclear hormone receptor PPAR-gamma in human pituitary tumors of different subtypes. PPAR-gamma activators (thiazolidinediones) induced G0-G1 cell-cycle arrest and apoptosis in human, and murine corticotroph, somatolactotroph, and gonadotroph pituitary tumor cells, and suppressed in vitro hormone secretion. In vivo development and growth of murine corticotroph, somatolactotroph and gonadotroph tumors, generated by subcutaneous injection of ACTH-secreting AtT20, PRL- and GH-secreting GH3, and LH-secreting LbetaT2, and alpha-T3 cells, was markedly suppressed in rosiglitazone treated mice, and plasma ACTH, and serum corticosterone, GH, PRL and LH levels were attenuated in all treated animals. PPAR-gamma is an important novel molecular target in pituitary adenoma cells and as PPAR-gamma ligands inhibit tumor cell growth and ACTH, GH, PRL and LH secretion in vitro and in vivo, thiazolidinediones are proposed as a novel oral medical management for pituitary tumors.     

Ovarian influence on gonadotropin and prolactin release in mated rabbits

In 17β-estradiol (E)-treated ovariectomized (OVX) rabbits, the coitus-induced luteinizing hormone (LH) surge is only one fourth that in ovarian-intact rabbits. In this study, we determined the pattern of the coitusinduced gonadotropin release, i.e., LH and folliclestimulating hormone (FSH), in OVX + E animals without or with continuous 3-wk treatment of 20-α-hydroxypregn-4-en-3-one (20αP). For positive and negative experimental controls, ovarian-intact rabbits were either mated or sham mated, respectively. The pituitary hormones prolactin (PRL) and growth hormone (GH) were measured to serve as collateral controls for gonadotropins. The addition of continuous 20αP in OVX + E does fail to stimulate a coitus-induced LH surge equal in magnitude and duration to the LH surge in ovarian-intact rabbits. Postcoital levels of FSH were greater in OVX + E + 20αP animals than those in OVX + E rabbits. Coitus induced a PRL surge in ovarianintact and OVX + steroid-treated females, but not in mated males, thereby suggesting a gender difference in this neuroendocrine circuit. Neither coitus nor steroids altered plasma GH values in female or male animals. We conclude that chronic administration of neither E nor E + 20αP can restore full-scale gonadotropin surges in OVX rabbits, whereas replacement of one or both of these steroids is sufficient for a coitusinduced PRL surge. Moreover, the presented observation that activin stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release suggests a possible involvement of ovarian proteins in the production of a full-scale coitus-induced GnRH/LH surge.