The identification of gonadal gonadotropin-releasing hormone receptors (GnRH-R) and evidence of direct inhibitory effects of GnRH agonists upon steroidogenesis in adult rat gonads, lend credence to a putative intragonadal role of a locally secreted GnRH or GnRH-like peptide. Using reverse transcription-polymerase chain reaction followed by Southern blot hybridization and sequencing, we identified, both in the ovary and in the testis of fetal and adult rats, a fully processed GnRH messenger RNA (mRNA), the sequence of which, in adult testis, was identical to that found in the hypothalamus. We also detected in the testis, but not in the ovary, a transcript containing the first intron. The ontogeny of GnRH and GnRH-R gene expression was studied in rat gonads from 14.5 to 21.5 days post-coitum (dpc), using dot blot hybridization of total RNA. During this period, the levels of cyclophilin mRNA normalized to total RNA remained unchanged. Thus, we used cyclophilin as an internal standard. GnRH mRNA was detected in the ovary at 18.5 dpc, four days later than in the testis, and similar levels were found in both sexes at birth. GnRH-R mRNA was present at 14.5 dpc in the testis and at 15.5 dpc in the ovary, with the levels at 21.5 dpc being 2.4 times higher in the testis than in the ovary. GnRH and GnRH-R mRNA levels increased in both sexes in late fetal development, but this increase appeared two days sooner in the ovary compared with the testis, thus supporting the hypothesis that expression of the GnRH and GnRH-R genes is regulated in a sex-dependent manner during fetal development. In all cases, expression of GnRH and GnRH-R preceded gonadotropin receptors in the gonads and initiation of gonadotropin secretion by the pituitary.

ABSTRACT

Ether stress applied at 10.00 h induced a 100% increase in serum prolactin in intact and ovariectomized androgenized rats. Ovariectomy significantly diminished the basal serum prolactin values observed in intact androgenized rats. Two doses of progesterone (5 mg) given to intact and ovariectomized androgenized rats 14 and 2 h before exposure to ether stress increased prolactin values in the control groups but completely prevented the effect of stress. Exposure to ether stress induced a 100% increase in serum prolactin values in androgenized rats with increased serum progesterone levels 4 days after the induction of ovulation and the luteal phase with human chorionic gonadotropin (hCG). A group of androgenized rats with induced maternal behaviour and which had been suckled for 6 days was given 100 i.u. hCG and suckled for another 6 days after the hCG-induced luteal phase had been established. The serum prolactin and progesterone values of these rats were significantly higher than those treated with hCG only and ether stress did not increase prolactin release. A greatly increased serum concentration of prolactin was obtained in pro-oestrous and oestrous virgin rats after exposure to ether stress. Serum prolactin was also increased by stress in male rats. Progesterone administration to these female and male rats prevented stress-induced prolactin release. To ascertain the part played by dopamine and serotonin in the effect of stress on prolactin release, groups of androgenized and oestrous female rats were treated with bromocriptine or p-chlorophenylalanine methylester hydrochloride (pCPA). The dopaminergic agonist bromocriptine markedly reduced prolactin levels in the unstressed androgenized rats, but did not prevent the prolactin increases induced by stress. Administration of pCPA had no effect on basal or stress-increased serum levels of prolactin. It is concluded that modifications of the ovarian steroid secretions, especially of progesterone, has profound effects on prolactin release in response to ether stress. The release of the hormone was not mediated by a dopaminergic or serotonergic regulatory pathway.

J. Endocr. (1986) 110, 423–428

The aim of our in vitro experiments with isolated porcine ovarian follicles was to study the effects of gonadotropins, GH, IGF-I and oxytocin (OT) on release of ovarian steroid, OT, IGF-I, insulin-like growth factor-binding protein-3 (IGFBP-3), prostaglandin F (PGF), prostaglandin E (PGE) and cAMP. It was found that quarters of ovarian follicles cultured for 8 days produced significant amounts of progesterone, estradiol-17 beta, OT and IGFBP-3 with peaks of accumulation from the 3rd to the 8th day of culture. Addition of serum promoted progesterone, estradiol and OT release, whilst accumulation of IGFBP-3 was maintained to a greater extent in serum-free medium. GH (10 ng/ml or above) was able to inhibit androstenedione, OT, PGF and IGFBP-3, to stimulate IGF-I and cAMP, and to alter testosterone and PGE release by follicles cultured in serum-supplemented and/or serum-free medium. IGF-I (10 ng/ml or more) inhibited androstenedione and PGF secretion, stimulated testosterone, estradiol, OT and cAMP production, but did not influence progesterone, IGFBP-3 or PGE output in these conditions. OT (100 ng/ml) was able to inhibit androstenedione and to stimulate testosterone, IGF-I, PGF and PGE, but not estradiol or IGFBP-3 release. A stimulatory effect of LH on progesterone and OT and an inhibitory influence of LH on estradiol secretion in the serum-supplemented medium were observed. FSH in these conditions stimulated OT, but not progesterone or estradiol secretion. The use of this experimental model suggests the involvement of gonadotropins, OT, GH and IGF-I in the control of ovarian steroid and nonapeptide hormone, growth factor, growth factor-binding protein, prostaglandin and cyclic nucleotide production. The stimulatory effect of GH on IGF-I, and the stimulatory influence of IGF-I on OT, as well as coincidence of the majority of effects of IGF-I and OT, suggest the existence of a GH-IGF-I-OT axis. On the other hand, the different patterns of action of GH and IGF-I on OT, estrogen and IGFBP-3 suggest that part of the GH effect on ovarian cells is IGF-I independent.

There is a 2- to 3-fold increase in luteinizing hormone-beta (LHbeta) or follicle-stimulating hormone-beta (FSHbeta) antigen-bearing gonadotropes during diestrus in preparation for the peak LH or FSH secretory activity. This coincides with an increase in cells bearing LHbeta or FSHbeta mRNA. Similarly, there is a 3- to 4-fold increase in the percentage of cells that bind GnRH. In 1994, we reported that this augmentation in gonadotropes may come partially from subsets of somatotropes that transitionally express LHbeta or FSHbeta mRNA and GnRH-binding sites. The next phase of the study focused on questions relating to the somatotropes themselves. Do these putative somatogonadotropes retain a somatotrope phenotype? As a part of ongoing studies that address this question, a biotinylated analog of GHRH was produced, separated by HPLC and characterized for its ability to elicit the release of GH as well as bind to pituitary target cells. The biotinylated analog (Bio-GHRH) was detected cytochemically by the avidin-peroxidase complex technique. It could be displaced by competition with 100-1000 nM GHRH but not corticotropin-releasing hormone or GnRH. In cells from male rats exposed to 1 nM Bio-GHRH, 28+/-6% (mean+/-s.d) of pituitary cells exhibited label for Bio-GHRH (compared with 0.8+/-0.6% in the controls). There were no differences in percentages of GHRH target cells in populations from proestrous (28+/-5%) and estrous (25+/-5%) rats. Maximal percentages of labeled cells were seen following addition of 1 nM analog for 10 min. In dual-labeled fields, GHRH target cells contained all major pituitary hormones, but their expression of ACTH and TRH was very low (less than 3% of the pituitary cell population) and the expression of prolactin (PRL) and gonadotropins varied with the sex and stage of the animal. In all experimental groups, 78-80% of Bio-GHRH-reactive cells contained GH (80-91% of GH cells). In male rats, 33+/-6% of GHRH target cells contained PRL (37+/-9% of PRL cells) and less than 20% of these GHRH-receptive cells contained gonadotropins (23+/-1% of LH and 31+/-9% of FSH cells). In contrast, expression of PRL and gonadotropins was found in over half of the GHRH target cells from proestrous female rats (55+/-10% contained PRL; 56+/-8% contained FSHbeta; and 66+/-1% contained LHbeta). This reflected GHRH binding by 71+/-2% PRL cells, 85+/-5% of LH cells and 83+/-9% of FSH cells. In estrous female rats, the hormonal storage patterns in GHRH target cells were similar to those in the male rat. Because the overall percentages of cells with Bio-GHRH or GH label do not vary among the three groups, the differences seen in the proestrous group reflect internal changes within a single group of somatotropes that retain their GHRH receptor phenotype. Hence, these data correlate with earlier findings that showed that somatotropes may be converted to transitional gonadotropes just before proestrus secretory activity. The LH and FSH antigen content of the GHRH target cells from proestrous rats demonstrates that the LHbeta and FSHbeta mRNAs are indeed translated. Furthermore, the increased expression of PRL antigens by these cells signifies that these convertible somatotropes may also be somatomammotropes.

Abstract

Nitric oxide (NO) is involved in the regulation of endocrine functions, but only a few studies have been reported about its role in placental hormone secretion. We investigated whether NO has any function in the release of human chorionic gonadotropin (hCG) in two different choriocarcinoma cell lines, JEG-3 and BeWo. First, nitric oxide synthase (NOS) was characterized in the choriocarcinoma cells. NOS activity was localized mainly in the particulate fraction and depended on calcium/calmodulin. Activity was inhibited by the presence of the l-arginine analog, N^G-monomethyl-l-arginine (L-NMMA; 1 × 10⁻⁴ m). Western blot analysis showed that the choriocarcinoma cells contained an endothelial isoform of NOS.

The NO donor, sodium nitroprusside (SNP; 1 × 10⁻⁵ and 1 × 10⁻⁴ m), significantly inhibited hCG secretion in both choriocarcinoma cell lines. The suppression of hCG release by SNP (1 × 10⁻⁵ m) was blocked by the addition of an NO scavenger, hemoglobin (1 × 10⁻⁶ m). l-Arginine (1 × 10⁻² m), a NOS substrate, inhibited basal hCG secretion in JEG-3 cells. Incubation of the cells with L-NMMA (1 × 10⁻⁴ and 1 × 10⁻³ m) significantly increased hCG release. Exposure of both cell lines to increasing concentrations of a cyclic GMP analog (8-bromo-cyclic GMP; 1 × 10⁻⁴ to 1 × 10⁻² m) caused a dose-dependent inhibition of hCG release. Cyclic GMP accumulation in response to SNP (1 × 10⁻⁴ m), however, was not detected in either JEG-3 or BeWo cells. These data demonstrated that the endothelial isoform of NOS and a functional l-arginine-NO pathway are present in the choriocarcinoma cell lines. In addition, these findings support the hypothesis that NO produced in these cell lines is involved in the regulation of hCG secretion. We assume that although cyclic GMP is likely to play a role as a second messenger, a cyclic GMP-independent pathway cannot be excluded as a possible physiological mechanism in the attenuation of hCG release by NO.

Journal of Endocrinology (1996) 150, 243–253

Excitatory amino acids, such as glutamate, constitute a major transmitter system in the control of hypothalamic-pituitary secretion. Different subtypes of glutamate receptors, such as NMDA (N-methyl-d-aspartic acid) and KA (kainate) receptors, are involved in the control of anterior pituitary secretion. Other receptor subtypes, such as AMPA (activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and metabotropic receptors, have been identified, although their role in the control of neuroendocrine function remains largely unknown. Recent reports have demonstrated the involvement of AMPA receptors in the control of the steroid-induced luteinizing hormone (LH) surge in female and growth hormone (GH) secretion in male rats. The aim of this study was to assess the potential role of AMPA receptors in the control of GH, prolactin (PRL), LH and follicle-stimulating hormone (FSH) secretion in prepubertal 23-day-old rats. To this end, prepubertal female rats were injected with AMPA (2.5 or 5 mg/kg i.p.) or the antagonist of AMPA receptors 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo (f) quinoxaline-7-sulfonamide (NBQX; 0.25 or 0.50 mg/kg i.p.). Serum LH and FSH concentrations and hypothalamic LH-releasing hormone (LHRH) content remained unchanged after AMPA or NBQX administration. In contrast, serum PRL levels significantly decreased 15, 30 and 60 min after i.p. administration of AMPA and increased 120 min after NBQX treatment, whereas serum GH levels increased after AMPA treatment and decreased after NBQX administration. Considering that AMPA has been shown to activate a subset of kainate receptors, its effects were compared with those elicited by 2.5 mg/kg KA in prepubertal female rats. At this age, however, KA was unable to reproduce the effects of AMPA on PRL and GH secretion, thus suggesting that the actions observed after AMPA administration were carried out specifically through AMPA receptors. In addition, as the effects of AMPA on LH secretion in adult females have been proved to be steroid-dependent, the effects of AMPA (2.5 mg/kg) and NBQX (0.5 mg/kg) were tested in prepubertal animals with different gonadal backgrounds, i.e. intact males, and intact and ovariectomized (OVX) females. The effects of AMPA in prepubertal females appeared to be modulated by ovarian secretion, as the inhibition of PRL secretion disappeared and LH secretion was partially suppressed by AMPA in OVX animals whereas the stimulatory effect on GH release was enhanced by ovariectomy. Furthermore, in male rats, AMPA administration significantly decreased PRL secretion and increased serum GH levels, the amplitude of the GH response being higher than in prepubertal females. To ascertain the pituitary component for the reported actions of AMPA, hemi-pituitaries of male rats were incubated in the presence of AMPA (10(-8)-10(-6) M). The results obtained showed no effect of AMPA on PRL, GH and gonadotropin secretion in vitro. Finally, we investigated the involvement of the dopaminergic (DA) system in the inhibitory action of AMPA on PRL secretion. Pre-treatment of prepubertal female rats with a dopamine receptor antagonist (domperidone: 1 mg/kg) resulted in the blockage of AMPA-mediated inhibition of PRL secretion, thus suggesting that this action is probably mediated by an increase in DA activity. In conclusion, we provide evidence for the physiological role of AMPA receptors in the control of PRL and GH secretion in prepubertal rats. In contrast, our data cast doubts on the involvement of AMPA receptors in the regulation of gonadotropin secretion at this age. The effects of AMPA reported herein were not mediated through activation of kainate receptors and were probably exerted at the hypothalamic or suprahypothalamic levels. In addition, we show that ovarian secretion actively modulates the effects of AMPA receptor activation on anterior pituitary secretion in prepubertal female rats.

Abstract

Many studies have consistently shown that castration induces a prompt increase in serum levels and pituitary content of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as a concomitant rise in steady state levels of the messenger RNAs directing their synthesis. The reports of effects of castration on the overall physiology of hypothalamic luteinizing hormone-releasing hormone (LHRH) — steady state levels of LHRH mRNA, post-translational processing and secretion — have, however, not been consistent.

The goal of the studies reported here was to provide the first analysis of the effect of castration, at multiple post-operative time points, on steady state levels of LHRH mRNA and on the levels of hypothalamic proLHRH. All these data are correlated with hypothalamic levels of the mature LHRH decapeptide and with serum and pituitary levels of immunoreactive LH and FSH.

Adult male rats were either castrated or sham-castrated (controls) and then sacrificed at 1, 3, 5, 7, 14, 21 or 28 days postoperatively. As expected, there was a prompt and sustained rise in serum immunoreactive LH and FSH in castrates compared with sham-operated animals. Intrapituitary LH levels rose above levels in the sham-operated animals by 14 days post castration. Intra-pituitary FSH showed a biphasic response, first falling significantly below control levels, then rising above control levels at 21 days.

Steady state levels of LHRH mRNA in castrates, measured by reverse transcription/polymerase chain reaction, were increased about 2-fold above control levels by 1 day postoperatively, but were virtually identical to control levels at each of the other time points despite marked changes in the gonadotropins.

ProLHRH content in castrates was 1·8-times that seen in controls at 1 day post castration (P<0·05), concomitant with the rise in steady state levels of LHRH mRNA at that time point. However, proLHRH content in castrates was no different from that seen in controls at each of the later time points examined. LHRH content was unchanged through 7 days after castration, but then fell significantly to 57% of control levels in hypothalami from animals gonadectomized 14 to 21 days previously (P<0·001 vs control), and to 54% of sham-operated levels at 28 days postoperatively (P<0·001).

We conclude that: (1) changes in steady state levels of LHRH mRNA after castration are small and transient and (2) increased proLHRH coupled with unchanged LHRH levels at 1 day post castration, and castrate animal pro-LHRH at control levels coupled with falling LHRH at later post-castration time points indicate that the effect of gonadectomy on post-translational processing of pro-LHRH to LHRH is, likewise, small and transient.

In aggregate our data suggest that most of the increase in serum LH and FSH seen in male rats after castration is not mediated at the hypothalamic level.

Journal of Endocrinology (1996) 148, 509–515

Abstract

The significance of stress-induced hypogonadism remains unclear. Since plasma testosterone and LH have renotropic activity that is other than reproductive, we hypothesize that stress-induced hypogonadism is an adaptive response to protect the kidney. To examine this hypothesis, we prepared hypogonadal male rats with different levels of LH and testosterone through orchiectomy (castration), through chronic treatment with a slowly secreted form of gonadotropin-releasing hormone agonist (GnRH_A; GnRH_A pretreatment), or through both treatments concomitantly (castration with GnRH_A pretreatment). Castrated rats had undetectable plasma testosterone and high plasma LH. GnRH_A-pretreated rats had low plasma testosterone and normal plasma LH. Castrated rats with GnRH_A pretreatment had undetectable plasma testosterone and normal plasma LH. We compared their sensitivity to HgCl₂ nephrotoxicity and found that, when a low dose of HgCl₂ (1·5 mg/kg body weight (BW)) was injected s.c. to induce acute renal failure, endogenous creatinine clearance (Ccr) decreased from 390 ± 30 to 94 ± 17 ml/h per kg BW in intact (unpretreated) rats. Such a decrease in Ccr was completely prevented in castrated rats (388 ± 30 ml/h per kg BW) and partially prevented in GnRH_A-pretreated rats (216 ± 40 ml/h per kg BW). When a high dose of HgCl₂ (2·25 mg/kg BW) was injected, half of the eight intact rats died but castrated rats and GnRH_A-pretreated rats survived (P<0·05). The elevated resistance in castrated rats was reduced when plasma LH was reduced with GnRH_A pretreatment, but was restored by additional pretreatment with ovine LH (40 μg/day), as evidenced by changes in Ccr. Elevated resistance in castrated rats was also reduced by the administration of testosterone propionate. In conclusion, hypogonadism activated the preventive and defensive mechanisms that protect the kidney through both decreased plasma testosterone and high or even normal plasma LH.

Journal of Endocrinology (1996) 148, 553–559

Gonadotropin releasing hormone (GnRH) regulates the reproductive system through a specific G-protein-coupled receptor (GPCR) in pituitary gonadotropes. The existence of two (or more) forms of GnRH in most vertebrates suggested the existence of GnRH receptor subtypes (I and II). Using sequence information for extracellular loop 3 of a putative Type II GnRH receptor from a reptile species, we have looked for a Type II GnRH receptor gene in the human genome EST (expressed sequence tag) database. A homolog was identified which has 45% and 41% amino acid identity with exons 2 and 3 of the known human GnRH pituitary receptor (designated Type I) and much lower homology with all other GPCRs. A total of 27 contiguous ESTs was found and comprised a continuous sequence of 1642 nucleotides. The EST sequences were confirmed in the cloned human gene and in PCR products of cDNA from several tissues. All EST transcripts detected were in the antisense orientation with respect to the novel GnRH receptor sequence and were highly expressed in a wide range of human brain and peripheral tissues. PCR of cDNA from a wide range of tissues revealed that intronic sequence equivalent to intron 2 of the Type I GnRH receptor was retained. The failure to splice out putative intron sequences in transcripts which spanned exon-intron boundaries is expected in antisense transcripts, as candidate donor and acceptor sites were only present in the gene when transcribed in the orientation encoding the GnRH receptor homolog. No transcripts extended 5' to the sequence corresponding to intron 2 of the Type I GnRH as the antisense transcripts terminated in poly A due to the presence of a polyadenylation signal sequence in the putative intron 2 when transcribed in the antisense orientation. These findings suggest that a Type II GnRH receptor gene has arisen during vertebrate evolution and is also present in the human. However, the receptor may have become vestigial in the human, possibly due to the abundant and universal tissue transcription of the opposite DNA strand to produce antisense RNA.