Gonadotrophin regulation in the lactating rat.

Post-partum lactation in the rat is associated with follicular quiescence and an attenuation of gonadotrophin secretion. The present study demonstrates that the lactating rat responds to exogenous LH-releasing hormone (LH-RH) in a manner similar to dioestrous rats. Oestrogen priming increases the LH and FSH response to LH-RH to a smaller degree in ovariectomized, lactating, than in non-lactating, ovariectomized rats. Pituitary LH levels throughout lactation did not seem to be related LH-RH-induced LH release. A diminished post-castration rise in both LH and FSH, and a diminished positive feedback response to oestrogen administration were also observed and may indicate a disruption of gonadotrophin regulation at both the hypothalamic and the pituitary level.     

Gonadotrophin subunit and GnRH receptor gene expression in the pars distalis of the equine pituitary

In the horse, pronounced changes in fertility occur annually in response to photoperiod. However, the mechanisms regulating gonadotrophin synthesis and release in this species remain unclear. Here, we investigated the expression of gonadotrophin subunits and GnRH receptor (GnRH-R) mRNA in the pituitary glands of Thoroughbred horses during the breeding (BS) and non-breeding (NBS) season. Seasonal effects on the prevalence of gonadotrophs in the pars distalis were also examined. GnRH-R and common α-, LHβ- and FSHβ-subunit mRNA contents were determined by Northern analysis and the prevalence of LH–gonadotrophs assessed by immunohistochemistry in pituitaries from sexually active females (mares) in the BS, and sexually inactive mares in the NBS. These variables were then measured in castrated male horses (geldings). In mares, pituitary content of FSHβ mRNA was significantly higher in the NBS (P < 0.01). Conversely, the content of common α-subunit mRNA was significantly higher during the BS (P < 0.05). In contrast, GnRH-R and LHβ mRNA abundance were unaffected by season. Interestingly, whereas no seasonal effects were apparent on the number of LH–gonadotrophs/field, the proportion of LH cells (in relation to all other cells) was higher in BS than NBS animals (P < 0.05); this resulted from an increased number of non-gonadotroph cells during the NBS (P < 0.05). In geldings, no significant seasonal effects were detected for any of the variables investigated (P > 0.05). These results reveal robust seasonal effects on common α-subunit and FSHβ gene expression in the pituitary of the mare, in the absence of detectable changes in the content of LHβ or GnRH-R mRNA.     

Clinical applications of GnRH and its analogues.

The chemical characterization of the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) has stimulated the development of analogues of GnRH with important clinical applications. Chronic administration of the GnRHanalogues nafarelin acetate and leuprolide acetate results in an initial stimulation of pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion, which is followed by a profound decrease in the secretion of LH and FSH. The decrease in the secretion of LH and FSH produces a hypogonadal state that is associated with an improvement in many sex-steroid-dependent disease processses. The GnRH analogues are clearly effective in the treatment of prostate cancer, endometriosis, uterine myomas, polycystic ovarian disease, and the premenstrual syndrome.     

GnRH analogues: applications in assisted reproductive techniques

The ability to prevent an endogenous LH surge revolutionised the efficacy of assisted reproductive techniques (ART) such that GnRH agonists were rapidly adopted in the 1980s. Prior to this, premature luteinisation occurred in up to 25% of superovulated cycles leading to cycle cancellation and severely compromised outcomes. Analogues have been applied in a variety of drug protocols (long, short flare) but there has been little research to moderate the degree of pituitary suppression. There has also been ongoing and unresolved debate about the role of LH in supporting follicular development. By 2001, the first GnRH antagonists were registered for use in ART. Their ability to cause immediate suppression of gonadotrophin (particularly LH) secretion means that they can be given after exogenous stimulation has begun and thereby dramatically shorten the total duration of a treatment cycle. After initial enthusiasm and then scepticism that pregnancy rates may not be as high as the established agonist regimens, these preparations are now being increasingly adopted with at least comparable outcomes in large trials. They are certainly favoured by patients for their reduced side-effect profile and particularly for the shortening of the total cycle length. This shift in practice is occurring alongside gathering momentum in favour of milder stimulation protocols and a new perception of what constitutes successful treatment. The focus is moving away from surrogate outcomes such as oocyte numbers and conception rates towards long-term outcomes for women and their offspring, namely the achievement of a live singleton birth per treatment started.     

Gonadotrophin glycosylation and function

This review emphasizes the heterogeneous structure of the gonadotrophin hormones and the influence of different oligosaccharide structures on the bioactivity of these hormones. A summary has been made of the changes in biopotency of the gonadotrophins throughout the life-cycle of the human and in different endocrine states in the rat. In general it appears that the charge of the gonadotrophin conferred by the acid radicals attached to the terminal groups on the oligosaccharide structures strongly influences biopotency. Basic structures have a greater potency in in-vitro assays, but a short half-life in the circulation, while acidic isoforms are less potent, but have a longer circulatory time and are thus more active in in-vivo estimations. More basic forms are secreted over the adult reproductive years compared with the prepubertal period and old age. The glycosyl structure of the carbohydrate groups also alters in different endocrine states and is probably also important for the bioactivity and potency of the hormone. Gonadotrophin-releasing hormone (GnRH) and gonadal steroids can influence the type of isoform synthesized and released, and therefore affect the function of gonadotrophins. GnRH enhances glycosylation, sulphation and biopotency. Oestradiol potentiates the glycosylation induced by GnRH and reduces sialylation, while testosterone increases sialylation.     

Dexamethasone suppresses gonadotropin-releasing hormone (GnRH) secretion and has direct pituitary effects in male rats: differential regulation of GnRH receptor and gonadotropin responses to GnRH

Endogenous or exogenous glucocorticoid excess leads to the development of hypogonadotropic hypogonadism, but the site(s) and mechanisms of glucocorticoid action are uncertain. We studied the effects of various doses of dexamethasone (Dex) on the hypothalamic-pituitary-gonadal axis in intact and castrate testosterone-replaced (cast + T) male rats and attempted to determine possible sites of Dex effects. A dose-dependent suppression of basal gonadotropin secretion was induced by 5 days of Dex treatment (20, 100, 500, or 2,500 micrograms/kg.day), and the highest dose completely abolished the postcastration rise in pituitary GnRH receptor number (GnRH-R) and serum gonadotropin levels. Administration of exogenous GnRH (0.02-200 micrograms/day over 2 days) resulted in a dose-dependent induction in GnRH-R in both intact and cast + T rats, but the effect was significantly (P less than 0.01) augmented in Dex-treated animals. In contrast, acute LH and FSH responses to GnRH (10, 25, 50, 100, or 250 ng, iv) were significantly blunted in Dex-treated rats. The data suggest that 1) Dex suppresses hypothalamic GnRH secretion, thereby preventing the postcastration rises in GnRH-R and gonadotropins; 2) at the pituitary level, Dex dissociates GnRH-R and gonadotropin responses to GnRH, augmenting GnRH-R induction by GnRH and suppressing gonadotropin responses to GnRH at a postreceptor site; and 3) the model of Dex-treated rats may be useful to study differential GnRH regulation of GnRH-R and gonadotropin secretion.     

A role for hypothalamic astrocytes in dehydroepiandrosterone and estradiol regulation of gonadotropin-releasing hormone (GnRH) release by GnRH neurons

Molecules of astrocyte origin influence gonadotropin-releasing hormone (GnRH) release and GnRH neuronal growth and differentiation. Furthermore, type 1 astrocytes express steroid receptors, presenting the possibility that steroid actions on GnRH neurons might occur via astrocytes. Utilizing GT1-7 cells, a GnRH-secreting cell line, the present study demonstrates that astrocytes mediate dehydroepiandrosterone (DHEA) or estradiol (E2) stimulated GnRH secretion. Conditioned media (CM) from astrocytes cultured for 48 h alone, with DHEA (DHEA-CM), or with E2 (E2-CM) were collected, treated with charcoal to remove steroids, and added to GT1-7 cells in culture for 12 h to test the effect on GnRH secretion. DHEA-CM and E2-CM stimulated GnRH secretion by GT1-7 cells by 4- and 3-fold, respectively. The effect of DHEA-CM on GnRH secretion by GT1-7 cells appears to be related to both DHEA and its metabolite, E2, since blocking the metabolism of DHEA into estrogen in the DHEA-treated astrocytes partially reversed the stimulatory effect of DHEA-CM. Addition of transforming growth factor (TGF)-beta1-neutralizing antibody to the astrocyte cultures reversed the stimulatory effects of both DHEA-CM and E2-CM on GnRH secretion by GT1-7 cells, suggesting that TGF-beta1 derived from astrocytes may be the principle mediator of E2 and DHEA effects. These data provide evidence for a novel mechanism by which circulating steroids and/or neurosteroids may modulate GnRH secretion.     

Insulin regulation of GnRH gene expression through MAP kinase signaling pathways

In mammals, reproduction is acutely regulated by metabolic status. Insulin is an important nutritional signal from the periphery that may regulate the reproductive axis. To determine whether insulin acts directly on the GnRH neuron, we performed studies in mouse-derived GnRH-expressing cell lines. Both insulin receptor protein and mRNA were detected in these cells. A saturation radioligand binding assay revealed high affinity, low capacity binding sites for insulin in GnRH neurons. Insulin also stimulated GnRH promoter activity in GnRH neurons. This effect was blocked by pretreatment with the MEK inhibitor, PD98059, indicating a role for MAP kinase signaling. In transient transfection studies, insulin treatment stimulated expression of a 1250 bp mouse GnRH gene promoter fragment four-fold when compared to promoter activity in untreated cells. In contrast, insulin did not stimulate activity of a 587 bp fragment of the mGnRH gene promoter, indicating that the promoter elements mediating insulin stimulation of the GnRH promoter are located between -1250 and -587 bp. Our studies suggest that insulin may regulate reproductive function by direct effects on the GnRH neurons and specifically by stimulating GnRH gene expression.     

Evidence for differential regulation of GnRH signaling via heterodimerization among GnRH receptor paralogs in the protochordate, Ciona intestinalis

The endocrine and neuroendocrine systems for reproductive functions have diversified as a result of the generation of species-specific paralogs of peptide hormones and their receptors including GnRH and their receptors (GnRHR), which belong to the class A G protein-coupled receptor family. A protochordate, Ciona intestinalis, has been found to possess seven GnRH (tGnRH-3 to -8 and Ci-GnRH-X) and four GnRHR (Ci-GnRHR1 to -4). Moreover, Ci-GnRHR4 (R4) does not bind to any Ciona GnRH and activate any signaling pathways. Here we show novel functional diversification of GnRH signaling pathways via G protein-coupled receptor heterodimerization among Ciona GnRHR. R4 was shown to heterodimerize with R2 specifically in test cells of vitellogenic oocytes by coimmunoprecipitation. The R2-R4 heterodimerization in human embryonic kidney 293 cells cotransfected with R2 and R4 was also observed by coimmunoprecipitation and fluorescent energy transfer analyses. Of particular interest is that the R2-R4 heterodimer decreases the cAMP production in a nonligand-selective manner via shift of activation of Gs protein to Gi protein by R2, compared with R2 monomer/homodimer. Considering that the R1-R4 heterodimer elicits 10-fold more potent Ca2? mobilization than R1 monomer/homodimer in a ligand-selective manner but does not affect cAMP production, these results indicate that R4 regulates differential GnRH signaling cascades via heterodimerization with R1 and R2 as an endogenous allosteric modulator. Collectively, the present study suggests that the heterodimerization among GnRHR paralogs, including the species-specific orphan receptor subtype, is involved in rigorous and diversified GnRHergic signaling of the protochordate, which lacks a hypothalamus-pituitary gonad axis.     

Opioid and neurotransmitter regulation of pituitary gonadotropin-releasing hormone (GnRH) receptors in the ovariectomized estradiol-treated rat: role of altered GnRH secretion

An acute transient fall in the number of pituitary GnRH receptors (GnRH-R) is observed before the preovulatory gonadotropin surge in cycling rats and before the afternoon daily gonadotropin surge in ovariectomized estradiol-treated rats. In the latter model, this fall can be reproduced by administration of the opioid antagonist naloxone, whereas the opioid agonist morphine acutely increases GnRH-R. In this study we investigated the mechanisms of this opioid effect and examined the effects of other neurotransmitter substances on modulation of pituitary GnRH-R. Administration of the dopaminergic agonists bromocriptine and L-dopa or the alpha-adrenergic receptor blocker phenoxybenzamine elevated GnRH-R acutely from average basal values of 240 +/- 22 and 254 +/- 21 fmol/mg protein to maximal values of 374 +/- 49, 441 +/- 67 and 461 +/- 75 fmol/mg, respectively, whereas the alpha-adrenergic agonist clonidine transiently decreased GnRH-R to 186 +/- 19 fmol/mg. Placement of radiofrequency lesions in the mediobasal hypothalamus or pretreatment with anti-GnRH serum completely abolished the ability of both morphine and naloxone to modulate the number of GnRH-R. These data indicate that the opioid-induced modulation of pituitary GnRH-R requires an intact hypothalamus and that both dopaminergic and alpha-adrenergic neurotransmitter systems may be involved. The final step of this action probably involves acute modulation of GnRH secretion (altered frequency and/or amplitude), which results in acute transient changes in the number of pituitary GnRH-R.     

GnRH release from the mediobasal hypothalamus. In vitro regulation by oxytocin

The effects of oxytocin (OT) on gonadotropin-releasing hormone (GnRH) release from the adult male rat mediobasal hypothalamus and median eminence were studied in an in vitro incubation system. OT induced a dose-related (10(-13) to 10(-9) M) inhibition of both basal and KCl-stimulated GnRH release from the mediobasal hypothalamus. OT, but not arginine vasopressin, also inhibited GnRH release from the isolated median eminence, and this inhibition was blocked by treatment with an OT receptor antagonist. Moreover, antagonist alone stimulated GnRH release from the isolated median eminence. These results demonstrate that OT can inhibit in vitro release of GnRH by an OT receptor mediated mechanism at the level of neurosecretory terminals in the median eminence and suggest that the hypothalamic OT neuronal system may play an inhibitory role in the modulation of GnRH secretion.     

Hormonal regulation of human trophoblast differentiation: a possible role for 17beta-estradiol and GnRH

We have examined the role of 17beta-estradiol and gonadotropin releasing hormone (GnRH) in the regulation of functional differentiation in human trophoblasts. In contrast to its recognized functions as a proliferation-promoting hormone in a variety of cell types, we found that 17beta-estradiol induced terminal differentiation in human trophoblastic cells, and that this event was estrogen-receptor-mediated. This process involved a loss in expression of Cyclins A2 and E, and a coincident increase in p27(Kip1). The anti-proliferative effects of 17beta-estradiol were annulled by specific transforming growth factor-beta 1 (TGFbeta1)-neutralizing antibody, suggesting that 17beta-estradiol may mediate its growth-inhibitory actions, through TGFbeta1 activity. Following exposure to Buserelin, cultured human trophoblastic cells stopped proliferating and formed functionally mature syncytiotrophoblasts. This differentiation event, that involved a drastic loss in expression of proliferating-cell-nuclear-antigen, could be blocked by Cetrorelix, suggesting the involvement of functional GnRH receptors. Preliminary studies on the characterization of the human placental GnRH receptor, indicate the presence of multiple receptor isoforms across human gestation.     

Sequence analysis, endocrine regulation, and signal transduction of GnRH receptors in teleost fish

Three gonadotropin-releasing hormones (GnRHs) and three cognate receptors have been identified in vertebrates, with distinct distributions and functions. According to their sequences, the receptors can be grouped into distinct classes: types I, II, and III. One branch contains all type-I GnRH receptors (GnRH-R-I) from mammals and fish; another branch clusters mainly amphibian and human type-II GnRH receptors; and a third branch includes evolved fish, mainly perciform species, type-III GnRH receptors. Taken tilapia GnRH receptors as a model, the present study summarizes the information regarding the amino-acid residues assumed to be involved in the receptors' structure, binding, activation, and intracellular signal transduction, including arrangement of the disulfide bonds, glycosylation sites, coupling to G proteins, and protein kinase A or protein kinase C phosphorylation sites.     

GnRH II and type II GnRH receptors.

Hypothalamic gonadotrophin-releasing hormone (GnRH I), which is of a variable structure in vertebrates, is the central regulator of the reproductive system through its stimulation of gonadotrophin release from the pituitary. A second form of GnRH (GnRH II) is ubiquitous and conserved in structure from fish to humans, suggesting that it has important functions and a discriminating receptor that selects against structural change. GnRH II is distributed in discrete regions of the central and peripheral nervous systems and in nonneural tissues. The cognate receptor for GnRH II has recently been cloned from amphibians and mammals. It is highly selective for GnRH II, has a similar distribution to GnRH II in the nervous system and, notably, in areas associated with sexual behaviour. It is also found in reproductive tissues. An established function of GnRH II is in the inhibition of M currents (K(+) channels) through the GnRH II receptor in the amphibian sympathetic ganglion, and it might act through this mechanism as a neuromodulator in the central nervous system. The conservation of structure over 500 million years and the wide tissue distribution of GnRH II suggest that it has a variety of reproductive and nonreproductive functions and will be a productive area of research.