Regulation Of Gonadotropin-releasing Hormone Research Articles

Fasting-induced suppression of LH secretion does not require activation of ATP-sensitive potassium channels

Reproductive hormone secretions are inhibited by fasting and restored by feeding. Metabolic signals mediating these effects include fluctuations in serum glucose, insulin, and leptin. Because ATP-sensitive potassium (K(ATP)) channels mediate glucose sensing and many actions of insulin and leptin in neurons, we assessed their role in suppressing LH secretion during food restriction. Vehicle or a K(ATP) channel blocker, tolbutamide, was infused into the lateral cerebroventricle in ovariectomized mice that were either fed or fasted for 48 h. Tolbutamide infusion resulted in a twofold increase in LH concentrations in both fed and fasted mice compared with both fed and fasted vehicle-treated mice. However, tolbutamide did not reverse the suppression of LH in the majority of fasted animals. In sulfonylurea (SUR)1-null mutant (SUR1(-/-)) mice, which are deficient in K(ATP) channels, and their wild-type (WT) littermates, a 48-h fast was found to reduce serum LH concentrations in both WT and SUR(-/-) mice. The present study demonstrates that 1) blockade of K(ATP) channels elevates LH secretion regardless of energy balance and 2) acute fasting suppresses LH secretion in both SUR1(-/-) and WT mice. These findings support the hypothesis that K(ATP) channels are linked to the regulation of gonadotropin-releasing hormone (GnRH) release but are not obligatory for mediating the effects of fasting on GnRH/LH secretion. Thus it is unlikely that the modulation of K(ATP) channels either as part of the classical glucose-sensing mechanism or as a component of insulin or leptin signaling plays a major role in the suppression of GnRH and LH secretion during food restriction.

Cloning and functional analysis of the proximal promoter region of the three GnRH genes from the silver sea bream ( Sparus sarba)

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a major role in releasing pituitary gonadotropin and controlling vertebrate reproduction. In this study, three GnRH cDNAs, GnRH-I (sbGnRH; 348 bp), GnRH-II (cGnRH-II; 557 bp), and GnRH-III (sGnRH; 483 bp), were cloned from the brain of the silver sea bream ( Sparus sarba). In order to understand how the expression of the GnRH isoforms was regulated in the brain, the promoter of each gene was cloned and analyzed. We found regulatory motifs in the promoters that were conserved in the GnRH promoters of tilapia and zebrafish, suggesting that these motifs play a critical role in GnRH regulation. We performed functional analyses and examined tissue-specific expression for each GnRH promoter using EGFP reporter fusions in zebrafish. The GnRH-I promoter was active in the forebrain area, including the olfactory bulb-terminal nerve area and peripheral preoptic areas; the GnRH-II promoter was active in the midbrain; and the GnRH-III promoter was active in the olfactory bulb. These results show that the GnRH promoters of the silver sea bream GnRH genes exhibit tissue-specific activity.

Immortalized hypothalamic gonadotropin-releasing hormone neurons.

The neuroendocrine hypothalamus has been intensively studied using whole animals and tissue slices. However, it has been difficult to approach questions at the molecular and cellular level. By targeting expression of the oncogene product, simian virus 40 T antigen, in transgenic mice using the regulatory domain of the rat gonadotropin-releasing hormone (GnRH) gene, we have produced specific hypothalamic tumours. These tumours have been cultured to produce clonal cell lines (GT-1 cells) that express T antigen, GnRH and many other neuronal markers, but do not express other hypothalamic hormones. These immortal cell lines have a distinctive neuronal phenotype, process the GnRH peptide accurately and secrete GnRH in a pulsatile pattern. Thus, by targeting oncogenesis to a defined population of neurons using the regulatory region of a gene that is expressed late in differentiation of that cell lineage, we have succeeded in immortalizing hypothalamic GnRH neurons. The GT-1 cell lines are an excellent model for future molecular, cell biological, physiological and biochemical investigations into the mechanisms involved in regulation of GnRH and the characteristics of an isolated central nervous system neuron. Their derivation demonstrates the utility of targeting tumorigenesis to specific differentiated neurons of the central nervous system in transgenic mice.

Gonadotropin-inhibitory hormone in seasonally-breeding songbirds: neuroanatomy and functional biology

The discovery of gonadotropin-inhibitory hormone (GnIH) in Japanese Quail Coturnix japonica has given us a new perspective on the neuroendocrine control of reproductive physiology and behavior. Using highly photoperiodic passerine songbirds as our model system, we have been able to investigate GnIH neuroanatomy and functional biology. The distribution of GnIH peptide is similar among the galliform and passerine species studied so far. We have identified GnIH cDNA and localized its transcript in Gambel’s White-crowned Sparrow Zonotrichia leucophrys gambelii. There is a high degree of homology between the galliform and passerine GnIH cDNA sequences and peptides. Based on transcript localization, GnIH precursor polypeptide is only produced in the paraventricular nucleus, yet GnIH-immunoreactive fibers project to multiple brain areas, suggesting multiple physiological and behavioral functions. GnIH immunoreactive neurons appear to contact gonadotropin-releasing hormone (GnRH)-I and -II neurons, raising the possibility of direct regulation of GnRH by GnIH. In a photoperiod experiment, GnIH neurons increased in area during the termination of reproduction in Song Sparrows, Melospiza melodia, implying temporal photoperiodic regulation of this neuropeptide. Using peripheral administration of GnIH in vivo, we demonstrated rapid inhibition of LH release in laboratory and field experiments. Furthermore, we sought to determine the effects of central infusions of GnIH. Centrally-administered GnIH rapidly reduced circulating LH and significantly reduced the number of copulation solicitations performed in response to song playback, with no reduction of locomotor activity. Central infusion of rhodaminated GnIH elucidated putative GnIH binding sites in the median eminence close to GnRH-I fiber terminals, and in the midbrain on or close to GnRH-II neurons. These data demonstrate direct effects of GnIH upon reproductive physiology and behavior, possibly at multiple physiological levels and over different time-frames. Thus, the discovery of GnIH has unearthed a complex and interesting system for the regulation of avian reproductive biology.

Regulation of Gonadotropin-Releasing Hormone in Nonhypothalamic Tissues

It is well established that hypothalamic gonadotropin-releasing hormone (GnRH) controls reproductive function by stimulating the production of gonadotropins from the pituitary. GNRH gene and its receptor (GnRHR) have also been detected outside the hypothalamus, and a growing body of literature supports an extrapituitary role for GnRH action. The exact function of GnRH in these tissues is not known, but GnRH expression has been described in reproductive tissues, including the ovary, placenta, breast, testes, and prostate. This article provides an overview of the regulation of GnRH gene expression in nonhypothalamic reproductive tissues. After GnRH gene structure is reviewed, the physiologic role of GnRH and regulation of its expression in several reproductive tissues are examined. When possible, transcriptional regulation is discussed, but due to low levels of expression, transcriptional regulation of GnRH in extrahypothalamic tissues has been extremely difficult to study. Consequently, the factors that mediate GnRH gene expression in these tissues are only beginning to be identified.

E-box regulation of gonadotropin-releasing hormone (GnRH) receptor expression in immortalized gonadotrope cells

The pituitary gland's ability to respond to the hypothalamic hormone GnRH (gonadotropin-releasing hormone) depends directly on the gonadotrope-specific expression of the GnRH receptor (GnRHR), a G-protein coupled transmembrane protein coded by the GnRHR gene. In the present study, we have investigated the potential regulatory role of seven noncanonical E-box enhancer sequences within the 856 bp proximal 5′-flanking region of the mGnRHR gene in regulating transcription. These sequences are known to mediate the action of clock gene proteins on the expression of a diverse array of genes both central and peripheral. In the present studies the expression of all of the cognate clock genes was identified in the αT3-1 gonadotrope cell line. Additionally, luteinizing hormone-immunoreactive cells in the adult rodent pituitary gland were also shown to co-express the PERIOD-1 protein. By means of chromatin immunoprecipitation of αT3-1 nuclear extracts we were able to capture promoter fragments of the GnRHR and Period-1 genes, indicating that E-boxes in these promoters bind the CLOCK protein. RNA interference experiments with αT3-1 cells in which Bmal1 expression was attenuated also confirmed the involvement of E-boxes in transcriptional regulation of the mGnRHR gene. Subsequent luciferase reporter assay experiments with GnRHR constructs possessing intact or mutated E-boxes confirmed the use of these sequences for the regulation of mGnRH-R/ luc expression. Transient overexpression of the dominant negative E-box-binding factor CLOCK-Δ19, or the inhibitory clock protein mPER1, markedly reduced CLOCK/BMAL1-driven mGnRH-R/ luc expression in a dose-dependent fashion. Our data implicate the clock genes as important factors controlling GnRHR expression in murine gonadotrope cells.

Involvement of NF‐κB subunit p65 and retinoic acid receptors, RARα and RXRα, in transcriptional regulation of the human GnRH II gene

Gonadotropin‐releasing hormone (GnRH) I and II are hypothalamic decapeptides with pivotal roles in the development of reproductive competence and regulation of reproductive events. In this study, transcriptional regulation of the human GnRH II gene was investigated. By scanning mutation analysis coupled with transient promoter assays, the motif at −641/−636 (CATGCC, designated GII‐Sil) was identified as a repressor element. Mutation of this motif led to full restoration of promoter activity in TE671 medulloblastoma and JEG‐3 placenta choriocarcinoma cells. Supershift and chromatin immunoprecipitation assays showed in vitro and in vivo binding of NF‐κB subunit p65 and the retinoic acid receptors, RARα and RXRα, to the promoter sequences. Over‐expression of these protein factors indicated that p65 is a potent repressor, and the RARα/RXRα heterodimer is involved in the differential regulation of the GnRH II gene in neuronal and placental cells. This was confirmed by quantitative real‐time PCR. Treatment of cells with the RARα/RXRα ligands, all‐trans retinoic acid and 9‐cis‐retinoic acid, reduced and increased GnRH II gene expression in TE671 and JEG‐3 cells, respectively. Taken together, these data demonstrate the differential roles of NF‐κB p65 and RARα/RXRα, interacting with the same sequence in the promoter of the human GnRH II gene to influence gene expression in a cell‐specific manner.

Sex Steroids Are Involved in the Regulation of Gonadotropin-Releasing Hormone and Dopamine D2 Receptors in Female Tilapia Pituitary1

Although molecular mechanisms underlying steroid effects on GnRH and dopamine receptors are well documented in mammals, little is known in fish. Herein, we describe the expression of pituitary GnRH and dopamine receptors relative to gonadotropin expression and release. We exposed female tilapia to graded doses of estradiol or 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) in vitro, and of estradiol in vivo, and determined mRNA levels of gnrhr1, gnrhr3, drd2, lhb, and fshb by real-time PCR. We also determined gonadotropin levels using specific ELISAs. Exposure to low doses of estradiol caused increased gnrhr3 mRNA levels in vivo and in vitro, probably related to positive feedback on FSH release. Increasing concentrations of estradiol resulted in increased drd2 mRNA levels in vivo and in vitro, inhibition of LH and FSH release, and inhibition of lhb mRNA levels in vivo, possibly related to negative feedback. At high doses of estradiol, FSH release increased in preparation for a new generation of follicles. Exposure to nanomolar doses of DHP resulted in increased drd2 mRNA levels, probably related to negative feedback on LH release. A decrease in drd2 levels at the micromolar range of DHP (concomitant with increased gnrhr3 and fshb mRNA levels) may be related to the recruitment of a new generation of oocytes. Exposure to DHP also resulted in increased lhb mRNA levels toward final oocyte maturation. Salmon GnRH analog (sGnRHa) increased mRNA levels of gnrh1and gnrh3; when combined with DHP, sGnRHa synergistically increased expression of gnrh3 only. These results emphasize the role of sex steroids on positive and negative feedbacks controlling the reproductive cycle.

Kisspepeptin-GPR54 signaling in the neuroendocrine reproductive axis

Kisspeptins, which are products of the Kiss1 gene, and their receptor, GPR54, have emerged as key players in the regulation of gonadotropin-releasing hormone (GnRH) secretion. Mutations or targeted deletions of GPR54 produce isolated hypogonadotropic hypogonadism in humans and mice, indicating that signaling through this receptor is a prerequisite for sexual maturation. Centrally administered kisspeptins stimulate GnRH and gonadotropin secretion in prepubertal and adult animals. Kisspeptin-expressing neurons are direct targets for the negative and positive feedback actions of sex steroids, which differentially regulate the expression of KiSS-1 mRNA in various regions of the forebrain. This review highlights what is currently known about kisspeptin-GPR54 signaling in the regulation of the neuroendocrine reproductive axis.

Role of metalloproteinase‐dependent EGF receptor activation in α1‐adrenoceptor‐stimulated MAP kinase phosphorylation in GT1‐7 neurons

Adrenoceptors (ARs) are involved in the regulation of gonadotropin-releasing hormone (GnRH) release from native and immortalized hypothalamic (GT1-7) neurons. However, the AR-mediated signaling mechanisms and their functional significance in these cells are not known. Stimulation of GT1-7 cells with the alpha1-AR agonist, phenylephrine (Phe), causes phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinases that is mediated by protein kinase C (PKC)-dependent transactivation of the epidermal growth factor receptor (EGF-R). Phe stimulation causes shedding of the soluble ligand, heparin-binding EGF (HB-EGF), as a consequence of matrix metalloproteinase (MMP) activation. Phe-induced phosphorylation of the EGF-R, and subsequently of Shc and ERK1/2, was attenuated by inhibition of MMP or HB-EGF with the selective inhibitor, CRM197, or by a neutralizing antibody. In contrast, phosphorylation of the EGF-R, Shc and ERK1/2 by EGF and HB-EGF was independent of PKC and MMP activity. Moreover, inhibition of Src attenuated ERK1/2 responses by Phe, but not by HB-EGF and EGF, indicating that Src acts upstream of the EGF-R. Consistent with a potential role of reactive oxygen species (ROS), Phe-induced phosphorylation of EGF-R was attenuated by the antioxidant, N-acetylcysteine. These data suggest that activation of the alpha1-AR causes phosphorylation of ERK1/2 through activation of PKC, ROS and Src, and shedding of HB-EGF, which binds to and activates the EGF-R.

Molecular cloning and gonadotropin-releasing hormone regulation of receptor activated for C protein kinase (RACK) in tilapia Oreochromis mossambicus

A receptor for activated C-kinase (RACK) cDNA in the Mozambique tilapia (Oreochromis mossambicus) was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), and it was compared with other vertebrates. Nucleotide sequence analysis revealed that the full length of cDNA (1109 bp) cloned encompasses 954 bp open reading frame encoding 317 amino acid residues. The deduced amino acid sequence shares 95–99% identity with the teleosts and 77–95% compared with other vertebrates. Like most RACK sequences in other vertebrates, seven repeats of the WD-40 motif are well-conserved in the sequence of the tilapia. Further, the effectiveness of gonadotropin-releasing hormone (GnRH) regulation of RACK LH, and FSH gene expression was further investigated by a single injection of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). The results showed that injection of tilapia brain with either sGnRH or cGnRH-II significantly increased mRNA levels of RACK, which then stimulate LH and FSH. This result suggests that RACK and protein kinase C (PKC) are involved in GnRH-mediated gonadotropin hormone (GtH) release. In addition, cGnRH-II enhanced the FSH and LH mRNA as well as sGnRH expression levels in tilapia; these results further demonstrate that both sGnRH and cGnRH regulate GtH through PKC.

Involvement of phospholipase C and intracellular calcium signaling in the gonadotropin-releasing hormone regulation of prolactin release from lactotrophs of tilapia ( Oreochromis mossambicus)

Gonadotropin-releasing hormone (GnRH) is a potent stimulator of prolactin (PRL) secretion in various vertebrates including the tilapia, Oreochromis mossambicus. The mechanism by which GnRH regulates lactotroph cell function is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated, we examined whether GnRH might stimulate PRL release through an increase in phospholipase C (PLC), inositol triphosphate (IP 3), and intracellular calcium ( Ca i 2 + ) signaling. Using Ca i 2 + imaging and the calcium-sensitive dye fura-2, we found that chicken GnRH-II (cGnRH-II) induced a rapid dose-dependent increase in Ca i 2 + in dispersed tilapia lactotrophs. The Ca i 2 + signal was abolished by U-73122, an inhibitor of PLC-dependent phosphoinositide hydrolysis. Correspondingly, cGnRH-II-induced tPRL 188 secretion was inhibited by U-73122, suggesting that activation of PLC mediates cGnRH-II’s stimulatory effect on PRL secretion. Pretreatment with 8-( N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of Ca 2+ release from intracellular stores, impeded the effect of cGnRH-II on Ca i 2 + . To further address the possible involvement of intracellular Ca 2+ stores, IP 3 concentrations in the tilapia rostral pars distalis (RPD containing 95–99% PRL cells) was determined by a radioreceptor assay. We found that GnRH-II induces a rapid (<5 min) and sustained increase in IP 3 concentration in the RPD. Secretion of tPRL 188 in response to cGnRH-II was suppressed by Ca 2+ antagonists (TMB-8 and nifedipine). These data, along with our previous findings that show PRL release increases with a rise in Ca i 2 + , suggest that GnRH may elicit its PRL releasing effect by increasing Ca i 2 + . Furthermore, the rise in Ca i 2 + may be derived from PLC/IP 3-induced mobilization of Ca 2+ from intracellular stores along with influx through L-type voltage-gated Ca 2+ channels.

Role of neuropeptide Y in the regulation of gonadotropin releasing hormone system in the forebrain of Clarias batrachus (Linn.): Immunocytochemistry and high performance liquid chromatography-electrospray ionization-mass spectrometric analysis

Although the importance of neuropeptide Y (NPY) in the regulation of gonadotropin releasing hormone (GnRH) and reproduction has been highlighted in recent years, the neuroanatomical substrate within which these substances might interact has not been fully elucidated. Present work was undertaken with a view to define the anatomical-physiological correlates underlying the role exercised by NPY in the regulation of GnRH in the forebrain of the teleost Clarias batrachus. Application of double immunocytochemistry revealed close associations as well as colocalizations of the two peptides in the olfactory receptor neurons (ORNs), olfactory nerve fibers and their terminals in the glomeruli, ganglion cells of nervus terminalis, medial olfactory tract, fibers in the area ventralis telencephali/pars supracommissuralis and cells as well as fibers in the pituitary. NPY containing axons were found to terminate in the vicinity of GnRH cells in the pituitary with light as well as electron microscopy. Double immunoelectron microscopy demonstrated gold particles for NPY and GnRH colocalized on the membrane and in dense core of the secretory granules in the cells distributed in all components of the pituitary gland. To assess the physiological implication of these observations, NPY was injected via the intracranial route and the response of GnRH immunoreactive system was evaluated by relative quantitative morphometry as well as high performance liquid chromatography (HPLC) analysis. Two hours following NPY (20 ng/g body weight) administration, a dramatic increase was observed in the GnRH immunoreactivity in the ORNs, in the fibers of the olfactory bulb (163%) and medial olfactory tract (351%). High performance liquid chromatography-electrospray ionization-mass spectrometric analysis confirmed the immunocytochemical data. Significant rise in the salmon GnRH (sGnRH)-like peptide content was observed in the olfactory organ (194.23%), olfactory bulb (146.64%), telencephalon+preoptic area (214.10%) and the pituitary (136.72%) of the NPY-treated fish. However, GnRH in the hypothalamus was below detection limit in the control as well as NPY-treated fish. Present results suggest the involvement of NPY in the up-regulation of sGnRH containing system at different level of neuraxis extending from the olfactory epithelium to the pituitary in the forebrain of C. batrachus.

Neuropeptide Y Gene Expression in Male Sheep: Influence of Photoperiod and Testosterone

The frequency of pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) is high in the breeding season and low in the nonbreeding season. These alterations in the patterns of GnRH and LH release are due to an interaction of daylength and gonadal steroid negative feedback. A vast amount of data indicates that steroid-responsive neural systems may play a role in regulating seasonal changes in GnRH release. One candidate system is neuropeptide Y (NPY). To determine the independent and interactive influences of photoperiod and steroid exposure on NPY mRNA levels, we used hypothalamic tissue from four groups (n = 4 per group) of castrated male sheep that were simultaneously housed in photochambers and exposed to: (1) a 16L:8D photoperiod (LD); (2) LD and implanted with testosterone (LD + T); (3) a 10L:14D photoperiod (SD), and (4) SD + T. Circulating levels of T averaged 2.8 ± 0.2 ng/ml in implanted animals, but were undetectable in nonimplanted males. Mean LH levels were significantly reduced (p < 0.01) in the LD + T group as compared with the other groups which did not differ from each other. The silver grain area per NPY neuron in the arcuate nucleus, as assessed by in situ hybridization, was inversely related to mean LH values, with the grain area per cell being significantly greater (p < 0.05) for LD + T males than for all other groups which did not differ from each other. NPY cell numbers were not significantly different (p > 0.10) among the treatment groups. These results show that NPY mRNA expression is increased in male sheep during a LD photoperiod in a T-dependent manner. Our data are consistent with the idea that NPY is involved in the seasonal regulation of GnRH and LH release in the male sheep.

A Novel AP-1 Site Is Critical for Maximal Induction of the Follicle-stimulating Hormone β Gene by Gonadotropin-releasing Hormone

Regulation of follicle-stimulating hormone (FSH) synthesis is a central point of convergence for signals controlling reproduction. The FSHbeta subunit is primarily regulated by gonadotropin-releasing hormone (GnRH), gonadal steroids, and activin. Here, we identify elements in the mouse FSHbeta promoter responsible for GnRH-mediated induction utilizing the LbetaT2 cell line that endogenously expresses FSH. The proximal 398 bp of the mouse FSHbeta promoter is sufficient for response to GnRH. This response localizes primarily to an AP-1 half-site (-72/-69) juxtaposed to a CCAAT box, which binds nuclear factor-Y. Both elements are required for AP-1 binding, creating a novel AP-1 site. Multimers of this site confer GnRH induction, and mutation or internal deletion of this site reduces GnRH induction by 35%. The same reduction was achieved using a dominant negative Fos protein. This is the only functional AP-1 site identified in the proximal 398 bp, since its mutation eliminates FSHbeta induction by c-Fos and c-Jun. GnRH regulation of the FSHbeta gene occurs through induction of multiple Fos and Jun isoforms, forming at least four different AP-1 molecules, all of which bind to this site. Mitogen-activated protein kinase activity is required for induction of FSHbeta and JunB protein. Finally, AP-1 interacts with nuclear factor-Y, which occupies its overlapping site in vivo.

Astrocytes and brain function: implications for reproduction.

Recent evidence suggests that astrocytes have important neuroregulatory functions in addition to their classic functions of support and segregation of neurons. These newly revealed functions include regulation of neuron communication, neurosecretion, and synaptic plasticity. Although these actions occur throughout the brain, this review will focus on astrocyte-neuron interactions in the hypothalamus, particularly with respect to their potential contribution to the regulation of gonadotropin-releasing hormone (GnRH) secretion and reproduction. Hypothalamic astrocytes have been documented to release a variety of neuroactive factors, including transforming growth factors-alpha and -beta, insulin-like growth factor-1, prostaglandin E2, and the neurosteroid, 3 alpha-hydroxy-5 alpha-pregnane-20-one. Each of these factors has been shown to stimulate GnRH release, and receptors for each factor have been documented on GnRH neurons. Astrocytes have also been implicated in the regulation of synaptic plasticity in key areas of the hypothalamus that control GnRH release, an effect achieved by extension and retraction of glial processes (i.e., glial ensheathment). Through this mechanism, the number of synapses on GnRH neurons and GnRH regulatory neurons can potentially be modulated, thereby influencing the activation state of GnRH neurons. The steroid hormone 17beta-estradiol, which triggers the GnRH and luteinizing hormone surge, has been shown to induce the astrocyte-regulated changes in hypothalamic synaptic plasticity, as well as enhance formation and release of the astrocyte neuroactive factors, thereby providing another potential mechanistic layer for astrocyte regulation of GnRH release. As a whole, these studies provide new insights into the diversity of astrocytes and their potential role in reproductive neuroendocrine function.

Differential regulation of gonadotropin-releasing hormone (GnRH)I and GnRHII messenger ribonucleic acid by gonadal steroids in human granulosa luteal cells.

In humans, reproduction was generally believed to be controlled by only one form of GnRH (called mammalian GnRH or GnRHI). However, recently, a second form of GnRH, analogous to chicken GnRHII, was discovered in several tissues, including the human ovary. The regulation and function of GnRHI in the hypothalamus has been well studied. However, the function and regulation of GnRHI, and particularly GnRHII in the ovary, is less well understood. Because gonadal sex steroids are one of the main regulators of reproduction, we investigated, in the present study, the regulation of GnRHI and GnRHII mRNA expression by 17beta-estradiol (E2) and RU486 (a progesterone antagonist) in human granulosa luteal cells (hGLCs). The levels of the mRNA transcripts encoding the two GnRH forms were examined using semiquantitative RT-PCR followed by Southern blot analysis. With time in culture, GnRHI and GnRHII mRNA levels significantly increased, by 120% and 210%, at d 8 and d 1, respectively. The levels remained elevated until the termination of these experiments at d 10. A 24-h treatment of hGLCs with E2 (10(-9) to 10(-7) M) resulted in a dose-dependent decrease and increase in mRNA expression of GnRHI and GnRHII, respectively. E2 (10(-9) M) significantly decreased GnRHI mRNA levels (by 55%) and increased GnRHII mRNA levels (by 294%). Time-course studies demonstrated that E2 (10(-9) M) significantly decreased GnRHI mRNA levels in a time-dependent manner, with maximal inhibition of 77% at 48 h. In contrast, GnRHII mRNA levels significantly increased in a time-dependent fashion, reaching a maximum level of 280% at 24 h. Cotreatment of hGLCs with E2 and tamoxifen (an E2 antagonist) reversed the inhibitory and stimulatory effects of E2 on the mRNA expression of GnRHI and GnRHII, respectively. Time- and dose-dependent treatment with RU486 did not affect GnRHI mRNA levels in hGLCs. In contrast, RU486 treatment significantly increased GnRHII mRNA levels in hGLCs in a time- and dose-dependent fashion, with a maximum increase being observed at 24 h (with 10(-5)M RU486). In summary, the present study demonstrated that the expression of GnRHI and GnRHII at the transcriptional level is differently regulated by E2 and P4 in hGLCs.

Social regulation of gonadotropin-releasing hormone.

Behavioral interactions among social animals can regulate both reproductive behavior and fertility. A prime example of socially regulated reproduction occurs in the cichlid fish Haplochromis burtoni, in which interactions between males dynamically regulate gonadal function throughout life. This plasticity is mediated by the brain, where neurons that contain the key reproductive regulatory peptide gonadotropin-releasing hormone (GnRH) change size reversibly depending on male social status. To understand how behavior controls the brain, we manipulated the social system of these fish, quantified their behavior and then assessed neural and physiological changes in the reproductive and stress axes. GnRH gene expression was assessed using molecular probes specific for the three GnRH forms in the brain of H. burtoni. We found that perception of social opportunity to increase status by a male leads to heightened aggressiveness, to increased expression of only one of the three GnRH forms and to increases in size of GnRH-containing neurons and of the gonads. The biological changes characteristic of social ascent happen faster than changes following social descent. Interestingly, behavioral changes show the reverse pattern: aggressive behaviors emerge more slowly in ascending animals than they disappear in descending animals. Although the gonads and GnRH neurons undergo similar changes in female H. burtoni, regulation occurs via endogenous rather than exogenous social signals. Our data show that recognition of social signals by males alters stress levels, which may contribute to the alteration in GnRH gene expression in particular neurons essential for the animal to perform in its new social status.

Regulation by Gonadal Steroids of the mRNA Encoding for a Type I Receptor for TGF-β in the Female Rat Hypothalamus

We have recently shown that the mRNA encoding for a type I receptor for transforming growth factor beta and activin – named B1 – is expressed in hypothalamic areas implicated in gonadotropin-releasing hormone regulation, particularly in estrogen-receptive regions. In the present study, we examined whether ovarian steroids may regulate expression of B1 mRNA in the hypothalamus. Comparing relative levels of B1 mRNA expression in ovariectomized (OVX), OVX + estradiol-treated, and OVX + estradiol + progesterone-treated female rats, we observed that estrogen significantly (p < 0.05) downregulated B1 mRNA levels in the anteroventral periventricular nucleus (by 12.5%), medial preoptic nucleus (by 27.5%), and arcuate nucleus (by 29.5%). In contrast, no effects of gonadal steroids were observed in the median preoptic nucleus. We next examined whether cells expressing B1 mRNA may be direct targets for the action of estrogen. Using an in situ hybridization coupled to immunohistochemical labeling, we found that many B1-mRNA-expressing cells also exhibited estrogen receptor alpha immunoreactvity in anteroventral periventricular nucleus, medial preoptic nucleus, and arcuate nucleus. Taken together, these results reveal that estrogen may directly modulate expression of B1 mRNA in the hypothalamus and support the idea that transforming growth factors beta play an important role in the hypothalamic control of gonadotropin-releasing hormone function.

Involvement of protein kinase C and arachidonic acid pathways in the gonadotropin-releasing hormone regulation of oocyte meiosis and follicular steroidogenesis in the goldfish ovary.

The involvement of protein kinase C (PKC) and arachidonic acid (AA) pathways were investigated in the GnRH regulation of oocyte meiosis and follicular testosterone production in the goldfish ovary. The results clearly demonstrate differences in the postreceptor mechanisms involving the stimulatory and inhibitory actions of GnRH peptides on basal and gonadotropin (GtH)-induced reinitiation of oocyte meiosis and steroidogenesis. In isolated goldfish follicles in vitro, the observed stimulatory effects of both salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) on germinal vesicle breakdown were completely blocked by addition of PKC inhibitors, suggesting the involvement of PKC, presumably through activation of phospholipase C/diacylglycerol pathways in the GnRH-induced reinitiation of oocyte meiosis. Administration of an AA metabolism inhibitor, however, only blocked the stimulatory effect of sGnRH without affecting cGnRH-II-induced meiosis. As observed previously, in the presence of GtH, sGnRH was found to inhibit GtH-induced resumption of meiosis and testosterone production, whereas cGnRH-II was without effect. The inhibitory effect of sGnRH on GtH-induced meiosis and steroidogenesis was completely reversed by addition an AA metabolism inhibitor, whereas PKC inhibitors had no effect. These findings provide functional evidence in support of the novel hypothesis that goldfish ovarian follicles contain GnRH-receptor subtypes with different ligand selectivity mediating stimulatory and inhibitory actions of sGnRH and cGnRH in the goldfish ovary.