The regulation of galanin gene expression in gonadotropin-releasing hormone neurons

In rats, galanin modulates luteinizing hormone (LH) secretion, and gonadotropin-releasing hormone (GnRH) neurons provide a possible source of this galanin. To understand galanin's physiological role in GnRH neurons, we used double-label in situ hybridization and computerized image analysis to examine the regulation of galanin message in GnRH neurons. We found that galanin gene expression in GnRH neurons is regulated by sex steroids, induced coincident with the LH surge, and persists well after the completion of the LH surge, and that the induction of galanin message in GnRH neurons coincident with the LH surge is sexually differentiated neonatally. We postulate that the rise in galanin gene expression in GnRH neurons at the time of the LH surge serves to replenish galanin released with GnRH that is needed for the production of the LH surge, or that galanin is involved in physiological events that occur subsequent to the LH surge.     

In vivo gonadotropin-releasing hormone secretion in female rats during peripubertal development and on proestrus.

Pubertal development in female rats is characterized by increased LH levels and the appearance of estrogen-dependent afternoon LH mini-surges. In these studies we performed the first analysis of GnRH patterns in peripubertal rats to determine whether there are similar changes in pulsatile GnRH release. Microdialysis samples were collected at 5-min intervals throughout a 5-h afternoon period from 22 rats sampled on a single day between 30-47 days of age. Adult female rats were sampled on proestrus for comparison. In 30- to 33-day-old rats, GnRH release was infrequent (2.7 pulses/5 h; n = 3), whereas intermediate pulse frequencies were observed in 34- to 37-day-old rats (6.4 pulses/5 h; n = 9) and 38- to 42-day-old (5.0 pulses/5 h; n = 5) rats. The highest GnRH pulse frequencies were observed in 43- to 47-day-old rats (9.4 pulses/5 h; n = 5). Mean GnRH pulse amplitude did not vary significantly with age. Animals sampled before vaginal opening (VO) exhibited significantly slower GnRH pulse frequencies than those sampled after vaginal opening (1.3 pulses/5 h pre-VO vs. 7.6 pulses/5 h post-VO; P = 0.01). An afternoon increase in GnRH secretion, defined operationally as a greater than 25% increase in mean GnRH levels in the last half of the sampling period and tentatively termed a mini-surge, was observed in 0%, 33%, 40%, and 60% of 30- to 33-, 34- to 37-, 38- to 42-, and 43- to 47-day-old rats, respectively. An overall increase in GnRH pulse frequency was observed in females displaying a mini-surge (9.0 pulses/5 h with mini-surge compared with 4.7 pulses/5 h with no mini-surge). The mini-surge itself, however, was associated with a late afternoon increase in GnRH pulse amplitude and not in pulse frequency. In adult proestrous rats, peak levels during the GnRH surge were an order of magnitude greater than those reached in pubertal animals. Our findings demonstrate that pubertal maturation in the female rat is associated with an acceleration of GnRH pulse generator activity and that later stages of pubertal maturation are characterized by the appearance of afternoon increases in GnRH release that may underlie previously reported mini-surges in LH.     

Age-related decreases in serum gonadotropin levels and gonadotropin-releasing hormone gene expression in the medial preoptic area of the male rat are dependent upon testicular feedback

In the male rat, age-associated reproductive decline is thought to be due in part to diminished GnRH secretion. We tested the hypothesis that the age-related decrease in GnRH secretion is due to decreased GnRH gene expression by comparing GnRH mRNA and peptide content in the anterior forebrain of intact young and old male rats. Since sex steroids modulate GnRH secretion, we also determined hypothalamic-pituitary responsiveness to removal of testicular feedback by comparing GnRH mRNA and gonadotropin levels in intact and orchidectomized young and old rats. In an initial study, 10 20-micron coronal sections from the medial preoptic area (MPOA) were anatomically matched and compared in intact young (3-month-old) and old (24-month-old) male F344 rats (n = 5/group). In another group of young and old male rats (n = 8-12/group), animals were randomly assigned to be either orchidectomized or sham operated. Rats were killed 21 days after surgery, and comparisons were made in 12 anatomically matched sections of MPOA and 4 matched sections of diagonal band of Broca. In both studies, GnRH mRNA was quantitated by in situ hybridization, using a 35S-labeled oligodeoxynucleotide probe complementary to rat prepro-GnRH mRNA and a computerized image analysis system. In a third study, GnRH content was measured by RIA in microdissected regions of the arcuate nucleus and median eminence in intact 3- and 24-month-old male rats (n = 10 and 8, respectively). Serum LH, FSH, and testosterone (T) levels were measured by RIA in trunk blood of all animals. The number of neurons expressing the GnRH gene in the MPOA was significantly lower in sham-operated old rats (mean +/- SEM, 10.5 +/- 0.5 cells/section) than in young rats (13.7 +/- 0.7 cells/section; P less than 0.01), while cellular GnRH mRNA content was unchanged with age (103 +/- 1 vs. 103 +/- 2 grains/cell). Similar results were obtained in intact old and young rats. GnRH peptide content was significantly decreased in the arcuate nucleus of intact old (0.5 +/- 0.08 ng/mg protein) compared to young animals (2.3 +/- 0.7 ng/mg protein; P less than 0.05), with a trend toward a decrease in the median eminence of old (53 +/- 2 ng/mg protein) vs. young rats (69 +/- 7 ng/mg protein; P = 0.06).(ABSTRACT TRUNCATED AT 400 WORDS)     

A subset of gonadotropin-releasing hormone neurons in the ovine medial basal hypothalamus is activated during increased pulsatile luteinizing hormone secretion

GnRH neurons active in the preovulatory LH surge have been identified in several species using the early intermediate gene product, Fos, but the GnRH neurons active during episodic LH secretion remain unknown. In this study, we have used Fos and Fos-related antigens (FRA) to determine whether a subset of GnRH neurons is active when pulsatile LH secretion is acutely stimulated in sheep. In experiment 1, episodic LH secretion was stimulated in five of six ewes by injection of an opioid antagonist to luteal phase ewes. These five ewes had a 6-fold increase in the percentage of GnRH neurons in the medial basal hypothalamus (MBH) expressing Fos/FRA, compared with control ewes that had no LH pulses before death. Fos/FRA expression was not increased in GnRH neurons found in any other area. In experiment 2, episodic LH secretion was induced in rams by introduction of estrous ewes. This treatment increased Fos/FRA expression in MBH GnRH neurons approximately 10-fold compared with control rams. Again, this increase in Fos/FRA expression in GnRH neurons was limited to the MBH. This selective activation of MBH GnRH neurons could reflect the preferential inhibition of these perikarya by endogenous opioid peptides. It also raises the possibility that a subset of GnRH neurons in the MBH may be responsible for episodic GnRH secretion in sheep.     

FOS expression in gonadotropin-releasing hormone neurons: enhancement by steroid treatment and mating.

Expression of the protooncoprotein FOS is now widely believed to be a marker for neuronal activation. In female rats, a steroid-induced LH surge is accompanied by an increase in FOS-positive GnRH neurons, especially in the region of the organum vasculosum of the lamina terminalis. The present study, conducted in mice, has examined the effects of both steroid hormone treatment and sexual behavior on the expression of FOS in GnRH neurons and their distribution in the central nervous system. Thirty-three ovariectomized mice, each bearing a sc priming capsule of 17 beta-estradiol, were divided into five groups, four of which were treated sequentially with estradiol benzoate (1 microgram) and progesterone (500 micrograms). In females maintained on 17 beta-estradiol only and killed between 1400-1530 h, only 1.3 +/- 0.7% of GnRH neurons contained FOS, while treatment with estradiol benzoate/progesterone increased FOS expression significantly to 31.7 +/- 8.5% in the same time period. In animals killed at 1530-1700 h, FOS expression declined in the absence of a male (13.8 +/- 2.2%) or when the male present in the cage displayed some sexual behavior but did not ejaculate (13.0 +/- 8.6%). Interestingly, the expression of FOS was maintained at a high level (42.3 +/- 11.4%) into the late afternoon in females paired with a reproductively successful (ejaculating) male. There was a positive correlation (r2 = 0.65; P < 0.01) between the level of LH and the number of FOS-positive GnRH neurons. Hence, the expression of FOS in GnRH neurons was enhanced by both a steroid regimen leading to a LH surge and an intense level of mating behavior. Mapping of the GnRH neurons indicates that in animals with the highest level of FOS expression, FOS-positive GnRH neurons were not confined to the region of the organum vasculosum of the lamina terminalis, but were found more widely distributed along the entire rostro-caudal axis of these cells.     

Progesterone-receptive beta-endorphin and dynorphin B neurons in the arcuate nucleus project to regions of high gonadotropin-releasing hormone neuron density in the ovine preoptic area

Progesterone inhibits gonadotropin-releasing hormone (GnRH) secretion through interneuronal systems located in the mediobasal hypothalamus in ewes. Endogenous opioid peptides are implicated in this inhibition of GnRH secretion. The distributions of endogenous opioid peptides are known to overlap with progesterone receptors (PR) in the arcuate nucleus. We investigated whether PR is expressed by beta-endorphin and dynorphin B neurons in the arcuate nucleus and if a subset of double-labeled cells projects to the preoptic area where most GnRH neurons are detected. Injection of a retrograde tracer, Fluorogold, into the rostral preoptic area was performed in ovariectomized ewes pretreated with estrogen and progesterone. Brain sections were processed using double immunocytochemistry. Only brains of ewes with an injection site encompassing at least 80 GnRH neurons were processed for PR and then either beta-endorphin or dynorphin B immunocytochemistry. Antigen retrieval is essential for PR detection but causes Fluorogold to fade. Thus, quantitative analysis was performed on photographs taken before and after antigen retrieval. We found that 25-30% of PR-containing neurons, 20% of beta-endorphin cells and 22% of dynorphin B neurons in the arcuate nucleus project toward the preoptic area. From the PR/beta-endorphin double-labeled cells that represent 25 and 36% of PR and beta-endorphin cells, respectively, 35% were labeled with Fluorogold. From the PR/dynorphin B double-labeled cells that account for 39 and 62% of PR and dynorphin B neurons, respectively, 26% contained Fluorogold. These data strongly support the hypothesis that progesterone acts in the arcuate nucleus through beta-endorphin and dynorphin B neurons to affect preoptic area GnRH neurons.     

Reproductive experience increases prolactin responsiveness in the medial preoptic area and arcuate nucleus of female rats

The experience of pregnancy plus lactation produces long-term enhancements in maternal behavior as well as reduced secretion of prolactin, a key hormone for the initial establishment of maternal care. Given that prolactin acts centrally to induce maternal care as well as regulate its own secretion, we tested whether prolactin receptors in brain regions known to regulate behavioral and neuroendocrine processes were up-regulated and more responsive to prolactin in reproductively experienced females. Diestrous primiparous (8 wk after weaning) and age-matched virgin rats were treated with 250 microg ovine prolactin sc or vehicle and the brains collected 2 h later for measurement of mRNA for genes involved in prolactin signaling. Reproductively experienced rats had lower serum prolactin concentrations, compared with virgin rats, suggesting enhanced prolactin feedback on the arcuate neurons regulating prolactin secretion. In the medial preoptic area and arcuate nucleus (regions involved in regulating maternal behavior and prolactin secretion, respectively), the level of long-form prolactin receptor mRNA was higher in primiparous rats, and prolactin treatment induced a further increase in receptor expression in these animals. In the same regions, suppressors of cytokine signaling-1 and -3 mRNA levels were also markedly increased after prolactin treatment in reproductively experienced but not virgin rats. These results support the idea that reproductive experience increases central prolactin responsiveness. The induction of prolactin receptors and enhanced prolactin responsiveness as a result of pregnancy and lactation may help account for the retention of maternal behavior and shifts in prolactin secretion in reproductively experienced females.     

The morphometry of astrocytes in the rostral preoptic area exhibits a diurnal rhythm on proestrus: relationship to the luteinizing hormone surge and effects of age

The morphometry of astrocytes in the arcuate nucleus exhibits cyclic changes during the estrous cycle leading to dynamic changes in the communication between neurotransmitters and neuropeptides that regulate pituitary hormone secretion. Data suggest that remodeling of direct and/or indirect inputs into GnRH neurons may influence the timing and/or amplitude of the preovulatory LH surge in young rats. We have previously found that aging alters the timing and amplitude of the LH surge. Therefore, the purpose of this study was to focus on the rostral preoptic area where GnRH cell bodies reside. We assessed the possibility that the morphometry of astrocytes in the rostral preoptic area displays time-related and age-dependent changes on proestrus. Our results demonstrate that, in young rats, astrocyte cell surface area decreases between 0800 h and 1200 h, before the initiation of the LH surge. Changes in surface area over the cycle were specific to astrocytes in close apposition to GnRH neurons. In contrast, in middle-aged rats astrocyte surface area was significantly less than in young rats and did not change during the day. These findings suggest that a loss of astrocyte plasticity could lead to the delayed and attenuated LH surge that has been previously observed in middle-aged rats.     

Metabolic syndrome induces inflammation and impairs gonadotropin-releasing hormone neurons in the preoptic area of the hypothalamus in rabbits

Rabbits with high fat diet (HFD)-induced metabolic syndrome (MetS) developed hypogonadotropic hypogonadism (HH) and showed a reduced gonadotropin-releasing hormone (GnRH) immunopositivity in the hypothalamus. This study investigated the relationship between MetS and hypothalamic alterations in HFD-rabbits. Gonadotropin levels decreased as a function of MetS severity, hypothalamic gene expression of glucose transporter 4 (GLUT4) and interleukin-6 (IL-6). HFD determined a low-grade inflammation in the hypothalamus, significantly inducing microglial activation, expression and immunopositivity of IL-6, as well as GLUT4 and reduced immunopositivity for KISS1 receptor, whose mRNA expression was negatively correlated to glucose intolerance. Correcting glucose metabolism with obetcholic acid improved hypothalamic alterations, reducing GLUT4 and IL-6 immunopositivity and significantly increasing GnRH mRNA, without, however, preventing HFD-related HH. No significant effects at the hypothalamic level were observed after systemic anti-inflammatory treatment (infliximab). Our results suggest that HFD-induced metabolic derangements negatively affect GnRH neuron function through an inflammatory injury at the hypothalamic level.     

Morphological evidence for direct interaction between arcuate nucleus neuropeptide Y (NPY) neurons and gonadotropin-releasing hormone neurons and the possible involvement of NPY Y1 receptors.

Neuropeptide Y (NPY) neurons in the arcuate nucleus of the hypothalamus (ARH) have been shown to play an important role in modulating LH secretion. One mechanism by which the ARH NPY system may regulate LH secretion is by modulating GnRH neuronal function. Thus, the present study examined whether the ARH NPY system provided direct input to GnRH cell bodies in the preoptic area (POA), as well as to their nerve terminals in the median eminence (ME). The possible involvement of the NPY Y1 receptor subtype in mediating the effects of NPY was also investigated. Lactating rats were used in these studies because they have increased hypothalamic NPY content, especially in the ARH/ME areas, making it easier to detect NPY fibers and terminals. The anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was iontophoresed into the ARH of lactating rats; and triple-label immunofluorescence was performed, with the aid of confocal microscopy, to visualize NPY, PHA-L, and GnRH. GnRH cell bodies were found scattered throughout the organum vasculosum laminae terminalis (OVLT)/POA region, and NPY/ PHA-L double-labeled fibers were found in very close proximity to numerous GnRH perikarya. In the ME, double-labeled NPY/PHA-L fibers were found in the inner and external zones, and they were found in close proximity to GnRH neuronal fibers. Using a NPY Y1 specific antibody, double-label immunofluorescence was performed to examine whether the Y1 receptor subtype was expressed in GnRH neurons. No convincing Y1-positive staining was found in GnRH cell bodies in the OVLT/POA region. However, abundant Y1-positive fiber and cell staining were observed throughout the region, and Y1-positive fibers were found in close apposition to GnRH cell bodies. In contrast, numerous GnRH nerve fibers and terminals in both the OVLT and ME were colocalized with Y1-positive staining. The results of this study suggest that ARH NPY neurons come in close contact with GnRH neurons and may provide direct input to both GnRH cell bodies in the POA region and to their nerve terminals in the ME. The Y1 receptor subtype may be directly involved in NPY modulation of GnRH secretion from its nerve terminals.     

Growth-associated protein-43 messenger ribonucleic acid expression in gonadotropin-releasing hormone neurons during the rat estrous cycle

We have shown previously at the ultrastructural level that morphological changes occur in the external zone of the median eminence allowing certain GnRH nerve terminals to contact the pericapillary space on the day of proestrus. The present study was designed to determine whether the intrinsic determinant of neuronal outgrowth, growth-associated protein-43 (GAP-43), was expressed in GnRH neurons of adult female rats, and whether its expression varied throughout the estrous cycle. To accomplish this, we perfusion-fixed groups of adult female rats at 0800 and 1600 h on diestrous day 2 (diestrous II), at 0800 h and 1600 h on proestrus, and at 0800 and 1600 h on estrus (n = 4 rats/group) and used double labeling in situ hybridization and quantification to compare the levels of GAP-43 messenger RNA (mRNA) in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense complementary RNA (cRNA) probe labeled with the hapten digoxigenin, whereas the GAP-43 cRNA probe was labeled with 35S and detected by autoradiography. In addition, GAP-43 protein was identified with immunohistochemistry in the median eminence. The results show that many GnRH neurons expressed GAP-43 mRNA and that GAP-43 protein was present in many GnRH axon terminals in the outer layer of the median eminence. The number of GnRH neurons expressing GAP-43 mRNA was significantly higher on proestrus (64 +/- 5%) than on diestrous II (40 +/- 2%; P < 0.001) or on estrus (45 +/- 8%; P < 0.05), and the GAP-43 mRNA levels in GnRH neurons also varied as a function of time of death during the estrous cycle. The GAP-43 mRNA levels in GnRH neurons were higher on proestrus and estrus than on diestrous II (P < 0.05). These data show that 1) GAP-43 is expressed in adult GnRH neurons; 2) GAP-43 mRNA expression in GnRH neurons fluctuates during the estrous cycle; and 3) GAP-43 mRNA content in GnRH neurons is highest on the day of proestrus, before and during the onset of the LH surge. These observations suggest that the increased GAP-43 mRNA expression in GnRH neurons on the day of proestrus could promote the outgrowth of GnRH axon terminals to establish direct neurovascular contacts in the external zone of the median eminence and thus facilitate GnRH release into the pituitary portal blood.     

A second gene for gonadotropin-releasing hormone: cDNA and expression pattern in the brain.

In vertebrates, the gonadotropin-releasing hormone(GnRH) decapeptide is secreted from hypothalamic nerve terminals to regulatereproduction via control of synthesis and release of pituitary gonadotropins.Only one GnRH peptide has been found in mammals, with one exception, althoughnumerous other vertebrate species express more than one of the eight knowndecapeptide forms as shown by immunocytochemical labeling of distinct cellgroups in the brain. However, neither the functional nor the evolutionaryrelationships among these GnRH forms are clear, because only one preprohormonegene sequence from any species has been reported. The most ubiquitousalternative form of GnRH is [His5,Trp7,Tyr8]GnRH (also referred to aschicken-II), which differs from the mammalian sequence at amino acids 5, 7, and8. This peptide has been shown to have the most potent releasing-hormoneactivity, although immunocytochemical staining has suggested it is synthesizedonly in the mesencephalon. Here we report the cloning and expression pattern ofthe gene for the precursor of this form from the teleost fish Haplochromisburtoni. This is the second GnRH-encoding gene to be characterized in thisspecies. The newly discovered preprohormone gene differs from that previouslyreported in two ways. First, whereas the original gene predicts only a singleassociated peptide, this one predicts two associated peptides, both of whichappear to be unique. Second, the gene for [His5,Trp7,Tyr8]GnRH is expressed inonly one cell group in the mesencephalon. In contrast, the previously reportedgene is expressed only in the terminal nerve. The striking differences betweenthe preprohormone structure and localization suggest that the genes coding forthe two known GnRH forms in H. burtoni did not arise from a recent duplicationevent. Interestingly, neither of the two genes found to date in this species isexpressed in cells which project from the hypothalamus to the pituitary,suggesting that yet a third gene coding for GnRH may exist.     

Triiodothyronine exerts direct cell-specific regulation of thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus

Thyroid hormone administered systemically exerts negative feedback control of biosynthesis of the TRH pro-hormone in the hypothalamic paraventricular nucleus (PVN), the origin of neurons that regulate anterior pituitary TSH secretion, but not in any other group of TRH-synthesizing neurons in the brain. To determine whether this response is mediated by direct effects on PVN neurons, we studied the effect of unilateral stereotaxic implants of L-T3 into the anterior hypothalamus on the concentration of pro-TRH mRNA and pro-TRH in the PVN of hypothyroid rats. Because hypothalamic-pituitary-thyroid function is also regulated by central catecholamines, we also determined the effect of unilateral ablation of ascending catecholaminergic fibers to one side of the PVN by stereotaxic injection of 6-hydroxydopamine or transection of ascending catecholaminergic pathways. T3-implanted hypothyroid animals showed a marked reduction in pro-TRH mRNA and immunoreactive pro-TRH in medial parvocellular neurons of the PVN on the same side as the implant, but not in contralateral PVN neurons or TRH-synthesizing neurons in other hypothalamic regions. In contrast, hypothyroid animals implanted with pellets of hormonally inactive 3,5-diiodo-L-thyronine showed intense symmetric hybridization and immunoreaction product in both wings of the PVN. Despite marked unilateral reduction in the catecholamine innervation to the PVN, no reduction in pro-TRH mRNA or immunoreactive pro-TRH was observed in the PVN on the affected side compared to that on the unaffected side. These studies demonstrate that negative feedback regulation of thyroid hormone occurs directly on TRH neurons and is restricted only to those in the PVN tuberoinfundibular system.     

Neuropeptide Y gene expression in the arcuate nucleus is increased during preovulatory luteinizing hormone surges.

Recent studies have suggested that neuropeptide Y (NPY) plays an important role in the induction of the preovulatory LH surge. The present study was performed in order to determine if a change in NPY gene expression within arcuate nucleus NPY neurons is associated with the generation of the preovulatory LH surge. In Exp 1, in situ hybridization was used to measure NPY messenger RNA (mRNA) levels in the arcuate nucleus of female rats at 0900 h and every 2 h from 1400-2200 h on the day of proestrus (PRO). Comparisons between groups showed a clear, stepwise increase in NPY gene expression throughout the day of PRO. At 1600 h, when LH values were significantly greater than 0900 h values, NPY mRNA labeling intensities in the arcuate nucleus were significantly greater than 0900 h levels (P < 0.01). By 1800 h, the time at which the LH surge peaked, NPY mRNA levels also peaked and were nearly 3-fold greater than levels observed at 0900 h (P < 0.01). NPY mRNA levels at 2000 h and 2200 h remained elevated above 0900 h levels (P < 0.01) but by 2000 h had decreased significantly from 1800 h levels (P < 0.05). In Exp 2, NPY mRNA levels were measured once again at 0900 h and 1800 h on PRO, and then at 0900 h and 1800 h on metestrus (MET), in order to determine if the change in gene expression seen in Exp 1 was unique to the day of PRO, or if it simply reflected a daily rhythm of gene expression in the nucleus. Analysis of mRNA levels showed no difference in NPY mRNA levels between 0900-1800 h on MET. Also, NPY mRNA levels at 0900 h and 1800 h on MET were significantly less than levels at 1800 h on PRO (P < 0.01). These results are consistent with the hypothesis that NPY neurons participate in the generation of LH surges through increased production of NPY and subsequent potentiation of the release and/or actions of LHRH.     

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.     

Neurosteroids alter gamma-aminobutyric acid postsynaptic currents in gonadotropin-releasing hormone neurons: a possible mechanism for direct steroidal control

Pulsatile GnRH release is required for fertility and is regulated by steroid feedback. Whether or not steroids or their metabolites act directly on GnRH neurons is not well established. In some neurons, steroid metabolites known as neurosteroids modulate the function of the GABAA receptor. Specifically, the progesterone derivative allopregnanolone is an allosteric agonist at this receptor, whereas the androgen dehydroepiandrosterone sulfate (DHEAS) is an allosteric antagonist. We hypothesized these metabolites act similarly on GnRH neurons to modify the response to GABA. Whole-cell voltage-clamp recordings of GABAergic miniature postsynaptic currents (mPSCs) were made from green fluorescent protein-identified GnRH neurons in brain slices from diestrous mice. Glutamatergic currents were blocked with antagonists and action potentials blocked with tetrodotoxin, minimizing presynaptic effects of treatments. Allopregnanolone (5 microm) increased mPSC rate of rise, amplitude and decay time by 15.9 +/- 6.1%, 16.5 +/- 6.3%, and 58.3 +/- 18.6%, respectively (n = 7 cells). DHEAS (5 microm) reduced mPSC rate of rise (32.1 +/- 5.7%) and amplitude (27.6 +/- 4.3%) but did not alter decay time (n = 8). Effects of both neurosteroids were dose dependent between 0.1 and 10 microm. In addition to independent actions, DHEAS also reversed effects of allopregnanolone on rate of rise and amplitude so that these parameters were returned to pretreatment baseline values (n = 6). These data indicate allopregnanolone increases and DHEAS decreases responsiveness of GnRH neurons to activation of GABAA receptors by differentially modulating current flow through GABAA receptor chloride channels. This provides one mechanism for direct steroid feedback to GnRH neurons.