Stimulation Of Luteinizing Hormone Secretion Research Articles

FRI290 Proteomic Identification Of Secretoneurin-Stimulated Proteins In Purified Mouse Gonadotropes

Abstract Disclosure: A. Herdman: None. A. Lagasse: None. A. Haney: None. S. Byrum: None. U. Boehm: None. M.C. MacNicol: None. A.M. MacNicol: None. G.V. Childs: None. A.K. Odle: None. Secretoneurin (SN) is a proteolytic product of secretogranin II (SCG2), a member of the granin family of proteins. Both SCG2 and SN have been localized in the anterior pituitary (AP), and SCG2 co-localizes to gonadotrope granules containing luteinizing hormone (LH). Preliminary studies from others have shown that exogenous SN is capable of stimulating LH secretion from the mouse LßT2 cell line and goldfish pituitaries, and SN may provide a paracrine signal from other AP cells to support LH production/secretion. Despite the implications for regulatory reproductive signaling, a comprehensive study of the effect of SN on female mammalian gonadotropes has not been conducted. To define the pathways and processes affected by exogenous secretoneurin (SN) on murine gonadotrope protein expression, pituitaries from 18 proestrous female mice expressing Gnrhr-driven Cre and a floxed fluorescent Cre reporter were collected and dispersed. Half of the dispersed pituitaries were stimulated with 1 μM SN, and all cells were incubated for 3 hours. Gonadotropes were purified via fluorescence-activated cell sorting. After pooling to optimize cell counts, control and SN-stimulated gonadotropes were submitted for mass-spectrometry proteomics. A differential expression analysis was performed to determine fold-change (FC) in proteins from SN-stimulated samples over control (unstimulated), and false discovery rate (FDR)-controlled adjusted p values were used to assign significance (Adj p≤0.05). A total of 948 proteins were identified in the purified gonadotrope pools. Of these, 136 proteins were significantly differentially expressed between control and SN-stimulated gonadotrope pools; 53 proteins were upregulated and 83 were downregulated in SN-stimulated gonadotropes. Upregulated targets of particular interest include the SN precursor SCG2 (FC = 12, p<0.05), as well as prohormone convertase 2 (PC2: FC = 8, p<0.05) which can cleave SCG2 to produce SN. Also upregulated is ELAV-like protein 1 (ELAVL1: FC=5, p<0.05), an RNA-binding protein known to regulate gonadotropin-releasing hormone receptor (GnRHR) expression and bind other gonadotrope-critical mRNAs. Although LHß was unchanged, the glycoprotein alpha subunit (CGA) was decreased 4-fold following SN stimulation (p = 0.05). This is consistent with previous reports showing decreased Cga mRNA following SN stimulation. In addition to the expected “cell signaling” and “response to stimulation”, gene ontology analysis showed that the major biological processes altered by SN stimulation include processing of RNA (including RNA binding, translation regulation, etc.) and regulation of cell structure. Indeed, several RNA-binding proteins and actin-related proteins were significantly changed. Our data confirm previous results from LßT2 cells and define new SN targets and pathways that may play a broader role in gonadotrope physiology. Presentation: Friday, June 16, 2023

Effect of a neurokinin 3 receptor-selective agonist administration on the embryos recovered from superovulated cows.

Superovulation (SOV) treatment of cows results in unovulated follicles and inconsistent quality of the recovered embryos. It has been demonstrated that luteinizing hormone (LH) secretion is suppressed during SOV treatment of cows, which may cause insufficient follicle development and variation in the development of recovered embryos and unovulated follicles. Pulsatile gonadotropin-releasing hormone/LH secretion is controlled by the activity of kisspeptin, neurokinin B and dynorphin (KNDy) neurons in the arcuate nucleus in many mammals. As neurokinin B promotes the activity of KNDy neurons, we hypothesized that senktide, a neurokinin B receptor agonist, has the potential as a therapeutic drug to improve the ovulation rate and quality of recovered embryos in SOV-treated cows via stimulation of LH secretion. Senktide was administered intravenously (30 or 300nmol/min) for 2h, beginning from 72h after the start of SOV treatment. LH secretion was examined before and after administration, and embryos were collected 7 d after estrus. Senktide administration increased LH secretion in SOV-treated cows. The ratios of code 1, code 1 and 2, and blastocyst stage embryos to recovered embryos were increased by senktide (300nmol/min) administration. Moreover, the mRNA levels of MTCO1, COX7C, and MTATP6 were upregulated in recovered embryos of senktide (300nmol/min)-administered animals. These results indicate that the administration of senktide to SOV-treated cows enhances LH secretion and upregulates the expression of genes involved in mitochondrial metabolism in embryos, thereby improving embryo development and embryo quality.

SAT-420 Gonadotropin-Releasing Hormone Stimulates Luteinizing Hormone Exocytosis without Elevating Lhb Expression

Continuous but not pulsatile delivery of GnRH leads to marked down regulation of luteinizing hormone (LH) secretion and blockade of reproductive functions. However, the relationship between LH beta subunit gene (Lhb) expression and LH secretion has not been studied in detail. Here we studied the relationship between Lhb expression, LH release and intracellular LH content under different experimental paradigms. Experiments were performed in vivo and in vitro using female and male rats and in cultured rat pituitary cells. In vitro experiments included evaluation of Lhb expression during the prolonged absence of GnRH, influence of culture medium on gene expression, continuous vs repetitive GnRH application, and ELISA vs Western blot analysis of LH content. Experiments showed that Lhb expression declined progressively after pituitary removal and cell dispersion, indicating the loss of regulated gene expression. Addition of GnRH receptor agonists did not rescue Lhb expression, suggesting that other factor(s) account(s) for regulated gene expression. On the other hand, GnRH stimulated LH secretion in vitro, leading to gradual depletion of the secretory pool during continuous receptor activation. Inhibition of transcription by actinomycin D had only a minor effect on in vitro LH secretion, in contrast to cycloheximide, a protein synthesis inhibitor. In vivo administration of a GnRH receptor agonist was accompanied with a rapid increase in serum LH levels and a progressive depletion of the intrapituitary LH levels, without affecting Lhb expression. These results indicate that GnRH controls LH synthesis and exocytosis post-transcriptionally and that pulsatile GnRH release protects gonadotrophs from depletion of the LH secretory pool.

Central RFRP-3 Stimulates LH Secretion in Male Mice and Has Cycle Stage-Dependent Inhibitory Effects in Females.

RFamide-related peptide (RFRP)-3 is a neuropeptide thought to play an inhibitory role in the regulation of reproduction in various mammalian species, although some stimulatory effects have been reported. To date, the effects of RFRP-3 on gonadotropin secretion have been scarcely studied in mice. The aim of the current study was to characterize the effect of RFRP-3 administration on gonadotropin secretion in male and female mice. Adult intact and castrated male mice received acute central injections of 0.5 to 5 nmol of RFRP-3, and luteinizing hormone (LH) concentration was assayed in tail-tip blood samples. RFRP-3 had a dose-dependent stimulatory effect on LH secretion when administered centrally to both intact and castrated mice, and this effect was diminished when RFRP-3 was administered to kisspeptin receptor knockout mice. In female mice, central RFRP-3 had an inhibitory effect on LH secretion when administered at the time of the preovulatory LH surge in intact mice, or of an estradiol-induced LH surge in ovariectomized mice. Conversely, central RFRP-3 administration had no effect on LH levels in intact diestrus or ovariectomized, low-dose estradiol-implanted mice. Finally, peripheral administration of RFRP-3 to intact males and to females at the time of the preovulatory LH surge or during diestrus had no effect on LH secretion. Taken together, these results provide a detailed description of sex- and cycle stage-dependent effects of RFRP-3 on gonadotrophin secretion in mice. Moreover, it appears that the stimulatory effects are mediated in part by the receptor for kisspeptin, a potent stimulator of the reproductive axis.

Differences in the reproductive hormone rhythm of tree sparrows (Passer montanus) from urban and rural sites in Beijing: The effect of anthropogenic light sources

The pervasiveness of anthropogenic light in urban environments has increased the exposure to light of many animals. Since photoperiod is a regulator of the timing of reproduction in most temperate region birds, such light sources could potentially change the timing of reproduction. We compared the luteinizing hormone (LH), testosterone (T), and estradiol (E2) levels of tree sparrow (Passer montanus) populations sampled at two urban and two rural sites in China, and also performed a controlled photoperiod experiment to determine the influence of artificial light on the endocrine rhythm of these populations. LH levels of urban tree sparrows increased earlier than those of rural ones, but rural populations had higher LH peaks. A linear mixed model (LMM) indicates that increased exposure to light at night (LAN) significantly influenced the LH, T and E2 concentrations of free-living tree sparrows in urban environments compared to their rural counterparts. The results of the controlled photoperiod experiment showed that tree sparrows that were exposed to 6lux of light during the dark phase of the artificial photoperiod began to secrete LH earlier, and had lower peak LH levels, than control birds. A LMM indicates that LAN had a significant effect on LH levels in this experiment. Although urban tree sparrows began to secrete LH earlier than their rural counterparts, we found no corresponding advance in T or E2 secretion. On the contrary, peak T and E2 levels of urban birds were lower than those of rural birds. These results suggest that although anthropogenic light sources appear to advance the onset of LH secretion in urban tree sparrow populations, they also lower peak LH, and consequently levels of T and E2. A possible explanation for these observations is that greater exposure to anthropogenic light in urban environments stimulates LH secretion and may influence photosensitivity, but further experimental work is required to test this hypothesis.

Influence of season and nutritional status on the direct effects of leptin, orexin-A and ghrelin on luteinizing hormone and growth hormone secretion in the ovine pituitary explant model

The aim of this study was to examine whether leptin (anorexigenic peptide), orexin-A, and ghrelin (orexigenic peptides) could directly (ie, independently of hypothalamic influences) affect the secretion of luteinizing hormone (LH) and growth hormone (GH) by adenohypophyseal (AP) explants obtained from normally fed or fasted (48 h) ewes during the breeding and nonbreeding seasons. In addition, a specific ovine super leptin antagonist (SLAN-3) was used to assess the interactions between leptin and ghrelin and/or orexin-A. Pituitary glands from 16 ovariectomized Polish Longwool ewes that had received estradiol-releasing subcutaneous implants were collected in the breeding (November; n = 8) and nonbreeding (May; n = 8) seasons. The AP explants were incubated for 240 min in a gas-liquid interface and treated with leptin (50 ng/mL), ghrelin (100 ng/mL), orexin-A (100 ng/mL), and SLAN-3 (500 ng/mL) with orexin-A or ghrelin. Treatments with leptin and SLAN-3 + orexin-A increased (P < 0.05) LH concentrations in the cultures of AP explants from fasted animals in the breeding season. Orexin-A increased (P < 0.05) LH secretion by AP explants from both fasted and fed animals in the breeding season. Ghrelin stimulated (P < 0.05) GH secretion by AP explants collected from fasted animals in nonbreeding season and from normally fed ewes in both seasons. Leptin decreased (P < 0.05) GH secretion by AP explants collected from fasted ewes in both seasons and from nonfasted ewes in the breeding season. However, the treatment with SLAN-3 + ghrelin resulted in greater (P < 0.05) GH concentrations compared with leptin treatment of AP explants from fasted ewes in the breeding season and from normally fed ewes in nonbreeding season. In summary, leptin, orexin-A, and ghrelin exerted direct effects on AP secretory function in an ex situ model and both the reproductive season and nutritional status of the animals impinged on the direct effects of the peptides on LH and GH release. Specifically, orexin-A was more potent than leptin in directly stimulating LH secretion in cycling ewes, whereas ghrelin and leptin generally had opposing effects on the secretory function of somatotrophs in sheep.

Effect of long-term dietary lead exposure on some maturation and reproductive parameters of a female Prussian carp (Carassius gibelio B.)

Lead is reported to be an endocrine disruptor. In the current study, we exposed female Prussian carp to artificial feed, supplemented with five doses of dietary Pb (0, 1 (control), 8, 13, 24, and 49 mg/kg) over either a 24-month period or a 12-month period, followed by further 12 months where the fish were fed the control diet. Periodically, oocyte maturation, gonadosomatic index (GSI), ability to secret luteinizing hormone (LH) as well as gonad Pb concentrations were measured. It was found that the reproductive system of the female Prussian carp is not indifferent to chronic exposure to lead. The negative effect was manifested by a decrease in the GSI after 12 months despite the fact that a higher proportion of oocytes at more advanced maturity stages were concurrently observed. After 12 months of exposure, the effect on LH secretion varied according to the dose. In the group exposed to the lowest dose (8 mg/kg), LH decreased spontaneously, and in the groups exposed to the highest two doses (24 and 48 mg/kg), a significantly higher LHRH-A-stimulated LH secretion was observed. After 24 months of lead exposure, the effects on oocyte maturation and size and on GSI values were not pronounced. Analysis of the effect of lead exposure on LH secretion showed that the relationships were similar to those observed after 12 months but nonsignificant. During chronic a 24-month period exposure to Pb, Prussian carp female appears to acclimate to Pb doses used in the experiment. After 12 months of exposure and 12 months of depuration, the levels of spontaneous and stimulated LH secretion observed in all the groups were similar to the control, which is evidence that depuration eliminates the previously observed effects of exposure to lead. Lead is easily accumulated in the ovary, reaching a fivefold higher level (0.8 mg/kg tissue) compared to the control (0.15 mg/kg tissue), but after discontinuation of exposure, this organ is quickly depurated. The results indicate that environmental Pb can be a potent endocrine disruptor affecting ovarian steroidogenesis, gametogenesis, and ovulation, which may lead to adverse impacts on fish reproduction and population density and that female Prussian carp become resistant to the negative effects of lead with advancing age, and their organs cope by reaching a state of homeostasis.

Effects of Aroclor 1254 on LH and 17,20β-P secretion in female Prussian carp (Carassius gibelio Bloch) in the spawning season

The effects of Aroclor 1254 on the secretion of luteinizing hormone (LH) and 17,20&amp;beta;-dihydroxy-4-pregnen-3-one (17,20&amp;beta;-P) in female Prussian carp (Carassius gibelio Bloch) at the time of their natural spawning were determined. Aroclor 1254 at doses of 0.01, 0.1, and 1 mg/kg body weight was dissolved in 500 ml of oil and was administered three times intraperitoneally or rectally to female Prussian carp every 24 h for three days. Blood samples were collected after 3 days of Aroclor 1254 administration to determine the LH and 17,20&amp;beta;-P concentrations using ELISA. At 6, 12, and 24 h after injection of gonadotropin-releasing hormone analogue (GnRH-A) blood samples were collected for stimulated LH secretion determination. Aroclor 1254 administered intraperitoneally (0.1 and 1 mg/kg)and rectally (0.01 and 1 mg/kg) significantly increased spontaneous LH secretion. In the case of GnRH-A-stimulated LH release, Aroclor 1254 (administered intraperitoneally only) at concentrations of 0.1 and 1 mg/kg significantly decreased gonadotropin release. The intraperitoneal injections of the lowest tested concentration of Aroclor 1254 also significantly decreased 17,20&amp;beta;-P secretion. The results show that Aroclor 1254 can affect the reproductive system of Prussian carp by changing the secretion of two very important hormones, LH and 17,20&amp;beta;-P, at the time of natural spawning. &amp;nbsp;

GPR54-Dependent Stimulation of Luteinizing Hormone Secretion by Neurokinin B in Prepubertal Rats

Kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn) are coexpressed within KNDy neurons that project from the hypothalamic arcuate nucleus (ARC) to GnRH neurons and numerous other hypothalamic targets. Each of the KNDy neuropeptides has been implicated in regulating pulsatile GnRH/LH secretion. In isolation, kisspeptin is generally known to stimulate, and Dyn to inhibit LH secretion. However, the NKB analog, senktide, has variously been reported to inhibit, stimulate or have no effect on LH secretion. In prepubertal mice, rats and monkeys, senktide stimulates LH secretion. Furthermore, in the monkey this effect is dependent on kisspeptin signaling through its receptor, GPR54. The present study tested the hypotheses that the stimulatory effects of NKB on LH secretion in intact rats are mediated by kisspeptin/GPR54 signaling and are independent of a Dyn tone. To test this, ovarian-intact prepubertal rats were subjected to frequent automated blood sampling before and after intracerebroventricular injections of KNDy neuropeptide analogs. Senktide robustly induced single LH pulses, while neither the GPR54 antagonist, Kp-234, nor the Dyn agonist and antagonist (U50488 and nor-BNI, respectively) had an effect on basal LH levels. However, Kp-234 potently blocked the senktide-induced LH pulses. Modulation of the Dyn tone by U50488 or nor-BNI did not affect the senktide-induced LH pulses. These data demonstrate that the stimulatory effect of NKB on LH secretion in intact female rats is dependent upon kisspeptin/GPR54 signaling, but not on Dyn signaling.

The response of luteinizing hormone secretion to photoperiod is modified by the level of nutrition in female Mediterranean goats

This paper reports the influence of nutrition on the photoperiodic control of luteinizing hormone (LH) secretion in female Mediterranean goats (i.e., goats from the Mediterranean area in general). Ovariectomized, oestradiol-treated goats were subjected to two consecutive intervals of 3 months of long days followed by 3 months of short days (group LDSD, N = 20), or vice versa (group SDLD, N = 20). The LDSD and SDLD does were also randomly assigned to one of two nutrition groups that received either 1.1 (H group, N = 10) or 0.7 (L group, N = 10) times their maintenance requirements. Live weight and body condition score were determined weekly and LH concentrations twice per week. To establish the pulsatility of secretion of LH, three periods of intensive sampling were undertaken. Melatonin was determined after a period of 45 short or long days. All photoperiod/nutrition groups showed large variations in LH concentrations according to photoperiod, with nutrition having a significant effect ( P < 0.001). The mean time between the shift from long to short days and the stimulation of LH secretion, and between the shift from short to long days and the inhibition of LH secretion, was different in each nutrition group (at least P < 0.05). No differences were seen in the frequency of LH pulses between the nutrition groups, but differences between sampling periods were observed ( P < 0.001). Melatonin secretion was not affected by food supply. These results confirm: (1) that Mediterranean female goats are sensitive to photoperiod, (2) that this environmental cue may control the timing of pituitary activity under natural conditions, and (3) suggest that nutrition plays an important role in the effect of photoperiod on LH secretion.

Interaction of ghrelin and opioids in luteinizing hormone (LH) secretion by pituitary cells of common carp, &lt;I&gt;Cyprinus carpio&lt;/I&gt; (Actinopterygii: Cypriniformes: Cyprinidae), cultured in vitro

Background. Ghrelin, a newly discovered hormone is involved mainly in the regulation of body energy homeostasis. It has also been proved that ghrelin affects many other processes including the control of the hypothalamo-pituitary-gonadal axis in vertebrates. Ghrelin interacts also with other peptides and neurotransmitters, which are involved in gonadotropin release, such as endogenous opioid peptides. The aim of the experiment was to compare the effects of ghrelin and naltrexone, an opioid antagonist, on LH secretion by pituitary cells of mature female and male common carp, Cyprinus carpio L. Materials and methods. In the in vitro experiment the time-dependent (10 and 24 h) action of ghrelin (10&ndash;7 or 10&ndash;6 M) and naltrexone at 10&ndash;6 M added alone or in combination on LH secretion by enzymatically dispersed pituitary cells of mature female and male carp (Cyprinus carpio L.) was studied. Results. Ghrelin alone at a dose of 10&ndash;6 M stimulated LH secretion after 10 h of female- but not male-derived cell incubations. Ghrelin at 10&ndash;7 M had no effect on LH secretion from either female or male cells. Naltrexone alone stimulated LH secretion only after 24 h of female cell culture. In male cells no significant changes in LH secretion in response to naltrexone alone were found after 10 or 24 h incubation period. Combined treatment (ghrelin 10&ndash;7 or 10&ndash;6 M and naltrexone) stimulated LH secretion in female and male cell incubations at 10 and 24 h: LH levels were significantly higher in comparison to control, to ghrelin alone (in cells of both sexes) and to naltrexone alone (in male cells only). Conclusion. The results suggest that opioids and ghrelin may control LH secretion in carp acting synergistically, probably through the same receptor type. The concept of opioid and ghrelin interaction in the gonadal steroid feedback on LH release is also discussed.

Weak evidence of bright light effects on human LH and FSH

BackgroundMost mammals are seasonal breeders whose gonads grow to anticipate reproduction in the spring and summer. As day length increases, secretion increases for two gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH). This response is largely controlled by light. Light effects on gonadotropins are mediated through effects on the suprachiasmatic nucleus and responses of the circadian system. There is some evidence that seasonal breeding in humans is regulated by similar mechanisms, and that light stimulates LH secretion, but primate responses seem complex.MethodsTo gain further information on effects of bright light on LH and FSH secretion in humans, we analyzed urine samples collected in three experiments conducted for other goals. First, volunteers ages 18-30 years and 60-75 commenced an ultra-short 90-min sleep-wake cycle, during which they were exposed to 3000 lux light for 3 hours at balanced times of day, repeated for 3 days. Urine samples were assayed to explore any LH phase response curve. Second, depressed participants 60-79 years of age were treated with bright light or dim placebo light for 28 days, with measurements of urinary LH and FSH before and after treatment. Third, women of ages 20-45 years with premenstrual dysphoric disorder (PMDD) were treated to one 3-hour exposure of morning light, measuring LH and FSH in urine before and after the treatments.ResultsTwo of the three studies showed significant increases in LH after light treatment, and FSH also tended to increase, but there were no significant contrasts with parallel placebo treatments and no significant time-of-day treatment effects.ConclusionsThese results gave some support for the hypothesis that bright light may augment LH secretion. Longer-duration studies may be needed to clarify the effects of light on human LH and FSH.

Immunoneutralization of Inhibin in Cycling Rats Increases Follicle-Stimulating Hormone Secretion, Stimulates the Ovary and Attenuates Progesterone Receptor-Dependent Preovulatory Luteinizing Hormone Secretion

Passive immunization against inhibin with an anti-inhibin serum (AIS) during the diestrous phase in cycling rats increased follicle-stimulating hormone secretion, stimulated the ovaries and reduced the magnitude of the luteinizing hormone (LH) surge in the afternoon of proestrus. The involvement of gonadotrope progesterone receptor (PR) expression/action in the inhibitory effects of the follicle-stimulating hormone-dependent putative ovarian factor gonadotropin surge-attenuating factor on preovulatory LH secretion was studied in the absence of circulating free inhibin. Proestrous pituitaries from rats injected with AIS or a non-immune serum (NIS) were studied for determination of PR-AB and PR-B mRNAs by RT-PCR and PR-B and PR-A isoform proteins by Western blot. In addition, pituitaries from AIS- and NIS-injected rats were incubated and studied for PR-dependent LH secretion parameters: LH-releasing hormone (LHRH)-stimulated LH secretion, progesterone-potentiated LHRH-stimulated LH secretion and LHRH self-priming. Also, the effects of the antiprogestagen RU486 on these LH secretion parameters were evaluated and compared with those of AIS. Finally, gonadotrope PR phosphorylation was evaluated by immunohistochemistry. Results showed that the hyperstimulated ovaries of AIS-injected rats produce a factor, different from inhibin, that blocked LHRH self-priming and P-potentiation of LHRH-stimulated LH secretion. These effects were not due to decreased pituitary PR mRNAs, PR protein expression or PR protein B/A ratio. The inhibitory effect of AIS on PR-dependent LH secretion seemed to be due to gonadotrope PR dephosphorylation. Taken together, the findings indicated that the putative gonadotropin surge-attenuating factor affected LH surge through an inhibition of PR phosphorylation/action but not PR expression.

Regional differences in the pituitary distribution of luteinizing hormone in the gonadectomized and proestrous female rat

Previous data have shown that regional differences in the presence of anterior pituitary luteinizing hormone (LH) generally correlate with the comparable disparities in distribution of gonadotropes throughout the gland. In female rats, the differences are apparent over the estrous cycle, but are more prominent during the hours preceding the proestrus surge of LH. The current experiments examined (1) if such regional disparities are present throughout the surge window, (2) if differences are mirrored by release of LH in vitro and (3) if the appearance of regional differences is altered in ovariectomized females. Results showed that a comparative elevation in the rostral portion of the pituitary during the pre-surge period diminishes and finally disappears concurrent with the rise in circulating LH. This increase in rostral LH concentrations is reflected in this region by a comparable effect in vitro on stimulated LH secretion from pituitary fragments, although the effect is somewhat diminished by referencing release against tissue concentrations of LH present in a contralateral rostral fragment. Ovariectomies conducted at 1500h on proestrus, at a time when a significant regional difference has faded, resulted in a prompt increase in LH across all areas of the pituitary, and the emergence of a marked augmentation in rostral concentrations over the ensuing 72h. The effect was not seen when ovariectomies were performed on estrus. These data show that, while a regional disparity in anterior pituitary LH is present as circulating concentrations of estradiol rise prior to the LH surge, the removal of this steroid feedback at a time when LH synthesis is normally amplified accentuates the difference between the rostral region and other areas of the pituitary.

Direct kisspeptin-10 stimulation on luteinizing hormone secretion from bovine and porcine anterior pituitary cells

Kisspeptins are peptide hormones encoded by the KiSS-1 gene and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. However, peripheral administration, as well as central administration, of kisspeptin stimulates luteinizing hormone (LH) secretion in some mammalian species. In order to evaluate the direct effects of kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) on LH secretion from bovine and porcine anterior pituitary (AP) cells, LH-releasing effects of kisspeptin-10 on AP cells were compared with GnRH in vitro. The AP cells were prepared from 1-month-old intact male calves, 8-month-old castrated male calves, or 6-month-old barrows, and then the cells were incubated for 2 h with the peptides. The 1000 nM and 10,000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from the bovine AP cells ( P < 0.05). The 100 nM and 1000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from porcine AP cells ( P < 0.05). As 10 nM of GnRH strongly stimulated LH secretion from all AP cells tested in this study, the present results suggest that kisspeptin-10 has a direct, but weak, stimulating effect on LH secretion in bovine and porcine AP cells. The present study is the first to examine the direct actions of kisspeptin on the bovine and porcine pituitary gland as far as we know. Kisspeptin might have other actions on the pituitary because the pituitary has multiple roles.

Kisspeptin Activation of Gonadotropin Releasing Hormone Neurons and Regulation of KiSS-1 mRNA in the Male Rat

The KiSS-1 gene codes for a family of neuropeptides called kisspeptins which bind to the G-protein-coupled receptor GPR54. To assess the possible effects of kisspeptins on gonadotropin secretion, we injected kisspeptin-52 into the lateral cerebral ventricles of adult male rats and found that kisspeptin-52 increased the serum levels of luteinizing hormone (p < 0.05). To determine whether the kisspeptin-52-induced stimulation of luteinizing hormone secretion was mediated by gonadotropin-releasing hormone (GnRH), we pretreated adult male rats with a GnRH antagonist (acyline), then challenged the animals with intracerebroventricularly administered kisspeptin-52. The GnRH antagonist blocked the kisspeptin-52-induced increase in luteinizing hormone. To examine whether kisspeptins stimulate transcriptional activity in GnRH neurons, we administered kisspeptin-52 intracerebroventricularly and found by immunocytochemistry that 86% of the GnRH neurons coexpressed Fos 2 h after the kisspeptin-52 challenge, whereas fewer than 1% of the GnRH neurons expressed Fos following injection of the vehicle alone (p < 0.001). To assess whether kisspeptins can directly act on GnRH neurons, we used double-label in situ hybridization and found that 77% of the GnRH neurons coexpress GPR54 mRNA. Finally, to determine whether KiSS-1 gene expression is regulated by gonadal hormones, we measured KiSS-1 mRNA levels by single-label in situ hybridization in intact and castrated males and found significantly higher levels in the arcuate nucleus of castrates. These results demonstrate that GnRH neurons are direct targets for regulation by kisspeptins and that KiSS-1 mRNA is regulated by gonadal hormones, suggesting that KiSS-1 neurons play an important role in the feedback regulation of gonadotropin secretion.

Involvement of serotonin 5HT 1 and 5HT 2 receptors and nitric oxide synthase in the medial preoptic area on gonadotropin secretion

Due to the stimulatory action of serotonin (5HT) and nitric oxide (NO) on the secretion of gonadotropins and PRL, this work aimed at investigating the participation of serotoninergic receptors 5HT 1 and 5HT 2 of the medial preoptic area (MPOA) in the control of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) secretion and the possible modulation by ovarian steroids as well as the possible participation of NO as a mediator of the stimulatory effects of serotonin in the MPOA on LH secretion. Microinjections of three different doses (0.02, 0.2, and 2 ug) of methiothepin, a serotoninergic 5HT 1 antagonist or ketanserin, a seretoninergic 5HT 2 antagonist, were carried out into the MPOA in ovariectomized rats treated or not with estrogen or estrogen plus progesterone. Other groups of ovariectomized rats treated with estrogen, estrogen plus progesterone or vehicle were prepared to evaluate NOS activity in the MPOA. Plasma LH, FSH, and PRL in ovariectomized rats were not altered by the microinjection of methiothepin or ketanserin in the MPOA. Methiothepin microinjection in the MPOA reduced LH but did not change plasma FSH and PRL in ovariectomized rats treated with estrogen or estrogen plus progesterone. On the other hand, ketanserin microinjection in the MPOA reduced plasma LH and FSH but did not change plasma PRL in the animals submitted to the same steroidal treatment. NOS activity in the MPOA was significantly reduced by methiothepin or ketanserin in ovariectomized rats treated with estrogen or estrogen plus progesterone. In conclusion, this work showed that in the studied conditions, serotonin in the MPOA: (1) does not work in the control of PRL secretion through 5HT 1 and 5HT 2 receptors; (2) integrates the control of FSH secretion by 5HT 2 receptors, but not 5HT 1; (3) in the presence of estrogen, stimulates LH secretion by 5HT 1 and 5HT 2 receptors, which can be differentially modulated by progesterone; (4) at least partly, stimulates LH secretion by nitric oxide activity.

Chemosensory stimulation of luteinizing hormone secretion in male Siberian hamsters (Phodopus sungorus).

Male Siberian hamsters (Phodopus sungorus) housed in long days (LD), but not short days (SD) release luteinizing hormone (LH) when exposed to females. This study examined whether this response is specific to a female and identifies the source of a stimulus that induces LH release. Serum concentrations of LH, testosterone (T), follicle stimulating hormone (FSH), and cortisol were examined in all experiments. T concentrations mirrored the LH response; FSH and cortisol were unchanged in response to all stimuli. Exposure to an LD female, irrespective of her reproductive status, but not an SD female, elicited LH release. Exposure to another male did not trigger LH release. Males released LH when allowed physical contact with an anesthetized female, but not when separated from a normally active female, suggesting that tactile or nonvolatile chemosensory stimuli elicit LH release. Urine and secretions collected from the vagina as well as oral, midventral, perineal, and rectal glands, elicited marked behavioral responses in male P. sungorus. Despite these behavioral responses, only feces from females elicited LH release in males. Males released LH in response to feces extracted from the rectum and to cotton swabs that had been rubbed against the rectal mucosa, suggesting that a component of rectal secretions may trigger LH release in male Siberian hamsters. Taken together, these data and previous data from our laboratory indicate that both the production of and the response to a pheromone that triggers the selective release of LH is regulated by day length.

Tamoxifen Induces Gonadotropin-Releasing Hormone Self-Priming through an Estrogen-Dependent Progesterone Receptor Expression in the Gonadotrope of the Rat

Tamoxifen (TX) is an antiestrogen with varying levels of antagonist/agonist activity on the reproductive axis of the rat. It has been reported that TX, in contrast to other selective estrogen receptor modulators (SERMs), increases the content of cytosolic estrogen receptors (ER) in the gonadotrope and induces gonadotropin releasing hormone (GnRH) self-priming in the absence of E. GnRH priming is believed to be a consequence of E-dependent progesterone receptor (PR) activation. The purpose of this study was to determine whether TX induces PR expression in the gonadotrope in an E-dependent manner, and whether the blockade of PR activation affects TX-dependent GnRH self-priming in ovariectomized (OVX) rats. Chronic OVX rats were injected (sc) over 3 days with 25 µg estradiol benzoate (EB), 3 mg TX, 0.5 mg RU58668, a ‘pure’ anti-E (aE), 2 mg RU38486, an anti-P at the receptor (aP), TX+aE and TX+aP. Controls were given 0.2 ml oil. While EB and TX increased mRNA for both PR A+B and PR B expression and the number and intensity of nuclei immunoreactive (IR) for PR in the gonadotrope, the aE and aP given alone had no effect on either PR mRNA levels or nuclear PR-IR. The aE reduced the effect of TX on PR expression (mRNA and nuclear IR) while the aP slightly reduced nuclear PR-IR only. In addition, pituitaries from each of the seven groups were incubated with: 10^–8M E₂, 10^–7M TX, 10^–8M aE, 10^–8M aP, TX+aE, TX+aP or medium alone, respectively. Pituitaries were tested for GnRH self-priming (two pulses of 15 min 1 h apart) and the secretion of LH and PRL determined by specific RIAs. Pituitaries from rats treated with EB and incubated with E₂ had increased basal and GnRH-stimulated luteinizing hormone (LH) and prolactin (PRL) secretion and GnRH self-priming. TX reduced basal and stimulated LH secretion, increased PRL secretion and induced a robust GnRH self-priming. All these effects of TX were blocked by the aE, while the aP blocked GnRH self-priming only. In conclusion, tamoxifen induced PR expression (mRNA and nuclear IR) in the gonadotrope in an E-dependent manner, while activation of these PR through intracellular signaling of GnRH induced GnRH self-priming.

Stimulation of luteinizing hormone secretion by N-methyl-D,L-aspartic acid in the adult male guinea-pig: incomplete blockade by gonadotropin-releasing hormone receptor antagonism.

Stimulation of luteinizing hormone (LH) secretion by N-methyl-D,L-aspartic acid (NMA), reported for several mammalian species, is generally accepted to be mediated through stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) release. In view of a previously reported unexpected inhibitory action of NMA on GnRH release from hypothalamic explant of intact male guinea-pigs, the aim of the present study was to assess the in vivo effects of NMA in the adult male guinea-pig. In the gonadally intact male, NMA (5 mg/animal) elicited a robust LH secretion, which was blocked by the N-methyl-D-asparte-receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP-5,12 mg/animal). In the castrated male, NMA elicited only a marginal and inconsistent LH secretion. Cetrorelix (CET), a GnRH receptor antagonist, administered intracardiacally 1 min or 45 min preceding bolus injection of NMA significantly reduced the LH response to NMA in the intact male. Surprisingly, following GnRH receptor blockade with CET, there still was a substantial residual serum LH response to NMA, while CET completely abolished the serum LH response to high dose (1 microg or 10 microg) guinea-pig GnRH (gpGnRH). These results indicate that NMA stimulates LH secretion in the gonadally intact male guinea-pig in vivo and that this effect is mediated in part through gpGnRH-independent mechanisms.