Gonadotropin-releasing Hormone mRNA Levels Research Articles

The Involvement of the microRNAs miR-466c and miR-340 in the Palmitate-Mediated Dysregulation of Gonadotropin-Releasing Hormone Gene Expression.

Diets high in saturated fatty acids are associated with obesity and infertility. Palmitate, the most prevalent circulating saturated fatty acid, is sensed by hypothalamic neurons, contributing to homeostatic dysregulation. Notably, palmitate elevates the mRNA levels of gonadotropin-releasing hormone (Gnrh) mRNA and its activating transcription factor, GATA binding protein 4 (Gata4). GATA4 is essential for basal Gnrh expression by binding to its enhancer region, with Oct-1 (Oct1) and CEBP-β (Cebpb) playing regulatory roles. The pre- and post-transcriptional control of Gnrh by palmitate have not been investigated. Given the ability of palmitate to alter microRNAs (miRNAs), we hypothesized that palmitate-mediated dysregulation of Gnrh mRNA involves specific miRNAs. In the mHypoA-GnRH/GFP neurons, palmitate significantly downregulated six miRNAs (miR-125a, miR-181b, miR-340, miR-351, miR-466c and miR-503), and the repression was attenuated by co-treatment with 100 μM of oleate. Subsequent mimic transfections revealed that miR-466c significantly downregulates Gnrh, Gata4, and Chop mRNA and increases Per2, whereas miR-340 upregulates Gnrh, Gata4, Oct1, Cebpb, and Per2 mRNA. Our findings suggest that palmitate may indirectly regulate Gnrh at both the pre- and post-transcriptional levels by altering miR-466c and miR-340, which in turn regulate transcription factor expression levels. In summary, palmitate-mediated dysregulation of Gnrh and, consequently, reproductive function involves parallel transcriptional mechanisms.

Aspartame Intake Delayed Puberty Onset in Female Offspring Rats and Girls.

The disturbance of the hypothalamic-pituitary-gonadal (HPG) axis, gut microbiota (GM) community, and short-chain fatty acids (SCFAs) is a triggering factor for pubertal onset. The study investigates the effects of the long-term intake of aspartame on puberty and GM in animals and humans. Aspartame-fed female offspring rats result in vaginal opening time prolongation, serum estrogen reduction, and serum luteinizing hormone elevation. , 60mgkg-1 aspartame treatment decreases the mRNA levels of gonadotropin-releasing hormone (GnRH), Kiss1, and G protein-coupled receptor 54 (GPR54), increases the mRNA level of RFamide-related peptide-3 (RFRP-3), and decreases the expression of GnRH neurons in the hypothalamus. Significant differences in relative bacterial abundance at the genus levels and decreased fecal SCFA levels are noted by 60mgkg-1 aspartame treatment. Among which, Escherichia-Shigella is negatively correlated with several SCFAs. In girls, high-dose aspartame consumption decreases the risk of precocious puberty. Aspartame reduces the chance of puberty occurring earlier than usual in female offspring and girls. Particularly, 60mgkg-1 aspartame-fed female offspring delays pubertal onset through the dysregulation of HPG axis and GM composition by inhibiting the Kiss1/GPR54 system and inducing the RFRP-3. An acceptable dose of aspartame should be recommended during childhood.

Exposure to Cadmium Alters the Population of Glial Cell Types and Disrupts the Regulatory Mechanisms of the HPG Axis in Prepubertal Female Rats.

Despite the fact that the brain is susceptible to neurotoxicity induced by cadmium (Cd), the effects of Cd on the neuroanatomical development in the hypothalamus and regulatory mechanisms of the hypothalamic-pituitary-gonadal (HPG) axis are not fully understood. To clarify this issue, we investigated the effects of 25mg/kg BW/day cadmium chloride (CdCl2) on neuroanatomical alterations in the hypothalamus of prepubertal female rats. Twenty-four Sprague-Dawley rats were randomly assigned to two groups (n = 12), and CdCl2 was administered via gavage from postnatal days (PND) 21 to PND35. The results of the stereological analysis demonstrated that prepubertal exposure to Cd reduced the number of neurons and oligodendrocytes in the arcuate (ARC) and dorsomedial hypothalamus nucleus (DMH) nuclei. In contrast, Cd exposure increased the number of microglial cells in the ARC and DMH nuclei. Cd exposure decreased the mRNA levels of gonadotropin-releasing hormone (GnRH) and increased the mRNA levels of RFamide-related peptide (RFRP-3), but not kisspeptin (Kiss1) in the hypothalamus. Moreover, hormonal assay showed that Cd exposure caused a reduction in the concentration of gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum. Immunohistochemical expression of RFRP-3 in neuronal cell bodies demonstrated that the mean number of RFRP-3 expressing neurons in the DMH nucleus of cadmium-treated rats was dramatically higher than the vehicle group. Overall, exposure to Cd during the prepubertal period alters the population of neurons and glial cell types in the hypothalamus. Additionally, Cd exposure disrupts the regulatory mechanisms of the HPG axis.

Chronic exposure to perfluorohexane sulfonate leads to a reproduction deficit by suppressing hypothalamic kisspeptin expression in mice

BackgroundPerfluorohexane sulfonate (PFHxS) is a six-carbon perfluoroalkyl sulfonic acid found as an environmental contaminant. This study aims to investigate the effects of PFHxS exposure on female reproduction and the underlying mechanism in mice.MethodsEight-week-old ICR mice were divided randomly into four groups administered corn oil (vehicle) and PFHxS at doses of 0.5, 5, and 50 mg/kg/day for 42 days by intragastric administration. Body weight, ovarian weight, estrous cycle, follicle counts, and serum sex hormone levels were evaluated. The expression of kisspeptin and gonadotropin releasing hormone (GnRH) in the hypothalamus was also detected.ResultsCompared to vehicle exposure, 5 mg/kg/day PFHxS treatment prolonged the estrous cycle, especially the duration of diestrus, after 42 days of treatment. The numbers of secondary follicles, antral follicles and corpus lutea were significantly reduced in the PFHxS-treated mice. Moreover, compared with the control mice, the PFHxS-treated mice showed decreases in the serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estrogen (E2), and reduced GnRH mRNA levels, along with the lack of an LH surge. Furthermore, the PFHxS-treated mice had lower levels of kisspeptin immunoreactivity and kiss-1 mRNA in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV) than the control mice. After intraventricular administration of kisspeptin-10, the numbers of secondary follicles, antral follicles and corpus lutea recovered, along with the levels of GnRH mRNA, FSH, and LH in the mice treated with 5 mg/kg/day PFHxS.ConclusionThese results indicate that chronic exposure of mice to 5 mg/kg/day PFHxS affects reproductive functions by inhibiting kisspeptin expression in the ARC and AVPV regions, leading to deficits in follicular development and ovulation.

A role for GABA agonist in controlling the reproduction of female rats via hypothalamic ghrelin, kisspeptin, and RFRP-3 gene expression

Kisspeptin stimulates gonadotropin releasing hormone (GnRH). The GnRH neurons receive inhibitory inputs from ghrelin, RFamide related peptide-3 (RFRP-3), and gamma-aminobutyric acid (GABA) neurons. Polycystic ovary syndrome (PCOS) is associated with increased levels of GnRH/LH and kisspeptin, and decreased release of GABA, ghrelin, and RFRP-3. In the present study, the effects of GABAB receptor agonist, baclofen, were investigated on GnRH, KiSS1, RFRP-3, and ghrelin gene expression in the hypothalamus of PCOS model rats. For induction of PCOS, female Wistar rats weighing 180-200g received intra-muscular injection of estradiol valerate. Fifteen PCOS rats in three groups received intraperitoneal injections of saline, 5, or 10 mg/kg baclofen for two weeks. The hypothalamic samples were dissected. Gene expression levels of GnRH, KiSS1, RFRP-3, and ghrelin were determined by real time qPCR method. Results revealed that baclofen significantly decreased the mean relative KiSS1 gene expression compared to PCOS group. Also, the mean relative RFRP-3 gene expression significantly increased in the baclofen-receiving rats in comparison to PCOS group. Furthermore, baclofen did not change GnRH or ghrelin mRNA levels in comparison to PCOS group. According to these results it can be concluded that in PCOS condition the GABAergic signaling pathway may suppress GnRH neural activity via down or up regulation of the intra-hypothalamic neuropeptides upstream of GnRH neurons.

Intracerebroventricular salusin-β infusion to rats increases hypothalamus-pituitary-testicular axis hormones

Salusin-β (Sal-β), which originates from preprosalusin, is a multifunctional hormone with a peptide structure. Sal-β exists in the hypothalamus and can stimulate the pituitary gland. The present study was conducted to determine the effects of Sal-β on hormones that play roles in the male reproductive system. Forty male Wistar Albino rats were used in the study. No infusions were performed on the control group, and infusions were applied to the infusion groups (artificial cerebrospinal fluid to the sham group, 2 and 20 nM Sal-β to the experimental group) through intracerebroventricular infusion for 7 days at 10 μl/hour rate. The animals were decapitated after 7 days of infusion; and the hypothalamus, testicles, and blood tissue samples were collected. The gonadotropin-releasing hormone (GnRH) mRNA levels were determined from the hypothalamus tissues by using the Real Time-PCR Method, and the serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone levels were determined using the ELISA method. Also, Hematoxylin-Eosin Staining Method was used for histopathological evaluations in the testicle tissues. As a result, Sal-β infusion increased GnRH mRNA levels in hypothalamus tissues (p < 0.05) besides, serum LH, FSH, and testosterone levels of the rats were higher at significant levels following Sal-β infusion compared to the control and sham group (p < 0.05). In the histological examination of the testicle tissues, Sal-β application was found to decrease the seminiferous tubule diameter and germinal epithelial thickness (p < 0.05). This evidence is the first, indicating that Sal-β, which is administered to male rats with central infusion, stimulates hypothalamus and pituitary tissues, and causes increased secretion of male reproductive hormones.

Changes in mRNA and protein levels of gonadotropin releasing hormone and receptor in ovine thymus, lymph node, spleen, and liver during early pregnancy

There exists maternal immunological modification in maternal immune organs during early pregnancy in mammals. Gonadotropin releasing hormone (GnRH) is widely distributed in vertebrate tissues, including immune organs. However, it is unclear that early pregnancy induces expression of GnRH and GnRH receptor (GnRHR) in ovine immune organs. The objective of this study was to explore the expression of GnRH and GnRHR in main immune organs (thymus, lymph node, spleen, and liver) during early pregnancy in sheep. Ovine thymus, lymph node, spleen and liver were sampled at day 16 of estrous cycle, and days 13, 16, and 25 of pregnancy. The expression of GnRH and GnRHR was detected through real-time quantitative PCR, Western blot and immunohistochemistry analysis. The results indicated that early pregnancy induced upregulation of mRNA and protein levels of GnRH and GnRHR in the maternal lymph node, spleen and liver, and mRNA and protein of GnRH in the maternal thymus, but mRNA and protein of GnRHR decreased in the maternal thymus during early pregnancy. In summary, the mRNA and protein levels of GnRH and GnRHR were changed in maternal thymus, lymph node, spleen and liver in a tissue specific manner during early pregnancy in sheep.

Effects of predator-induced stress during pregnancy on reproductive output and offspring quality in Brandt’s voles (Lasiopodomys brandtii)

Animals may use different reproductive strategies depending on environmental conditions. This study investigated the effects of maternal stress induced by exposure to predator odors on reproductive output and adult offspring quality in pregnant Brandt’s voles (Lasiopodomys brandtii). We exposed pregnant Brandt’s voles to cat urine, rabbit urine, or distilled water for 18 days (1 h/day). Our results indicated that pregnant voles in the cat odor-exposed group tended to produce more offspring and the number of viable female offspring was larger. However, we did not observe any differences in the sex ratios of vole offspring among the three treatment groups. Compared with the control (distilled water) group, female offspring of voles in the cat odor-exposed group had lower body weights, lengths, and smaller body weights in relation to body length, whereas they had larger ovaries in relation to body weight. Furthermore, the female offspring of the voles exposed to the cat odor had higher concentrations of serum estradiol and higher levels of gonadotropin-releasing hormone mRNA in the hypothalamus compared with the female offspring of the voles in the control group. However, we observed no differences among the male offspring in the three treatment groups. Our findings suggest that pregnant Brandt’s voles suffering from chronic exposure to predator odors will produce greater numbers of low-weight female offspring that probably possess higher breeding potential to improve the fitness via regulation of the hypothalamic-pituitary-gonadal axis.

Vasopressin regulates hypothalamic GnRH synthesis: Histomorphological evidence in hypothalamus and biological effects in GT1-7 cells

AimsTo investigate the direct histomorphological clues and observe the biological effects of VP acting on gonadotropin-releasing hormone (GnRH) secretion. Main methodsImmunofluorescence was conducted to investigate the expressions of GnRH and VP in experimental left varicocele (ELV) rats and ELV repair rats. The colocalization of GnRH and VP was observed by electron microscopy immunohistochemistry. The protein-protein interaction between GnRH and VP was tested by co-immunoprecipitation (co-IP) and the proximity ligation assay (PLA). The effects of intracellular and extracellular VP on GnRH and relative transcription factors (Oct-1, Otx2, Pbx1b and DREAM) were respectively evaluated in VP overexpressed and VP treated GT1-7 cells. Key findingsBoth hypothalamic GnRH and VP decreased in ELV rats and recovered by ELV repair. The overlapped immunolocalizations of GnRH and VP mainly distributed in the lateral part of the arcuate nucleus (ArcL) and median eminence (ME) with a Manders' overlap coefficient of 0.743 ± 0.117. Immunoreactive substances of GnRH and VP existed in the same and adjacent terminals. VP overexpression did not cause any significant effects on the expressions of GnRH and Oct-1, as well as GnRH promoter activity. While 50–200 pg/ml VP treatments increased GnRH mRNA levels in a dose- and time-dependent manner in GT1-7 cells. Additionally, 200 pg/ml VP triggered a marked promotion of expressions of GnRH, Oct-1, Oxt2 Pbx1b and DREAM, as well as GnRH promoter activity (P < 0.05). SignificanceThe results reveal the colocalization and interaction of VP and GnRH, which will be conducive to explain the effects and mechanisms of VP acting on reproduction.

SUN-470 The Homeodomain Transcription Factors Six3 and OTX2 Act on Mouse and Human Kisspeptin Promoters to Repress Transcription

Idiopathic hypogonadotropic hypogonadism (IHH) is a gonadotropin-releasing hormone (GnRH) deficiency disorder that, in a subset of individuals, can occur alongside anosmia (Kallmann Syndrome). Known genetic causes for these disorders account for only 45% of affected individuals, leaving the majority of unknown origin. In mice, the homeodomain transcription factors Six3 and OTX2 are necessary for a normal number of GnRH neurons, GnRH neuron migration, and/or GnRH mRNA levels. The loss of just a single allele results in subfertility, hypogonadism, and/or anosmia, making them promising candidates for dysregulation in IHH and Kallman Syndrome. Previous work has shown the importance of these factors within GnRH neurons, but little is known about their roles in upstream cell types, such as kisspeptin neurons. Using qPCR and RNA-seq we have verified that Six3 and OTX2 are present in both an arcuate population (KTaR) and AVPV population (KTaV) of immortalized kisspeptin neurons. This led us to hypothesize that Six3 and OTX2 are critical regulators of kisspeptin gene transcription. To test this hypothesis we performed transient transfections in KTaR and KTaV cell lines with Six3 and OTX2 expression vectors along with either a mouse or human kisspeptin luciferase reporter. We found that in both populations, Six3 repressed both mouse (p < 0.05) and human (p < 0.01) kisspeptin transcription. We also found that OTX2 repressed both mouse and human kisspeptin transcription in the KTaV cell line (p < 0.05), but did not alter transcription in the KTaR cell line. These results suggest that Six3 and OTX2 can regulate the kisspeptin promoter in vitro and produce a response that is consistent between both mouse and human promoters. Effects of Six3 are similar between kisspeptin populations, while OTX2 may function in a population-specific manner.

Effects of puerarin on estrogen-regulated gene expression in gonadotropin-releasing hormone pulse generator of ovariectomized rats

Effects of puerarin on the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator function is investigated, for the first time, in ovariectomized rats at the level of mRNA expression of estrogen-responsive genes, e.g., estrogen receptor (ER), GnRH and its receptor (GnRHR). Rats were treated orally for 90 days either with a soy-free diet containing two different doses of puerarin (low dose of 600 mg/kg and high dose of 3000 mg/kg) or estradiol benzoate (E2B) at either low dose (4.3 mg/kg) or high dose (17.3 mg/kg). Levels of mRNA expression in the medial preoptic area/anterior hypothalamus (MPOA/AH), mediobasal hypothalamus/median eminence (MBH/ME) and adenohypophysis were measured by quantitative TaqMan® real-time RT-PCR. Plasma levels of luteinizing hormone (LH) and prolactin (PRL) were measured by radioimmunoassay. In the MPOA/AH, both puerarin and E2B decreased ERα mRNA levels without any significant changes in ERβ and GnRH mRNA levels. Both puerarin and E2B did not significantly alter the expression levels of ERα, ERβ and GnRHR in the MBH/ME. E2B exerted significant effects on the down-regulation of adenohypophyseal GnRHR mRNA transcripts and serum LH levels. Puerarin did not cause significant changes in pituitary GnRHR mRNA transcripts and serum LH and PRL levels. This is the first study to demonstrate that in ovariectomized rat models of ovarian hormone deprivation, puerarin acted as a weak estrogen-active compound in the hypothalamic GnRH pulse generator through the downregulation of MPOA/AH ERα mRNA expression.

Role of leptin and the leptin receptor in the pathogenesis of varicocele-induced testicular dysfunction.

The present study investigated the expression of leptin and its receptor in the left testis and hypothalamus of rats with varicocele and clarified their roles in the pathogenesis of varicocele-induced testicular dysfunction. A total of 40 male rats were divided randomly into four groups. Groups 1 (G1) and 3 (G3) underwent a sham operation. Groups 2 (G2) and 4 (G4) underwent operations to form a varicocele created by partial ligation of the left renal vein. G1 and G2 rats were euthanized 4 weeks after the operation while G3 and G4 rats were euthanized at 8 weeks. The expression of leptin and its receptor was analyzed by immunohistochemistry. The mRNA levels of leptin, its receptor, kisspeptin (KiSS-1), G-protein coupled receptor 54 (GPR54), gonadotropin releasing hormone (GnRH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured by reverse transcription-quantitative polymerase chain reaction. Testicular spermatogenesis function and gonadal hormone levels were measured. Compared with G1 and G3, the expression of leptin and its receptor in rat testis was significantly higher in G2 and G4, respectively. Leptin expression was inversely associated with the number of sperm in the left epididymis, thickness of the seminiferous epithelium and the diameter of seminiferous tubules. The expression of leptin receptors in the hypothalamus of G2 and G4 was significantly increased compared with that in G1 and G3, respectively. The mRNA levels of KiSS-1, GPR54, GnRH, LH and FSH in G2 and G4 were significantly increased compared with that in G1 and G3, respectively. Serum testosterone levels in G2 and G4 rats were significantly lower than those in G1 and G3 rats, respectively. There was no significant difference between the serum levels of FSH, LH and leptin. These results suggest that leptin and its receptor may serve significant roles in the pathogenesis of varicocele-induced testicular dysfunction.

Identification and functional characterization of two Secretogranin II genes in orange-spotted grouper (Epinephelus coioides)

Secretoneurin (SN) is an important stimulator of pituitary luteinizing hormone (LH) synthesis and secretion in goldfish. It is unknown whether this neuropeptide performs the same role in other fish species. In this study, the full-length cDNAs encoding Secretogranin IIa (SgIIa) and b (SgIIb) were cloned from the brain of orange-spotted grouper. Sequence analysis showed that a 34-amino acid SN peptide (SNa) is present in SgIIa proprotein, and a 33-amino acid SN peptide (SNb) is present in SgIIb proprotein. The two SN peptides share a low degree of similarity but contain the signature YTPQ-X-LA-X7-EL sequence. Real-time PCR showed that two SgII genes are mainly expressed in the brain and pituitary. During ovarian development, the expression levels of two SgII genes in the hypothalamus and pituitary were significantly reduced at the stage when the ovary contained full-grown oocytes. The biological functions of the two SN peptides were further investigated in vitro and in vivo. Both SN peptides could significantly elevate the mRNA levels of Gonadotropin-Releasing Hormone 1 (GnRH1) and 3 (GnRH3) in the hypothalamic fragments and upregulated the expression of Follicle-Stimulating Hormone beta (FSHb) and Luteinizing Hormone beta (LHb) in the pituitary cells. The stimulatory effects on the expression of GnRHs and Gonadotropins were also observed after intraperitoneal injection of SN peptides. Our study indicated that the SgII/SN system has stimulatory effects on the reproductive axis of orange-spotted grouper.

Effects of gonadotropin-releasing hormone agonist on human chorionic gonadotropin activity in granulosa cells of immature female rats.

Although the expression of gonadotropin-releasing hormone (GnRH) in the ovaries is well established, its physiological role remains unknown. The aim of this study was to determine whether ovarian GnRH mediates the actions of human chorionic gonadotropin (hCG) in the granulosa cells of immature female rats. Follicular growth was induced by administration of pregnant mare serum gonadotropin (PMSG, 15 IU/0.15 ml) on day 25 after birth, and hCG (20 IU/0.2 ml) was administered on day 27 revealing the increase of plasma progesterone level. Primary cultures of granulosa cells were established from large follicles 2 days after PMSG treatment. Progesterone synthesis was augmented by hCG in a dose-dependent manner. Annexin A5 (ANXA5), a biomarker of GnRH, was expressed in the granulosa-luteal cells after hCG treatment, as shown by immunohistochemistry, suggesting that hCG treatment induced GnRH action. The GnRH mRNA level was increased by hCG, and treatment with GnRH agonist (GnRHa) increased ANXA5 mRNA levels in the primary cultures of granulosa cells. Concomitant incubation of GnRH (10–7 M) or GnRHa (fertirelin acetate, 10–8 M) with hCG suppressed progesterone synthesis during a 3 h incubation period. The mRNA expression of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) was synergistically stimulated and suppressed by hCG and GnRHa, respectively. GnRHa stimulated p21 expression, and GnRHa and hCG synergistically reduced the mRNA expression levels of p27 and FOXO1. These data suggest that GnRH induced by LH may have a role for the LH-mediated luteinization of granulosa cells. In addition, ANXA5 may be involved in GnRH action. GnRH-ANXA5 would be an important mechanism in cell differentiation.

GnRH mRNA levels in male three-spined sticklebacks, Gasterosteus aculeatus, under different reproductive conditions

In vertebrates, reproduction is regulated by the brain–pituitary–gonad (BPG) axis, where the gonadotropin-releasing hormone (GnRH) is one of the key components. However, very little is known about the possible role of GnRH in the environmental and feedback control of fish reproduction. To investigate this, full-length gnrh2 (chicken GnRH II) and gnrh3 (salmon GnRH) sequences of male three-spined sticklebacks (Gasterosteus aculeatus), which are clustered with the taxa of the same GnRH type as other Euteleostei, were cloned and annotated. gnrh1 is absent in this species. The mRNA levels of gnrh2 and gnrh3 in the sticklebacks' brain were measured under breeding and post-breeding conditions as well as in castrated and sham-operated breeding fish and castrated/sham-operated fish kept under long-day (LD 16:8) and short-day (LD 8:16) conditions. Fully breeding males had considerably higher mRNA levels of gnrh2 and gnrh3 in the thalamus (Th) and in the telencephalon and preoptic area (T+POA), respectively, than post-breeding males. Sham-operated breeding males have higher gnrh3 mRNA levels than the corresponding castrated males. Moreover, higher gnrh2 mRNA levels in the Th and higher gnrh3 mRNA levels in the T+POA and hypothalamus (HypTh) were also found in long-day sham-operated males than in sham-operated fish kept under an inhibitory short day photoperiod. Nevertheless, gnrh2 and gnrh3 mRNA levels were not up-regulated in castrated males kept under long-day photoperiod, which suggests that positive feedbacks on the brain-pituitary-gonad axis are necessary for this response.

A Critical Point of Male Gonad Development: Neuroendocrine Correlates of Accelerated Testicular Growth in Rats during Early Life

Testis growth during early life is important for future male fertility and shows acceleration during the first months of life in humans. This acceleration coincides with the peak in gonadotropic hormones in the blood, while the role of hypothalamic factors remains vague. Using neonatal rats to assess this issue, we found that day 9 of life is likely critical for testis development in rats. Before this day, testicular growth was proportional to body weight gain, but after that the testes showed accelerated growth. Hypothalamic kisspeptin and its receptor mRNA levels begin to elevate 2 days later, at day 11. A significant increase in the mRNA levels for gonadotropin-releasing hormone (GnRH) receptors in the hypothalamus between days 5 and 7 was followed by a 3-fold decrease in GnRH mRNA levels in this brain region during the next 2 days. Starting from day 9, hypothalamic GnRH mRNA levels increased significantly and positively correlated with accelerated testicular growth. Triptorelin, an agonist of GnRH, at a dose that had no effect on testicular growth during “proportional” period, increased testis weights during the period of accelerated growth. The insensitivity of testicular growth to GnRH during “proportional” period was supported by inability of a 2.5-fold siRNA knockdown of GnRH expression in the hypothalamus of the 7-day-old animals to produce any effect on their testis weights. GnRH receptor blockade with cetrorelix was also without effect on testis weights during “proportional” period but the same doses of this GnRH antagonist significantly inhibited “accelerated” testicular growth. GnRH receptor mRNA levels in the pituitary as well as plasma LH concentrations were higher during “accelerated” period of testicular growth than during “proportional” period. In general, our data defined two distinct periods in rat testicular development that are primarily characterized by different responses to GnRH signaling.

Molecular cloning of cDNA of gonadotropin-releasing hormones in the Chinese sturgeon (Acipenser sinensis) and the effect of 17β-estradiol on gene expression

The Chinese sturgeon, Acipenser sinensis, is a rare and large-sized anadromous bony fish and understanding of its reproductive regulation is a precondition for controlled reproduction. In this study, two gonadotropin-releasing hormone (GnRH) precursor cDNAs, AsGnRH1 (mammalian type) and AsGnRH2 (chicken type 2), were sequenced in A. sinensis. The precursor cDNAs of the AsGnRH1 and AsGnRH2 are 381 and 649 base pairs (bp), encoding signal peptide plus precursors of 92 and 86 amino acids, respectively. Multiple sequence alignment suggests that AsGnRH1 and AsGnRH2 decapeptides are highly conserved among vertebrates. Besides, AsGnRH1 had closer evolutionary relationship with tetrapods, while AsGnRH2 was conservatively grouped with teleosts in the phylogenetic analysis. Tissue distribution analysis shows that AsGnRH2 is exclusively transcribed in the brain, whereas AsGnRH1 exhibits more extensive tissue distribution including brain, liver, spleen and gonad. Furthermore, Chinese sturgeons were subcutaneously implanted with 17β-estradiol (E2) and the effect of E2 on brain GnRH mRNA levels was evaluated by real-time PCR. A significant increase in AsGnRH1 and AsGnRH2 mRNA levels is detected in fish receiving E2 implantation compared to controls after one month (P<0.05). These results indicate that E2 exerts positive feedback effects on the transcription of the two GnRHs in immature Chinese sturgeon.

The phytoestrogen daidzein may affect reproductive performance of Zhedong White geese by regulating gene mRNA levels in the HPG axis

1. The effect of daidzein, a naturally occurring phytoestrogen, on the reproductive performance of 120 female Zhedong White geese was determined. The geese were divided into 4 groups which were fed on diets containing 0 (Control), 10 (Da1), 20 (Da2) and 30 (Da3) mg daidzein per kg diet. Egg production and weight, fertility and hatchability rates, concentrations of estradiol (E2), triiodothyronine (T3), progesterone (P4), thyroxine (T4) and growth hormone (GH) in serum, and mRNA levels of gonadotropin releasing hormone (GnRH), β-follicle stimulating hormone (FSHβ), follicle stimulating hormone receptor (FSHR), oestrogen receptor1 (ESR1), oestrogen receptor2 (ESR2), prolactin (PRL), prolactin receptor (PRLR), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the hypothalamus-pituitary-gonadal axis (HPGA) were measured.2. Daidzein increased egg weight and fertility but had no detectable effect on egg production and hatchability.3. Daidzein affected serum P4 and GH concentrations and T4 rhythm, up-regulated GnRH mRNA and PRLR mRNA levels in the hypothalamus, down-regulated PRLR mRNA in the hypothalamus, PRL mRNA in the pituitary, and ESR2 mRNA levels in the ovary, respectively. The mRNA rhythms of PRLR in the hypothalamus, PRL, PRLR and FSHβ in the pituitary, FSHR, ESR1 and ESR2 in the ovary were significantly changed in the Da2 group.4. It is suggested that an appropriate dose of daidzein might improve reproductive performance by affecting serum hormone concentrations and rhythms and regulating gene mRNA levels in the HPGA of female Zhedong White geese.

Early-life citalopram-induced impairments in sexual behavior and the role of androgen receptor

Postnatal treatment with selective serotonin reuptake inhibitors (SSRIs) has been found to affect brain development and the regulation of reproduction in rodent models. The normal masculinization process in the brain requires a transient decrease in serotonin (5-HT) levels in the brain during the second postnatal week. Strict regulation of androgen receptor (AR) and gonadotropin-releasing hormone (GnRH) expression is important to control male reproductive activity. Therefore, this study was designed to examine the effects of a potent SSRI (citalopram) on male sexual behavior and expression levels of AR and GnRH in adult male mice receiving either vehicle or citalopram (10mg/kg) daily during postnatal days 8–21. The citalopram-treated male mice showed altered sexual behavior, specifically a significant reduction in the number of intromissions preceding ejaculation compared with the vehicle-treated mice. The citalopram-treated male mice displayed elevated anxiety-like behavior in an open field test and lower locomotor activity in their home cage during the subjective night. Although there was no change in GnRH and AR mRNA levels in the preoptic area (POA), quantified by real-time polymerase chain reaction, immunostained AR cell numbers in the medial POA were decreased in the citalopram-treated male mice. These results suggest that the early-life inhibition of 5-HT transporters alters the regulation of AR expression in the medial POA, likely causing decreased sexual behavior and altered home cage activity in the subjective night.

Screening estrogenic activity of environmental contaminants and water samples using a transgenic medaka embryo bioassay

Many natural or synthetic chemicals may act as exogenous estrogens and affect the reproductive health of humans and wildlife. Since these xenoestrogens are ubiquitous, it is essential to monitor their presence in the environment.Hence, we developed a bioassay using the transgenic medaka (Oryzias latipes) embryo, in which the green fluorescent protein (GFP) was placed under the control of the gnrh3 promoter, one of the three paralogous gonadotropin-releasing hormone (GnRH) genes that regulate reproductive function and behavior. As medaka embryos are transparent, the fluorescent expression of GFP can be easily observed in vivo during development.We exposed newly fertilized medaka embryos to varying solutions of bisphenol A (BPA), nonylphenol (NP), 17β-estradiol (E2), or a river water sample, and monitored their development. During embryonic development, the mRNA levels of GnRHs, GnRH receptors, and estrogen receptors (ERs) were measured with quantitative real-time reverse transcription-PCR.Our results showed that the chemicals and the river water significantly decreased the fluorescent intensity of the GnRH3 neurons, postponed the eye development, and retarded the growth of the embryos. The three xenoestrogens also lowered the heart rate, lengthened the time to hatch, suppressed the expression of the three GnRH genes, and up-regulated the ERα mRNA level. In addition, the GnRH3 mRNA level was significantly correlated with the fluorescence intensity of the GnRH neurons.We concluded that the transgenic medaka embryo is a rapid and sensitive bioassay for screening environmental water samples. We also found that xenoestrogens had significant effects on GnRH gene expression and embryonic development.