Synthesis Of Gonadotropin-releasing Hormone Research Articles

Integrative proteomic and transcriptomic analysis in the female goat ovary to explore the onset of puberty

Puberty is considered a prerequisite for affecting reproductive performance and productivity. Little was known about molecular changes in pubertal goat ovaries. Therefore, we measured and performed a correlation analysis of the mRNA and proteins changes in the pre-pubertal and pubertal goat ovaries. The results showed that only six differentially expressed genes and differentially abundant proteins out of 18,139 genes and 7550 proteins quantified had significant correlations. CNTN2 and THBS1, discovered in the mRNA-mRNA interaction network, probably participated in pubertal and reproductive regulation by influencing GnRH receptor signals, follicular development, and ovulation. The predicted core transcription factors may either promote or inhibit the expression of reproductive genes and act synergistically to maintain normal reproductive function in animals. The interaction between PKM and TIMP3 with other proteins may impact animal puberty through energy metabolism and ovarian hormone secretion. Pathway enrichment analyses revealed that the co-associated key pathways between ovarian genes and proteins at puberty included calcium signalling pathway and olfactory transduction. These pathways were associated with gonadotropin-releasing hormone synthesis and secretion, signal transmission, and cell proliferation. In summary, these results enriched the potential molecules and signalling pathways that affect puberty and provided new insights for regulating and promoting the onset of puberty. SignificanceThis study conducted the first transcriptomic and proteomic correlation analysis of pre-pubertal and pubertal goat ovaries and identified six significantly correlated molecules at both the gene and protein levels. Meanwhile, we were drawn to several molecules and signalling pathways that may play a regulatory role in the onset of puberty and reproduction by influencing reproductive-related gene expression, GnRH receptor signals, energy metabolism, ovarian hormone secretion, follicular development, and ovulation. This information contributed to identify potential biomarkers in pubertal goat ovaries, which was vital for predicting the onset of puberty and improving livestock performance.

A classification of genes involved in normal and delayed male puberty.

Puberty is a pivotal biological process that completes sexual maturation to achieve full reproductive capability. It is a major transformational period of life, whose timing is strongly affected by genetic makeup of the individual, along with various internal and external factors. Although the exact mechanism for initiation of the cascade of molecular events that culminate in puberty is not yet known, the process of pubertal onset involves interaction of numerous complex signaling pathways of hypothalamo-pituitary-testicular (HPT) axis. We developed a classification of the mechanisms involved in male puberty that allowed placing many genes into physiological context. These include (i) hypothalamic development during embryogenesis, (ii) synaptogenesis where gonadotropin releasing hormone (GnRH) neurons form neuronal connections with suprahypothalamic neurons, (iii) maintenance of neuron homeostasis, (iv) regulation of synthesis and secretion of GnRH, (v) appropriate receptors/proteins on neurons governing GnRH production and release, (vi) signaling molecules activated by the receptors, (vii) the synthesis and release of GnRH, (viii) the production and release of gonadotropins, (ix) testicular development, (x) synthesis and release of steroid hormones from testes, and (xi)the action of steroid hormones in downstream effector tissues. Defects in components of this system during embryonic development, childhood/adolescence, or adulthood may disrupt/nullify puberty, leading to long-term male infertility and/or hypogonadism. This review provides a list of 598 genes involved in the development of HPT axis and classified according to this schema. Furthermore, this review identifies a subset of 75 genes for which genetic mutations are reported to delay or disrupt male puberty.

Photoperiodic regulation of avian physiology: From external coincidence to seasonal reproduction.

Seasonal cycles of environmental cues generate variation in the timing of life-history transition events across taxa. It is through the entrainment of internal, endogenous rhythms of organisms to these external, exogenous rhythms in environment, such as cycling temperature and daylight, by which organisms can regulate and time life history transitions. Here, we review the current understanding of how photoperiod both stimulates and terminates seasonal reproduction in birds. The review describes the role of external coincidence timing, the process by which photoperiod is proposed to stimulate reproductive development. Then, the molecular basis of light detection and the photoperiodic regulation of neuroendocrine timing of seasonal reproduction in birds is presented. Current data indicates that vertebrate ancient opsin is the predominant photoreceptor for light detection by the hypothalamus, compared to neuropsin and rhodopsin. The review then connects light detection to well-characterized hypothalamic and pituitary gland molecules involved in the photoperiodic regulation of reproduction. In birds, Gonadotropin-releasing hormone synthesis and release are controlled by photoperiodic cues via thyrotropin-stimulating hormone-β (TSHβ) independent and dependent pathways, respectively. The review then highlights the role of D-box and E-box binding motifs in the promoter regions of photoperiodic genes, in particular Eyes-absent 3, as the key link between circadian clock function and photoperiodic time measurement. Based on the available evidence, the review proposes that at least two molecular programs form the basis for external coincidence timing in birds: photoperiodic responsiveness by TSHβ pathways and endogenous internal timing by gonadotropin synthesis.

Effects of melatonin on the production of GnRH and LH in luteal cells of pregnant sows.

Effects of melatonin on the release and synthesis of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) at the hypothalamus and pituitary levels have been explored in some species, but a similar study in the corpora lutea (CL) has not yet been conducted. In this study, the immunostaining for GnRH and LH was observed in luteal cells of porcine CL during pregnancy, and a significant effect of pregnant stage on the level of GnRH and LH was found; higher values for GnRH and LH immunostaining and mRNA were detected in the early and mid-stages CL than in the later-stage CL (P < 0.01). Furthermore, the patterns of melatonin membrane receptors (MT1 and MT2) expression were consistent with those of GnRH and LH expression in the CL of pregnant sows; the relative levels of MT1 and MT2 in the early and mid-stages were significantly higher than those in the later-stage (P < 0.01). In luteal cells, melatonin dose-dependently increased in GnRH and LH secretion and mRNA expression. Melatonin also increased the GnRH-induced accumulation of LH and the LH-induced secretion of P4 in luteal cells. Additionally, the effects of melatonin on luteal GnRH and LH production were blocked by luzindole, a non-selective MT1 and MT2 receptor antagonist. Our results demonstrate the stimulatory effects of melatonin on GnRH and LH production in luteal cells of pregnant sows, suggesting a potential role for melatonin in luteal function through regulating the release and synthesis of GnRH and LH in luteal cells.

Gonadotropin-releasing hormone stimulation of annexin A5 expression in the thymus of male rats.

As gonadotropin-releasing hormone (GnRH) is expressed in the thymus, its direct action on thymic cells, including thymic involution, has been suggested. Annexin A5 (ANXA5), a biomarker of GnRH, was used to determine whether GnRH affects the thymus of male rats. Immunohistochemistry showed positive reactions for ANXA5 in large medullary epithelial cells at 30 days of age, and the expression continued until 180 days of age. Organ culture of thymus pieces was performed to examine the direct action of a GnRH agonist (GnRHa) on the expression of Anxa5 and Gnrh mRNA. Thymus tissues obtained from male rats (40–60 days old) were cut into small pieces (2–3 mm3) and incubated for 3 hr with the GnRHa. The expression levels of Anxa5 and Gnrh mRNA were augmented by the GnRHa. Immunohistochemistry of these tissue fragments showed that ANXA5 expression was enhanced, especially in medullary epithelial cells. These results revealed that GnRH synthesis in the thymus could affect thymic epithelial cells after puberty.

Aging reduces kisspeptin receptor (GPR54) expression levels in the hypothalamus and extra-hypothalamic brain regions.

Aging leads to the diminished pulsatile secretion of hypothalamic gonadotropin-releasing hormone (GnRH). Kisspeptin (Kp), the upstream regulator of the hypothalamic-pituitary-gonadal (HPG) axis, regulates GnRH synthesis and release through its cognate receptor, G-protein coupled receptor 54 (GPR54). In turn, GnRH regulates GPR54 expression. GnRH administration into the third ventricle has been shown to induce neurogenesis in different brain regions in old age. However, aging-associated changes in hypothalamic and extra-hypothalamic GPR54 expression were unclear. Therefore, the expression levels of GPR54 were evaluated in various brain regions of adult (age, 3-4 months) and old (age, 20-24 months) male Wistar rats in the present study. In the hypothalamus, mRNA and protein levels of Kp and GPR54 were identified to be significantly decreased in old age. Furthermore, GnRH1 expression in the hypothalamus was analyzed to observe the functional consequence of a reduced Kp-GPR54 system in the hypothalamus. It was found that hypothalamic GnRH1 levels were significantly decreased in old age. As GnRH regulates GPR54 levels, GPR54 was examined in extra-hypothalamic regions. GPR54 levels were found to be significantly decreased in the hippocampus and medulla and pons in old-age rats when compared to adult rats. Notably, GPR54 expression was observed in the frontal lobe, cortex, midbrain and cerebellum of adult and old-age rats; however, the difference between the two groups was not statistically significant. To the best of our knowledge, this is the first study that provides the quantitative distribution of GPR54 in different brain regions during aging. Thus, the reduced levels of Kp and its receptor, GPR54 in the hypothalamus could be cumulatively responsible for reduced levels of GnRH observed in old age.

Plasma level and expression of visfatin in the porcine hypothalamus during the estrous cycle and early pregnancy

Visfatin appears to be an energy sensor involved in the regulation of female fertility, which creates a hormonal link integrating the control of energy homeostasis and reproduction. This study evaluates the expression levels of visfatin gene and protein in selected areas of the porcine hypothalamus responsible for gonadotropin-releasing hormone synthesis: the mediobasal hypothalamus (MBH) and preoptic area (POA), and visfatin concentrations in the blood plasma. The tissue samples were harvested from gilts on days 2–3, 10–12, 14–16, and 17–19 of the estrous cycle, and on days 10–11, 12–13, 15–16, 27–28 of pregnancy. Visfatin was localized in the cytoplasm and nucleus of cells creating both studied hypothalamic structures. The study demonstrated that visfatin gene and protein expression in MBH and POA depends on hormonal status related to the phase of the estrous cycle or early pregnancy. Blood plasma concentrations of visfatin during the estrous cycle were higher on days 2–3 in relation to other studied phases of the cycle, while during early pregnancy, the highest visfatin contents were observed on days 12–13. This study demonstrated visfatin expression in the porcine hypothalamus and its dependence on the hormonal milieu related to the estrous cycle and early pregnancy.

Anatomopathological Evaluation of Young Bulls (Bos taurus) Testicles Submitted to Immunocastration

Background: Immunocastration is a less invasive and painless procedure compared to surgical castration, which causes greater stress to animals, especially when performed improperly. Immunocastration stimulates the production of antibodies against Gonadotropin-Releasing Hormone (GnRH), temporarily blocking the hormone production by the male gonads. Anatomopathological evaluation of animals submitted to immunocastration with the establishment of different degrees of testicular degeneration helps to evaluate the efficacy of the different dose ranges used for this procedure. Because of the scarcity of information about this procedure in young bulls, the study aimed to compare the immunocastrated and non-castrated animals.Materials, Methods &amp; Results: Eighteen Angus-Limousin bulls were used from weaning (7 ± 1 months) to slaughtering (15 ± 1 months) with the establishment of two groups composed of nine non-castrated (NC) and nine immunocastrated (IC) animals. In the IC group, three doses of GnRH synthesis inhibitor vaccine (Bopriva®) were applied on days 0 (weaning), 87 and 223. At slaughter, scrotal circumference, weight (kg), width (cm), length (cm) and total size (width x length) of the testicles were obtained, then were routinely processed for histopathological analysis. Four degrees of testicular degeneration was established: grade 0 (no changes), grade 1 (mild), grade 2 (moderate) and grade 3 (severe). Statistical analysis was performed using Student's t-test to compare the means of macroscopic variables. The scrotal circumference started to decrease in the IC animals on day 132, with a certain growth from day 194. There was a significant difference between the two groups for width (P = 0.001), length (P = 0.004), total size (P = 0.003) and scrotal circumference (P = 0.04). Testicles of the IC group tended to be lighter (P = 0.06). The final difference between the groups was 6.3 cm for scrotal perimeter, with respective averages of 27.44 cm and 33.77 cm for the IC and NC group, respectively. In the microscopic evaluation, NC animals obtained grade 0. In the IC animals, 33.33% (3/9) had degeneration grade 1 with slight basement membrane tortuosity and Sertoli cell atrophy, 33.33% (3/9) obtained grade 2 with moderate tortuosity and thickening of seminiferous tubules, Sertoli and Leydig cell atrophy, and absence of spermatogenesis and 33.33% (3/9) reached grade 3, with decreased seminiferous tubules and a severe and diffuse atrophy of Leydig and Sertoli cells and complete absence of spermatogenesis, with a 1:5 ratio in seminiferous tubule diameter between NC and IC grade 3 animals, respectively.Discussion: There was a significant difference in several testicular macroscopic aspects between the two groups, similar to values using eight months old field-bred animals, with a difference of 8.78 cm between groups, also with the use of three doses of Bopriva. The only macroscopic variable that showed just a tendency of difference between the groups was testicular weight, whereas, in a study with crossbred animals with nine months old, there was a difference of 152 g in testicular weight between groups. Histologically, in immunocastrated two-year-old Nelore cattle, 85% demonstrated a total absence of spermatogenesis and 15% had a partial reduction, suggesting functional recovery of Sertoli cells after the last vaccination booster or partial inhibition of spermatogenesis. In this study, 90-day dose intervals produced effective immunization during 105 days, which is the suggested period for the third dose. The anatomopathological analysis proved to be effective for the comparison between the groups and the degree of the promoted degeneration.

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.

Identification of a promoter element mediating kisspeptin-induced increases in GnRH gene expression in sheep

Gonadotropin-releasing hormone (GnRH) plays an important role in regulating the activities of other components downstream of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining the normal reproductive cycle of animals. However, the molecular mechanisms by which GnRH synthesis and secretion are regulated in sheep remains unclear. In this study, a series of eight recombinant vectors with deletion fragments were constructed and cotransfected with pGL3-Basic and pRL-SV40 into sheep hypothalamic neuronal cells. After treatment with 1 nM kisspeptin, the core promoter of the sheep GnRH gene was identified to be in the region of −1912 bp to −1461 bp by dual-luciferase reporter assay. Bioinformatics analysis showed that there was a binding site for the transcription factor Otx-2 in the core promoter region (−1786 to −1770 bp) that was highly conserved among different species. The expression patterns of Kiss-1, Otx-2 and GnRH in the sheep hypothalamus were the same, and the expression of Kiss-1, Otx-2 and GnRH was significantly higher in the breeding season than in nonbreeding season (P < 0.01). In addition, when hypothalamic neurons were cultured in vitro with kisspeptin, kisspeptin induced the expression of GnRH and Otx-2. In conclusion, these results provide evidence that the core promoter region (−1786 to −1770 bp) of the GnRH gene is involved in the regulation of hypothalamic activity by kisspeptin and that binding of the transcription factor Otx-2 mediates this activation.

The effect of manganese exposure on GnRH secretion via Nrf2/mGluR5/COX-2/PGE2/signaling pathway.

There are limited studies focused on the precise mechanism of gonadotropin-releasing hormone (GnRH) secretion dysfunction after overexposure to manganese (Mn). The objective of the present study was to explore the mechanism of Mn disruption of GnRH synthesis via nuclear factor erythroid-2-related factor-2 (Nrf2)/metabotropic glutamate receptor-5 (mGluR5)/cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) signaling pathway in vitro and in vivo. Primary astrocytes were cultured and treated with different doses of Mn, tert-butylhydroquinonet (tBHQ; Nrf2 agonists), 3-[(2-methyl-4-thaizolyl) ethynyl] pyridine (MTEP; mGluR5 inhibitor), and celecoxib (COX-2 inhibitor) to measure the levels of COX-2, mGluR5, Nrf2, and Nrf2 target genes. Mice were randomly divided into 11 groups, of which included the control group, 12.5-, 25-, and 50-mg/kg MnCl2 group, dimethyl sulfoxide (DMSO) group, tBHQ control group, tBHQ pretreatment group, MTEP control group, MTEP pretreatment group, celecoxib control group, and celecoxib pretreatment group. The injection was administered every day for 2 weeks. Then, levels of GnRH, PGE2, COX-2, mGluR5, Nrf2, Nrf2 target genes, and morphological changes in the hypothalamus of mice were measured. Mn reduced protein levels of Nrf2 and mRNA expression of Nrf2 target genes and increased mGluR5, COX-2, PGE2, and GnRH levels. Meanwhile, injury-related histomorphology changes in the hypothalamus of mice were significantly present. In conclusion, excessive exposure to Mn disrupts GnRH secretion through Nrf2/mGluR5/COX-2/PGE2 signaling pathway.

Microcystin-leucine arginine inhibits gonadotropin-releasing hormone synthesis in mice hypothalamus

Microcystin-leucine arginine (MC-LR) causes serum testosterone declines and male reproductive disorders. However, the molecular mechanisms underlying the pathological changes are still unclear. In the present study, we aimed to investigate the toxic effects of MC-LR on gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus. Our results demonstrated that MC-LR could enter GnRH neurons and inhibit GnRH synthesis, resulting in the decrease of serum GnRH and testosterone levels. The inhibitory effects of MC-LR on GnRH synthesis were identified to be associated with activation of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB)/c-Fos signaling pathway. With miRNA microarray analyses, we found that miR-329-3p was down-regulated most dramatically in MC-LR-treated GT1–7 cells. We then further identified that miR-329-3p regulated PRKAR1A and PRKACB expression and thus influenced GnRH synthesis. This is the first study to explore the molecular mechanism underlying the inhibitory effects of MC-LR on GnRH synthesis in the hypothalamus. Our data have provided a new perspective in the development of diagnosis and treatment strategies for male infertility as a result of dysfunction of the hypothalamic-pituitary-gonadal axis.

Microcystin-LR reduces the synthesis of gonadotropin-releasing hormone by activating multiple signaling pathways resulting in decrease of testosterone in mice

We previously reported Microcystin-LR (MC-LR) could enter the hypothalamus, reduce the expression of gonadotropin-releasing hormone (GnRH), and induce male reproductive barriers. However, the molecular mechanisms underlying in the hypothalamus have not been elucidated in detail. In this study, we further showed that MC-LR inhibited the synthesis of GnRH in GnRH neurons via activating protein kinase a (PKA), cAMP-response element binding protein (Creb), protein kinase c (PKC), nuclear factor kappa B (NF-κB), extracellular regulated protein kinases (Erk) and P38 protein, and thus resulted in the change of activity of transcriptional enhancers or suppressors such as Oct-1, Otx-2, Pbx1a, Dlx-2, c-Jun and c-Fos. Following exposure, MC-LR-treated mice exhibited decreased GnRH level. Our data demonstrated that MC-LR can stimulate intracellular Ca2+ and cAMP to activate PKC, PKA and MAPK signaling pathways in GnRH neurons, and then inhibit Pbx1a, Oct-1, Dlx-2, Otx-2 and upregulate c-Jun and c-Fos to initiate the transcription of GnRH, which provides novel insights to explore the mechanism associated with MC-LR-induced male reproductive barriers.

Histological and Morphometric Examination of the Testes of Boars and Male Pigs Immunocastrated with Improvac®

Background: Surgical castration of male piglets is frequently performed to prevent development of an unpleasant taste and flavor in meat of mature males, called boar taint. Disadvantages of this method are primarily related to the lower feed efficiency of surgically castrated male pigs, production of less lean meat and more fat on the carcasses than boars, as well as ethical issues associated with the procedure itself. Immunocastration is a method of immunization against gonadotropinreleasing hormone (GnRH) and represents an alternative to avoid boar taint. This study aimed to examine the influence of immunocastration on histological and morphometric parameters of male pig testes.Materials, Methods &amp; Results: A total of 60 crossbred male pigs selected from 30 litters were assigned to two experimental groups: boars (n = 30) and immunocastrated males (n = 30). Two doses (2 mL per dose) of the Improvac® vaccine were given to pigs scheduled for immunocastration. The first injection was given at 10 weeks of age, and the second injection at 20 weeks of age. All pigs were fed the same commercial diet ad libitum, group-housed (15 pigs/pen) and slaughtered at 25 weeks of age. After slaughter, testicle weights were measured and samples of testicular tissue were taken for histological examination. Immunocastrated males had lower testicle weights than boars (P &lt; 0.01). Furthermore, structural damage to the germinal epithelium was visible in the seminiferous tubules of vaccinated pigs. In the testicular interstitium of these animals, the number of Leydig cells were reduced; almost all of these cells exhibited different degrees of abnormality. Moreover, the diameter of the seminiferous tubules and thickness of the germinal epithelium were greater (P &lt; 0.01), and the percentage of germinal epithelium in the seminiferous tubules as well as percentage of blood vessels in the interstitium were higher in boars as compared to immunocastrated males (P &lt; 0.01). Seminiferous tubule lumen diameter was greater (P &lt; 0.01), and percentage of lumen in the seminiferous tubules as well as percentage of non-blood vessel tissue in the interstitium were higher in immunocastrated males than boars (P &lt; 0.01).Discussion: Vaccination against GnRH, as used in this study, reduced the synthesis of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which led to decreased spermatogenesis and androgen production. The smaller diameter of the seminiferous tubules as well as germinal epithelium thickness, and lower percentage of germinal epithelium in the seminiferous tubules in immunocastrated males could be explained by reduced FSH synthesis, and subsequent reduced spermatogenesis. The described testicular malformations in immunocastrated animals caused their lower weight, and greater diameter of the lumen as well as a higher lumen percentage of the seminiferous tubules. The vaccine reduced LH synthesis, so the Leydig cells of immunocastrated animals had lower activity than the same cells in boars. The percentage of blood vessels in the interstitium of boar testes was higher, which was related to the blood supply required for normal function of Leydig cells. It was concluded that differences existed in the structure of the seminiferous tubules and interstitium between boars and immunocastrated males , which were reflected in values of the examined histological and morphometric parameters. The development of these differences was related to the inhibition of GnRH synthesis and secretion.

ERα and GnRH co-localize in the hypothalamic neurons of the South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

Gonadotropin-releasing hormone (GnRH) is the key regulator of the hypothalamic-pituitary-gonadal axis. Estradiol (E2) affects GnRH synthesis and delivery. Hypothalamic estrogen receptors (ER) modulate GnRH expression acting as transcription factors. The South American plains vizcacha, Lagostomus maximus, is able to ovulate up to 800 oocytes per reproductive cycle, and shows continuous folliculogenesis with pre-ovulatory follicle formation and an ovulatory event at mid-gestation. The aim of this work was to analyze the hypothalamic expression of ER in the vizcacha at different gestational time-points, and its relationship with GnRH expression, serum luteinizing hormone (LH) and E2. The hormonal pattern of mid-gestating vizcachas was comparable to ovulating-females with significant increases in GnRH, LH and E2. Hypothalamic protein and mRNA expression of ERα varied during pregnancy with a significant increase at mid-gestation whereas ERβ mRNA expression did not show significant variations. Hypothalamic immunolocalization of ERα was observed in neurons of the diagonal band of Brocca, medial preoptic area (mPOA), periventricular, suprachiasmatic, supraoptic (SON), ventromedial, and arcuate nuclei, and medial eminence, with a similar distribution throughout gestation. In addition, all GnRH neurons of the mPOA and SON showed ERα expression with no differences across the reproductive status. The correlation between GnRH and ERα at mid-gestation, and their co-localization in the hypothalamic neurons of the vizcacha, provides novel information compared with other mammals suggesting a direct action of estrogen as part of a differential reproductive strategy to assure GnRH synthesis during pregnancy.

Peripheral Inhibitor of AChE, Neostigmine, Prevents the Inflammatory Dependent Suppression of GnRH/LH Secretion during the Follicular Phase of the Estrous Cycle.

The study was designed to test the hypothesis that the inhibition of acetylcholinesterase (AChE) activity at the periphery by Neostigmine (0.5 mg/animal) will be sufficient to prevent inflammatory dependent suppression of the gonadotropin-releasing hormone (GnRH)/luteinising hormone (LH) secretion in ewes in the follicular phase of the estrous cycle, and this effect will be comparable with the systemic AChE inhibitor, Donepezil (2.5 mg/animal). An immune/inflammatory challenge was induced by peripheral administration of lipopolysaccharide (LPS; 400 ng/kg). Peripheral treatment with Donepezil and Neostigmine prevented the LPS-induced decrease (P < 0.05) in LHβ gene expression in the anterior pituitary gland (AP) and in LH release. Moreover, Donepezil completely abolished (P < 0.05) the suppressory effect of inflammation on GnRH synthesis in the preoptic area, when pretreatment with Neostigmine reduced (P < 0.05) the decrease in GnRH content in this hypothalamic structure. Moreover, administration of both AChE inhibitors diminished (P < 0.05) the inhibitory effect of LPS treatment on the expression of GnRH receptor in the AP. Our study shows that inflammatory dependent changes in the GnRH/LH secretion may be eliminated or reduced by AChE inhibitors suppressing inflammatory reaction only at the periphery such as Neostigmine, without the need for interfering in the central nervous system.

Microcystin-LR causes sexual hormone disturbance in male rat by targeting gonadotropin-releasing hormone neurons

Microcystin-LR (MC-LR), which generates strong reproductive toxicity, reduces serum testosterone level without directly damaging Leydig cells. The purpose of this study was to identify the target cells of MC-LR in the rat hypothalamic-pituitary axis and to investigate the underlying mechanisms. We found that the gonadotropin-releasing hormone (GnRH) neurons were the direct target of MC-LR. In vivo results showed that upon exposure to rats, cells around the third ventricle of hypothalamus underwent apoptosis. Gnrh1 expression was steadily decreased. The serum levels of GnRH, luteinizing hormone, follicle-stimulating hormone and testosterone showed a similar pattern of early-stage increase and late-stage decline. After treatment with MC-LR in immortalized hypothalamic (GT1-7) neurons, increase in cellular Ca2+ and ATP resulted in downregulation of the transcriptional activators Oct-1, Otx-2, Pbx1a and Dlx2, and upregulation of the transcriptional repressor c-Jun, which may mechanistically account for the downregulation of GnRH synthesis. Ca2+ and ATP could also stimulate the release of GnRH, contributing to its unique serum release pattern.

Fgf8-Deficient Mice Compensate for Reduced GnRH Neuronal Population and Exhibit Normal Testicular Function.

Gonadotropin-releasing hormone (GnRH) is critical for the onset and maintenance of reproduction in vertebrates. The development of GnRH neurons is highly dependent on fibroblast growth factor (Fgf) signaling. Mice with a hypomorphic Fgf8 allele (Fgf8 Het) exhibited a ~50% reduction in GnRH neuron number at birth. Female Fgf8 Het mice were fertile but showed significantly delayed puberty. However, it was unclear if these mice suffered additional loss of GnRH neurons after birth, and if male Fgf8 Het mice had normal pubertal transition and testicular function. In this study, we examined postnatal GnRH neuron number and hypothalamic GnRH content in Fgf8 Het mice from birth to 120 days of age. Further, we examined seminal vesicle and testicular growth, testicular histology, and circulating luteinizing hormone (LH) around and after pubertal transition. Our results showed that GnRH neuron numbers were significantly and consistently reduced in Fgf8 Het mice of both sexes in all ages examined, suggesting these animals were born with an inherently defective GnRH system, and no further postnatal loss of GnRH neurons had occurred. Despite an innately compromised GnRH system, male and female Fgf8 mice exhibited normal levels of immunoassayable hypothalamic GnRH peptide at all ages examined except on 60 days of age, suggesting increased GnRH synthesis or reduced turnover as a compensatory mechanism. Fgf8 Het males also had normal seminal vesicle and testicular mass/body mass ratios, testicular histology, and circulating LH. Overall, our data speak to the extraordinary ability of a GnRH system permanently compromised by developmental defect to overcome pre-existing deficiencies to ensure pubertal progression and reproduction.

Serotonin reuptake inhibitor citalopram inhibits GnRH synthesis and spermatogenesis in the male zebrafish.

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants for the treatment of depression. However, SSRIs cause sexual side effects such as anorgasmia, erectile dysfunction, and diminished libido that are thought to be mediated through the serotonin (5-hydroxytryptamine, 5-HT) system. In vertebrates, gonadotropin-releasing hormone (GnRH) neurons play an important role in the control of reproduction. To elucidate the neuroendocrine mechanisms of SSRI-induced reproductive failure, we examined the neuronal association between 5-HT and GnRH (GnRH2 and GnRH3) systems in the male zebrafish. Double-label immunofluorescence and confocal laser microscopy followed by three-dimensional construction analysis showed close associations between 5-HT fibers with GnRH3 fibers and preoptic-GnRH3 cell bodies, but there was no association with GnRH2 cell bodies and fibers. Quantitative real-time PCR showed that short-term treatment (2 wk) with low to medium doses (4 and 40 μg/L, respectively) of citalopram significantly decreased mRNA levels of gnrh3, gonadotropins (lhb and fshb) and 5-HT-related genes (tph2 and sert) in the male zebrafish. In addition, short-term citalopram treatment significantly decreased the fluorescence density of 5-HT and GnRH3 fibers compared with controls. Short-term treatment with low, medium, and high (100 μg/L) citalopram doses had no effects on the profiles of different stages of spermatogenesis, while long-term (1 mo) citalopram treatment with medium and high doses significantly inhibited the different stages of spermatogenesis. These results show morphological and functional associations between the 5-HT and the hypophysiotropic GnHR3 system, which involve SSRI-induced reproductive failures.

GnRH level regulation in the hypothalamus of female rats of different age

In recent years the study of age-related derangements of the gonadotropin releasing hormone (GnRH) synthesis and secretion resulting from both the GnRH gene expression changes and interaction of glia with GnRH-ergic neurons of the hypothalamus is a focus of attention. It was demonstrated earlier that this could result from the decreased activity of monoaminergic and peptidergic systems that control the GnRH preovulatory secretion surge initiation, specifically from the loss of the signal coming from the suprachiasmatic nuclei (SCN) of the hypothalamus. This signal is critical for the emerging of the GnRH regular cyclic secretion, which is mediated prominently by vasoactive intestinal peptide (VIP). We have studied age-related changes in the biogenic amines and VIP content in the hypothalamic structures responsible for the GnRH synthesis and secretion. It has been shown by us that the GnRH level in the median eminence with the arcuate nuclei (ME-Arc) of the hypothalamus of 22-month-old rats is half as high compared to that of 7-8-month-old animals. Beside that, the VIP level in the SCN tended to decrease, with the norepinephrine, dopamine, and 5-hydroxyindoleacetic acid levels decreased significantly in the median preoptic area of the hypothalamus responsible for the GnRH synthesis and in the ME-Arc exercising its secretion into the portal vein of the pituitary. It has been shown that the initial phase of the reproductive failure with 13-14-month-old animals having irregular estrous cycles is characterized by gradual disappearance of the normal biogenic amine diurnal dynamics in the studied hypothalamic structures, which could be due to the loss of regulatory signals coming from the SCN.