Unraveling Hypothalamus-Pituitary dysregulation: Hypergonadotropism in F1 progeny due to prenatal exposure to hexavalent chromium.

The endocrine disruptor hexavalent chromium [Cr(VI)] is a proven reproductive toxicant. We recently demonstrated that prenatal Cr(VI) exposure causes testicular resistance to gonadotropins, resulting in hypergonadotropic hypoandrogenism in F1 rats. However, the mechanism driving hypergonadotropism in F1 rats exposed to Cr(VI) prenatally remains an enigma. Therefore, we hypothesized that 'Prenatal Cr(VI) exposure may disrupt steroid hormones-mediated negative feedback regulation of the hypothalamic GnRH, and its receptor in the pituitary of F1 rats, leading to hypergonadotropism.' We administered potassium dichromate (50, 100, or 200 mg/L) to pregnant rats through drinking water between days 9 and 14, and their male F1 offspring were euthanized at 60 days of age. Prenatal Cr(VI) exposure in F1 rats resulted in the accumulation of Cr in the hypothalamus and pituitary. Western blot detected decreased hypothalamic GnRH, Kisspeptin1, and its receptor GPR54, along with diminished ERα, AR, aromatase, and 5α reductase, and GnRH regulatory transcription factors Pit-1 and GATA-4 proteins. Immunohistochemical studies revealed increased immunopositivity of GnRH receptor, AR, 5α reductase, ERα, ERß, and aromatase proteins in the pituitary, whereas decreased Kisspeptin1, GPR54, and inhibin ß. Our findings imply that Cr(VI) exposure during the prenatal period disrupts the hypothalamic Kisspeptin-GPR54-Pit-1/GATA4-GnRH network, boosting the pituitary GnRH receptor. We conclude that prenatal exposure to Cr(VI) alters GnRH expression in the hypothalamus and its receptor in the pituitary of F1 progeny through interfering with the negative feedback effect of androgens and estrogens.

Direct evidence that KNDy neurons maintain gonadotropin pulses and folliculogenesis as the GnRH pulse generator.

The gonadotropin-releasing hormone (GnRH) pulse is fundamental for mammalian reproduction: GnRH pulse regimens are needed as therapies for infertile women as continuous GnRH treatment paradoxically inhibits gonadotropin release. Circumstantial evidence suggests that the hypothalamic arcuate KNDy neurons expressing kisspeptin (encoded by Kiss1), neurokinin B (encoded by Tac3), and dynorphin A serve as a GnRH pulse generator; however, no direct evidence is currently available. Here, we show that rescuing >20% KNDy neurons by transfecting Kiss1 inside arcuate Tac3 neurons, but not outside of these neurons, recovered folliculogenesis and luteinizing hormone (LH) pulses, an indicator of GnRH pulses, in female global Kiss1 knockout (KO) rats and that >90% conditional arcuate Kiss1 KO in newly generated Kiss1-floxed rats completely suppressed LH pulses. These results first provide direct evidence that KNDy neurons are the GnRH pulse generator, and at least 20% of KNDy neurons are sufficient to maintain folliculogenesis via generating GnRH/gonadotropin pulses.

AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) receptors are essential for reproduction and are expressed in numerous urogenital, reproductive, and non-reproductive cancers. In addition to canonical G protein-coupled receptor signaling, GnRH receptors functionally interact with several receptor tyrosine kinases. AXL is a receptor tyrosine kinase expressed in numerous tissues as well as multiple tumors. Here we tested the hypothesis that AXL, along with its endogenous ligand Gas6, impacts GnRH receptor signaling. METHODS: We used clonal murine pituitary αT3-1 and LßT2 gonadotrope cell lines to examine the effect of AXL activation on GnRH receptor-dependent signaling outcomes. ELISA and immunofluorescence were used to observe AXL and GnRH receptor expression in αT3-1 and LßT2 cells, as well as in murine and human pituitary sections. We also used ELISA to measure changes in ERK phosphorylation, pro-MMP9 production, and release of LHß. Digital droplet PCR was used to measure the abundance of Egr-1 transcripts. A transwell migration assay was used to measure αT3-1 and LßT2 migration responses to GnRH and AXL. RESULTS: We observed AXL, along with the GnRH receptor, expression in αT3-1 and LßT2 gonadotrope cell lines, as well as in murine and human pituitary sections. Consistent with a potentiating role of AXL, Gas6 enhanced GnRH-dependent ERK phosphorylation in αT3-1 and LßT2 cells. Further, and consistent with enhanced post-transcriptional GnRH receptor responses, we found that Gas6 increased the abundance of Egr-1 transcripts. Suggesting functional significance, in LßT2 cells, Gas6/AXL signaling stimulated LHß production and enhanced GnRH receptor-dependent generation of pro-MMP9 protein and promoted cell migration. CONCLUSIONS: Altogether, these data describe a novel role for AXL as a modulator of GnRH receptor signaling. Video Abstract.

Gonadotropin-Releasing Hormone Receptor (GnRHR) and Hypogonadotropic Hypogonadism.

Human sexual and reproductive development is regulated by the hypothalamic-pituitary-gonadal (HPG) axis, which is primarily controlled by the gonadotropin-releasing hormone (GnRH) acting on its receptor (GnRHR). Dysregulation of the axis leads to conditions such as congenital hypogonadotropic hypogonadism (CHH) and delayed puberty. The pathophysiology of GnRHR makes it a potential target for treatments in several reproductive diseases and in congenital adrenal hyperplasia. GnRHR belongs to the G protein-coupled receptor family and its GnRH ligand, when bound, activates several complex and tissue-specific signaling pathways. In the pituitary gonadotrope cells, it triggers the G protein subunit dissociation and initiates a cascade of events that lead to the production and secretion of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) accompanied with the phospholipase C, inositol phosphate production, and protein kinase C activation. Pharmacologically, GnRHR can be modulated by synthetic analogues. Such analogues include the agonists, antagonists, and the pharmacoperones. The agonists stimulate the gonadotropin release and lead to receptor desensitization with prolonged use while the antagonists directly block the GnRHR and rapidly reduce the sex hormone production. Pharmacoperones include the most recent GnRHR therapeutic approaches that directly correct the misfolded GnRHRs, which are caused by genetic mutations and hold serious promise for CHH treatment. Understanding of the GnRHR's genomic and protein structure is crucial for the most appropriate assessing of the mutation impact. Such mutations in the GNRHR are linked to normosmic hypogonadotropic hypogonadism and lead to various clinical symptoms, including delayed puberty, infertility, and impaired sexual development. These mutations vary regarding their mode of inheritance and can be found in the homozygous, compound heterozygous, or in the digenic state. GnRHR expression extends beyond the pituitary gland, and is found in reproductive tissues such as ovaries, uterus, and prostate and non-reproductive tissues such as heart, muscles, liver and melanoma cells. This comprehensive review explores GnRHR's multifaceted role in human reproduction and its clinical implications for reproductive disorders.

Rational Design, Synthesis and Binding Affinity Studies of Anthraquinone Derivatives Conjugated to Gonadotropin-Releasing Hormone (GnRH) Analogues towards Selective Immunosuppression of Hormone-Dependent Cancer.

Gonadotropin-releasing hormone (GnRH) is pivotal in regulating human reproduction and fertility through its specific receptors. Among these, gonadotropin-releasing hormone receptor type I (GnRHR I), which is a member of the G-protein-coupled receptor family, is expressed on the surface of both healthy and malignant cells. Its presence in cancer cells has positioned this receptor as a primary target for the development of novel anti-cancer agents. Moreover, the extensive regulatory functions of GnRH have underscored decapeptide as a prominent vehicle for targeted drug delivery, which is accomplished through the design of appropriate conjugates. On this basis, a rationally designed series of anthraquinone/mitoxantrone-GnRH conjugates (con1-con8) has been synthesized herein. Their in vitro binding affinities range from 0.06 to 3.42 nM, with six of them (con2-con7) demonstrating higher affinities for GnRH than the established drug leuprolide (0.64 nM). Among the mitoxantrone based GnRH conjugates, con3 and con7 show the highest affinities at 0.07 and 0.06 nM, respectively, while the disulfide bond present in the conjugates is found to be readily reduced by the thioredoxin (Trx) system. These findings are promising for further pharmacological evaluation of the synthesized conjugates with the prospect of performing future clinical studies.

EGF-Enhanced GnRH-II Regulation in Decidual Stromal Cell Motility through Twist and N-Cadherin Signaling.

Crucial roles in embryo implantation and placentation in humans include the invasion of the maternal decidua by extravillous trophoblasts and the motile behavior of decidual endometrial stromal cells. The effects of the epidermal growth factor (EGF) and GnRH-II in the endometrium take part in early pregnancy. In the present study, we demonstrated the coaction of EGF- and GnRH-II-promoted motility of human decidual endometrial stromal cells, indicating the possible roles of EGF and GnRH-II in embryo implantation and early pregnancy. After obtaining informed consent, we obtained human decidual endometrial stromal cells from decidual tissues from normal pregnancies at 6 to 12 weeks of gestation in healthy women undergoing suction dilation and curettage. Cell motility was evaluated with invasion and migration assays. The mechanisms of EGF and GnRH-II were performed using real-time PCR and immunoblot analysis. The results showed that human decidual tissue and stromal cells expressed the EGF and GnRH-I receptors. GnRH-II-mediated cell motility was enhanced by EGF and was suppressed by the knockdown of the endogenous GnRH-I receptor and EGF receptor with siRNA, revealing that GnRH-II promoted the cell motility of human decidual endometrial stromal cells through the GnRH-I receptor and the activation of Twist and N-cadherin signaling. This new concept regarding the coaction of EGF- and GnRH-promoted cell motility suggests that EGF and GnRH-II potentially affect embryo implantation and the decidual programming of human pregnancy.

The roles of GnRH in the human central nervous system.

It is widely known that GnRH plays a role in facilitating reproductive function via the HPG axis, and this was once believed to be its only function. However, over the last several decades important neuromodulatory roles of GnRH in multiple brain functions have been elucidated. Multiple GnRH isoforms and receptors have been detected outside the HPG-axis across different species. In this review, we focus on the human CNS where GnRH I and II isoforms and a functional GnRH I receptor have been isolated. We first describe the traditional understanding of GnRH within the hypothalamus and the pituitary and current clinical use of GnRH analogues. We then review the location and function of GnRH-producing neurons and receptors located outside the HPG axis. We next review the GnRH I and II neuron location and quantity and GnRH I receptor gene expression throughout the human brain, using the Allen Brain Map Atlas. This analysis demonstrates a wide expression of GnRH throughout the brain, including prominent expression in the basal forebrain and cerebellum. Lastly, we examine the potential role of GnRH in aging and inflammation and its therapeutic potential for neurodegenerative disease and spinal cord lesions.

ERQC autophagy: Yet another way to die.

A novel autophagy pathway eliminates nonnative polytopic membrane proteins from the endoplasmic reticulum that evade degradation by the ubiquitin proteasome system.

Quality control autophagy degrades soluble ERAD-resistant conformers of the misfolded membrane protein GnRHR.

Molecular chaperones triage misfolded proteins via action as substrate selectors for quality control (QC) machines that fold or degrade clients. Herein, the endoplasmic reticulum (ER)-associated Hsp40 JB12 is reported to participate in partitioning mutant conformers of gonadotropin-releasing hormone receptor (GnRHR), a G protein-coupled receptor, between ER-associated degradation (ERAD) and an ERQC autophagy pathway. ERQC autophagy degrades E90K-GnRHR because pools of its partially folded and detergent-soluble degradation intermediates are resistant to ERAD. S168R-GnRHR is globally misfolded and disposed of via ERAD, but inhibition of p97, the protein retrotranslocation motor, shunts S168R-GnRHR from ERAD to ERQC autophagy. Partially folded and grossly misfolded forms of GnRHR associate with JB12 and Hsp70. Elevation of JB12 promotes ERAD of S168R-GnRHR, with E90K-GnRHR being resistant. E90K-GnRHR elicits association of the Vps34 autophagy initiation complex with JB12. Interaction between ER-associated Hsp40s and the Vps34 complex permits the selective degradation of ERAD-resistant membrane proteins via ERQC autophagy.

GnRH receptor mediates lipid storage in female adipocytes via AMPK pathway.

Objective: Due to high levels of serum gonadotropin-releasing hormone (GnRH), perimenopausal or menopausal women, girls with central precocious puberty, women of polycystic ovary syndrome, and females receiving long-term GnRH agonist (GnRHa) treatment are at substantially higher risk of developing obesity. However, it remains poorly understood how GnRH affects body weight. Here, we explored whether the gonadotropin-releasing hormone receptor (GnRHR) was expressed in adipocytes and how GnRHR mediated lipid accumulation and the development of obesity. Methods: The samples were from 18 patients with benign tumors operated between 01/2018 and 06/2018 at the Women's Hospital School of Medicine Zhejiang University. Immunofluorescence, Western Blotting, and RT-PCR were used to detect whether the GnRH receptor was expressed in the specimens and human preadipocytes-subcutaneous (HPA-s). The GnRH receptor agonist diphereline with different concentrations was used to stimulate the HPA-s cells for 24, 48, and CCK-8 was used to detect cell proliferation. Oil red-O staining was used to detect lipid droplets in mature adipocytes. The phosphorylation of AMPK-Ser485/Thr172 was detected by Western Blotting. Results: GnRH receptor was expressed in all 18 human subcutaneous adipose tissue specimens. Cultured HPA-s expressed the GnRH receptor, and the expression increased during the process of cell maturation. The GnRH receptor agonist diphereline can stimulate the proliferation of HPA-s cells, which can advance the earliest occurrence of lipid droplets in HPA-s cells and the occurrence of lipid droplets in 50% cells by 1-2 days. Diphereline can stimulate the increase in the number of lipid droplets in mature adipocytes. The phosphorylation level of AMPK-Ser485/Thr172 in mature adipocytes was decreased by diphereline. Conclusion: The GnRH receptor was expressed in adipocytes. As adipocytes mature, GnRH receptor expression gradually increased. GnRHa stimulates the proliferation of HPA-s, promotes adipocyte maturation, increases the formation of lipid droplets in mature adipocytes, and inhibits the activation of the AMPK pathway in adipocytes. Our findings may elucidate the mechanism of obesity in these female populations and provide some evidence on how GnRH contributes to obesity. Additionally, these results provide theoretical support for further research on the mechanisms of obesity, thus enhancing our understanding of the functional diversity of GnRH and establishing a new theoretical basis for the impact of GnRH on metabolism.

Spike protein of SARS-CoV-2 suppresses gonadotrophin secretion from bovine anterior pituitaries.

Coronavirus disease (COVID-19), the ongoing global pandemic, is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Recent evidence shows that the virus utilizes angiotensin-converting enzyme 2 (ACE2) as a spike protein receptor for entry into target host cells. The bovine ACE2 contains key residues for binding to the spike protein receptor-binding domain. This study evaluated the hypothesis that bovine gonadotroph expresses ACE2, and spike protein suppresses luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from cultured bovine anterior pituitary (AP) cells. ACE2 mRNA expression and ACE2 protein expression were detected in the bovine AP cells using reverse transcription PCR and western blot analysis. Immunofluorescence microscopy analysis with the anti-ACE2 antibody revealed the co-localization of ACE2 and gonadotropin-releasing hormone (GnRH) receptor on the gonadotroph plasma membrane. Approximately 90% of GnRH receptor-positive cells expressed ACE2, and approximately 46% of ACE2-positive cells expressed the GnRH receptor. We cultured bovine AP cells for 3.5 days and treated them with increasing concentrations (0, 0.07, 0.7, or 7 pM) of recombinant spike protein having both S1 and S2 regions. The spike protein (0.07-7 pM) suppressed both basal and GnRH-induced LH secretion (P < 0.05). Spike protein (0.7-7 pM) suppressed GnRH-induced (P < 0.05), but not basal FSH secretion. In contrast, pre-treatment with ERK 1/2/5 inhibitor (U0126) partially restored the GnRH-induced LH and FSH secretion from the spike protein suppression. Collectively, the results indicate that gonadotrophs express ACE2, a receptor for coronavirus 2 spike protein, which in turn suppresses LH and FSH secretion from AP cells.

miR-31-5p from placental and peripheral blood exosomes is a potential biomarker to diagnose preeclampsia.

BACKGROUND: Preeclampsia, a multisystem disorder of unknown etiology, is one of the leading causes of maternal and perinatal morbidity and mortality. Identifying sensitive, noninvasive markers can aid its prevention and improve prognosis. microRNAs (miRs), which function as negative regulators of gene expression, are closely related to preeclampsia occurrence and development. Herein we investigated the relationship between the DLK1-Dio3 imprinted miR cluster derived from placental and peripheral blood exosomes of pregnant women with preeclampsia and routine clinical diagnostic indicators, and also determined its potential as a noninvasive diagnostic marker. METHODS: Exosomes were extracted from the placenta and peripheral blood of pregnant women with preeclampsia. RESULTS: qPCR data indicated that the expression level of miRs, such as miR-134, miR-31-5p, miR-655, miR-412, miR-539, miR-409, and miR-496, in pregnant women with preeclampsia was significantly lower than that in healthy controls; miR-31-5p expression was the most different. Gene ontology analysis predicted that genes negatively regulated by miR-31-5p were mainly enriched in cellular entity, cellular process, and binding; moreover, Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that genes were involved in gonadotropin-releasing hormone receptor pathway and other signaling pathways. Correlation analysis revealed that miR-31-5p was significantly negatively correlated with clinical indicators of preeclampsia, such as systolic and diastolic pressure, lactate dehydrogenase, and proteinuria. CONCLUSION: We believe that exosome-derived miR-31-5p can serve as an effective and sensitive biomarker to determine the course of preeclampsia in pregnant women.

Transcriptomic evidence of luteinizing hormone-releasing hormone agonist (LHRH-A) regulation on lipid metabolism in grass carp (Ctenopharyngodon idella).

In this study, RNA sequencing was used to identify the hepatic gene expression profile in grass carp associated with luteinizing hormone-releasing hormone agonist (LHRH-A) treatment. A total of 93,912,172 reads were generated by HiSeq 4000 sequencing platform. After filtering, 83,450,860 clean reads were mapped to the reference genome. By calculating the FPKM of genes, 1475 differentially expressed genes were identified. PPAR signaling pathway was enriched with upregulated genes in LHRH-A injection group showing the regulation of the lipid metabolism by LHRH-A. The expression of eight key genes in PPAR signaling pathway was confirmed by qPCR and the results suggested that ACSL4A, ACSL4B, ANGPTL4, LPL, RXRBA and SLC27A1B were significantly stimulated by LHRH-A injection. This investigation provides the evidence that LHRH-A could play a role in lipid metabolism.

Roles of Pyk2 in signal transduction after gonadotropin-releasing hormone receptor stimulation.

The receptor for gonadotropin-releasing hormone (GnRH) is highly expressed in hypothalamic neurons. It has been reported that GnRH treatment of cultured GnRH neurons (GT1-7 cells) activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in the activation of extracellular signal-regulated protein kinase 1 (ERK1) and ERK2 (ERK1/2). In the present study, we first examined the possibility that GnRH treatment might activate epidermal growth factor receptor (EGFR). We found that activation of EGFR after GnRH treatment for 5 min was much less than after EGF or heparin-binding EGF treatment. Next, we examined whether or not Pyk2 bound to growth factor receptor-binding protein 2 (Grb2). We overexpressed FLAG-fused Pyk2 in GT1-7 cells, and immunoprecipitated Pyk2 using an anti-FLAG antibody. The binding of Pyk2 to Grb2 was detected only after GnRH treatment. In contrast, a site-directed mutant of Pyk2 wherein tyrosine 881 was mutated to phenylalanine did not bind to Grb2. Studies with small interfering RNA and inhibitors indicated that the activation of Grb2/Ras/Raf/MEK was a major pathway to ERK1/2 activation after the short-term treatment of GT1-7 cells with GnRH.

Functional differences in the hypothalamic-pituitary-gonadal axis are associated with alternative reproductive tactics based on an inversion polymorphism.

The evolution of social behavior depends on genetic changes, yet, how genomic variation manifests itself in behavioral diversity is still largely unresolved. Chromosomal inversions can play a pivotal role in producing distinct behavioral phenotypes, in particular, when inversion genes are functionally associated with hormone synthesis and signaling. Male ruffs exhibit alternative reproductive tactics (ARTs) with an autosomal inversion determining two alternative morphs with clear behavioral and hormonal differences from the ancestral morph. We investigated hormonal and transcriptomic differences in the pituitary and gonads. Using a GnRH challenge, we found that the ability to synthesize testosterone in inversion carriers is severely constrained, whereas the synthesis of androstenedione, a testosterone precursor, is not. Inversion morphs were able to produce a transient increase in androstenedione following the GnRH injection, supporting the view that pituitary sensitivity to GnRH is comparable to that of the ancestral morph. We then performed gene expression analyses in a second set of untreated birds and found no evidence of alterations to pituitary sensitivity, gonadotropin production or gonad sensitivity to luteinizing hormone or follicle-stimulating hormone across morphs. Inversion morphs also showed reduced progesterone receptor expression in the pituitary. Strikingly, in the gonads, inversion morphs over-expressed STAR, a gene that is located outside of the inversion and responsible for providing the cholesterol substrate required for the synthesis of sex hormones. In conclusion, our results suggest that the gonads determine morph-specific differences in hormonal regulation.

Gonadotropin-Releasing Hormone Receptor Expression in Human Spinal Cord.

The expression of the gonadotrophin-releasing hormone receptor expression on pituitary gonadotrophs in humans is well characterized. In nervous system they have also been found in hippocampi and cerebral cortex. However, gonadotrophin-releasing hormone receptor expression in human spinal cord has not been reported. This study was to analyze the gonadotrophin-releasing hormone receptor expression in human spinal cord by immunohistochemistry, mRNAs by reverse transcriptase polymerase chain reaction, cDNA cloning and Western blot. The results show immunoreactive material to gonadotrophin-releasing hormone receptor in motoneurons of the spinal cord. Further, the study revealed that spinal cord expressed the gonadotrophin-releasing hormone receptor mRNA. The amplicon sequence corresponds to 100% of identity to GenBank. In Western blot, a band of 37 kDa were found in extracts of spinal cord and placenta as a control. In conclusion, human spinal cord expresses gonadotrophin-releasing hormone receptor analyzed through immunohistochemistry, the expression of its mRNA, cloning its cDNA and Western blot analysis. The presence of gonadotrophin-releasing hormone receptor in the spinal cord suggests the possibility of an extrapituitary functional role independent of reproductive system.

A multicenter open-label randomized phase II trial of paclitaxel plus EP-100, a novel LHRH receptor-targeted, membrane-disrupting peptide, versus paclitaxel alone for refractory or recurrent ovarian cancer.

OBJECTIVE: This randomized open-label phase II study evaluated the safety and clinical activity of EP-100 plus weekly paclitaxel in patients with recurrent ovarian cancer expressing positive LHRH receptor. METHODS: In a limited "run-in" dose escalation phase for EP-100, six patients were treated with ascending dose levels (13 mg/m2, 20 mg/m2, 30 mg/m2). In the randomized phase, patients received weekly paclitaxel (80 mg/m2 intravenously) plus twice weekly EP-100 (30 mg/m2 intravenously; combination arm) or weekly paclitaxel alone (80 mg/m2 intravenously; paclitaxel arm). The primary study endpoint was overall response rate (ORR). RESULTS: Forty-four patients were then randomized to either the experimental combination arm (n = 23) or the standard of care paclitaxel monotherapy arm (n = 21). The ORR was 35% (95%CI 16%-57%) for the combination arm and 33% (95% CI 15%-57%) for the paclitaxel arm. An interesting observation from an unplanned analysis was that a subset of patients with target liver lesions showed a greater overall response rate to the combination (69%) compared to paclitaxel alone (16%). The frequency of treatment-related grade 3-4 adverse events was similar between treatment arms: 48% vs 43% for the combination and paclitaxel arms, respectively. CONCLUSIONS: ORR in the EP-100 combination arm was similar to that in the group treated with paclitaxel alone; however, a subset of patients with liver metastases appeared to benefit from the combination. The addition of EP-100 did not appear to augment the adverse event profile of paclitaxel and was well tolerated.

Cardiac and Brainstem Neuroinflammatory Pathways Account for Androgenic Incitement of Cardiovascular and Autonomic Manifestations in Endotoxic Male Rats.

ABSTRACT: Inconsistent reports are available on the role of testosterone in end-organ damage caused by endotoxemia. Here, pharmacologic, surgical, and molecular studies were employed to assess the testosterone modulation of cardiovascular, autonomic, and peripheral and central inflammatory derangements caused by endotoxemia. Studies were performed in conscious male rats preinstrumented with femoral indwelling catheters for the measurement of blood pressure and subjected to castration or pharmacologic interventions that interrupt the biosynthetic cascade of testosterone. Compared with the effects of lipopolysaccharide (10 mg/kg intravenously) in sham operated rats, 2-week castration reduced the lipopolysaccharide-evoked (1) falls in blood pressure, (2) decreases in time- and frequency-domain indices of heart rate variability, (3) shifts in spectral measures of cardiac sympathovagal balance toward parasympathetic dominance, and (4) increases in protein expressions of toll-like receptor-4 and monocyte chemoattractant protein-1 in heart and medullary neurons of the nucleus tractus solitarius and rostral ventrolateral medulla. While the ameliorating actions of castration on endotoxic cardiovascular manifestations were maintained after testosterone replacement, the concomitant inflammatory signals were restored to near-sham levels. The favorable influences of castration on inflammatory and cardiovascular abnormalities of endotoxemia were replicated in intact rats pretreated with degarelix (gonadotropin-releasing hormone receptor blocker) or finasteride (5α-reductase inhibitor) but not formestane (aromatase inhibitor). The data signifies the importance of androgens and its biosynthetic enzymes in cardiovascular and autonomic insults induced by the endotoxic inflammatory response. Clinically, the interruption of testosterone biosynthesis could offer a potential strategy for endotoxemia management.

The expression of type I and II gonadotropin-releasing hormone receptors transcripts in Vervet monkey (Chlorocebus aethiops) spermatozoa.

Gonadotropin-Releasing Hormone (GnRH), acting via the GnRH receptor (GnRHR), and a member of G-protein coupled receptor (GPCR), plays an essential role in the control of reproduction while operating primarily at the hypothalamic level of the gonadotropic axis. GnRH and its receptor are co-expressed in certain specific cells, suggesting an autocrine regulation of such cells. In the male reproductive system, two forms of GnRH (I and II) and its receptors (GnRHR) are present in the human and non-human primate (NHP) testis, prostate, epididymis, seminal vesicle, and human spermatozoa. In humans, the GnRHR-II receptor gene is disrupted by a frameshift in exon 1 and a stop codon in exon 2, rendering the receptor non-functional, whereas a fully functional GnRHR-II receptor is present in New-World and Old-World monkeys. There is no evidence of the existence of a GnRH receptor in NHP sperm. Since the NHP has a phylogenetic relationship to man and is often used as models in reproductive physiology, this present study aimed to determine GnRHR-I and GnRHR-II in Vervet monkey (Chlorocebus aethiops) spermatozoa. A total of 24 semen samples were obtained from four adult Vervet monkeys through electro-ejaculation and utilized for genotyping and gene expression analysis of GnRHR-I and II. Here we report that both receptors were successfully identified in the Vervet monkey sperm with the abundance of GnRHR-I gene expression compared to GnRHR-II. In comparison to the human, there is no evidence of such a stop codon at position 179 in exon 2 of the Vervet GnRHR-II. These findings suggest that both receptors are transcriptionally functional in Vervet spermatozoa.

Immunization with oral KISS1 DNA vaccine inhibits testicular Leydig cell proliferation mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes in goats.

This study aimed to analyze the effect and mechanism of immunization of oral KISS1 DNA vaccine on the proliferation of goat testicular Leydig cells. Ten 8-week-old male goats were randomly divided into KISS1 DNA vaccine and control groups for immunization (five goats each group). These goats were sacrificed at 8 weeks after primary immunization, and the tissue samples of hypothalamus, pituitary, and testis and Leydig cell samples were collected for RT-PCR and CCK8 assay. Immunization with the oral KISS1 DNA vaccine effectively inhibited the proliferation of Leydig cells, the expression of hypothalamus KISS1, GPR54, and GnRH mRNA, pituitary GnRHR and LH mRNA, testicular LHR mRNA, and apoptosis-inhibitory gene Bcl-2 mRNA in Leydig cells. By contrast, the immunization enhanced the mRNA expression of apoptosis-promoting gene Bax and Clusterin in Leydig cells. These findings indicate that immunization with the oral KISS1 DNA vaccine can inhibit the proliferation of goat testicular Leydig cells mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes.