RF9 Rescues Cortisol-Induced Repression of Testosterone Levels in Adult Male Macaques.

Cortisol inhibits hypothalamic-pituitary-gonadal (HPG) axis whereas RF9, a potent agonist of kisspeptin receptor (GPR54) activates HPG-axis during fasting-induced stress and under normal physiological conditions. However, the effect of RF9 on the cortisol-induced repressed HPG-axis is not studied yet. This study investigated whether exogenous cortisol-induced repression of the HPG-axis can be rescued by RF9. Six intact adult male rhesus monkeys (Macaca mulatta) habituated to chair-restraint were administered hydrocortisone sodium succinate at a rate of 20 mg/kg of body weight (BW) per day for 12 days. Single blood sample was taken by venipuncture from each animal on alternate days for hormones analyses. On experimental day 12, hydrocortisone treated monkeys received a single intravenous bolus of RF9 (n = 3) and vehicle (n = 3). The animals were bled for a period of 4 h at 60 min intervals from an indwelling cannula in the saphenous vein. RF9 was administered intravenously at the dose of 0.1 mg/kg BW immediately after taking 0 min sample. Plasma cortisol and testosterone concentrations were measured by using specific enzyme immunoassays. Hydrocortisone treatment increased plasma cortisol levels (P ≤ 0.0001) and decreased plasma testosterone (P ≤ 0.0127) levels. Interestingly, compared to vehicle, RF9 treatment significantly increased plasma testosterone levels at 120 min (P ≤ 0.0037), 180 min (P ≤ 0.0016), and 240 min (P ≤ 0.0001) intervals in the hydrocortisone treated monkeys. From these results, we concluded that RF9 administration relieves the suppressed HPG-axis in term of plasma testosterone levels in the cortisol treated monkeys.

CD83, a Novel MAPK Signaling Pathway Interactor, Determines Ovarian Cancer Cell Fate.

Ovarian cancer is a leading cause of death from gynecologic malignancies worldwide. Although CD83 is widely described as a solid marker for mature dendritic cells, emerging pieces of evidence indicate the expression of membrane protein CD83 by various tumor cells, including ovarian cancer cells. However, the potential role of CD83 in ovarian cancer cell properties and development remains absolutely unknown. By using human CD83 stable overexpression and knockdown sublines of several ovarian cancer cells, we observed that CD83 advanced the growth proliferation, colony formation ability, spheroid formation, and in vivo tumorigenicity of ovarian cancer cells; surprisingly, CD83 limited their migration and invasion potentials. Positive regulation of proliferation/stemness factors (e.g., cyclin-CDKs and KIT/CD44) but negative regulation of matrix metallopeptidases (e.g., MMP1 and 7) by CD83 were revealed by the integrated analysis of transcriptome and proteome. Furthermore, immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP) first identified the association of CD83 with MAP3K7 (also known as TAK1) and MAP3K7-binding protein TAB1 on the cell membrane. Moreover, CD83 functions through the activation of MAP3K7-MEK1/2-ERK1/2 cascades to further regulate downstream FOXO1/p21/CDK2/CCNB1 and STAT3/DKK1 signaling pathways, thus activating proliferation and spheroid formation of ovarian cancer cells, respectively. Collectively, our findings define a CD83-MAPK pathway in the regulation of proliferation and stemness in ovarian cancer cells, with potential therapeutic applications in blocking their progression.

Recent advances in the regulation of testicular germ cell tumors by microRNAs.

Testicular germ cell tumors (TGCTs) are generally rare but represent the most common solid tumors in young men. They are classified broadly into seminoma, which resemble primordial germ cells (PGCs), and non-seminoma, which are either undifferentiated (embryonic carcinoma) or differentiated (teratoma, yolk sac tumor, choriocarcinomas) patterning. A widespread role for microRNAs (miRNAs), in diverse molecular processes driving initiation and progression of various types of TGCTs has been recently studied. We discuss the involvement of different miRNAs in the development and progression of different types of TGCTs. Moreover, we highlight the aberrant expression of miRNAs in TGCTs and several targets, which may define miRNAs as oncomiRs or tumor suppressors. A better understanding of miRNA biology may ultimately yield further insight into the molecular mechanisms of tumorigenesis and new therapeutic strategies against TGCTs.

Role of EZH2 in cell lineage determination and relative signaling pathways.

Enhancer of zeste homolog 2 (EZH2), a catalytic component of polycomb repressive complex 2 (PRC2), epigenetically regulates chromatin structure and gene expression through trimethylation at histone H3K27 and recruitment of DNA methyltransferases for gene silencing. Despite extensive studies of the role of EZH2 in cancer progression and malignancy, increasing evidences suggest that EZH2 plays a critical role in stem cells renewal, maintenance, and differentiation into specific cell lineages. Here, we reviewed the update information regarding how EZH2 contributes to stem cell maintenance and cell lineage determination (including gonadogenesis, neurogenesis, myogenesis, osteogenesis, hematopoiesis, lymphopoiesis, adipogenesis, epidermal differentiation and hepatogenesis), and the regulation of EZH2 by phosphorylation and different signaling pathways.

Distinct Metabolic Features of Seminoma and Embryonal Carcinoma Revealed by Combined Transcriptome and Metabolome Analyses.

Seminoma and embryonal carcinoma (EC), two typical types of testicular germ cell tumors (TGCTs), present significant differences in growth behavior, expression characteristics, differentiation potential, clinical features, therapy, and prognosis. The purpose of this study was to compare the distinctive or preference metabolic pathways between seminoma and EC. The Cancer Genome Atlas revealed that many genes encoding metabolic enzymes could distinguish between seminoma and EC. Using well-characterized cell line models for seminoma (Tcam-2 cells) and EC (NT2 cells), we characterized their metabolite profiles using ultraperformance liquid chromatography coupled to Q-TOF mass spectrometry (UPLC/Q-TOF MS). In general, the integrated results from transcriptome and metabolite profiling revealed that seminoma and EC exhibited distinctive characteristics in the metabolisms of amino acids, glucose, fatty acids, sphingolipids, nucleotides, and drugs. Notably, an attenuation of citric acid cycle/mitochondrial oxidative phosphorylation and sphingolipid biosynthesis as well as an increase in arachidonic acid metabolism and (very) long-chain fatty acid abundance occurred in seminoma as compared with EC. Our study suggests histologic subtype-dependent metabolic reprogramming in TGCTs and will lead to a better understanding of the metabolic signatures and biology of TGCT subtypes.

Testicular germ cell tumor: a comprehensive review.

Testicular tumors are the most common tumors in adolescent and young men and germ cell tumors (TGCTs) account for most of all testicular cancers. Increasing incidence of TGCTs among males provides strong motivation to understand its biological and genetic basis. Gains of chromosome arm 12p and aneuploidy are nearly universal in TGCTs, but TGCTs have low point mutation rate. It is thought that TGCTs develop from premalignant intratubular germ cell neoplasia that is believed to arise from the failure of normal maturation of gonocytes during fetal or postnatal development. Progression toward invasive TGCTs (seminoma and nonseminoma) then occurs after puberty. Both inherited genetic factors and environmental risk factors emerge as important contributors to TGCT susceptibility. Genome-wide association studies have so far identified more than 30 risk loci for TGCTs, suggesting that a polygenic model fits better with the genetic landscape of the disease. Despite high cure rates because of its particular sensitivity to platinum-based chemotherapy, exploration of mechanisms underlying the occurrence, progression, metastasis, recurrence, chemotherapeutic resistance, early diagnosis and optional clinical therapeutics without long-term side effects are urgently needed to reduce the cancer burden in this underserved age group. Herein, we present an up-to-date review on clinical challenges, origin and progression, risk factors, TGCT mouse models, serum diagnostic markers, resistance mechanisms, miRNA regulation, and database resources of TGCTs. We appeal that more attention should be paid to the basic research and clinical diagnosis and treatment of TGCTs.

GATA4 is a negative regulator of contractility in mouse testicular peritubular myoid cells

Reduced contractility of the testicular peritubular myoid (PTM) cells may contribute to human male subfertility or infertility. Transcription factor GATA4 in Sertoli and Leydig cells is essential for murine spermatogenesis, but limited attention has been paid to the potential role of GATA4 in PTM cells. In primary cultures of mouse PTM cells, siRNA knockdown of GATA4 increased the contractile activity, while GATA4 overexpression significantly attenuated the contractility of PTM cells using a collagen gel contraction assay. Using RNA sequencing and qRT-PCR, we identified a set of genes that exhibited opposite expressional alternation between Gata4 siRNA vs nontargeting siRNA-treated PTM cells and Gata4 adenovirus vs control adenovirus-treated PTM cells. Notably, ion channels, smooth muscle function, cytokines and chemokines, cytoskeleton, adhesion and extracellular matrix were the top four enriched pathways, as revealed by cluster analysis. Natriuretic peptide type B (NPPB) content was significantly upregulated by GATA4 overexpression in both PTM cells and their culture supernatant. More importantly, the addition of 100 µM NPPB could abolish the promoting effect of Gata4 silencing on PTM cell contraction. Taken together, we suggest that the inhibitory action of GATA4 on PTM cell contraction is mediated at least partly by regulating genes belonging to smooth muscle contraction pathway (e.g. Nppb).

A miR-125b/CSF1-CX3CL1/tumor-associated macrophage recruitment axis controls testicular germ cell tumor growth.

Tumor growth is modulated by crosstalk between cancer cells and the tumor microenvironment. Recent advances have shown that miRNA dysfunction in tumor cells can modulate the tumor microenvironment to indirectly determine their progression. However, this process is poorly understood in testicular germ cell tumors (TGCTs). We reported here that miR-125b was repressed in TGCT samples by epigenetic modifications rather than genetic alternations. Furthermore, miR-125b overexpression significantly alleviated the tumor growth in two NCCIT human embryonic carcinoma xenograft models in vivo, whereas miR-125b did not stimulate autonomous tumor cell growth in vitro. Notably, forced expression of miR-125b in NCCIT embryonic carcinoma cells decreased the abundance of host tumor-associated macrophages (TAMs) within tumor microenvironment. Selective deletion of host macrophages by clodronate abolished the anti-tumoral ability of miR-125b in xenograft models. By RNA profiling, Western blot and luciferase reporter assay, we further observed that miR-125b directly regulated tumor cell-derived chemokine CSF1 and CX3CL1, which are known to control the recruitment of TAMs to tumor sites. Lastly, we found that one set of miRNAs, which are under the regulation of miR-125b, might convergently target CSF1/CX3CL1 in NCCIT cells using miRNA profiling. These findings uncover the anticancer effect of miR-125b via mediating tumor-stroma crosstalk in xenograft models of TGCTs and raise the possibility of targeting miR-125b as miRNA therapeutics.

Melatonin promotes sheep Leydig cell testosterone secretion in a co-culture with Sertoli cells.

Leydig cells synthesize and secrete testosterone, and are regulated by Sertoli cells. These two cell types may work together to regulate testicular androgen production. Studies have shown that Leydig cell androgen synthesis can be dramatically enhanced by Sertoli cells in the presence of melatonin, which can regulate the secretory function of Leydig and Sertoli cells. However, the molecular mechanism of melatonin-regulated Leydig cell androgen production via Sertoli cells remains unclear. Here, we found that 10-7 M melatonin increased testosterone production in co-cultured Leydig and Sertoli cells isolated from sheep. Melatonin increased the expression of stem cell factor and insulin-like growth factor-1 and decreased estrogen synthesis in Sertoli cells. Melatonin promoted insulin-like growth factor-1 and decreased estrogen content via the membrane melatonin receptor 1. It also enhanced stem cell factor expression via the retinoic acid receptor-related orphan receptor alpha. Addition of PD98059, a MEK inhibitor, to Sertoli cell culture demonstrated that the melatonin upregulation of insulin-like growth factor-1 and downregulation of estrogen may be through the MEK/extracellular signal-regulated kinase pathway. Together, these results suggest that melatonin may function through modulating melatonin receptor 1-regulated insulin-like growth factor-1 expression, as well as melatonin receptor 1-induced suppression of estrogen synthesis to increase androgen production in co-cultured Leydig and Sertoli cells.

Requirement for CCNB1 in mouse spermatogenesis.

Spermatogenesis, which involves mitosis and meiosis of male germ cells, is a highly complicated and coordinately ordered process. Cyclin B1 (CCNB1), an important regulator in cell cycle machinery, is proved essential for mouse embryonic development. However, the role of CCNB1 in mammalian spermatogenesis remains unclear. Here we tested the requirement for CCNB1 using conditional knockout mice lacking CCNB1 in male germ cells. We found that ablation of CCNB1 in gonocytes and spermatogonia led to mouse sterile caused by the male germ cells' depletion. Gonocyte and spermatogonia without CCNB1 is unable to proliferate normally and apoptosis increased. Moreover, CCNB1 ablation in spermatogonia may promote their differentiation by downregulating Lin28a and upregulating let-7 miRNA. However, ablation of CCNB1 in premeiotic male germ cells did not have an effect on meiosis of spermatocytes and male fertility, suggesting that CCNB1 may be dispensable for meiosis of spermatocytes. Collectively, these results indicate that CCNB1 is critically required for the proliferation of gonocytes and spermatogonia but may be redundant in meiosis of spermatocytes in mouse spermatogenesis.

Gonadotropin inhibitory hormone and RF9 stimulate hypothalamic-pituitary-adrenal axis in adult male rhesus monkeys.

Stress activates gonadotropin inhibitory hormone (GnIH), hypothalamic-pituitary-adrenal axis (HPA-axis) and represses hypothalamic-pituitary-gonadal axis (HPG-axis) but RF9 administration relieves stress-induced repression of the HPG-axis. Importantly, it was not known whether GnIH signaling and RF9 synthetic peptide modulate the HPA axis. To assess this, mammalian orthologs of GnIH (RFRP-1 and RFRP-3) and RF9 were administered to intact adult male rhesus monkeys. RFRP-1 (125µg/animal), RFRP-3 (250µg/animal) and RF9 (0.1mg/kg BW) were intravenously (iv) injected into normal fed (n=4) monkeys. Additionally, a single bolus iv injection of RF9 (0.1mg/kg BW) was also administered to 48h fasted monkeys (n=4) to check the effects of RF9 signaling on an activated HPA-axis. Serial blood samples were collected, centrifuged and the obtained plasma was used for the analysis of cortisol by specific enzyme immunoassay. RFRP-1 treatment significantly increased cortisol levels while RFRP-3 increased the plasma cortisol, but the effect was non-significant. RF9 treatment significantly increased cortisol levels in normal fed animals. In contrast, RF9 injection did not significantly alter circulating cortisol in fasted monkeys. In conclusion, our results suggest stimulatory action of RFRPs and RF9 on the HPA axis in the adult male monkeys. However, the mechanism and site of action of RFRP-1 and RF9 along the HPA-axis is still unknown. Therefore, further studies are needed to decipher the mechanism and site of action of RFRPs and RF9 on the HPA axis in primates.

The control of male fertility by spermatid-specific factors: searching for contraceptive targets from spermatozoon's head to tail.

Male infertility due to abnormal spermatozoa has been reported in both animals and humans, but its pathogenic causes, including genetic abnormalities, remain largely unknown. On the other hand, contraceptive options for men are limited, and a specific, reversible and safe method of male contraception has been a long-standing quest in medicine. Some progress has recently been made in exploring the effects of spermatid-specifical genetic factors in controlling male fertility. A comprehensive search of PubMed for articles and reviews published in English before July 2016 was carried out using the search terms 'spermiogenesis failure', 'globozoospermia', 'spermatid-specific', 'acrosome', 'infertile', 'manchette', 'sperm connecting piece', 'sperm annulus', 'sperm ADAMs', 'flagellar abnormalities', 'sperm motility loss', 'sperm ion exchanger' and 'contraceptive targets'. Importantly, we have opted to focus on articles regarding spermatid-specific factors. Genetic studies to define the structure and physiology of sperm have shown that spermatozoa appear to be one of the most promising contraceptive targets. Here we summarize how these spermatid-specific factors regulate spermiogenesis and categorize them according to their localization and function from spermatid head to tail (e.g., acrosome, manchette, head-tail conjunction, annulus, principal piece of tail). In addition, we emphatically introduce small-molecule contraceptives, such as BRDT and PPP3CC/PPP3R2, which are currently being developed to target spermatogenic-specific proteins. We suggest that blocking the differentiation of haploid germ cells, which rarely affects early spermatogenic cell types and the testicular microenvironment, is a better choice than spermatogenic-specific proteins. The studies described here provide valuable information regarding the genetic and molecular defects causing male mouse infertility to improve our understanding of the importance of spermatid-specific factors in controlling fertility. Although a male contraceptive 'pill' is still many years away, research into the production of new small-molecule contraceptives targeting spermatid-specific proteins is the right avenue.