Dopamine receptor D2 regulates genes involved in germ cell movement and sperm motility in rat testes†.

The function of dopamine receptor D2 (D2R) is well associated with sperm motility; however, the physiological role of D2R present on testicular cells remains elusive. The aim of the present study is to delineate the function of testicular D2R. Serum dopamine levels were found to decrease with age, whereas testicular D2R expression increased. In rat testicular sections, D2R immunolabeling was observed in interstitial cells, spermatogonia, spermatocytes and mature elongated spermatids, whereas tyrosine hydroxylase immunolabeling was selectively detected in Leydig cells. In vitro seminiferous tubule culture following bromocriptine (D2R agonist) treatment resulted in decreased cAMP levels. Microarray identified 1077 differentially expressed genes (511 up-regulated, 566 down-regulated). The majority of differentially expressed genes were present in post-meiotic cells including early and late spermatids, and sperm. Gene ontology elucidated processes related to extra-cellular matrix to be enriched and was supported by differential expression of various collagens and laminins, thereby indicating a role of dopamine in extra-cellular matrix integrity and transport of spermatids across the seminiferous epithelium. Gene ontology and enrichment map also highlighted cell/sperm motility to be significantly enriched. Therefore, genes involved in sperm motility functions were further validated by RT-qPCR. Seven genes (Akap4, Ccnyl1, Iqcf1, Klc3, Prss55, Tbc1d21, Tl18) were significantly up-regulated, whereas four genes (Dnah1, Dnah5, Clxn, Fsip2) were significantly down-regulated by bromocriptine treatment. The bromocriptine-stimulated reduction in seminiferous tubule cyclic AMP and associated changes in spermatid gene expression suggests that dopamine regulates both spermatogenesis and spermiogenesis within the seminiferous epithelium, and spermatozoa motility following spermiation, as essential processes for fertility.

DNA methylation biomarkers to identify epigenetically abnormal spermatozoa in male partners from couples experiencing recurrent pregnancy loss.

Previously, we showed that DNA methylation defects in spermatozoa from male partners of couples undergoing recurrent pregnancy loss (RPL) could be a contributing paternal factor. In the present study, we aimed to determine whether the methylation levels of selected imprinted genes can be used as diagnostic markers to identify epigenetically abnormal spermatozoa sample in these cases. The methylation levels of selected imprinted genes in spermatozoa, which were previously found to be differentially methylated, were combined into a probability score (between 0-1) using multiple logistic regression. Different combinations of these genes were investigated using Receiver Operating Characteristic analysis, and the threshold values were experimentally validated in an independent cohort of 38 control and 45 RPL spermatozoa samples. Among the different combinations investigated, a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 (AUC = 0.88) with a threshold value of 0.61 was selected with a specificity of 90.41% and sensitivity of 70%. The results from the validation study indicated that 97% of the control samples had probability scores below this threshold, whereas 40% of the RPL samples were above this threshold with a post-hoc power of 97.8%. Thus, this combination can correctly classify control samples and potentially identify epigenetically abnormal spermatozoa samples in the male partners of couples undergoing RPL. We propose that the combined DNA methylation levels of these imprinted genes can be used as a diagnostic tool to identify spermatozoa samples with epigenetic defects which could contribute to the pathophysiology of RPL and the couple could be counselled appropriately.

Prolactin Regulates Testicular Gene Expression and Cell Cycle Processes Predominantly via JAK2/STAT5 Pathway in the Male Rat.

Hyperprolactinemia is prevalent in up to 16% of infertile males. Although the prolactin receptor (PRLR) is present on various testicular cells, the physiological role of this receptor in spermatogenesis remains elusive. The aim of this study is to delineate prolactin actions in rat testicular tissue. Serum prolactin, developmental expression of PRLR, signaling pathways associated, and gene transcription regulation in the testes were investigated. Serum prolactin and testicular PRLR expression was found to be significantly increased at pubertal and adult ages as compared to prepubertal. Further, PRLR activated the JAK2/STAT5 pathway, but not the MAPK/ERK and PI3K/AKT pathway in the testicular cells. Gene expression profiling following prolactin treatment in seminiferous tubule culture resulted in a total of 692 differentially expressed genes, of which 405 were upregulated and 287 were downregulated. Enrichment map analysis showed that prolactin target genes are involved in processes such as cell cycle, male reproduction, chromatin remodeling, and cytoskeletal organization. Novel gene targets of prolactin whose role in testes is unexplored were obtained and validated by qPCR. Additionally, 10 genes involved in cell cycle process were also validated; 6 genes (Ccna1, Ccnb1, Ccnb2, Cdc25a, Cdc27, Plk1) were found to be significantly upregulated, whereas 4 genes (Ccar2, Nudc, Tuba1c, Tubb2a) were found to be significantly downregulated in testes after treatment with prolactin. Taken together, the findings from this study suggest a crucial role of prolactin in male reproduction and identified target genes regulated by prolactin in the testes.

Synthesis and biological evaluation of benzamide-chalcone hybrids as potential c-Met kinase and COX-2 inhibitors.

c-Met kinase and cyclooxygenase 2 (COX-2) enzymes are two significant targets in tumor progression. Chalcone and benzamide moieties were combined using molecular hybridization to assess their potential as c-Met kinase and COX-2 inhibitors. 4-Methylbenzamide and 4-chlorobenzamide chalcone analogs were synthesized, characterized, and evaluated for antiproliferative activity on Michigan Cancer Foundation-7 (MCF-7), HT-29, MDA-MB-231, COLO-205, and A549 cell lines by sulforhodamine-B stain (SRB) assay. Following the SRB assay, compounds were evaluated for their c-Met kinase and COX-2 inhibitory potential. All compounds inhibited COX-2 with half-maximal inhibitory concentration (IC50 ) <10 µM. Compounds 7h, 7i, 7j, 8f, and 8j inhibited c-Met with IC50 <10 µM. Compound 7h was evaluated for its long-term antiproliferative and anti-migratory effects by colony formation and wound healing assay. It exerted these effects in a concentration-dependent manner. Compounds 7j and 8j were further evaluated for in vitro antiangiogenic effects. Compound 7j exhibited moderate antiangiogenic effect while compound 8j exhibited strong effect. Compounds 7h, 7i, 7j, 8f, and 8j were evaluated for the serum protein binding, using the in vitro bovine serum albumin binding assay. The results indicated that the tested compounds bind to bovine serum albumin (BSA) and can be further explored by other studies.

Sex hormones regulate lipid metabolism in adult Sertoli cells: A genome-wide study of estrogen and androgen receptor binding sites.

Optimal functioning of Sertoli cells is crucial for spermatogenesis which is under tight regulation of sex hormones, estrogen and androgen. Adult rat Sertoli cells expresses estrogen receptor beta (ERß) and androgen receptor (AR), both of which regulate gene transcription by binding to the DNA. The present study is aimed to acquire a genome-wide map of estrogen- and androgen-regulated genes in adult Sertoli cells. ChIP-Seq was performed for ERß and AR in Sertoli cells under physiological conditions. 30,859 peaks in ERß and 9,594 peaks in AR were identified with a fold enrichment >2 fold. Pathway analysis for the genes revealed metabolic pathways to be significantly enriched. Since Sertoli cells have supportive functions and provide energy substrates to germ cells during spermatogenesis, significantly enriched metabolic pathways were explored further. Peaks of the genes involved in lipid metabolism, like fatty acid, glyceride, leucine, and sphingosine metabolism were validated. Motif analysis confirmed the presence of estrogen- and androgen-response elements (EREs and AREs). Moreover, transcript levels of enzymes involved in the lipid metabolic pathways were significantly altered in cultured Sertoli cells treated with estrogen and androgen receptor agonists, demonstrating functional significance of these binding sites. This study elucidates a mechanism by which sex hormones regulate lipid metabolism in Sertoli cells by transcriptionally controlling the expression of these genes, thereby shedding light on the roles of these hormones in male fertility.

DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss.

STUDY QUESTION: What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? SUMMARY ANSWER: Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST), zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. WHAT IS KNOWN ALREADY: RPL is defined as loss of two or more pregnancies, affecting 1-2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative paternal factors that could be contributing towards pregnancy loss in cases of idiopathic RPL. STUDY DESIGN, SIZE, DURATION: In this case-control study, 112 couples undergoing RPL with no identifiable cause were recruited from September 2015 to May 2018. The control group comprised of 106 healthy proven fertile couples with no history of infertility or miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS: In this study, we investigated the paternal genetic and epigenetic factors that could be associated with RPL. We studied DNA methylation, by pyrosequencing, of selected imprinted genes implicated in embryo development, such as IGF2-H19 DMR, IG-DMR, MEST, ZAC, KvDMR, PEG3, PEG10 and small nuclear ribonucleoprotein polypeptide N (SNRPN) in sperm of men whose partners present RPL. Global DNA methylation in sperm was evaluated by studying 5mC content and long interspersed nuclear element 1 (LINE1) promoter methylation. We also studied polymorphisms by pyrosequencing in the IGF2-H19 DMR as well in the IGF2 promoter in both groups. MAIN RESULTS AND THE ROLE OF CHANCE: In the RPL group, we found a significant decrease in the global sperm 5mC levels and significant decrease in DNA methylation at three CpG sites in LINE1 promoter. For IGF2-H19 DMR and IG-DMR, a significant decrease in sperm DNA methylation at specific CpG sites was observed in RPL group. For maternally imprinted genes like MEST, ZAC, KvDMR, PEG3 and PEG10 hypermethylation was noted. Polymorphism studies for IGF2-H19 DMR and IGF2 revealed significant differences in the genotypic frequencies in males. LIMITATIONS, REASONS FOR CAUTION: In this study, we analysed the methylation levels of selected candidate imprinted genes implicated in embryo development. Detection of methylation changes occurring at the genome-wide level may reveal further candidate genes having a better distinction between the control and study groups. WIDER IMPLICATIONS OF THE FINDINGS: Our study demonstrates that certain polymorphisms and aberrant sperm methylation status in imprinted genes are associated with RPL and could contribute to the aetiology of RPL. This study suggests that investigation of paternal genetic and epigenetic factors could be useful in identification of possible causes of idiopathic RPL. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Department of Science and Technology-Science and Engineering Research Board (EMR/2014/000145) and National Institute for Research in Reproductive Health intramural funds (RA/872/01-2020). All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Genome-wide identification of estrogen receptor binding sites reveals novel estrogen-responsive pathways in adult male germ cells.

Spermatogenesis occurs in the seminiferous epithelium that shows the presence of estrogen receptors alpha (ERα) and beta (ERß), both of which regulate gene transcription by binding to the DNA. Estrogen responsive phases of spermatogenesis are well documented; however, the genes regulated remain inexplicit. To study the regulation of genes by estrogen in male germ cells, we performed chromatin immunoprecipitation (ChIP) sequencing for ERα and ERß under normal physiological conditions. A total of 27 221 DNA binding regions were enriched with ERα and 20 926 binding sites with ERß. Majority of the peaks were present in the intronic regions and located 20 kb upstream or downstream from the transcription start site (TSS). Pathway analysis of the genes enriched by ChIP-Seq showed involvement in several biological pathways. Genes involved in pathways whose role in spermatogenesis is unexplored were validated; these included prolactin, GnRH, and oxytocin signaling. All the selected genes showed the presence of estrogen response elements (EREs) in their binding region and were also found to be significantly enriched by ChIP-qPCR. Functional validation using seminiferous tubule culture after treatment with estrogen receptor subtype-specific agonist and antagonist confirmed the regulation of these genes by estrogen through its receptors. The genes involved in these pathways were also found to be regulated by the respective receptor subtypes at the testicular level in our in vivo estrogen receptor agonist rat models. Our study provides a genome-wide map of ERα and ERß binding sites and identifies the genes regulated by them in the male germ cells under normal physiological conditions.

Unveiling the Role of Prolactin and its Receptor in Male Reproduction.

Prolactin is a peptide hormone known to have multiple functions. However, the role of prolactin has been extensively studied only in female physiology and its function in male reproduction still remains majorly unexplored. Studies in rodents and humans have demonstrated the presence of prolactin and its receptor in testes, thereby suggesting a possible role during spermatogenesis. Experimental evidences from prolactin and prolactin receptor deficient male rodent models as well as studies done in hypo- and hyper-prolactinemic males hint at neuroendocrine and reproductive abnormalities. Nonetheless, there still remains a lot of ambiguity on the exact role of prolactin and its receptor in male reproduction. This review summarizes in depth on the role of prolactin in spermatogenesis.

Delineating the regulation of estrogen and androgen receptor expression by sex steroids during rat spermatogenesis.

Estrogen receptors (ERα and ß) and androgen receptor (AR) regulate various critical processes during spermatogenesis. Since spermatogenesis is very sensitive to hormonal stimuli and perturbations, it is important to understand the regulation of expression of these receptors by sex steroid hormones. Although many studies have reported deregulation of steroid receptors on endocrine disruption, there is no consensus on the regulation of their expression by steroid hormones during spermatogenesis, and a lack of clear understanding of the mechanism of regulation. Here, we evaluated the receptor expressions in a well-established exogenous estradiol administration model. We then investigated the mechanisms by which the individual receptors regulate their expression by binding to the respective hormone response elements upstream of these receptor genes. By further employing in vitro and in vivo models of ER and AR stimulation or antagonism, we delineated their regulation in a receptor subtype-specific manner. Our results indicate that ERα positively regulates expression of both the ERs; whereas, ERß and AR negatively regulate expression of both ERß and AR by direct binding to upstream regulatory regions. The perturbations in the levels of steroid receptors could be an important factor contributing to spermatogenic defects and male sub-fertility after estradiol and ER agonist treatment. Our study delineates the direct contribution of the individual steroid receptors in the regulation of their own expression.