The human genome project has identified, besides ovarian relaxin (RLN), six other relaxin-like molecules (RLN3, H1-RLN, INSL3-6), most of which appear to be expressed in the testis and/or male reproductive system, together with four different G-protein-coupled receptors responsive to one or other of these peptides. Earlier work on relaxin in the male assumed the simplistic hypothesis of only a single relaxin-like entity. This review systematically examines the expression and physiology of relaxin-like molecules in the male reproductive system in order to reappraise the importance of this hormone system for male reproductive function. Although there are important species differences, only INSL3 and INSL6 appear to be generally expressed at a moderately high level within the testis, whereas ovarian RLN is consistently a major secretory product of the prostate epithelium. However, all members of this relaxin-like family appear to be expressed also at a low level in different organs of the male reproductive system, suggesting possible autocrine/paracrine effects. The four receptors (RXFP1-4) for these peptides are also expressed to differing levels in both somatic and seminiferous compartments of the testis and in the prostate, supporting relevant functions for most members of this interesting peptide family. Recent studies of relaxin family peptides in prostate pathology highlight their functional importance in the clinical context as potential causative, diagnostic and therapeutic agents and warrant more specific and detailed studies of their roles also in regard to male fertility and other aspects of male reproductive function.

Detection of Cx43 expression in equine testicular, epididymal, and prostatic cells is important for a better understanding of the role of intercellular membrane channels in direct cell communication within the reproductive tract of stallions.

Leydig cell tumors (LCT) are the most common type of testicular stromal tumor. Herein, we investigate the G protein-coupled estrogen receptor (GPER) and peroxisome proliferator-activated receptor (PPAR) implication in regulation of lipid homeostasis including the expression of steroidogenesis-controlling molecules in clinical specimens of LCTs and tumor Leydig cells (MA-10). We showed the general structure and morphology of LCTs by scanning electron and light microscopy. In LCTs, mRNA and protein analyses revealed increased expression of GPER and decreased expression of PPARα, β, and γ. Concomitantly, changes in expression pattern of the lutropin receptor (LHR), protein kinase A (PKA), perilipin (PLIN), hormone sensitive lipase (HSL), steroidogenic acute regulatory protein (StAR), translocator protein (TSPO), HMG-CoA synthase, and reductase (HMGCS, HMGCR) were observed. Using MA-10 cells treated with GPER and PPAR antagonists (alone and in combination), we demonstrated GPER-PPAR-mediated control of estradiol secretion via GPER-PPARα and cyclic guanosine monophosphate (cGMP) concentration via GPER-PPARγ. It is assumed that GPER and PPAR can crosstalk, and this can be altered in LCT, resulting in a perturbed lipid balance and steroidogenesis. In LCTs, the phosphatidylinositol-3-kinase (PI3K)-Akt-mTOR pathway was disturbed. Thus, PI3K-Akt-mTOR with cGMP can play a role in LCT outcome and biology including lipid metabolism.

BackgroundThe dynamic cross-talk between epididymal cells is hormonally regulated and, in part, through direct cell-to-cell interactions. To date, no information is available regarding possible impact of anti-androgens on the proteins involved in the gap junctional communication within the boar epididymis. Thus, a question arised whether prenatal or postnatal exposure to an anti-androgen flutamide alters the expression of gap junction protein - connexin43 (Cx43) and androgen receptor (AR) expression in the caput, corpus and cauda epididymis and leads to delayed effects on morphology and function of adult pig epididymis.MethodsFirst two experimental groups received flutamide prenatally on gestational days 20-28 and 80-88 (GD20 and GD80) and further two groups were exposed to flutamide postanatally on days 2-10 and 90-98 after birth (PD2 and PD90). Epididymides were collected from adult boars. Routine histology was performed using hematoxylin-eosin staining. The expression of Cx43 and AR were analyzed using immunohistochemistry and Western blotting. Both analyses were supported by quantitative approaches to demonstrate the variations of the expression levels following the treatment. Apoptotic cells were identified using TUNEL assay.ResultsHistological examination revealed differences in epididymal morphology of flutamide-exposed boars when compared to controls. Scarce spermatic content were seen within the corpus and cauda lumina of GD20, PD2 and PD90 groups. Concomitantly, frequency of epididymal cell apoptosis was significantly higher (p < 0.05) after exposure to flutamide at GD20. Moreover, in GD20, PD2, and PD90 groups, significantly lower AR expression (p < 0.05) was found in the principal and basal cells of the corpus and cauda regions, while in the stromal cells AR expression was significantly reduced (p < 0.05) along the epididymal duct. Concomitantly, a decrease in Cx43 expression (p < 0.05) was noticed in the stromal cells of the cauda region of GD20 and PD2 groups. This indicates high sensitivity of the stromal cells to androgen withdrawal.ConclusionsThe region-specific alterations in the epididymis morphology and scarce spermatic content within the lumina of the corpus and cauda indicate that flutamide can induce delayed effects on the epididymal function of the adult boar by decrease in AR protein levels that results in altered androgen signaling. This may cause disturbances in androgen-dependent processes including Cx43 (de)regulation, however, we can not exclude the possibility that in response to flutamide decreased Cx43 expression may represent one mechanism responsible for functional disturbance of the boar epididymis.

Placentation requires the production of numerous growth factors, hormones and transcription factors. Many of them, like the adipose tissue-derived leptin or adiponectin, have been identified in the placenta and their role has been established in the proliferation and subsequent development of the placenta. Apelin is another adipokine known for proliferative effects in different cell types. PcR, immunoblotting and immunocytochemistry were used to study mRNA and protein expression of apelin and its receptor (APJ) in syncytiotrophoblast (BeWo) and cytotrophoblast (JEG-3) cells as well in immunohistochemistry in human normal placenta slides. The effect of apelin on cell proliferation study was investigated by alamarBlue ® and cell counting Kit-8 assays, the cell cycle by the flow cytometry method and the protein expression of cyclins and phosphorylation level of extracellular signal-regulated kinases (ERK)1/2, phosphatidylinositol 3'-kinase/protein kinase B (Akt), signal transducer and activator of transcription 3 (Stat3) and 5'-monophosphate-activated protein kinase (AMPKα) were studied by western blotting. Apelin was increased in JEG-3 compared with in BeWo cells, while APJ was the same in both placenta cell lines. Immunocytochemical analyses revealed high cytoplasmic and/or membrane apelin localisation in JEG-3, while BeWo cells exhibited markedly weaker apelin signal in the cytoplasm. Apelin increased cell proliferation as well as the percentage of cells in the G2/M phase of the cell cycle, cyclin proteins and the expression of all kinases mentioned above. In conclusion, apelin by promotion of trophoblast cell proliferation by APJ and ERK1/2, Stat3 and AMPKα signalling could be a new important adipokine in the regulation of early placental development.

Several recent studies have indicated that androgen disruption induced by the administration of anti-androgen flutamide during critical developmental stages results in various reproductive abnormalities, mainly in rodents. However, scarce data are available regarding the alterations caused by this toxicant on cell-cell adhesion molecules. Of note, there is no report on the expression and regulation of tight and adherens junction proteins in the boar. Therefore, the purpose of this study was to analyse whether foetal and neonatal exposure to flutamide affects the expression and distribution of ZO-1, occludin, β-catenin, and N-cadherin in testes of adult pigs. Moreover, to evaluate whether androgen signal was altered in the boar, testicular levels of testosterone and oestradiol and the expression of androgen receptor were examined. Flutamide (50 mg/kg bw) was injected into pregnant gilts during gestational days 20-28 and 80-88 (GD20, GD80), and into male piglets on postnatal days 2-10 (PD2). In the testes of all flutamide-exposed boars, expressions of ZO-1, N-cadherin and β-catenin were significantly decreased at mRNA and protein level, whereas expression of occludin was unchanged when compared with the controls. In addition, in severely damaged seminiferous tubules of PD2 pigs, mislocalization of ZO-1, N-cadherin and β-catenin was observed. Changes in junction protein expressions were accompanied by disturbed intratesticular androgen-oestrogen balance, although androgen receptor expression was not altered. Taken together, these results demonstrate that blockade of androgen action by flutamide during both gestational and neonatal periods affects the expression of ZO-1, N-cadherin and β-catenin in adult pig testes. Of concern, neonatal window seems to be most critical for the organization of BTB and consequently for normal spermatogenesis in the boar. It is likely that altered expression of junction proteins is related to insufficient testosterone production and/or excessive oestradiol synthesis, which may result from impaired Leydig cell function.

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Background Notch signaling pathway is involved in contact‐dependent communication between the cells of seminiferous epithelium, and its proper activity is important for undisturbed spermatogenesis. Objectives The aim was to assess the effect of Notch pathway inhibition on the expression of nuclear (AR) and membrane (ZIP9) androgen receptors and androgen‐regulated genes, claudin‐5 and claudin‐11, in TM4 mouse Sertoli cell line. Materials and methods DAPT (γ‐secretase inhibitor) treatment and recombination signal binding protein silencing were employed to reduce Notch signaling, whereas immobilized ligands were used to activate Notch pathway in TM4 cells. To reveal specific effect of each androgen receptor, AR or ZIP9 silencing was performed. Results Notch pathway inhibition increased the expression of AR and ZIP9 mRNA and proteins (p < 0.01; p < 0.05) in TM4 cells, whereas incubation with Notch ligands, rDLL1 or rJAG1, reduced AR (p < 0.01; p < 0.001) and ZIP9 (p < 0.05; p < 0.01) expressions, respectively. Testosterone enhanced the expression of both receptors (p < 0.05; p < 0.01). Androgen‐regulated claudin‐5 and claudin‐11 (p < 0.01; p < 0.001) and cAMP (p < 0.001) were elevated in Notch‐inhibited cells, while activation of Notch signaling by DLL1 or JAG1 reduced claudin‐11 or claudin‐5 level (p < 0.01; p < 0.001), respectively. Discussion Our findings indicate opposite effect of Notch and androgen signaling on the expression of androgen receptors in TM4 cells. We demonstrated that AR expression is regulated by DLL1‐mediated Notch signaling, whereas JAG1 is involved in the regulation of ZIP9. The expression of both claudins and cAMP production is under inhibitory influence of Notch pathway. The effects of Notch signaling on claudin‐5 and claudin‐11 expression are mediated by ZIP9 and AR, respectively. Conclusion Notch signaling may be considered as an important pathway controlling Sertoli cell physiology, and its alterations may contribute to disturbed response of Sertoli cells to androgens.