Mouse testicular transcriptome after modulation of non-canonical oestrogen receptor activity.

The aims of this study were to shed light on the role of G-protein-coupled membrane oestrogen receptor (GPER) and oestrogen-related receptor (ERR) in mouse testis function at the gene expression level, as well as the involvement of GPER and ERR in cellular and molecular processes. Male mice were injected (50µg kg-1,s.c.) with the GPER antagonist G-15, the ERRα inverse agonist XCT790 or the ERRß/ERRγ agonist DY131. Next-generation sequencing (RNA-Seq) was used to evaluate gene expression. Bioinformatic analysis of read abundance revealed that 50, 86 and 171 transcripts were differentially expressed in the G-15-, XCT790- and DY131-treated groups respectively compared with the control group. Annotated genes and their protein products were categorised regarding their associated biological processes and molecular functions. In the XCT790-treated group, genes involved in immunological processes were upregulated. In the DY131-treated group, genes with increased expression were primarily engaged in protein modification (protein folding and small protein conjugation). In addition, the expression of genes recognised as oncogenes, such as BMI1 proto-oncogene, polycomb ring finger (Bmi1) and nucleophosphin 1 (Npm1), was significantly increased in all experimental groups. This study provides detailed information regarding the genetic changes in the testicular transcriptome of the mouse in response to modulation of non-canonical oestrogen receptor activity.

Differential expression of cell-cell junction proteins in the testis, epididymis, and ductus deferens of domestic turkeys (Meleagris gallopavo) with white and yellow semen.

Tight, adherens, and gap junctions are involved in the regulation of reproductive tissue function in male mammals. In birds, including domestic turkeys, intercellular interactions performed by junctional networks have not yet been studied. Furthermore, the cellular and molecular basis of yellow semen syndrome (YSS) in the turkey population remains poorly understood. Thus, the aim of the present study was 2-fold: first, to provide new information on the localization and expression of cell-cell junction proteins in the testis, epididymis, and ductus deferens of domestic turkeys and second, to compare expression of junctional protein genes between 2 turkey population, one that produces white normal semen (WNS) and the other that produces yellow abnormal semen. Expression of occludin, zonula occludens-1 (ZO-1), connexin 43 (Cx43), N- and E-cadherin, and ß-catenin genes were investigated using 3 complementary techniques: quantitative real-time PCR, western blot, and immunohistochemistry. Compared to WNS testis, epididymis, and ductus deferens, YSS tissues exhibited downregulation of occludin and ß-catenin mRNA (P < 0.05) and protein (P < 0.05 and P < 0.01, respectively) and upregulation of N- and E-cadherin mRNA (P < 0.001, P < 0.05, P < 0.01, respectively) and protein (P < 0.01, P < 0.05, and P < 0.05, respectively). In contrast, ZO-1 and Cx43 mRNA and protein were upregulated in YSS testis (P < 0.05 and P < 0.001, respectively) but not in epididymis and ductus deferens; both mRNAs and proteins were downregulated (P < 0.05) compared to the respective WNS epididymis and ductus deferens. Altered staining intensity of immunoreactive proteins in YSS vs. WNS reproductive tissue sections confirmed the gene expression results. The present study is the first to demonstrate altered levels of junctional protein gene expression in reproductive tissues of male YSS turkeys. These findings may suggest that subtle changes in junctional protein expression affect the microenvironment in which spermatozoa develop and mature and thus may have an impact on the appearance of yellow semen in domestic turkeys.

Towards understanding leydigioma: do G protein-coupled estrogen receptor and peroxisome proliferator-activated receptor regulate lipid metabolism and steroidogenesis in Leydig cell tumors?

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, ß, 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.

The meaning of non-classical estrogen receptors and peroxisome proliferator-activated receptor for boar Leydig cell of immature testis.

Communication in biological systems involves diverse-types of cell-cell interaction including cross-talk between receptors expressed by the target cells. Recently, novel sort of estrogen receptors (G protein - coupled estrogen receptor; GPER and estrogen-related receptor; ERR) that signal directly via estrogen binding and/or via mutual interaction-regulated estrogen signaling were reported in various organs including testis. Peroxisome proliferator - activated receptor (PPAR) is responsible for maintaining of lipid homeostasis that is critical for sex steroid production in the testis. Here, we investigated the role of interaction between GPER, ERRß and PPARγ in steroidogenic Leydig cells of immature boar testis. Testicular fragments cultured ex vivo were treated with GPER or PPARγ antagonists. Then, cell ultrastructure, expression and localization of GPER, ERRß, PPARγ together with the molecular receptor mechanism, through cyclic AMP and Raf/Ras/extracellular signal activated kinases (ERK), in the control of cholesterol concentration and estrogen production by Leydig cells were studied. In the ultrastructure of antagonist-treated Leydig cells, mitochondria were not branched and not bifurcated as they were found in control. Additionally, in PPARγ-blocked Leydig cells changes in the number of lipid droplets were revealed. Independent of used antagonist, western blot revealed decreased co-expression of GPER, ERRß, PPARγ with exception of increased expression of ERRß after PPARγ blockage. Immunohistochemistry confirmed presence of all receptors partially located in the nucleus or cytoplasm of Leydig cells of both control and treated testes. Changes in receptor expression, decreased cholesterol and increased estradiol tissue concentrations occurred through decreased cAMP level (with exception after GPER blockage) as well as Raf/Ras/ERK pathway expression. These all findings indicate that GPER-ERRß-PPARγ interaction exists in immature boar testis and regulates Leydig cell function. Further detailed studies and considerations on GPER-ERRß-PPARγ as possible diagnosis/therapy target in disturbances of testis steroidogenic function are needed.

Notch signaling regulates nuclear androgen receptor AR and membrane androgen receptor ZIP9 in mouse Sertoli cells.

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.

Leydig cell tumorigenesis - implication of G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and xenoestrogen exposure. In vivo and in vitro appraisal.

The etiology and molecular characteristics of Leydig cell tumor (LCT) are scarcely known. From the research data stems that estrogen can be implicated in LCT induction and development, however it is not investigated in detail. Considering the above, herein we analyzed the relation between G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and insulin-like family peptides (insulin-like 3 peptide; INSL3 and relaxin; RLN) expressions as well as estrogen level with impact of xenoestrogen (bisphenol A; BPA, tetrabromobisphenol A; TBBPA, and tetrachlorobisphenol A; TCBPA). While in our previous studies altered GPER-PPAR partnership was found in human LCT being a possible cause and/or additionally effecting on LCT development, here mouse testes with experimentally induced LCT and mouse tumor Leydig cell (MA-10) treated with BPA chemicals were examined. We revealed either diverse changes in expression or co-expression of GPER and PPAR in mouse LCT as well as in MA-10 cells after BPA analogues when compared to human LCT. Relationships between expression of INSL3, RLN, including co-expression, and estrogen level in human LCT, mouse LCT and MA-10 cells xenoestrogen-treated were found. Moreover, involvement of PI3K-Akt-mTOR pathway or only mTOR in the interactions of examined receptors and hormones was showed. Taken together, species, cell of origin, experimental system used and type of used chemical differences may result in diverse molecular characteristics of LCT. Estrogen/xenoestrogen may play a role in tumor Leydig cell proliferation and biochemical nature but this issue requires further studies. Experimentally-induced LCT in mouse testis and MA-10 cells after BPA exposure seem to be additional models for understanding some aspects of human LCT biology.

Do G-protein coupled estrogen receptor and bisphenol A analogs influence on Leydig cell epigenetic regulation in immature boar testis ex vivo?

Organotypic culture of testicular fragments from 7-day-old male pigs (Polish White Large) was used. Tissues were treated with an antagonist of G-protein coupled estrogen receptor (GPER) (G-15; 10 nM), and bisphenol A (BPA), and its analogs (TBBPA, TCBPA; 10 nM) alone or in combination and analyzed using electron and light (stainings for collagen fibers, lipid droplet and autophagy markers) microscopes. In addition, mRNA and protein abundances and localization of molecules required for miRNA biogenesis and function (Drosha, Exportin 5; EXPO5, Dicer, and Argonaute 2; AGO2) were assessed together with calcium ion (Ca2+) and estradiol concentrations. Regardless of GPER blockade and/or treatment with BPA, TBBPA and TCBPA, there were no changes in Leydig cell morphology. Also, there were no changes in lipid droplet content and distribution but there were changes in lipid and autophagy protein abundance. In the interstitial tissue, there was an increase of collagen content, especially after treatment with BPA analogs and G-15 + BPA. Independent of the treatment, there was downregulation of EXPO5 and Dicer genes but the Drosha and AGO2 genes were markedly upregulated as a result of treatment with G-15 + BPA and TCBPA, respectively. There was always a lesser abundance of EXPO5 and AGO2 proteins regardless of treatment. There was markedly greater abundances of Drosha after G-15 + BPA treatment, and this also occurred for Dicer after treatment with G-15 + TCBPA. Immunolocalization of miRNA proteins indicated there was a cytoplasmic-nuclear pattern in control and treated cells. There was an increase of Ca2+ concentrations after treatment with G-15 and BPA analogs. Estradiol secretion decreased after antagonist and chemical treatments when these were administered alone, however, there was an increase in estradiol secretion after treatment with combinations of these compounds.

Do estrogens regulate lipid status in testicular steroidogenic Leydig cell?

In this study mouse Leydig cell (MA-10) were treated with G-protein coupled membrane estrogen receptor antagonist (G-15; 10 nM). Cells were analyzed by Western blotting for expression of estrogen-related receptors (ERRα, ß and γ), steroidogenic markers (lutropin receptor; LHR and 3ß-hydroxysteroid dehydrogenase; 3ß-HSD) and lipid droplet markers (perilipin; PLIN and microtubule-associated protein 1 A/1B-light chain 3; LC3). Concomitantly, microscopic analyses by light microscope (immunofluorescent staining for lipid droplets, PLIN and LC3) as well as by electron microscope (for lipid droplet ultrastructure) were utilized. For analysis of cholesterol content, cAMP level and progesterone secretion, G-15, estrogen receptor (ER) antagonist (ICI 182,780; 10 µM), 17ß-estradiol (10 mM) and, bisphenol A (BPA; 10 nM) were used alone or in combinations. We revealed no changes in ERRs expression but alterations in ERRß and γ localization in G-15-treated cells when compared to control. Partial translocation of ERRß and γ from the cell nucleus to cytoplasm was observed. Decreased expression of LHR, 3ß-HSD, PLIN and LC3 was detected. Moreover, in treated cells large lipid droplets and differences in their distribution were found. Very strong signal of co-localization for PLIN and LC3 was found in treated cells when compared to control. In ultrastructure of treated cells, degenerating lipid droplets and double membrane indicating on presence of lipophagosome were observed. We found, that only (i) BPA and G-15 did not effect on cholesterol content, (ii) BPA, G-15 and ICI did not effect on cAMP level and (iii) BPA, ICI alone and in combination, and BPA with G-15 did not modulate progesterone secretion. These findings showed complex and diverse estrogen effects on mouse Leydig cells at various steps of steroid hormone production (cholesterol storage, release and processing). Lipid homeostasis and metabolism in these cells were affected by endogenous and exogenous estrogen, interactions of receptors (GPER, ER and ERR) and GPER and ER antagonists.

Expression and secretion of albumin in male turkey (Meleagris gallopavo) reproductive tract in relation to yellow semen syndrome1.

Yellow semen syndrome (YSS) is the most widely recognized problem among male turkeys. Yellow semen is of low quality and, when used for insemination, results in reduction of fertility and hatchability. Elevated level of serum albumin-like protein accession no. XP_003205725 is a characteristic feature of yellow seminal plasma suggesting albumin role in YSS pathology. However, knowledge regarding the expression of albumin in the reproductive tract in relation to YSS is very limited. The aim of this study was to identify albumin secretion and localization sites in the turkey reproductive tract in relation to YSS. Reproductive tract tissues and liver originating from turkeys producing white semen (WS) and YSS were used for analysis of albumin mRNA expression and its localization using immunohistochemistry. Moreover, albumin abundance in tissues, blood and seminal plasma was analyzed using two dimensional electrophoresis and western blot analysis. Albumin mRNA expression was found in all parts of the reproductive tract. Apart from the liver, the highest expression of albumin was found in the ductus deferens in YSS turkeys. The testicular spermatids, Leydig, and myoid cells and the epithelium of the epididymis and ductus deferens were the main secretion sites of albumin in the reproductive tract in turkeys. Higher albumin abundance was found in the reproductive tract and seminal plasma of YSS toms compared to WS toms. Our results demonstrated that germ cells from spermatocytes to spermatids, Leydig cells, and myoid cells synthesized and secreted albumin in turkey testis, and epithelial cells are the main secretion sites in epididymis and ductus deferens. Ductus deferens secretion of albumin seems to be mostly responsible for YSS. Over-secretion by the ductus deferens may be the main origin of albumin abundance in YSS semen. Knowledge regarding disturbances of albumin secretion in relation to YSS may be useful for future work on studies related to better understanding the molecular basis of YSS.

Regulation of steroidogenic function of mouse Leydig cells: G-coupled membrane estrogen receptor and peroxisome proliferator-activated receptor partnership.

We tested whether G-coupled membrane estrogen receptor (GPER) and peroxisome proliferator activated receptor (PPAR) partnership exists and whether this interaction regulates mouse Leydig cell function. Mature and aged mice were treated with the antagonist of GPER (G-15; 50 µg/kg b.w). Leydig cells (MA-10) were treated with G-15 (10 nM) alone or in combination with peroxisome proliferator-activated receptor α or γ antagonists, respectively (PPARα, 10 µM; PPARγ, 10 µM). GPER blockage affected testis steroidogenic status via changes in lutropin and cholesterol levels as well as protein expression alterations of the lutropin receptor, acute steroidogenesis activating protein, translocator protein, and protein kinase A in mouse Leydig cells both in vivo and in vitro. Inactivation of both GPER and PPAR in vitro revealed expressional modulation of other steroidogenesis-controlling molecules acting on various steps of lipid homeostasis e.g. cytochrome P450scc, perilipin, hormone sensitive lipase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase. Concomitantly, microscopic analysis of cells treated with antagonists showed changes in morphology, migration competences and cytoskeleton structure. In the above processes, the action of GPER and PPARα was regulated through the PI3K/Akt pathway, while PPARγ was mediated by the Ras/Raf pathway. In addition, GPER and PPARs specifically controlled individual signaling proteins. For the first time, we report here the importance of GPER-PPARα and -PPARγ 'neopartnership' in maintenance of Leydig cell morpho-functional status.

The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function-in vivo and in vitro evaluation.

In this study, G-coupled estrogen receptor (GPER) was inactivated, by treatment with antagonist (G-15), in testes of C57BL/6 mice: immature (3 weeks old), mature (3 months old) and aged (1.5 years old) (50 µg/kg bw), as well as MA-10 mouse Leydig cells (10 nM/24 h) alone or in combination with 17ß-estradiol or antiestrogen (ICI 182,780). In G-15-treated mice, overgrowth of interstitial tissue was found in both mature and aged testes. Depending on age, differences in structure and distribution of various Leydig cell organelles were observed. Concomitantly, modulation of activity of the mitochondria and tubulin microfibers was revealed. Diverse and complex GPER regulation at the mRNA level and protein of estrogen signaling molecules (estrogen receptor α and ß; ERα, ERß and cytochrome P450 aromatase; P450arom) in G-15 Leydig cells was found in relation to age and the experimental system utilized (in vivo and in vitro). Changes in expression patterns of ERs and P450arom, as well as steroid secretion, reflected Leydig cell heterogeneity to estrogen regulation throughout male life including cell physiological status.We show, for the first time, GPER with ERs and P450arom work in tandem to maintain Leydig cell architecture and supervise its steroidogenic function by estrogen during male life. Full set of estrogen signaling molecules, with involvement of GPER, is crucial for proper Leydig cell function where each molecule acts in a specific and/or complementary manner. Further understanding of the mechanisms by which GPER controls Leydig cells with special regard to male age, cell of origin and experimental system used is critical for predicting and preventing testis steroidogenic disorders based on perturbations in estrogen signaling.

Differences in aromatase expression and steroid hormone concentrations in the reproductive tissues of male domestic turkeys (Meleagris gallopavo) with white and yellow semen.

1. To show hormonal differences between male turkeys with yellow semen syndrome (YSS) and white, normal semen (WNS), the expression of aromatase, oestrogen receptor α (ERα), and oestrogen receptor ß (ERß) as well as testosterone and oestradiol concentrations in YSS and WNS testes, epididymis, and ductus deferens were examined. 2. To measure gene expression levels of aromatase and oestrogen receptors (ERs), three complementary techniques (real-time PCR, Western blot, and immunohistochemistry) were used, whereas steroid hormone levels were determined radio-immunologically. 3. Upregulation of aromatase and ERα mRNAs in YSS testes (P < 0.05; P < 0.01), epididymis (P < 0.001; P < 0.001), and ductus deferens (P < 0.05; P < 0.01) compared to those of WNS tissues was detected. Significant increases in the levels of aromatase and ERα proteins were detected in YSS testes (P < 0.001; P < 0.05), epididymis (P < 0.001; P < 0.001), and ductus deferens (P < 0.001; P < 0.05). The expression of ERß mRNA and protein level was upregulated in the testes (P < 0.05; P < 0.01) and epididymis (P < 0.001; P < 0.01) but not in ductus deferens where it was downregulated (P < 0.01; P < 0.01). Increased intensity of immunoreactive proteins in YSS versus WNS reproductive tissues corroborated gene expression results. 4. Testosterone concentration diminished in YSS epididymis (P < 0.05) and ductus deferens (P < 0.05), but not in the testes, remaining at high level (P < 0.05) compared to WNS values. Concomitantly, increased oestradiol concentration was found in YSS testes (P < 0.05) and epididymis (P < 0.05) but decreased in the ductus deferens (P < 0.05). 5. From the published literature, this study is the first to demonstrate the ability for androgen aromatisation in the turkey reproductive tissues and to show the cellular targets for locally produced oestrogens. The data suggested that the androgen/oestrogen ratio is a mechanistic basis for amplification of differences between turkeys with white and yellow semen and that these results can have a relevance in applied sciences to widen the knowledge on domestic bird reproduction.

Interplay between estrogen-related receptors and steroidogenesis-controlling molecules in adrenals. In vivo and in vitro study.

Estrogen-related receptors (ERRs) α, ß and γ appear to be novel molecules implicated in estrogen signaling. We blocked and activated ERRs in mouse (C57BL/6) adrenals and adrenocortical cells (H295R) using pharmacological agents XCT 790 (ERRα antagonist) and DY131 (ERRß/γ agonist), respectively. Mice were injected with XCT 790 or DY131 (5 µg/kg bw) while cells were exposed to XCT 790 or DY131 (0.5 µg/L). Irrespectively of the agent used, changes in adrenocortical cell morphology along with changes in lutropin, cholesterol levels and estrogen production were found. Diverse and complex ERRs regulation of multilevel-acting steroidogenic proteins (perilipin; PLIN, cytochrome P450 side-chain cleavage; P450scc, translocator protein; TSPO, steroidogenic acute regulatory protein; StAR, hormone sensitive lipase; HSL and HMG-CoA reductase; HMGCR) was revealed. Blockage of ERRα decreased P450scc, StAR and TSPO expressions. Activation of ERRß/γ increased P450scc, StAR and HMGCR while decreased HSL expressions. PLIN expression increased either after XCT 790 or DY131 treatment. Additionally, treatment with both XCT 790 or DY131 decreased activity of Ras/Raf, Erk and Akt indicating their involvement in control of morphology and steroidogenic function of cortex cells. ERRs are important in maintaining morpho-function of cortex cells through action in specific, opposite, or common manner on steroidogenic molecules.

Does signaling of estrogen-related receptors affect structure and function of bank vole Leydig cells?

To get a deeper insight into the function of estrogen-related receptors (ERRs) and dissect underlying mechanism in Leydig cells, ERRs (type α, ß and γ) were blocked or activated in testes of adult bank voles (Myodes glareolus) which show seasonal changes in the intratesticular sex hormones level. Both actively reproducing animals (long day conditions; LD) and those with regression of the reproductive system (short day conditions; SD) received intraperitoneal injections of selective ERRα antagonist 3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide (XCT 790) or selective ERRß/ERRγ agonist N-(4-(Diethylaminobenzylidenyl)-N'-(4-hydroxybenzoyl)-hydrazine (DY131) (50 µ/kg bw; six doses every other day). Markedly more, XCT 790 (P < 0.05) but also DY131 affected interstitial tissue histology whose volume increased in both LD and SD males while seminiferous epithelium structure was untouched. Ultrastructure analysis revealed alterations in mitochondria number as well as endoplasmic reticulum and Golgi complexes volume and structure especially after ERRα blockage. Diverse and complex ERRs regulation at mRNA level and protein expression (P < 0.05; P < 0.01 and P < 0.001) of steroidogenic (lutropin receptor (LHR), translocator protein (TSPO), steroidogenic acute regulatory protein (StAR)) and secretory (insulin-like protein 3 (INSL3) and relaxin (RLN)) molecules were revealed in relations to endogenous estrogen level in treated males. Notably, immunolocalization of ERRs and above proteins, exclusively in Leydig cells, indicated their involvement in Leydig cell function control based on interactions with endogenous estrogen level and/or estrogen signaling via ERRs. Treatment with XCT 790 or DY131 significantly decreased (P < 0.05; P < 0.01 and P < 0.001) intratesticular estrogens concentration, with exception in SD DY131 males. In addition, androgens level was decreased, but not in LD DY131 voles. Similarly, ERRßγ activation significantly reduced (P < 0.05; P < 0.01 and P < 0.001) cAMP and calcium ions (Ca2+) concentrations particularly in DY131 voles. Overall, for the first time, we have shown that ERRs are involved in maintenance of Leydig cell architecture and supervision of its steroidogenic and secretory activity that is closely related to endogenous estrogen status in the testis. Further understanding of mechanism(s) by which individual types of ERRs can control Leydig cell function is relevant for predicting and preventing steroidogenic and spermatogenic disorders.

Maternal high-fat diet during pregnancy and lactation had gender difference effect on adiponectin in rat offspring.

This study investigated the effect of a high-fat (HF) diet on plasma adiponectin and steroid hormones levels, and the protein expression of adiponectin and its receptors, in the gonads and gonadal (periovarian and epididymal) white adipose tissue (WAT) of dams and their offspring. Female Wistar rats were fed a HF diet (30% fat) or a standard breeding (BD) diet (5% fat) during pregnancy and lactation. At 21 days of lactation, mothers and both sexes of prepubertal offspring were killed by decapitation. Plasma adiponectin, testosterone (T) and oestradiol (E2) levels were analyzed by ELISA. The protein expression of adiponectin and its receptors 1 (AdipoR1) and 2 (AdipoR2) was assayed by Western blot and immunohistochemistry. Plasma adiponectin levels in HF dams were lower compared to BD dams, and correlated with protein expression of adiponectin and its receptors, but not with steroid hormone levels. Female HF offspring had lower plasma adiponectin levels, reduced intensity of adiponectin and AdipoR1 in the ovary, and decreased E2 in parallel with increased T. In contrast, male HF offspring had higher plasma adiponectin levels, increased intensity of adiponectin and AdipoR1 in the testis, and decreased T in parallel with increased E2. In conclusion, feeding a HF diet to dams during pregnancy and lactation disturbs plasma adiponectin levels and protein expression, both in female and male offspring; it lowers adiponectin secretion and protein expression in the female whereas in male it is increased. As a consequence, there is disruption of steroid secretion in offspring, towards T in females, and E2 in males.

Primary and tumor mouse Leydig cells exposed to polychlorinated naphthalenes mixture: Effect on estrogen related-receptors expression, intracellular calcium level and sex hormones secretion.

We report the effects of polychlorinated napthalanes (PCNs) on the mRNA expression of estrogen-related receptors (ERRs) α, ß and γ, calcium (Ca2+) concentration, and sex steroid secretion in mouse primary and tumor Leydig cells. The cells were exposed to a mixture of PCNs (10nM) alone or in combination with one of sex steroid receptor antagonists; 182,780 (ICI; 10µM); hydroxyflutamide (HF; 10(-4)M) and G-coupled estrogen receptor antagonist (G15; 10nM) respectively. The expression of mRNAs and protein for ERRα, ß, and γ was detected in primary and tumor Leydig cells. The expression of ERRs was always lower in primary Leydig cells. Exposure of Leydig cells to PCNs significantly increased the expression of ERRs mRNA irrespective of the cell type. Concomitantly, an increased concentration of Ca2+ and sex steroids was revealed in exposed cells. After ICI, HF or G15 was added no changes in expression of ERRs was found. In Leydig cells changes in ERRs expression at mRNA level are clearly linked to changes in Ca2+ level and steroid secretion. Estrogen and androgen receptors are not involved in PCNs action in Leydig cells. The effect of PCNs on mouse Leydig cells is independent on the cell of origin (primary or tumor).

Flutamide alters the distribution of c-Src and affects the N-cadherin-ß-catenin complex in the seminiferous epithelium of adult rat.

This study was undertaken to explore interactions between c-Src kinase and the N-cadherin-ß-catenin complex in seminiferous tubules of flutamide-treated rats. An anti-androgen flutamide (50 mg/kg bw) was injected daily into adult rats from postnatal days 82 to 88. Testes from 90-day-old control and flutamide-treated rats were used for experiments. Flutamide did not affect testis morphology, but impaired connexin43 immunoexpression between Sertoli cells at the blood-testis barrier (BTB) region, indicating the BTB as a sensitive target for flutamide. Real-time RT-PCR and western blot analyses revealed upregulation of N-cadherin at the mRNA and protein level after flutamide exposure (p < 0.05), whereas no changes in ß-catenin and c-Src expression were observed. Notably, membranous ß-catenin immunolocalization indicated its involvement in the cell adhesion complex rather than its contribution to the Wnt signaling pathway. As we used an exposure regime which avoided germ cell loss, it is likely that changes in the N-cadherin-ß-catenin complex are a primary effect of androgen signaling disruption by flutamide. Immunohistochemistry revealed a diffusion of N-cadherin and ß-catenin signals away from the BTB with concomitant disruption of c-Src staining pattern. As detected by immunofluorescence and coimmunoprecipitation, flutamide promoted disassembly of the N-cadherin-ß-catenin complex, induced N-cadherin to dissociate from c-Src at the BTB site, and altered interactions between the cell junction proteins and/or c-Src. Equally important, increased levels of p-N-cadherin-Tyr860 and p-ß-catenin-Tyr654 (p < 0.05) pointed to a mechanism related to adhesion complex disassembly and suggested a potential role of c-Src in the control of the protein-protein dynamics. Overall, for the first time we have shown that flutamide alters the distribution of c-Src and affects N-cadherin-ß-catenin interactions at the BTB. Understanding mechanism(s) by which anti-androgens can affect intercellular adhesion within the testis is relevant for predicting and preventing reproductive disorders affecting male fertility.

Postnatal exposure to flutamide affects CDH1 and CTNNB1 gene expression in adult pig epididymis and prostate and alters metabolism of testosterone.

In both epididymis and prostate the dynamic cross-talk between the cells is hormonally regulated and, in part, through direct cell-to-cell interactions. Functionality of the male reproductive organs may be affected by exposure to specific chemicals, so-called 'reprotoxicants'. In this study we tested whether early postnatal and prepubertal exposure to anti-androgen flutamide altered the expression of adherens junction genes encoding E-cadherin (CDH1) and ß-catenin (CTNNB1) in adult pig epididymis and prostate. In addition, the expression of mRNAs and proteins for 5α-reductase (ST5AR2) and aromatase (CYP19A1) were examined to show whether flutamide alters metabolism of testosterone. Thus, flutamide was injected into male piglets between Days 2 and 10 and between Days 90 and 98 postnatally (PD2 and PD90; 50 mg/kg bw), tissues that were obtained on postnatal Day 270. To assess the expression of the genes and proteins, real-time RT-PCR and Western blot were performed respectively. Moreover, adherens junction proteins were localized by immunohistochemistry. In response to flutamide, CDH1 and CTNNB1 expressions were down-regulated along the epididymis, mostly in PD2 group (p < 0.001, p < 0.01). In the prostate, CDH1 mRNA and protein expressions were significantly down-regulated (p < 0.01), whereas CTNNB1 mRNA was slightly up-regulated in both flutamide-treated groups. CTNNB1 protein level was markedly elevated in both PD2 (p < 0.001) and PD90 (p < 0.01) groups. In the epididymis, the expression of ST5AR2 and CYP19A1 was down- and up-regulated, respectively (p < 0.05), whereas in the prostate evident decrease in CYP19A1 expression (p < 0.001, p < 0.01, p < 0.05) was demonstrated. In both tissues, membranous immunolocalization of CTNNB1 suggests its involvement in cell-cell adhesion. Overall, flutamide administration resulted in suppression of androgen action in the epididymis and prostate leading to deregulation of CDH1 and CTNNB1 gene expressions which is probably caused by the alterations in the expression of ST5AR2 and CYP19A1 in both reproductive organs.

Detection of aromatase, androgen, and estrogen receptors in bank vole spermatozoa.

Spermatozoa are highly specialized cells which transport a single-copy haploid genome to the site of fertilization. Before this, spermatozoa undergo a series of biochemical and functional modifications. In recent years, the crucial role of androgens and estrogens in proper germ cell differentiation during spermatogenesis has been demonstrated. However, their implication in the biology of mature male gametes is still to be defined. Our study provides evidence for the first time that aromatase, the androgen receptor (AR), as well as the estrogen receptors α and ß (ERα and ERß), are present in bank vole spermatozoa. We demonstrated the region-specific localization of these proteins in bank vole spermatozoa using confocal microscopy. Immunoreactive aromatase was observed in the proximal head region and in both the proximal and distal tail regions, whereas steroid hormone receptors were found only in the proximal region of the sperm head. Protein expression in sperm lysates was detected by Western blot analysis. Immunohistochemical results were analyzed quantitatively. Our results show that bank vole spermatozoa are both a source of estrogens and a target for steroid hormone action. Moreover, the presence of aromatase and steroid hormone receptors in the bank vole spermatozoa indicates a potential function of these proteins during capacitation and/or the acrosome reaction.

Prenatal and neonatal exposure to flutamide affects function of Leydig cells in adult boar.

In this study, flutamide, an androgen receptor antagonist, was used as a tool to better understand the role of androgen receptor signaling and androgen signaling disruption during fetal and neonatal periods on porcine Leydig cell development and function. Flutamide, 50 mg kg(-1) d(-1) was administered into pregnant gilts during gestational days 20 to 28 and days 80 to 88 and into male piglets on postnatal days 2 to 10 (PD2). Leydig cells of flutamide-exposed boars, especially those of PD2 males, displayed morphologic alterations, increased size, and occupied increased area (P < 0.001) of the testes when compared with the control. Despite this, testosterone concentrations were reduced significantly in comparison with those of controls (P < 0.05, P < 0.001). Reduced testosterone production in response to flutamide exposure appeared to be related to changes in testosterone metabolism, as shown by increased aromatase mRNA (P < 0.05, P < 0.01), protein expression (P < 0.01, P < 0.001), and elevated estradiol concentrations (P < 0.001). Moreover, impaired Leydig cell responsiveness to LH was indicated by the decreased expression of LH receptor (P < 0.05, P < 0.001). No significant effect of flutamide was found on LH and FSH concentrations. Taken together, our data indicate that flutamide when administered during prenatal or neonatal period have a long-term effect on Leydig cell structure and function, leading to androgen-estrogen imbalance. Leydig cell failure was most evident in adult boars neonatally exposed to flutamide, suggesting that androgen action during neonatal development is of pivotal importance for the differentiation and function of porcine adult Leydig cell population.