Modulatory Effects of Estradiol and Its Mixtures with Ligands of GPER and PPAR on MAPK and PI3K/Akt Signaling Pathways and Tumorigenic Factors in Mouse Testis Explants and Mouse Tumor Leydig Cells.

The present study was designed to evaluate how estradiol alone or in combination with G protein-coupled estrogen receptor (GPER) agonists and GPER and peroxisome proliferator-activated receptor (PPAR) antagonists alter the expression of tumor growth factor ß (TGF-ß), cyclooxygenase-2 (COX-2), hypoxia inducible factor 1-alpha (HIF-1α), and vascular endothelial growth factor (VEGF) in mouse testis explants and MA-10 mouse tumor Leydig cells. In order to define the hormone-associated signaling pathway, the expression of MAPK and PI3K/Akt was also examined. Tissue explants and cells were treated with estradiol as well as GPER agonist (ICI 182,780), GPER antagonist (G-15), PPARα antagonist (GW6471), and PPARγ antagonist (T00709072) in various combinations. First, we showed that in testis explants GPER and PPARα expressions were activated by the GPER agonist and estradiol (either alone or in mixtures), whereas PPARγ expression was activated only by GPER agonist. Second, increased TGF-ß expression and decreased COX-2 expression were found in all experimental groups of testicular explants and MA-10 cells, except for up-regulated COX-2 expression in estradiol-treated cells, compared to respective controls. Third, estradiol treatment led to elevated expression of HIF-1α and VEGF, while their lower levels versus control were noted in the remaining groups of explants. Finally, we demonstrated the up-regulation of MAPK and PI3Kp85/Akt expressions in estradiol-treated groups of both ex vivo and in vitro models, whereas estradiol in mixtures with compounds of agonistic or antagonistic properties either up-regulated or down-regulated signaling kinase expression levels. Our results suggest that a balanced estrogen level and its action together with proper GPER and PPAR signaling play a key role in the maintenance of testis homeostasis. Moreover, changes in TGF-ß and COX-2 expressions (that disrupted estrogen pathway) as well as disturbed GPER-PPAR signaling observed after estradiol treatment may be involved in testicular tumorigenesis.

Senescent cells in rabbit, nutria and chinchilla testes-Results from histochemical and immunohistochemical studies.

Rabbit, nutria and chinchilla testes were evaluated to compare testicular cellular senescence. There were no major species-specific differences in structure of either seminiferous tubules or interstitial tissue. There, however, were occasional abnormalities in seminiferous tubule structure with there being multinucleated and exfoliated cells present in rabbit testes. Furthermore, there were seminiferous tubules without a lumen that were filled with premeiotic/meiotic cells in nutria; and tubules with vacuolization with there being no post-meiotic cells in chinchillas. There were no differences in distribution or content of acids, total proteins and polysaccharides in the testis of any of the three species. Results using comparative immunohistochemistry procedures indicated the testes contained a few senescent cells in seminiferous tubules with typical morphology and there was a large number of senescent cells in seminiferous tubules of nutrias and chinchillas that had an abnormal structure (P <0.001). Compared to rabbit testes, in which there was the least number of senescent cells in seminiferous tubules, there was a greater abundance of senescence markers in both nutria and chinchilla testes (P < 0.05; P < 0.001, respectively). Furthermore, there were small abundances of caspase 3 and LC3 in the testes of all species. In chinchilla testes, there was a lesser concentration of cholesterol (P < 0.001) and testosterone compared with the other species. Cellular senescence in testes, therefore, can be assessed by detection of morpho-functional disorders of the testis of the three species evaluated in the present study.

Implication of Membrane Androgen Receptor (ZIP9) in Cell Senescence in Regressed Testes of the Bank Vole.

Here, we studied the impact of exposure to short daylight conditions on the expression of senescence marker (p16), membrane androgen receptor (ZIP9) and extracellular signal-regulated kinase (ERK 1/2), as well as cyclic AMP (cAMP) and testosterone levels in the testes of mature bank voles. Animals were assigned to groups based on an analysis of testis diameter, weight, seminiferous tubule diameter and the interstitial tissue area: group 1, not fully regressed (the highest parameters); group 2 (medium parameters); or group 3, regressed (the lowest parameters). Cells positive for p16 were observed only in the seminiferous tubule epithelium. However, in groups 1 and 2, these were mostly cells sloughed into the tubule lumen. In group 3, senescent cells resided in between cells of the seminiferous epithelium. Staining for ZIP9 was found in Sertoli cells. Western blot analysis showed a trend towards a decreased expression of p16 and ZIP9 in the testes of the voles in groups 2 and 3, compared to group 1. In addition, a trend towards an increased expression of ERK, as well as an increase of cAMP and testosterone levels, was revealed in group 2. In the regressed testes, a functional link exists between senescence and androgen levels with implication of ZIP9 and cAMP/ERK signaling pathways.

Levels of the neuropeptide phoenixin-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue in rat model of polycystic ovary syndrome.

Phoenixin (PNX) is a newly discovered peptide produced by proteolytic cleavage of a small integral membrane protein 20 (Smim20), which acts as an important regulator of energy homeostasis and reproduction. Since dysfunction of reproduction is characteristic in polycystic ovarian syndrome (PCOS), the role of PNX in pathogenesis of PCOS needs further investigation. The objective of this study was to determine expression of Smim20, PNX-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue (PAT) of letrozole induced PCOS rats. Phosphorylation of extracellular signal-regulated kinase (ERK1/2), protein kinases A (PKA) and B (Akt) were also estimated. We observed that PCOS rats had high weight gain and a number of ovarian cyst, high levels of testosterone, luteinizing hormone and PNX-14, while low estradiol. Smim20 mRNA expression was higher in the ovary and PAT, while PNX-14 peptide production was higher only in the ovary of PCOS rat. Moreover, in PCOS rats Gpr173 level was lower in PAT but at the protein level increased only in the ovary. Depending on the tissues, kinases phosphorylation were significantly differ in PCOS rats. Our results showed higher levels of PNX-14 in PCOS rats and indicated some novel findings regarding the mechanisms of PCOS pathophysiology.

Interstitial Leydig Cell Tumorigenesis-Leptin and Adiponectin Signaling in Relation to Aromatase Expression in the Human Testis.

Although epidemiological studies from the last years report an increase in the incidences of Leydig cell tumors (previously thought to be a rare disease), the biochemical characteristics of that tumor important for understanding its etiology, diagnosis, and therapy still remains not completely characterized. Our prior studies reported G-protein coupled estrogen receptor signaling and estrogen level disturbances in Leydig cell tumors. In addition, we found that expressions of multi-level-acting lipid balance- and steroidogenesis-controlling proteins including peroxisome proliferator-activated receptor are altered in this tumor. In order to get deeper into the other molecular mechanisms that regulate lipid homeostasis in the Leydig cell tumor, here we investigate the presence and expression of newly-described hormones responsible for lipid homeostasis balancing (leptin and adiponectin), together with expression of estrogen synthase (aromatase). Samples of Leydig cell tumors (n = 20) were obtained from patients (31-45 years old) and used for light and transmission electron microscopic, western blotting, and immunohistochemical analyses. In addition, body mass index (BMI) was calculated. In tumor mass, abundant lipid accumulation in Leydig cells and various alterations of Leydig cell shape, as well as the presence of adipocyte-like cells, were observed. Marked lipid content and various lipid droplet size, especially in obese patients, may indicate alterations in lipid homeostasis, lipid processing, and steroidogenic organelle function in response to interstitial tissue pathological changes. We revealed significantly increased expression of leptin, adiponectin and their receptors, as well as aromatase in Leydig cell tumors in comparison to control. The majority of patients (n = 13) were overweight as indicated by their BMI. Moreover, a significant increase in expression of phospholipase C (PLC), and kinases Raf, ERK which are part of adipokine transductional pathways, was demonstrated. These data expand our previous findings suggesting that in human Leydig cell tumors, estrogen level and signaling, together with lipid status, are related to each other. Increased BMI may contribute to certain biochemical characteristics and function of the Leydig cell in infertile patients with a tumor. In addition, altered adipokine-estrogen microenvironment can have an effect on proliferation, growth, and metastasis of tumor cells. We report here various targets (receptors, enzymes, hormones) controlling lipid balance and estrogen action in Leydig cell tumors indicating their possible usefulness for diagnostics and therapy.

Effect of estrogen-related receptor silencing on miRNA protein machinery expression, global methylation, and deacetylation in bank vole (Myodes glareolus) and mouse tumor Leydig cells.

The function of estrogen-related receptor (ERR) in testicular cells is at the beginning of exploration. Our previous findings showed that expression pattern of estrogen-related receptor (ERR) in mouse Leydig cell depends on physiological status of the cell. Exogenous hormones/hormonally active chemicals affect ERR expression. In Leydig cells in vitro, ERRα and ERRγ show opposing regulatory properties. The aim of this study was to examine the role of ERR in epigenetic processes in cells with altered level of secreted estrogens; mouse tumor Leydig cells and bank vole Leydig cells, respectively. In Leydig cells, ERRα and ERRγ were silenced via siRNA. mRNA and protein expression and protein localization of molecules required for miRNA biogenesis and function (Exportin 5, Dicer, Drosha and Argonaute 2; Ago2) were studied with the use of qRT-PCR, Western blotting, and immunohistochemistry. Global DNA methylation and histone deacetylation status together with estradiol secretion were determined with fluorometric, and immunoenzymatic assays. Regardless of ERR type knockdown in tumor Leydig cells, downregulation (P < 0.05; P < 0.01; P < 0.001) of Exportin5, Dicer, Drosha but not Ago2 was revealed while at protein level only Drosha was downregulated (P < 0.01) by both ERRα and ERRγ. Oppositely, Exportin5, Dicer and Ago2 showed ERR type-dependent regulation (downregulation; P < 0.01 by ERRα and upregulation; P < 0.01; P < 0.001 by ERRγ). In ERR-silenced vole Leydig cells, expression of Exportin5, endonucleases and Ago2 was not changed. Immunolocalization of Dicer and Ago2 was independent of the cell origin in contrast to localization of Exportin5 and Drosha which was dependent on the cell origin and ERR type knockdown. Absence of ERR effected on cell methylation status (ERRα increased it; P < 0.01 while ERRγ decreased it; P < 0.01, P < 0.001) but it not changed histone deacetylates activity. ERRα and ERRγ silencing decreased (P < 0.01, P < 0.001) estradiol secretion in both tumor and vole Leydig cells. In mouse and bank vole Leydig cell, Exportin5, Dicer, Drosha and Ago2 expression as well as methylation status are regulated by ERR in a manner related to receptor type, molecule type, cell origin and level of secreted estrogen.

Telocytes are localized to testis of the bank vole (Myodes glareolus) and are affected by lighting conditions and G-coupled membrane estrogen receptor (GPER) signaling.

We aim to explore the presence of a novel cell type, telocytes (TCs), in the bank vole testis interstitium following G-coupled membrane estrogen receptor (GPER) signaling withdrawal. In addition, the involvement of interstitial cells in lipid homeostasis was investigated. Bank voles (actively reproducing or regressed) were administered with GPER antagonist (G-15; 50 µg/kg bw) injections. To examine TC distribution, ultrastructure, function, and their connotation in the interstitial tissue lipid balance, electron microscopic observations were implemented. Immunohistochemistry and Western blot for the TC marker, CD34, and lipid balance molecules: leptin, adiponectin, and perilipin were performed. Photoperiod-regulated testis steroidogenic function was estimated via serum melatonin level and intratesticular cholesterol concentrations in immunoenzymatic assays. We demonstrate the presence of TCs in bank vole testis interstitium. Distinctive TC morphology: small cell bodies with very long, slender prolongations, constituting a three-dimensional network around the interstitial cells was seen. Ultrastructurally, scarce mitochondria, a few cisternae of the endoplasmic reticulum, and lipid droplets indicated possible TC implications in lipid homeostasis. Changes in CD34 expression in TCs were seen in relation to GPER disturbances. In GPER-blocked testis, single TCs were present in the LD interstitium when in SD ones they were occasionally absent. Moreover, in TCs of SD voles, a lack of lipid droplets was revealed, likely reflecting attenuated TC function during regression. However, melatonin levels decreased in GPER-blocked LD and SD. Concomitantly, leptin, adiponectin, and perilipin expressions together with cholesterol content varied after blockage. Based on our results we suggest TCs are an important component of the bank vole testis interstitium as they are implicated in ultramorphology maintenance, protein interactions, and lipid homeostasis.

Telocytes in the mouse testicular interstitium: implications of G-protein-coupled estrogen receptor (GPER) and estrogen-related receptor (ERR) in the regulation of mouse testicular interstitial cells.

Telocytes (TCs), a novel type of interstitial cells, are involved in tissue homeostasis maintenance. This study aimed to investigate TC presence in the interstitium of mouse testis. Additionally, inactivation of the G-coupled membrane estrogen receptor (GPER) in the testis was performed to obtain insight into TC function, regulation, and interaction with other interstitial cells. Mice were injected with a GPER antagonist (G-15; 50 µg/kg bw), and the GPER-signaling effect on TC distribution, ultrastructure, and function, as well as the interstitial tissue interaction of GPER with estrogen-related receptors (ERRs), was examined. Microscopic observations of TC morphology were performed with the use of scanning and transmission electron microscopes. Telocyte functional markers (CD34; c-kit; platelet-derived growth factor receptors α and ß, PDGFRα and ß; vascular endothelial growth factor, VEGF; and vimentin) were analyzed by immunohistochemistry/immunofluorescence and Western blot. mRNA expression of CD34 as well as ERR α, ß, and γ was measured by qRT-PCR. Relaxin and Ca2+ concentrations were analyzed by immunoenzymatic and colorimetric assays, respectively. For the first time, we reveal the presence of TCs in the interstitium together with the peritubular area of mouse testis. Telocytes were characterized by specific features such as a small cell body and extremely long prolongations, constituting a three-dimensional network mainly around the interstitial cells. Expression of all TC protein markers was confirmed. Based on scanning electron microscopic observation in GPER-blocked testis, groups of TCs were frequently seen. No changes were found in TC ultrastructure in GPER-blocked testis when compared to the control. However, tendency to TC number change (increase) after the blockage was observed. Concomitantly, no changes in mRNA CD34 expression and increase in ERR expression were detected in GPER-blocked testes. In addition, Ca2+ was unchanged; however, an increase in relaxin concentration was observed. Telocytes are an important component of the mouse testicular interstitium, possibly taking part in maintaining its microenvironment as well as contractile and secretory functions (via themselves or via controlling of other interstitial cells). These cells should be considered a unique and useful target cell type for the prevention and treatment of testicular interstitial tissue disorders based on estrogen-signaling disturbances.

Bisphenol A and dibutyl phthalate affect the expression of juxtacrine signaling factors in rat testis.

The study was designed to examine the effects of model plastic derived compounds, bisphenol A (BPA) and dibutyl phthalate (DBP), on juxtacrine communication in adult rat testis, by evaluating the expression of Notch pathway components. Testicular explant were exposed in vitro to BPA (5 × 10-6 M, 2.5 × 10-5 M, 5 × 10-5 M) or DBP (10-6 M, 10-5 M, 10-4 M) for 24 h. To determine the expression of Notch1, Dll4, Hey1, Hes1 and Hey5 real-time RT-PCR was used. Protein levels and localization of NOTCH1 receptor, its ligand DLL4 as well as HEY1, HES1 and HEY5 factors were detected by western blot analysis and immunohistochemistry, respectively. Upregulation of Notch1, Dll4 and Hey1 at the mRNA and protein level was demonstrated in testis explants after BPA and DBP treatment (p < 0.05; p < 0.01; p < 0.001). Hes5 expression decreased after BPA (p < 0.05; p < 0.01; p < 0.001), whereas Hes1 expression was not altered by either BPA or DBP. Tested chemicals altered immunoexpression of activated NOTCH1, DLL4, HEY1 and HES5 both in seminiferous epithelium and interstitial tissue, exerting differential effects on particular cell types. In conclusion, BPA and DBP affect Notch signaling pathway in rat testis, which indicates that juxtacrine communication is a potential target for the action of plastic derived compounds in male gonad.

Anti-androgen 2-hydroxyflutamide modulates cadherin, catenin and androgen receptor phosphorylation in androgen-sensitive LNCaP and androgen-independent PC3 prostate cancer cell lines acting via PI3K/Akt and MAPK/ERK1/2 pathways.

This study aimed to investigate rapid effect of anti-androgen 2-hydroxyflutamide (HF) on cadherin/catenin complex and androgen receptor (AR) phosphorylation in prostate cancer cell lines. In addition, a role of PI3K/Akt and MAPK/ERK1/2 pathways in mediating these effects was explored. We have demonstrated that in androgen-sensitive LNCaP cells HF induced rapid increase of E-cadherin phosphorylation at Ser 838/840 (p<0.05) in MAPK/ERK1/2-dependent manner, whereas phosphorylation of ß-catenin at Tyr 654 was unchanged. Concomitantly, the reduction of the level of AR phosphorylated at Ser210/213 was found (p<0.01). In androgen-independent PC3 cells HF decreased Tyr 860 N-cadherin and Tyr 645 ß-catenin phosphorylation (p<0.01), acting via both MAPK/ERK1/2 and PI3K/Akt pathways. Further, we evidenced that MAPK/ERK1/2 and PI3K/Akt pathways were differentially influenced by HF in LNCaP and PC3 cells. In LNCaP cells, both Akt (p<0.01) and ERK1/2 (p<0.001) phosphorylation were negatively regulated and this effect was mediated by Raf-1 (p<0.05). In contrast, in PC3 cells HF stimulated Akt (p<0.001) and ERK1/2 (p<0.001) activation, but had no effect on the crosstalk between PI3K/Akt and MEK/ERK1/2 pathways at the Raf-1 kinase level. Our findings expand the role of anti-androgen into non-genomic signaling, creating a link between anti-androgen action and phosphorylation of adherens junction proteins in prostate cancer cells.

Are aryl hydrocarbon receptor and G-protein-coupled receptor 30 involved in the regulation of seasonal testis activity in photosensitive rodent-the bank vole (Myodes glareolus)?

Within the reproductive system both aryl hydrocarbon receptor (AHR) and G-protein-coupled receptor 30 (GPR30) contribute to estrogen signaling and controlling of reproductive physiology. The specific question is whether and how AHR and GPR30 are involved in regulation of testis function in seasonally breeding rodents. Bank vole testes were obtained from animals reared under 18 hours light:6 hours dark (LD) and 6 hours light:18 hours dark (SD) conditions. Aryl hydrocarbon receptor and GPR30 expression were analyzed by quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry and/or immunofluorescent staining. In addition, the activity of enzymes involved in the intracellular signal transduction; extracellular signal-regulated kinase (ERK), protein kinase (PKA), matrix metalloproteinase 9 (MMP 9) and the concentrations of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and calcium (Ca(2+)) were examined by immunohistochemical, immunoenzymatic, and colorimetric assays, respectively. Aryl hydrocarbon receptor and GPR30 were expressed in testes of actively reproducing voles and regressed ones although their expression at the messenger RNA and AHR also at protein level appeared to be photoperiod-dependent. A specific cellular localization and expression of AHR and GPR30 correlated with the expression of ERK, PKA, and MMP 9. Moreover, we found robust differences in the levels of cAMP, cGMP, and Ca(2+) in testicular homogenates between LD and SD voles. In the testes of LD voles, the levels of second messengers were always higher compared to SD. In vole testis, AHR and GPR30 can induce signaling pathways that involve ERK, PKA, MMP 9 and cAMP, cGMP, Ca(2+). In addition, in AHR, signaling the engagement of both photoperiod and estrogens, whereas in GPR30, signaling only estrogens is reported. It is likely that in vole, because of a differential activity of signaling molecules, signal transduction via AHR rather than through GPR30 plays a role in regulation of seasonal changes of testis physiology.