Androgens and Notch signaling cooperate in seminiferous epithelium to regulate genes related to germ cell development and apoptosis.

It was reported previously that in adult males disruption of both androgen and Notch signaling impairs spermatid development and germ cell survival in rodent seminiferous epithelium. To explain the molecular mechanisms of these effects, we focused on the interaction between Notch signaling and androgen receptor (AR) in Sertoli cells and investigate its role in the control of proteins involved in apical ectoplasmic specializations, actin remodeling during spermiogenesis, and induction of germ cell apoptosis. First, it was revealed that in rat testicular explants ex vivo both testosterone and Notch signaling modulate AR expression and cooperate in the regulation of spermiogenesis-related genes (Nectin2, Afdn, Arp2, Eps8) and apoptosis-related genes (Fasl, Fas, Bax, Bcl2). Further, altered expression of these genes was found following exposure of Sertoli cells (TM4 cell line) and germ cells (GC-2 cell line) to ligands for Notch receptors (Delta-like1, Delta-like4, and Jagged1) and/or Notch pathway inhibition. Finally, direct interactions of Notch effector, Hairy/enhancer-of-split related with YRPW motif protein 1, and the promoter of Ar gene or AR protein were revealed in TM4 Sertoli cells. In conclusion, Notch pathway activity in Sertoli and germ cells regulates genes related to germ cell development and apoptosis acting both directly and indirectly by influencing androgen signaling in Sertoli cells.

Liquid semen storage-induced alteration in the protein composition of turkey (Meleagris gallopavo) spermatozoa.

Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca2+-binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology.

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.

Follicle-stimulating hormone regulates Notch signalling in the seminiferous epithelium of continuously and seasonally breeding rodents.

CONTEXT: Juxtacrine (contact-dependent) communication between the cells of seminiferous epithelium mediated by Notch signalling is of importance for the proper course of spermatogenesis in mammals. AIMS: The present study was designed to evaluate the role of follicle-stimulating hormone (FSH) in the regulation of Notch signalling in rodent seminiferous epithelium. METHODS: We explored the effects (1) of pharmacological inhibition of the hypothalamus-pituitary-gonadal (HPG) axis and FSH replacement in pubertal rats, and (2) of photoinhibition of HPG axis followed by FSH substitution in seasonally breeding rodents, bank voles, on Notch pathway activity. Experiments on isolated rat Sertoli cells exposed to FSH were also performed. Gene and protein expressions of Notch pathway components were analysed using RT-qPCR, western blot and immunohistochemistry/immunofluorescence. KEY RESULTS: Distribution patterns of Notch pathway proteins in bank vole and rat seminiferous epithelium were comparable; however, levels of activated Notch1 and Notch3, hairy/enhancer of split 1 (HES1) and hairy/enhancer of split-related with YRPW motif 1 (HEY1) in bank voles were dependent on the length of the photoperiod. In response to FSH similar changes in these proteins were found in both species, indicating that FSH is a negative regulator of Notch pathway activity in seminiferous epithelium. CONCLUSIONS: Our results support a common mechanism of FSH action on Notch pathway during onset and recrudescence of spermatogenesis in rodents. IMPLICATIONS: Interaction between FSH signalling and Notch pathway in Sertoli cells may be involved in spermatogenic activity changes of the testes occurring during puberty or photoperiod shift in continuously and seasonally breeding rodents, respectively.

Nuclear and Membrane Receptors for Sex Steroids Are Involved in the Regulation of Delta/Serrate/LAG-2 Proteins in Rodent Sertoli Cells.

Delta/Serrate/LAG-2 (DSL) proteins, which serve as ligands for Notch receptors, mediate direct cell-cell interactions involved in the determination of cell fate and functioning. The present study aimed to explore the role of androgens and estrogens, and their receptors in the regulation of DSL proteins in Sertoli cells. To this end, primary rat Sertoli cells and TM4 Sertoli cell line were treated with either testosterone or 17ß-estradiol and antagonists of their receptors. To confirm the role of particular receptors, knockdown experiments were performed. mRNA and protein expressions of Jagged1 (JAG1), Delta-like1 (DLL1), and Delta-like4 (DLL4) were analyzed using RT-qPCR, Western blot, and immunofluorescence. Testosterone caused downregulation of JAG1 and DLL1 expression, acting through membrane androgen receptor ZRT- and Irt-like protein 9 (ZIP9) or nuclear androgen receptor (AR), respectively. DLL4 was stimulated by testosterone in the manner independent of AR and ZIP9 in Sertoli cells. The expression of all studied DSL proteins was upregulated by 17ß-estradiol. Estrogen action on JAG1 and DLL1 was mediated chiefly via estrogen receptor α (ERα), while DLL4 was controlled via estrogen receptor ß (ERß) and membrane G-protein-coupled estrogen receptor (GPER). To summarize, the co-operation of nuclear and membrane receptors for sex steroids controls DSL proteins in Sertoli cells, contributing to balanced Notch signaling activity in seminiferous epithelium.

Transcriptome and Proteome Analysis Revealed Key Pathways Regulating Final Stage of Oocyte Maturation of the Turkey (Meleagris gallopavo).

In birds, the zona pellucida (ZP) matrix that surrounds the ovulated oocyte-called the inner perivitelline layer-is involved in sperm-zona interaction and successful fertilization. To identify the important genes and proteins connected with the final step of egg development, next-generation sequencing and two-dimensional electrophoresis, combined with mass spectrometry, were used for the analysis of mature oocytes at the F1 developmental stage. A total of 8161 genes and 228 proteins were annotated. Six subfamilies of genes, with codes ZP, ZP1-4, ZPD, and ZPAX, were identified, with the dominant expression of ZPD. The main expression site for ZP1 was the liver; however, granulosa cells may also participate in local ZP1 secretion. A ubiquitination system was identified in mature oocytes, where ZP1 was found to be the main ubiquitinated protein. Analysis of transcripts classified in estrogen receptor (ESR) signaling indicated the presence of ESR1 and ESR2, as well as a set of estrogen-dependent genes involved in both genomic and nongenomic mechanisms for the regulation of gene expression by estrogen. Oxidative phosphorylation was found to be a possible source of adenosine triphosphate, and the nuclear factor erythroid 2-related factor 2 signaling pathway could be involved in the response against oxidative stress. Oocyte-granulosa cell communication by tight, adherens, and gap junctions seems to be essential for the final step of oocyte maturation.

Transcriptome analysis of human Leydig cell tumours reveals potential mechanisms underlying its development.

Leydig cell tumours are the most common sex cord-stromal tumours. In the last years, apparent increased incidence is noted while aetiology of the tumour is still unknown. Therefore, here, we focused on the genetics of Leydig cell tumours using the next-generation sequencing. Leydig cell micronodules were revealed in patients with azoospermia who were qualified for testicular biopsy. Complete gene set of Leydig cell tumours was compared with transcriptome of healthy Leydig cells obtained from donors. Bioinformatic analysis of the obtained sequencing data revealed alterations in expression of 219 transcripts. We showed, for the first time, that a significant proportion of differentially expressed genes is directly involved in regulation of apoptotic process, which downregulation might be important to Leydig cell tumour development. Additionally, we found a significant upregulation of heat shock protein genes that might be a unique feature of Leydig cell tumours when compared to other tumour types. Our study offers fundamental transcriptomic data for future studies on human Leydig cell tumour that are crucial to determine its causes. Moreover, presented here the in-depth analysis and discussion of alterations observed in tumour transcriptome may be important for the diagnosis and therapy of this pathology.

Flutamide treatment reveals a relationship between steroidogenic activity of Leydig cells and ultrastructure of their mitochondria.

Our present knowledge on interrelation between morphology/ultrastructure of mitochondria of the Leydig cell and its steroidogenic function is far from satisfactory and needs additional studies. Here, we analyzed the effects of blockade of androgen receptor, triggered by exposure to flutamide, on the expression of steroidogenic proteins (1) and ultrastructure of Leydig cells' constituents (2). We demonstrated that increase in the expression level of steroidogenic (StAR, CYP11A1, 3ß-HSD, and CYP19A1) proteins (and respective mRNAs) in rat testicular tissue as well as elevation of intratesticular sex steroid hormone (testosterone and estradiol) levels observed in treated animals correspond well to morphological alterations of the Leydig cell ultrastructure. Most importantly, up-regulation of steroidogenic proteins' expression apparently correlates with considerable multiplication of Leydig cell mitochondria and subsequent formation of local mitochondrial networks. Interestingly, we showed also that the above-mentioned processes were associated with elevated transcription of Drp1 and Mfn2 genes, encoding proteins implicated in mitochondrial dynamics. Collectively, our studies emphasize the importance of mitochondrial homeostasis to the steroidogenic function of Leydig cells.

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma†.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties, and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRVP X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

Pde8b haploinsufficiency in mice is associated with modest adrenal defects, impaired steroidogenesis, and male infertility, unaltered by concurrent PKA or Wnt activation.

PDE8B, PRKAR1A and the Wnt/ß-catenin signaling are involved in endocrine disorders. However, how PDEB8B interacts with both Wnt and protein kinase A (PKA) signaling in vivo remains unknown. We created a novel Pde8b knockout mouse line (Pde8b-/-); Pde8b haploinsufficient (Pde8b+/-) mice were then crossed with mice harboring: (1) constitutive beta-catenin activation (Pde8b+/-;ΔCat) and (2) Prkar1a haploinsufficieny (Pde8b+/-;Prkar1a+/-). Adrenals and testes from mice (3-12-mo) were evaluated in addition to plasma corticosterone, aldosterone and Dkk3 concentrations, and the examination of expression of steroidogenesis-, Wnt- and cAMP/PKA-related genes. Pde8b-/- male mice were infertile with down-regulation of the Wnt/ß-catenin pathway which did not change significantly in the Pde8b+/-;ΔCat mice. Prkar1a haploinsufficiency also did not change the phenotype significantly. In vitro studies showed that PDE8B knockdown upregulated the Wnt pathway and increased proliferation in CTNNB1-mutant cells, whereas it downregulated the Wnt pathway in PRKAR1A-mutant cells. These data support an overall weak, if any, role for PDE8B in adrenocortical tumorigenesis, even when co-altered with Wnt signaling or PKA upregulation; on the other hand, PDE8B appears to play a significant role in male fertility.

Characterization of carp seminal plasma Wap65-2 and its participation in the testicular immune response and temperature acclimation.

Two functionally distinct isoforms of warm-temperature acclimation related 65-kDa protein (Wap65-1 and Wap65-2) with a role in the immune response are present in fish. To our knowledge, contrary to Wap65-1, Wap65-2 has neither been isolated nor functionally characterized in carp especially in reproductive system. The aim of this study was to characterize Wap65-2 and ascertain its functions in immune response and temperature acclimation within reproductive system. Wap65-2 corresponded to one of the most abundant proteins in carp seminal plasma, with a high immunologic similarity to their counterparts in seminal plasma of other fish species and a wide tissue distribution, with predominant expression in the liver. The immunohistochemical localization of Wap65-2 to spermatogonia, Leydig cells, and the epithelium of blood vessels within the testis suggests its role in iron metabolism during spermatogenesis and maintenance of blood-testis barrier integrity. Wap65-2 secretion by the epithelial cells of the spermatic duct and its presence around spermatozoa suggests its involvement in the protection of spermatozoa against damage caused by heme released from erythrocytes following hemorrhage and inflammation. Our results revealed an isoform-specific response of Wap65 to temperature acclimation and Aeromonas salmonicida infection which alters blood-testis barrier integrity. Wap65-2 seems to be related to the immune response against bacteria, while Wap65-1 seems to be involved in temperature acclimation. This study expands the understanding of the mechanism of carp testicular immunity against bacterial challenge and temperature changes, in which Wap65-2 seems to be involved and highlights their potential usefulness as biomarkers of inflammation and temperature acclimation.

Crosstalk between Androgen-ZIP9 Signaling and Notch Pathway in Rodent Sertoli Cells.

Our recent study demonstrated altered expression of Notch ligands, receptors, and effector genes in testes of pubertal rats following reduced androgen production or signaling. Herein we aimed to explore the role of nuclear androgen receptor (AR) and membrane androgen receptor (Zrt- and Irt-like protein 9; ZIP9) in the regulation of Notch pathway activation in rodent Sertoli cells. Experiments were performed using TM4 and 15P-1 Sertoli cell lines and rat primary Sertoli cells (PSC). We found that testosterone (10-8 M-10-6 M) increased the expression of Notch1 receptor, its active form Notch1 intracellular domain (N1ICD) (p < 0.05, p < 0.01, p < 0.001), and the effector genes Hey1 (p < 0.05, p < 0.01, p < 0.001) and Hes1 (p < 0.05, p < 0.001) in Sertoli cells. Knockdown of AR or ZIP9 as well as antiandrogen exposure experiments revealed that (i) action of androgens via both AR and ZIP9 controls Notch1/N1ICD expression and transcriptional activity of recombination signal binding protein (RBP-J), (ii) AR-dependent signaling regulates Hey1 expression, (iii) ZIP9-dependent pathway regulates Hes1 expression. Our findings indicate a crosstalk between androgen and Notch signaling in Sertoli cells and point to cooperation of classical and non-classical androgen signaling pathways in controlling Sertoli cell function.

Flutamide Alters the Expression of Chemerin, Apelin, and Vaspin and Their Respective Receptors in the Testes of Adult Rats.

Adipokines influence energy metabolism and have effects on male reproduction, including spermatogenesis and/or Sertoli cell maturation; however, the relationship between these active proteins and androgens in testicular cells is limited. Here, we studied the impact of short-term exposure to flutamide (an anti-androgen that blocks androgen receptors) on the expression of chemerin, apelin, vaspin and their receptors (CCRL2, CMKLR1, GPR1, APLNR, GRP78, respectively) in adult rat testes. Moreover, the levels of expression of lipid metabolism-modulating proteins (PLIN1, perilipin1; TSPO, translocator protein) and intercellular adherens junction proteins (nectin-2 and afadin) were determined in testicular cells. Plasma levels of adipokines, testosterone and cholesterol were also evaluated. Gene expression techniques used included the quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB) and immunohistochemistry (IHC). The androgen-mediated effects observed post-flutamide treatment were found at the gonadal level as chemerin, apelin, and vaspin gene expression alterations at mRNA and protein levels were detected, whereas the cellular targets for these adipokines were recognised by localisation of respective receptors in testicular cells. Plasma concentrations of all adipokines were unchanged, whereas plasma cholesterol content and testosterone level increased after flutamide exposure. Differential distribution of adipokine receptors indicates potential para- or autocrine action of the adipokines within the rat testes. Additionally, changes in the expression of PLIN1 and TSPO, involved in the initial step of testosterone synthesis in Leydig cells, suggest that testicular cells represent a target of flutamide action. Increase in the gene expression of PLIN1 and TSPO and higher total plasma cholesterol content indicates enhanced availability of cholesterol in Leydig cells as a result of androgen-mediated effects of flutamide. Alterations in adherens junction protein expression in the testis confirm the flutamide efficacy in disruption of androgen signalling and presumably lead to impaired para- and autocrine communication, important for proper functioning of adipokines.

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.

Disruption of androgen signaling during puberty affects Notch pathway in rat seminiferous epithelium.

BACKGROUND: Onset of spermatogenesis at puberty is critically dependent on the activity of hypothalamic-pituitary-gonadal axis and testosterone production by Leydig cells. The aim of this study was to examine whether activation of Notch receptors and expression of Notch ligands and effector genes in rat seminiferous epithelium are controlled by androgen signaling during puberty. METHODS: Peripubertal (5-week-old) Wistar rats received injections of flutamide (50 mg/kg bw) daily for 7 days to reduce androgen receptor (AR) signaling or a single injection of ethanedimethane sulphonate (EDS; 75 mg/kg bw) to reduce testosterone production. Gene and protein expressions were analyzed by real-time RT-PCR and western blotting, respectively, protein distribution by immunohistochemistry, and steroid hormone concentrations by enzyme-linked immunosorbent assay. Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test or by Kruskal-Wallis test, followed by Dunn's test. RESULTS: In both experimental models changes of a similar nature in the expression of Notch pathway components were found. Androgen deprivation caused the reduction of mRNA and protein expression of DLL4 ligand, activated forms of Notch1 and Notch2 receptors and HES1 and HEY1 effector genes (p < 0.05, p < 0.01, p < 0.001). In contrast, DLL1, JAG1 and HES5 expressions increased in seminiferous epithelium of both flutamide and EDS-treated rats (p < 0.05, p < 0.01, p < 0.001). CONCLUSIONS: Androgens and androgen receptor signaling may be considered as factors regulating Notch pathway activity and the expression of Hes and Hey genes in rat seminiferous epithelium during pubertal development. Further studies should focus on functional significance of androgen-Notch signaling cross-talk in the initiation and maintenance of spermatogenesis.

Evolutionary origin and functioning of pregenital abdominal outgrowths in a viviparous insect, Arixenia esau.

Although pregenital abdominal outgrowths occur only rarely in pterygote insects, they are interesting from the evolutionary viewpoint because of their potential homology to wings. Our previous studies of early development of an epizoic dermapteran, Arixenia esau revealed that abdominal segments of the advanced embryos and larvae, growing inside a mother's uterus, are equipped with paired serial outgrowths. Here, we focus on the origin and functioning of these outgrowths. We demonstrate that they bud from the lateral parts of the abdominal nota, persist till the end of intrauterine development, and remain in contact with the uterus wall. We also show that the bundles of muscle fibers associated with the abdominal outgrowths may facilitate flow of the haemolymph from the outgrowths' lumen to the larval body cavity. Following completion of the intrauterine development, abdominal outgrowths are shed together with the larval cuticle during the first molt after the larva birth. Using immunohistochemical and biochemical approaches, we demonstrate that the Arixenia abdominal outgrowths represent an evolutionary novelty, presumably related to intrauterine development, and suggest that they are not related to serial wing homologs.

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.

The effects of flutamide on cell-cell junctions in the testis, epididymis, and prostate.

In this review, we summarize recent findings on the effect of the anti-androgen flutamide on cell-cell junctions in the male reproductive system. We outline developmental aspects of flutamide action on the testis, epididymis, and prostate, and describe changes in junction protein expression and organization of junctional complexes in the adult boar following prenatal and postnatal exposure. We also discuss findings on the mechanisms by which flutamide induces alterations in cell-cell junctions in reproductive tissues of adult males, with special emphasis on cytoplasmic effects. Based on the results from in vivo and in vitro studies in the rat, we propose that flutamide affects the expression of junction proteins and junction complex structure not only by inhibiting androgen receptor activity, but equally important by modulating protein kinase-dependent signaling in testicular cells. Additionally, results from studies on prostate cancer cell lines point to a role for the cellular molecular outfit in response to flutamide.