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Porker immunocastration against gonadoliberin (GnRH) secretion has been utilized since 2009; however, consumers are still skeptical of it. This is due to not having full information available on the problem of a boar taint, as well as a lack of research on morphological and molecular changes that may occur in the animal reproductive system and other body systems. The present study aimed to explore the functional status of steroidogenic Leydig cells of the testicular interstitial tissue in immunocastrated Polish Landrace pigs. Analyses were performed using Western blot, immunohistochemistry for relaxin (RLN), insulin-like 3 protein (INSL3), pelleted growth factor receptor α (PDGFRα), cytochrome P450scc, 3β- and 17β-hydroxysteroid dehydrogenases (3β-HSD, 17β-HSD), cytochrome P450arom, and 5α-reductase (5α-RED). Immunoassay ELISA was used to measure the androstenone, testosterone, and estradiol levels in the testis and serum of immunocastrates. We revealed disturbances in the distribution and expression of (i) RLN, indicating an inflammatory reaction in the interstitial tissue; (ii) INSL3 and PDGFRα, indicating alterations in the differentiation and function of fetal, perinatal, or adult Leydig cell populations; (iii) P450scc, 3β-HSD, 17β-HSD, P450arom, and 5α-RED, indicating disturbances in the sex steroid hormone production and disturbed functional status of Leydig cells; as well as (iv) decreased levels of androstenone, testosterone, and estradiol in testicular tissue and serum, indicating the dedicated action of Improvac to reduce boar taint at both the hypothalamic–hypophysis–gonadal axis and local level (Leydig cells). In summary, our study provides a significant portion of knowledge on the function of Leydig cells after immunocastration, which is also important for the diagnosis and therapy of testis dysfunction due to GnRH action failure and/or Leydig cell differentiational–functional alterations.

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.

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

With estrogen regulation of the reproductive system, G-protein-coupled membrane estrogen receptor (GPER) and estrogen-related receptors (ERRs) are implicated. Non-canonical receptors can bind estrogens such as environmental and pharmacological chemicals. These compounds induce rapid non-genomic pathways or receptor interaction including autoactivation. Testicular tumors occur in dogs more frequently than in other domestic animals. Also, in recent decades there were increased occurrences of various tumor types in dogs. Using qRT-PCR, Western blot and immunohistochemistry procedures in the present study, there was determination of abundance pattern of GPER, ERRα, β and γ in dog tests when there were intratubular germ cell tumors. There was quantitation of estradiol, cyclic GMP and calcium ions (Ca2+). There were changes (P

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, 3β-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.

Cryptorchidism in horses is a commonly occurring malformation. The molecular basis of this pathology is not fully known. In addition, the origins of high intratesticular estrogen levels in horses remain obscure. In order to investigate the role of the G-protein-coupled membrane estrogen receptor (GPER) and establish histological and biochemical cryptorchid testis status, healthy and cryptorchid horse testes were subjected to scanning electron microscopy analysis, histochemical staining for total protein (with naphthol blue black, NBB), acid content (with toluidine blue O, TBO), and polysaccharide content (with periodic acid–Schiff, PAS). The expression of GPER was analyzed by immunohistochemistry and Western blot. GPER-mediated intracellular cAMP and calcium (Ca2+) signaling were measured immunoenzymatically or colorimetrically. Our data revealed changes in the distribution of polysaccharide content but not the protein and acid content in the cryptorchid testis. Polysaccharides seemed to be partially translocated from the interstitial compartment to the seminiferous tubule compartment. Moreover, the markedly decreased expression of GPER and GPER downstream molecules, cAMP and Ca2+, suggests their potential role in testis pathology. Increased estrogen levels in cryptorchid conditions may be linked to disturbed GPER signaling. We postulate that GPER is a prominent key player in testis development and function and may be used as a new biomarker of horse testis in health and disease.

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.Summary sentenceTurkey seminal plasma cysteine-rich venom protein containing feature motifs of CRISP family proteins is secreted within the reproductive tract, stimulates sperm motility, and participates in sperm-egg interaction.Graphical Abstract

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 p p p p p 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.

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 β (ERβ) 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

To characterize polychlorinated biphenyls (PCBs) action on Leydig cells, PCBs congeners, low-chlorinated (delor 103; d103) and high-chlorinated ones (delor 106; d106) were selected. The cells were treated according to PCBs dose (d103 or d106 0.2 ng/ml in low doses:, or 2 ng/ml in high doses) and type (d103 + d106 in low doses or 103 + 106 in high doses). After 24 h treatment with PCBs, a distinct increase in estrogen-related receptors (ERRs type α, β and γ) expression was revealed. However, the dose- and type-dependent PCBs effect was mostly exerted on ERRα expression. A similar increase in ERRs expression was demonstrated by estradiol but not testosterone, which was without an effect on ERRs. PCBs caused no decrease in the membrane potential status of Leydig cells (either in dose or type schedule) but had severe effects on the mitochondria number and structure. Moreover, PCBs markedly increased calcium (Ca2+) concentration and sex steroid secretion (both androgens and estrogens were elevated). These findings suggest a similar estrogenic action of PCBs congeners (d103 and d106) on Leydig cell function. We report dose- and type-specific effects of PCBs only on Leydig cell ERRs expression. Both delors showed common effects on the mitochondria ultrastructural and functional status. Based on our results, ERRα seems to be the most sensitive to hormonal modulation. The increases in Ca2+ and sex steroid secretion may be due to the activation of ERRs by PCBs binding and/or direct effect of PCBs on ERRs mRNA/protein expression. Nevertheless, to confirm the existence of possible relationships between ERRs signaling (including PCBs as ligands) and mitochondria function in Leydig cells, further intensive studies are needed.

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.

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.

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 β-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 β-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.

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

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 (p0.05) in MAPK/ERK1/2-dependent manner, whereas phosphorylation of β-catenin at Tyr 654 was unchanged. Concomitantly, the reduction of the level of AR phosphorylated at Ser210/213 was found (p0.01). In androgen-independent PC3 cells HF decreased Tyr 860 N-cadherin and Tyr 645 β-catenin phosphorylation (p0.01), 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 (p0.01) and ERK1/2 (p0.001) phosphorylation were negatively regulated and this effect was mediated by Raf-1 (p0.05). In contrast, in PC3 cells HF stimulated Akt (p0.001) and ERK1/2 (p0.001) 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.

We report the effects of polychlorinated napthalanes (PCNs) on the mRNA expression of estrogen-related receptors (ERRs) α, β 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 (10 nM) alone or in combination with one of sex steroid receptor antagonists; 182,780 (ICI; 10 μM); hydroxyflutamide (HF; 10 −4 M) and G-coupled estrogen receptor antagonist (G15; 10 nM) respectively. The expression of mRNAs and protein for ERRα, β, 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).

Background Present study was designed to establish a causal connection between changes in the cell-cell junction protein expression at the blood-testis barrier and alterations in the adult rat testis histology following an anti-androgen flutamide exposure. Particular emphasis was placed on the basal ectoplasmic specialization (ES) in the seminiferous epithelium and expression of gap junction protein, connexin 43 (Cx43). Methods Flutamide (50 mg/kg body weight) was administered to male rats daily from 82 to 88 postnatal day. Testes from 90-day-old control and flutamide-exposed rats were used for all analyses. Testis morphology was analyzed using light and electron microscopy. Gene and protein expressions were analyzed by real-time RT-PCR and Western blotting, respectively, protein distribution by immunohistochemistry, and steroid hormone concentrations by radioimmunoassay. Results Seminiferous epithelium of both groups of rats displayed normal histology without any loss of germ cells. In accord, no difference in the apoptosis and proliferation level was found between control and treated groups. As shown by examination of semi-thin and ultrathin sections, cell surface occupied by the basal ES connecting neighboring Sertoli cells and the number of gap and tight junctions coexisting with the basal ES were apparently reduced in flutamide-treated rats. Moreover, the appearance of unconventional circular ES suggests enhanced internalization and degradation of the basal ES. These changes were accompanied by decreased Cx43 and ZO-1 expression (p

This study was undertaken to explore interactions between c-Src kinase and the N-cadherin-β-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 β-catenin and c-Src expression were observed. Notably, membranous β-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-β-catenin complex are a primary effect of androgen signaling disruption by flutamide. Immunohistochemistry revealed a diffusion of N-cadherin and β-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-s-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-β-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-β-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.