Senescence and autophagy relation with the expressional status of non-canonical estrogen receptors in testes and adrenals of roe deer (Capreolus capreolus) during the pre-rut period.

The roe deer bucks represent a spontaneous model to study the synchronized testicular involution and recrudescence cycles. However, cellular processes and hormonal control of steroidogenic glands are scarcely known. For the present study testes and adrenal glands obtained from roe deer during the pre-rut season were used. We aimed to determine (i) senescence and autophagy involvement in testis atrophy (immunohistochemical analysis for tumor suppressor protein encoded by the cyclin-dependent kinase inhibitor 2A; p16 and microtubule-associated protein 1A/1B-light chain 3; LC3, respectively), (ii) the size of the adrenal cortex and medulla (morphometric analysis), (iii) G-protein coupled estrogen receptor (GPER) and estrogen-related receptors (ERRs; type α, ß, and Y) distribution and expression (qRT-PCR and immunohistochemical analyses) and (iv) serum testosterone and estradiol levels (immunoassay ELISA). This study revealed pre-rut characteristics of testis structure with the presence of both senescence and autophagy-positive cells and higher involvement of senescence, especially in spermatogenic cells (P < 0.05). In the adrenal cortex, groups of cells exhibiting shrinkage were observed. The presence of ERRs in cells of the seminiferous epithelium and interstitial Leydig cells and GPER presence distinctly in Leydig cells was revealed. In adrenals, these receptors were localized in groups of normal-looking cells and those with shrinkage. Morphometric analysis showed differences in cortex width which was smaller (P < 0.05) than that of the medulla. A weak immunohistochemical signal was observed for ERRß when compared to ERRα and ERRγ. The mRNA expression level of ERRα and ERRγ was lower (P < 0.001 and P < 0.05, respectively) while ERRß was higher (P < 0.001) in adrenals when compared to testes. mRNA GPER expression was similar in both glands. In the pre-rut season, the testosterone level was 4.89 ng/ml while the estradiol level was 0.234 ng/ml. We postulate that: (i) senescence and autophagy may be involved in both reinitiation of testis function and/or induction of abnormal processes, (ii) hormonal modulation of testis inactivity may affect adrenal cortex causing cell shrinkage, (iii) ERRs and GPER localization in spermatogenic cells and interstitial cells, as well as cortex cells, may maintain and control the morpho-functional status of both glands, and (iv) androgens and estrogens (via ERRs and GPER) drive cellular processes in the testis and adrenal pre-rut physiology.

Leydig Cells in Immunocastrated Polish Landrace Pig Testis: Differentiation Status and Steroid Enzyme Expression Status.

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, 3ß- and 17ß-hydroxysteroid dehydrogenases (3ß-HSD, 17ß-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, 3ß-HSD, 17ß-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.

Abundance of estrogen receptors involved in non-canonical signaling in the dog testis.

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, ß 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 < 0.01; P < 0.001) in GPER, ERRα and ß in both mRNA transcript and protein abundances including less (P < 0.001) co-abundance of ERRγ mRNA transcript and protein. Receptors were mainly located in Leydig cells with there being receptor delocalization to the cell cytoplasm or occasionally detections in the seminiferous tubule epithelia, especially of testicular tumor tissues. There were also greater estradiol (P < 0.05) and lesser cGMP and Ca2+ concentrations in testicular tumor tissues indicating there was a disrupted sex steroid milieu and tumor cell metastasis. Results from the present study provide further evidence that ERRγ has marked actions in testicular germ cell tumor initiation and development and in further structural-functional disruptions of dog testis. Concomitantly, abundance pattern of GPER and ERRs, relative to concentrations of cGMP and Ca2+, may be an additional indicator of intratubular germ cell tumors in dogs.

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.

The G-Protein-Coupled Membrane Estrogen Receptor Is Present in Horse Cryptorchid Testes and Mediates Downstream Pathways.

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.

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.

Chlorinated biphenyls effect on estrogen-related receptor expression, steroid secretion, mitochondria ultrastructure but not on mitochondrial membrane potential in Leydig cells.

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, ß 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.

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.

Flutamide induces alterations in the cell-cell junction ultrastructure and reduces the expression of Cx43 at the blood-testis barrier with no disturbance in the rat seminiferous tubule morphology.

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 < 0.01) and a loss of linear distribution of these proteins at the region of the blood-testis barrier. On the other hand, Cx43 expression in the interstitial tissue of flutamide-treated rats increased (p < 0.01), which could be associated with Leydig cell hypertrophy. Concomitantly, both intratesticular testosterone and estradiol concentrations were elevated (p < 0.01), but testosterone to estradiol ratio decreased significantly (p < 0.05) in flutamide-treated rats compared to the controls. CONCLUSIONS: Short-term treatment with flutamide applied to adult rats exerts its primary effect on the basal ES, coexisting junctional complexes and their constituent proteins Cx43 and ZO-1, without any apparent morphological alterations in the seminiferous epithelium. In the interstitial compartment, however, short-term exposure leads to both histological and functional changes of the Leydig cells.