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Visfatin is an adipokine involved in energy metabolism, insulin resistance, inflammation, and female reproduction. Due to limited data about its action in the human placenta, the aims of our studies included the analysis of visfatin expression and immunolocalization in trophoblast cell lines JEG-3 and BeWo as well as in human placentas from normal and pathological pregnancies. Moreover, we also checked the hormonal regulation of visfatin levels and the molecular mechanism of observed changes in JEG-3 cells. Cell culture and placental fragments collection along with statistical analysis were performed using standard laboratory procedures also described in our previous papers. We demonstrated an increased gene and protein expression of visfatin in JEG-3, BeWo cells, while variable expression in maternal and fetal parts of normal/ pathological pregnancy placentas. In addition, the immunolocalization of visfatin was observed in the cytoplasm of both cell lines, the capillary epithelium of the maternal part and syncytiotrophoblasts of the placental fetal part; in all tested pathologies, the signal was also detected in decidual cells. Furthermore, we demonstrated that hormones: progesterone, estradiol, human chorionic gonadotropin, and insulin increased the visfatin levels in JEG-3 cells with the involvement of specific signaling pathways. Taken together, differences in the expression and localization of visfatin between normal and pathological placentas suggested that visfatin may be a potential marker for the diagnosis of pregnancy disorders. In addition, we found that placental levels of visfatin can be regulated by hormones known to modulate the function of placental cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dawid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The importance and regulation of adrenal androgen production and signaling are not completely understood and are scarcely studied. In addition, there is still a search for appropriate animal models and experimental systems for the investigation of adrenal physiology and disease. Therefore, the main objective of the study was to evaluate the effect of luteinizing hormone (LH) signaling and selenium (Se2+) exposure on androgen adrenal signaling via canonical androgen receptor (AR), and membrane androgen receptor acting as zinc transporter (zinc- and iron-like protein 9; ZIP9). For herein evaluations, adrenals isolated from transgenic mice with elevated LH receptor signaling (KiLHRD 582G ) and adrenals obtained from rabbits used for ex vivo adenal cortex culture and exposure to Se2+ were utilized. Tissues were assessed for morphological, morphometric, and Western blot analyses and testosterone and zinc level measurements.Comparison of adrenal cortex histology and morphometric analysis in KiLHRD 582G mice and Se2+-treated rabbits revealed cell hypertrophy. No changes in the expression of proliferating cell nuclear antigen (PCNA) were found. In addition, AR expression was decreased (p < 0.001) in both KiLHRD 582G mouse and Se2+-treated rabbit adrenal cortex while expression of ZIP9 showed diverse changes. Its expression was increased (P < 0.001) in KiLHRD 582G mice and decreased (P < 0.001) in Se2+-treated rabbits but only at the dose 10 ug/100 mg/ tissue. Moreover, increased testosterone levels (P < 0.05) and zinc levels were detected in the adrenal cortex of KiLHRD 582G mice whereas in rabbit adrenal cortex treated with Se2+, the effect was the opposite (P < 0.001)., (© 2023. The Author(s).)

Telocytes represent a relatively recently discovered population of interstitial cells with a unique morphological structure that distinguishes them from other neighboring cells. Through their long protrusions extending from the cell body, telocytes create microenvironments via tissue compartmentalization and create homo- and hetero-cellular junctions. These establish a three-dimensional network enabling the maintenance of interstitial compartment homeostasis through regulation of extracellular matrix organization and activity, structural support, paracrine and juxtracrine communication, immunomodulation, immune surveillance, cell survival, and apoptosis. The presence of telocytes has also been confirmed in testicular interstitial tissue of many species of animals. The objective of this review is to summarize recent findings on telocytes in the male gonad, on which conclusions have been deduced that indicate the involvement of telocytes in maintaining the cytoarchitecture of the testicular interstitial tissue, in the processes of spermatogenesis and steroidogenesis, and photoperiod-mediated changes in the testes in seasonally reproductive animals., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Indoxyl sulfates are uremic indolic toxins known to participate in the pathogenesis of cardiovascular diseases during chronic kidney disease in humans and some animal species. However, nothing is known about the indoxyl sulfate effect on the thyroid gland which is especially responsible for the general organism metabolism. This study determines the morpho-functional status of the thyroid gland after exposure to indoxyl sulfate (10, 25, and 50 mM) with the use of an ex vivo system and rabbit (n=10) as an experimental model thyroid gland histology, immunoexpression of thyrotropin receptor (TSHR), and concentrations of thyroxine (T4) and triiodothyronine (T3) were evaluated. Statistical analyses were performed using one-way analysis of the variance (ANOVA) followed by Tukey's post hoc comparison test. Minor alterations in thyroid tissue structure e.g. very rare exfoliated epithelial cells, condensed colloid fluid, or slight loosening of the epithelium were found. In addition, modulated dose dependent-expression of TSHR (p<0.01, p<0.001) together with a decreased level of T4 and T3 (p<0.001, p<0.01) exception of an increased level of T4 after the middle dose of indoxyl sulfate were revealed. We report here, for the first time, that indoxyl sulfate affects the thyroid gland mainly at the molecular level. The rabbit thyroid gland ex vivo system seems to be suitable for further studies on the thyroid gland in health and disease. However, the effect of TSH-TSHR signaling at ultrastructural, and epigenetic levels needs supplementary appraisal.

Spexin (SPX) is a novel neuropeptide and adipokine negatively correlated with obesity and insulin resistance. A recent study investigated expression and regulatory function of SPX in the hypothalamus and pituitary; however, the effect on ovarian function is still unknown. The aim of this study was to characterize the expression of SPX and its receptors, galanin receptors 2 and 3 (GALR2/3), in the human ovary and to study its in vitro effect on granulosa cells (GC) function. Follicular fluid (FF) and GC were obtained from normal weight and obese healthy and diagnosed with polycystic ovarian syndrome (PCOS) women. Expression of SPX and GALR2/3 in the ovary was studied by qPCR, western blot, and immunohistochemistry. The level of SPX in FF was assessed by enzyme-linked immunosorbent assay. The in vitro effect of recombinant human SPX on GC proliferation, steroidogenesis, and signaling pathways (MAP3/1, STAT3, AKT, PKA) was analyzed. Moreover, GC proliferation and estradiol (E2) secretion were measured with and without an siRNA against GALR2/3 and pharmacological inhibition of the above kinases. The results showed that both the SPX concentration in FF and its gene expression were decreased in GC of obese and PCOS women, while the protein expression of GALR2/3 was increased. We noted that SPX reduced GC proliferation and steroidogenesis; these effects were mediated by GALR2/3 and kinases MAP3/1, AKT, and STAT3 for proliferation or kinases MAP3/1 and PKA for E2 secretion. The obtained data clearly documented that SPX is a novel regulator of human ovarian physiology and possibly plays a role in PCOS pathogenesis., (© The Author(s) 2023. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

The trace element selenium (Se) is essential for the maintenance of spermatogenesis and fertility. A growing volume of evidence shows that Se is necessary for testosterone synthesis, and Se can stimulate Leydig cell proliferation. However, Se can also act as a metalloestrogen, which can mimic estrogen and activate the estrogen receptors. This study aimed to investigate Se effect on estrogen signaling and the epigenetic status of Leydig cells. Mouse Leydig cells (MA-10) were cultured in a medium supplemented with different Se concentrations (4, 8 µM) for 24 h. Next, cells were assessed for morphological and molecular (qRT PCR, western blot, immunofluorescence) analyses. Immunofluorescence revealed strong immunosignal for 5-methylcytosine in both control and treated cells, with a stronger signal in the 8 μM treated group. qRT-PCR confirmed an increased expression of methyltransferase 3 beta (Dnmt3b) in 8 μM cells. Analysis of the expression of γH2AX (a marker for double-stranded DNA breaks) revealed an increase in the DNA breaks in cells exposed to 8 μM Se. Selenium exposure did not affect the expression of canonical estrogen receptors (ERα and ERβ), however, an increase in membrane estrogen receptor G-protein coupled (GPER) protein expression was observed.To sum up, in a high concentration (8 μM) Se affects GPER expression (non-genomic estrogen signaling) in Leydig cells possibly via acting on receptor protein and/or its binding. This causes DNA breaks and induces changes in Leydig cell methylation status, especially in de novo methylation which is mediated by Dnmt3b., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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, β, 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 ERRβ 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 ERRβ 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., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Research has linked hormones to behavioral outcomes in intricate ways, often moderated by psychological dispositions. The associations between testosterone and antisocial or prosocial outcomes also depend on dispositions relevant to status and dominance. In two studies (N1 = 68, N2 = 83), we investigated whether endogenous testosterone, measured in saliva, and narcissism, a psychological variable highly relevant to status motivation, interactively predicted men's preferences regarding resource allocation. Narcissism moderated the links between testosterone and social value orientation: among low narcissists testosterone negatively predicted generosity in resource allocation and probability of endorsing a prosocial (vs. pro-self) value orientation, whereas among high narcissists testosterone tended to positively predict generosity and the probability of endorsing a prosocial (vs. pro-self) value orientation. We discuss these results as examples of calibrating effects of testosterone on human behavior, serving to increase and maintain social status. We advocate the relevance of psychological dispositions, alongside situations, when examining the role of T in social outcomes., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Spexin (SPX) is a newly identified neuropeptide, a natural ligand for the galanin receptors (GALR) 2/3, which is involved in maintaining physiological functions including female reproduction. One of the most common endocrine disorder in reproductive system is polycystic ovary syndrome (PCOS), however the role of SPX in PCOS is still unknown. The objective of this study was to determine the expression of mRNA and peptide levels of SPX and its receptors GALR2/3 in the hypothalamus and ovary (by real time PCR and Western blot) as well as plasma levels of SPX (ELISA) in letrozole - induced PCOS rats. We observed that SPX plasma level does not change in PCOS rats. In the hypothalamus transcript level of Spx and Galr3 were significantly higher in PCOS rats compared to the control, while mRNA of Galr2 and protein expression of GALR2/3 were lower. Moreover, expression of Spx and Galr2/3 mRNA as well as GALR2/3 peptide production were lower in the ovary of PCOS rats. In summary, while our results did not show differences in plasma SPX levels, we observed tissue-dependent significant differences in the SPX/GALR2/3 levels between PCOS and control rats, what indicates possible new mechanisms of PCOS neuroendocrinology., Competing Interests: Declaration of competing interest The authors have no financial or commercial conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.

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.

Telocytes (TCs) are a new type of mesenchymal cells that have been discovered recently in many organs and tissues. However, studies of TCs in the avian reproductive system are still at the beginning. Chickens are one of the world's most popular domesticated animals, providing inexpensive but valuable proteins and nutrients from chickens and eggs to nourish the human bodies. Chickens have important scientific value; thus, understanding the reproductive system regulations seems to be important. The utero-vaginal junction is involved in the regulation of sperm storage. The sperm storage tube (SST) in the utero-vaginal junction stores sperm. The purpose of this study was to investigate the existence of TCs in the utero-vaginal junction of the chicken, and their structural relationships with the sperm storage tube and surrounding cell types. We studied the morphology, ultrastructure, and immune characterization of TCs., Methods: The utero-vaginal junction of 4-month-old healthy adult chickens ( n = 10) were used for Masson's staining, fluorescent in situ hybridization technique (FISH), and transmission electron microscopy (TEM) analysis. The results showed that TCs were present in the utero-vaginal junction. TCs appear as CD34 immunopositive and C-kit immunopositive. They were identified especially via small-body and long-protrusion telopodes (Tps) containing Podomers (Pm) and Podoms (Pd). The Tps were bent, folded, and intertwined with each other, sometimes in the shape of a labyrinth. The Tps were embedded between collagen fiber bundles, smooth muscle bundles, and around blood vessels and releasing vesicles. TCs surround these glands, forming heteromorphic cell connections with surrounding lymphocytes and plasma cells, smooth muscle cells, blood vessels, collagen fibers, and fibroblast-formed homotypic or allotypic connections in a complex three-dimensional network structure. This study provides a morphological basis for the possible role of TCs in regulating the utero-vaginal junction physiological role and in intercellular communication., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhu, Wang, Pawlicki, Wang, Pawlicka, Meng, Feng and Yang.)

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 < 0.01; P < 0.001) in GPER, ERRα and β 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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Porcine tissue gene expression is highly similar to the expression of homologous genes in humans. Based on this fact, the studies on porcine tissues can be employed to understand human physiology and to predict or treat diseases. Our prior studies clearly showed that there was a regulatory partnership of the peroxisome proliferator-activated receptor (PPAR) and the G-protein coupled membrane estrogen receptor (GPER) that relied upon the tumorigenesis of human and mouse testicular interstitial cells, as well as the PPAR-estrogen related receptor and GPER-xenoestrogen relationships which affected the functional status of immature boar testes. The main objective of this study was to identify the biological processes and signaling pathways governed by PPARα, PPARγ and GPER in the immature testes of seven-day-old boars after pharmacological receptor ligand treatment. Boar testicular tissues were cultured in an organotypic system with the respective PPARα, PPARγ or GPER antagonists. To evaluate the effect of the individual receptor deprivation in testicular tissue on global gene expression, Next Generation Sequencing was performed. Bioinformatic analysis revealed 382 transcripts with altered expression. While tissues treated with PPARα or GPER antagonists showed little significance in the enrichment analysis, the antagonists challenged with the PPARγ antagonist displayed significant alterations in biological processes such as: drug metabolism, adhesion and tubule development. Diverse disruption in the Notch signaling pathway was also observed. The findings of our study proposed that neither PPARα nor GPER, but PPARγ alone seemed to be the main player in the regulation of boar testes functioning during early the postnatal developmental window.

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.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Leydig cell tumors (LCT) are the most common type of testicular stromal tumor. Herein, we investigate the G protein-coupled estrogen receptor (GPER) and peroxisome proliferator-activated receptor (PPAR) implication in regulation of lipid homeostasis including the expression of steroidogenesis-controlling molecules in clinical specimens of LCTs and tumor Leydig cells (MA-10). We showed the general structure and morphology of LCTs by scanning electron and light microscopy. In LCTs, mRNA and protein analyses revealed increased expression of GPER and decreased expression of PPARα, β, and γ. Concomitantly, changes in expression pattern of the lutropin receptor (LHR), protein kinase A (PKA), perilipin (PLIN), hormone sensitive lipase (HSL), steroidogenic acute regulatory protein (StAR), translocator protein (TSPO), HMG-CoA synthase, and reductase (HMGCS, HMGCR) were observed. Using MA-10 cells treated with GPER and PPAR antagonists (alone and in combination), we demonstrated GPER-PPAR-mediated control of estradiol secretion via GPER-PPARα and cyclic guanosine monophosphate (cGMP) concentration via GPER-PPARγ. It is assumed that GPER and PPAR can crosstalk, and this can be altered in LCT, resulting in a perturbed lipid balance and steroidogenesis. In LCTs, the phosphatidylinositol-3-kinase (PI3K)-Akt-mTOR pathway was disturbed. Thus, PI3K-Akt-mTOR with cGMP can play a role in LCT outcome and biology including lipid metabolism.

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

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.

Communication in biological systems involves diverse-types of cell-cell interaction including cross-talk between receptors expressed by the target cells. Recently, novel sort of estrogen receptors (G protein - coupled estrogen receptor; GPER and estrogen-related receptor; ERR) that signal directly via estrogen binding and/or via mutual interaction-regulated estrogen signaling were reported in various organs including testis. Peroxisome proliferator - activated receptor (PPAR) is responsible for maintaining of lipid homeostasis that is critical for sex steroid production in the testis. Here, we investigated the role of interaction between GPER, ERRβ and PPARγ in steroidogenic Leydig cells of immature boar testis. Testicular fragments cultured ex vivo were treated with GPER or PPARγ antagonists. Then, cell ultrastructure, expression and localization of GPER, ERRβ, PPARγ together with the molecular receptor mechanism, through cyclic AMP and Raf/Ras/extracellular signal activated kinases (ERK), in the control of cholesterol concentration and estrogen production by Leydig cells were studied. In the ultrastructure of antagonist-treated Leydig cells, mitochondria were not branched and not bifurcated as they were found in control. Additionally, in PPARγ-blocked Leydig cells changes in the number of lipid droplets were revealed. Independent of used antagonist, western blot revealed decreased co-expression of GPER, ERRβ, PPARγ with exception of increased expression of ERRβ after PPARγ blockage. Immunohistochemistry confirmed presence of all receptors partially located in the nucleus or cytoplasm of Leydig cells of both control and treated testes. Changes in receptor expression, decreased cholesterol and increased estradiol tissue concentrations occurred through decreased cAMP level (with exception after GPER blockage) as well as Raf/Ras/ERK pathway expression. These all findings indicate that GPER-ERRβ-PPARγ interaction exists in immature boar testis and regulates Leydig cell function. Further detailed studies and considerations on GPER-ERRβ-PPARγ as possible diagnosis/therapy target in disturbances of testis steroidogenic function are needed., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Organotypic culture of testicular fragments from 7-day-old male pigs (Polish White Large) was used. Tissues were treated with an antagonist of G-protein coupled estrogen receptor (GPER) (G-15; 10 nM), and bisphenol A (BPA), and its analogs (TBBPA, TCBPA; 10 nM) alone or in combination and analyzed using electron and light (stainings for collagen fibers, lipid droplet and autophagy markers) microscopes. In addition, mRNA and protein abundances and localization of molecules required for miRNA biogenesis and function (Drosha, Exportin 5; EXPO5, Dicer, and Argonaute 2; AGO2) were assessed together with calcium ion (Ca 2+ ) and estradiol concentrations. Regardless of GPER blockade and/or treatment with BPA, TBBPA and TCBPA, there were no changes in Leydig cell morphology. Also, there were no changes in lipid droplet content and distribution but there were changes in lipid and autophagy protein abundance. In the interstitial tissue, there was an increase of collagen content, especially after treatment with BPA analogs and G-15 + BPA. Independent of the treatment, there was downregulation of EXPO5 and Dicer genes but the Drosha and AGO2 genes were markedly upregulated as a result of treatment with G-15 + BPA and TCBPA, respectively. There was always a lesser abundance of EXPO5 and AGO2 proteins regardless of treatment. There was markedly greater abundances of Drosha after G-15 + BPA treatment, and this also occurred for Dicer after treatment with G-15 + TCBPA. Immunolocalization of miRNA proteins indicated there was a cytoplasmic-nuclear pattern in control and treated cells. There was an increase of Ca 2+ concentrations after treatment with G-15 and BPA analogs. Estradiol secretion decreased after antagonist and chemical treatments when these were administered alone, however, there was an increase in estradiol secretion after treatment with combinations of these compounds., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this study mouse Leydig cell (MA-10) were treated with G-protein coupled membrane estrogen receptor antagonist (G-15; 10 nM). Cells were analyzed by Western blotting for expression of estrogen-related receptors (ERRα, β and γ), steroidogenic markers (lutropin receptor; LHR and 3β-hydroxysteroid dehydrogenase; 3β-HSD) and lipid droplet markers (perilipin; PLIN and microtubule-associated protein 1 A/1B-light chain 3; LC3). Concomitantly, microscopic analyses by light microscope (immunofluorescent staining for lipid droplets, PLIN and LC3) as well as by electron microscope (for lipid droplet ultrastructure) were utilized. For analysis of cholesterol content, cAMP level and progesterone secretion, G-15, estrogen receptor (ER) antagonist (ICI 182,780; 10 μM), 17β-estradiol (10 mM) and, bisphenol A (BPA; 10 nM) were used alone or in combinations. We revealed no changes in ERRs expression but alterations in ERRβ and γ localization in G-15-treated cells when compared to control. Partial translocation of ERRβ and γ from the cell nucleus to cytoplasm was observed. Decreased expression of LHR, 3β-HSD, PLIN and LC3 was detected. Moreover, in treated cells large lipid droplets and differences in their distribution were found. Very strong signal of co-localization for PLIN and LC3 was found in treated cells when compared to control. In ultrastructure of treated cells, degenerating lipid droplets and double membrane indicating on presence of lipophagosome were observed. We found, that only (i) BPA and G-15 did not effect on cholesterol content, (ii) BPA, G-15 and ICI did not effect on cAMP level and (iii) BPA, ICI alone and in combination, and BPA with G-15 did not modulate progesterone secretion. These findings showed complex and diverse estrogen effects on mouse Leydig cells at various steps of steroid hormone production (cholesterol storage, release and processing). Lipid homeostasis and metabolism in these cells were affected by endogenous and exogenous estrogen, interactions of receptors (GPER, ER and ERR) and GPER and ER antagonists., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

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 β, PDGFRα and β; vascular endothelial growth factor, VEGF; and vimentin) were analyzed by immunohistochemistry/immunofluorescence and Western blot. mRNA expression of CD34 as well as ERR α, β, and γ was measured by qRT-PCR. Relaxin and Ca 2+ 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, Ca 2+ 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.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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

Estrogen-related receptors (ERRs) α, β and γ appear to be novel molecules implicated in estrogen signaling. We blocked and activated ERRs in mouse (C57BL/6) adrenals and adrenocortical cells (H295R) using pharmacological agents XCT 790 (ERRα antagonist) and DY131 (ERRβ/γ agonist), respectively. Mice were injected with XCT 790 or DY131 (5 μg/kg bw) while cells were exposed to XCT 790 or DY131 (0.5 μg/L). Irrespectively of the agent used, changes in adrenocortical cell morphology along with changes in lutropin, cholesterol levels and estrogen production were found. Diverse and complex ERRs regulation of multilevel-acting steroidogenic proteins (perilipin; PLIN, cytochrome P450 side-chain cleavage; P450scc, translocator protein; TSPO, steroidogenic acute regulatory protein; StAR, hormone sensitive lipase; HSL and HMG-CoA reductase; HMGCR) was revealed. Blockage of ERRα decreased P450scc, StAR and TSPO expressions. Activation of ERRβ/γ increased P450scc, StAR and HMGCR while decreased HSL expressions. PLIN expression increased either after XCT 790 or DY131 treatment. Additionally, treatment with both XCT 790 or DY131 decreased activity of Ras/Raf, Erk and Akt indicating their involvement in control of morphology and steroidogenic function of cortex cells. ERRs are important in maintaining morpho-function of cortex cells through action in specific, opposite, or common manner on steroidogenic molecules., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

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

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