Spexin role in human granulosa cells physiology and PCOS: expression and negative impact on steroidogenesis and proliferation†.

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.

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.

Levels of spexin and its receptors GALR2 and GALR3 in the hypothalamus and ovary of letrozole-induced polycystic ovary syndrome in rats.

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.

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.

Peroxisome Proliferator-Activated Receptor γ, but Not α or G-Protein Coupled Estrogen Receptor Drives Functioning of Postnatal Boar Testis-Next Generation Sequencing Analysis.

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.

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.

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

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

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

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

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

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

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

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

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

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

Insights into the role of estrogen-related receptors α, ß and γ in tumor Leydig cells.

In this study, we demonstrate, for the first time, estrogen-related receptor (ERR) regulation of the physiological and biochemical status of testicular tumor Leydig cells. In a mouse tumor Leydig cells, ERRs (α, ß, and γ) were silenced via siRNA. Cell morphology and cell physiology (proliferation and observation of monolayer formation) were performed by inverted phase-contrast microscope. Leydig cell functional markers (steroid receptors and signaling molecules) were examined by immunofluorescence and Western blotting. Additionally, progesterone secretion was assessed. Mitochondrial mass and membrane potential were analyzed by flow-cytometry while cGMP and Ca2+ concentrations were analyzed using immunoenzymatic and colorimetric assays, respectively. These results revealed, ERRs indirectly regulate Leydig cell proliferation while ERRα and ß affect cell monolayer formation. ERRs interact with canonical and membrane estrogen receptors (ERα, ERß, and GPER), androgen receptor, metalloproteinase (MMP 9), protein kinase A (PKA), extracellular-regulated kinase (ERK), and neurogenic locus notch homolog protein 2 (Notch2). Depending on the type of ERR knocked down, coupled with estradiol treatment, changes in progesterone concentration and cGMP and Ca2+ concentrations constitute a microenvironment that may effect tumor Leydig cell characteristics. ERRs should be considered important factors in developing of innovating approaches that target pathological processes of testicular Leydig cells.