Primary Rat Sertoli Cells Research Articles

Transmigration of macrophages through primary adult rat Sertoli cells

ABSTRACT The blood testis barrier (BTB) is often studied with isolated immature Sertoli cells (SCs), transepithelial resistance (TER) measurements and FITC dextran diffusion assays. Recently, it was found that even in the absence of SCs, only few immune cells enter the seminiferous tubules. Thus, in this study, we evaluated the testicular immunological barrier (TIB) in vitro by transmigration of macrophages through SCs with and without peritubular cells (PCs) and with or without matrigel (MG). Primary PCs were isolated from adult rat testis and kept in mono- or co-cultures with the conditionally reprogrammed primary adult Sertoli cell line (PASC1) from rat that has been recently generated by our group. Rat monocytes isolated from fresh blood were differentiated into M0 macrophages, and after polarization to M1 or M2 macrophages characterized by gene expression of CXCL11 and TNF-α for M1, or CCL17 and CCL22 for M2. Transmigration of LeukoTracker-labeled M0, M1, and M2 macrophages through mono- and co-cultures of PCs/SCs with and without MG demonstrated that SCs are the main constituent of the TIB in vitro with only a negligible contribution of PCs or MG. Moreover, M2 macrophages showed less migration activity compared to M0 or M1. Treatment of SCs with testosterone (T) showed positive effects on the barrier in contrast to negative effects by interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α). The new transmigration model is suitable to evaluate transmigration of macrophages through a barrier consisting of testicular cells and can be applied to study the integrity of testicular barriers with respect to immunological aspects.

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

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

Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5‐year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood–testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6‐nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6‐nitrobenzylthioinosine 5′‐monophosphate (NBMPR‐P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first‐line treatment to avoid testicular relapse should be considered.

Seminal exosomal miR-210-3p as a potential marker of Sertoli cell damage in Varicocele.

Varicocoele-associated stressors, such as hypoxia and heat, can damage cell function and viability, and some exosomal biomarkers released from impaired cells may reflect the cell status in testis. To find if seminal exosomal microRNAs can reflect the Sertoli cell function in varicocoele. Experimental left varicocoele rat model was established (n=24), and patients with different grades of varicocoele (n=104) were enrolled. Primary rat Sertoli cells were isolated with enzymatic hydrolysis. Exosomes were isolated from primary rat Sertoli cells, rat epididymis tissue, and human seminal plasma with polymer-based precipitation method. Exosomal microRNAs were quantified with qPCR. Inhibin-B was detected with enzyme immunoassay. The correlation analysis between microRNA and inhibin-B was evaluated with Spearman's correlation. We screened 12 previously reported hypoxia-responsive microRNAs in the primary rat Sertoli cells and found that 4 exosomal microRNAs increased significantly in response to in vitro hypoxia treatment (P<.05). Of the 4 microRNAs, only miR-210-3p was upregulated in the rats with experimental varicocoele (P<.01). In the patients with varicocoele, we found that seminal exosomal miR-210-3p significantly increased in patients with grade II and III varicocoele (P<.01), and miR-210-3p negatively correlated with sperm count (P<.01) and seminal inhibin-B expression (r=-0.39, P<.01). For the 30 patients with microsurgical varicocelectomy, the operation notably decreased miR-210-3p (P<.01). Seminal exosomal miR-210-3p may be a novel, sensitive, and non-invasive biomarker of Sertoli cell damage in varicocoele.

Effects of Boron on Cytotoxicity, Apoptosis, and Cell Cycle of Cultured Rat Sertoli Cells In vitro.

The present study aimed to investigate the effects of the administration of boron on viability, apoptosis, and cell cycle of primary rat Sertoli cells (SCs) in vitro. SCs were aseptically isolated from 18-22-day-old male Sprague-Dawley (SD) rats. SCs were identified with immunofluorescence using anti-vimentin antibody. Further, to investigate the effects of boron on Sertoli cells, SCs of the boron treatment group were exposed to different concentrations (0.25, 0.5, 1, 5, 10, 40, and 80mmol/L) of boric acid. Using MTT and Cell Counting Kit-8 assays, the impact of boron on SCs viability was analyzed. Cell apoptosis and cycle of SCs were analyzed using flow cytometry. A concentration of 0.5mmol/L boric acid resulted in the highest viability and lowest necrosis and apoptosis. Above this concentration (even 1.0mmol/L) showed lower viability and higher levels of necrosis and apoptosis. Administration of < 0.5mmol/L boron significantly promoted the viability of Sertoli cells (P < 0.01); however, the exposure to high dose (> 10mmol/L) of boron exhibited significant adverse effects on Sertoli cells (P < 0.01) and even toxic effects, inhibiting cell viability compared to the control group. Flow cytometry analysis showed that treatment with 0.5mmol/L of boron significantly inhibited the apoptosis of Sertoli cells and the proportion of cells in S and G2/M phases was markedly increased; however, a higher concentration of 40 and 80mmol/L of boron promoted Sertoli cell apoptosis and cells were completely arrested at G0/G1 phase. Boron at doses below 0.5mmol/L could significantly improve the viable capacity of testicular Sertoli cells in vitro and inhibit their apoptosis. However, high dose of boron (at a concentration higher than 5.0mmol/L) exhibited noticeable toxic effects, inhibiting cell viability, accelerating apoptosis of Sertoli cells, and arresting cell cycle at G0/G1 phase.

Sertoli Cells Avert Neuroinflammation-Induced Cell Death and Improve Motor Function and Striatal Atrophy in Rat Model of Huntington Disease.

Huntington's disease (HD) is a genetically heritable disorder, linked with continuing cell loss and degeneration mostly in the striatum. Currently, cell therapy approaches in HD have essentially been focused on replenishing or shielding cells lost over the period of the disease. Herein, we sought to explore the in vitro and in vivo efficacy of primary rat Sertoli cells (SCs) and their paracrine effect against oxidative stress with emphasis on HD. Initially, SCs were isolated and immunophenotypically characterized by positive expression of GATA4. Besides, synthesis of neurotrophic factors of glial cell-derived neurotrophic factor and VEGF by SCs were proved. Next, PC12 cells were exposed to hydrogen peroxide in the presence of conditioned media (CM) collected from SC (SC-CM) and cell viability and neuritogenesis were determined. Bilateral striatal implantation of SC in 3-nitropropionic acid (3-NP)-lesioned rat models was performed, and 1month later, post-graft analysis was done. According to our in vitro results, the CM of SC protected PC12 cells against oxidative stress and remarkably augmented cell viability and neurite outgrowth. Moreover, grafted SCs survived, exhibited decreases in both gliosis and inflammatory cytokine levels, and ameliorated motor coordination and muscle activity, together with an increase in striatal volume as well as in dendritic length of the striatum in HD rats. In conclusion, our results indicate that SCs provide a supportive environment, with potential therapeutic benefits aimed at HD.

Hydroxyflutamide affects connexin 43 via the activation of PI3K/Akt-dependent pathway but has no effect on the crosstalk between PI3K/Akt and ERK1/2 pathways at the Raf-1 kinase level in primary rat Sertoli cells

We investigated the effects of 2-hydroxyflutamide (HF), an active metabolite of the anti-androgen flutamide, on the activation of the phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) in rat Sertoli cells. Sertoli cells, isolated from 20-day-old rat testes, were cultured in vitro and treated with HF, testosterone, or HF+testosterone. Studies by western blotting demonstrated that HF inhibited the testosterone-mediated increase in c-Src activity (p<0.05). In contrast, Akt phosphorylation was augmented within 5min after HF treatment (p<0.01). This effect was accompanied by a rapid upregulation in PTEN phosphorylation (p<0.001). Despite no changes in Raf-1 phosphorylation, HF increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (p<0.001), indicating that the effect of the anti-androgen on ERK1/2 was independent of PI3K/Akt-pathway activation at this level. Since HF inhibited the testosterone-mediated increase in c-Src activity, it is likely that activation of both Akt and ERK1/2 occurred in a p-Src independent manner. Activation of PI3K/Akt-pathway by HF resulted in the reduced level of Sertoli cell functional marker, connexin 43 (p<0.01). Collectively, these data provide evidence that HF rapidly and transiently affects the protein kinase-dependent signaling pathways, acting both as an antagonist and agonist. Moreover, using testes of flutamide-treated rats for 7days, we demonstrated that the anti-androgen can modulate the protein kinase-dependent pathways in long term by enhancing Akt and ERK1/2 protein expression (p<0.05).

Retinoic acid promotes Sertoli cell differentiation and antagonises activin-induced proliferation

From puberty and throughout adult spermatogenesis, retinoid signalling is essential for germ cell differentiation and male fertility. The initiation of spermatogonial differentiation and germ cell meiosis occurs under the direction of local retinoid signalling in the testis, and corresponds with the final phase of somatic Sertoli cell differentiation at puberty. Here, we consider the cellular and molecular basis of retinoid actions upon Sertoli cell differentiation. Primary rat Sertoli cells were isolated during the pubertal proliferative and quiescent phases at postnatal days 10- and 20- respectively, and cultured with all-trans-retinoic acid. We show that retinoid signalling can potently suppress activin-induced proliferation by antagonising G1 phase progression and entry into the cell cycle. Retinoid signalling was also found to initiate tight junction formation in primary Sertoli cells, consistent with a pro-differentiative role. This study implicates retinoid signalling in the differentiation of both somatic and germ cells in the testis at puberty.

The Tyrosine Phosphatase SHP2 Regulates Sertoli Cell Junction Complexes1

The blood-testis barrier (BTB) is a large junctional complex composed of tight junctions, adherens junctions, and gap junctions between adjacent Sertoli cells in the seminiferous tubules of the testis. Maintenance of the BTB as well as the controlled disruption and reformation of the barrier is essential for spermatogenesis and male fertility. Tyrosine phosphorylation of BTB proteins is known to regulate the integrity of adherens and tight junctions found at the BTB. SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) and a key regulator of growth factor-mediated tyrosine kinase signaling pathways. We found that SHP2 is localized to Sertoli-Sertoli cell junctions in rat testis. The overexpression of a constitutive active SHP2 mutant, SHP2 Q79R, up-regulated the BTB disruptor ERK1/2 via Src kinase in primary rat Sertoli cells in culture. Furthermore, focal adhesion kinase (FAK), which also supports BTB integrity, was found to interact with SHP2 and constitutive activation of SHP2 decreased FAK tyrosine phosphorylation. Expression of the SHP2 Q79R mutant in primary cultured Sertoli cells also resulted in the loss of tight junction and adherens junction integrity that corresponded with the disruption of the actin cytoskeleton and mislocalization of adherens junction and tight junction proteins N-cadherin, β-catenin, and ZO-1 away from the plasma membrane. These results suggest that SHP2 is a key regulator of BTB integrity and Sertoli cell support of spermatogenesis and fertility.

Cryptorchidism-induced CFTR down-regulation results in disruption of testicular tight junctions through up-regulation of NF- B/COX-2/PGE2

Does elevated temperature-induced cystic fibrosis transmembrane conductance regulator (CFTR) down-regulation in Sertoli cells in cryptorchid testis disrupt testicular tight junctions (TJs) through the nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)) pathway? Our results suggest that CFTR may be involved in regulating testicular TJs and the blood-testis barrier (BTB) through its negative regulation of the NF-κB/COX-2/PGE(2) pathway in Sertoli cells, a defect of which may result in the spermatogenesis defect in cryptorchidism. Cryptorchidism, or undescended testes, is known to result in defective spermatogenesis. Although an elevated testicular temperature is regarded as an important factor affecting spermatogenesis in cryptorchidism, the exact mechanism remains elusive. It is known that the expression of functional CFTR is temperature sensitive. Our previous study has demonstrated that CFTR negatively regulates NF-κB/COX-2/PGE(2) in bronchial epithelial cells. Disruption of TJs by COX-2/PGE(2) has been found in tumour cells. Expression of CFTR, NF-κB, COX-2 and TJ proteins was examined in the testes of a surgical-induced cryptorchidism mouse model and a testicular hyperthermia mouse model, as well as in control or CFTR-inhibited/knocked down primary rat Sertoli cells. PGE(2) production was measured by ELISA. Sertoli cell barrier function was determined by transepethelial resistance (TER) measurements in rat Sertoli cell primary cultures. BTB integrity in the cryptorchidism model was monitored by examining tracker dye injected into seminiferous tubules. Down-regulation of CFTR accompanied by activation of NF-κB, up-regulation of COX-2 and down-regulation of TJ proteins, including ZO-1 and occludin, was observed in a cryptorchidism mouse model. BTB leakage revealed impaired BTB integrity in cryptorchid testes, confirming the destruction of TJs. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and CFTR knockout mouse model. Culturing primary Sertoli cells at 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression and PGE(2) production compared with the culture at the physiological 32°C. Inhibition or knockdown of CFTR led to increased COX-2 but decreased ZO-1 and occludin expression in Sertoli cells, which could be mimicked by PGE(2), but reversed by NF-κB or COX-2 inhibitor, suggesting that the regulation of TJs by CFTR is mediated by a NF-κB/COX-2/PGE(2) pathway. Inhibition of CFTR or administration of PGE(2) significantly decreased Sertoli cell TER. This study has tested only the CFTR/NF-κB/COX-2/PGE(2) pathway in mouse testes in vivo and in rat Sertoli cells in vitro, and thus, it has some limitations. Further investigations in other species, especially humans, are needed. Our study may shed more light on one of the aspects of the complicated underlying mechanisms of defective spermatogenesis induced by cryptorchidism.

Transporter‐mediated mechanism of nucleoside penetration of the blood‐testis barrier

The blood‐testis barrier (BTB) prevents the entry of many therapeutics into the lumen of the seminiferous tubules (STs) shielding semen from chemical exposure. The BTB is formed by tight junctions between Sertoli cells. One drug class of HIV therapeutics, nucleoside analogs (NSAs), can penetrate the BTB and be detected in the semen. The purpose of this study is to determine the mechanisms by which NSAs are transported across the BTB. Transport studies in isolated rodent STs using H3uridine (Kt 89.72uM) as a model nucleoside substrate indicate that Sertoli cells take up nucleosides almost exclusively via equilibrative nucleoside transporter 1 (ENT1). The IC50 for NBMPR, an ENT inhibitor, is 12.9 nM. These data correspond with immunohistochemical staining of rat testes showing ENT1 on the basolateral membrane, whereas ENT2 is on the apical membrane of Sertoli cells. This localization suggests that ENT1 acts as an uptake transporter and ENT2 may facilitate the efflux of NSAs into the lumen of STs. Uptake of didanosine, a commonly used NSA, in HEK293 cells can be blocked by ENT1 inhibition. Trans‐epithelial transport of uridine by primary rat Sertoli cells can also be blocked by NBMPR. These data indicate a novel ENT1 and 2‐dominant mechanism for the transepithelial transport of nucleoside analogs across the BTB. AI083927.

MRNA-Selective Translation Induced by FSH in Primary Sertoli Cells

FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m(7)GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5'cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far.

Effect of H2-Gamendazole and Other Indazole Carboxylic Acid (ICA) Analogs on Primary Rat Sertoli Cells Cytoskeletal Structure and Elongation Factor 1 Alpha (EEF1A1) Expression and Function.

Effect of H2-Gamendazole and Other Indazole Carboxylic Acid (ICA) Analogs on Primary Rat Sertoli Cells Cytoskeletal Structure and Elongation Factor 1 Alpha (EEF1A1) Expression and Function.

Developmental patterns of PPAR and RXR gene expression during spermatogenesis

Members of the family of nuclear receptors that include peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are important mediators of selective gene activation during development and cellular differentiation. In this study, developmentally-specific PPAR and RXR patterns of expression that occur in somatic and germ cell populations in the testis were determined using quantative real-time PCR (qRT-PCR) studies on RNAs that were isolated from StaPut purified mouse germ cells and primary rat Sertoli cells. These qRT-PCR studies indicate that transcripts encoding the PPAR-Alpha (α), -Beta (β), and -Gamma (γ) and RXR -Alpha (α), -Beta (β), and -Gamma (γ) are developmentally expressed in both differentiating germ and Sertoli cells. In further experiments aimed at deciphering the physiological role that PPAR-Gamma (γ) plays in Sertoli cells, 15-day primary rat Sertoli cells were infected with recombinant adenoviral vectors containing PPAR-Gamma (γ) cDNA and PPAR-Gamma (γ) RNAi constructs. Affymetrix microarray analysis and qRT-PCR validation studies using total RNA isolated from these transfected cells indicated that PPAR-Gamma regulates the pattern of expression of key lipid metabolic genes in Sertoli cells.

Developmental regulation of p70 S6 kinase by a G protein-coupled receptor dynamically modelized in primary cells.

The mechanisms whereby G protein-coupled receptors (GPCR) activate signalling pathways involved in mRNA translation are ill-defined, in contrast to tyrosine kinase receptors (TKR). We compared a GPCR and a TKR, both endogenously expressed, for their ability to mediate phosphorylation of 70-kDa ribosomal S6 kinase p70S6K in primary rat Sertoli cells at two developmental stages. In proliferating cells stimulated with follicle-stimulating hormone (FSH), active p70S6K was phosphorylated on T389 and T421/S424, through cAMP-dependent kinase (PKA) and phosphatidyl-inositide-3 kinase (PI3K) antagonizing actions. In FSH-stimulated differentiating cells, active p70S6K was phosphorylated solely on T389, PKA and PI3K independently enhancing its activity. At both developmental stages, insulin-induced p70S6K regulation was consistent with reported data. Therefore, TKR and GPCR trigger distinct p70S6K active conformations. p70S6K developmental regulation was formalized in a dynamic mathematical model fitting the data, which led to experimentally inaccessible predictions on p70S6K phosphorylation rate.

A time course study on the "in vitro" effects of T3 and testosterone on androgen and estrogen receptors in peripuberal primary rat Sertoli cells.

The effect of Tri-iodothyronine (T3) administration leading to the precocius differentiation of Sertoli cell in prepuberal rats has been previously shown. The functional maturation of Sertoli cells is associated with changes in androgen metabolism. We have recently demonstrated that T3 influences androgen metabolism in Sertoli cells by inhibiting aromatase activity and reduces drastically the ER contents in peripubertal hypothyroid rats. To better understand the role of T3 in modulating steroid action on Sertoli cells, we performed a time course study evaluating the in vitro effects of T3 and testosterone (T) on androgen (ARs) and estrogen (ERs) receptor content in Sertoli cells isolated from two weeks old Wistar rats. ARs and ERs basal levels did not change during the time course study indicating that the exposure to culture medium per se did not affect either receptor type. After 24 hrs of incubation with either T3 or T, a decrease of ERs in both nucleus and cytosol was observed. Such a decrease was augmented by the simultaneous administration of both hormones. ARs displayed a different temporal pattern in the two cellular compartments and exhibited an earlier rise in the cytosol induced by either T3 or T. At 36 hrs, ARs were significantly enhanced in both compartments in response to either T or T3 exposure while combined hormonal treatment caused an additive increase compared with the single treatment group. As a consequence of the opposite behaviour pattern displayed by ARs and ERs, the ratio between total ARs and ERs contents was increased after 24 hrs of exposure to hormonal treatment. To evaluate if treatments performed induced a functional maturation of Sertoli cells, transferrin levels in culture medium were measured. The increase of this protein paralleled that of ARs content as well as that of ARs/ERs ratio. This study demonstrates that thyroid hormone induces a progressive increase of (AR)/(ER) ratio in the differentiating Sertoli cells bringing them to a prevalent androgen dependency along their functional maturation.

148. HORMONAL REGULATION OF miRNA IN THE TESTIS

Acute suppression of circulating reproductive hormones (FSH and testosterone) inhibits sperm release (spermiation) (1), although the molecular mechanisms of spermiation failure are poorly understood. Micro-RNAs (miRNAs) are small non-coding RNAs that regulate protein expression, and are essential for normal spermatogenesis. Recent studies suggest that miRNAs are exquisitely sensitive to hormonal control by FSH, LH and testosterone (2–4). This suggests that hormonal regulation of miRNAs in the testis following acute hormonal suppression may contribute to spermiation failure. Therefore, we hypothesised that gonadotrophin regulated miRNAs control spermiation outcome. We used array analysis to show that miRNA expression is hormonally regulated by FSH and testosterone in our rat in vivo model of spermiation failure and also in primary rat Sertoli cells by. qPCR validation revealed that miR-7b, -23a, -30c, -125b, -148b, -197, -483, -592, and -690 are all hormonally sensitive testicular miRNAs. Bioinformatic analyses of potential gene targets of these miRNAs predicted numerous protein components localised in the testicular tubulobulbar complex (TBC). The TBC is a podosome-like structure found between Sertoli cells and adjacent germ cells in the testis, and is thought to internalise intact inter-cellular structures and regulate spermatid head shape prior to spermiation. WASP, a TBC protein that regulates actin filament dynamics, contained a conserved binding site for miR-690 within its 3'UTR. Increased miR-690 expression following hormone suppression corresponded to a decrease in WASP protein expression in vivo and in vitro. In addition, transfection of miR-690 into HEK293T cells down-regulated WASP protein. Our results suggest that following hormone suppression, miR-690 is stimulated in the Sertoli cell, thereby inhibiting WASP protein expression. We conclude that miRNA-mediated disruption of TBC integrity potentially regulates spermatid disengagement. This study describes new molecular mechanisms in the testis that may control spermiation outcome of potential significance in male hormonal contraception.

Regulation of Neurotrophin 3 Expression by SRY During Male Sex Determination.

Neurotrophin 3 (Nt3) plays a significant role in male gonadal development. It is required for mesonephric cell migration into the testis for testis cord formation. The expression profiles for Nt3 are consistent with those expected for a gene directly regulated by SRY, and potential SRY binding HMG box elements were identified in the Nt3 promoter. Therefore, Nt3 regulation by SRY was investigated. Nt3 expression profiles in rat were verified spatially and temporally by immunohistochemical localization and Nt3 was found to be in the developing testis cords in Sertoli cells of rat gonads. Nt3 promoter constructs with luciferase reporters were made using mouse and rat Nt3 promoters and HMG boxes mutated by site specific mutagenesis. Promoter activity was monitored using transfection assays in primary P20 rat Sertoli cells and in E13 rat testis cell lines. SRY element site specific mutant promoters had reduced luciferase reporter activity compared to wild type promoters. Since SRY and SOX9 are important for sex determination and SOX9 binds to DNA sequences highly similar to those bound by SRY, the potential roles of both SRY and SOX9 in regulation of the Nt3 promoter were investigated. The potential for SRY and SOX9 to affect Nt3 promoter activity was examined in co-transfection assays using SRY and SOX9 expression vectors. Results indicate that both SRY and SOX9 can stimulate the Nt3 promoter in transfection assays. Further analysis is required to reveal the key players in vivo: Knock down of Sry and Sox9 expression is being used to determine if there is a change in Nt3 expression when Sry or Sox9 are silenced at the initiation of gonadal differentiation. Gel shifts and high sensitivity ChIP assays are being utilized to determine the relative binding affinities of SRY and SOX9 to HMG boxes in the Nt3 promoter and investigate binding to the Nt3 promoter in vivo. These studies have helped clarify the regulation of Nt3 in the gonad during sex determination and reveal the potential for Nt3 to be regulated directly by SRY.

Transcriptional Regulation of the Estrogen Receptor α Gene by Testosterone in Cultures of Primary Rat Sertoli Cells

Background: We wanted to identify the presence of the estrogen receptor (ER) α in Sertoli cells and gain insight on the regulation of the ERα gene expression by testosterone in Sertoli cells. The transcriptional regulation of the ERα gene was investigated in primary Sertoli cell cultures by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). Methods: Primary Sertoli cell culture was performed. The expression levels of ERα and ERβ mRNA in Sertoli cells were detected by Northern blot, RT-PCR, immunocytochemistry and in situ hybridization. Results: The ovary, testis and epididymis showed a moderate to high expression of ERα while the prostate, ovary and LNCap cells showed the ERβ expression. ERα mRNA and protein were detected in the germ cells and Sertoli cells by in situ hybridization and immunocytochemistry. The level of ERα mRNA was gradually decreased in a time-dependent manner after testosterone treatment, and the changes of ERα mRNA were dependent on the concentration of testosterone. Androgen binding protein and testosterone-repressive prostate message-2 (TRPM-2) mRNA were reduced at 24 hour by estradiol, while the transferrin mRNA was not affected. ERα mRNA was strongly detectable in the testes of 7 days-old-rats, but it was gradually decreased from 14 to 21 days of age. The primary Sertoli cells also showed the same pattern. The ERα gene expression was also regulated by testosterone in the Sertoli cells prepared from the 14- and 21-day old rats. Conclusions: These results suggest that ER α is transcriptionally regulated by testosterone and it may play some role in the Sertoli cells. (J Kor Soc Endocrinol 21:106~115, 2006)

G protein-coupled receptor kinases and beta arrestins are relocalized and attenuate cyclic 3',5'-adenosine monophosphate response to follicle-stimulating hormone in rat primary Sertoli cells.

The FSH receptor (FSH-R) is a member of the rhodopsin-like subfamily of G protein-coupled receptors that undergoes homologous desensitization upon agonist stimulation. In immortalized cell lines overexpressing the FSH-R, G protein-coupled receptor kinases (GRKs) and beta-arrestins are involved in the phosphorylation, uncoupling, and internalization of this receptor. In an effort to appreciate the physiological relevance of GRK/beta-arrestin actions in natural FSH-R-bearing cells, we used primary rat Sertoli cells as a model. GRK2, -3, -5, -6a, and -6b and beta-arrestins 1 and 2 were expressed in primary rat Sertoli cells. Overexpression of these different GRKs and beta-arrestins in primary rat Sertoli cells significantly attenuated the FSH-induced cAMP response, and FSH rapidly triggered a relocalization of endogenously expressed GRK2, -3, -5, and -6 and beta-arrestins 1 and 2 from the cytosol to the membranes. These results highlight the relationship existing between the GRK/beta-arrestin regulatory system and the FSH-R signaling machinery in a physiological model.