Insulin is essential for the regulation of glucose homeostasis. Insulin dysfunction occurs in several pathologies, such as diabetes mellitus, which is associated with fertility problems. Somatic Sertoli cells (SCs) not only metabolize glucose to lactate, which is the central energy source used by developing germ cells, but also determine the germ cell population size. If a deregulation in SCs apoptosis occurs, it will affect germ cells, compromising spermatogenesis. As SCs apoptotic signaling is a hormonally regulated process, we hypothesized that the lack of insulin could lead to alterations in apoptotic signaling. Therefore, we examined the effect of insulin deprivation on several markers of apoptotic signaling in cultured rat SCs. We determined mRNA and protein expression of apoptotic markers as well as caspase-3 activity. SCs cultured in insulin deprivation demonstrated a significant decrease on mRNA levels of p53, Bax, caspase-9, and caspase-3 followed by a significant increase of Bax and decrease of caspase-9 protein levels relatively to the control. Caspase-3 activity was also decreased in SCs cultured in insulin deprivation conditions. Our results show that insulin deprivation decreases caspase-dependent apoptotic signaling in cultured rat SCs evidencing a possible mechanism by which lack of insulin can affect spermatogenesis and fertility.

The understanding of main mechanisms that determine the ability of immune privilege related to Sertoli cells (SCs) will provide clues for promoting a local tolerogenic environment. In this study, we evaluated the property of humoral and cellular immune response modulation provided by porcine SCs. Porcine SCs were resistant to human antibody and complement-mediated formation of the membrane attack complex (38.41+/-2.77% vs. 55.02+/-5.44%, p=0.027) and cell lysis (42.95+/-1.75% vs. 87.99 +/-2.25%, p<0.001) compared to immortalized aortic endothelial cells, suggesting that porcine SCs are able to escape cellular lysis associated with complement activation by producing one or more immunoprotective factors that may be capable of inhibiting membrane attack complex formation. On the other hand, porcine SCs and their culture supernatant suppressed the up-regulation of CD40 expression (p<0.05) on DCs in the presence of LPS stimulation. These novel findings, as we know, suggest that immune modulatory effects of porcine SCs in the presence of other antigen can be obtained from the first step of antigen presentation. These might open optimistic perspectives for the use of porcine SCs in tolerance induction eliminating the need for chronic immunosuppressive drugs.

Spermatogenesis is a process by which haploid cells differentiate from germ cells in the seminiferous tubules of the testes. TLE3, a transcriptional co-regulator that interacts with DNA-binding factors, plays a role in the development of somatic cells. However, no studies have shown its role during germ cell development in the testes. Here, we examined TLE3 expression in the testes during spermatogenesis. TLE3 was highly expressed in mouse testes and was dynamically regulated in different cell types of the seminiferous tubules, spermatogonia, spermatids, and Sertoli cells, but not in the spermatocytes. Interestingly, TLE3 was not detected in Sertoli cells on postnatal day 7 (P7) but was expressed from P10 onward. The microarray analysis showed that the expression of numerous genes changed upon TLE3 knockdown in a Sertoli cell line TM4. These include 1597 up-regulated genes and 1452 down-regulated genes in TLE3-knockdown TM4 cells. Ingenuity Pathway Analysis (IPA) showed that three factors were up-regulated and two genes were down-regulated upon TLE3 knockdown in TM4 cells. The abnormal expression of the three factors is associated with cellular malfunctions such as abnormal differentiation and Sertoli cell formation. Thus, TLE3 is differentially expressed in Sertoli cells and plays a crucial role in regulating cell-specific genes involved in the differentiation and formation of Sertoli cells during testicular development.

1. Cultured epithelia of Sertoli cells from prepubertal rats were grown on Matrigel-coated millipore filters for short-circuit current (Isc) measurements. Under basal conditions, these epithelia exhibited a 'zero' transepithelial potential difference, a 'zero' short-circuit current and a transepithelial resistance of 60 Omega cm2. 2. Forskolin (100 microM) and 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) (100 microM) added to the apical side stimulated the Isc (forskolin, peak DeltaIsc = 1.32 +/- 0.16 microA cm-1; cpt-cAMP, peak DeltaIsc = 0.88 +/- 0.16 microA cm-2). 3. ATP (100 microM) added apically elicited a Isc response (peak DeltaIsc = 6.45 +/- 0. 28 microA cm-2) which was similar in magnitude to that of 1 microM thapsigargin (peak DeltaIsc = 6.09 +/- 0.44 microA cm-2). The potency of the responses to other nucleotides: UTP >= ATP > ADP >> AMP = adenosine indicates the involvement of a mixture of P2Y receptors. 4. Removal of extracellular Cl- and HCO3- reduced the Isc response to ATP by 70 % and 40 %, respectively. Removal of K+ had no effect, whereas removal of Na+ attenuated the Isc response. 5. The response to ATP was insensitive to agents known to block anion secretion (except apical diphenylamine-2-carboxylate (DPC) and DIDS). The resistance to perturbation by pharmacological agents may be a unique property of the seminiferous epithelium. 6. Whole-cell current recordings in cultured rat Sertoli cells demonstrated a DIDS-sensitive outwardly rectifying Cl- conductance with activating and inactivating characteristics at depolarizing and hyperpolarizing voltages, respectively. 7. The stimulation of electrogenic ion transport by ATP may be part of a complex mechanism regulating fluid secretion by the testis. Cultured Sertoli cell epithelia are shown to provide a useful model to investigate transepithelial transport in the seminiferous epithelium.

Heat stress reduces the number of Sertoli cells and impairs spermatogenesis. Mounting evidence indicates that lipid homeostasis is fundamental to cell survival. However, little is known about the role of lipid peroxides in the heat stress-induced apoptosis of Sertoli cells. In the present study, we used metabolomics to explore the changes of lipid peroxides in porcine Sertoli cells under heat stress using liquid chromatograph-mass spectrometry. These results showed a notable increase in the content of 8-hydroxyeicosatetraenoic acid (8-HETE), and 15-hydroxyeicosatetraenoic acid (15-HETE). Furthermore, we found that among arachidonate lipoxygenases, heat stress significantly increased the expression of arachidonate 15-lipoxygenase type B (ALOX15B). Moreover, baicalein, a specific inhibitor of ALOX15B, reduced the content of 8-HETE and 15-HETE, and decreased the apoptosis rate in heat stress-treated porcine Sertoli cells. In addition, baicalein and small interfering RNAs targeting ALOX15B increased the content of 8-HETE and 15-HETE, and activated the p38-p53 pathway, causing apoptosis in heat stress treated porcine Sertoli cells. Interestingly, a p38 inhibitor decreased the expression of ALOX15B, reduced the content of 8-HETE and 15-HETE, and decreased the expression of p53 and the apoptosis rate in heat stress treated porcine Sertoli cells. A p53 inhibitor had similar effect on Sertoli cells. These results indicated that heat stress enhanced the expression of ALOX15B, increased the content of 8-HETE and 15-HETE, and activated the p38-p53 pathway to cause apoptosis. ALOX15B and lipid peroxides obtained feedback from the p38-p53 pathway. Our findings will help to reveal the mechanism of lipid metabolism in Sertoli cells, and could provide a new targeted substrate for anti-heat stress strategies.

Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells.

Primary cell culture is an established and widely used technique to study Sertoli cell function in vitro. However, the relative difficulty of stably overexpressing or knocking down genes in Sertoli cell culture has limited progress in the field. In this technical report, we present a method to transduce 20 dpp rat Sertoli cell cultures with VSV-G pseudotyped lentiviral based vectors at a high rate (~80%), with stable reporter gene expression. Although high transgene expression is desirable, it was noted that at transduction rates > 60% inter-Sertoli cell tight junction integrity and, hence, Sertoli cell function, were transiently compromised. We envisage that this optimized procedure has the potential to stimulate Sertoli cell research, and motivate the use of Sertoli cells in various cell therapy applications.

Sertoli cells produce anti-Müllerian hormone (AMH), a glycoprotein belonging to the transforming growth factor-beta family. AMH mediates the regression of Müllerian ducts in the developing male fetus. However, the role of AMH in the regulation of primary Sertoli cells remains unclear. The present study was designed to investigate the effect of AMH on the viability and proliferation of Sertoli cells, with an additional focus on stem cell factor (SCF). Treatment of Sertoli cells with increasing concentrations of rh-AMH (0, 10, 50, 100, and 800ng/ml) for two days revealed that AMH, at high concentrations, increased apoptosis. These results were confirmed by a significant increase in Caspase-3 and Bax and a decrease in Bcl-2 protein and mRNA expression (P<0.01). Paradoxically, treatment with a low concentration of rh-AMH (10ng/ml), but not higher concentrations (50-800ng/ml), promoted Sertoli cell proliferation, which was verified by an increase in PCNA mRNA (P<0.05). Furthermore, only low concentrations of rh-AMH activated the non-canonical ERK signaling pathway. Similarly, low concentrations of rh-AMH (10-50ng/ml) significantly increased (P<0.05) SCF mRNA and SCF protein levels. These findings indicate that AMH differentially regulates the fate of Sertoli cells in vitro by promoting proliferation at low concentrations and apoptosis at high concentrations. In addition, AMH increased the expression of SCF, an important regulator of Sertoli cell development. Therefore, AMH may play a role in Sertoli cell development.

Regulation of Sertoli cell number is a key event to determine normal spermatogenesis. We have previously shown that relaxin and its G-protein coupled receptor RXFP1 are expressed in rat Sertoli cells, and that relaxin stimulates Sertoli cell proliferation. This study examined the mechanisms underlying the mitogenic effect of relaxin in a primary culture of Sertoli cells removed from testes of immature rats. Stimulation with exogenous relaxin increased Sertoli cell number and the expression of the proliferating cell nuclear antigen (PCNA), but did not affect the mRNA level of the differentiation markers cadherins 1 and 2. Relaxin-induced Sertoli cell proliferation was blocked by inhibition of MEK/ERK1/2 or PI3K/AKT pathways, but not by inhibition of PKC or EGFR activity. Relaxin induced a rapid and transient activation of ERK1/2 phosphorylation, which was MEK and SRC-dependent, and involved upstream activation of G(i). AKT activation could be detected 5 min after relaxin stimulation, and was still detected after 24h of stimulation with relaxin. Relaxin-induced AKT phosphorylation was G(i)- but not PKA-dependent, and it was blocked by both PI3K and MEK inhibitors. In conclusion, the mitogenic effect of relaxin in Sertoli cell involves coupling to G(i) and activation of both MEK/ERK1/2 and PI3K/AKT pathways.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors of transcription factors composed of three family members: PPARα, PPARβ/δ and PPARγ. This study was aimed to evaluate the role of PPARs in the estradiol production via follicle stimulating hormone (FSH) in the ovine Sertoli cells. At the first step, transcripts of PPARα, PPARβ /δ and PPARγ were evaluated by quantitative real time PCR (qRT-PCR) in the ovine Sertoli cells in vitro after FSH treatment. PPARγ transcript was increased in FSH-treated cells while PPARα and PPAR β /δ transcripts were unchanged. At the second step, Pioglitazone as PPARγ agonist and 2-chloro-5-nitrobenzanilide (GW9662) as PPARγ antagonist were used in the FSH-treated Sertoli cells and then, the estradiol production and aromatase transcript were evaluated. Aromatase transcript was increased by pioglitazone in the FSH-treated Sertoli cells while GW9662 did not change its transcript. The estradiol production was increased by low concentrations of pioglitazone in FSH-treated Sertoli cells while the production of this hormone was decreased by the high concentration of Pioglitazone. The GW9662 did not change the production of estradiol in FSH-treated Sertoli cells. It is concluded that FSH regulates the estradiol production and aromatase expression in a way independently of PPARβ/δ and PPARα activation, although FSH increases the transcript of PPARγ and in this way, it could affect (mostly increase) aromatase transcript and estradiol production. Probably, this effect of FSH in the estradiol production via PPARγ is only a servo-assist mechanism which if it was inhibited, the estradiol production was not considerably affected.

Male spermatogenesis is a complex biological process that is regulated by hormonal signals from the hypothalamus (GnRH), the pituitary gonadotropins (LH and FSH) and the testis (androgens, inhibin). The two key somatic cell types of the testis, Leydig and Sertoli cells, respond to gonadotropins and androgens and regulate the development and maturation of fertilization competent spermatozoa. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. In order to address this problem we have applied an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the "translatome" (polysome-associated mRNAs) of Leydig or Sertoli cells in vivo. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated. Modulation of a small subset of Sertoli cell genes occurred after FSH and testosterone stimulation. However, Leydig cells responded robustly to gonadotropin deprivation and LH restoration with acute changes in polysome-associated mRNAs. These studies identified the transcription factors that are induced by LH stimulation, uncovered novel potential regulators of LH signaling and steroidogenesis, and demonstrate the effects of LH on the translational machinery in vivo in the Leydig cell.

Notch signaling components have long been detected in Sertoli and germ cells in the developing and mature testis. However, the role of this pathway in testis development and spermatogenesis remains unknown. Using reporter mice expressing green fluorescent protein following Notch receptor activation, we found that Notch signaling was active in Sertoli cells at various fetal, neonatal, and adult stages. Since Notch signaling specifies stem cell fate in many developing and mature organ systems, we hypothesized that maintenance and differentiation of gonocytes and/or spermatogonial stem cells would be modulated through this pathway in Sertoli cells. To this end, we generated mutant mice constitutively expressing the active, intracellular domain of NOTCH1 (NICD1) in Sertoli cells. We found that mutant Sertoli cells were morphologically normal before and after birth, but presented a number of functional changes that drastically affected gonocyte numbers and physiology. We observed aberrant exit of gonocytes from mitotic arrest, migration toward cord periphery, and premature differentiation before birth. These events, presumably unsupported by the cellular microenvironment, were followed by gonocyte apoptosis and near complete disappearance of the gonocytes by day 2 after birth. Molecular analysis demonstrated that these effects are correlated with a dysregulation of Sertoli-expressed genes that are required for germ cell maintenance, such as Cyp26b1 and Gdnf. Taken together, our results demonstrate that Notch signaling is active in Sertoli cells throughout development and that proper regulation of Notch signaling in Sertoli cells is required for the maintenance of gonocytes in an undifferentiated state during fetal development.

Sertoli cells (SCs) possess inherent immunosuppressive properties and are major contributors to the immunoprivileged status of mammalian testis. SCs have been reported to inhibit the activation of B cells, T cells and natural killer cells but not dendritic cells (DCs). Herein, we present evidence that co-culture with SCs results in a persistent state of DC immaturity characterized by down-regulation of the surface molecules I-A/E, CD80, CD83, CD86, CCR7 and CD11c, as well as reduced production of pro-inflammatory cytokines. SC-conditioned DCs (SC-DCs) displayed low immunogenicity and enhanced immunoregulatory functions, including the inhibition of T-cell proliferation and the promotion of Foxp3(+) regulatory T-cell development. Mechanistically, the activation of p38, extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription 3 was suppressed in SC-DCs. More importantly, we demonstrate that galectin-1 secreted by SCs plays a pivotal role in the differentiation of functionally tolerogenic SC-DCs. These findings further support the role of SCs in maintaining the immunoprivileged environment of the testis and provide a novel approach to derive tolerogenic DCs, which may lead to alternative therapeutic strategies for the treatment of immunopathogenic diseases.

Accumulating evidence indicates a key role of Sertoli cell (SC) malfunction in spermatogenesis impairment induced by obesity. Nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) is expressed in SCs, but the role of NLRP3 in the pathological process of obesity-induced male infertility remains unclear.

Epithelial-mesenchymal transition (EMT) is a fundamental process in embryonic development by which sessile epithelial cells are converted into migratory mesenchymal cells. Our laboratory has been successful in the establishment of Xenopus tropicalis immature Sertoli cells (XtiSCs) with the restricted differentiation potential. The aim of this study is the determination of factors responsible for EMT activation in XtiSCs and stemness window acquisition where cells possess the broadest differentiation potential. For this purpose, we tested three potent EMT inducers-GSK-3 inhibitor (CHIR99021), FGF2, and/or TGF-β1 ligand. XtiSCs underwent full EMT after 3-day treatment with CHIR99021 and partial EMT with FGF2 but not with TGF-β1. The morphological change of CHIR-treated XtiSCs to the typical spindle-like cell shape was associated with the upregulation of mesenchymal markers and the downregulation of epithelial markers. Moreover, only CHIR-treated XtiSCs were able to differentiate into chondrocytes in vitro and cardiomyocytes in vivo. Interestingly, EMT-shifted cells could migrate towards cancer cells (HeLa) in vitro and to the injury site in vivo. The results provide a better understanding of signaling pathways underlying the generation of testis-derived stem cells.

Conditional deletion of Gata4 in Sertoli cells (SCs) of adult mice has been shown to increase permeability of the blood-testis barrier (BTB) and disrupt spermatogenesis. To gain insight into the molecular underpinnings of these phenotypic abnormalities, we assessed the impact of Gata4 gene silencing in cell culture models. Microarray hybridization identified genes dysregulated by siRNA-mediated inhibition of Gata4 in TM4 cells, an immortalized mouse SC line. Differentially expressed genes were validated by quantitative RT-PCR analysis of primary cultures of Gata4(flox/flox) mouse SCs that had been subjected to cre-mediated recombination in vitro. Depletion of GATA4 in TM4 cells and primary SCs was associated with altered expression of genes involved in key facets of BTB maintenance, including tight/adherens junction formation (Tjp1, Cldn12, Vcl, Tnc, Csk) and extracellular matrix reorganization (Lamc1, Col4a1, Col4a5, Mmp10, Mmp23, Timp2). Western blotting and immunocytochemistry demonstrated reduced levels of tight junction protein-1, a prototypical tight junction protein, in GATA4-depleted cells. These changes were accompanied by a loss of morphologically recognizable junctional complexes and a decline in epithelial membrane resistance. Furthermore, Gata4 gene silencing was associated with altered expression of Hk1, Gpi1, Pfkp, Pgam1, Gls2, Pdk3, Pkd4, and Ldhb, genes regulating the production of lactate, a key nutrient that SCs provide to developing germ cells. Comprehensive metabolomic profiling demonstrated impaired lactate production in GATA4-deficient SCs. We conclude that GATA4 plays a pivotal role in the regulation of BTB function and lactate metabolism in mouse SCs.

The retinoblastoma protein (RB) is essential for normal cell cycle control. RB function depends, at least in part, on interactions with the E2F family of DNA-binding transcription factors (E2Fs). To study the role of RB in the adult testis, a Sertoli cell (SC)-specific Rb knockout mouse line (SC-RbKO) was generated using the Cre/loxP recombination system. SC-RbKO mice exhibited an age-dependent testicular atrophy, impaired fertility, severe SC dysfunction, and spermatogenic defects. Removal of Rb in SC induced aberrant SC cycling, dedifferentiation, and apoptosis. Here we show that E2F3 is the only E2F expressed in mouse SCs and that RB interacts with E2F3 during mouse testicular development. In the absence of RB, the other retinoblastoma family members p107 and p130 began interacting with E2F3 in the adult testes. In vivo silencing of E2F3 partially restored the SC maturation and survival as well as spermatogenesis in the SC-RbKO mice. These results point to RB as a key regulator of SC function in adult mice and that the RB/E2F3 pathway directs SC maturation, cell cycle quiescence, and RB protects SC from apoptosis.

Dmrt1 has been suggested to play significant roles in sex determination and differentiation, but various expression patterns and cell types have been observed in the testis of vertebrates. Polyploid gibel carp, because of the multiple modes of unisexual gynogenesis and sexual reproduction, has become a unique case to explore the evolution of sex determination and differentiation. However, the sex-determination related genes in gibel carp have remained unknown. In this study, we identified and characterized 4 cDNAs of Dmrt1 genes. Subsequently, a polyclonal antibody specific to CagDMRT1 was prepared to examine its expression and distribution patterns at protein level. Significantly, both relative real-time PCR and Western blot detection confirmed predominant expression of CagDmrt1 in the adult testis of gibel carp. Moreover, the intensive expression of CagDMRT1 around spermatogenic cysts was revealed during spermatogenesis. And, following immunofluorescence co-localization of CagDMRT1 and CagVASA, a prominent CagDMRT1 expression in Sertoli cells and a mild CagDMRT1 expression in spermatogenic cells including spermatogonia and primary spermatocytes were clearly characterized. The CagDMRT1 signal in Sertoli cells is extensively distributed in both nuclei and cytoplasm, while the CagDMRT1 in spermatogonia and primary spermatocytes is mainly expressed in nuclei, and there is only the remained CagDMRT1 signal in the cytoplasm of secondary spermatocytes. These findings suggest that DMRT1 should be related to testis differentiation and spermatogenesis in gibel carp.

Pediatric chemotherapy treatments can impair long-term male fertility. Unfortunately, no fertility preservation solution is available for pre-pubertal boys. Studies suggest that doxorubicin, used against pediatric cancers, induces oxidative stress in the testis. However, the targeted testicular cell types remain unknown. The goal of this study was to determine whether doxorubicin can induce oxidative stress in rat spermatogonia (GC-6Spg) and immature Sertoli (Ser-W3) cell lines, and to assess their protection by antioxidants. Using the MTT assay, we have shown that doxorubicin induces a time- and dose-dependent cytotoxicity in these two cell lines, Ser-W3 being more sensitive than GC-6Spg. After 3 h of treatment, reactive oxygen species and nuclear 8-oxo-deoxyguanosine increase in Ser-W3, but not in GC-6Spg. Moreover, after 6 h of treatment, intracellular reduced glutathione levels decrease significantly in Ser-W3 cells. These results show that doxorubicin induces oxidative stress in the Ser-W3 cell line. However, a depletion in glutathione does not affect their survival, and supplementation only offers a weak protection after exposure to doxorubicin, suggesting that the glutathione system is not essential for Ser-W3 cell line's defense against doxorubicin. On the other hand, among four antioxidants selected from the literature, none reduces the cytotoxicity of doxorubicin in Ser-W3 cells. Together, our data suggest that oxidative stress may not be a major pathway for doxorubicin's cytotoxicity in GC-6Spg and Ser-W3 lines. This study provides new insights in the mechanisms by which chemotherapies affect the pre-pubertal testis, with the long-term goal to help improve the quality of life of pediatric cancer survivors.

Meiosis begins at puberty and relies on several factors, including androgens and retinoic acid in the mouse testis. CYP26B1 degrades retinoic acid in the testis during prenatal development preventing meiosis initiation. Given the concurrence of meiotic entry and completion of Sertoli cell maturation in response to androgens at puberty in the mouse, we proposed that CYP26B1 is downregulated by androgens in the Sertoli cell during this period. By immunohistochemistry, we showed that CYP26B1 declines in Sertoli cells after birth. However, luciferase reporter assays and quantitative reverse transcription-polymerase chain reaction performed in the prepubertal mouse Sertoli cell line SMAT1 revealed no changes in Cyp26b1 expression in response to androgen treatment. Furthermore, studies carried out using primary Sertoli cells of 10-day-old mice showed no changes in either Cyp26b1 or CYP26B1 expression in response to androgen treatment. In summary, the hereby reported decline in CYP26B1 expression in Sertoli cells towards pubertal onset does not appear to be caused by a direct inhibitory effect of androgens on Sertoli cells in the mouse.