Cytoplasmic retention of the sex-determining factor SOX9 via the microtubule network

SOX9 is a sex-determining factor which induces Sertoli cell differentiation and subsequent testis cord formation. It is expressed both in male and female undifferentiated gonads in the cytoplasmic compartment of pre-Sertoli cells. At the time of sexual differentiation, SOX9 moves into the nucleus of male pre-Sertoli cells whereas in female, it remains in the cytoplasm and then its expression decreases. To study the cytoplasmic localization of SOX9, we have analyzed its interaction with the cytoskeleton components. By treatment of NT2/D1 and transfected NIH3T3 cell lines and embryonic gonads with nocodazole, a drug depolymerizing the microtubules, we show that cytoplasmic retention of SOX9 requires the integrity of the microtubule network. Using biochemical experiments, we demonstrated that SOX9 is able to interact with microtubules in vitro and in vivo. On the other hand, we observed a complete male-specific reorganization of the microtubule network in epithelial Sertoli cells of the male embryonic gonad at the time of sexual differentiation and testis cord formation.     

SOX9 regulates prostaglandin D synthase gene transcription in vivo to ensure testis development

In mammals, male sex is determined by the Y-chromosomal gene Sry (sex-determining region of Y chromosome). The expression of Sry and subsequently Sox9 (SRY box containing gene 9) in precursors of the supporting cell lineage results in the differentiation of these cells into Sertoli cells. Sertoli cells in turn orchestrate the development of all other male-specific cell types. To ensure that Sertoli cells differentiate in sufficient numbers to induce normal testis development, the early testis produces prostaglandin D(2) (PGD(2)), which recruits cells of the supporting cell lineage to a Sertoli cell fate. Here we show that the gene encoding prostaglandin D synthase (Pgds), the enzyme that produces PGD(2), is expressed in Sertoli cells immediately after the onset of Sox9 expression. Promoter analysis in silico and in vitro identified a paired SOX/SRY binding site. Interestingly, only SOX9, and not SRY, was able to bind as a dimer to this site and transactivate the Pgds promoter. In line with this, a transgenic mouse model showed that Pgds expression is not affected by ectopic Sry expression. Finally, chromatin immunoprecipitation proved that SOX9 but not SRY binds to the Pgds promoter in vivo.     

Fgf9 induces proliferation and nuclear localization of FGFR2 in Sertoli precursors during male sex determination

Recently, we demonstrated that loss of Fgf9 results in a block of testis development and a male to female sex-reversed phenotype; however, the function of Fgf9 in sex determination was unknown. We now show that Fgf9 is necessary for two steps of testis development just downstream of the male sex-determining gene, Sry: (1) for the proliferation of a population of cells that give rise to Sertoli progenitors; and (2) for the nuclear localization of an FGF receptor (FGFR2) in Sertoli cell precursors. The nuclear localization of FGFR2 coincides with the initiation of Sry expression and the nuclear localization of SOX9 during the early differentiation of Sertoli cells and the determination of male fate.     

The p38 MAPK pathway mediates interleukin-1-induced Sertoli cell proliferation

We have reported earlier that interleukin-1 (IL-1) is a potent growth factor for immature Sertoli cells (somatic cells in the testis required for testicular development and later spermatogenesis) and that this effect is synergistic with the mitogenic effect of follicle-stimulating hormone (FSH). The aim of the present study was to determine whether MAPK pathways are involved in mediating the mitogenic effect of IL-1 on Sertoli cells. Western blotting revealed that IL-1alpha activated p38 MAPK and JNK/SAPK, but not ERK, in Sertoli cells from 8- or 9-day-old rat. The inhibitor of p38 MAPK SB203580 attenuated the IL-1alpha-induced proliferation of Sertoli cells, as assessed by (3)H-thymidine incorporation and supravital staining as well as by direct cell counting. We conclude that the p38 MAPK pathway mediates the proliferative effect of IL-1alpha on immature Sertoli cells in vitro. Since the mitogenic effect of FSH is mediated via ERK, the synergistic action of IL-1alpha and FSH may be explained by their different intracellular signalling pathways. Induction of IL-1 by inflammation, infection or other tissue injuries may result in testicular damage by interfering with normal Sertoli cell development and thus future spermatogenesis.     

Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule- associated protein (cytoplasmic dynein)

Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.     

Global Genome Analysis of the Downstream Binding Targets of Testis Determining Factor SRY and SOX9

A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination.     

Sox9-related signaling controls zebrafish juvenile ovary–testis transformation

In almost all vertebrates, the downstream of the sox9 signaling axis is well conserved for testis differentiation. The upstream genes of this pathway vary from species to species during evolution. Yet, little is known about how these signaling cascades are regulated and what cellular processes are dominant in ovary–testis transformation in juvenile zebrafish. In this study, we find that the transforming gonads undergo activation of sox9a-expressing stromal cells with increased deposition of extracellular matrix and formation of degenerative compartments. This leads to follicle disassembly, oocyte degeneration, follicle cell-cyp19a1a-amh conversions, and, eventually, formation of the testis cord. In vitro primary culture of juvenile ovary tissue in gonadotropins increases cytoplasmic accumulation of sox9a and p-Erk1/2, and induces mesenchymal morphology. MAPK inhibitors (MKI), a mixture of PD98059 and U0216, eliminate the cytoplasmic distribution but do not eradicate nuclear localization of sox9a and p-Erk1/2. Nuclear p53 are relatively increased in MKI-treated cells that exhibit less spreading and reduced proliferation. Despite uniform nuclear condensation, only a fraction of cells displayed the apoptotic phenotype. These results suggest that high levels of cytoplasmic sox9a and p-Erk1/2 activity activate stromal cells and enhance the production of extracellular matrix required for testis cord formation, whereas deregulation and translocation of sox9a and p-Erk1/2 induce follicle disassembly and incomplete apoptosis associated with nuclear p53. Together with the established FSH/cAMP/MAPK/AMH pathway in mammalian granulosa and Sertoli cells, we demonstrated that the sox9 axis signaling that determines testis formation in mammals also induces zebrafish ovary–testis transition, and adds to its conserved role in sex reversal.     

Antagonism of the testis- and ovary-determining pathways during ovotestis development in mice

Ovotestis development in B6-XY^POS mice provides a rare opportunity to study the interaction of the testis- and ovary-determining pathways in the same tissue. We studied expression of several markers of mouse fetal testis (SRY, SOX9) or ovary (FOXL2, Rspo1) development in B6-XY^POS ovotestes by immunofluorescence, using normal testes and ovaries as controls. In ovotestes, SOX9 was expressed only in the central region where SRY is expressed earliest, resulting in testis cord formation. Surprisingly, FOXL2-expressing cells also were found in this region, but individual cells expressed either FOXL2 or SOX9, not both. At the poles, even though SOX9 was not up-regulated, SRY expression was down-regulated normally as in XY testes, and FOXL2 was expressed from an early stage, demonstrating ovarian differentiation in these areas. Our data (1) show that SRY must act within a specific developmental window to activate Sox9; (2) challenge the established view that SOX9 is responsible for down-regulating Sry expression; (3) disprove the concept that testicular and ovarian cells occupy discrete domains in ovotestes; and (4) suggest that FOXL2 is actively suppressed in Sertoli cell precursors by the action of SOX9. Together these findings provide important new insights into the molecular regulation of testis and ovary development.     

Microtubule polarity in Sertoli cells: a model for microtubule-based spermatid transport

Bundles of microtubules occur adjacent to ectoplasmic specializations (ESs) that line Sertoli cell crypts and support developing spermatids. These microtubules are oriented parallel to the direction of spermatid movement during spermatogenesis. We propose a model in which ESs function as vehicles, and microtubules as tracks, for microtubule-based transport of spermatids through the seminiferous epithelium. Microtubule polarity provides the basis for the direction of force generation by available mechanoenzymes. As part of a more general study designed to investigate the potential role of microtubule-based transport during spermatogenesis, we have studied the polarity of cytoplasmic microtubules of Sertoli cells. Rat testis blocks were incubated in a lysis/decoration buffer, with and without exogenous purified bovine brain tubulin. This treatment results in the decoration of endogenous microtubules with curved tubulin protofilament sheets (seen as hooks in cross section). The direction of curvature of the hooks indicates microtubule polarity; that is, clockwise hooks are seen when viewing microtubules from the plus to the minus end. We found that, in Sertoli cells, most of the hooks were orientated in the same direction. Significantly, when viewed from the base of the epithelium, hooks pointed in a clockwise direction. The clockwise direction of dynein arms on axonemes of sperm tails, in the same section, provided an internal check of the section orientation. Electron micrographs of fields of seminiferous epithelium were assembled into montages for quantitative analysis of microtubule polarity. Our data indicate that Sertoli cell cytoplasmic microtubules are of uniform polarity and are orientated with their minus ends toward the cell periphery. These observations have significant implications for our proposed model of microtubule-based transport of spermatids through the seminiferous epithelium.     

Distribution of the microtubule-dependent motors cytoplasmic dynein and kinesin in rat testis.

To examine the possible role of microtubule-based transport in testicular function, we used immunofluorescent techniques to study the presence and localization of the microtubule mechanoenzymes cytoplasmic dynein (a slow-growing end-directed motor) and kinesin (a fast-growing end-directed motor) within rat testis. Cytoplasmic dynein immunofluorescence was observed in Sertoli cells during all stages of spermatogenesis, with a peak in apical cytoplasm during stages IX-XIV. Cytoplasmic dynein immunofluorescence was also localized within Sertoli cells to steps 9-14 (stages IX-XIV) germ cell-associated ectoplasmic specializations. In germ cells, cytoplasmic dynein immunofluorescence was observed in manchettes of steps 15-17 (stages I-IV) spermatids, and small, hollow circular structures were seen in the cytoplasm of step 17 and step 18 spermatids during stages V and VI. Kinesin immunofluorescence was observed in manchettes of steps 10-18 spermatids (stages X-VI). The stage-dependent apical Sertoli cell cytoplasmic dynein immunofluorescence, in conjunction with the previously reported orientation of Sertoli cell microtubules (slow-growing ends toward the lumen) and peak secretion of androgen-binding protein and transferrin, is consistent with the hypothesis that cytoplasmic dynein is involved in Sertoli cell protein transport and secretion. Further, the localization of cytoplasmic dynein and kinesin to manchettes is consistent with current hypotheses concerning manchette function.     

Follicle-stimulating hormone-induced aromatase in immature rat Sertoli cells requires an active phosphatidylinositol 3-kinase pathway and is inhibited via the mitogen-activated protein kinase signaling pathway

Postnatal development and function of testicular Sertoli cells are regulated primarily by FSH. During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development. Growth factors like epidermal growth factor (EGF) are produced in the testis and play a role in regulation of estradiol production and male fertility. Although these divergent factors modulate gonadal function, little is known about their mechanism of action in Sertoli cells. The present study investigates the intracellular events that take place down-stream of FSH and EGF receptors in Sertoli cells isolated from immature (10-d-old) rats, and examines which intracellular signals may be involved in their effects on aromatase activity and estradiol production in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were treated with FSH in combination with EGF and signaling pathway-specific inhibitors. Levels of estradiol production, aromatase mRNA (Cyp19a1), and aromatase protein (CYP19A1) were determined. Western blot analysis was performed to determine the effects of FSH and EGF on levels of activated (phosphorylated) AKT1 and p42 ERK2 and p44 ERK1, also named MAPK1 and MAPK3, respectively. The stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production were blocked by inhibition of the phosphatidylinositol 3-kinase/AKT1 signaling pathway. In contrast, inhibition of ERK signaling augmented the stimulatory effects of FSH on estradiol production, aromatase mRNA, and protein levels. Furthermore, EGF inhibited the expression of aromatase mRNA and protein in response to FSH, and these inhibitory effects of EGF were critically dependent on the activation of the ERK signaling pathway. We conclude that an active phosphatidylinositol 3-kinase /AKT signaling pathway is required for the stimulatory actions of FSH, whereas an active ERK/MAPK pathway inhibits estradiol production and aromatase expression in immature Sertoli cells.     

The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules

Sertoli cells of testis belong to a unique type of polarized epithelial cells and are essential for spermatogenesis. They form the blood-testis barrier at the base of seminiferous tubule. Their numerous long, microtubule-rich processes extend inward and associate with developing germ cells to sustain germ cell growth and differentiation. How Sertoli cells develop and maintain their elaborate processes has been an intriguing question. Here we showed that, by microinjecting lentiviral preparations into mouse testes of 29 days postpartum, we were able to specifically label individual Sertoli cells with GFP, thus achieving a clear view of their natural configurations together with associated germ cells in situ. Moreover, compared to other microtubule plus end-tracking proteins such as CLIP-170 and p150(Glued), EB1 was highly expressed in Sertoli cells and located along microtubule bundles in Sertoli cell processes. Stable overexpression of a GFP-tagged dominant-negative EB1 mutant disrupted microtubule organizations in cultured Sertoli cells. Furthermore, its overexpression in testis Sertoli cells altered their shapes. Sertoli cells in situ became rod-like, with decreased basal and lateral cell processes. Seminiferous tubule circularity and germ cell number were also reduced. These data indicate a requirement of proper microtubule arrays for Sertoli cell plasticity and function in testis.