Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation

It is well established that the c-kit gene plays an essential role in the proliferation of differentiating spermatogonia in prepuberal mice. However, the mechanisms that regulate the onset of spermatogenesis, i.e. differentiation of spermatogonial stem cells and c-kit expression, are poorly understood. Here we identify a novel signal transduction system in mouse prepuberal testis regulating this developmental event, involving bone morphogenetic protein 4 (BMP4) and its transduction machinery. BMP4 is produced by Sertoli cells very early in the postnatal life and is successively down regulated in peri-puberal Sertoli cells. Its receptor Alk3 and the R-Smad Smad5 are specifically expressed both in proliferating primordial germ cells and in postnatal spermatogonia. BMP4 stimulation of cultured spermatogonia induces Smad4/5 nuclear translocation and the formation of a DNA-binding complex with the transcriptional coactivator p300/CBP. In vitro exposure of undifferentiated spermatogonia to BMP4 exerts both mitogenic and differentiative effects, inducing [3H]thymidine incorporation and Kit expression. As a result of the latter event, Kit-negative spermatogonia acquire sensitivity to Stem Cell Factor

UV and genotoxic stress induce ATR relocalization in mouse spermatocytes

During meiosis, phosphorylation of H2AX is one of the earliest cellular responses to the generation of DNA double-strand breaks (DSBs) by the SPO11 topoisomerase. ATM is the kinase which mediates the formation of phosphorylated H2AX (H2AX) meiotic foci, while ATR is the kinase which signals chromosome asynapsis at the level of the XY bivalent. To investigate the possible role of ATR also in DNA damage signalling in meiotic cells, we studied the effect of UV radiation and chemotherapy drugs on H2AX phosphorylation and ATR relocalization in mouse pachytene spermatocytes. Here, we report that UV, a single strand break DNA-damaging agent, induces ATR relocalization from the XY sex body to nuclear foci and intense H2AX phosphorylation. Other DNA damage proteins such as MDC1, NBS1 and 53BP1 showed a similar relocalization following UVA microirradiation of spermatocytes. We found that DNA damage induced by UV increased the intensity and the number of H2AX foci also in Atm null spermatocytes. Inhibition of RNA synthesis was found to induce the formation of H2AX foci, but it did not influence the DNA damage response to UV irradiation. Finally, exposure of spermatocytes to double strand break DNA-damaging agents such as cisplatin, bleomycin or etoposide also induced ATR relocalization and intense H2AX phosphorylation and led to anomalies in synaptonemal assembly. Our results demonstrate that DNA damage induced by genotoxic stress can activate ATR and influence meiotic chromatin remodelling through H2AX phosphorylation, likely as part of a response which normally ensures germ cell genomic integrity

Transcriptome analysis of differentiating spermatogonia stimulated with kit ligand

Kit ligand (KL) is a survival factor and a mitogenic stimulus for differentiating spermatogonia. However, it is not known whether KL also plays a role in the differentiative events that lead to meiotic entry of these cells. We performed a wide genome analysis of difference in gene expression induced by treatment with KL of spermatogonia from 7-day-old mice, using gene chips spanning the whole mouse genome. The analysis revealed that the pattern of RNA expression induced by KL is compatible with the qualitative changes of the cell cycle that occur during the subsequent cell divisions in type A and B spermatogonia, i.e. the progressive lengthening of the S phase and the shortening of the G2/M transition. Moreover, KL up-regulates in differentiating spermatogonia the expression of early meiotic genes (for instance: Lhx8, Nek1, Rnf141, Xrcc3, Tpo1, Tbca, Xrcc2, Mesp1, Phf7, Rtel1), whereas it down-regulates typical spermatogonial markers (for instance: Pole, Ptgs2, Zfpm2, Egr2, Egr3, Gsk3b, Hnrpa1, Fst, Ptch2). Since KL modifies the expression of several genes known to be up-regulated or down-regulated in spermatogonia during the transition from the mitotic to the meiotic cell cycle, these results are consistent with a role of the KL/kit interaction in the induction of their meiotic differentiation

SOHLH1 and SOHLH2 control Kit expression during postnatal male germ cell development

How Kit expression is regulated in the germline remains unknown. SOHLH1 and SOHLH2, two bHLH transcription factors specifically expressed in germ cells, are involved in spermatogonia and oocyte differentiation. In the male, deletion of each factor causes loss of Kit-expressing spermatogonia in the prepuberal testis. In the female, SOHLH1 and SOHLH2 ablations cause oocyte loss in the neonatal ovary. To investigate whether Kit expression is regulated by these two factors in male germ cells, we examined SOHLH1 and SOHLH2 expression during fetal and postnatal mouse development. We found a strong positive correlation between Kit and the two transcription factors only in postnatal spermatogonia. SOHLH2 was enriched in undifferentiated spermatogonia, whereas SOHLH1 expression was maximal at Kit-dependent stages. Expression of SOHLH1, but not SOHLH2, was increased in postnatal mitotic germ cells by treatment with all-trans retinoic acid. We found that E-box sequences within the Kit promoter and its first intron can be transactivated in transfection experiments overexpressing Sohlh1 or Sohlh2. Co-transfection of both factors showed a cooperative effect. EMSA experiments showed that SOHLH1 and SOHLH2 can independently and cooperatively bind an E-box-containing probe. In vivo co-immunoprecipitations indicated that the two proteins interact and overexpression of both factors increases endogenous Kit expression in embryonic stem cells. SOHLH1 was found by ChIP analysis to occupy an E-box-containing region within the Kit promoter in spermatogonia chromatin. Our results suggest that SOHLH1 and SOHLH2 directly stimulate Kit transcription in postnatal spermatogonia, thus activating the signaling involved in spermatogonia differentiation and spermatogenetic progression

Leukemia inhibitory factor sustains the survival of mouse primordial germ cells cultured on TM4 feeder layers

Various growth factors and cytokines were tested for their effects on survival and proliferation of mouse primordial germ cells (PGCs) cultured on TM4 cell feeder layers. Leukemia inhibitory factor was able to sustain the survival of PGCs from 10.5 dpc embryos for at least 3 days and to slow down degeneration of PGCs from 11.5 dpc embryos cultured on TM4 feeder layers

In or out stemness: comparing growth factor signalling in mouse embryonic stem cells and primordial germ cells

Embryonic stem (ES) cells do not exist in nature but, usually produced from the inner cell mass (ICM) of the blastocyst, are considered equivalent to ICM cells captured during a short period of transient self-renewal and pluripotency capability. Although, artificial, ES cells represent a formidable model to investigate fundamental aspects of cell stemness and early embryo development. ES cells are indeed the only stem cell type able to indefinite self-renewal and to differentiate into cellular derivates of ectodermal, mesodermal and endodermal lineages. Recent extensive studies have revealed that ES cells maintain self-renewal and pluripotency because of a self-organizing network of transcription factors and intracellular pathways activated by extracellular signalling that together prevent their differentiation and promote their proliferation, and because of epigenetic processes that maintain the chromatin in a plastic differentiation status. Primordial germ cells (PGCs), the embryonic precursors of gametes, because of their unique ability to retain true developmental totipotency, are considered the mother of all stem cells. Despite several similarities with ES cells, they display only transient self-renewal capability and distinct lineage-specific characteristics. In fact, in normal condition PGCs are believed to differentiate into germ cells only, oogonia/oocytes in the female, and prospermatogonia in the male which ultimately produce eggs and sperm, respectively. It is not until the fertilization of the egg or parthenogenesis that the intrinsic germ cell totipotency program is revealed. Many aspects of the extrinsic factors and signalling required for ES cell self-renewal and pluripotency have been identified and dissected. On the other hand, several extrinsic factors controlling PGC development have been identified, but the underlying molecular signalling remains little defined. In the present review, by comparing the available information about signalling elicited by four growth factors such as leukaemia inhibitory factor (LIF), bone morphogenic protein 4 (BMP4), fibroblast growth factor 2 (FGF2) and kit ligand (KL) in mouse ES cells and PGCs, on which most of such studies have been performed, we aimed to give clues for the molecular understanding of the similarities and differences between these two unique cell types and to explain how apparent contradictory properties such as lineage-specific characteristics and pluripotency may coexist within PGCs. The first two growth factors have been demonstrated to control key aspects of the self-renewal and pluripotency of ES cells. BMP4 and KL are known for their crucial role in regulating various process of PGC development in the embryo from the formation of PGC precursors and PGC specification (BMP4) to their survival, proliferation and migration (KL). Moreover, the combined action of LIF, FGF2 and KL is necessary and sufficient for PGC transformation into ES-like cells termed embryonic germ (EG) cells

Developmental regulation of the thyroid hormone receptor alpha 1 mRNA expression in the rat testis

The multiplicity of thyroid hormone (TH) effects appears to be mediated by two TH receptors (THRs) encoded by two genes, alpha and beta, and, perhaps, by their various isoforms. The expression of THR beta is correlated with the presence of high affinity binding sites for TH, and all the mutations which cause the syndrome of generalized thyroid hormone resistance occur in THR beta. The function of THR alpha has not been clearly defined as yet. Another enigma in TH action is the effect on the testis. It has been shown that the testis of the adult rat does not respond to TH as measured by an increase in oxygen consumption. Furthermore, it has not been possible to demonstrate the presence of a nuclear high affinity binding site for TH in adult testis. To resolve these problems were measured the levels of THR alpha, its nonhormone binding variant, and THR beta mRNA in the testis at various stages of development. We discovered that the beta-message is absent at all times, whereas the alpha-message is expressed only from fetal through prepubertal stages and is absent in adult testis. THR alpha, but not the beta-mRNA, was detected in immature Sertoli cells in culture, and neither was found in adult Sertoli cell-enriched cultures. Furthermore, THR alpha and its variant mRNA was found, using in situ hybridization, in the seminiferous cords and seminiferous tubules of fetal and prepubertal testis.(ABSTRACT TRUNCATED AT 250 WORDS

Molecular mechanisms utilized by alternative c-kit gene products in the control of spermatogonial proliferation and sperm-mediated egg activation

The c-kit proto-oncogene plays a dual role in the control of male fertility in mice through two alternative gene products: (1). c-kit [the transmembrane tyrosine kinase receptor for stem cell factor (SCF)], which is expressed and functional in differentiating spermatogonia of the postnatal testis, in which c-kit is essential for pre-meiotic proliferation; and (2). tr-kit, an intracellular protein which is specifically accumulated during spermiogenesis through the use of an alternative intronic promoter, and which is able to trigger mouse egg activation when microinjected into the cytoplasm of metaphase II arrested oocytes. Here, we summarize the most recent findings about the molecular pathways through which c-kit regulates cell cycle progression in mitotic germ cells, and those through which sperm-derived tr-kit triggers parthenogenetic completion of meiosis II and pronuclear formation in microinjected mouse eggs

Putative second messengers affect cell coupling in the seminiferous tubules

Junctional transfer of ions in monolayer primary cultures of Sertoli cells and in intact seminiferous tubules from 20 day-old rats has been investigated by using electrophysiological techniques. The electrotonic coupling of both intact tubule and monolayer culture was inhibited by the presence of dibutyryl cyclic AMP (10(-4) M) in the medium. In contrast, 1-oleoyl-2-acetylglycerol (10(-5) M) and 12-O-tetradecanoyl-phorbol-13-acetate (10(-7) M) decreased the junctional resistivity and increased the extent of the coupling in immature tubules. The significance of this modulation of the cell coupling in the seminiferous epithelium is discussed

SOHLH1 and SOHLH2 directly down-regulate Stimulated by Retinoic Acid 8 (STRA8) expression.

As the name implies, Stimulated by Retinoic Acid 8 is an early retinoic acid (RA) responsive gene pivotal for the beginning of meiosis in female and male germ cells. Its expression is strictly time-dependent and cell-specific (premeiotic germ cells) and likely requires a complex mechanism of regulation. In this study, we demonstrate a direct negative control of SOHLH1 and SOHLH2, 2 germ cell specific bHLH transcription factors, on Stra8 expression. We observed a negative correlation between STRA8 and SOHLH1 expression in prepuberal differentiating mouse KIT+ spermatogonia and found that SOHLH1 and SOHLH2 were able to directly and cooperatively repress STRA8 expression in cell lines in vitro through binding to its promoter. We also identified 2 canonical E-Box motives in the Stra8 promoter that mediated the negative regulation of SOHLH1 and SOHLH2 on these gene both in the cell lines and KIT+ spermatogonia. We hypothesize that this novel negative activity of SOHLH1and SOHLH2 in male cooperates with that of other transcription factors to coordinate spermatogonia differentiation and the RA-induced meiosis and in female ensures STRA8 down-regulation at mid-end stages of meiotic prophase I