BRCA1 establishes DNA damage signaling and pericentric heterochromatin of the X chromosome in male meiosis

During meiosis, DNA damage response (DDR) proteins induce transcriptional silencing of unsynapsed chromatin, including the constitutively unsynapsed XY chromosomes in males. DDR proteins are also implicated in double strand break repair during meiotic recombination. Here, we address the function of the breast cancer susceptibility gene Brca1 in meiotic silencing and recombination in mice. Unlike in somatic cells, in which homologous recombination defects of Brca1 mutants are rescued by 53bp1 deletion, the absence of 53BP1 did not rescue the meiotic failure seen in Brca1 mutant males. Further, BRCA1 promotes amplification and spreading of DDR components, including ATR and TOPBP1, along XY chromosome axes and promotes establishment of pericentric heterochromatin on the X chromosome. We propose that BRCA1-dependent establishment of X-pericentric heterochromatin is critical for XY body morphogenesis and subsequent meiotic progression. In contrast, BRCA1 plays a relatively minor role in meiotic recombination, and female Brca1 mutants are fertile. We infer that the major meiotic role of BRCA1 is to promote the dramatic chromatin changes required for formation and function of the XY body

MEK/ERK signaling directly and indirectly contributes to the cyclical self‐renewal of spermatogonial stem cells

Coordination of stem cell fate is regulated by extrinsic niche signals and stem cell intrinsic factors. In mammalian testes, spermatogonial stem cells maintain constant production of abundant spermatozoa by alternating between self-renewal and differentiation at regular intervals according to a periodical program known as the seminiferous epithelial cycle. Although retinoic acid (RA) signaling has been suggested to direct the cyclical differentiation of spermatogonial stem cells, it remains largely unclear how their cycle-dependent self-renewal/proliferation is regulated. Here, we show that MEK/ERK signaling contributes to the cyclical activity of spermatogonial stem cells. We found that ERK1/2 is periodically activated in Sertoli cells during the stem cell self-renewal/proliferation phase, and that MEK/ERK signaling is required for the stage-related expression of the critical niche factor GDNF. In addition, ERK1/2 is activated in GFRα1-positive spermatogonial stem cells under the control of GDNF and prevent them from being differentiated. These results suggest that MEK/ERK signaling directly and indirectly maintains spermatogonial stem cells by mediating a signal that promotes their periodical self-renewal/proliferation. Conversely, RA signaling directly and indirectly induces differentiation of spermatogonial stem cells. We propose that temporally regulated activations of RA signaling and a signal regulating MEK/ERK antagonistically coordinates the cycle-related activity of spermatogonial stem cells

Regulation of cyclic gene expression change in Sertoli cells during mouse spermatogenesis

　　Mammalian spermatogenesis is a highly organized system that can produce largenumber of spermatozoa continuously. Spermatogenesis takes place within theseminiferous tubules that are composed of germ cells and Sertoli cells that aremorphologically and functionally unique somatic cells specialized to supportspermatogenesis. Sertoli cells directly contact with germ cells, and regulate their properlocalization and release into lumen. They also provide nutrients and growth factorsessential for the differentiation of germ cells and the stem cell maintenance. Furthermore, Sertoli cells play a pivotal role for maintaining the integrity of the seminiferous epithelium by forming tight junction. Consequently, dysfunction of Sertoli cells leads to the disruption of spermatogenesis and male infertility. Therefore, understanding of the Sertoli cell’s function is crucial to clarify the microenvironment for spermatogenesis.　　Spermatogenesis progresses in a spatially and temporally regulated manner, known as spermatogenic wave. During spermatogenesis, germ cells in different developmental stages form groups and synchronously differentiate. In the mouse testis, twelve germ cell groups, known as seminiferous epithelial stages I- XII, can be recognized and they are arranged in order along the tubule length showing the spatial continuity. This cyclical program in spermatogenesis is called “seminiferous epithelial cycle”. One cycle takes 8.6 days in the mouse and 12.9 days in the rat. When rat germ cells are transferred to mouse testis, spermatogenesis proceed with the cycle characteristic of rat germ cells. Therefore it is believed that the cycle length is primarily regulated by germ cells.　　It has been known that Sertoli cells change their gene expression patterns in accordance with the continuous alteration of the epithelial stages. Because heterogeneous gene expression in Sertoli cells was observed even in testes lacking differentiating germ cells, it is suggested that cyclic gene expression in Sertoli cells can occur independently of synchronous spermatogenic differentiation. Furthermore, in the early phase of the first round spermatogenesis when intact seminiferous epithelial cycle was not established, the wave-like expression in Sertoli cells was associated with the timing of spermatogonia differentiation. Growing evidence suggests interesting possibility that Sertoli cells support stage-specific events of spermatogenesis by creating stage-specific microenvironments. 　　It has been suggested that retinoic acid (RA) signaling is involved in the regulation of seminiferous epithelial cycle. In vitamin A-deficient (VAD) mice, spermatogenesis was arrested at spermatogonia stage, and the injection of vitamin A (retinol) or RA into VAD mice triggers initiation of synchronized spermatogenesis in all seminiferous tubules. Moreover, it was reported that Sertoli cell-specific deletion of RARα resulted in disruption of the stage-dependent gene expression in Sertoli cells. These evidences suggest the involvement of RA signaling in the stage-dependent expression of Sertoli cells. However, it remains elusive how RA signaling controls the periodicity of Sertoli cells and whether other signaling pathway(s) is involved in the regulation. Furthermore, importance of periodicity in Sertoli cells for spermatogenesis is also unknown. 　　In part I, to understand the periodicity of Sertoli cells, I performed comprehensive analysis of stage-dependent gene expression in Sertoli cells by using microarray, and identified 419 stage-dependent genes. In part II, I investigated implication of Notch signaling in Sertoli cells, because the list of stage-dependent genes contained Notch1 receptor. I found that the Notch ligand, Jagged2 was expressed in germ cells, and Notch1 receptor was expressed and activated in Sertoli cells in stage-dependent manner. To examine the involvement of Notch signaling in periodicity of Sertoli cells and spermatogenesis, I inactivated Notch signaling in Sertoli cells by using cre-loxP system.However, my data demonstrated that genetic ablation of Notch signaling in Sertoli cells did not affect spermatogenesis nor stage-dependent gene expression in Sertoli cells as long as I examined, suggesting that Notch signaling in Sertoli cells is dispensable for mouse spermatogenesis and the stage-dependent expression. 　　In part III, I studied relationship between stage-dependent gene expression in Sertoli cells and RA signaling. I found that RA signaling was periodically activated in stage VII-XII seminiferous tubules, and stage-dependent genes showing peak at stage I-VI and VII-XII tended to be suppressed and activated by RA signaling, respectively. To examine the significance of the periodicity in Sertoli cells for spermatogenesis, I disrupted stage-dependent gene expression in Sertoli cells by means of lentivirus mediated suppression of RA signaling, and found it led to stage-specific defects in germ cell differentiation, delayed recovery of tight junction component and abnormal morphology of Sertoli cells. Taken together, the stage-dependent gene expression in Sertoli cells is primarily regulated by periodic activation of RA signaling, and is important for stage-specific events of spermatogenesis

Poised chromatin and bivalent domains facilitate the mitosis-to-meiosis transition in the male germline

BackgroundThe male germline transcriptome changes dramatically during the mitosis-to-meiosis transition to activate late spermatogenesis genes and to transiently suppress genes commonly expressed in somatic lineages and spermatogenesis progenitor cells, termed somatic/progenitor genes.ResultsThese changes reflect epigenetic regulation. Induction of late spermatogenesis genes during spermatogenesis is facilitated by poised chromatin established in the stem cell phases of spermatogonia, whereas silencing of somatic/progenitor genes during meiosis and postmeiosis is associated with formation of bivalent domains which also allows the recovery of the somatic/progenitor program after fertilization. Importantly, during spermatogenesis mechanisms of epigenetic regulation on sex chromosomes are different from autosomes: X-linked somatic/progenitor genes are suppressed by meiotic sex chromosome inactivation without deposition of H3K27me3.ConclusionsOur results suggest that bivalent H3K27me3 and H3K4me2/3 domains are not limited to developmental promoters (which maintain bivalent domains that are silent throughout the reproductive cycle), but also underlie reversible silencing of somatic/progenitor genes during the mitosis-to-meiosis transition in late spermatogenesis

Purification of GFRα1+ and GFRα1– Spermatogonial Stem Cells Reveals a Niche-Dependent Mechanism for Fate Determination

Summary: Undifferentiated spermatogonia comprise a pool of stem cells and progenitor cells that show heterogeneous expression of markers, including the cell surface receptor GFRα1. Technical challenges in isolation of GFRα1+ versus GFRα1– undifferentiated spermatogonia have precluded the comparative molecular characterization of these subpopulations and their functional evaluation as stem cells. Here, we develop a method to purify these subpopulations by fluorescence-activated cell sorting and show that GFRα1+ and GFRα1– undifferentiated spermatogonia both demonstrate elevated transplantation activity, while differing principally in receptor tyrosine kinase signaling and cell cycle. We identify the cell surface molecule melanocyte cell adhesion molecule (MCAM) as differentially expressed in these populations and show that antibodies to MCAM allow isolation of highly enriched populations of GFRα1+ and GFRα1– spermatogonia from adult, wild-type mice. In germ cell culture, GFRα1– cells upregulate MCAM expression in response to glial cell line-derived neurotrophic factor (GDNF)/fibroblast growth factor (FGF) stimulation. In transplanted hosts, GFRα1– spermatogonia yield GFRα1+ spermatogonia and restore spermatogenesis, albeit at lower rates than their GFRα1+ counterparts. Together, these data provide support for a model of a stem cell pool in which the GFRα1+ and GFRα1– cells are closely related but show key cell-intrinsic differences and can interconvert between the two states based, in part, on access to niche factors. : In this article, Garbuzov and colleagues devise a new strategy for isolating pure populations of GFRα1+ and GFRα1– undifferentiated spermatogonia from adult testis of TertTomato reporter mice based on expression of telomerase and GFRα1. Transcriptional profiling showed a remarkable similarity between GFRα1+ and GFRα1– cells, and both populations showed elevated stem cell activity by transplantation. Keywords: spermatogonial stem cells, germ cells, telomerase, germ line, stem cells, niche, transplantation, RNA-seq, FAC