Histone methyltransferase DOT1L is essential for self-renewal of germline stem cells.

Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at HoxC9 and HoxC10 genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.

Long-Term Ex Vivo Expansion of Murine Spermatogonial Stem Cells in a Simple Serum-Free Medium.

Spermatogonial stem cells (SSCs) possess both self-renewal and differentiation abilities to sustain lifelong production of enormous numbers of spermatozoa in males. SSCs hold a unique position among tissue-specific stem cells in adults because of their ability to transmit the genetic information to subsequent generations. Ex vivo expansion of SSCs in conjunction with their transplantation is highly invaluable to study SSCs and develop new reproductive technologies for therapeutic applications. In this chapter, we describe a culture system involving a simple serum-free medium for mouse SSCs. Elimination of the serum from the culture is important to enhance the effects of exogenous factors, which are rather masked by the serum, and to avert the serum-induced inflammatory responses of testicular mesenchymal cells, which cause adverse effects on SSC proliferation. Consequently, using this culture system has proven for the first time that glial cell line-derived neurotrophic factor (GDNF) was found to be the key factor to drive the self-renewing proliferation of SSCs, and fibroblast growth factor 2 enhanced the GDNF-dependent proliferation of SSCs. Besides determining these two key cytokines, the simplicity of the system enabled individual modification of its components to develop long-term cultures of rat and rabbit SSCs. The basics of these culture systems will enable development of the culture conditions for human and other mammalian SSCs in the near future.

Conserved and non-conserved characteristics of porcine glial cell line-derived neurotrophic factor expressed in the testis.

Glial cell line-derived neurotrophic factor (GDNF) is essential for the self-renewal and proliferation of spermatogonial stem cells (SSCs) in mice, rats, and rabbits. Although the key extrinsic factors essential for spermatogonial proliferation in other mammals have not been determined, GDNF is one of the potential candidates. In this study, we isolated porcine GDNF (pGDNF) cDNAs from neonatal testis and generated recombinant pGDNF to investigate its biological activity on gonocytes/undifferentiated spermatogonia, including SSCs. In porcine testis, long and short forms of GDNF transcripts, the counterparts of pre-(α)pro and pre-(ß)pro GDNF identified in humans and rodents, were expressed. The two transcripts encode identical mature proteins. Recombinant pGDNF supported proliferation of murine SSCs in culture, and their stem cell activity was confirmed by a transplantation assay. Subsequently, porcine gonocytes/undifferentiated spermatogonia were cultured with pGDNF; however, pGDNF did not affect their proliferation. Furthermore, GDNF expression was localised to the vascular smooth muscle cells, and its cognate receptor GFRA1 expression was negligible during spermatogonial proliferation in the testes. These results indicate that although pGDNF retains structural similarity with those of other mammals and conserves the biological activity on the self-renewal of murine SSCs, porcine SSCs likely require extrinsic factors other than GDNF for their proliferation.

Cell-surface DEAD-box polypeptide 4-immunoreactive cells and gonocytes are two distinct populations in postnatal porcine testes.

DEAD-box polypeptide 4 (DDX4) is an evolutionally conserved ATP-dependent RNA helicase that is exclusively expressed in germ cell lineage. Although DDX4 is believed to reside and function in the cytoplasm, recent studies in mice and humans suggest that its epitope is expressed on the cell surface of a small subpopulation in the ovary, putative oogonial stem cells. No study has examined whether such cell-surface DDX4(+) cells exist in the testes of any species. In this study, we explored cell-surface DDX4(+) cells in postnatal porcine testes before the onset of spermatogenesis, where gonocytes, which are the precursors of spermatogonial stem cells, are the only germ cell population. Transfection experiments demonstrated that recombinant porcine DDX4 can be expressed on the cell surface, and cell-surface DDX4-immunoreactive cells were identified in the testis by flow cytometry. Although the DDX4-expressing cells identified in the testis were indeed gonocytes, the cell-surface DDX4-immunoreactive cells expressed negligible DDX4 mRNA and protein levels. Furthermore, they did not express other germ cell markers, such as ZBTB16, NANOS2, and DAZL, but prominently expressed early primordial germ cell markers, such as PRDM1, IFITM3, and EPCAM. Nonetheless, the cell-surface DDX4-immunoreactive cells generated neither germ cell colonies nor teratomas following transplantation into immunocompromised mouse testes. Taken together, these results demonstrate that testicular cell-surface DDX4-immunoreactive cells are not germ cells and constitute a distinct subpopulation that is different from gonocytes. Moreover, the subpopulation in porcine testes might be species specific because no DDX4-immunoreactive cells were found in postnatal mouse testes.

Proteomic analysis of in vivo 14-3-3 interactions in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae produces two 14-3-3 proteins, Bmh1 and Bmh2, whose exact functions have remained unclear. Here, we performed a comprehensive proteomic analysis using multistep immunoaffinity purification and mass spectrometry and identified 271 yeast proteins that specifically bind to Bmh1 and -2 in a phosphorylation-dependent manner. The identified proteins have diverse biochemical functions and cellular roles, including cell signaling, metabolism, and cell cycle regulation. Importantly, there are a number of protein subsets that are involved in the regulation of yeast physiology through a variety of cell signaling pathways, including stress-induced transcription, cell division, and chitin synthesis at the cell wall. In fact, we found that a yeast mutant deficient in Bmh1 and -2 had defects in signal-dependent response of the MAPK (Hog1 and Mpk1) cascade and exhibited an abnormal accumulation of chitin at the bud neck. We propose that Bmh1 and -2 are common regulators of many cell signaling modules and pathways mediated by protein phosphorylation and regulate a variety of biological events by coordinately controlling the identified multiplex phosphoprotein components.

14-3-3 proteins modulate the expression of epithelial Na+ channels by phosphorylation-dependent interaction with Nedd4-2 ubiquitin ligase.

The ubiquitin E3 protein ligase Nedd4-2 is a physiological regulator of the epithelial sodium channel ENaC, which is essential for transepithelial Na+ transport and is linked to Liddle's syndrome, an autosomal dominant disorder of human salt-sensitive hypertension. Nedd4-2 function is negatively regulated by phosphorylation via a serum- and glucocorticoid-inducible protein kinase (Sgk1), which serves as a mechanism to inhibit the ubiquitination-dependent degradation of ENaC. We report here that 14-3-3 proteins participate in this regulatory process through a direct interaction with a phosphorylated form of human Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2). The interaction is dependent on Sgk1-catalyzed phosphorylation of hNedd4-2 at Ser-468. We found that this interaction preserved the activity of the Sgk1-stimulated ENaC-dependent Na+ current while disrupting the interaction decreased ENaC density on the Xenopus laevis oocytes surface possibly by enhancing Nedd4-2-mediated ubiquitination that leads to ENaC degradation. Our findings suggest that 14-3-3 proteins modulate the cell surface density of ENaC cooperatively with Sgk1 kinase by maintaining hNedd4-2 in an inactive phosphorylated state.

Transcriptomic and proteomic analysis of a 14-3-3 gene-deficient yeast.

BMH1 and BMH2 encode Saccharomyces cerevisiae 14-3-3 homologues whose exact functions have remained unclear. The present work compares the transcriptomic and proteomic profiles of the wild type and a BMH1/2-deficient S. cerevisiae mutant (bmhDelta) using DNA microarrays and two-dimensional polyacrylamide gel electrophoresis. It is reported here that, although the global patterns of gene and protein expression are very similar between the two types of yeast cells, a subset of genes and proteins (a total of 220 genes) is significantly induced or reduced in the absence of Bmh1/2p. These genes include approximately 60 elements that could be linked to the reported phenotypes of the bmhDelta mutant (e.g., accumulation of glycogen and hypersensitivity to environmental stress) and/or could be the potential downstream targets of interacting partners of Bmh1/2p such as Msn2p and Rtg3p. Importantly, >30% of the identified genes (71 genes) were found to be associated with carbon (C) and nitrogen (N) metabolism and transport, thereby suggesting that Bmh1/2p may play a major role in the regulation of C/N-responsive cellular processes. This study presents the first comprehensive overview of the genes and proteins that are affected by the depletion of Bmh1/2p and extends the scope of knowledge of the regulatory roles of Bmh1/2p in S. cerevisiae.