Mouse CHD4-NURD is Required for Neonatal Spermatogonia Survival and Normal Gonad Development.

Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of CHD4 and CHD3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of Chd4 early in gametogenesis, but not Chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that CHD4 controls expression of Dmrt1 and its downstream target Plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of CHD4 in mammalian gametogenesis pointing to functions in gene expression early in the process.

An Efficient In Vitro Culture System To Amplify Spermatogonia Stem Cell Markers

Introduction: Proliferation of spermatogonial stem cells (SSCs) can be a treatment for infertile men. Here, we design an efficient method based on culturing in the presence of Sertoli cells to improve the expression level of some specific spermatogonia stem cell genes during two weeks post culture. Materials and Methods: Cells were derived from neonatal (2-6 days old) mice testes and were cultured in DMEM medium with FBS. The colonization of cultured SSCs in days 4, 7, and 14 of culture was counted via phase-contrast microscope and Image J software. Methyl thiazolyl tetrazolium (MTT) test was performed to evaluate the viability of cultured SSCs in days 3, 7, and 14 of culture. The expression level and the alteration pattern of specific spermatogonial markers, i.e., Stra8, DAZL, and Piwill2 was examined via real-time polymerase chain reaction (PCR) during two weeks post culture. Results: The number and the diameters of colonies showed a significant increase in cultured cells. MTT results proved the higher viability of testicular cells during the culture period. The results of ALP staining detected a positive reaction in spermatogonia colonies. Real-time PCR data showed that culturing SSCs in the presence of interstitial cells of the testis, amplified the level and alteration pattern of specific spermatogonia stem cells genes beneficial in the enrichment of SSCs propagation. Conclusion: Providing a similar culture environment to testicular niche increases viability, forms SSCs colonies, and regulates the level and alteration pattern of spermatogonia stem cell genes.

438. PLZF is a spermatogonia stem cell-specific marker in the sheep testis: application to enrichment of ovine spermatogonial stem cell

Identification and isolation of spermatogonial stem cells (SSCs) are prerequisite for long-term culture, genetic manipulation, and transplantation research. The promyelocytic leukemia zinc-finger (PLZF) has been identified as a spermatogonia stem cell marker in rodent and other species, however its expression in sheep testis has not been reported yet. In this study, we validated an antibody that specifically binds to spermatogonia stem cell in sheep testis, thus demonstrated that PLZF is a spermatogonia stem cell marker and can be used for its identification. Testes from 12 Merino rams were selected to represent four stages of testis development at testis weights of 3–5 g (neonatal), 30 g (peripubertal), 50 g (prepubertal) and 100 g (mature). Three testes sections from 4 different developmental stage were stained with PLZF antibody and 25 individual tubules in each section were counted. In the sections, the percentage of PLZF positive cells/per tubule was increased nearly 2-fold from neonatal (6. 4 ± 0. 4%) to peripubertal (1 2.2 ± 2.8%), and then the percentage begin to decline in prepubertal (4.6 ± 0. 7%) and mature testes (3.1 ± 0.6%). A single cell suspension of testicular cells was generated by a two step enzymic digestion (n = 4) and spermatogonia stem cells were enriched by overnight differential plating with 0.2% gelatine coated flask. The percentages of spermatogonia stem cells in the single cell suspensions were assessed by PLZF antibody staining of smears. Compared with the initial isolation (3.1 ± 0.6%), spermatogonia were enriched 11-fold in overnight differential plating (34.0 ± 5.7,%) (P < 0.05). These data provide the basis for future studies aimed at refining conditions of spermatogonial stem cell culture and manipulation before male germ stem cell transplantation in sheep.