Male germ-line stem cell potential is predicted by morphology of cells in neonatal rat testes.

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 10/2002; 99(18):11706-11. DOI: 10.1073/pnas.182412099
Source: PubMed

ABSTRACT Gonocytes are a transient population of male germ-line stem cells that are derived from primordial germ cells in the embryo and give rise to spermatogonial stem cells, which establish and maintain spermatogenesis in the postnatal testis. In contrast to spermatogonial stem cells, gonocytes can be identified easily in neonatal rat testis cell suspensions based on their large size and distinct morphology. Furthermore, histological analysis of testes from neonatal transgenic rats demonstrated that gonocytes are the only cells that express a lacZ reporter transgene. Two gonocyte subpopulations, designated pseudopod and round, were identified and isolated from neonatal (0-4 days postpartum) rat testis cell suspensions. Male germ-line stem cells, identified by their ability to produce and maintain colonies of spermatogenesis upon transplantation into infertile recipient testes, were present almost exclusively in the pseudopod gonocyte subpopulation. In contrast, annexin V staining indicated that the majority of round gonocytes undergo apoptosis. These results indicate that a nearly pure population of male germ-line stem cells can be prospectively identified in neonatal rat testis cell suspensions by morphological criteria. Together, the pseudopod and round gonocyte populations will provide powerful tools for the study of cellular mechanisms that control cell fates and the establishment of spermatogenesis in the postnatal testis.

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    ABSTRACT: 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 is 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.
    Biology of Reproduction 03/2014; 90(4). DOI:10.1095/biolreprod.113.114405 · 3.45 Impact Factor
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    ABSTRACT: Neonatal testicular germline stem cell suspension prepared from twenty 3-5 day old albino rats were incubated in normal saline or in NC-01in triplicate groups. A control group of saline alone (without cell suspension) was also prepared. The cell-containing groups and control were incubated for 21 days at room temperature, stained with 50% Mezo (organic) and visually studied daily with a view to histological characterizing the changes in cellular aggregation, morphology and coloration during incubation. Images of dense oval cells and cellular developmental transformation into mixed populations of these testicular cells showed clearly identifiable patterns of clump formation. Saline alone (control) did not show the presence of any cells during the period of incubation. In vitro expansion of neonatal germline stem cells within 21 days of incubation was associated with multiple divisions into numerous structurally similar cells and transformation into clusters of multi-colored component rod-like cells. A many-fold increase in the number of dense oval cells was observed in the NC-01 culture medium than in the normal saline. Using these morphological criteria and functional characteristics, we suggest a many-fold increased presence of rat neonatal spermatogonial stem cells capable of cellular division in NC-01 culture medium than in normal saline.
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    ABSTRACT: The production of spermatozoa relies on a pool of spermatogonial stem cells (SSCs), formed in infancy from the differentiation of their precursor cells, the gonocytes. Throughout adult life, SSCs will either self-renew or differentiate, in order to maintain a stem cell reserve while providing cells to the spermatogenic cycle. By contrast, gonocytes represent a transient and finite phase of development leading to the formation of SSCs or spermatogonia of the first spermatogenic wave. Gonocyte development involves phases of quiescence, cell proliferation, migration, and differentiation. Spermatogonia, on the other hand, remain located at the basement membrane of the seminiferous tubules throughout their successive phases of proliferation and differentiation. Apoptosis is an integral part of both developmental phases, allowing for the removal of defective cells and the maintenance of proper germ-Sertoli cell ratios. While gonocytes and spermatogonia mitosis are regulated by distinct factors, they both undergo differentiation in response to retinoic acid. In contrast to postpubertal spermatogenesis, the early steps of germ cell development have only recently attracted attention, unveiling genes and pathways regulating SSC self-renewal and proliferation. Yet, less is known on the mechanisms regulating differentiation. The processes leading from gonocytes to spermatogonia have been seldom investigated. While the formation of abnormal gonocytes or SSCs could lead to infertility, defective gonocyte differentiation might be at the origin of testicular germ cell tumors. Thus, it is important to better understand the molecular mechanisms regulating these processes. This review summarizes and compares the present knowledge on the mechanisms regulating mammalian gonocyte and spermatogonial differentiation. © 2015 Society for Reproduction and Fertility.


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