PROSPECTIVE IDENTIFICATION AND ISOLATION OF TYPE-A SPERMATOGONIA FROM JUVENILE RHESUS MACAQUE TESTES

Abstract

Spermatogonial stem cells (SSCs) are undifferentiated germ cells that balance self-renewal and differentiation to support sperm production in the mammalian testis. Non-human primate testes contain two types of histologically distinguishable undifferentiated spermatogonia, designated Adark and Apale based on nuclear morphology. Classical and contemporary models suggest that Adark and Apale spermatogonia are reserve and renewing stem cells, respectively, although these designations are debatable. To gain insight into the biology of rhesus spermatogonia we prospectively identified and isolated two types of germ cells from juvenile rhesus testes (15–20 months of age). The only germ cells present at this stage of rhesus testis development are Adark and Apale spermatogonia. Putative germ cells were selected from heterogeneous juvenile testis cell suspensions using an inverted phase contrast microscope equipped with a micromanipulator and pulled-glass capillary pipette. Germ cells were distinguished from other testicular cell types based on their large size and characteristic color. hile both subpopulations of picked germ cells were relatively large (>12 μm), one was larger (large cells) than the other (small cells). Picking accuracy was determined by immunofluorescent staining for Vasa, which marks all germ cells in the juvenile rhesus testis. While 2.4% of testis cells in the heterogeneous suspensions expressed Vasa, each picked subpopulation was comprised of nearly pure germ cells (small=96.9%; large=97.3%). Hematoxylin staining was more intense in the nuclei of small germ cells than large germ cells, suggesting that they may represent Adark and Apale spermatogonia, respectively. To determine if either or both populations contain functional stem cells, we performed rhesus to nude mouse testis xenotransplantation. Two months after transplantation, recipient nude mouse testes were evaluated in whole mount by staining with an antibody that recognizes rhesus testis cells. Preliminary results indicated that the large germ cells produced chains of rhesus spermatogonia connected by intracytoplasmic bridges on the basement membrane of mouse seminiferous tubules. The small germ cells failed to produce similar foci of colonization. Thus, our data indicate that two phenotypically and functionally distinct germ cell types can be identified and isolated from juvenile rhesus testis cell suspensions, providing an opportunity to study their molecular characteristics. Supported by National Institutes of Health grants U54 HD08160 to TMP and KEO, R01 RR18500 to KEO, an institutional postdoctoral fellowship HD007332 to BPH, and the Magee-Womens Research Institute and Foundation. (poster)