Labeling was conducted 24 h after the first of two total i.p. neighboring SNAP91+ A-single spermatogonia, potentially indicative of niche saturation. ERBB3+ spermatogonia also synchronize their cell cycles with epithelium stages VIIICIX, where they form physical associations with preleptotene spermatocytes transiting the blood-testis barrier and Sertoli cells undergoing sperm release. Thus, A-single spermatogonia heterogeneity within this short-lived and reoccurring microenvironment invokes novel theories on how cellular niches integrate with testicular physiology to orchestrate sperm development in mammals. testes has further shown fragmentation of spermatogonial syncytia containing 4C16 cells in the vicinity of germline stem N-Bis(2-hydroxypropyl)nitrosamine cell niches after experimentally inducing severe germ cell loss [17]. Interestingly, syncytial fragmentation under these conditions yielded predominantly paired spermatogonia that reoccupied vacant germline stem cell niches [17]. Still, defining the associated cellular components that comprise a germline stem cell niche within mammalian gonads continues to evade scientists [4, 18]. This inability to pinpoint how spermatogonial stem cell fate is regulated at an anatomical level in mammals prohibits genetic analyses to more precisely elucidate how spermatogenesis is maintained and initiated in vivo. Given the cyclical nature of the seminiferous epithelium [5], extrinsic factors critical for maintenance of stem spermatogonia [19], and dependence of spermatogonial stem cell numbers on Sertoli cell numbers [20], it is reasonable to hypothesize that highly structured niches do regulate sperm stem cell fate in mammals. Moreover, in mammals, genetic or chemical depletion of endogenous germline stem cells is required for donor spermatogonia to effectively colonize recipient testes and maintain spermatogenesis [4]. This concept is clearly supported by discoveries in where early differentiating progenitors re-fill vacant niches and become germline stem cells lacking syncytia [21, 22]. Thus, based on modeling in both invertebrates and vertebrates, germline stem cell niches in mammals would theoretically function to regulate the fate of A-single spermatogonia. Here, we identify a factor related to the neuregulin receptor, ERBB3, that is transiently detected during a 1- to 2-day period each 12.9-day rat spermatogenic cycle in a rare subset of SNAP91+, ZBTB16+, SALL4+ A-single spermatogonia. Along a rat spermatogenic wave, the ERBB3+ and ERBB3? A-single spermatogonia colocalize specifically to epithelial N-Bis(2-hydroxypropyl)nitrosamine segments of stage VIIICIX seminiferous tubules undergoing sperm release. Therein, ERBB3+ spermatogonia form direct contacts with Sertoli cells and transitioning preleptotene spermatocytes, thus mapping this novel spermatogonial type to definable microanatomy at the basement membrane of the rat seminiferous epithelium. Accordingly, selective induction of early spermatozoan progenitors from one A-single spermatogonial pool N-Bis(2-hydroxypropyl)nitrosamine within this ephemeral environment presents a model where remaining A-single spermatogonia act as stem cells to support subsequent rounds of spermatogenesis. MATERIALS AND METHODS Animal Protocols Protocols for use of wild-type (Harlan Co.) and tg[23] Sprague-Dawley rats in the present study were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern (UTSW) Medical Center in Dallas, as certified by the Association for Assessment and Accreditation of Laboratory Animal Care International. Analysis of N-Bis(2-hydroxypropyl)nitrosamine A-Single Spermatogonial Subtypes Immunofluorescence-based data on numbers of spermatogonia were collected in testis sections and seminiferous tubule whole mounts (0.5- to 2.5-cm pieces) after labeling with antibodies to spermatogonial markers, as detailed below under and [23] Sprague-Dawley rats and fixed for approximately 18 h at 4C in 0.1 M sodium phosphate buffer (pH 7.2) containing 4% paraformaldehyde. Fixed testes were equilibrated through a 10%, 18%, and 25% sucrose (w/v, dissolved in 1 PBS [catalog no. 14040-182; Invitrogen, Inc.]) gradient by sequential overnight incubations (24 h) at 4C in 15 ml of each respective sucrose solution. Once equilibrated to 25% sucrose, testes were embedded N-Bis(2-hydroxypropyl)nitrosamine in tissue freezing medium (catalog no. 72592; Electron Microscopy Sciences, Inc.) and frozen using a cryobath (catalog no. 45972; Shandon Lipshaw). Frozen testes were used to prepare a parallel series of cryosections (section thickness, 8 m). Frozen sections were stored at ?40C until use in antibody-labeling assays or stained by the periodic acid-fuchsin sulfurous acid technique described above. Before antibody labeling, sections were equilibrated in air to approximately 22C24C for 15 min, hydrated in PBS Rabbit polyclonal to EREG (catalog no. D8537; Sigma) at 22C24C for 10 min, heat-treated at 80C for 8 min.