Isolation of undifferentiated and early differentiating type A spermatogonia from Pou5f1-GFP reporter mice

Abstract

Limited understanding of the mechanisms underlying self-renewal and differentiation of spermatogonial stem cells hampers our ability to develop new therapeutic and contraceptive approaches. Mouse models of spermatogonial stem cell development are key to developing new insights into the biology of both the normal and diseased testis. Advances in transgenic reporter mice have enabled the isolation, molecular characterization, and functional analysis of mouse Type A spermatogonia subpopulations from the normal testis, including populations enriched for spermatogonial stem cells. Application of these reporters both to the normal testis and to gene-deficient and over-expression models will promote a better understanding of the earliest steps of spermatogenesis, and the role of spermatogonial stem cells in germ cell tumor.

Figure 1

Schematic diagram of spermatogenesis in the prepubertal and adult mouse testis showing the expression time-course of Pou5f1 and Kit. Spermatogenesis begins with mitotic proliferation and differentiation of diploid, undifferentiated spermatogonia. Differentiating spermatogonia unidirectionally continue differentiation into spermatocytes, which undergo two meiotic divisions to form haploid spermatids. Haploid spermatids undergo dramatic structural modifications leading up to the formation of spermatozoa. The time from the initiation of differentiation to the formation of spermatozoa is a period of several weeks. Day 7 testis only contain spermatogenic cells up to Type B spermatogonia; Day 10 testis only contain spermatogenic cells up to leptotene spermatocytes (28). Diagrams do not depict the relative proportion of each cell population.

Figure 2

Fluorescence micrograph and flow cytometric evaluation of freshly isolated tubules and cells isolated from Pou5f1-GFP^Mann mice. (A–B) Logitudinal cross-section adjacent to basement membrane (A) and through lumen (B) of a live, freshly isolated Pou5f1-GFP^Mann seminiferous tubule, demonstrating distribution of GFP+ spermatogonia along the basement membrane at 10 dpp. (C) GFP+ cells (arrows) in a freshly isolated, non-enriched primary cell population from 10 dpp Pou5f1-GFP^Mann mice. (D) Flow cytometry fluorescence intensity profile of wild-type (control) and Pou5f1-GFP^Mann freshly isolated cells from 10 dpp mice demonstrating a large population of GFP+ spermatogonia available for specific isolation through Fluorescence Activated Cell Sorting (FACS). (E) Representative fluorescence micrograph of a freshly isolated tubule isolated from Pou5f1-GFP^Mann mice at 100 dpp demonstrating A_aligned spermatogonia.

Figure 3

Undifferentiated and early differentiating spermatogonia isolated through Fluorescence Activated Cell Sorting (FACS). (A) Flow-cytometric analysis and sorting of wild-type and Pou5f1-GFP^Mann cells stained with APC-Kit antibody. (B) Real-time-PCR analysis of Kit and canonical markers of undifferentiated spermatogonia in Pou5f1+/Kit- and Pou5f1+/Kit+ cells sorted from A. Cells were processed for RT-PCR using the TaqMan Gene Expression Cells-to-CT Kit (Applied Biosystems, Carlsbad, CA) according to manufacturer’s instructions. The expression value of each gene was normalized to the amount of an internal control gene (Eif3land Rps3) (29) and a relative quantitative fold change was determined using the ΔΔ Ct method. Experiments were performed in triplicate and data are represented as mean ± standard error. Taqman Gene Expression Assays (Applied Biosystems, Carlsbad, CA) used for specific transcripts were: Mm00460859_m1 (Eif3l), Mm00833897_m1 (Gfra1), Mm00445212_m1 (Kit), Mm00437606_s1 (Neurog3), Mm00658129_gH (Pou5f1), Mm00656272_m1 (Rps3), Mm00493681_m1 (Thy1), and Mm01176868_m1 (Zbtb16). Normalization to Eif3l and Rps3 produced identical results.