Cell-cycle-dependent colonization of mouse spermatogonial stem cells after transplantation into seminiferous tubules

Abstract

Spermatogonial stem cells (SSCs) migrate to the niche upon introduction into the seminiferous tubules of the testis of infertile animals. However, only 5-10% of the transplanted cells colonize recipient testes. In this study, we analyzed the impact of cell cycle on spermatogonial transplantation. We used fluorescent ubiquitination-based cell cycle indicator transgenic mice to examine the influence of cell cycle on SSC activity of mouse germline stem (GS) cells, a population of cultured spermatogonia enriched for SSCs. GS cells in the G1 phase are more efficient than those in the S/G2-M phase in colonizing the seminiferous tubules of adult mice. Cells in the G1 phase not only showed higher expression levels of GFRA1, a component of the GDNF self-renewal factor receptor, but also adhered more efficiently to laminin-coated plates. Furthermore, this cell cycle-dependency was not observed when cells were transplanted into immature pup recipients, which do not have the blood-testis barrier (BTB) between Sertoli cells, suggesting that cells in the G1 phase may passage through the BTB more readily than cells in the S/G2-M phase. Thus cell cycle status is an important factor in regulating SSC migration to the niche.

Fig. 1.

Expression of Fucci transgenes in the undifferentiated spermatogonia compartment. A: Histological sections of Cdt1-KO2 and Gem-AG transgenic mouse testes. Cdt1-KO2 is expressed predominantly in cells on the basement membrane, while Gem-AG expression is rarely found in the same region. B: Immunostaining of testes using antibodies against markers of undifferentiated spermatogonia. Antibodies against the indicated antigens were used to stain testes of Cdt1-KO2 and Gem-AG transgenic mice. Counterstained with Hoechst 33342 (blue). C: Quantification of cells with undifferentiated spermatogonia marker expression. At least 80 cells with each indicated spermatogonia marker were counted. Bars = 50 μm (A), 20 μm (B).

Fig. 2.

Derivation of GS cells from Fucci mice. A: Appearance of GS cells derived from a transgenic mouse pup containing both Cdt1-KO2 and Gem-AG transgenes. B: Flow cytometric analysis of Fucci transgene expression in GS cells. C: Flow cytometric analysis of the cell cycle distribution of Fucci-ROSA GS cells using Hoechst 33342. GS cells were stained with Hoechst 33342 for analysis of DNA content. D: Relative increase in adhesion of Cdt1-KO2⁺ GS cells to laminin-coated plates. Logarithmically growing GS cells were dissociated by trypsin and incubated on laminin-coated plates for the indicated time. E: Quantification of cells with Cdt1-KO2 or Gem-AG fluorescence that attached to laminin. At least 112 cells in 15 random fields were counted in four experiments. Cells were incubated on laminin-coated plates for the indicated time, and were recovered with trypsin for cell counting. The proportion of cells with Cdt1-KO2 or Gem-AG fluorescence is indicated. The results were compared with logarithmically growing cells (Before). F: Recovery of GS cells from laminin-coated plates (n = 4). The cells were incubated for the indicated time, and total adherent cell number was determined after collecting attached cells with trypsin. G: Flow cytometric analysis of cell surface marker expression. GS cells with Cdt1-KO2 or Gem-AG fluorescence were gated and analyzed for the expression of surface antigens. H: Real-time PCR analysis of GS cells. Fucci-ROSA GS cells were sorted according to their transgene expression patterns, and mRNA from each fraction was collected for real-time PCR (n = 3). Bar = 20 μm (A, D).

Fig. 3.

Colonization of recipient mouse seminiferous tubules by Fucci-ROSA GS cells at intervals up to 2 months after transplantation. A: Whole mount appearance of seminiferous tubules that received transplantation of Fucci-ROSA GS cells. Seminiferous tubules were collected at the indicated time and were analyzed under UV light. Note the decrease in the proportion of cells with Gem-AG fluorescence. B: Doublet (top) and chain (bottom) of spermatogonia on the basement membrane at 2 weeks after transplantation. C: Flow cytometric analysis of recipient testes. Recipient testes were dissociated into single cells, and expression patterns of the Fucci transgenes were compared. Forward scatter and side scatter profiles change as donor cells differentiate into haploid cells. D: Quantification of cells with Cdt1-KO2⁺ or Gem-AG⁺ fluorescence by flow cytometry. The proportions of cells expressing Cdt1-KO2 or Gem-AG fluorescence were plotted using data from flow cytometric analysis at the indicated time points. E: Fucci transgene expression in the central and edge regions of germ cell colonies 2 months after transplantation. F: Immunostaining of recipient testes 2 months after transplantation. Antibodies against indicated antigens were used to stain testes of Cdt1-KO2 and Gem-AG transgenic mice. Counterstained with Hoechst 33342 (blue). G: Quantification of cells expressing undifferentiated spermatogonia markers. At least 178 cells with each indicated spermatogonia marker were counted. Bar = 20 μm (A, B, E, F).

Fig. 4.

Functional analysis of SSC activity by spermatogonial transplantation into adult recipients. A: Macroscopic appearance of recipient testes following transplantation of Fucci-ROSA GS cells after cell sorting. Equal numbers of cells were transplanted, and the testes were recovered for LacZ staining 2 months after transplantation. Individual blue tubules indicate colonies of spermatogenesis arising from donor stem cells. B: Colony number. Results of three transplantation experiments using 12 recipient testes. C: Histological appearance of a recipient testis that received transplantation of Cdt1-KO2⁺/Gem-AG^– cells. Bars = 1 mm (A), 20 μm (C).

Fig. 5.

Functional analysis of SSC activity by spermatogonial transplantation into pup recipients. A: Macroscopic appearance of recipient testes following transplantation of Fucci-ROSA GS cells after cell sorting. Equal numbers of cells were transplanted, and the testes were recovered for LacZ staining 2 months after transplantation. Individual blue tubules indicate colonies of spermatogenesis arising from donor stem cells. B: Colony number. Results of seven transplantation experiments involving at least 11 recipient testes. C: Immunostaining of GS cells using antibodies against activated RAC1, CLDN3, CLDN7, and CLDN8. Logarithmically growing GS cells were centrifuged by cytospin and stained with the indicated antibodies. Bars = 1 mm (A), 20 μm (C).