A transit-amplifying population underpins the efficient regenerative capacity of the testis

Abstract

The spermatogonial stem cell (SSC) that supports spermatogenesis throughout adult life resides within the GFRα1-expressing A type undifferentiated spermatogonia. The decision to commit to spermatogenic differentiation coincides with the loss of GFRα1 and reciprocal gain of Ngn3 (Neurog3) expression. Through the analysis of the piRNA factor Miwi2 (Piwil4), we identify a novel population of Ngn3-expressing spermatogonia that are essential for efficient testicular regeneration after injury. Depletion of Miwi2-expressing cells results in a transient impact on testicular homeostasis, with this population behaving strictly as transit amplifying cells under homeostatic conditions. However, upon injury, Miwi2-expressing cells are essential for the efficient regenerative capacity of the testis, and also display facultative stem activity in transplantation assays. In summary, the mouse testis has adopted a regenerative strategy to expand stem cell activity by incorporating a transit-amplifying population to the effective stem cell pool, thus ensuring rapid and efficient tissue repair.

Figure 1.

Miwi2 Tomato expression defines a small population of undifferentiated spermatogonia. (A) Schematic over of the 5′ region of the Miwi2 locus (top) and the Miwi2^Tom transcriptional reporter allele (bottom). (B) Representative FACS analysis of live CD45^Neg CD51^Neg gated cells in testicular populations of wild-type and Miwi2^Tom/+ mice. Numbers indicate the percentages of cells of the defined subpopulations. (C) qRT-PCR expression analysis of Miwi2 in Miwi2-Tom^Pos c-kit^Neg and Miwi2-Tom^Pos c-kit^Pos populations (n = 3). (D) Enumeration of testicular CD45^Neg CD51^Neg Miwi2-Tom^Pos c-kit^Neg cells per testis is shown (n = 15). (E) Cell cycle parameters of CD45^Neg CD51^Neg Miwi2-Tom^Pos c-Kit^Neg cells as determined by DNA content. (F) Cell surface expression by FACS of the indicated markers in CD45^Neg CD51^Neg Miwi2-Tom^Pos c-Kit^Neg are shown, as well as isotype control staining. (G) Representative images of Miwi2^Tom/+ seminiferous tubules stained with α-GFRα1 (Green), α-tdTomato (Red), and α-Plzf (Blue). Representative examples of Miwi2-Tom^Hi GFRα1^Neg (red box), Miwi2-Tom^Neg GFRα1^Pos (green box), and Miwi2-Tom^Lo GFRα1^Pos (white box) populations are highlighted. Bar, 25 µm. (H) Enumeration of testicular the populations defined in G. Numbers represents total PLZF^Pos cells in each category normalized to 1,000 sertoli cells (n = 5). Error bars represent SEM.

Figure 2.

Miwi2-Tom^Hi c-Kit^Neg cells are a subpopulation of Ngn3-positive spermatogonia. (A) Scatter plot of gene expression from biological quadruplicates of CD45^Neg CD51^Neg Miwi2-Tom^Pos c-Kit^Neg (n = 4) and CD45^Neg CD51^Neg Miwi2-Tom^Pos c-Kit^Pos populations (n = 4). Genes highlighted in red show a fold change ≥2 and a significance of ≤0.05. Number of genes up- and down-regulated in the respective populations are indicated. (B) Enrichment and depletion of select genes in CD45^Neg CD51^Neg Miwi2-Tom^Pos c-Kit^Neg population. (C) Fluidigm qRT-PCR of Miwi2-Tom^Pos cells as indicated. Each row corresponds to a specific gene; each column corresponds to a single cell; color code of expressions levels indicated in log₂ expression. (D) Representative FACS analysis of testicular cells from Miwi2^Tom/+; Ngn3^GFP/+ mice. CD45^Neg CD51^Neg gated cells are shown. Numbers indicate the percentages of cells of the defined subpopulations. (E) Representative images of Miwi2^Tom/+; Ngn3^GFP/+ seminiferous tubules stained with α-GFP (green) and α-tdTomato (red). White arrow indicates Miwi2-Tom and Ngn3-GFP double-positive spermatogonia. Solid white arrow indicates Ngn3-GFP single-positive spermatogonia. (F) Enumeration of Ngn3^Pos Miwi2^Neg GFRα1^Neg and Ngn3^Pos Miwi2^Pos GFRα1^Neg cells expressed as number of cells per 1,000 sertoli cells (n = 4). Error bars represent SEM. Bar, 25 µm.

Figure 3.

Miwi2-expressing cells are transit amplifying and do not contribute to the long-term maintenance of spermatogenesis. (A) Schematic overview of the Miwi2^DTR allele. (B) Representative FACS analysis of live CD45^Neg CD51^Neg gated cells is shown of testicular populations of wild-type and Miwi2^DTR/+ mice stained with c-kit and DTR antibodies (top) and after 3x DTx administrations analyzed 1 d after the last injection (bottom). Numbers indicate the percentages of cells of the defined subpopulations. (C) Enumeration of the GFRα1^Pos populations in adult Miwi2^DTR/+ mice that were untreated (n = 3) or treated with 3x DTx injections and analyzed 1 d (n = 4) or 1 wk (W; n = 4) after the last injection. Numbers represent cells of indicated chain length normalized to 1,000 sertoli cells. (D) Histology of testis; sections stained with H&E at time points indicated (1–16 wk [W]) after DTx administration are shown. Bar, 10 µm. (E) Testicular weight of the indicated cohorts at indicated time points. n, number of mice analyzed per time point. P-values are shown; n.s., no statistical significance. (F) Enumeration of fully spermatogenic tubules in wild-type and Miwi2^DTR/+ mice at the final 16 wk time point after DTx administration is shown. (G) Fertility as represented by pups per plug of the indicated cohorts at 6 and 12 wk after DTx or vehicle control injection. Black and red represent wild-type and Miwi2^DTR/+ Dtx-treated mice, respectively. Error bars represent SEM.

Figure 4.

Miwi2-expressing cells are crucial for the regenerative capacity of the testis after injury. (A) Experimental overview of DTx and busulfan injections, as well as time points analyzed. (B) Testicular weight of the indicated cohorts overtime is shown. n, number of mice analyzed per time point for the DTx-injected mice. P-values are shown; n.s., no statistical significance. (C) Histology of testis section stained with H&E for the same time points as indicated in A. Bar, 100 µm. (D) Enumeration of fully spermatogenic tubules in DTx-injected wild-type and Miwi^DTR/+ mice for each stage during the time course (n = 3 for each genotype and time point). P-values are shown; n.s., no statistical significance. (E) Representative image of reconstitution from CD45^Neg CD51^Neg Miwi2-Tom^Pos c-kit^Neg transplanted cell isolated from Miwi2^Tom/+; Rosa26^YFP/+ mice. Colonies derived from the YFP-positive donor cells are shown. Bar, 1 mm. (F) Quantitation of colony formation for the indicated populations analyzed 3 mo after transplantation. Fold enrichment are calculated as colony forming units normalized per 10⁵ cells for the 10⁴ sorted CD45^Neg CD51^Neg Miwi2-Tom^Pos c-kit^Neg (n = 12) cells and 10⁶ testicular control cells transplanted (n = 4). Error bars represent SEM. (G) Immunofluorescence analysis of a transplanted YFP⁺ colony, spermatogonia that express GFRα1 indicated (white boxes) after reconstitution. Bar, 50 µm (H) Total number of A_s and A_pr spermatogonia per 1,000 of sertoli cells before (CTR) and at day 5, 8, and 15 after busulfan treatment (left). Enumeration of Miwi2-Tom^Hi GFRα1^Neg, Miwi2-Tom^Neg GFRα1^Pos, and Miwi2-Tom^LowGFRα1^Pos populations is shown for the same time points (right). Stacked columns represent normalized percentages of the indicated populations per time point. Error bars represent SEM of three (CTR and +15D) and 4 (+5D and +8D) mice analyzed. Significance between CTR and +8D Miwi2-Tom^LowGFRα1^Pos populations is indicated; *, P < 0.05; n.s., P > 0.05. (I) Normalized percentages of A_al8 at indicated time points after busulfan treatment (left) as presented in H. Significance between CTR and the indicated populations is shown; ***, P < 0.001. Representative images of Miwi2-Tom^LowGFRα1^Pos and Miwi2-Tom^Pos c-kit^Neg A_al8 are shown for +8D (top) and +15D (bottom), respectively. Bar, 25 µm.