Two-step oligoclonal development of male germ cells

Abstract

During mouse development, primordial germ cells (PGCs) that give rise to the entire germ line are first identified within the proximal epiblast. However, long-term tracing of the fate of the cells has not been done wherein all cells in and around the germ-cell lineage are identified. Also, quantitative estimates of the number of founder PGCs using different models have come up with various numbers. Here, we use tetrachimeric mice to show that the progenitor numbers for the entire germ line in adult testis, and for the initiating embryonic PGCs, are both 4 cells. Although they proliferate to form polyclonal germ-cell populations in fetal and neonatal testes, germ cells that actually contribute to adult spermatogenesis originate from a small number of secondary founder cells that originate in the fetal period. The rest of the "deciduous" germ cells are lost, most likely by apoptosis, before the reproductive period. The second "actual" founder germ cells generally form small numbers of large monoclonal areas in testes by the reproductive period. Our results also demonstrate that there is no contribution of somatic cells to the male germ cell pool during development or in adulthood. These results suggest a model of 2-step oligoclonal development of male germ cells in mice, the second step distinguishing the heritable germ line from cells selected not to participate in forming the next generation.

Fig. 1.

Germ cells in testis form large single-color areas after reproductive period. (A) Normal male karyotype of R1 ES cells used in this study. The image of the SKY chromosome painting of wild-type R1 cells used to generate Rosa 26 fluorescent knock-in clones is shown. (B) An example of 3-color (mRFP1, EGFP, and nonfluorescent) germ cells in a testis of a tetrachimeric mouse. (Left) Distribution of colored germ cells observed in Center. (Right) A magnified image of the white box shown in Center. Note that the interstitial cells have green, blue, and red cells. (C) Single-color (green) patches of germ cells occupying seminiferous tubules in a tetrachimeric mouse. (Left) Fluorescent image of the single-color domain formed by green germ cells. (Center) Immunostaining with TRA98, a germ cell marker. (Right) Nuclear staining with Hoechst 33342. (D) A longitudinal section of seminiferous tubules. Sertoli cells (arrowheads) and interstitial cells have green, red, and blue cells. (E) Tetrachimeric Leydig cells (arrowheads) in the testis of a tetrachimeric mouse. In the right of D and E, Nuclear counter-stainings with Hoechst 33342 are shown. (F) Examples of tetrachimeric epididymal epithelial cells from a tetrachimeric mouse. (Scale bars, 1 mm in B; 100 μm in C–F.)

Fig. 2.

Germ cells in a testis generally have a fewer colors than the mouse. (A–D) Bilateral testes generally have the same combination of colors. (A) Single-color (EGFP) testes germ cells in a tetrachimeric mouse. (B) Two-color (ECFP and nonfluorescent) testes germ cells in a tetrachimeric mouse. (C) Three-color (mRFP1, ECFP, and nonfluorescent) testes germ cells in a tetrachimeric mouse. (D) Three-color (mRFP1, ECFP, and EGFP chimeric germ cells) testes germ cells in a tetrachimeric mouse. (E) Difference of distribution of mRFP1 (R)-, ECFP (C), and EGFP (G)-expressing cells in adult male chimeric mice was not significant. (F) Difference of distribution of R-, C-, and G- expressing germ cells in adult male testes was not significant (ns). (G) The relationship between age and number of colors that testes that do not have is not significant. (H) Numbers of patches observed in sections of 2-color (red squares) and 3-color (blue rhomboids) testes do not correlate with age. (Scale bars, 250 μm in A–D.)

Fig. 3.

PGCs during migration from allantois and those within genital ridges are observed as a mixed population of different colors. (A) Migrating PGCs observed in the hindgut region of an E8.5 tetrachimeric embryo. Red (mRFP1 positive) and white (nonfluorescent) arrowheads indicate PGCs. (B) PGCs in the genital ridge of an E12.5 tetrachimeric embryo. (Lower) A magnified image of a white box shown in Upper Left. Arrows indicate fluorescent PGCs. Dashed lines indicate locations of TNAP-positive cells. (A Right and B Right) TNAP staining of the same sections shown in Left. (C) PGCs in the genital ridge of an E13.5 tetrachimeric embryo. Arrows indicate fluorescent PGCs. (Scale bars, 100 μm in A and B; 250 μm in C.)

Fig. 4.

Formation of single-color domains of germ cells during neonatal to prepubertal period. (A) An example of a testis of a newborn tetrachimeric mouse. (B) An example of a chimeric testis at P7. TRA98-positive (green) germ cells show a mixture of blue and red cells (colored arrowheads). (C–E) An example of a green and red chimeric testis at P14. In this example, single-color patches of different colors have started forming in distinct areas of a testis. (D and E) Magnified pictures of areas in white rectangles shown in C, in which germ cells are stained with TRA98 (white). (D) In the region, red germ cell patches are forming, although a small number of green germ cells (green arrowheads) are observed. (E) In the region green germ cell patches are forming, although a small number of red germ cells (red arrowheads) are observed. (F) NGN3-positive cells form a green patch in the region where green germ cells are selected. (G) TUNEL staining of the region where green germ cells are selected. TUNEL-positive apoptotic cells (purple) are exclusively observed in red germ cells. (H) NGN3-positive germ cells form single-color patches (colored arrowheads) in green and blue chimeric testis of a P3 tetrachimeric mouse. (Right) Magnified picture of the white rectangle shown in Left. (Scale bars, 250 μm in C; 100 μm in A and B; 50 μm in D–H.)

Fig. 5.

Our model of 2-step oligoclonal development of testis germ cells. Male germ line starts from 4 cells. They proliferate, migrate, and split to bilateral genital ridges. However, germ cells actually contributing adult spermatogenesis during the reproductive period originate from a small number of a second founder population that initially seed onto genital ridges. The rest of germ cells (deciduous germ cells) are removed, most likely by apoptosis, before the reproductive period.