Marker expression reveals heterogeneity of spermatogonia in the neonatal mouse testis

Abstract

Prospermatogonia transition to type A spermatogonia, which provide the source for the spermatogonial stem cell (SSC) pool. A percentage of these type A spermatogonia then differentiate to enter meiosis as spermatocytes by ∼P10. It is currently unclear as to when these distinct populations are initially formed in the neonatal testis, and when the expression of markers both characteristic of and required for the adult undifferentiated and differentiating states is established. In this study, we compared expression of known spermatogonial cell fate markers during normal development and in response to the differentiation signal provided by retinoic acid (RA). We found that some markers for the undifferentiated state (ZBTB16/PLZF and CDH1) were expressed in nearly all spermatogonia from P1 through P7. In contrast, differentiation markers (STRA8 and KIT) appeared in a subset of spermatogonia at P4, coincident with the onset of RA signaling. GFRA1, which was present in nearly all prospermatogonia at P1, was only retained in STRA8/KIT- spermatogonia. From P4 through P10, there was a great deal of heterogeneity in the male germ cell population in terms of expression of markers, as markers characteristic of the undifferentiated (except GFRA1) and differentiating states were co-expressed through this interval. After P10, these fate markers diverged to mark distinct populations of undifferentiated and differentiating spermatogonia, and this pattern was maintained in juvenile (P18) and adult (P>60) testes. Taken together, these results reveal that the spermatogonia population is heterogeneous during the first wave of spermatogenesis, and indicate that neonatal spermatogonia may not serve as an ideal substitute for studying the function of adult spermatogonia.

Figure 1. Spermatogonia exhibit morphological heterogeneity by P4

Sections from Bouin’s-fixed testes from mice at P1 (A–B) and P4 (C–D) were stained with H & E. Blue arrows identify prospermatogonia (A–B) and spermatogonia, the latter of which exhibited a number of different nuclear sizes and morphologies (C–D). Green arrows (B, D) indicate representative Sertoli cells. Scale bars = 60 µm.

Figure 2. Fate marker detection in germ cells of the neonatal testis at P1

Phalloidin was used to label F-actin (blue) in all images. Each column contains one representative section labeled with antibodies against the proteins listed at the top. The first row is green + blue channels, the second is red + blue, and the bottom row is a merged image of green + red + blue. White arrows indicate KIT+ interstitial cells, which are present throughout testis development (Rothschild et al. 2003). Scale bar = 60 µm.

Figure 3. Fate marker detection in germ cells of the neonatal testis at P4

Phalloidin was used to label F-actin (blue) in all images. Each column contains one representative section labeled with antibodies against the proteins listed at the top. The first row is green + blue channels, the second is red + blue, and the bottom row is a merged image of green + red + blue. Cords containing ZBTB16+ spermatogonia were either KIT+ (yellow dashed lines) or KIT− (white dashed lines). Green arrows indicate KIT+ spermatogonia and red arrows point to GFRA1+ spermatogonia. Scale bar = 60 µm.

Figure 4. Fate marker detection in germ cells of the neonatal testis at P7

Phalloidin was used to label F-actin (blue) in all images. Each column contains one representative section labeled with antibodies against the proteins listed at the top. The first row is green + blue channels, the second is red + blue, and the bottom row is a merged image of green + red + blue. Red arrows indicate GRFA1+/KIT− spermatogonia, green arrows indicate GFRA1−/KIT+ spermatogonia, and the yellow arrow points to a rare GFRA1+/KIT+ spermatogonium. Scale bar = 60 µm.

Figure 5. Fate marker detection in germ cells of the neonatal testis at P10

Phalloidin was used to label F-actin (blue) in all images. Each column contains one representative section labeled with antibodies against the proteins listed at the top. The first row is green + blue channels, the second is red + blue, and the bottom row is a merged image of green + red + blue. Red arrows indicate GRFA1+/KIT− spermatogonia and green arrows indicate GFRA1−/KIT+ spermatogonia. Scale bar = 60 µm.

Figure 6. Fate marker detection in germ cells of the neonatal testis at P18

Phalloidin was used to label F-actin (blue) in all images. Each column contains one representative section labeled with antibodies against the proteins listed at the top. The first row is green + blue channels, the second is red + blue, and the bottom row is a merged image of green + red + blue. Scale bar = 60 µm.

Figure 7. Fate marker changes in germ cells of the neonatal testis following RA-induced differentiation

Phalloidin was used to label F-actin (blue) in all images. Each row contains a temporal detection of RA-induced changes in testis sections. KIT is labeled green in all images, while STRA8 (top row, A–D), CDH1 (middle row, E–H), and ZBTB16 (bottom row, I–L) are labeled in red. The hours after RA injection are indicated on each image, and the actual age is in parentheses. Scale bar = 60 µm.

Figure 8. Model for spermatogonial development in the neonatal testis

(A) At P1, prospermatogonia express undifferentiated markers such as CDH1, ZBTB16, and GFRA1. Following continuous or multiple exposures to RA, differentiating spermatogonia express STRA8 and KIT; a single dosage of RA is not sufficient for differentiation, and spermatogonia appear to revert to the undifferentiated state. (B) Prospermatogonia (white) transition to a heterogeneous population of differentiating and undifferentiated (putative stem cell) spermatogonia. Populations of differentiating spermatogonia enter meiosis, while the SSCs remain undifferentiated to provide a consistent source of differentiated spermatogonia after the first wave of spermatogenesis.