Riding the spermatogenic wave: profiling gene expression within neonatal germ and sertoli cells during a synchronized initial wave of spermatogenesis in mice

Abstract

Continual sperm production relies on germ cells undergoing spermatogenesis asynchronously. As a result, the testis always contains a mixed population of germ cells at different stages of their differentiation process. The heterogeneous nature of the testis makes profiling gene expression within Sertoli cells or specific populations of germ cells impossible when a wild-type testis is assessed. We recently reported a unique method for synchronizing spermatogenesis without affecting fertility by manipulating RA levels within the neonatal testis. Using this protocol, combined with the RiboTag transgenic mouse line, we have mapped the Sertoli and germ cell translatome during the initial synchronized wave of spermatogenesis. Using microarray analysis, we identified 392 and 194 germ cell and Sertoli cells transcripts, respectively, that dynamically change during spermatogonial differentiation, division, and the onset of meiosis. Functional annotation clustering revealed that transcripts enriched in germ cells were mostly associated with meiosis (21 transcripts), chromatin organization (12 transcripts), and cell cycle (3 transcripts). In addition, glycoproteins (65 transcripts), cell adhesion (15 transcripts), and cell junction (13 transcripts) transcripts were overrepresented in the Sertoli cell-enriched list. These datasets represent the first transcriptional analysis of spermatogonial differentiation, division, and meiotic onset. These data suggest that several of the genes encoding meiotic proteins are expressed and are actively being translated well before germ cells enter meiosis. In addition, this study provides novel candidate genes, Asf1b and Esyt3, that may be involved in the regulation of spermatogonial chromatin reorganization, germ-Sertoli cell interactions, and/or blood-testis barrier formation.

Keywords: Sertoli cells; gene expression; germ cells; retinoic acid; retinoids; spermatogonia; testis; translational profiling.

FIG. 1

WIN 18,446/RA treatment of neonatal mice during a synchronized first wave of spermatogenesis. Neonatal mice were fed vehicle (gum tragacanth) or WIN 18,446 for nine consecutive days and either sacrificed or injected with vehicle (dimethylsulfoxide) or RA. STRA8 localization in cross-sections of vehicle control (top) and WIN 18,446/RA (bottom) treated animals. Testes were collected prior to injection (0 h) or 1, 2, 4, 6, and 8 days postinjection. Green arrows denote STRA8-negative spermatogonia, red arrows denote STRA8-positive spermatogonia, and black arrows denote STRA8-positive preleptotene spermatocytes. Bars = 100 μm.

FIG. 2

Activation of the RiboTag in germ or Sertoli cells of the testis. RiboTag mice were bred with either a germ cell-specific cre line (Stra8-cre; A) or a Sertoli cell-specific cre line (Amh-cre; B). RiboTag activation was verified via immunohistochemistry using an anti-HA antibody in synchronized neonatal testis sections. Black arrows represent immunopositive spermatogonia, and purple arrows represent immunopositive Sertoli cell cytoplasm. No primary antibody negative control is represented within the inset. Bars = 50 μm. C) Microarray analysis results confirm the enrichment for well-known germ, Sertoli, Leydig, or housekeeping genes in the IP from RiboTag/Stra8-cre (top) or RiboTag/Amh-cre (bottom) mouse lines.

FIG. 3

Heat map of germ cell- or Sertoli cell-enriched transcripts during the first wave of spermatogenesis. Relative expression levels of 392 germ cell-enriched (A) and 192 Sertoli cell-enriched (B) transcripts that significantly change during a synchronized first wave of spermatogenesis. Significance was determined by ANOVA (P < 0.05) and a greater than 2-fold change compared to WIN 18,446-only (0 h) treated mice. Expression was normalized to the mean intensity of the probe set, and these probes were ordered by expression (green = low; red = high). Data were visualized in GeneSpring GX Version 12.5; h = hour, d = days.

FIG. 4

Identification of candidate blood-testis barrier transcripts within Sertoli cells. Graphs depicting the microarray expression pattern of Sertoli cell-enriched transcripts that are associated processes involved in blood-testis barrier formation. Raw expression values are represented on the y-axis, and time post-RA injection is on the x-axis.

FIG. 5

Identification of candidate chromatin modifying transcripts within germ cells. Graphs depicting the microarray expression pattern of germ cell-enriched transcripts that are associated with DNA packaging and chromatin organization (A) and histone modifications (B) across the first wave of synchronized spermatogenesis. Raw expression values are represented on the y-axis, and time post-RA injection is given on the x-axis.

FIG. 6

Retinoic acid (RA)-regulated germ and Sertoli cell transcripts. Venn diagram for all RA up-regulated (A) or down-regulated (B) germ (RiboTag/Ngn3-cre) or Sertoli (RiboTag/Amh-cre) cell transcripts. The Sertoli cell-enriched transcripts were determined by comparing the IP mRNA to the total mRNA in all the time points assayed, whereas germ cell-enriched transcripts were identified by comparing WIN 18,446-only and WIN 18,446/RA-treated testes left to recover for 4 h. C) Graph depicting the microarray expression pattern of the top five retinoid metabolism transcripts that significantly change in expression during the first wave of synchronized spermatogenesis. Significance was determined by an ANOVA test (P < 0.05) compared to WIN 18,446-only (0 h) treatment. Raw expression values are represented on the y-axis, and time post-RA injection is given on the x-axis.

FIG. 7

Retinoid metabolism transcripts within germ and Sertoli cell. A Venn diagram depicting all the known retinoid metabolism transcripts (red circle) that are enriched in germ cells (RiboTag/Stra8-cre; blue circle) or Sertoli cells (RiboTag/Amh-cre; green circle).

FIG. 8

Actively translated cell cycle transcripts during the first wave of spermatogenesis. Graphs depicting the microarray expression patterns of actively translated germ cell-enriched cyclins (A) and actively translated Sertoli cell cycle transcripts, Tgfb1 and Smad3, (B) across the first wave of synchronized spermatogenesis. Raw expression values are represented on the y-axis, and time post-RA injection is given on the x-axis.

FIG. 9

Actively translated meiotic transcripts during the first wave of spermatogenesis. Graph depicting the microarray expression pattern of known meiotic transcripts that are actively translated within germ cell across the first wave of synchronized spermatogenesis. Raw expression values are represented on the y-axis, and time post-RA injection is given on the x-axis.