Characterizing the Spermatogonial Response to Retinoic Acid During the Onset of Spermatogenesis and Following Synchronization in the Neonatal Mouse Testis

Abstract

Retinoic acid (RA), the active metabolite of vitamin A, is known to be required for the differentiation of spermatogonia. The first round of spermatogenesis initiates in response to RA and occurs in patches along the length of the seminiferous tubule. However, very little is known about the individual differentiating spermatogonial populations and their progression through the cell cycle due to the heterogeneous nature of the onset of spermatogenesis. In this study, we utilized WIN 18,446 and RA as tools to generate testes enriched with different populations of spermatogonia to further investigate 1) the undifferentiated to differentiating spermatogonial transition, 2) the progression of the differentiating spermatogonia through the cell cycle, and 3) Sertoli cell number in response to altered RA levels. WIN 18,446/RA-treated neonatal mice were used to determine when synchronous S phases occurred in the differentiating spermatogonial population following treatment. Five differentiating spermatogonial S phase windows were identified between spermatogonial differentiation and formation of preleptotene spermatocytes. In addition, a slight increase in Sertoli cell number was observed following RA treatment, possibly implicating a role for RA in Sertoli cell cycle progression. This study has enhanced our understanding of the spermatogonial populations present in the neonatal testis during the onset of spermatogenesis by mapping the cell cycle kinetics of both the undifferentiated and the differentiating spermatogonial populations and identifying the precise timing of when specific individual differentiating spermatogonial populations are enriched within the testis following synchrony, thus providing an essential tool for further study of the differentiating spermatogonia.

Keywords: cell cycle; retinoic acid; spermatogenesis; spermatogonia; testis.

FIG. 1

LIN28A and ZBTB16 expression is not restricted to the undifferentiated spermatogonial population in the postnatal testis. Images depict representative sections of whole-mounted tubules from 10-dpp wild-type mouse testes (A–C) stained for ZBTB16 and LIN28A (A), STRA8 and LIN28A (B), and STRA8 and GFRA1 (C). Pink arrows denote ZBTB16 and LIN28A copositive spermatogonia, blue arrows indicate STRA8 and LIN28A copositive spermatogonia, purple arrows designate STRA8 only-positive spermatogonia, and orange arrows depict GFRA1 only-positive spermatogonia. Bar = 50 μm.

FIG. 2

GFRA1 and STRA8 efficiently mark the undifferentiated-to-differentiating spermatogonial transition in the neonatal testis. Images depict representative sections of whole-mounted tubules from 3-dpp (A), 6-dpp (B), and 10-dpp (C) wild-type mouse testes stained for STRA8 and GFRA1. Blue arrows denote GFRA1 only-positive germ cells, purple arrows indicate STRA8 and GFRA1 copositive germ cells, and orange arrows designate STRA8 only-positive spermatogonia. Bar = 50 μm.

FIG. 3

STRA8 is expressed in patches of seminiferous tubules during the initiation of spermatogenesis. Image depicts a representative section of whole-mounted tubule from a 6-dpp mouse testis stained for STRA8 shown in green and GFRA1 shown in red (A). STRA8-only expression is displayed in the zoomed-in sections of tubule (B, C) to demonstrate where STRA8 expression begins and ends along the length of this tubule. A model for the asynchronous onset of spermatogenesis denotes STRA8 expression in spermatogonia designated by the green cells, and the established RA signaling boundaries are indicated by the pink lines (D). Bars = 100 μm.

FIG. 4

The transition from undifferentiated to differentiating spermatogonia is fluid following the RA trigger. Images depict representative sections of whole-mounted tubules taken from mice treated with WIN 18,446-only (A) or WIN 18,446 followed by an RA injection and allowed to recover for 8 h (B) and 24 h (C) and stained for STRA8 and GFRA1. White arrows denote GFRA1 only-positive spermatogonia, orange arrows designate STRA8 and GFRA1 copositive spermatogonia, and purple arrows indicate STRA8-positive spermatogonia. Bar = 50 μm. Bar graph depicts the average percentage of GFRA1 only-positive, STRA8 only-positive, and GFRA1- and STRA8-copositive spermatogonia from testes of mice following synchronization, displayed in D. Error bars represent SEM.

FIG. 5

WIN 18,446 treatment halts the majority of undifferentiated spermatogonia in G0. Images depict representative cross sections of seminiferous tubules taken from mice treated with WIN 18,446-only and an injection of EdU 12 h prior to euthanization and stained for GCNA and either MKI67 (A) or EdU incorporation (B). White arrows denote GCNA only-positive spermatogonia, orange arrows indicate MKI67 only-positive cells, and blue arrows denote EdU-incorporated cells. Bars = 25 μm.

FIG. 6

MKI67 is not expressed in spermatogonia immediately following WIN 18,446/RA exposure. Images depicts representative cross sections of seminiferous tubules taken from mice treated with WIN 18,446 followed by an RA injection and allowed to recover for 16 h (A), 46 h (B), and 72 h (C) and stained for GCNA and MKI67. White arrows denote GCNA only-positive spermatogonia, orange arrows indicate MKI67 only-positive cells, and purple arrows depict GCNA- and MKI67-copositive spermatogonia. Bars = 25 μm. Bar graph indicates the average percentage of GCNA only-positive and GCNA- and MKI67-copositive germ cells per round tubule from testes of mice following synchronization, displayed in D. * denotes a P-value < 0.0005. Error bars represent SEM.

FIG. 7

The onset of spermatogenesis following WIN 18,446/RA synchronization mimics the timing of the first round of spermatogenesis. Images depict representative cross sections of testes from mice treated with WIN 18,446 followed by an RA injection and given 8 h (A), 16–18 h (B), 28 h (C), 46–48 h (D), 56 h (E), 70–72 h (F), 94–96 h (G), 120–122 h (H), and 144–146 h (I, J) of recovery and stained for either STRA8 (A–F, I) or KIT (G, H). Cells positive for EdU incorporation are shown in green (A–I). Five synchronized differentiating spermatogonial S phases are denoted by colocalization of either STRA8 (B, D, F) or KIT (G, H) with EdU. Preleptotene spermatocyte formation occurred 144–146 h post-RA treatment, depicted in fluorescence by colocalization of STRA8 and EdU (I) and by the round nuclear morphology and expression of STRA8 shown by immunohistochemistry (J). Differentiating spermatogonia not undergoing S phase are indicated by STRA8 only-positive expression (A, C, E). A model for STRA8 and KIT expression during the timing of spermatogonial S phases following the onset of synchronized spermatogenesis is diagrammed in K. Black bar = 50 μm.

FIG. 8

Undifferentiated A spermatogonia transition to become either A1 or A2 differentiating spermatogonia during the onset of synchronized spermatogenesis. TEM images depict representative cross sections of testes from mice treated with WIN 18,446-only (A) or with WIN 18,446 followed by an RA injection and given 18 h (B, C), 48 h (D), 72 h (E), 96 h (F), 120 h (G), and 144 h (H) of recovery. White arrows denote heterochromatin present along the nuclear envelope within the undifferentiated and differentiating spermatogonia and preleptotene spermatocytes. A model for the progression of spermatogonial differentiation from undifferentiated spermatogonia following the onset of synchronized spermatogenesis indicating that either the population of A1 or A2 differentiating spermatogonia is skipped is diagrammed in I. Bars = 2 μm.

FIG. 9

Excess RA exposure slightly increased the number of Sertoli cells in the postnatal testis. Immunofluorescent images depict representative cross sections of testes from mice treated with the WIN 18,446 vehicle control only (GD) (A), WIN 18,446-only (B), GD followed by RA vehicle (DMSO) (D), WIN 18,446 followed by an injection of RA (E), and GD followed by an injection of RA (F) stained for SOX9 expression. Bar graphs indicate the average number of SOX9-positive Sertoli cells per round tubule from testes of mice treated with either WIN 18,446-only compared to GD-only, displayed in C, or from mice treated with either WIN 18,446 followed by an injection of RA or GD followed by an injection of RA compared to GD followed by an injection of DMSO, displayed in G. * denotes a P-value = 0.01. Error bars represent SEM. White bars = 25 μm.