Med1 regulates meiotic progression during spermatogenesis in mice

Abstract

Spermatogenesis is a highly coordinated process. Signaling from nuclear hormone receptors, like those for retinoic acid (RA), is important for normal spermatogenesis. However, the mechanisms regulating these signals are poorly understood. Mediator complex subunit 1 (MED1) is a transcriptional enhancer that directly modulates transcription from nuclear hormone receptors. MED1 is present in male germ cells throughout mammalian development, but its function during spermatogenesis is unknown. To determine its role, we generated mice lacking Med1 specifically in their germ cells beginning just before birth. Conditional Med1 knockout males are fertile, exhibiting normal testis weights and siring ordinary numbers of offspring. RA-responsive gene products stimulated by RA gene 8 (Stra8) and synaptonemal complex protein 3 (Sycp3) are first detected in knockout spermatogonia at the expected time points during the first wave of spermatogenesis, and persist with normal patterns of cellular distribution in adult knockout testes. Meiotic progression, however, is altered in the absence of Med1. At postnatal day 7 (P7), zygotene-stage knockout spermatocytes are already detected, unlike in control testes, with fewer pre-leptotene-stage cells and more leptotene spermatocytes observed in the knockouts. At P9, Med1 knockout spermatocytes prematurely enter pachynema. Once formed, greater numbers of knockout spermatocytes remain in pachynema relative to the other stages of meiosis throughout testis development and its maintenance in the adult. Meiotic exit is not inhibited. We conclude that MED1 regulates the temporal progression of primary spermatocytes through meiosis, with its absence resulting in abbreviated pre-leptotene, leptotene, and zygotene stages, and a prolonged pachytene stage.

Figure 1. Vasa-cre-mediated Med1 knockout (VMKO) testes exhibit normal morphology and weight

A. Cross-sections of 6-week-old VMKO (left) and control (right) testes immunostained for MED1 (green) and the pan-germ cell marker TRA98 (red), and exposed to DNA stain DAPI (blue). Arrows identify Sertoli cell nuclei positive for MED1 but negative for TRA98. Scale bars = 50μm. B. Cross-sections of 6-week-old VMKO (left) and control (right) testes stained with hematoxylin and eosin. Scale bars = 50μm. C. Assessment of testis weight:body weight measurements of 10-week-old VMKO (left, N=9) and control (right, N=25) male mice. Differences are not statistically significant (Student’s t-test).

Figure 2. VMKO male mice exhibit normal fertility

A. VMKO and control males were mated with wild type female mice for a period of 5 months. Average litter size for each group is shown. Graph exhibits mean values ± SEM. Difference is not statistically significant (N.S., Student’s t-test). B. The cumulative number of pups sired by each animal over the entire breeding period.

Figure 3. Adult VMKO testes contain normal numbers of retinoic-acid responsive germ cells

A. Cross-sections of 6-week-old VMKO (left) and control (right) testes immunostained for STRA8 (green) and exposed to DAPI (blue). Arrows identify STRA8 positive cells. Scale bars = 50μm. B. Cross-sections of 6-week-old VMKO (left) and control (right) testes immunostained for SYCP3 (green) and exposed to DAPI (blue). Arrows identify SYCP3 positive cells. Scale bars = 50μm. C. Quantitation of STRA8+ and SYCP3+ cells per seminiferous tubule cross-section in 6-week-old VMKO and control testes. Graph exhibits mean values ± SEM. Differences are not statistically significant (N.S., Student’s t-test).

Figure 4. Neonatal and juvenile VMKO testes contain normal numbers of retinoic-acid responsive germ cells

A. Cross-sections of 2-day-old (P2, left) and P4 (right) VMKO and control testes immunostained for STRA8 (top) and exposed to DAPI (bottom). Arrows identify STRA8 positive cells. Scale bars = 50μm. B. Quantitation of STRA8+ (P4 testis) and SOHLH1+ (P7 testis) cells per seminiferous tubule cross-section in VMKO and control testes. Graph exhibits mean values ± SEM. Differences are not statistically significant (N.S., Student’s t-test). C. Cross-sections of P7 VMKO and control testes immunostained for SOHLH1 (top) and exposed to DAPI (bottom). Scale bars = 50μm. D. Cross-sections of P9 (left) and P12 (right) VMKO and control testes immunostained for SYCP3 (top) and exposed to DAPI (bottom). Scale bars = 50μm.

Figure 5. VMKO male germ cells prematurely enter zygonema and pachynema in the first meiotic prophase

A–B. Meiotic chromosome spreads were prepared from P7 (A) and P9 (B) VMKO and control testes and immunostained for SYCP3 (green) and γH2AX (red). Representative images are shown for all observed meiotic stages. Scale bars = 10 μM. C. Quantification of the percentage of P7 cells in the chromosome spreads that were SYCP3 positive. Difference is not statistically significant (N.S., Student’s t-test). D–E. Quantification of the percentage of P7 (D) and P14 (E) cells in the chromosome spreads that were pre-leptotene, leptotene, zygotene and pachytene. ***p<0.001; statistical significance calculated using Student’s t-test. N.O. = not observed. All graphs exhibit mean values ± SEM.

Figure 6. VMKO testes exhibit a higher proportion of pachytene cells relative to other meiotic stages

A–B. Cross-sections of P28 (A) and P30 (B) VMKO and control testes immunostained for γH2AX (top) and exposed to DAPI (bottom). Asterisks demarcate seminiferous tubules enriched with pachytene cells (concentrated foci of γH2AX). Arrows identify leptotene and zygotene spermatocytes. Scale bars = 50μm. C–D. Quantification of the percentage of P28 (C) and 6-week-old (D) cells in the chromosome spreads that were pre-leptotene, leptotene, zygotene and pachytene. **p<0.01, ***p<0.001; statistical significance calculated using Student’s t-test. Graphs exhibit mean values ± SEM.

Figure 7. VMKO testes exhibit normal post-meiotic germ cell development

Cross-sections of 6-week-old VMKO (left) and control (right) testes immunostained for BOULE (green) and exposed to DAPI (blue). Scale bars = 25μm.