E2F1 controls germ cell apoptosis during the first wave of spermatogenesis

Abstract

Cell cycle control during spermatogenesis is a highly complex process owing to the control of the mitotic expansion of the spermatogonial cell population and following meiosis, induction of DNA breaks during meiosis and the high levels of physiological germ-cell apoptosis. We set out to study how E2F1, a key controller of cell cycle, apoptosis, and DNA damage responses, functions in the developing and adult testis. We first analyzed the expression pattern of E2f1 during post-natal testis development using RNA in situ hybridization, which showed a differential expression pattern of E2f1 in the adult and juvenile mouse testes. To study the function of E2f1, we took advantage of the E2F1(-/-) mouse line, which was back-crossed to C57Bl/6J genetic background. E2f1 loss led to a severe progressive testicular atrophy beginning at the age of 20 days. Spermatogonial apoptosis during the first wave of spermatogenesis was decreased. However, already in the first wave of spermatogenesis an extensive apoptosis of spermatocytes was observed. In the adult E2F1(-/-) testes, the atrophy due to loss of spermatocytes was further exacerbated by loss of spermatogonial stem cells. Surprisingly, only subtle changes in global gene expression array profiling were observed in E2F1(-/-) testis at PND20. To dissect the changes in each testicular cell type, an additional comparative analysis of the array data was performed making use of previously published data on transcriptomes of the individual testicular cell types. Taken together, our data indicate that E2F1 has a differential role during first wave of spermatogenesis and in the adult testis, which emphasizes the complex nature of cell cycle control in the developing testis.

Keywords: E2F1; apoptosis; cell cycle; testis.

Figure 1

E2f1 is expressed in a dynamic manner during post‐natal mouse testis development. RNAScope 2.0 assay was used to localize E2f1 mRNA on testicular sections; positive signal is cytoplasmic brown precipitate. An immunohistochemical detection of Plzf and pH2AX was coupled to the RNAscope assay to aid identification of the different E2f1‐positive germ‐cell subtypes (right panels, E2f1 in brown, pH2AX and PLZF blue). (A) E2f1 mRNA is detected at PND 10 in all germ cells (green arrow), and a subset of PMCs (black arrow) and interstitial cells (open arrow head). SCs (purple arrow) are negative for E2f1 mRNA. PC = positive control with mmPpib probe, NC = negative control with Dapb probe against a bacterial transcript. PLZF‐positive undifferentiated spermatogonia express E2f1. (B) At P20 E2f1 mRNA is no longer detected in PMCs (black arrow). The Pachytene spermatocytes (dashed circle) had very low or no E2f1 expression. Spermatogonia (green arrow) and the Plzf‐positive E2f1 were detected in the Plzf‐positive spermatogonia. The Pachytene spermatocytes have a positive blue pH2AX‐signal in the sex body. And the leptotene and zygotene spermatocytes (red arrow, dispersed nuclear blue pH2AX‐signal) are positive. (C) At P40 a stage‐specific E2f1 expression pattern is observed with E2f1 expression in similar cell types as P20. Elongating spermatocytes (asterisk) have a positive cytoplasmic signal. A false‐positive background is observed in round spermatid nucleoli. Black square marks the area of the picture in the higher magnification. Scale bar 50 μm in RNAScope only panels and 20 μm in RNAscope and immunohistochemistry panels.

Figure 2

Loss of E2f1 in C57 genetic background leads to decreased testicular size from PND20 onwards. (A) Body weights of E2f1^+/+, E2f1^+/−,\ and E2f1^−/− animals at different time points n = 4–6. There is significant decrease in bodyweight at the age of 270 days (9 months) in the E2f1^−/−. (B) Relative testis weights (mg of testis/g of bodyweight) of E2f1^+/+, E2f1^+/−, and E2f1^−/− animals at different time points. A statistically significant decrease in relative testis weight can be observed at PND20 in E2F1^−/−. (C) Fertility test was performed by breeding E2f1^+/+ and E2f1^−/− males (n = 3–4) with four different C57 females at different time points. The number of pups per litter was recorded. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D–F) Classification of seminiferous tubule morphology to normal, tubules with germ‐cell loss, and tubules with only SCs.

Figure 3

Histological alterations in E2F1^−/− testis already at P20. (A) Loss of meiotic germ cells can be observed on a microscopic level already at P20 in E2F1^−/− testis (asterisk). (B) At P90 SCO‐seminiferous‐tubule cross sections (open arrow head) and loss of meiotic germ cells (asterisk) are observed. There was evidence of previous successful rounds of spermatogenesis even in the disrupted tubules, since elongated spermatids could be found (asterisk). Scale bar represents 100 μm.

Figure 4

Decreased apoptosis of the first‐wave spermatogonia and increased apoptosis of meiotic germ cells in the E2F1^−/− testis. TUNEL assay coupled to detection of MAGE‐B4 positive spermatogonia was performed. *p < 0.05, ***p > 0.001. (A) Quantitation of TUNEL‐positive cells. (B) Quantitation of apoptotic spermatogonia in E2F1^+/+ and E2F1^−/− at the ages of P10 and P40. N.O. = not observed. (C) Representative pictures from the TUNEL/MAGE‐B4‐assay testes at P40. MAGE‐B4‐positive spermatogonia = white arrow head. MAGE‐B4 = green, TUNEL = red. Inset: Positive control for TUNEL assay with DNAse treatment. (D) Phosphorylated γH2AX (pH2AX) localization was assessed in P40 E2F1^+/+ and ^−/− testis together with PLZF (a marker for undifferentiated spermatogonia). pH2AX staining was comparable in control and mutant. There was a strong nuclear pH2AX signal in the meiotic cells undergoing meiotic DSB formation (the leptotene and zygotene spermatocytes, white arrow heads). No positive foci (indicating DNA damage) are detected outside the sex body in the pachytene spermatocytes (open arrow head). PLZF‐positive undifferentiated spermatogonia remain negative for pH2AX in the E2F1^−/− testis (double arrow head). pH2AX = orange, PLZF = green. Scale bar represents 50 μm in C and D.

Figure 5

Loss of undifferentiated spermatogonia in the absence of E2f1. (A) Relative mRNA levels of germ‐cell markers for undifferentiated spermatogonia (Oct4, Ngn3, Plzf), differentiating spermatogonia (Stra8) and meiotic cells (Spo11). (B) Quantitation of proliferating spermatogonia in E2F1^+/+ and E2F1^−/− at the ages of P10 and P40. MAGE‐B4 (spermatogonia marker) and BrdU (proliferation marker) positive cells were count. N.O. = not observed * = p < 0.05, ** = p < 0.01.

Figure 6

Sertoli cells and peritubular myoid cells are not affected by E2f1 loss. The number and proliferation of Sertoli cells is not altered in E2F1^−/− at P10 and P40. (A–B) WT1 (SC marker) and BrdU (proliferation marker) positive cells were analyzed in P10 and in P40 control and E2F1^−/− testes. n = 4. (C–D) Relative mRNA levels of Claudin11 (a SC tight‐junction marker) and Sma (a peritubular myoid cell marker) were analyzed from whole testis RNA at different time points.

Figure 7

E2f1^−/− mice have an unaltered hypothalamo‐pituitary‐gonadal axis. (A) Serum hormone levels of 90‐day‐old E2f1^+/+, and E2f1^−/− animals. FSH (ng/mL), LH (ng/mL), testosterone (pg/μL), n = 5–6. (B) Relative mRNA levels of genes related to androgen signaling in E2f1^+/+ and E2f1^−/− testis at ages of PND6, 20 and 90, n = 4. Ar, Spinlw2 (Eppin) mRNA levels and Rhox5 mRNA levels. *p < 0.05, **p < 0.01.

Figure 8

Ablation of E2f1 leads to upregulation of transcripts related to spermatogonia and SCs. (A) A schematic representation of the RNA microarray data analysis workflow. The list of differentially expressed genes from the E2F1‐array was compared against the cell‐type‐specific transcriptome data from Soumillon et al. to assess which cell types are responsible for the transcript level changes in the array. (B) A Venn diagram of the different cell‐type‐weighed transcript groups showing no overlap between different ell types in the analysis. (C) Graph representing the number of up‐ and downregulated transcripts from the E2F1‐array grouped according to the cell‐type‐weighed analysis. Majority of the DE genes are upregulated in E2f1^−/− testis at P20.