E4 Transcription Factor 1 (E4F1) Regulates Sertoli Cell Proliferation and Fertility in Mice

Abstract

In the mammalian testes, Sertoli cells are the only somatic cells in the seminiferous tubules that provide structural, nutritional and regulatory support for developing spermatogenic cells. Sertoli cells only proliferate during the fetal and neonatal periods and enter a quiescent state after puberty. Functional evidences suggest that the size of Sertoli cell population determines sperm production and fertility. However, factors that direct Sertoli cell proliferation and maturation are not fully understood. Transcription factor E4F1 is a multifunctional protein that serves essential roles in cell fate decisions and because it interacts with pRB, a master regulator of Sertoli cell function, we hypothesized that E4F1 may have a functional role in Sertoli cells. E4f1 mRNA was present in murine testis and immunohistochemical staining confirmed that E4F1 was enriched in mature Sertoli cells. We generated a conditional knockout mouse model using Amh-cre and E4f1^flox/flox lines to study E4F1 fucntion in Sertoli cells and the results showed that E4f1 deletion caused a significant reduction in testis size and fertility. Further analyses revealed that meiosis progression and spermiogenesis were normal, however, Sertoli cell proliferation was impaired and germ cell apoptosis was elevated in the testis of E4f1 conditional knockout mice. On the basis of these findings, we concluded that E4F1 was expressed in murine Sertoli cells and served important functions in regulating Sertoli cell proliferation and fertility.

Keywords: E4F1; fertility; proliferation; sertoli cells; spermatogenesis.

Figure 1

The relative mRNA expression and protein localization of E4F1 in postnatal mouse testes. (a) Quantification of mRNA expression of E4f1 in testes of mice at different development stages. Data were analyzed by mean ± SEM for 3 mice per stage. * denote significantly at p < 0.05. (b) Immunohistochemical staining of E4F1 in testes at PD6 (scale bar = 20 µm) and adult (scale bar = 1 00 µm) male mice. Black arrow indicates spermatogonia and red arrow indicates Sertoli cells.

Figure 2

Loss of E4f1 in Sertoli cells resulted in reduced testis size and Subfertility (a) E4f1 expression level in control and E4f1 cKO testes. n = 3 (b) Reprehensive images of immunohistochemistry staining for E4F1 in control and E4f1 cKO testes. Black arrow indicates spermatogonia and red arrow indicates Sertoli cells. Scale bar = 50 µm. (c) Representative images of the testes from control and E4f1 cKO mice at PD120. (d) Testis/Body weight ratio of wildtype (n = 7), control (n = 6) and E4f1 cKO (n = 8) mice at PD120. (e,f) Representative images of hematoxylin and eosin (H&E) stained testes of control and E4f1 cKO male mice at PD35 and PD120. Scale bar = 100µm. (g) Quantification of litter size per mice of control and E4f1 cKO male mice. n = 7. (h) Quantification of sperm concentration per mice of control and E4f1 cKO male mice. n = 3. *, **** denote significantly different at p < 0.05 and p < 0.0001.

Figure 3

E4f1 deletion in Sertoli cells did not affect meiosis progression and spermiogenesis (a) Immunofluorescent staining for SOX9 and LIN28 in cross-sections of testes from controls and E4f1 cKO male mice. Scale bar = 100 µm. (b) Quantification of the number of spermatogonia per Sertoli cell in testes from controls and E4f1 cKO male mice. n = 3. (c) Immunofluorescent staining for SYCP3 in spread spermatocyte from controls and E4f1 cKO male mice. Scale bar = 10 µm. (d) Quantification of the number of different stages spermatocyte of meiosis per mice of controls and E4f1 cKO male mice. n = 3. (e) Immunofluorescent staining for PNA in cross-sections of testes from controls and E4f1 cKO male mice. Scale bar = 50 µm. (f) Quantification of the number of different stages spermatozoa of meiosis per mice of control and E4f1 cKO male mice. n = 3.

Figure 4

Loss of E4f1 resulted in decreased Sertoli cell population. (a) Quantification of seminiferous tubules diameter per tubule of controls and E4f1 cKO male mice. n = 3. (b) Immunofluorescence staining for TUNEL in cross-section from controls and E4f1 cKO male mice. Scale bar = 100 µm. (c) The percentage of TUNEL positive tubules in testes of controls and E4f1 cKO male mice. n = 3. (d) Quantification of TUNEL positive cells per TUNEL positive tubules in testes of controls and E4f1 cKO male mice. n = 3. (e) Immunofluorescence staining for SOX9 and TRA98 in cross-sections of testes from controls and E4f1 cKO male mice. Scale bar = 100 µm. (f) Quantification of germ cells per tubule in VII and VIII stages of the testes from controls and E4f1 cKO male mice. n = 3. (g) Quantification of Sertoli cells per cord in VII and VIII stages of testes from controls and E4f1 cKO male mice. n = 3. (h) Quantification of germ cells per Sertoli cell in VII and VIII stages of the testes from controls and E4f1 cKO male mice. n = 3. *, ** and *** denotes significantly different at p < 0.05, p < 0.01, and p < 0.001.

Figure 5

E4f1 deletion affected proliferation and apoptosis in Sertoli cells. (a) Immunofluorescence staining of EdU and SOX9 in cross-sections of testes from control and E4f1 cKO males at PD0. Scale bar = 50 µm. (b) Percentages of proliferative Serotli cells in cross-sections of testes from control and E4f1 cKO males at PD0. n = 3. (c) TUNEL staining in cross-sections of testes from control and E4f1 cKO males at PD0. Scale bar = 50 µm. (d) Quantification of TUNEL⁺ cells in control and E4f1 cKO males. n = 3. (e) Immunofluorescence co-staining of TUNEL and SOX9 in cross-sections of testes from control and E4f1 cKO males at PD0. Scale bar = 50 µm. (f) Quantification of apoptotic Sertoli cells in testes from PD0 controls and E4f1 cKO male mice. n = 3. * and ** denotes significantly different at p < 0.05 and p < 0.01.