Chromatin associated Sin3A is essential for male germ cell lineage in the mouse

Abstract

Spermatogenesis is a complex process that requires coordinated proliferation and differentiation of male germ cells. The molecular events that dictate this process are largely unknown, but are likely to involve highly regulated transcriptional control. In this study, we investigate the contribution of chromatin associated Sin3A in mouse germ cell lineage development. Genetic inactivation of Sin3A in the male germline leads to sterility that results from the early and penetrant apoptotic death observed in Sin3A-deleted germ cells, coincident with the reentry in mitosis. Sin3A-deleted testes exhibit a Sertoli-cell only phenotype, consistent with the absolute requirement for Sin3A in germ cells' development and/or viability. Interestingly, transcripts analysis revealed that the expression program of Sertoli cells is altered upon inactivation of Sin3A in germ cells. These studies identified a central role for the mammalian Sin3-HDAC complex in the germ cell lineage, and point to an exquisite transcriptional crosstalk between germ cells and their niche to support fertility in mammals.

Figure 1. Deletion of Sin3A in male germ cells results in Sertoli cell only phenotype

(a.) Representative testes from adult (24-week old) Sin3A^gc+/− and Sin3A^gc−/− male mice. (b.) Weights (mg) of the testes from Sin3A^gc+/− (black) and Sin3A^gc−/− males (grey) at day 10 (D10, n=3 for each genotype, p<0.028), D14 (n=3, p<0.01), D21 day (n=6, p<0.008), adult (n=3, p<0.007). Error bars represent the standard deviation (c.) Representative Hematoxylin and eosin-stained cross sections of testes from D10, D14 and D21 and adult Sin3A^gc+/− (left) or Sin3A^gc−/− mice (right). Scale bar =50μm. (d.) Representative immunostaining with an anti-Sin3A antibody on cross sections from D10, D14, D21 and adult testis of the indicated genotype. Scale bar =50μm. Inset boxes correspond to higher magnification.

Figure 2. VasaCre-mediated deletion of Sin3A leads to a loss of germ cells

(a.) Representative immunostaining with an anti-TRA98 antibody on cross sections from D10, D14, D21 day old and adult testis from Sin3A^gc−/− and Sin3A^gc+/− mice. Scale bar =50 μm. Inset boxes correspond to higher magnification. (b.) Percentage of seminiferous tubules with positive staining for TRA98. Shown is the average result from two independent experiments (c-d.) Relative expression determined by quantitative RT-PCR in of Sin3A^gc+/− and Sin3A^gc−/− testes at indicated time points for (c) germ cell specific transcripts MVH, plzf, RBM, and ngf and (d) proliferation marker pcna. Transcript abundance is expressed as relative to housekeeping B2M transcript abundance and normalized to the corresponding Sin3A^gc+/− testes. Shown is the average result from two independent experiments performed in duplicate.

Figure 3. Early postnatal germ cells undergo apoptosis in the absence of Sin3A

(a.) Representative immunostaining on cross sections of testes from D1, D3 and D10 mice of the indicated genotype with an anti-TRA98 or an anti-Cleaved Caspase 3 antibody. Inset boxes show higher magnification of the corresponding image. Scale bar =50 μm. (b-c.) Percentage of tubules with no positive staining for TRA98 (b.) and tubules with positive staining for cleaved Caspase 3 (c.) from testes of the indicated age and genotype. Note that no TRA98-megative tubules were detected in Sin3A^gc/+ testes after D1. Shown is the average of two independent experiments performed in duplicate.

Figure 4. Sin3A mediated loss of germ cells affects Sertoli cells

(a.) Representative immunostaining on cross sections of testes from D10, D14 and D21 mice of the indicated genotype with an anti-GATA1 antibody. Scale bar =50 μm. Inset boxes correspond to higher magnification (b.) Relative expression of Sertoli cell-specific transcripts, determined by quantitative RT-PCR in testis of Sin3A^gc+/− and Sin3A^gc−/− mice at indicated time points. Expression is relative to housekeeping gene B2M and normalized to corresponding Sin3A^gc+/− testis. Shown is the average of two independent experiments performed on two independent mice per genotype in duplicate.