Increased sex chromosome expression and epigenetic abnormalities in spermatids from male mice with Y chromosome deletions

Abstract

During male meiosis, the X and Y chromosomes are transcriptionally silenced, a process termed meiotic sex chromosome inactivation (MSCI). Recent studies have shown that the sex chromosomes remain substantially transcriptionally repressed after meiosis in round spermatids, but the mechanisms involved in this later repression are poorly understood. Mice with deletions of the Y chromosome long arm (MSYq-) have increased spermatid expression of multicopy X and Y genes, and so represent a model for studying post-meiotic sex chromosome repression. Here, we show that the increase in sex chromosome transcription in spermatids from MSYq- mice affects not only multicopy but also single-copy XY genes, as well as an X-linked reporter gene. This increase in transcription is accompanied by specific changes in the sex chromosome histone code, including almost complete loss of H4K8Ac and reduction of H3K9me3 and CBX1. Together, these data show that an MSYq gene regulates sex chromosome gene expression as well as chromatin remodelling in spermatids.

Fig. 1.

Increased transcription of sex-linked genes in round spermatids from mice with MSYq deletions. Gene-specific RNA FISH was performed on spermatogenic cells from mice with two-thirds (2/3MSYq–) or complete (MSYq–) deletion of the Y long arm, with wild-type XY mice as a control. (A) Table showing the average percentage of round spermatids expressing a given gene, on the basis of the presence of an RNA FISH signal. After angular transformation of the individual percentages, an ANOVA test was used to determine whether the difference between the three genotypes was significant at the P=0.05 level. sc, single copy gene; mc, multicopy gene. More details on gene copy number are given in supplementary material Fig. S1. (B) RNA FISH on XY (left panel), 2/3MSYq– (middle panel) and MSYq– (right panel) round spermatids using probes for Fmr1, Ddx3x, Slx and Ube1y1/Zfy1. DNA is stained with DAPI (blue) and the RNA FISH signal is in red. Scale bar: 5 μm. For Slx and Ube1y1/Zfy1, there is an increase in the number of RNA FISH signals per spermatid in the 2/3MSYq– and MSYq– deletion models compared with the XY control. (C) Quantification of the number of Ube1y1/Zfy1 RNA FISH signals per expressing round spermatid from XY and MSYq– mice.

Fig. 2.

Increased testicular levels of GFP and SLX in mice with partial deletions of the Yq. (A) Western blot analysis of testicular GFP levels from XY and 2/3MSYq– mice carrying an X-linked reporter GFP transgene. Membranes were hybridised with an anti-GFP antibody (top panel), then washed and reprobed for actin (bottom panel) as a loading control. (B) Quantification of whole testis GFP levels from the western blot in A using ImageJ software. The level of GFP is represented as the ratio of GFP to actin and is given an arbitrary value of one in the XY testis. Error bars represent the s.e.m. The difference in testis GFP levels between the two genotypes was statistically significant (P<0.001, Student's t-test). (C) GFP immunostaining of testis sections from XY (top panel) and 2/3MSYq– (bottom panel) mice carrying an X-linked GFP transgene. The GFP protein is present in the cytoplasm of spermatogonia (asterisks), early meiotic cells, and elongating spermatids (arrows). There is an increase in GFP levels in the spermatid cytoplasm in the 2/3MSYq– testis compared to the XY control, but the spermatogonial levels of GFP are unchanged. (D) Whole testis western blot analysis of SLX levels in XY and 2/3MSYq– mice. Actin was used as a whole testis loading control. (E) Quantification of SLX levels in the testis of XY and 2/3MSYq– males relative to actin from the western blot in D using ImageJ software. Error bars represent the s.e.m. The difference in SLX levels between the two genotypes was found to be statistically significant (P<0.005, Student's t-test). (F) SLX immunostaining of spermatogenesis stage VIII seminiferous tubules from XY (top panel), 2/3MSYq– (middle panel) and MSYq– (bottom panel) mice. SLX is present in the cytoplasm of round and elongating spermatids from all three genotypes, although there is an increase in SLX levels in the two Yq deletion models compared with the XY control.

Fig. 3.

The epigenetic marks associated with PMSC are altered in MSYq– spermatids. (A) The DAPI-dense heterochromatic PMSC (arrows) still forms in round spermatids from the 2/3MSYq– and MSYq– mice. (B) The histone modification H48Ac is present on PMSC in round spermatids from XY (top row, arrows) and 2/3MSYq– (middle row, arrows) mice, but is lost from PMSC in the majority of round spermatids from MSYq– mice (bottom row, arrows). Far left: a stage I seminiferous tubule stained for H4K8Ac (red), DAPI (blue) and γH2AX (green). Left and right panel: higher magnification of round spermatids from a stage I tubule. H4K8Ac is present throughout the nucleus and is enriched on PMSC (arrows) but excluded from the chromocentre in spermatids from XY and 2/3MSYq– mice. H4K8Ac is still present in the nucleus of MSYq– spermatids but is not enriched on PMSC. Far right panel: H4K8Ac immunostaining of surface-spread round spermatids. H4K8Ac was enriched on PMSC in 36.6% of spermatids from XY mice but only 6.7% of MSYq– spermatids, where it is reduced compared to the XY control (see Table 1).

Fig. 4.

Decreased H3K9me3 and CBX1 enrichment on PMSC in spermatids from MSYq– mice. (A) H3K9me3 is reduced on PMSC but not the chromocentre of MSYq– spermatids. Far left panel: a stage VIII seminiferous tubule stained for H3K9me3 (red), DAPI (blue) and the acrosomal protein DKKL1 (green). Left and right panel: higher magnification of round spermatids from a stage VIII tubule. H3K9me3 enrichment on the PMSC (arrows) is reduced in the MSYq– spermatids (bottom row) compared to XY (top row) and 2/3MSYq– (middle row) spermatids. Far right panel: H3K9me3 immunostaining of surface-spread round spermatids reveals that H3K9me3 enrichment on PMSC (arrows) is reduced in MSYq– spermatids relative to the XY control. (B) Antibody staining for the heterochromatin protein CBX1 (green) reveals that this protein is reduced on PMSC of MSYq– spermatids (bottom row, arrows) compared to XY (top row, arrows) and 2/3MSYq– spermatids (middle row, arrows).