Loss of function of male-specific lethal 3 (Msl3) does not affect spermatogenesis in rodents

Abstract

Background: Male-specific lethal 3 (Msl3) is a member of the chromatin-associated male-specific lethal MSL complex, which is responsible for the transcriptional upregulation of genes on the X chromosome in males of Drosophila. Although the dosage complex operates differently in mammals, the Msl3 gene is conserved from flies to humans. Msl3 is required for meiotic entry during Drosophila oogenesis. Recent reports indicate that also in primates, Msl3 is expressed in undifferentiated germline cells before meiotic entry. However, if Msl3 plays a role in the meiotic entry of mammals has yet to be explored.

Results: To understand, if Msl3a plays a role in the meiotic entry of mammals, we used mouse spermatogenesis as a study model. Analyses of single-cell RNA-seq data revealed that, in mice, Msl3 is mostly expressed in meiotic cells. To test the role of Msl3 in meiosis, we used a male germline-specific Stra8-iCre driver and a newly generated Msl3^flox conditional knock-out mouse line. Msl3 conditional loss-of-function in spermatogonia did not cause spermatogenesis defects or changes in the expression of genes related to meiosis.

Conclusions: Our data suggest that, in mice, Msl3 exhibits delayed expression compared to Drosophila and primates, and loss-of-function mutations disrupting the chromodomain of Msl3 alone do not impede meiotic entry in rodents.

Keywords: male‐specific lethal 3 (Msl3); meiosis; meiotic entry; spermatogenesis.

Figure 1.

Single-cell RNA sequencing of mouse testes. A) PCA plot of different germ cell population markers for spermatogonia, spermatocytes, and round/elongating spermatids to identify the germ cell type of each cluster. B) Clusters grouped into spermatogonia, transition states, spermatocytes, and spermatids based on the marker genes they expressed to establish the trajectory of spermatogenesis. C) Msl3 is expressed in spermatocytes and continues into spermatids, then ceases as spermatids transition into elongating spermatids. D) Correlation map of different marker genes showing that Msl3 expression most closely correlates with that of Tbpl1 and Spag6, which are markers of spermatocytes.

Figure 2.

Immunofluorescence staining against Stra8 and tdTomato at p30. Stra8 in green, tdTomato in red. Immunofluorescence shows that in Stra8iCre^+/−R26tdTomato^+/− mice, Stra8 and tdTomato staining colocalize, while in tdTomato^+/− controls, Stra8 positive cells are negative for tdTomato. tdTomato is only expressed in Stra8iCre cells. Scale bar = 50μm.

Figure 3.

The strategy adopted for Msl3 conditional knock-out mouse line generation. CRISPR/Cas9 was used to insert loxP sites upstream of exon 2 and downstream of exon 4 of the Msl3 chromodomain. Upon Cre recombinase activity, exons 2–4 are excised from the Msl3 gene, generating a constitutive knockout allele.

Figure 4.

A.) Image of Msl3 wildtype (WT) (left) and Msl3 cKO (right) testes at P45. The image shows comparable gross morphology, size, and shape. B,C.) Immunofluorescence staining of coronal WT and Msl3 cKO testes: anti SP-10 staining highlighting rounds and elongated spermatids in magenta (white arrowheads), DAPI staining in gray (Scale bars 50μm). D.) Bar graph illustrating the weight of pairs of testes for Msl3 WT and Msl3 cKO mice values +/− SD, n=3. E.) Bar graph showing the average diameter of the seminiferous tubules for controls (Msl3 WT) and Msl3 cKO mice; values +/− SD (10 seminiferous tubules per animal, n=3 animals per genotype).

Figure 5.

A.) Meiotic chromosome spread of spermatocytes from P17–21 Msl3 WT and cKO testes stained with antibodies against SYCP3. Meiosis prophase I was examined between mutants and controls. Spermatocytes were characterized as leptonema, zygonema, pachynema or diplonema. Scale bar=25um B.) 209 spermatocytes were counted for WT controls and 195 for Msl3 cKO. Percentage of Spermatocytes in different meiotic subphases of meiosis prophase-I show similar meiotic progression in Msl3 WT and Msl3 cKO. N=3 per genotype

Figure 6.

A) Read counts between Msl3 WT and Msl3 cKO show little to no reads for exons 2–4, validating their excision from the gene in our cKO model. B.) Volcano plot showing significantly upregulated and downregulated genes in our Msl3 cKO model.