Dosage Compensation of the X Chromosome during Sheep Testis Development Revealed by Single-Cell RNA Sequencing

SIMPLE SUMMARY: Male and female mammals carry the same complement of autosomes but differ with respect to their sex chromosomes: females carry XX chromosomes and males carry XY chromosomes. The evolutionary loss of genes from the Y chromosome led to a disparity in the dosage of X chromosomes versus autosomal genes, with males becoming monosomic for X-linked gene products. An imbalance in gene expression may have detrimental consequences. In males, X-linked genes need to be upregulated to levels equal to those of females, which is called dosage compensation. The existence of dosage compensation in germ cells is controversial. In testis, dosage compensation is thought to cease during meiosis. Some studies showed that the X chromosome is inactivated during meiosis and premature transcriptional inactivation of the X and Y chromosome during mid-spermatogenesis is essential for fertility. However, some studies failed to find support for male germline X inactivation. Using single-cell RNA seq data, in this study, we presented a comprehensive transcriptional map of sheep testes at different developmental stages and found that germ cell types in sheep testes show X-chromosome expression similar to that in the autosomes. The dosage compensation of germ cells at different stages was analyzed. MSL complex members are expressed in female flies and orthologs exist in many species, where dosage compensation mechanisms are absent or fundamentally different. This suggests that the MSL complex members also function outside of the dosage compensation machinery. Studies have shown that MSL complex can regulate mammalian X inactivation and activation. ABSTRACT: Dosage compensation is a mechanism first proposed by Susumu Ohno, whereby X inactivation balances X gene output between males (XY) and females (XX), while X upregulation balances X genes with autosomal gene output. These mechanisms have been actively studied in Drosophila and mice, but research regarding them lags behind in domestic species. It is unclear how the X chromosome is regulated in the sheep male germline. To address this, using single-cell RNA sequencing, we analyzed testes in three important developmental stages of sheep. We observed that the total RNA per cell from X and autosomes peaked in SSCs and spermatogonia and was then reduced in early spermatocytes. Furthermore, we counted the detected reads per gene in each cell type for X and autosomes. In cells experiencing dose compensation, close proximity to MSL (male-specific lethal), which is regulated the active X chromosome and was observed. Our results suggest that there is no dose compensation in the pre-meiotic germ cells of sheep testes and, in addition, MSL1 and MSL2 are expressed in early germ cells and involved in regulating mammalian X-chromosome inactivation and activation.