X Chromosome Inactivation during Drosophila Spermatogenesis

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Abstract
Genes with male- and testis-enriched expression are under-represented on the Drosophila melanogaster X chromosome. There is also an excess of retrotransposed genes, many of which are expressed in testis, that have “escaped” the X chromosome and moved to the autosomes. It has been proposed that inactivation of the X chromosome during spermatogenesis contributes to these patterns: genes with a beneficial function late in spermatogenesis should be selectively favored to be autosomal in order to avoid inactivation. However, conclusive evidence for X inactivation in the male germline has been lacking. To test for such inactivation, we used a transgenic construct in which expression of a lacZ reporter gene was driven by the promoter sequence of the autosomal, testis-specific ocnus gene. Autosomal insertions of this transgene showed the expected pattern of male- and testis-specific expression. X-linked insertions, in contrast, showed only very low levels of reporter gene expression. Thus, we find that X linkage inhibits the activity of a testis-specific promoter. We obtained the same result using a vector in which the transgene was flanked by chromosomal insulator sequences. These results are consistent with global inactivation of the X chromosome in the male germline and support a selective explanation for X chromosome avoidance of genes with beneficial effects late in spermatogenesis. During spermatogenesis, the X chromosome is inactivated in the male germline (sperm cells), thereby silencing, or inactivating, genes residing on the X chromosome. X chromosome silencing is thought to be common among species with XY sex determination and has important implications for genome evolution. For example, genes with increased expression in the male tend to be under-represented on the X chromosome, and many testes-specific genes have been “retrotransposed,” or moved, from the sex to autosomal chromosomes. However, compelling evidence for X chromosome inactivation in the fruit fly Drosophila has been lacking. Here, we used a transgenic technique to test for male germline X inactivation in this important model organism. We randomly inserted a “reporter gene” whose expression requires a regulatory element for an autosomal testis-specific gene into multiple autosomal and X-chromosomal locations. We found that autosomal insertions of the reporter gene have significantly higher expression in the male germline than X-linked insertions. This pattern holds for two different transgenes with nearly 50 independent insertions, providing strong evidence for X chromosome inactivation during spermatogenesis. The silencing of X-linked gene expression in the male germline may contribute to the observed paucity of male-expressed genes on the X chromosome and the excess of retrotransposed genes that have moved from the X chromosome to the autosomes.