In most mammals, the male to female sex ratio of offspring is about 50% because half of the sperm contain either the Y chromosome or X chromosome. In mice, the Y chromosome encodes fewer than 700 genes, whereas the X chromosome encodes over 3,000 genes. Although overall gene expression is lower in sperm than in somatic cells, transcription is activated selectively in round spermatids. By regulating the expression of specific genes, we hypothesized that the X chromosome might exert functional differences in sperm that are usually masked during fertilization. In this study, we found that Toll-like receptors 7/8 (TLR7/ 8) coding the X chromosome were expressed by approximately 50% of the round spermatids in testis and in approximately 50% of the epididymal sperm. Especially, TLR7 was localized to the tail, and TLR8 was localized to the midpiece. Ligand activation of TLR7/8 selectively suppressed the mobility of the X chromosome-bearing sperm (X-sperm) but not the Y-sperm without altering sperm viability or acrosome formation. The difference in sperm motility allowed for the separation of Y-sperm from X-sperm. Following in vitro fertilization using the ligand-selected high-mobility sperm, 90% of the embryos were XY male. Likewise, 83% of the pups obtained following embryo transfer were XY males. Conversely, the TLR7/8-activated, slow mobility sperm produced embryos and pups that were 81% XX females. Therefore, the functional differences between Y-sperm and X-sperm motility were revealed and related to different gene expression patterns, specifically TLR7/8 on X-sperm. Activation of Toll-like receptor 7/8 encoded by the X chromosome alters sperm motility and provides a novel simple technology for sexing sperm. PLoS Biol 17(8): e3000398. https://doi.including the nuclear condensation, acrosome formation, and the elongation of the sperm flagella [3,4]. As indicated by studies using knockout mouse models, the morphological changes associated with spermiogenesis are associated with the expression of various genes, including Tekt, Tnp, and Gba2 in the round spermatids [5][6][7]. Thus, active gene transcription occurs on chromosomes, including sex chromosomes, in haploid male germ cells, and some of them are essential for cell survival [8]. Using the cytoplasmic bridges between spermatids, cytoplasm including RNAs and proteins are shared to rescue Y chromosome bearing sperm (Y-sperm) [8,9]. It has been reported that the bridge works in spermatid at early stage of spermiogenesis, and the high levels of RNA polymerase II are detected in this stage [10,11]. However, RNA polymerase II is still detected in later stages of spermiogenesis [11], indicating that the unique features of sperm can be distinguished not only by the presence of the X or Y chromosome but also by expression of distinct genes encoded by each sex chromosome.However, mouse Y chromosome encodes fewer than 700 genes, whereas mouse X chromosome encodes over 3,000 genes [12,13]. The X chromosome contains some unique genes, such as Taz encoding tafazzin that is a t...
