142 Evidence against the role of Toll-like receptors 7/8 in sex selection in the mouse

Sex selection is of enormous economic value in agriculture production. Treating sperm with R848, a ligand for the X-linked Toll-like receptor 7 and 8 (TLR7/8) in mice, has been shown to reduce the motility of X sperm, allowing the separation of X- and Y-bearing sperm through the swim-up procedure, which subsequently afforded sex selection (Umehara et al. 2019 PLoS Biol. 17, e3000398). We aimed to utilise this finding in our studies. However, we were not able to repeat the prior published results. First, we tried to locate TLR7/8 on X-sperm in the mouse, human, and bovine by immunostaining. Over 90% of the murine and bovine sperm stained positively with the Bioss and Abcam anti-TLR7 antibodies. Specifically, staining with the Abcam anti-TLR7 antibody revealed only signals in the mouse sperm tails. For TLR8, 100% of murine and bovine sperm were stained positively in either the whole sperm, or all parts of the sperm except for the mid-piece. In human sperm, 75% showed positive signals for TLR7 while only 6.56% were positive for TLR8. In the TLR7-positive human sperm, as many as 57.14% were Y-bearing as shown by oligo fluorescence in situ hybridization. Second, we treated mouse sperm with TLR7/8 ligand R848 (0, 0.3, 0.03 μM) followed by the swim-up procedure and determined X- and Y-sperm ratios in the upper, middle, and lower layers by TaqMan quantitative real-time PCR. No significant differences in the ratios were found between the control and the treated sperm, and all layers showed ~1:1 ratios of X- and Y-sperm. Finally, we conducted IVF (n = 5) using superovulated murine oocytes and sperm of the upper and lower layers after R848 treatment (0, 0.03 μM). Blastocysts were collected and subjected to sexing PCR analysis. While the embryo sex ratios of some experiments were skewed towards either male or female, they were not consistently related to either the upper or lower layer. Overall, sex ratios of embryos fertilized with sperm of the upper and lower layer were 41.9% (39/93) and 39.34% (24/61), respectively. There was no significant difference between controls and treated, or between upper and lower layers (P > 0.05). In conclusion, we found that TLR7/8 could not be utilised for sex selection in the mouse. The sperm of the mouse, human, and bovine lacked X-sperm-specific expression patterns for TLR7/8.