TLR7/8 signalling affects X-sperm motility via the GSK3 α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos

Abstract

Background Goat milk is very similar to human milk in terms of its abundant nutrients and ease of digestion. To derive greater economic benefit, farmers require more female offspring (does); however, the buck-to-doe offspring sex ratio is approximately 50%. At present, artificial insemination after the separation of X/Y sperm using flow cytometry is the primary means of controlling the sex of livestock offspring. However, flow cytometry has not been successfully utilised for the separation of X/Y sperm aimed at sexing control in dairy goats. Results In this study, a novel, simple goat sperm sexing technology that activates the toll-like receptor 7/8 (TLR7/8), thereby inhibiting X-sperm motility, was investigated. Our results showed that the TLR7/8 coding goat X-chromosome was expressed in approximately 50% of round spermatids in the testis and sperm, as measured from cross-sections of the epididymis and ejaculate, respectively. Importantly, TLR7/8 was located at the tail of the X-sperm. Upon TLR7/8 activation, phosphorylated forms of glycogen synthase kinase α/β (GSK3 α/β) and nuclear factor kappa-B (NF-κB) were detected in the X-sperm, causing reduced mitochondrial activity, ATP levels, and sperm motility. High-motility Y-sperm segregated to the upper layer and the low-motility X-sperm, to the lower layer. Following in vitro fertilisation using the TLR7/8-activated sperm from the lower layer, 80.52 ± 6.75% of the embryos were XX females. The TLR7/8-activated sperm were subsequently used for in vivo embryo production via the superovulatory response; nine embryos were collected from the uterus of two does that conceived. Eight of these were XX embryos, and one was an XY embryo. Conclusions Our study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3 α/β-hexokinase pathway; this technique could be used to facilitate the efficient production of sexed dairy goat embryos.

Gender Control of Mouse Embryos by Activation of TLR7/8 on X Sperm via Ligands dsRNA-40 and dsRNA-DR

“…In 2022, Huang [ 20 ] combined R848 with an alkaline semen diluent, achieving the successful isolation and enrichment of goat X sperm. This sex control technique, relying on the R848 activation of X sperm TLR7/8, was successfully validated in several mammals, including mice, cattle, and sheep, confirming the robustness of this sex control technology [ 17 , 18 , 19 , 20 , 21 ].…”

“…The findings of Umehara [ 17 ] and Renfa [ 19 ] have shown that, after sperm treatment with R848, highly motile Y-bearing sperm are primarily located in the upper layer, while the lower layer predominantly contains slow-moving X-bearing sperm. To investigate whether the treatment of sperm with dsRNA-40 or dsRNA-DR can alter the ratio of X/Y sperm in the upper and lower layers, this study employed quantitative PCR based on the X-chromosome-specific gene Akap4 and the Y-chromosome-specific gene ddx3y to assess the percentage of Y and X sperm in the upper and lower layers.…”

“…Umehara later applied this system to separate frozen cattle semen, with similar results [ 18 ], offering a fresh perspective on animal sex control technology. In 2021, Chinese scientists led by Ren [ 19 ] applied this approach to treat goat sperm with R848, resulting in successful artificial insemination that produced sexually oriented young. Their research elucidated that R848 treatment inhibited X sperm motility in goats through the TLR7/8 signaling pathway, affecting X sperm ATP levels via the GSK3α/β–hexokinase pathway.…”

Single-cell transcriptomics reveals male germ cells and Sertoli cells developmental patterns in dairy goats

“…Testis samples were performed immunofluorescence as described (Ren et al, 2021). Briefly, the sections were deparaffinized, rehydrated, and infiltrated with 0.5% Triton X-100 for 10 min.…”

“…Owing to their environmental adaptability, these goats can increase the income of farmers. Moreover, the nutritional value of goat milk is high owing to its ease of digestion and absorption (Ren et al, 2021). This makes the dairy goat industry a major player in the dairy industry (Clark and Mora García, 2017).…”