Quantitative phosphoproteomics analyses reveal the regulatory mechanisms related to frozen-thawed sperm capacitation and acrosome reaction in yak (Bos grunniens)

Renzheng Zhang; Chunnian Liang; Xian Guo; Pengjia Bao; Jie Pei; Fude Wu; Mancai Yin; Min Chu; Ping Yan

doi:10.3389/fphys.2022.1013082

Quantitative phosphoproteomics analyses reveal the regulatory mechanisms related to frozen-thawed sperm capacitation and acrosome reaction in yak (Bos grunniens)

Front Physiol. 2022 Oct 6:13:1013082. doi: 10.3389/fphys.2022.1013082. eCollection 2022.

Authors

Renzheng Zhang^{1

2}, Chunnian Liang¹, Xian Guo¹, Pengjia Bao¹, Jie Pei¹, Fude Wu³, Mancai Yin³, Min Chu¹, Ping Yan¹

Affiliations

¹ Key Laboratory of Animal Genetics and Breeding on Tibetan Plateau, Ministry of Agriculture and Rural Affairs, Key Laboratory of Yak Breeding Engineering of Gansu Province, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou, China.
² College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, China.
³ Yak Breeding and Extension Service Center in in Qinghai Province, Xining, China.

Abstract

Mammalian spermatozoa are not mature after ejaculation and must undergo additional functional and structural changes within female reproductive tracts to achieve subsequent fertilization, including both capacitation and acrosome reaction (AR), which are dominated by post-translational modifications (PTMs), especially phosphorylation. However, the mechanism of protein phosphorylation during frozen-thawed sperm capacitation and AR has not been well studied. In this study, the phosphoproteomics approach was employed based on tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy to analyze frozen-thawed sperm in Ashidan yak under three sequential conditions (density gradient centrifugation-based purification, incubation in the capacitation medium and induction of AR processes by the calcium ionophore A23187 treatment). The identification of 1,377 proteins with 5,509 phosphorylation sites revealed changes in phosphorylation levels of sperm-specific proteins involved in regulation of spermatogenesis, sperm motility, energy metabolism, cilium movement, capacitation and AR. Some phosphorylated proteins, such as AKAP3, AKAP4, SPA17, PDMD11, CABYR, PRKAR1A, and PRKAR2A were found to regulate yak sperm capacitation and AR though the cAMP/PKA signaling pathway cascades. Notably, the phosphorylation level of SPA17 at Y156 increased in capacitated sperm, suggesting that it is also a novel functional protein besides AKAPs during sperm capacitation. Furthermore, the results of this study suggested that the phosphorylation of PRKAR1A and PRKAR2A, and the dephosphorylation of CABYR both play key regulatory role in yak sperm AR process. Protein-protein interaction analysis revealed that differentially phosphorylated proteins (AKAP3, AKAP4, FSIP2, PSMD11, CABYR, and TPPP2) related to capacitation and AR process played a key role in protein kinase A binding, sperm motility, reproductive process, cytoskeleton and sperm flagella function. Taken together, these data provide not only a solid foundation for further exploring phosphoproteome of sperm in yak, but an efficient way to identify sperm fertility-related marker phosphorylated proteins.

Keywords: acrosome reaction; capacitation; phosphorylation; post-translational modifications; yak sperm.