TMT-based quantitative N-glycoproteomic analysis reveals glycoprotein protection can improve the quality of frozen bovine sperm

Cryopreservation of bovine semen plays a vital role in accelerating genetic improvement and elite breeding, but it has a detrimental effect on sperm quality, resulting in the decline of the reproductive efficiency. The glycosylation modification of protein has irreplaceable roles in spermatozoa. Herein, the effect of cryopreservation on glycoproteins of bovine spermatozoa has been studied for the first time using a tandem mass tag (TMT)-labeled quantitative glycoproteome. A total of 2598 proteins and 492 glycoproteins were identified, including 83 different expression proteins (DEPs) and 44 different expression glycosylated proteins (DEGPs) between fresh and frozen spermatozoa. Thirty-three DEPs are glycoproteins, which demonstrates that glycoproteins of bovine sperm were seriously affected by cryopreservation. Moreover, the effects include glycoprotein expression, glycosylation modification, and substructure localization for proteins such as glycoproteins TEX101, ACRBP, and IZOMU4. The biologic functions of the 115 changed proteins are mainly involved in sperm capacitation, migration in female genitalia, and sperm–egg interaction. Mostly key regulators were identified to be glycoproteins, which confirms that glycosylated proteins played important roles in bovine sperm. This comprehensive study of sperm glycoproteins helps to unravel the cryoinjury mechanisms, thus implying that glycoprotein protection should be an effective way to improve the quality of frozen sperm.