Cryodamage is a major problem in semen cryopreservation, causing changes in the levels of proteins that influence the function and motility of spermatozoa. In this study, protein samples prepared from fresh and frozen–thawed boar spermatozoa were compared using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled to 2D LC–MS/MS analysis. A total of 41 differentially expressed proteins were identified and quantified, including 35 proteins that were present at higher levels and six proteins that were present at lower levels in frozen–thawed spermatozoa by at least a mean of 1.79-fold (P<0.05). On classifying into ten distinct categories using bioinformatic analysis, most of the 41 differentially expressed proteins were found to be closely relevant to sperm premature capacitation, adhesions, energy supply, and sperm–oocyte binding and fusion. The expression of four of these proteins, SOD1, TPI1, ODF2, and AKAP3, was verified by western blot analysis. We propose that alterations in these identified proteins affect the quality of cryopreserved semen and ultimately lower its fertilizing capacity. This is the first study to compare protein levels in fresh and frozen–thawed spermatozoa using the iTRAQ technology. Our preliminary results provide an overview of the molecular mechanisms of cryodamage in frozen–thawed spermatozoa and theoretical guidance to improve the cryopreservation of boar semen.
Xiaoli Chen, Huabin Zhu, Chuanhuo Hu, Haisheng Hao, Junfang Zhang, Kunpeng Li, Xueming Zhao, Tong Qin, Kan Zhao, Huishan Zhu and Dong Wang
Cheng Zeng, Pei-Li Wu, Zhao-Tong Dong, Xin Li, Ying-Fang Zhou and Qing Xue
Endometriosis is an estrogen-dependent disease, and estrogen receptor 2 (ESR2) plays a critical role in the pathogenesis of ovarian endometriosis by promoting cell invasion. Yes-associated protein 1 (YAP1) plays suppressive roles in several types of tumors. However, the relationship between YAP1 and ESR2 is not fully understood. The aim of this study was to investigate the regulatory mechanism of YAP1 in terms of ESR2 and YAP1 regulation of endometriotic stromal cell (ECSC) invasion in ovarian endometriosis. Our results demonstrated that YAP1 mRNA and protein levels in eutopic endometrium (EU) tissues were higher than those in paired ectopic endometrium (EC) tissues. ECSCs transfected with siYAP1 exhibited a significant increase in both ESR2 mRNA levels and protein expression. Simultaneously, YAP1 overexpression in ECSCs yielded the opposite results. Co-IP assays demonstrated YAP1-NuRD complex formation by YAP1, CHD4 and MTA1 in ECSCs. YAP1 bound to two sites, (-539, -533) and (-158, -152), upstream of the ESR2 transcription initiation site. YAP1 binding to the two sites of the ESR2 promoter in ECSCs was significantly lower than that in eutopic endometrial stromal cells (EUSCs) from EU tissues. ECSCs transfected with siYAP1 exhibited increased invasion activity, while ECSCs transfected with siESR2 showed inhibition of invasion. However, transfection with siYAP1 and siESR2 together decreased the number of invading cells compared with transfection with siYAP1 alone. Therefore, we conclude that decreased levels of YAP1 in ovarian endometriomas enhance ESR2 expression via formation of a YAP1-NuRD complex, which further binds to the ESR2 promoters. Furthermore, YAP1 inhibits ECSCs invasion.