Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, the Netherlands.
Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.
Biol Reprod. 2022 Nov 14;107(5):1242-1253. doi: 10.1093/biolre/ioac169.
In a previous study, we reported that porcine sperm cysteine-rich secretory protein 2 (CRISP2) is localized in the post-acrosomal sheath-perinuclear theca (PT) as reduction-sensitive oligomers. In the current study, the decondensation and removal of CRISP2 was investigated during in vitro sperm capacitation, after both the induction of the acrosome reaction and in vitro fertilization. Confocal immunofluorescent imaging revealed that additional CRISP2 fluorescence appeared on the apical ridge and on the equatorial segment (EqS) of the sperm head following capacitation, likely due to cholesterol removal. After an ionophore A23187-induced acrosome reaction, CRISP2 immunofluorescence disappeared from the apical ridge and the EqS area partly not only owing to the removal of the acrosomal shroud vesicles, but to its presence in a subdomain of EqS. The fate of sperm head CRISP2 was further examined post-fertilization. In vitro matured porcine oocytes were co-incubated with boar sperm cells for 6-8 h and the zygotes were processed for CRISP2 immunofluorescent staining. Notably, decondensation of CRISP2, and thus of the sperm PT, occurred while the sperm nucleus was still fully condensed. CRISP2 was no longer detectable in fertilized oocytes in which sperm nuclear decondensation and paternal pronucleus formation were apparent. This rapid dispersal of CRISP2 in the PT is likely regulated by redox reactions for which its cysteine-rich domain is sensitive. Reduction of disulfide bridges within CRISP2 oligomers may be instrumental for PT dispersal and elimination.
在之前的研究中，我们报道了猪精子富含半胱氨酸的分泌蛋白 2（CRISP2）作为还原敏感的低聚物定位于顶体后-致密纤维鞘-核周鞘（PT）中。在本研究中，我们研究了在体外获能过程中 CRISP2 的去浓缩和去除，该过程在顶体反应的诱导和体外受精之后进行。共聚焦免疫荧光成像显示，在获能后，顶体反应和赤道段（EqS）上出现了更多的 CRISP2 荧光，可能是由于胆固醇的去除。在离子载体 A23187 诱导的顶体反应后，CRISP2 免疫荧光从顶体反应嵴和 EqS 区域部分消失，不仅是由于顶体帽囊泡的去除，而且还由于其存在于 EqS 的亚域中。进一步研究了精子头部 CRISP2 的命运。体外成熟的猪卵母细胞与公猪精子共同孵育 6-8 小时，然后对受精卵进行 CRISP2 免疫荧光染色。值得注意的是，在精子核仍完全浓缩的情况下，CRISP2 发生去浓缩，即精子 PT 发生去浓缩。在可见精子核去浓缩和雄性原核形成的受精卵母细胞中，不再检测到 CRISP2。PT 中 CRISP2 的这种快速分散可能受其富含半胱氨酸的结构域敏感的氧化还原反应调控。CRISP2 低聚物中二硫键的还原可能是 PT 分散和消除的关键。
