Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
Andrology. 2024 May;12(4):918-931. doi: 10.1111/andr.13517. Epub 2023 Aug 22.
Phospholipase C zeta (PLCZ1) is considered the major sperm-borne oocyte activation factor. Cryopreserved stallion spermatozoa are commonly used for intracytoplasmic sperm injection (ICSI). However, plasma membrane damage and protein modifications caused by cryopreservation could impair sperm structure and function, leading to a reduction of PLCZ1 and oocyte activation after ICSI.
We compared membrane integrity and PLCZ1 abundance in populations for fresh, frozen, and refrozen stallion spermatozoa, either thawed and refrozen at room or low temperature; and examined the effect of relative PLCZ1 content on cleavage after ICSI.
Western blotting, ELISA, and immunofluorescence were conducted in stallion spermatozoa, freezing extenders, and detergent-extracted sperm fractions to detect and quantify PLCZ1. Retrospectively, PLCZ1 content and cleavage rate were analyzed. Fresh, frozen, and refrozen at room and low temperatures spermatozoa were evaluated for acrosomal and plasma membrane integrity and PLCZ1 content using flow cytometry.
Western blotting, ELISA, and immunofluorescence revealed significant reduction of PLCZ1 in spermatozoa after cryopreservation and confirmed PLCZ1 detection in extenders. After detergent extraction, a PLCZ1-nonextractable fraction remained in the postacrosomal region of spermatozoa. Plasma membrane integrity was significantly reduced after freezing. Acrosomal and plasma membrane integrity were similar between frozen and refrozen samples at low temperature, but both were significantly higher than samples refrozen at room temperature. Acrosomal and plasma membrane integrity significantly correlated to PLCZ1 content. Percentages of PLCZ1-labeled spermatozoa and PLCZ1 content were reduced after freezing but not after refreezing. Relative content and localization of PLCZ1 were associated with cleavage rates after ICSI.
Sperm PLCZ1 content associates with cleavage rates after ICSI. Cryopreservation is detrimental to sperm plasma membrane integrity and PLCZ1 retention. However, refreezing did not result in additional PLCZ1 loss. Refreezing stallion spermatozoa at a low temperature resulted in better survival but did not improve PLCZ1 retention.
磷酯酶 C ζ（PLCZ1）被认为是主要的精子源性卵母细胞激活因子。冷冻保存的种马精子常用于胞质内精子注射（ICSI）。然而，冷冻保存引起的质膜损伤和蛋白质修饰可能会损害精子结构和功能，导致 PLCZ1 减少，并降低 ICSI 后的卵母细胞激活率。
我们比较了新鲜、冷冻和反复冷冻的种马精子的质膜完整性和 PLCZ1 丰度，这些精子在室温或低温下解冻和再次冷冻；并研究了相对 PLCZ1 含量对 ICSI 后卵裂的影响。
使用 Western blot、ELISA 和免疫荧光法检测和定量精子、冷冻液和去污剂提取的精子部分中的 PLCZ1。回顾性分析 PLCZ1 含量和卵裂率。使用流式细胞术评估新鲜、冷冻和反复冷冻（室温或低温）精子的顶体和质膜完整性及 PLCZ1 含量。
Western blot、ELISA 和免疫荧光法显示，冷冻后精子中的 PLCZ1 显著减少，并证实了 PLCZ1 在扩展剂中的检测。去污剂提取后，PLCZ1 不可提取部分仍存在于精子的顶体后区。冷冻后质膜完整性显著降低。低温下冷冻和反复冷冻的样本顶体和质膜完整性相似，但均显著高于室温下反复冷冻的样本。顶体和质膜完整性与 PLCZ1 含量显著相关。冷冻后 PLCZ1 标记精子的比例和 PLCZ1 含量降低，但反复冷冻后无变化。PLCZ1 的相对含量和定位与 ICSI 后的卵裂率相关。
精子 PLCZ1 含量与 ICSI 后的卵裂率相关。冷冻保存对精子质膜完整性和 PLCZ1 保留有害。然而，反复冷冻并没有导致 PLCZ1 进一步丢失。低温下反复冷冻种马精子可提高精子存活率，但不能改善 PLCZ1 保留。
