Department of Veterinary Biochemistry, College of Veterinary & Animal Science, Rajasthan University of Animal and Veterinary Sciences, Bikaner, Rajasthan, India.
Division of Animal Physiology & Biochemistry, ICAR-Central Sheep and Wool Research Institute, Avikanagar, Rajasthan, India.
Cell Biochem Funct. 2024 Jan;42(1):e3930. doi: 10.1002/cbf.3930.
Mammalian sperm remain quiescent but fertile for several weeks in cauda epididymis. Although several sperm quiescent factors of epididymal plasma have been identified in goat, pig and cattle; however, little is known in sheep. In the present study, purification and characterization of a novel sperm quiescent protein of ovine cauda epididymal plasma (CEP) was carried out. The sperm quiescent protein was partially purified by hydroxyapatite gel adsorption chromatography followed by DEAE-sepharose® anion exchange chromatography. In the latter, the sperm quiescent activity was eluted both in 0.05 and 0.2 M potassium phosphate buffer (pH 7.5) fractions having a predominant protein of about 80 and 70 kDa with 87% and 63% homogeneity, respectively. The proteins were designated as motility-inhibitory factor of sheep I and II (MIFS-I and II), respectively. Significant (about 60%) inhibition of sperm motility was observed following treatment of cauda epididymal sperm with 6 and 12 µg/mL of partially purified MIFS-I and II, respectively. Specific activities of the partially purified MIFS-I and II were 563 and 261 U/mg of protein, while the fold-purification of the activity were 5119 and 2373, respectively. Both the proteins were heat-labile and the activity was completely lost following incubation at 100°C for 5 min. Further, the partially purified MIFS-I (5 µg/mL) caused significant reduction in in vitro sperm capacitation and slight decline in tyrosine phosphorylated p72 and p52 proteins; however the protein was nontoxic to sperm. Mass spectrometric analysis of MIFS-I revealed significant identity with human semaphorin 3D. Both dot blot and western blot analysis demonstrated cross-reactivity of MIFS-I with polyclonal anti-human SEMA3D antibody. It was concluded that the MIFS-I of ovine CEP was putative ovine semaphorin 3D protein having potent sperm quiescent and decapacitating activities and it possibly acts through inhibition of protein tyrosine phosphorylation.
哺乳动物精子在附睾尾部保持静止但仍具有生育能力，可存活数周。尽管已经在山羊、猪和牛中鉴定出几种附睾血浆中的精子静止因子，但在绵羊中知之甚少。在本研究中，对绵羊附睾尾部血浆（CEP）的一种新型精子静止蛋白进行了分离和鉴定。该蛋白通过羟磷灰石凝胶吸附色谱法初步纯化，然后通过 DEAE-琼脂糖®阴离子交换色谱法进一步纯化。在后一种方法中，精子静止活性在 0.05 和 0.2 M 磷酸钾缓冲液（pH 7.5）洗脱液中洗脱，两种洗脱液中均含有约 80 和 70 kDa 的主要蛋白，分别具有 87%和 63%的纯度。这些蛋白分别被命名为绵羊 I 和 II 的运动抑制因子（MIFS-I 和 II）。用部分纯化的 MIFS-I 和 II 处理附睾尾部精子后，精子运动显著抑制（约 60%），浓度分别为 6 和 12 μg/mL。部分纯化的 MIFS-I 和 II 的比活性分别为 563 和 261 U/mg 蛋白，活性的比纯化倍数分别为 5119 和 2373。两种蛋白均不耐热，在 100°C 孵育 5 min 后，活性完全丧失。此外，部分纯化的 MIFS-I（5 μg/mL）可显著降低体外精子获能，并轻微降低酪氨酸磷酸化的 p72 和 p52 蛋白，但该蛋白对精子无毒性。MIFS-I 的质谱分析显示与人 Sema3D 有显著的同一性。斑点印迹和 Western blot 分析表明 MIFS-I 与多克隆抗人 SEMA3D 抗体发生交叉反应。综上所述，绵羊 CEP 的 MIFS-I 可能是绵羊 Sema3D 蛋白，具有潜在的精子静止和脱能活性，可能通过抑制蛋白酪氨酸磷酸化起作用。
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