Rajamanickam Gayathri D, Kastelic John P, Thundathil Jacob C
Faculty of Veterinary Medicine, Department of Production Animal Health, University of Calgary, Calgary, Alberta, Canada.
Mol Reprod Dev. 2017 Nov;84(11):1168-1182. doi: 10.1002/mrd.22879. Epub 2017 Sep 27.
Highly dynamic lipid microdomains (rafts) in the sperm plasma membrane contain several signaling proteins that regulate sperm capacitation. Na/K-ATPase isoforms (testis-specific isoform ATP1A4 and ubiquitous isoform ATP1A1) are abundant in bovine sperm plasma membrane. We previously reported that incubation of bovine sperm with ouabain, a specific Na/K-ATPase ligand, induced tyrosine phosphorylation of several sperm proteins during capacitation. The objective of this study was to investigate the roles of lipid rafts and non-rafts in Na/K-ATPase enzyme activity and signaling during bovine sperm capacitation. Content of ATP1A4 and, to a lesser extent, ATP1A1 was increased in raft and non-raft fractions of capacitated sperm, although non-raft enzyme activities of both isoforms were higher than the corresponding activities in rafts from capacitated sperm. Yet, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in both rafts and non-rafts. A comparative increase in phosphorylation of signaling molecules was observed in both raft (CAV1) and non-raft (EGFR and ERK1/2) membrane fractions during capacitation. Although SRC was phosphorylated in both membrane fractions, the non-raft fraction possessed more of this activated form. We also inferred, by immunoprecipitation, that ATP1A4 interacted with CAV1 and EGFR in the raft fraction, whereas interactions of ATP1A4 with SRC, EGFR, and ERK1/2 occurred in the non-raft fraction of ouabain-capacitated sperm; conversely, ATP1A1 interacted only with CAV1 in both fractions of uncapacitated and capacitated sperm. In conclusion, both raft and non-raft cohorts of Na/K-ATPase isoforms contributed to phosphorylation of signaling molecules during bovine sperm capacitation.
精子质膜中高度动态的脂质微区（脂筏）包含多种调节精子获能的信号蛋白。钠钾ATP酶同工型（睾丸特异性同工型ATP1A4和普遍存在的同工型ATP1A1）在牛精子质膜中含量丰富。我们之前报道，用哇巴因（一种特异性钠钾ATP酶配体）孵育牛精子，可在获能过程中诱导多种精子蛋白的酪氨酸磷酸化。本研究的目的是探讨脂筏和非脂筏在牛精子获能过程中对钠钾ATP酶活性及信号传导的作用。尽管两种同工型的非脂筏酶活性均高于获能精子脂筏中的相应活性，但获能精子的脂筏和非脂筏部分中ATP1A4的含量增加，ATP1A1的含量在较小程度上有所增加。然而，ATP1A4是脂筏和非脂筏中总钠钾ATP酶活性的主要同工型。在获能过程中，脂筏（CAV1）和非脂筏（EGFR和ERK1/2）膜部分的信号分子磷酸化均出现了相对增加。尽管SRC在两个膜部分均被磷酸化，但非脂筏部分中这种活化形式的含量更多。我们还通过免疫沉淀推断，在脂筏部分中ATP1A4与CAV1和EGFR相互作用，而在哇巴因诱导获能的精子的非脂筏部分中，ATP1A4与SRC、EGFR和ERK1/2发生相互作用；相反，在未获能和获能精子的两个部分中，ATP1A1仅与CAV1相互作用。总之，钠钾ATP酶同工型的脂筏和非脂筏群体在牛精子获能过程中均对信号分子的磷酸化有贡献。
