Department of Physiology, Veterinary Faculty, University of Murcia, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), Murcia, Spain.
Institute for Biomedical Research of Murcia (IMIB), Murcia, Spain.
J Assist Reprod Genet. 2019 Aug;36(8):1721-1736. doi: 10.1007/s10815-019-01526-6. Epub 2019 Jul 19.
Nitric oxide (NO) is a free radical synthesized mainly by nitric oxide synthases (NOSs). NO regulates many aspects in sperm physiology in different species. However, in vitro studies investigating NOS distribution, and how NO influences sperm capacitation and fertilization (IVF) in porcine, have been lacking. Therefore, our study aimed to clarify these aspects.
Two main experiments were conducted: (i) boar spermatozoa were capacitated in the presence/absence of S-nitrosoglutathione (GSNO), a NO donor, and two NOS inhibitors, N-nitro-L-arginine methyl ester hydrochloride (L-NAME) and aminoguanidine hemisulfate salt (AG), and (ii) IVF was performed in the presence or not of these supplements, but neither the oocytes nor the sperm were previously incubated in the supplemented media.
Our results suggest that NOS distribution could be connected to pathways which lead to capacitation. Treatments showed significant differences after 30 min of incubation, compared to time zero in almost all motility parameters (P < 0.05). When NOSs were inhibited, three protein kinase A (PKA) substrates (~ 75, ~ 55, and ~50 kDa) showed lower phosphorylation levels between treatments (P < 0.05). No differences were observed in total tyrosine phosphorylation levels evaluated by Western blotting nor in situ. The percentage of acrosome-reacted sperm and phosphatidylserine translocation was significantly lower with L-NAME. Both inhibitors reduced sperm intracellular calcium concentration and IVF parameters, but L-NAME impaired sperm ability to penetrate denuded oocytes.
These findings point out to the importance of both sperm and cumulus-oocyte-derived NO in the IVF outcome in porcine.
一氧化氮（NO）是一种主要由一氧化氮合酶（NOS）合成的自由基。NO 调节不同物种精子生理的许多方面。然而，在体外研究中，NOS 的分布以及 NO 如何影响猪精子获能和受精（IVF）的研究还很缺乏。因此，本研究旨在阐明这些方面。
进行了两项主要实验：（i）在存在/不存在 S-亚硝基谷胱甘肽（GSNO）、一氧化氮供体、两种 NOS 抑制剂 N-硝基-L-精氨酸甲酯盐酸盐（L-NAME）和氨基胍半硫酸盐盐（AG）的情况下，对猪精子进行获能；（ii）在存在或不存在这些补充剂的情况下进行 IVF，但卵母细胞和精子都没有在补充培养基中预先孵育。
我们的结果表明，NOS 的分布可能与导致获能的途径有关。与零时相比，在几乎所有运动参数中，处理后 30 分钟的差异均具有统计学意义（P<0.05）。当 NOS 被抑制时，三种蛋白激酶 A（PKA）底物（75、55 和~50 kDa）在处理之间的磷酸化水平较低（P<0.05）。Western blot 检测到的总酪氨酸磷酸化水平或原位均未观察到差异。与 L-NAME 相比，顶体反应精子和磷酯酰丝氨酸转位的比例明显较低。两种抑制剂均降低了精子细胞内钙离子浓度和 IVF 参数，但 L-NAME 损害了精子穿透去卵丘卵母细胞的能力。
这些发现指出了猪精子和卵丘-卵母细胞来源的 NO 在 IVF 结果中的重要性。
