Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, Girona, Spain.
Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, Girona, Spain.
Equine Vet J. 2022 Mar;54(2):415-426. doi: 10.1111/evj.13457. Epub 2021 Jun 1.
In several mammalian species, acute endometritis driven by the recruitment of polymorphonuclear cells (PMN) occurs in response to semen. These PMNs release DNA to form neutrophil extracellular traps (NETs) in cattle, horse and human, leading to sperm entrapment. While there is no evidence of this phenomenon occurring in donkeys, artificial insemination (AI) with frozen-thawed semen, which results in very poor pregnancy rates, leads to a large PMN recruitment to the uterus.
To investigate whether donkey semen can trigger NET release (NETosis) and if excessive NETosis occurs in response to frozen-thawed semen.
In vitro experiments.
Jenny PMNs were exposed to jackass fresh or frozen-thawed semen, isolated sperm or seminal plasma (SP), over the course of three experiments. NET formation in response to different treatments was assessed through manual quantification of stained slides. A one-way analysis of variance (ANOVA), followed by a post hoc Sidak test, was carried out to determine statistical significance.
NET release occurred in a semen concentration- and incubation-time-dependent manner. Surprisingly, frozen-thawed donkey sperm did not increase NETosis rate in comparison with the control (23 ± 2.5% vs. 31 ± 3.7%; P > .05), whereas fresh semen exposure did (78 ± 5.7% vs. 26 ± 3.2%, P < .01). NETosis increased in the presence of SP, regardless of the presence or absence of sperm, in comparison with the control in both fresh (84 ± 5.2% and 77 ± 5.0% vs. 12 ± 2.7%, respectively; P < .01) and frozen (95 ± 2.2% and 94 ± 2.9% vs. 14 ± 3.8%, respectively; P < .01) samples. Moreover, exposure of PMN to viable and motile sperm, in the absence of SP, did not increase NETosis rates (P > .05).
Donkey SP, and not sperm-intrinsic factors, is able to trigger NETosis in both time- and semen concentration-dependent manner. The physiological relevance of such response against semen in the donkey remains to be elucidated.
在几种哺乳动物中，多形核细胞（PMN）募集引起的急性子宫内膜炎是对精液的反应。这些PMN 在牛、马和人中释放 DNA 形成中性粒细胞细胞外陷阱（NETs），导致精子捕获。虽然没有证据表明这种现象在驴中发生，但冷冻解冻精液的人工授精（AI）导致非常低的妊娠率，导致大量PMN 募集到子宫。
研究驴精液是否能触发 NET 释放（NETosis），以及冷冻解冻精液是否会引起过度的 NETosis。
体外实验。
在三个实验中，将 Jenny PMN 暴露于公驴新鲜或冷冻解冻精液、分离精子或精浆（SP）中。通过手动定量染色载玻片评估不同处理下 NET 的形成。采用单因素方差分析（ANOVA），然后是事后 Sidak 检验，以确定统计学意义。
NET 释放呈精液浓度和孵育时间依赖性。令人惊讶的是，与对照组相比，冷冻解冻的驴精子并没有增加 NETosis 率（23 ± 2.5%比 31 ± 3.7%；P >.05），而新鲜精液暴露则增加了（78 ± 5.7%比 26 ± 3.2%，P <.01）。无论是否存在精子，SP 的存在都会导致 NETosis 增加，与新鲜（84 ± 5.2%和 77 ± 5.0%比 12 ± 2.7%，分别；P <.01）和冷冻（95 ± 2.2%和 94 ± 2.9%比 14 ± 3.8%，分别；P <.01）样本中的对照组相比。此外，在不存在 SP 的情况下，PMN 暴露于有活力和有活力的精子并不会增加 NETosis 率（P >.05）。
驴 SP，而不是精子内在因素，能够以时间和精液浓度依赖的方式触发 NETosis。这种针对精液的反应在驴中的生理相关性仍有待阐明。
