Ram sperm are particularly sensitive to freeze-thawing mainly due to their lipid composition, limiting their use in artificial insemination programs. We evaluated the extent of cholesterol and desmosterol incorporation into ram sperm through incubation with increasing concentrations of methyl-β-cyclodextrin (MβCD)-sterol complexes, and its effect on membrane biophysical properties, membrane lateral organization and cryopreservation outcome. Sterols were effectively incorporated into the sperm membrane at 10 and 25 mM MβCD-sterols, similarly increasing membrane lipid order at physiological temperature and during temperature decrease. Differential ordering effect of sterols in ternary-mixture model membranes revealed a reduced tendency of desmosterol of segregating into ordered domains. Live cell imaging of fluorescent cholesterol showed sterol incorporation and evidenced the presence of sperm sub-populations compatible with different sterol contents and a high concentration of sterol rich-ordered domains mainly at the acrosome plasma membrane. Lateral organization of the plasma membrane, assessed by identification of GM1-related rafts, was preserved after sterol incorporation except when high levels of sterols (25 mM MβCD-desmosterol) were incorporated. Ram sperm incubation with 10 mM MβCD-sterols prior to cryopreservation in a cholesterol-free extender improved sperm quality parameters after cooling and freezing. While treatment with 10 mM MβCD-cholesterol increased sperm motility, membrane integrity and tolerance to osmotic stress after thawing, incorporation of desmosterol increased the ability of ram sperm to overcome osmotic stress. Our research provides evidence on the effective incorporation and biophysical behavior of cholesterol and desmosterol in ram sperm membranes and on their consequences in improving functional parameters of sperm after temperature decrease and freezing.

中文翻译：

---

冷冻前将胆固醇和去骨甾醇掺入ram精子膜中：对膜生物物理特性和精子质量的影响。

主要由于其脂质成分，Ram精子对冻融特别敏感，从而限制了它们在人工授精程序中的使用。我们通过与浓度不断增加的甲基-β-环糊精（MβCD）-甾醇复合物孵育，评估了胆固醇和去骨甾醇掺入ram精子中的程度，以及其对膜生物物理特性，膜侧向组织和冷冻保存结果的影响。在10和25 mM的MβCD-固醇下，将甾醇有效地掺入精子膜中，在生理温度和温度降低过程中，类似地增加了膜脂质的顺序。三元混合物模型膜中固醇的不同有序作用表明减少了去甾醇分离成有序域的趋势。荧光胆固醇的活细胞成像显示了固醇的掺入，并证明了与不同固醇含量兼容的精子亚群的存在，以及主要在顶体质膜上存在高浓度的富含固醇的有序域。除掺入高水平的固醇（25 mMMβCD-去甾醇）外，在固醇掺入后，通过鉴定GM1相关筏确定了质膜的侧向组织。在无胆固醇的补充剂中冷冻保存之前，将Ram精子与10 mMMβCD-固醇一起孵育可改善精子质量参数，使其冷却和冷冻。尽管用10 mMMβCD-胆固醇治疗可提高精子运动能力，膜完整性和融化后对渗透压的耐受性，掺入去氢雌甾醇可提高ram精子克服渗透压的能力。我们的研究为降低精子温度和冷冻后胆固醇和去甾醇在精子细胞膜中的有效掺入和生物物理行为以及它们对改善精子功能参数的后果提供了证据。