Lukoseviciute K, Zilinskas H, Januskauskas A
Animal Reproduction Laboratory, Department of Obstetrics and Gynecology, Lithuanian Veterinary Academy, Kaunas, Lithuania.
Reprod Domest Anim. 2005 Apr;40(2):100-7. doi: 10.1111/j.1439-0531.2004.00559.x.
The present experiment was designed to determine the effects of various biologically active substances, such as oestradiol (OE), progesterone (P4) and heparin (Hep) alone or in combination on sperm plasma membrane scrambling, capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa. Spermatozoa were incubated for 180 min in capacitation medium supplemented with (i) 1 mug/ml OE; (ii) 1 mug/ml P4; (iii) 1 mug/ml OE and 1 mug/ml P4; (iv) 1 mug/ml OE and 5 mug/ml Hep; (v) 1 mug/ml P4 and 5 mug/ml Hep; (vi) 1 mug/ml OE, 1 mug/ml P4 and 5 mug/ml Hep. At predetermined time intervals aliquots were taken to assess sperm plasma membrane scrambling, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment was aimed to study the effects of OE, P4 and OE/P4 as potential inducers of AR in Hep-capacitated spermatozoa. Plasma membrane scrambling was assessed by a flow cytometer, using Merocyanine staining. Acrosomal status and viability of spermatozoa were evaluated under epifluorescence microscope with Ethidium homodimer-1/peanut agglutinin fluorescein isothiocyanate staining method (EthD-1/PNA-FITC). The results show that OE, P4 and a combination of OE/P4 at concentrations used did not affect sperm viability. Heparin significantly (p < 0.001) increased sperm plasma membrane scrambling of OE and P4-treated spermatozoa. P4 significantly affected the rate of sperm capacitation (p < 0.001) and AR (p < 0.05), but OE expressed membrane-stabilizing properties (p < 0.05). It can be concluded that in frozen-thawed bovine spermatozoa OE presents plasma membrane stabilizing properties that can be abolished by Hep, but not by P4. Progesterone possesses capacitating and AR-inducing properties in frozen-thawed bovine spermatozoa that can be alleviated by OE.
本实验旨在确定各种生物活性物质，如雌二醇（OE）、孕酮（P4）和肝素（Hep）单独或联合使用对解冻后牛精子质膜 scrambling、获能和顶体反应（AR）的影响。精子在补充有以下成分的获能培养基中孵育180分钟：（i）1微克/毫升OE；（ii）1微克/毫升P4；（iii）1微克/毫升OE和1微克/毫升P4；（iv）1微克/毫升OE和5微克/毫升Hep；（v）1微克/毫升P4和5微克/毫升Hep；（vi）1微克/毫升OE、1微克/毫升P4和5微克/毫升Hep。在预定的时间间隔取等分试样，以评估精子质膜scrambling或精子的获能（由溶血磷脂酰胆碱诱导的AR）。第二个实验旨在研究OE、P4和OE/P4作为Hep获能精子中AR潜在诱导剂的作用。使用部花青染色通过流式细胞仪评估质膜scrambling。在落射荧光显微镜下用乙锭同二聚体-1/花生凝集素异硫氰酸荧光素染色法（EthD-1/PNA-FITC）评估精子的顶体状态和活力。结果表明，所用浓度的OE、P4和OE/P4组合不影响精子活力。肝素显著（p < 0.001）增加了OE和P4处理精子的质膜scrambling。P4显著影响精子获能率（p < 0.001）和AR（p < 0.05），但OE表现出膜稳定特性（p < 0.05）。可以得出结论，在冻融牛精子中，OE具有质膜稳定特性，可被Hep消除，但不能被P4消除。孕酮在冻融牛精子中具有获能和诱导AR的特性，可被OE缓解。
