Imam S, Ansari M R, Ahmed N, Kumaresan A
Artificial Insemination Laboratory, Division of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India.
Theriogenology. 2008 May;69(8):925-31. doi: 10.1016/j.theriogenology.2007.05.066. Epub 2008 Mar 21.
The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS-PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen-thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P<0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P<0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity.
目的是确定输卵管蛋白对精子功能的影响。用磷酸盐缓冲液（PBS）冲洗从屠宰场获取的水牛输卵管；回收的液体（蛋白质浓度为2.3 mg/mL；平均每条输卵管含3.5 mg蛋白质）经离心、透析和澄清处理，然后将上清液应用于肝素-琼脂糖亲和柱。收集未结合部分，并用洗脱缓冲液分别洗脱结合的蛋白质。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中，在未结合肝素（H-未结合）部分检测到8条不同的蛋白带（12至177 kDa），在结合肝素（H-结合）部分检测到15条不同的蛋白带（12至165 kDa）。来自4头公水牛的精液分为三部分：第1部分和第2部分分别用肝素结合（H-结合）和非肝素结合（H-未结合）的输卵管蛋白处理，而第3部分作为未处理的对照。对平衡后并经冻融的精液进行活力、生存力、顶体完整率、黏液穿透距离和低渗肿胀试验评估。H-结合的输卵管液蛋白可提高（P<0.05）在冷冻保存期间呈进行性运动、存活、顶体完整且质膜功能正常（低渗肿胀试验）的精子比例，以及在宫颈黏液精子穿透试验中覆盖的距离。添加H-未结合的输卵管蛋白部分并未增加精子活力和穿透距离，但增加了（P<0.05）存活、顶体完整且质膜功能正常（低渗肿胀试验）的精子比例。我们得出结论，水牛输卵管液蛋白的H-结合部分在冷冻保存期间维持精子活力、生存力和膜完整性，而H-未结合蛋白维持精子生存力和膜完整性。
