Lefebvre R, Suarez S S
Department of Anatomy, Cornell University, Ithaca, New York 14853, USA.
Biol Reprod. 1996 Mar;54(3):575-82. doi: 10.1095/biolreprod54.3.575.
Sperm binding to oviductal epithelium is thought to be an important mechanism regulating sperm reservoir formation in the oviduct. On the basis of evidence in the hamster, we hypothesized that capacitation affects release of bovine sperm, allowing them to fertilize. Oviducts were obtained from the ovulatory side of estrous Holstein heifers. The isthmic and ampullar epithelia were milked out and reduced to fragments, which formed everted vesicles (explants). Explants were placed in tissue culture wells in TALP medium and incubated at 39 degrees C in 5% CO2. Frozen-thawed sperm were prepared by swim-up in TALP and capacitated by incubation for 4 h in TALP with 20 micrograms/ml heparin (without glucose). Uncapacitated sperm were used immediately after dilution into capacitation medium. Within 2 h of surgery, sperm were added to the explants and incubated with them for 15 min. Sperm and explants were videotaped, and the tapes were analyzed to determine the numbers of sperm bound per surface area. ANOVA did not show a difference between the number of sperm bound/0.1 mm2 in the isthmus and ampulla (p > 0.05); however, an effect of capacitation was detected (p = 0.0015). Also, the percentage of capacitated sperm, determined by chlortetracycline labeling, was greater in sperm that remained free-swimming in the presence of explants than in the absence of explants (p = 0.001). In conclusion, capacitation appears to be involved in the release of bovine sperm from oviductal epithelium and therefore could enable sperm to leave the reservoir and fertilize oocytes.
精子与输卵管上皮的结合被认为是调节输卵管中精子储存库形成的重要机制。基于仓鼠的相关证据，我们推测获能会影响牛精子的释放，使其能够受精。输卵管取自发情期荷斯坦小母牛排卵侧。将峡部和壶腹部上皮挤出并剪成碎片，这些碎片形成外翻囊泡（外植体）。将外植体置于含有输卵管液（TALP）培养基的组织培养孔中，在39℃、5%二氧化碳条件下孵育。冻融精子通过在TALP中上浮制备，并在含有20微克/毫升肝素（无葡萄糖）的TALP中孵育4小时进行获能。未获能精子稀释到获能培养基后立即使用。在手术2小时内，将精子加入外植体并与它们一起孵育15分钟。对精子和外植体进行录像，并分析录像以确定每单位表面积结合的精子数量。方差分析未显示峡部和壶腹部每0.1平方毫米结合的精子数量有差异（p>0.05）；然而，检测到获能的影响（p = 0.0015）。此外，通过金霉素标记确定的获能精子百分比，在有外植体存在时仍能自由游动的精子中比没有外植体时更高（p = 0.001）。总之，获能似乎参与了牛精子从输卵管上皮的释放，因此可能使精子离开储存库并使卵子受精。
