Andrews J C, Bavister B D
Department of Veterinary Science, University of Wisconsin, Madison 53706.
Biol Reprod. 1989 Jul;41(1):117-22. doi: 10.1095/biolreprod41.1.117.
Cauda epididymal hamster spermatozoa were capacitated with D-penicillamine in a chemically defined (protein-free) medium (= "chemical" capacitation). Hamster zonae pellucidae were incapable of inducing functional acrosome reactions in chemically capacitated hamster sperm in a protein-free medium during sperm-egg coincubation. The culture medium used throughout incubation was a modified Tyrode's solution containing 10 mM sodium lactate, 100 microM sodium pyruvate and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm motility was maintained in all media with PHE (20 microM penicillamine, 100 microM hypotaurine, and 1.0 microM epinephrine). Additional D-penicillamine (125 or 500 microM) or 3 mg/ml bovine serum albumin (high control: TALP-PVA) was used to capacitate sperm during preincubation at 1-2 X 10(6) sperm/ml for 4.0 h at 37 degrees C in 5% CO2 in air. Sperm were then coincubated (2 X 10(4) sperm/ml) with cumulus-free hamster eggs in TALP-PVA or TLP-PVA +/- additional D-penicillamine (total: 500 or 125 microM) for 1.5 or 6.0 h. Percent egg penetration was used as the definitive index of sperm capacitation and functional acrosome reactions. Chemically capacitated sperm did not penetrate eggs (0.0 +/- 0.0%) in the absence of albumin during 1.5 h of sperm-egg coincubation. When sperm were chemically capacitated with 125 microM or 500 microM D-penicillamine, then coincubated with eggs for 6.0 h in the absence of albumin, only 18.8 +/- 28.6% and 23.7 +/- 29.7%, respectively, of eggs were penetrated. Significantly (p less than 0.05) more eggs (67.7 +/- 22.4%) were penetrated when coincubated with chemically capacitated sperm for 1.5 h in medium containing albumin. These results demonstrate that zonae pellucidae of hamster eggs require the presence of albumin to efficiently induce functional acrosome reactions in sperm that are chemically capacitated with D-penicillamine.
将附睾尾部的仓鼠精子在化学成分明确(无蛋白)的培养基中用D - 青霉胺进行获能(即“化学”获能)。在精子与卵子共孵育期间,仓鼠透明带在无蛋白培养基中无法诱导经化学获能的仓鼠精子发生功能性顶体反应。整个孵育过程中使用的培养基是改良的台氏液,含有10 mM乳酸钠、100 μM丙酮酸钠和1.0 mg/ml聚乙烯醇(TLP - PVA)。在所有含有PHE(20 μM青霉胺、100 μM次牛磺酸和1.0 μM肾上腺素)的培养基中精子活力得以维持。在预孵育期间,额外添加D - 青霉胺(125或500 μM)或3 mg/ml牛血清白蛋白(高对照:TALP - PVA),以1 - 2×10⁶精子/ml的浓度在37℃、5%二氧化碳的空气中孵育4.0小时使精子获能。然后将精子(2×10⁴精子/ml)与去除卵丘的仓鼠卵子在TALP - PVA或TLP - PVA +/ - 额外的D - 青霉胺(总量:500或125 μM)中共孵育1.5或6.0小时。卵子穿透率用作精子获能和功能性顶体反应的决定性指标。在精子与卵子共孵育1.5小时期间,在无白蛋白的情况下,经化学获能的精子无法穿透卵子(0.0±0.0%)。当精子用125 μM或500 μM D - 青霉胺进行化学获能,然后在无白蛋白的情况下与卵子共孵育6.0小时时,分别只有18.8±28.6%和23.7±29.7%的卵子被穿透。当在含有白蛋白的培养基中与经化学获能的精子共孵育1.5小时时,显著(p<0.05)更多的卵子(67.7±22.4%)被穿透。这些结果表明,仓鼠卵子的透明带需要白蛋白的存在才能有效地诱导经D - 青霉胺化学获能的精子发生功能性顶体反应。