Cheng F P, Fazeli A R, Voorhout W F, Tremoleda J L, Bevers M M, Colenbrander B
Department of Herd Health & Reproduction, Veterinary Faculty, Utrecht University, The Netherlands.
Int J Androl. 1998 Apr;21(2):57-66. doi: 10.1046/j.1365-2605.1998.00096.x.
The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore, the effect of this component on sperm-zona binding and the subsequent AR was studied. Pooled FF, aspirated from the preovulatory follicles of mares in oestrous, was used and aliquots of the fluid were treated with charcoal to remove steroids (CFF). Charcoal treatment reduced the progesterone concentration in FF from 153 to < 2 ng/mL. Spermatozoa from fertile stallions collected by a swim-up procedure were preincubated in modified Tyrode's medium for 5 h and then incubated for 30 min at 37 degrees C with either (1) 50% FF + 50% CFF, (2) 50% FF + 50% CFF + 150 ng/mL progesterone, (3) 50% CFF + 150 ng/mL progesterone, (4)150 ng/mL progesterone or (5) modified Tyrode's medium alone. The sperm-hemizona assay was applied: (a) to compare the number of spermatozoa bound to a hemizona in the presence and absence of 1.5, 15 or 150 ng/mL progesterone after 1 h co-incubation of spermatozoa and hemizonae, (b) to compare the incidence of the AR in sperm-hemizona complexes incubated for 1 h in the presence and absence of 1 microgram/mL progesterone. Both spermatozoa in suspension and bound to a hemizona were treated with the supravital dye Ethidium homodimer and fixed. Their plasma membranes were permeabilized, and the outer acrosomal membranes were labelled with FITC-PNA. Viable spermatozoa without the outer acrosomal membrane were considered as physiologically acrosome-reacted. Results showed that (1) FF induced a higher percentage of AR than did CFF or modified Tyrode's medium, (2) addition of 150 ng/mL progesterone to CFF restored 77% of the AR-inducing activity and (3) CFF and modified Tyrode's medium both induced the AR to a similar extent when supplemented with 150 ng/mL progesterone. Neither FF nor progesterone treatment affected sperm viability severely. The number of spermatozoa bound to a hemizona in the presence of 15 and 150 ng/mL progesterone was significantly higher (p < 0.05) than the number of spermatozoa bound in the absence of progesterone. A higher incidence of the AR was found in sperm-hemizona complexes incubated in the presence of progesterone (55.6 +/- 3.4% vs. 27.1 +/- 4.3%, in the presence and absence of progesterone, respectively) (n = 15, p < 0.05). It is concluded that mare FF can induce the AR in stallion spermatozoa. Progesterone is the physiological component responsible for this AR-inducing capacity. Progesterone enhances sperm-zona binding activity and exerts an additive effect on the zona-induced AR.
本研究的目的是调查母马卵泡液（FF）是否能诱导公马精子发生顶体反应（AR），如果可以，则鉴定卵泡液中负责此作用的成分。此外，还研究了该成分对精子与透明带结合及随后顶体反应的影响。使用从发情期母马排卵前卵泡抽吸的混合卵泡液，并将部分卵泡液用活性炭处理以去除类固醇（CFF）。活性炭处理使卵泡液中的孕酮浓度从153 ng/mL降至<2 ng/mL。通过上浮法收集的可育公马精子，先在改良的Tyrode's培养基中预孵育5小时，然后在37℃下与以下物质孵育30分钟：（1）50%卵泡液+50% CFF，（2）50%卵泡液+50% CFF + 150 ng/mL孕酮，（3）50% CFF + 150 ng/mL孕酮，（4）150 ng/mL孕酮或（5）单独的改良Tyrode's培养基。应用精子-半透明带试验：（a）在精子与半透明带共同孵育1小时后，比较存在和不存在1.5、15或150 ng/mL孕酮时与半透明带结合的精子数量，（b）比较在存在和不存在1 μg/mL孕酮的情况下孵育1小时的精子-半透明带复合物中顶体反应的发生率。悬浮的精子和与半透明带结合的精子均用活体染料溴化乙锭同二聚体处理并固定。使它们的质膜通透化，并用异硫氰酸荧光素标记的花生凝集素（FITC-PNA）标记顶体外膜。没有顶体外膜的活精子被视为发生了生理性顶体反应。结果表明：（1）卵泡液诱导顶体反应的百分比高于CFF或改良的Tyrode's培养基，（2）向CFF中添加150 ng/mL孕酮可恢复77%的顶体反应诱导活性，（3）当添加150 ng/mL孕酮时，CFF和改良的Tyrode's培养基诱导顶体反应的程度相似。卵泡液和孕酮处理均未严重影响精子活力。在存在15和150 ng/mL孕酮时与半透明带结合的精子数量显著高于（p<0.05）不存在孕酮时结合的精子数量。在存在孕酮的情况下孵育的精子-半透明带复合物中发现顶体反应的发生率更高（分别为55.6±3.4%和27.1±4.3%，存在和不存在孕酮时）（n = 15，p<0.05）。结论是母马卵泡液可诱导公马精子发生顶体反应。孕酮是负责这种顶体反应诱导能力的生理成分。孕酮增强精子与透明带的结合活性，并对透明带诱导的顶体反应发挥相加作用。
