Pérez L J, Valcárcel A, de las Heras M A, Moses D F, Baldassarre H
Centro de Investigaciones Reproductivas Perez Companc, Av. De Mayo 701 Piso 14, (1084) Buenos Aires, Argentina.
Theriogenology. 1996 Apr 1;45(5):1037-46. doi: 10.1016/0093-691x(96)00031-3.
We have described the different patterns of chlortetracycline (CTC) binding to ram spermatozoa, immediately after ejaculation and upon in vitro capacitation and calcium ionophore-induced acrosomal exocytosis. Four different forms of CTC distribution were found. Form I showed an even distribution of fluorescence over the entire head, with a brighter band in the equatorial region. In Form II, uniform fluorescence was observed without equatorial band. Form III consisted of fluorescence in the anterior portion of the head. Form IV showed no fluorescence over the head. In all cases, fluorescence in the middle piece of the flagellum was observed as well. Immediately after ejaculation, Form I was the most abundant one (78%) in fresh semen with Forms II and III being relatively scarce (less than 15%). Form IV was virtually absent or appeared only occasionally. Incubation under in vitro capacitating conditions led to a significant decrease in Form I and to a significant increase in Forms II and III. Form II was mainly associated to intact acrosomes, while most spermatozoa in Form III showed intermediate forms of acrosomal status. Incubation of spermatozoa with the calcium ionophore A23187 resulted in 55% of spermatozoa showing Form IV, suggesting that it represents the acrosome-reacted stage. Form I was abruptly decreased at 30 min of incubation and was neglectible after 60 min. In contrast, Forms II and III increased at 30 min but decreased later on, suggesting that both forms represent intermediate stages before the acrosomal exocytosis. Analysis of acrosomal status in spermatozoa from individual CTC forms revealed that all spermatozoa that remained in Form II after incubation had intact acrosomes. Intermediate stages were predominant in Form III-spermatozoa, while most Form IV-spermatozoa underwent full acrosomal exocytosis. These results show that CTC binding can be used to monitor changes in ram spermatozoa during capacitation and acrosome-reaction.
我们已经描述了金霉素（CTC）在射精后、体外获能以及钙离子载体诱导顶体胞吐作用后与公羊精子结合的不同模式。发现了四种不同形式的CTC分布。形式I在整个头部显示出均匀的荧光分布，赤道区域有一条较亮的带。在形式II中，观察到均匀的荧光，没有赤道带。形式III由头部前部的荧光组成。形式IV在头部没有荧光。在所有情况下，鞭毛中段也观察到荧光。射精后立即，形式I是新鲜精液中最丰富的一种（78%），形式II和III相对较少（少于15%）。形式IV几乎不存在或仅偶尔出现。在体外获能条件下孵育导致形式I显著减少，形式II和III显著增加。形式II主要与完整的顶体相关，而形式III中的大多数精子显示顶体状态的中间形式。用钙离子载体A23187孵育精子导致55%的精子显示形式IV，表明它代表顶体反应阶段。孵育30分钟时形式I突然减少，60分钟后可忽略不计。相反，形式II和III在30分钟时增加，但随后减少，表明这两种形式都代表顶体胞吐作用前的中间阶段。对来自单个CTC形式的精子顶体状态的分析表明，孵育后仍处于形式II的所有精子都有完整的顶体。中间阶段在形式III精子中占主导地位，而大多数形式IV精子经历了完全的顶体胞吐作用。这些结果表明，CTC结合可用于监测公羊精子在获能和顶体反应过程中的变化。
