Chromatin decondensation and DNA synthesis in human sperm activated in vitro by using Xenopus laevis egg extracts.
Chromatin decondensation and DNA synthesis in human sperm activated in vitro by using Xenopus laevis egg extracts.
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使用非洲爪蟾卵提取物在体外激活人类精子中的染色质解缩和 DNA 合成。
DOI：
10.1002/jez.1402420213
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Ruddle,FH
中科院分区：
文献类型：
--
作者：
Brown,DB;Blake,EJ;Wolgemuth,DJ;Gordon,K;Ruddle,FH
使用体外精子活化系统研究核肿胀-染色质去凝聚和DNA合成;受精后体内发生的过程。将溶血素透化的人精子在非洲爪蟾提取物中孵育，并使用相差光学显微镜、电子显微镜和放射自显影进行检查。在3小时的孵育过程中，活化的精子核染色质经历了一个去凝聚-再凝聚循环，在此期间合成了DNA。在加入精子之前，对卵子提取物进行3小时预孵育时也会发生这种情况，这表明启动去凝聚-再凝聚周期所需的因素与精子相关。由于核肿胀和DNA合成都被发现是可重复的和可量化的，我们研究了各种试剂对这两个过程的影响，表征了鸡蛋提取物中诱导这些事件的关键成分。发现EGTA对激活精子中发生的诱导核肿胀或DNA合成没有影响。冷冻和解冻提取物或用阿非迪霉素处理提取物也对随后的核肿胀没有影响;然而，DNA合成活性被阻断。在碱性磷酸酶（AP）处理的提取物中孵育的精子既有核肿胀，又有DNA合成受阻。然而，如果精子用DTT预处理，然后与AP处理的提取物一起孵育，则只有提取物的DNA合成活性被阻断。当用丝氨酸蛋白酶抑制剂（PMSF、大豆胰蛋白酶抑制剂或α 2-巨球蛋白）处理提取物时，发生核肿胀;然而，DNA合成被阻断。这些数据表明，磷蛋白参与一个或多个激活事件，丝氨酸蛋白酶参与DNA的合成。
An in vitro sperm activation system was used to study nuclear swelling‐chromatin decondensation and DNA synthesis; processes that occur in vivo following fertilization. Lysolecithin‐permeabilized human sperm were incubated inXenopus laevisegg extract and examined by using phase‐contrast light microscopy, electron microscopy, and autoradiography. During a 3‐hour incubation, the activated sperm nuclear chromatin underwent a decondensation‐recondensation cycle during which DNA was synthesized. This also occurred when egg extract was given a 3‐hour preincubation before the addition of the sperm, suggesting that the factor(s) required for initiating the decondensation‐recondensation cycle is associated with the sperm. Because both nuclear swelling and DNA synthesis were found to be reproducible and quantifiable, we studied the effect of various agents on the two processes, characterizing the critical component(s) in the egg extract that induces these events. EGTA was found to have no effect on the induced nuclear swelling or DNA synthesis that occurs in the activated sperm. Freezing and thawing the extract or treating the extract with aphidicolin also had no effect on subsequent nuclear swelling; however, the DNA synthesis activity was blocked. Sperm incubated in extract treated with alkaline phosphatase (AP) had both nuclear swelling and DNA synthesis blocked. However, if the sperm were pretreated with DTT, and then incubated with the AP‐treated extract, only the DNA synthesis activity of the extract was blocked. When the extract was treated with serine protease inhibitors (PMSF, soybean trypsin inhibitor, or α‐2‐macroglobulin), nuclear swelling occurred; however, DNA synthesis was blocked. These data suggest that phosphoproteins are involved in one or more of the activation events and that a serine protease(s) is involved in the synthesis of DNA.