Goud P T, Goud A P, Rybouchkin A V, De Sutter P, Dhont M
Department of Obstetrics and Gynaecology, University Hospital of Ghent, Belgium.
Hum Reprod. 1998 May;13(5):1336-45. doi: 10.1093/humrep/13.5.1336.
Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO2) and (ii) omission of Ca2+ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca2+) or Ca2+-free injection medium (experiment set 2, n=278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca2+-free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca2+ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.
将人类精子显微注射到叙利亚金黄地鼠卵母细胞后获取核型是困难的，迄今报道的结果并不理想。这可能与注射和培养技术有关，也可能与地鼠卵母细胞对孤雌激活的高度敏感性有关，或者两者都有关系。因此，我们采用以下两种方法研究了地鼠卵母细胞-人类精子显微注射模型：（i）应用当代注射技术（触碰精子尾部）和培养技术（地鼠胚胎培养基，HECM-3，10%二氧化碳），以及（ii）在注射培养基中省略钙离子。因此，在第一系列实验中，向252个地鼠卵母细胞注射了人类精子。在注射后存活的219个（87%）卵母细胞中，雄性原核形成[两个原核（2PN），两个极体（PB）]、有丝分裂中期进入和精子染色体铺展的平均百分比分别为41.4%、27.8%和18.2%。对注射后未能形成雄性原核的卵母细胞进行分析发现，其中大多数仅形成了地鼠雌性原核，而精子核要么完整要么肿胀（部分解聚），这表明卵母细胞激活失败不太可能是这些卵母细胞中雄性原核形成失败的原因。在接下来的系列实验中，将同胞卵母细胞交替注射悬浮在常规（1.9 mM钙离子）或无钙离子注射培养基中的精子（实验组2，n = 278）。与常规组相比，无钙离子组中具有2PN、2PB、中期进入和精子染色体铺展的卵母细胞平均百分比有显著改善（2PN、2PB：51%对36.6%，中期进入：36.3%对26.9%，精子染色体铺展：28%对20.4%；所有P < 0.04）。因此，孤雌激活似乎是异种地鼠卵胞浆内单精子注射后雄性原核形成失败的促成因素之一。从这些实验可以得出结论，应用先进的注射和培养技术以及在注射培养基中省略钙离子，对于将地鼠卵母细胞显微注射用于对受精能力差的人类精子进行核型分析的常规应用是有前景的。
