Sutovsky P, Schatten G
Department of Zoology, University of Wisconsin, Madison 53706, USA.
Biol Reprod. 1997 Jun;56(6):1503-12. doi: 10.1095/biolreprod56.6.1503.
Oocyte-produced glutathione (the tripeptide gamma-glutamyl-cysteinyl-glycine; GSH) has been implicated in the reduction of disulfide bonds in the sperm nucleus during fertilization and thus in the development of the male pronucleus (PN). In this study, we show that the depletion of endogenous glutathione by 10 mM buthionine sulfoximine (BSO; specific inhibitor of GSH synthesis) during bovine oocyte maturation (24 h in vitro; represents prophase I to metaphase II transition in this species) blocks the formation of a male PN in > 85% of treated oocytes (vs. 6.8% in controls) and prevents the assembly of the sperm aster microtubules in approximately 35%. Consequently, the pronuclear migration and apposition do not occur. Ultrastructural observations suggest that the effect of BSO on pronuclear apposition might be due to incomplete disassembly of the sperm tail connecting piece, which normally leads to the release of the sperm centriole and to the reconstitution of the zygotic centrosome during fertilization. The sperm nucleus decondensation and migration blocks were reversed by the treatment of the GSH-depleted oocytes with 1-10 mM dithiothreitol (a disulfide bond-reducing agent) applied 8 h after insemination: 82% of these oocytes exhibited a normal male PN and pronuclear apposition 20 h after insemination. The pool of glutathione seems to be generated during oocyte maturation since > 80% of oocytes that were matured in the absence of BSO displayed a normal male PN, as apposed to a female PN, when inseminated and cultured in the presence of 10 mM BSO. These data suggest that the reduction of disulfide bonds in the sperm after incorporation is important for the formation of the male PN, as well as for the disassembly of the sperm tail connecting piece and pronuclear apposition. The lack of disulfide-reducing power in the GSH-depleted oocytes can be reversed by treatment with disulfide bond-reducing agents.
卵母细胞产生的谷胱甘肽（三肽γ-谷氨酰-半胱氨酰-甘氨酸；GSH）被认为在受精过程中参与精子细胞核中二硫键的还原，从而在雄原核（PN）的发育中发挥作用。在本研究中，我们发现，在牛卵母细胞成熟过程中（体外培养24小时；代表该物种从减数第一次分裂前期到中期II的转变），用10 mM丁硫氨酸亚砜胺（BSO；GSH合成的特异性抑制剂）耗尽内源性谷胱甘肽，会阻止超过85%的处理过的卵母细胞形成雄原核（对照组为6.8%），并使约35%的精子星体微管组装受阻。因此，原核迁移和并列无法发生。超微结构观察表明，BSO对原核并列的影响可能是由于精子尾部连接段的不完全解体，正常情况下，这会导致精子中心粒的释放以及受精过程中合子中心体的重建。用1-10 mM二硫苏糖醇（一种二硫键还原剂）处理受精后8小时的谷胱甘肽耗尽的卵母细胞，可逆转精子细胞核解聚和迁移受阻的情况：这些卵母细胞中有82%在受精后20小时显示出正常的雄原核和原核并列。谷胱甘肽池似乎是在卵母细胞成熟过程中产生的，因为在不存在BSO的情况下成熟的卵母细胞中，超过80%在10 mM BSO存在下受精和培养时，会显示出与雌原核相对的正常雄原核。这些数据表明，精子进入后二硫键的还原对于雄原核的形成、精子尾部连接段的解体以及原核并列都很重要。用二硫键还原剂处理可以逆转谷胱甘肽耗尽的卵母细胞中缺乏二硫键还原能力的情况。
