Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes
Recombinant fetuin-B protein maintains high fertilization rate in cumulus cell-free mouse oocytes
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DOI：
10.1093/molehr/gaw067
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发表时间：
2017-01-01
影响因子：
4
通讯作者：
Jahnen-Dechent, W.
中科院分区：
文献类型：
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作者：
Dietzel, E.;Floehr, J.;Jahnen-Dechent, W.
研究问题：胎球蛋白B是否能抑制小鼠卵母细胞在体外过早透明质膜（ZP）硬化，从而提高IVF率？简要答复：用重组小鼠胎球蛋白-B（rmFetuB）补充卵母细胞体外成熟（IVM）培养液可提高受精率，而不影响小鼠胚胎发育成囊胚。已知：缺乏胎球蛋白-B的小鼠由于过早的ZP硬化而不育。过早的ZP硬化也发生在卵母细胞IVM导致受精率下降。研究设计，大小，持续时间：我们受精批次的20-30小鼠中期II（MII）阶段的卵母细胞从C57 BL/6小鼠新鲜精子，并研究早期胚胎发育，直到囊胚hatching.PARTICIPANTS/MATERIALS，设置，方法：卵母细胞维持或不与rmFetuB在IVM和IVF。在卵母细胞分离之前，将外源rmFetuB添加到培养基中。ZP硬化通过糜蛋白酶消化时间和计数附着的spermes.Main结果和机会的作用进行定量：在卵丘细胞的情况下，rmFetuB剂量依赖性抑制ZP硬化和IVF率增加（P = 0.039）。胎球蛋白-B在>= 0.03 mg/ml时也抑制生理性受精触发的ZP硬化（由精子结合增加表示，P = 0.0002），而不增加胚胎死亡。外源rmFetuB增加IVF率达5小时的IVM（在1小时时P = 0.02，在5小时IVM时P = 0.01）。因此，卵母细胞受到保护，防止过早ZP硬化内源性胎球蛋白B。在人类和家畜中，通常在排卵前通过卵泡穿刺分离卵母细胞。在这种情况下，内源性胎球蛋白-B的剥夺将更早发生，并且IVF培养基中外源性胎球蛋白-B的作用可能更加显著。受精触发的ZP硬化对胚胎发育至关重要，但在本研究中，胎球蛋白-B补充的影响仅研究到囊胚期。添加胎球蛋白-B对移植后胚胎发育的任何影响仍有待确定。更广泛的影响的发现：该astacin型蛋白酶ovastacin触发明确的ZP硬化裂解透明质酸蛋白2。已知动物血清抑制过早的ZP硬化。在卵母细胞培养液中添加rFetuB可通过抑制ZP过早硬化而提高IVF率。在这方面，结果可能是有用的临床活动。大规模数据：无。
STUDY QUESTION: Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate?SUMMARY ANSWER: Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts.WHAT IS KNOWN ALREADY: Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate.STUDY DESIGN, SIZE, DURATION: We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching.PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm.MAIN RESULTS AND THE ROLE OF CHANCE: In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at >= 0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM).LIMITATIONS, REASONS FOR CAUTION: Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined.WIDER IMPLICATIONS OF THE FINDINGS: The astacin-type protease ovastacin triggers definitive ZP hardening by cleaving the zona pellucida protein 2. Animal sera are known to inhibit premature ZP hardening. The addition of rFetuB to the culture medium of oocytes could increase IVF rates by the inhibition of premature ZP hardening. In this regard, the results could be useful for clinical activity.LARGE SCALE DATA: None.