Wessel G M, Zaydfudim V, Hsu Y J, Laidlaw M, Brooks J M
Department of Molecular and Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
Dev Growth Differ. 2000 Oct;42(5):507-17. doi: 10.1046/j.1440-169x.2000.00534.x.
The regulation of yolk storage in oocytes and subsequent utilization in embryos is critical for embryogenesis. In sea urchins, the major yolk protein is made in the intestines, transported to the ovaries and accumulated in developing oocytes within membrane-bound vesicles comprising approximately 10% of the mass of an egg. Here, a non-yolk protein that accumulates specifically in yolk granules is reported. This protein was identified by cDNA cloning and, by use of antibodies to the recombinant protein, it was shown that this molecule is stored selectively in yolk granules of oocytes and embryos. No accumulation was seen in the accessory cells, testis, or intestines. In situ ribonucleic acid (RNA) hybridizations showed that the transcript accumulated only in oocytes, and was more highly concentrated in young oocytes. However, later in oogenesis, the messenger ribonucleic acid (mRNA) levels decreased significantly so that no signal was detectable in mature haploid eggs or at any later stage in development. However, by immunofluorescence and western blot analysis, the 30 kDa band was present throughout development. The predicted sequence of this protein shows that it is a member of the bep, HLC-32, EBP family of sea urchin proteins, but as it does not accumulate at the cell surface, nor in the hyaline layer in the two species studied here, as do other members of the family, it has been referred to as YP30 (30 kDa protein of the yolk platelet). To address its potential function, yeast two-hybrid analysis was performed to screen for proteins that potentially interact with YP30. It was found that it binds itself, and forms strongly interacting dimers. It is hypothesized that YP30 participates in the packaging and storage of major yolk protein during oogenesis, or in the utilization of the major yolk protein in development.
卵母细胞中卵黄储存的调控以及随后在胚胎中的利用对于胚胎发育至关重要。在海胆中，主要的卵黄蛋白在肠道中合成，运输到卵巢，并在发育中的卵母细胞内积累在膜结合囊泡中，这些囊泡约占卵子质量的10%。在此，报道了一种特异性积累在卵黄颗粒中的非卵黄蛋白。该蛋白通过cDNA克隆鉴定，并且通过使用针对重组蛋白的抗体表明，该分子选择性地储存在卵母细胞和胚胎的卵黄颗粒中。在辅助细胞、睾丸或肠道中未观察到积累。原位核糖核酸（RNA）杂交显示转录本仅在卵母细胞中积累，并且在年轻卵母细胞中浓度更高。然而，在卵子发生后期，信使核糖核酸（mRNA）水平显著下降，以至于在成熟单倍体卵子或发育的任何后期阶段都检测不到信号。然而，通过免疫荧光和蛋白质印迹分析，30 kDa条带在整个发育过程中都存在。该蛋白的预测序列表明它是海胆蛋白bep、HLC - 32、EBP家族的成员，但由于它不像该家族的其他成员那样在细胞表面或在此研究的两个物种中的透明层中积累，因此被称为YP30（卵黄小板的30 kDa蛋白）。为了研究其潜在功能，进行了酵母双杂交分析以筛选可能与YP30相互作用的蛋白质。发现它能与自身结合，并形成强相互作用的二聚体。据推测，YP30在卵子发生过程中参与主要卵黄蛋白的包装和储存，或在发育过程中参与主要卵黄蛋白的利用。
