Manandhar G, Feng D, Yi Y-J, Lai L, Letko J, Laurincik J, Sutovsky M, Salisbury J L, Prather R S, Schatten H, Sutovsky P
Department of Animal Sciences, University of Missouri, S-141 ASRC, 920 E Campus Drive, Columbia, Missouri 65211, USA.
Reproduction. 2006 Sep;132(3):423-34. doi: 10.1530/rep.1.00983.
Centrin is an evolutionarily conserved 20 kDa, Ca+2-binding, calmodulin-related protein associated with centrioles and basal bodies of phylogenetically diverse eukaryotic cells. Earlier studies have shown that residual centrosomes of non-rodent mammalian spermatozoa retain centrin and, in theory, could contribute this protein for the reconstruction of the zygotic centrosome after fertilization. The present work shows that CEN2 and CEN3 mRNA were detected in germinal vesicle-stage (GV) oocytes, MII oocytes, and pre-implantation embryos from the two-cell through the blastocyst stage, but not in spermatozoa. Boar ejaculated spermatozoa possess centrin as revealed by immunofluorescence microscopy and western blotting. Immature, GV oocytes possess speckles of centrin particles in the perinuclear area, visualized by immunofluorescence microscopy and exhibit a 19 kDa band revealed by western blotting. Mature MII stage oocytes lacked centrin that could be detected by immunofluorescence or western blotting. The sperm centrin was lost in zygotes after in vitro fertilization. It was not detectable in embryos by immunofluorescence microscopy until the late blastocyst stage. Embryonic centrin first appeared as fine speckles in the perinuclear area of some interphase blastocyst cells and as putative centrosomes of the spindle poles of dividing cells. The cells of the hatched blastocysts developed centrin spots comparable with those of the cultured cells. Some blastomeres displayed undefined curved plate-like centrin-labeled structures. Anti-centrin antibody labeled interphase centrosomes of cultured pig embryonic fibroblast cells as distinct spots in the juxtanuclear area. Enucleated pig oocytes reconstructed by electrofusion with pig fibroblasts displayed centrin of the donor cell during the early stages of nuclear decondensation but became undetectable in the late pronuclear or cleavage stages. These observations suggest that porcine zygotes and pre-blastocyst embryonic cells lack centrin and do not retain exogenously incorporated centrin. The early embryonic centrosomes function without centrin. Centrin in the blastocyst stage embryos is likely a result of de novo synthesis at the onset of differentiation of the pluripotent blastomeres.
中心蛋白是一种进化上保守的20 kDa、与钙调蛋白相关的钙结合蛋白，与系统发育上不同的真核细胞的中心粒和基体相关。早期研究表明，非啮齿类哺乳动物精子的残余中心体保留了中心蛋白，理论上，这种蛋白可能有助于受精后合子中心体的重建。目前的研究表明，在生发泡期（GV）卵母细胞、MII期卵母细胞以及从二细胞期到囊胚期的植入前胚胎中检测到了CEN2和CEN3 mRNA，但在精子中未检测到。免疫荧光显微镜和蛋白质印迹法显示，公猪射出的精子含有中心蛋白。未成熟的GV期卵母细胞在核周区域有中心蛋白颗粒斑点，通过免疫荧光显微镜可见，蛋白质印迹法显示有一条19 kDa的条带。成熟的MII期卵母细胞缺乏可通过免疫荧光或蛋白质印迹法检测到的中心蛋白。体外受精后，合子中的精子中心蛋白消失。直到囊胚晚期，通过免疫荧光显微镜在胚胎中都检测不到它。胚胎中心蛋白最初在一些间期囊胚细胞的核周区域以细小斑点的形式出现，并在分裂细胞的纺锤体极处作为假定的中心体出现。孵化囊胚的细胞形成了与培养细胞相当的中心蛋白斑点。一些卵裂球显示出不明确的弯曲板状中心蛋白标记结构。抗中心蛋白抗体将培养的猪胚胎成纤维细胞的间期中心体标记为近核区域的明显斑点。通过电融合与猪成纤维细胞重建的去核猪卵母细胞在核去凝聚的早期阶段显示出供体细胞的中心蛋白，但在原核晚期或分裂期变得无法检测到。这些观察结果表明，猪合子和囊胚前胚胎细胞缺乏中心蛋白，并且不保留外源掺入的中心蛋白。早期胚胎中心体在没有中心蛋白的情况下发挥功能。囊胚期胚胎中的中心蛋白可能是多能卵裂球分化开始时从头合成的结果。
