Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos

Abstract

Centrin is an evolutionarily conserved 20 kDa, Ca C2-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.

Somatic cell nuclear transfer efficiency: How can it be improved through nuclear remodeling and reprogramming?

“…Different figures and colors are used to depict the location and concentration of a variety of proteins that have been localized in donor nuclei, zygotes and SCNT zygotes (ZP, zona pellucida; PM, plasma membrane; NE, nuclear envelope). Data are based upon the following manuscripts: using a blastomere as a nuclear donor cell (Prather et al, 1989, 1990, 1991, 2000; Kubiak et al, 1991; Prather and Rickords, 1992; Parry and Prather, 1995) or fetal fibroblast as a nuclear donor cell (Park et al, 2001b; Moreira et al, 2003; Gao et al, 2004; Mananadhar et al, 2004; Novak et al, 2004; Teranishi et al, 2004; Sutovsky et al, 2005; Liu et al, 2006; Antelman et al, 2008; Chang et al, 2010; Zhao et al, 2010a). A more complete description of differences in histone acetylation and DNA methylation in donor cells, zygotes and SCNT embryos has recently been published (Zhao et al, 2010b).…”

Expression of mitochondrial transcription factor A (TFAM) during porcine gametogenesis and preimplantation embryo development

“…A standard laboratory Western blotting protocol was applied (Manandhar et al, 2006). Spermatozoa were pelleted, washed with PBS, and extracted by boiling for 5 min with loading buffer (2% sodium dodecyl sulfate, 20% glycerol, 0.002% bromophenol blue, and 5% b-mercaptoethanol).…”

“…Oocyte and embryo culture procedures were done by following our standard lab protocol (Manandhar et al, 2006). Cumulus-oocyte complexes (COCs) were aspirated from antral follicles (3 to 6 mm size) and washed in HEPES-buffered Tyrode lactate medium containing 0.1% (w/v) polyvinyl alcohol (TL-HEPES-PVA) for a total of three times.…”

Centrosomes in the zebrafish (Danio rerio): a review including the related basal body

“…In mammalian cells, centrin 2 is required for centriole duplication [81], and use of GFP-centrin constructs have shown differential behavior of mother and daughter centrioles [82,83]. Some centrins are usually associated with centrosomes, except in preimplantation porcine embryos [84], and are also found in basal bodies, including the connecting cilium of photoreceptors [79]. Centrin 2 has been shown to self-assemble into fibers in the presence of Ca 2+ reminiscent of those associated with basal bodies [85].…”