Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos.
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.
Manandhar
G
G
Department of Animal Sciences, University of Missouri, S-141 ASRC, 920 E Campus Drive, Columbia, Missouri 65211, USA.
Feng
D
D
Yi
Y-J
YJ
Lai
L
L
Letko
J
J
Laurincik
J
J
Sutovsky
M
M
Salisbury
J L
JL
Prather
R S
RS
Schatten
H
H
Sutovsky
P
P
D000818:Animals; D001755:Blastocyst / Q000737:chemistry*; D015153:Blotting, Western / Q000379:methods; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000235:genetics; D002478:Cells, Cultured; D018385:Centrosome / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000235:genetics; D019976:Cloning, Organism; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D005455:Fluorescent Antibody Technique; D008297:Male; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020554:Sperm Injections, Intracytoplasmic; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism; D013552:Swine / Q000502:physiology*; D015053:Zygote / Q000737:chemistry*
D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D012333:RNA, Messenger; C056608:caltractin