Cloning, sequencing, and expression analysis of mouse glucosamine-6-phosphate deaminase (GNPDA/oscillin).
It was reported that a hamster protein, called "oscillin," with a sequence related to that of an Escherichia coli GNPDA triggered Ca(2+) oscillations in mammalian oocytes when introduced into their cytoplasm upon fertilization. Recently, it was shown that GNPDA/oscillin is ubiquitously expressed in rat tissues and that a recombinant hamster GNPDA/oscillin protein does not exhibit oscillin activity when injected into oocytes. In the mouse, the nature and role of such a GNPDA/oscillin is not known, but another candidate protein, tr-kit, has been proposed as a sperm factor causing oocyte activation. In order to clarify this issue, we have characterized the mouse homolog of hamster and human GNPDA/oscillin, and examined its expression along with that of tr-kit, in parallel. We report here the molecular cloning and sequencing of mouse GNPDA/oscillin, which shows over 96% identity with the hamster and human homologs. Using specific primers, we performed an RT-PCR analysis to determine the tissue distribution of mouse GNPDA/oscillin mRNA. Unlike tr-kit mRNA which is expressed solely in mouse testis, GNPDA/oscillin mRNA is detected in unfertilized oocytes and in all tissues examined including testis, heart, thymus, liver, ovary, uterus, kidney, spleen, and lung. The protein itself is also detected in all tissues examined by Western blots. Indirect immunofluorescence studies, using an antibody raised against hamster GNPDA, demonstrate that GNPDA is lost with the acrosome reaction of mouse spermatozoa, is localized in the equatorial and neck regions of the human spermatozoa and the post-acrosomal region of the hamster spermatozoa. Our results thus indicate that mouse GNPDA/oscillin, the homolog of hamster oscillin, unlike tr-kit, does not exhibit some of the required characteristics expected from a putative sperm-derived oocyte-activating factor.
Amireault
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P
Département d'Obstétrique-Gynécologie, Université de Montréal and Centre de Recherche du CHUM, Hôpital St-Luc, Montréal, Québec, Canada.
Dubé
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F
D019747:Aldose-Ketose Isomerases / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D019009:Proto-Oncogene Proteins c-kit; D012333:RNA, Messenger; D051381:Rats; D017422:Sequence Analysis, DNA; D013094:Spermatozoa / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D011506:Proteins; D012333:RNA, Messenger; C097705:oscillin; D019009:Proto-Oncogene Proteins c-kit; C020582:glucosamine-6-phosphate isomerase; D019747:Aldose-Ketose Isomerases