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(77) TWO ISOFORMS OF THE ACTIVIN A SUBUNIT IN ZEBRAFISH OVARY: mRNA EXPRESSION AND REGULATION BY GONADOTROPIN.
DiMuccio, Tamara1, Mukai, Spencer1, Kohli, Nikki1, Clelland, Eric1, Wu, Tingting1, Peng, Chun1, 1 Department of Biology, Toronto, CA
ABSTRACT- Activins are dimeric proteins consisting of two inhibin subunits. Homo- and hetero-dimerizations of two subunits, A and B, produce three forms of activins, activin-A, -B, and -AB. Our recent studies have suggested that activin-A mediates gonadotropin-induced final oocyte maturation in the zebrafish. To further examine the structure and function of activin-A in the zebrafish ovary, we used primers for conserved regions of the mammalian activin A mRNA to amplify zebrafish ovarian cDNA. An expected size of PCR product was ligated into TOPO PCR2.1 vector and several clones were sequenced. Two distinct sets of sequences were obtained, both of which show highest homologies to activin A subunits of other species. They were therefore designated as activin A isoform 1 and 2. The identities of the two isoforms in the mature peptide region are: 84% at the nucleotide level and 93% at the amino acid level. Isoform 1 shows lower homology with A subunits from other species than isoform 2. To determine if mRNA levels of activin A isoforms vary in different stages of follicles and if they are regulated by gonadotropin, real-time PCR was performed using the ABI PRISM Sequence Detection System, with SYBR Green I as the reporter fluorescent dye. Analyses of total RNA extracted from different stages of follicles reveal that both isoform mRNAs are expressed in previtellogenic, vitellogenic, and mature follicles, with vitellogenic follicles showing highest mRNA levels. Treatment of follicles with human chorionic gonadotropin (hCG) significantly increased both isoform mRNAs levels. This study demonstrates that there are two activin A subunit isoforms expressed in the zebrafish ovary. The finding that activin A mRNA levels are up-regulated by hCG further supports the notion that activin-A mediates the action of gonadotropin in the ovary. (Supported by a grant from Natural Science and Engineering Research Council of Canada to CP)
KEY WORDS: activin, ovary, zebrafish