Molecular Cloning of Cytochrome P450 Aromatase Complementary Deoxyribonucleic Acid from Periimplantation Porcine and Equine Blastocysts Identifies Multiple Novel 5′-Untranslated Exons Expressed in Embryos, Endometrium, and Placenta

Abstract

To facilitate studies on the molecular mechanisms involved in the unique temporal expression of the P450 aromatase gene in porcine periimplantation embryos, a complementary DNA (cDNA) library was prepared from day 12 porcine embryo messenger RNAs (mRNAs) and screened with a cDNA fragment encoding the amino-terminus of human aromatase. Two size variants of nearly full-length cDNA clones (designated clones 33F and 34B; 2470 and 2588 bp, respectively) were isolated and sequenced. Clones 33F and 34B encoded identical aromatase proteins of 503 amino acids, but differed in size due to alternative polyadenylation signal usage for the corresponding mRNAs. Using the 5′-rapid amplification of cDNA ends procedure, two classes of cDNA clones that contained distinct putative exon 1 sequences (E1A and E1B, respectively), but were otherwise identical in exon 2 and 3 sequences, were isolated. The E1A DNA sequence was identical to the 5′-end specified by clones 33F and 34B, except for an additional 23 nucleotides. The E1A and E1B exons showed no significant homology with each other or with the aromatase cDNA sequences from other species, including the human. Expression of E1A-containing aromatase mRNA was higher than that of E1B in day 12 blastocysts, although both mRNAs were expressed at low levels in porcine endometrium and placenta of early pregnancy and in ovary at periestrus. To determine whether the E1A or E1B sequence was common to aromatase mRNAs in other steroidogenic preimplantation embryos, aromatase cDNA clones spanning exons 1-3 were isolated from equine embryos by a combination of RT-PCR and 5′-rapid amplification of cDNA ends. Equine embryonic exon 1 cDNA sequence had no homology with corresponding regions in aromatase transcripts of pigs or other species; in contrast, exon 2 and 3 cDNA sequences predicted an amino acid sequence with significant homology to aromatase. Results from the present study demonstrate alternative splicing of a novel first exon(s) in P450 aromatase gene transcripts in porcine preimplantation embryos and in endometrium and placenta of pregnancy. The lack of homology among equine, porcine, human, and bovine aromatase 5′-untranslated exons may indicate divergence in the corresponding regulatory motifs of these regions among mammalian species. These observations suggest the potential significance of activation of the E1A promoter and/or splicing of novel aromatase 5′-exons in the transient production of estrogens by periimplantation blastocysts.