Multiple isoforms of porcine aromatase are encoded by three distinct genes

Cytochrome P450 aromatase, a product of the CYP 19 gene and the terminal en zyme in the estrogen biosynthetic pathway, is synthesized by the ovary, end ometrium, placenta, and peri-implantation embryos in the pig and other mamm als, albeit to varying levels, implying its functional role(s) in pregnancy events. The aromatase produced by the pig tissues exists as three distinct isoforms (type I - ovary, type II - placenta, and type III - embryo), with presumed differences in substrate specificities, expression levels, activi ty, and mode of regulation. In order to delineate the molecular mechanisms whereby estrogen synthesis is regulated in these diverse tissues, the prese nt study examined if these aromatase isoforms represent products of multipl e genes or of a single gene via complex splicing mechanisms. Porcine genomi c DNA from a single animal was used as a template in the polymerase chain r eaction (PCR) to amplify isoform-specific sequences corresponding to exons 4 and 7, respectively. Nucleotide sequence analysis of the generated fragme nts revealed the presence of only clones corresponding to the three known a romatase types. Screening a porcine Bacterial Artificial Chromosome (BAC) l ibrary for aromatase gene by PCR yielded a single clone similar to 80 kb in length. Southern blot analysis, using probes specific for exons 1A-1B, 2-3 , 4-9, and 10 sequences indicated that the BAC genomic clone contains the e ntirety of the coding exons as well as the proximal promoter region. Sequen ce analysis of the fragment generated with exon 4 primers determined that t his BAC clone contains only the type II gene. The presence and relative ori entation of the untranslated 5'- exons 1A and 1B, previously demonstrated f or the type III isoform were evaluated in the BAC clone and genomic DNA by PCR, The 265 bp fragment generated from both PCR reactions was confirmed by sequence analysis to contain exons 1A and 1B that are located contiguous t o each other and separated by only three bp. A diagnostic procedure for typ ing aromatase isoforms was developed, based on the presence of specific res triction sites within isoform-specific exons. The use of this protocol conf irmed the existence of only three aromatase isoforms in the porcine genome and indicated changes in aromatase types expressed by the uterine endometri um as a function of pregnancy stage, The presence of distinct genes encodin g each of the aromatase isoform predicts important differences in the mecha nisms underlying the molecular evolution and regulation of porcine aromatas e, unique from those of other mammals, and suggests a critical role for P45 0 aromatase steroidal products in uterine functions related to pregnancy ev ents. (C) 2000 Elsevier Science Ltd, All rights reserved.