Cloning of oestrogen receptor beta from Old and New World primates: identification of splice variants and functional analysis.

Oestrogens have a major impact on reproductive function in both males and females. Two oestrogen receptor genes known as ERalpha (ESR1NR3A1) and ERbeta (ESR2NR3A2) have been cloned. Splice variant isoforms of the ERbeta gene have been identified in human, bovine and rodents and it has been suggested that the existence of these forms can influence oestrogen responsiveness. In the human, splicing of an alternative eighth exon results in the formation of a C-terminal variant called hERbetacx, or hERbeta2, but this isoform has not been identified in other species. The aim of the present study was to clone ERbeta cDNAs from primates so as to determine how closely they resembled the ERbeta isoforms found in the human. The two species studied were the stump-tailed macaque (Macaca arctoides), an Old World primate, and the common marmoset (Callithrix jacchus jacchus), a New World primate. Full length ERbeta (wild type, ERbeta1) cDNAs were cloned from macaque and marmoset; they encoded proteins of similar size to those found in human (59 and 54 kDa, long and short forms respectively) and shared significant sequence homology (97.5% in macaque and 93.8% in marmoset) with the human peptide sequence. Full length cDNAs homologous to the hERbeta2 variant were identified in both primates. Marmoset ERbeta2 was slightly shorter than that of human ERbeta2 (54 kDa compared with 55 kDa) and did not contain the peptide sequence used to raise an anti-hERbeta2 antibody. All the macaque ERbeta2 cDNAs contained 56 bp of intronic sequence which included an in-frame stop codon resulting in translation of a truncated protein ( approximately 35 kDa). In all three species, truncated, alternatively spliced mRNAs lacking exon 5 were isolated on multiple occasions from all tissue extracts. In transient transfection assays, ERbeta2-containing constructs were unable to induce transcription of an oestrogen response element (ERE) reporter plasmid in the presence of oestradiol. ERbeta1 from human, macaque and marmoset exhibited minor differences in their ability to induce transcription of the ERE reporter when incubated with different ligands (oestradiol, PPT, DPN, 5-alpha-androstane-3-beta, 17beta-diol (3betaAdiol), genistein) and this may be due to amino acid substitutions within their ligand binding domains. In conclusion, we have identified and cloned wild type ERbeta (ERbeta1) from macaque and marmoset and demonstrated that splice variant mRNAs homologous to hERbeta2 are formed in both species. The marmoset monkey, therefore, provides a suitable animal model in which to investigate the impact of ERbeta variant expression on tissue responsiveness to oestrogens.