A novel progestogen receptor subtype in the Japanese eel, Anguilla japonica¹

Abstract

A cDNA encoding a second type of a progestogen receptor (ePR2) was isolated from the same library as we had previously cloned a functional PR (ePR1) in eel testis. The amino acid sequence of the ePR2 shows low homology with ePR1 (34%), but both PRs showed progestogen-dependent transactivation in transfection experiment. Tissue distribution of ePR2 mRNA was clearly different from that of ePR1. Protein interaction between two PRs was demonstrated in vitro by a glutathione S-transferase pull-down assay. These results indicate that ePR2 is also a functional PR. This is the first isolation of two different functional PR molecules from a vertebrate. ß

Progestin is an essential factor for the initiation of the meiosis in spermatogenetic cells of the eel

“…After DNase I treatment, 5 g of RNA was transcribed by Superscript II (Invitrogen) by using an oligo (dT) primer. The resulting cDNA was amplified by PCR with ePR I-specific primers (sense primer, 5Ј-CAACTTGGATATGGCGAACAGC-3Ј; antisense primer, 5Ј-TGGCACCAAAACGGAGAATCCG-3Ј) and ePR II-specific primers (sense primer, 5Ј-CGACTTCGAACAGCTCGTA-TGG-3Ј; antisense primer, 5Ј-CTTTGAGATTCTGGAGAC-GAG-3Ј), which were designed based on published eel PR I and II sequences (36,37). The PCR cycling parameters were as follows: 25 cycles of 94°C for 30 sec, 58°C for 30 sec, and 72°C for 30 sec.…”

Molecular approaches related to the European eel (Anguilla anguilla) reproductive process

“…In this study, we identified duplicated nuclear progestin receptor genes (pgr1 and pgr2) in the genomes of the European and Japanese eels, in agreement with the previous cloning of two progestin receptors from Japanese eel testis (ePR1 and ePR2 Todo et al, 2000;Ikeuchi et al, 2002). In contrast, a single PGR has been reported in pufferfish (Takifugu rubripes), tetraodon (Tetraodon nigroviridis), zebrafish, medaka (Oryzias latipes), and stickleback (Hanna et al, 2010).…”

“…Both were mainly expressed in the pituitary and in the brain in female eel, while in males they were mainly expressed in the gonads and in other tissues outside the BPG axis (pgr1 in kidney and muscle, pgr2 in the gill). The tissue distribution of nPRs in the European eel is similar to what was found in the Japanese eel (Ikeuchi et al, 2002). Nevertheless, the different nPR tissue distribution found among teleosts may indicate species-specific differences of the nPR function.…”

“…Regarding the vertebrate PGR amino acid sequences, alignment clearly showed that DBD and LBD domains were well conserved among vertebrates, whereas A/B domain had lower identity. Almost all PGR residues critical for progestin binding (Williams et al, 1998) were conserved in the LBD of both Japanese eel (Todo et al, 2000;Ikeuchi et al, 2002;Hanna et al, 2010) and European eel PGR1 and PGR2, except for a leucine which was replaced by a valine residue in the PGR2 amino acid sequences.…”

“…The mPRs were first discovered and characterized in fish ovaries (Zhu et al, 2003a), and five isoforms (mPRα, mPRβ, mPRγ, mPRδ, mPRε) were subsequently identified in humans and other vertebrates (Zhu et al, 2003b;Thomas et al, 2012;Peterson et al, 2013). Both nPRs and mPRs are highly expressed in testis (Ikeuchi et al, 2002;Hanna et al, 2009) and brain (Thomas et al, 2012;Peterson et al, 2013), but the functions mediated by them are still unclear. In Japanese eel, progestin receptor 1 (pgr1) is expressed in testis germ cells, Sertoli cells, and testis interstitial cells, whereas progestin receptor 2 (pgr2) mRNA has been detected only in testis germ cells .…”