Using polymerase chain reaction and two degenerate primers whose designs were based on the two best conserved regions of the DNA-binding domain of the nuclear receptor superfamily, we identified and cloned a novel orphan receptor, named TAK1. The open reading frame of TAK1 encodes a protein of 596 amino acid residues. Based on the modular structure and the presence of a DNA-binding domain containing two zinc fingers TAK1 belongs to the steroid/thyroid hormone receptor superfamily. The amino acid sequence of TAK1 is most closely related to the orphan receptor TR2-11. Their overall sequence homology is 64%, with the highest similarity (82%) being observed in the DNA-binding domain. Northern blot analysis using RNA from multiple human tissues showed that a 9.4 kilobase TAK1 transcript was expressed ubiquitously and that the presence of a 2.8 kilobase mRNA was largely restricted to the testis. In situ hybridization using sections of rat and mouse testes and Northern blot analysis using RNA from testes of rats at various ages revealed that TAK1 is most abundantly expressed in spermatocytes whereas little expression was observed in other germ cells or somatic cells. In situ hybridization using other mouse and rat tissues revealed cell type-specific expression of TAK1 in several tissues. Our observations suggest a role for this putative transcription factor in the regulation of gene expression in specific cell types. In the testis, TAK1 appears to control gene expression during spermatogenesis, particularly during the meiotic phase.

Subtractive hybridization was used to isolate cDNAs highly expressed in stages IX-XI of the cycle of the seminiferous epithelium in the rat. One of the cloned cDNAs was sequenced and shown to be homologous to a previously described cDNA encoding rat prohibitin. Northern blot analyses showed that 1.9- and 1.2-kb transcripts were present in Sertoli cells whereas 1.5-, 1.2-, and 0.7-kb transcripts were expressed in germ cells. Western blot analyses with anti-peptide antibody to prohibitin revealed only a single 30-kDa protein in testis. Immunocytochemistry demonstrated that prohibitin protein was expressed constitutively in adult Leydig cells and Sertoli cells at all stages. Immunoreactivity of prohibition was very low in preleptotene spermatocytes, very high in leptotene spermatocytes, and very low in zygotene spermatocytes. In pachytene spermatocytes, immunoreactivity was very high in stages VII-XI and was minimal during stages XII and XIV. No protein was detected in spermatogonia and spermatocytes undergoing mitotic and meiotic divisions, respectively. These studies show that the prohibitin gene is expressed differentially in testis. The expression pattern of the prohibitin gene in rat testis appears to correlate with a proposed antiproliferative role of prohibitin.