Specific substance-P immunoreactivity can be detected in the Leydig cells, particularly of human testes, and to a lesser degree in mouse Leydig cells, but not in the rat. Using a modified polymerase chain reaction (PCR) assay, preprotachykinin-A (substance-P) mRNA could be detected in extracts of human, mouse, and bovine testes, but not in rat or boar testes or in bovine thyroid or corpus luteum used as negative controls. This assay is able to discriminate among the alpha, beta, and gamma transcripts of the gene and shows that only the beta and gamma transcripts are present in the testes. Sequencing analysis of the PCR products from bovine hypothalamus, mouse brain, and human testis confirmed the structure of these transcripts, which encode both substance-P and neurokinin-A (substance-K) neuropeptide hormones. Using a variant of this assay it was possible to identify tachykinin transcripts in as few as 500 freshly prepared purified mouse Leydig cells. In parallel studies PCR analysis was also able to confirm the presence of mRNA for both substance-P and neurokinin-A receptors in human testes. Thus, the tachykinins substance-P and neurokinin-A must now be added to the list of potentially paracrine substances regulating intratesticular function.

A variety of molecular techniques were used to search human and baboon gonadal tissues for evidence of transcription of the genes for the peptide hormones oxytocin and vasopressin. Only a highly sensitive assay based on a modification of the polymerase chain reaction succeeded in detecting mRNA copies of the oxytocin gene in both human and baboon corpus luteum. Vasopressin gene transcription was not detected in human testis and corpus luteum and was found only once in four different experiments in baboon corpus luteum. Evidence for oxytocin gene transcription in the human testis was found in three of five experiments. The method employed and subsequent sequence analysis of the polymerase products verified the presence of oxytocin mRNA with normal hypothalamic-type exonic structure in primate corpus luteum. Nevertheless, the very low levels of mRNA present are unlikely to support other than local functions for the encoded nonapeptide hormones.

In view of the small number of hormone-producing cells, the factors regulating oxytocin gene expression in the classic site of synthesis, in the magnocellular neurons of the hypothalamus, have not yet been characterized. In the early bovine corpus luteurn there is a tissue-specific oxytocin expression involving many more cells. This tissue therefore was chosen as a experimental system to identify deoxyribonucleic acid elements and nuclear proteins involved in the regulation of oxytocin gene expression. 3.2 kb from the 5' non-coding region of the bovine oxytocin gene have been sequenced and subcloned fragments used as probes for gel retardation and footprinting experiments. Binding sites for luteal as well as more ubiquitous proteins were detected in the oxytocin promoter region and in an artiodactyl-specific dispersed repeated deoxyribonucleic acid element. A binding site in the promoter region with a superficial similarity to an estrogen-responsive element (-159 to -152) was shown not to bind this steroid hormone receptor but to bind two nuclear proteins alternatively. One is a luteal protein, the other a more general transcription factor belonging to the steroid hormone receptor superfamily and similar, if not identical to the COUP protein. This alternative binding of a tissue-and phase-specifically expressed protein or an ubiquitous factor to the same site in the oxytocin promoter suggests a role for these two proteins in the transient up-regulation and subsequent down-regulation of the oxytocin gene during the differentiation of the bovine corpus luteum.