Thyrotropin-releasing hormone (TRH) plays the central regulatory role in the hypothalamic-pituitary-thyroid axis, but is also present in many extra-hypothalamic loci. The adult rat testis has been identified previously as a source of hypothalamic neuropeptides including TRH. To investigate whether the TRH gene is transcribed in testis, the identification and localization of prepro(pp) TRH mRNA and TRH were studied. Northern blot analyses of ppTRH mRNA in the adult rat testis showed a 2.0 kb band, hybridized with a ppTRH cRNA probe. This band was 0.4 kb greater than the 1.6 kb hypothalamic band. The concentration of ppTRH mRNA in the adult testis was approximately 13% of that found in the hypothalamus. Developmental studies of testicular ppTRH mRNA revealed that no ppTRH mRNA could be detected at the earliest stage (day 8). However, hybridization signals were detected on day 20 and increased progressively on days 35, 45 and 70 by 5.8, 6.4, and 9.8-fold, respectively. In addition, ppTRH mRNA was determined in Leydig cells by Northern analyses of elutriated testicular cell fractions. TRH was also measured in the rat testes at different developmental stages by RIA. TRH concentrations paralleled ppTRH mRNA during development. TRH was localized to Leydig cells by immunohistochemistry. These results indicate that ppTRH mRNA and TRH are present in the rat testis, especially in the Leydig cells. The changes of ppTRH gene expression and the concentration of TRH in the rat testis are developmentally dependent. TRH may function as a new paracrine or autocrine regulator of testicular function.

Our laboratory has demonstrated recently that thyrotropin (TSH) secretion is regulated in part by thyroid hormone inhibition of TSH-releasing hormone (TRH) synthesis and secretion, both in vitro and in vivo. Minute amounts of triiodothyronine (T3) can suppress TSH secretion when administered intracerebraventricularly, whereas identical quantities of T3 administered by a peripheral route do not lower circu-lating TSH concentrations (1). In addition, TRH secretion is augmented in vitro from experimentally hypothyroid rats and secretion is below normal from thyrotoxic rat hypothalami, and T3 (10-9-10-1 M) intro-duced into incubational media can prevent ouabain-activated TRH secretion in vitro (2). Most recently, we have demonstrated that there is augmented TRH messenger RNA in experimentally hypothyroid rats and reduced messenger RNA in the hypothalamic paraventricular nucleus in thyrotoxic rats, whereas no changes were seen in either group when whole hypothalamic RNA was hybridized (1). On the basis of these considerations, we wanted to determine whether thyroid hormones were involved also in the inhibitory regulation of the human TRH gene. Two X 106 plaques of a human lung fibroblast Lambda Fix genomic library were screened with a 32P-cDNA rat preproTRH (ppTRH) gene probe, which included the last three TRH coding sequences of the third exon. Under conditions of moderate stringency, an initial 15 kb DNA fragment was identified, which included the complete ppTRH genomic sequence of 3.3 kb, containing three exons separated by two introns of 1,050 and 650 base pairs respectively Figure 1 (3). The inferred cDNA sequence was confirmed by PCR amplification of hypothalamic

PreproTRH mRNA has been identified in rat cardiac tissues by Northern analyses and RNase protection assays with a specific rat 32P-TRH cRNA probe. Densitometric analyses revealed that atrial ppTRH mRNA concentrations were approximately five-fold greater than those of the ventricles. TRH concentrations (RIA), by contrast, were two-fold higher in ventricles. These data suggest that TRH and TRH mRNA are present in the rat heart, but their concentrations are dissociated, possibly because of differential post-transcriptional or post-translational processing. TRH is postulated to play an autocrine or paracrine role in cardiac physiology.