Expression Of Thyrotropin-Releasing Hormone Receptors In Rat Testis And The Effect Of TRH On Isolated Leydig Cells

BackgroundsThyrotropin-releasing hormone (TRH) is a tri-peptide (pyro-Glu-His-Pro-NH2) originally discovered as the central regulator of the hypothalamic-pituitary-thyroid (HPT) axis, controlling the pituitary synthesis and release of thyrotropin, which, in turn, stimulates the synthesis and release of thyroid hormones. Two specific receptors for TRH, TRH receptor type 1 (TRH-R1) and TRH receptor type 2 (TRH-R2), have been cloned from mammals. Amino acid sequence alignments of the two types of TRH receptors from the same species reveal a 50% overall identity. These receptors belong to the G protein-coupled receptor (GPCR) superfamily. TRH and its specific receptors were later found to be present in many regions of brain, exceeding its function from hormone to neuromodulator or neurotransmitter. Besides extrahypothalamus identified in a number of peripheral tissues, including pancreas, gastrointestinal tract and male reproductive system, etc. Although, previous studies have shown that TRH-R1 and TRH-R2 exhibit some differences in anatomic distribution both in central nervous system and peripheral tissues, testis is one of few peripheral tissues that can express both of the two receptors.Previous studies have shown that the presence of TRH-R1 and TRH-R2 were localized exclusively in adult Leydig cells (ALCs). However, few morphological studies have been done about the expression and localization of TRH-R1, especially, TRH-R2 protein in rat testis during postnatal development and in EDS-treated rat testis. Now, TRH has gone beyond its initial role in HPT, its extrahypothalamic functions have reviewed, which include seizure modulation, autonomic nervous system function, food intake, activation of arousal, and so on. It has recently been proposed that the main neurobiological function of TRH is to promote homeostasis. Yet, a role for TRH within the testis, which secretes TRH and expresses two types of TRH receptors, remains to be determined. So, the aim of this study was to elucidate the probable effects of TRH on testicular function.Research Contents and Methods1. Observation of the expression of TRH-R2, as well as TRH-R1, in postnatal (8d, 14d, 21d, 35d, 60d and 90d old) rat testes by means of Western blotting and immunohistochemistry. Changes in the intensity of TRH-R1 and TRH-R2 immunoreactive substance were measure using image analysis.2. Isolation and purification of mature ALCs from 90-day-old SD rats through continuous Percoll density gradient centrifugation. After administration of different doses of TRH to primary cultured ALCs, the effect of TRH on basal and hCG stimulated testosterone secretion in vitro was evaluated. 3. Establishment of EDS-induced Leydig cell apoptosis model. To investigate the expression of TRH-R1 and TRH-R2 during the regeneration of adult type Leydig cells by Western blotting and immunohistochemistry.4. Application of BrdU incorporation test to evaluate the effect of TRH on DNA synthesis of PLCs, isolated from 21-day-old SD rats through continuous Percoll density gradient centrifugation. BrdU incorporated cells were determined by immunocytochemistry for BrdU and the incorporation rates were analyzed by Chi-square (x2) test.Results1. Immunoblot and morphological results indicated that both TRH-R1 and TRH-R2 expressed and localized in the Leydig cells of rat testis during postnatal development and the same Leydig cell expressing TRH-R2 protein could also express TRH-R1 protein simultaneously. With the aid of image analysis, we found that the expression level of TRH-R1 and TRH-R2 in Leydig cells at different stages varied. The maximum expression content of TRH-R2 was at day 35 postpartum, when the testis contains mostly newly formed and immature Leydig cells, while TRH-R1 got a relatively high level after maturation of rat at postnatal day 60 to day 90.2. When the purified ALCs were treated with different doses of TRH in vitro, the basal and hCG-stimulated testosterone secretion was affected in a dose-dependent manner. Low concentrations of TRH (0.01 and 0.1 ng/ml) inhibited the basal and hCG-stimulated testosterone release of ALCs, however, relatively high doses of TRH (1 and 10 ng/ml) could increased hCG-stimulated testosterone production. Even the basal testosterone production in 1 ng/ml TRH treated group was a little higher than 0.1 ng/ml TRH treated group, yet there was no statistical difference between them. 3. After receving a single i.p. injection of EDS (75 mg/kg body weight) in dimethylsulphoxide-water (1:3 v/v), rats were sacrificed at 1, 2, 7, 14, 21 and 28 days post-treatmen. Results of serum testosterone measurement, histochemistry for 3β-HSD enzyme activity, immunohistochemistry for 3β-HSD and HE staining confirmed the validation of animal model. Leydig cell degeneration occurs within 24 hr of a single intraperitoneal injection of EDS, and 2–3 days after treatment, all Leydig cells are eliminated. On day 21 after EDS treatment, spindle-shaped Leydig cells began to reappear in the peritubular region of interstitium. Leydig cells increased a lot on day 28 after EDS treatment and a number of them move from peritubular origin toward the central interstitium accompanied by the change in cell shape from spindle-shaped to polygonal.The expression and localization of TRH-R1 and TRH-R2 in EDS treated rats were detected using immunohistochemistry and Western blotting in post-EDS 21 day and 28 day rats testis. Immunofluorescence double labeling indicated that TRH-R1 and TRH-R2 postively stained cells were exactly the newly regenerated Leydig cells.4. PLCs were isolated and purified through continuous Percoll density gradient centrifugation and maintained in short term primary culture. After incubation with low concentration of TRH (0.001-0.1 ng/ml) for 12 hrs, the BrdU incorporated cells increased (from 8.2% to over 11%), which indicated the DNA synthesis of Leydig cells from 21-day-old rats was increased. While a relatively high dose of TRH (1 ng/ml) were unable to stimulate the DNA synthesis, or even slightly decreased (10 ng/ml TRH) the number of BrdU labeled cells, although there were no statistical difference (p=0.094). When incubated with BrdU for 18 hrs and 36 hrs, the number and rates of BrdU-labelled cells in control and TRH (0.1 ng/ml) treated groups increased greatly. Compared with blank control group, TRH (0.1 ng/ml) could promote the DNA synthesis of PLCs (P<0.01).Conclusions1. We succeeded in isolation and purification progenitor and mature Leydig cells.2. We synthesized EDS and established EDS induced Leydig cell apoptosis animal model.3. The expression of TRH-R1 and TRH-R2 in postnatal rat testis and EDS-treated rats demonstrated the presence of them in Leydig cell lineage. Furthermore, in primary cultured cells, TRH could affect the testosterone synthesis of ALCs and the DNA synthesis of PLCs. TRH might partially participate in the regulation of development and function of Leydig cell lineage.