FGIN-1-27 administration versus TRT; effects on Leydig cell T production and on serum and intratesticular T concentrations

Young (N = 6) and old (N = 6) rats received FGIN-1-27 (1 mg/kg body weight dissolved in 10% DMSO in PBS) via daily ip injection for 10 days [22]. Control rats (N = 6) were injected daily for 10 days with vehicle. In some studies, the effects of FGIN-1-27 on T production in vivo were assessed after blocking LH secretion with centrorelix, a GnRH antagonist [8]. For the latter analyses, young rats (N = 6) were injected with cetrorelix (0.5 mg/animal dissolved in 10% methanol). One day later, rats began daily ip injections of FGIN-1-27 along with cetrorelix. On day 14, T was measured in duplicate samples of serum and intratesticular fluid (IF) by radioimmunoassay (RIA), according to methods described previously [50]. The assay sensitivity was 10 pg/tube, with intra-assay and interassay coefficients of variation of 11.2 and 9.6%, respectively. For studies of the effects of exogenous T administration, young and old rats (N = 6/group) were administrated subdermal 2 cm T-containing or empty silastic implants for 10 days [51].

Primary Leydig cells were isolated by established procedures [52]. In brief, rats were euthanized by decapitation, and the testes were immediately placed in cold dissociation buffer (M-199 medium with 2.1 g/L HEPES, 2.2 g/L sodium bicarbonate, 1.0 g/L BSA, 25 mg/L trypsin inhibitor, pH 7.4). The testicular artery was cannulated, and testes were perfused with type III collagenase (1 mg/mL) in dissociation buffer to clear testicular blood. The testes then were placed in dissociation buffer containing collagenase (0.25 mg/mL) at 34 °C and shaken at 90–100 rpm for 30 min. Digested testes were passed through a 100-μm nylon mesh to remove the tissue clumps, and the interstitial cells were pelleted by centrifugation at 1500 rpm for 5 min. Leydig cells were purified by Percoll gradient separation at 15 000 rpm, 4 °C for 1 h. The final purity of the Leydig cells, determined by staining the cells for 3beta-hydroxysteroid dehydrogenase activity, was consistently approximately 90%. To culture Leydig cells, isolated cells were resuspended in M-199 culture medium, and the cells (5 × 10⁵) were added to a 24-well culture plate and cultured for 2 h in the absence or presence of LH (1 ng/mL) at 34 °C in 5% CO₂ and 95% air [53]. After 2 h incubation, the media were collected and stored at −80 °C for T measurement by RIA.

Serum and IF were collected for T measurement by RIA, as described above. IF was collected as follows: The tunica albuginea was incised at one pole, and testes were centrifuged at low speed (50 × g; 15 min; 4 °C) to drain interstitial fluid. The seminiferous tubules were then extruded through the hub of a syringe, and the preparation was centrifuged (6000 × g; 15 min; 4 °C) to collect seminiferous tubule fluid (STF) as a supernatant above the collapsed seminiferous tubules [43]. Immediately after collection, STF was snap-frozen in liquid nitrogen and subsequently stored frozen at −80 °C before assay for T. LH concentration in serum was determined using rat LH ELISA kits (Abnova Corp., Taipei, Taiwan) following the manufacturer’s instructions. The intra- and interassay variabilities were 5.4 and 5.17% coefficient of variation. The detection range of the assay was 0–50 ng/mL.