Endocrine Abstracts

JOINT1306

Introduction: Patients with CAIS have a genetic defect in the androgen receptor, resulting in an external female genitalia with the presence of intra-abdominal testicles. While these testes do not exhibit functional spermatogenesis, they seem to serve as an important source of steroid hormones that also induce puberty. However, it remains unclear in how far the defective AR-mediated signalling impacts Leydig cell steroidogenic activity and function.

Research Aim: Here we aim to characterize the production of steroid hormones in a Leydig cell line and the responsiveness to different stimuli to establish a robust model system to test possible autocrine mechanisms.

Material and Methods: The MLTC-1 cell line was stimulated with progesterone, 8-Br-cAMP, hCG, LH, and a combination of LH and hCG or inhibited with ketoconazole. Moreover, the cells were transfected with plasmids encoding a dominant negative androgen receptor as well as enzymes affecting steroid production. Supernatants were collected at 4 and 24 hours post-stimulation, and androgen concentrations were quantified using liquid chromatography–mass spectrometry (LC-MS). Additionally, RNA and protein expression of steroidogenic enzymes were analysed 24 hours after stimulation.

Results: Stimulation of MLTC-1 cells generally resulted in a significant increase in androgen concentrations at the 4-hour time compared to 24 hours, suggesting that the initial stimulatory effect diminishes over time. Stimulation with LH exhibited the most pronounced effect, increasing androgen concentrations in the supernatant 50-fold compared to untreated control cells. In contrast, progesterone and 8-Br-cAMP did not induce a significant increase in androgen production. The inhibitor ketoconazole, however, effectively reduced androgen concentrations at 1 µM and 10 µM without compromising cell viability, whereas 100 µM negatively impacting cell growth. Transfection with plasmids expressing a dominant negative androgen receptor or enzymes modulating steroid hormone production demonstrated that the steroid profile could be successfully modulated in these cells.

Conclusion and Outlook: Our findings demonstrate that MLTC-1 cells can be used as a robust Leydig cell model for steroidogenesis, as evidenced by their responsiveness to LH. Moreover, transfecting these cells as a first step towards a gene therapy to modulate steroid production in CAIS patients is generally feasible.