It is well established that pituitary gonadotrophins provide central regulation of testicular function and that luteinizing hormone stimulated steroid production by the Leydig cells. The less well understood regulators of steroid production include a wide range of implicated autocrine/paracrine factors. One of these leptin has been shown to affect both basal and gonadotrophin-stimulated testosterone production. The aim of this study was to investigate the effect of factors secreted into culture media by pubertal Leydig cells on subsequent testosterone production by Leydig cells in vitro.
Testicular interstitial cells were isolated using a Percoll gradient from pubertal male Swiss mice aged 28-34 days. Cell count and viability were ascertained. The Leydig cell fraction (> 96%) were cultured for 3 hours at 32ºC with 5.0% CO2 under basal or equine chorionic gonadotrophin (eCG) stimulated conditions. In addition they were treated with either anti-leptin, or anti-leptin receptor antibodies. Conditioned media from each treatment group was then added to freshly purified Leydig cells and cultured for 3h. The direct effect of anti-leptin and anti-leptin receptor antibodies was also examined on these cells. Testosterone concentration in the media was determined by RIA.
Anti-leptin treatment significantly stimulated testosterone production 10 fold, while anti-leptin receptor stimulated testosterone production 2 fold. Basal and anti-leptin conditioned media significantly stimulated testosterone production (>80 fold), while anti-leptin receptor media had no effect. On the other hand eCG and eCG and anti-leptin treatment conditioned media inhibited testosterone production, while eCG and anti-leptin receptor treatment was significantly stimulatory (1.5 fold).
In conclusion, this study supports previous studies by our laboratory and others that leptin modulates Leydig cell steroidogenesis, however the results with an anti-leptin receptor suggests that leptin may signal through alternate pathways not involving the leptin receptor.