Insulin-like growth factor-I (IGF-I) protects cultured equine Leydig cells from undergoing apoptosis

Leydig cells located in the interstitial space of the testicular parenchyma produce testosterone which plays a critical role in the maintenance and restoration of spermatogenesis in many species, including horses. For normal spermatogenesis, maintaining Leydig cells is critical to provide an optimal and constant level of testosterone. Recently, an anti-apoptotic effect of IGF-I in testicular cells in rats has been reported, but a similar effect of IGF-I on equine Leydig cells remains to be elucidated. If IGF-I also protects stallion testicular cells from undergoing apoptosis, then IGF-I may have potential as a treatment regime to prevent testicular degeneration. The present study was designed to evaluate the anti-apoptotic effect of IGF-I on cultured equine Leydig cells. Testes were collected from 5 post-pubertal stallions (2-4 years old) during routine castrations. A highly purified preparation of equine Leydig cells was obtained from a discontinuous Percoll gradient. Purity of equine Leydig cells was assessed using histochemical 3β-HSD staining. Equine Leydig cells and selected doses of recombinant human IGF-1 (rhIGF-I; Parlow A.F., National Hormone and Peptide Program, Harbor-UCLA Medical Center) were added to wells of 24 or 96 well culture plates in triplicate and cultured for 24 or 48h under 95% air:5% CO₂ at 34°C. After 24 or 48h incubation, apoptotic rate was assessed using a Cell Death Detection ELISA kit. Significantly lower apoptotic rates were observed in equine Leydig cells cultured with 5, 10, or 50ng/ml of rhIGF-I compared with control cells cultured without rhIGF-I for 24h. Exposure to 1, 5, 10 or 50ng/ml of rhIGF-I significantly decreased apoptotic rate in equine Leydig cells cultured for 48h. After 48h incubation, cells were labeled with Annexin V and propodium iodine to determine the populations of healthy, apoptotic, and necrotic cells by counting stained cells using a Nikon Eclipse inverted fluorescence microscope. As a percentage of the total cells counted, significantly lower numbers of apoptotic cells were observed in cells treated with 10 (9%) or 50ng/ml (10%) of rhIGF-I compared with cells cultured without rhIGF-I (control, 22%). In this study, the results from the two assays indicated that rhIGF-I protected equine Leydig cells from undergoing apoptosis during cell culture for 24h or 48h. In conclusion, IGF-I may be an important paracrine/autocrine factor in protecting equine Leydig cells from undergoing apoptosis.