Establishment And Evaluation Of A Stable Steroidogenic Goat Leydig Cell Line

Leydig cells are interstitial cells located in the loose connective tissues adjacent to the seminiferous tubules in the testes and accounts for 2%~4% of the total cells. The function of the Leydig cells is to synthesize and secrete androgen. Leydig cells also have regulatory functions that can indirectly affect Sertoli cells through paracrine mechanisms in reproductive tissues to regulate spermatogenesis. There are many physiological, endocrinal, pharmacological and toxicological events and compounds that can interfere with the normal function of Leydig cells and affect testosterone secretion that results in sexual dysfunction in mammals. Though we can use primary Leydig cells to elucidate the molecular mechanisms that underlie the regulation of testosterone secretion, the ability of normal Leydig cells to proliferate in vitro limits many studies particularly those related to the long-term effects of physiological, endocrinal, pharmacological and toxicological events and compounds. The aim of the present study was to immortalize Leydig cells by human telomerase reverse transcriptase(hTERT), using hTERT gene can effectively avoid the changes in the biological characteristics of cells during the process of immortalization. We obtained results as follows:1. Using collagenase digestion culture method, 2.5 mg/mL collagenase digestion for 1 hour, then filtering, low speed centrifuging, natural sedimentation for individual cells. Cultured cells have the characteristics of fibrous form, typical intercellular contact, clear cell boundary, uniform morphology, active proliferation ability, and positive of Leydig cells specific 3β-HSD stain.2. Using liposome transfecting the exogenous gene pCI-neo-hTERT into 4th generation goat Leydig cells, using 500 μg/mL G-418 screening for a positive clones, and the cells we acquired has spread to 85 generations in a good condition, with active proliferation ability. hTERT RT-PCR and immunofluorescence detection have shown as positive for hTERT-GLCs at 30 and 50 generations, whereas the primary Leydig cells were negative, proving that hTERT has successfully transfected and expressed in hTERT-GLCs.3. RT-PCR detection of hTERT-GLCs at 30 and 50 generations can express key enzymes and receptors of testosterone synthesis pathway: LH-R, StAR, P450 scc, 3β-HSD. Testosterone secretion capacity was positive by ELISA test and no obvious difference between the primary Leydig cells and hTERT-GLCs, proving that hTERT-GLCs has normal testosterone secretion capacity.4. Determination of the cell growth curve and cell cycle compared to the primary leydig cells, have shown that, the hTERT-GLCs has a faster growth rate and longer S period, agreed with the research results that hTERT can promote cell proliferation. Karyotype analysis shown that hTERT-GLCs has a normal chromosome karyotype(2n=60). Telomere length detections shown that hTERT-GLCs has a longer telomere length compared to the primary Leydig cells, and no obvious difference compared to the length and the Hela cells, shows that its stable expression of telomerase. Soft agar experiment test shown that hTERT-GLCs cannot form any cells clones, proving that hTERT-GLCs are still anchorage-dependent cells, having not tumorigenicity.To sum up, we have isolated and purified high quality of primary goat Leydig cells, and successfully inducing the activation of telomerase, preventing the shortening of the telomere and maintaining a sufficient and stable length, and finally established a stable goat Leydig cell line. This cell line exhibited an extended replicative lifespan, retained typical steroidogenic properties and was free of neoplastic transformations, and these candidate cell line might be an excellent tool for the studies of development, function and regulation of normal Leydig cells.