Regulation of DNA synthesis in Leydig cells obtained from neonatal pig testes

Three distinct waves of Leydig cell development are found in the pig testes, which occur during fetal, perinatal, and prepubertal periods. Proliferation of Leydig cells is primarily regulated by luteinizing hormone (LH); however, effects of LH on proliferation of immature rat Leydig cells are mediated by specific growth factors and cytokines such as transforming growth factor-alpha (TGFalpha), insulin-like growth factor-1 (IGF-1), interleukin-1beta (IL-1beta), steroidogenesis-inducing protein (SIP), and TGFbeta. The objective of the present study was to identify growth factors that could possibly be involved in the proliferation of Leydig cells in the neonatal pig testis. Leydig cells were isolated from 3- to 5-d-old pig testes, cultured for 48 hr in serum-free media, washed, and treated with hCG and/or IGF-1, epidermal growth factor (EGF), IL-1beta, SIP, and TGFbeta for 18 hr. Tritiated thymidine incorporation into DNA was assessed over a subsequent 4-hr period. Incorporation of [3H]-thymidine was stimulated by hCG treatment with a 2.3-fold increase over control cultures. SIP also induced a significant increase (P < 0.0001) in the incorporation of [3H]thymidine into Leydig cell DNA. Similarly, EGF and IGF-1 also increased DNA synthesis in neonatal porcine Leydig cells, whereas IL-1beta had no effect. TGFbeta had very little, if any, effect on DNA synthesis when added alone, but inhibited the stimulatory effects of other mitogens (SIP, hCG, EGF/TGFalpha, and IGF-1). Our results indicate that these growth factors may play a role in the LH/hCG-dependent proliferation of Leydig cells during the perinatal period of development.