Development of in vitro culture of rat Leydig cells after purification with Nycodenz gradient

Ekayanti Mulyawati Kaiin, Ita Djuwita, Tuty Laswardi Yusuf, Mohamad Agus Setiadi

Department of Anatomy, Physiology and Pharmacology, Faculty of Veterinary Medicine, Bogor Agriculture University (IPB), Jl. Agatis Kampus Darmaga, Bogor, Indonesia.
Department of Veterinary Clinic, Reproduction and Pathology, Faculty of Veterinary Medicine, Bogor Agriculture University (IPB), Jl. Agatis Kampus Darmaga, Bogor, Indonesia.
Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia.

DOI: 10.4236/ojas.2013.34044                

Abstract

The study examined the effect of human Chorionic Gonadotrophin (hCG) and/or Insulin Transferring Sodium Selenite (ITS) on the proliferation and development of rat Leydig cells purified by Nycodenz gradient. Leydig cells purity, viability and proliferation after purification and 3 days of cultured were evaluated. Leydig cells 1 × 106 cells/ml were cultured in DMEM containing 10% Newborn Calf Serum (NBCS) and divided into four kinds of treatments 1) as a control, 2) control + 2.5 IU/ml hCG, 3) control + ITS (5 μg/ml insulin, 10 μg/ml transferrin, 5 μg/ml Se), and 4) control + hCG + ITS. Leydig cells purification results showed 91.40% of purity, viability was 98.17% and concentration 7.03 × 106 cells/ml. The addition of ITS and hCG + ITS in DMEM produced Leydig cell proliferation by 88.35% and 90.64% higher than in controls (86.82%) (p < 0.05). The addition of hCG did not increase Leydig cell proliferation (86.99%). In the primary culture of Leydig cells, the population doubling time (PDT) was 1.03 days, similar with the addition of hCG treatment (1.02 day). Declined in value of PDT significantly (p < 0.05) occurred in DMEM supplemented with ITS (0.97 day) and hCG + ITS (0.88 day). This result was also seen in the first and second Leydig cell lines. DMEM added with hCG or hCG + ITS resulted in a higher amount of testosterone (5.06 ng/ml; 5.25 ng/ml)) than the culture medium without hCG (2.46 ng/ml) (p < 0.05). It can be concluded that the combination of hCG + ITS can increase the number of Leydig cells and testosterone levels in the culture medium.

Keywords

Leydig; In Vitro; Rat; Nycodenz; hCG; ITS; Testosterone

Conflicts of Interest

The authors declare no conflicts of interest.

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