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


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.

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Kaiin, E. , Djuwita, I. , Yusuf, T. and Setiadi, M. (2013) Development of in vitro culture of rat Leydig cells after purification with Nycodenz gradient. Open Journal of Animal Sciences, 3, 299-304. doi: 10.4236/ojas.2013.34044.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Chen, G.-R., Ge, R.-S., Lin, H., Dong, L., Sottas, C.M. and Hardy, M.P. (2007) Development of a cryopreservation protocol for Leydig cells. Human Reproduction, 22, 2160-2168.
[2] Behre, H.M., Kliesch, S., Leifke E., Link T.M. and Nieschlag, E. (1997) Long-term effect of testosterone therapy on bone mineral density in hypogonadal men. Journal of Clinical Endocrinology and Metabolism, 82, 2386-2390.
[3] Bhasin, S., Singh, A.B., Mac, R.P., Carter, B., Lee, M.I. and Cunningham, G.R. (2003) Managing the risk of prostate disease during testosterone replacement therapy in older men: recommendations for a standardized monitoring plan. Journal of Andrology, 24, 299-311.
[4] Kaiin, E.M., Djuwita, I., Yusuf, T.L. and Setiadi, M.A. (2013) Concentration, purity and viability of Leydig cells after purification using Nycodenz and in vitro culture. Jurnal Kedokteran Hewan FKH Unsiyah (Journal of Veterinary Medicine), 7, 75-80. (in Indonesian language)
[5] Miller, S.R. (2006) Assessment of Nycodenz gradient on enrichment and culture of perinatal porcine spermatogonial stem cell. Thesis (pdf). The Graduate Faculty of North Carolina State University.
[6] Evans, W.H. and Flint, N. (1985) Subfraction of hepatic endosomes in Nycodenz gradients and by free electrophoresis. Biochemistry Journal, 232, 25-32.
[7] Manayagi, T., Kurosawa, R., Ohnuma, K., Ueyama, A., Ito, K. and Takahashi, J. (2003) Purification of mouse primordial germ cells by Nycodenz. Reproduction, 125, 667-675.
[8] Nakayama, Y., Yamamoto, T. and Abe, S. (1999) IGF-I, IGF-II and insulin promote differentiation of spermatogonia to primary spermatocytes in organ culture of newt testes. International Journal Development Biology, 43, 343-347.
[9] Chemes, H., Cigorraga, S., Bergada, C., Schteingart, H., Rey, R. and Pellizzari, E. (1992) Isolation of human Leydig cell mesenchymal precursors from patients with the androgen sensitivity syndrome: Testosterone production and response to human chorionic gonadotrophin stimulation in culture. Biology of Reproduction, 46, 793-801.
[10] Cudicini, C., Lejeune, H., Gomez, E., Bosmans, E., Ballet, F., Saez, J. and Jegou, B. (1997) Human Leydigs cells and Sertoli cells are producers of Interleukin-1 and -6. Journal of Clinical Endocrinology and Metabolism, 82, 1426-1433.
[11] Hu, J., You, S., Li, W., Wang, D., Nagpal, M.L., Mi, Y., Liang, P. and Lin, T. (1998) Expression and regulation of interferon-γ-inducible protein 10 genes in rat Leydig cells. Endocrinology, 139, 3637-3645.
[12] Saez, J.M. (1994) Leydig Cells: Endocrine, paracrine and autocrine regulation. Endocrine Reviews, 15, 574-626.
[13] El-Sherbiny, A.M., Amin, S.O., Hernandez, C. and Carreau, S. (1994) The immature rabbit testis: Presence of two distinct populations of Leydig cells. World Rabbit Science, 2, 141-146.
[14] Pointis, G., Rao, B., Latreille, M.T. and Cedard, L. (1984) Hormonal regulation of testosteron in short term primary culture of fetal mouse Leydig cells. Journal of Steroid Biochemistry, 20, 525-528.
[15] Bernier, M., Gibb, W., Haour, F., Collu, R., Saez, J. M. and Ducharme, J.R. (1983) Studies with purified immature porcine Leydig cells in primary culture. Biology of Reproduction, 29, 1172-1178.
[16] Risbridger, G.P. and Hedger, M.P. (1992) Adult rat Leydig cell cultures: Minimum requirement for maintenance of luteinizing hormone respon-siveness and testosterone production. Molecular and Cellular Endocrinology, 83, 125-132.
[17] Yang, J.-M., Arnush, M., Chen, Q.-Y., Wu, X.-D., Pang, B. and Jiang, X.-Z. (2003) Cadmium-induced damaged to primary cultures of rat Leydig cells. Reproductive Toxicology , 17, 553-560.
[18] Mader, S.S. (2000) Human biology. McGraw Hill, Iowa.
[19] Butler, M. (2004) Animal cell culture & technology. 2nd Edition, Bios Scientific Publisher, Taylor & Francis Group, London.
[20] Freshney, R.I. (2005) Culture of animal cells: A manual of basic technique. John Wiley & Sons Inc, Hoboken.
[21] Hebert, R., Lejeune, H. and Saez, J.M. (2001) Origin, differentiation and regulation of fetal and adult Leydig cells. Molecular and Cellular Endocrinology, 179, 47-74.
[22] Browning, J.Y., Heindel, J.J. and Grotjan, Jr. H.E. (1982) Method for primary culture of purified Leydig cells isolated from adult rat testes. Journal of Tissue Culture Methods, 7(2), 55-58.
[23] Klinefelter, G.R., Hall, P.F. and Ewing, L.L. (1987) Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biology of Reproduction, 36, 769-783.
[24] Renlund, N. (2006) Hormonal and paracrine influences on Leydig cell steroidogenesis Dissertation. Karolinska Institutet, Stockholm.
[25] Seedelaar, J.P.M. and Isaacs, J.T. (2009) Tissue culture media supplemented with 10% fetal calf serum contains a castrate level of testosterone. Prostate, 69, 1724-1729.
[26] Hendlund, T.E. and Miller, G.J. (1994) A serum-free defined medium capable of supporting growth of four established human prostatic carcinoma cell lines. Prostate 24, 221-228.
[27] Freshney, R.I. 1987. Animal cell culture: A practical approach. IRL Press, Washington.

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