Prepubertal Buffalo (Bubalus bubalis) Leydig Cells: Isolation, Culture and Characterization
DOI:
https://doi.org/10.6000/1927-520X.2013.02.01.5Keywords:
Male buffalo, testis, Leydig cells, cell culture, CYP11A1, testosteroneAbstract
Water buffalo (Bubalus bubalis) is an economically important livestock species in India. Male buffaloes display delayed sexual maturity as compared to the bulls (Bos taurus). Serum testosterone level, the key regulator of sexual maturity of males, is reported to be low in male buffaloes in comparison to bulls. Testosterone secretion and progression of spermatogenesis is mediated essentially by Leydig cells in the males. Establishment of primary culture for buffalo Leydig cells can provide an excellent tool to investigate the factors which regulate testicular steroidogenesis. Therefore, the objectives of the present study were to isolate, culture and characterize buffalo Leydig cells. Immunohistological analysis revealed that cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) specifically mark the Leydig cells in prepubertal buffalo testis. Using enzymatic digestion and Percoll density gradient centrifugation, a cell population that consisted of approximately 95% pure Leydig cells was obtained as indicated by CYP11A1 staining. Purified Leydig cells were cultured in DMEM/F12 supplemented with 10% foetal bovine serum (FBS) for 72 h. The cultured Leydig cells proliferated, expressed Leydig-cell specific transcripts (STAR, HSD3B1, HSD3B6, and CYP17A1) and proteins (CYP11A1, HSD3B and LHCGR), and secreted testosterone. It was concluded from the present study that buffalo Leydig cells can be maintained in culture for 72 h. The primary culture of buffalo Leydig cells can be used for studying acute responses, biochemical properties and other factors regulating testicular steroidogenesis, independent of other testicular cell types.
References
Nagarajan M, Kumar N, Nishanth G, Haribaskar R, Paranthaman K, Gupta J, et al. Microsatellite markers in buffalo. BMC Genet 2009; 10: 68. http://dx.doi.org/10.1186/1471-2156-10-68 DOI: https://doi.org/10.1186/1471-2156-10-68
Srivastava AK. Dairy scenario, In: Mallik M. Ed. National Dairy Research Institute Vision 2030, 2011; p. 1-7. Available from: URL http://www.ndri.res.in/ndri/Design/Vision.pdf.
Mudgal VO. Comparative efficiency for milk production of buffaloes and cattle in the tropics. Proceedings of II World Buffalo Congress. New Delhi, India 1988; Vol II, Part II, pp. 454-462.
Madan ML, Prakash BS. Reproductive endocrinology. Soc Reprod Fertil Suppl 2007; 64: 261-81. DOI: https://doi.org/10.5661/RDR-VI-261
Ahmad N, Umair S, Shahab M, Arslan M. Testicular development. Theriogenology 2010; 73: 20-25. http://dx.doi.org/10.1016/j.theriogenology.2009.06.034 DOI: https://doi.org/10.1016/j.theriogenology.2009.06.034
da Luz PA, Andrighetto C, Santos PR, Jorge A, Constantino MV, Pereira FT, et al. Daily sperm production. Spermatogenesis 2012; 2: 88-93. http://dx.doi.org/10.4161/spmg.20220 DOI: https://doi.org/10.4161/spmg.20220
Sharma IJ, Agarwal SP, Agarwal VK, Dwaraknath PK. Changes in profiles of serum sex steroids. Theriogenology 1984; 22: 175-86. http://dx.doi.org/10.1016/0093-691X(84)90430-8 DOI: https://doi.org/10.1016/0093-691X(84)90430-8
Gulia S, Sarkar M, Kumar V, Meyer HH, Prakash BS. Divergent development of testosterone secretion. Trop Anim Health Prod 2010; 42: 1143-48. http://dx.doi.org/10.1007/s11250-010-9538-x DOI: https://doi.org/10.1007/s11250-010-9538-x
Hemeida NA, el-Baghdady YR, el-Fadaly MA. Serum profiles of androstenedione, testosterone and LH. J Reprod Fertil 1985; 74: 311-16. http://dx.doi.org/10.1530/jrf.0.0740311 DOI: https://doi.org/10.1530/jrf.0.0740311
Dixit VD, Singh B, Singh P, Georgie GC, Galhotra MM, Dixit VP. Circadian and pulsatile variations. Theriogenology 1998; 50: 283-92. http://dx.doi.org/10.1016/S0093-691X(98)00136-8 DOI: https://doi.org/10.1016/S0093-691X(98)00136-8
Aumuller G, Seitz J. Protein secretion. Int Rev Cytol 1990; 121: 127-31. DOI: https://doi.org/10.1016/S0074-7696(08)60660-9
Stocco DM. The role of the StAR protein. J Endocrinol 2000; 164: 247-53. http://dx.doi.org/10.1677/joe.0.1640247 DOI: https://doi.org/10.1677/joe.0.1640247
Payne AH, Hales DB. Overview of steroidogenic enzymes. Endocr Rev 2004; 25: 947-70. http://dx.doi.org/10.1210/er.2003-0030 DOI: https://doi.org/10.1210/er.2003-0030
Payne AH, Youngblood GL. Regulation of expression. Biol Reprod 1995; 52: 217-25. http://dx.doi.org/10.1095/biolreprod52.2.217 DOI: https://doi.org/10.1095/biolreprod52.2.217
Lervik S, von Krogh K, Karlsson C, Olsaker I, Andresen O, Dahl E, et al. Steroidogenesis in primary cultures. Theriogenology 2011; 76: 1058-69. http://dx.doi.org/10.1016/j.theriogenology.2011.05.011 DOI: https://doi.org/10.1016/j.theriogenology.2011.05.011
Goel S, Reddy N, Mandal S, Fujihara M, Kim SM, Imai H. Spermatogonia-specific proteins. Theriogenology 2010; 74: 1221-32. http://dx.doi.org/10.1016/j.theriogenology.2010.05.025 DOI: https://doi.org/10.1016/j.theriogenology.2010.05.025
Moundipa PF, Beboy NS, Zelefack F, Ngouela S, Tsamo E, Schill WB, et al. Effects of Basella alba. Asian J Androl 2005; 7: 411-17. http://dx.doi.org/10.1111/j.1745-7262.2005.00056.x DOI: https://doi.org/10.1111/j.1745-7262.2005.00056.x
Lin D, Gitelman SE, Saenger P, Miller WL. Normal genes. J Clin Invest 1991; 88: 1955-62. http://dx.doi.org/10.1172/JCI115520 DOI: https://doi.org/10.1172/JCI115520
Sun XR, Risbridger GP. Site of macrophage. Biol Reprod 1994; 50: 363-67. http://dx.doi.org/10.1095/biolreprod50.2.363 DOI: https://doi.org/10.1095/biolreprod50.2.363
Moran FM, Ford JJ, Corbin CJ, Mapes SM, Njar VC, Brodie AM, et al. Regulation of microsomal. Endocrinology 2002; 143: 3361-69. http://dx.doi.org/10.1210/en.2002-220329 DOI: https://doi.org/10.1210/en.2002-220329
Weng Q, Medan MS, Watanabe G, Tsubota T, Tanioka Y, Taya K. Immunolocalisation of steroidogenic enzymes. J Reprod Dev 2005; 51: 299-304. http://dx.doi.org/10.1262/jrd.16077 DOI: https://doi.org/10.1262/jrd.16077
Choi I, Kim JY, Lee FJ, Kim YY, Chung CS, Chang J, et al. Ontogeny of expression. J Androl 2009; 30: 57-74. http://dx.doi.org/10.2164/jandrol.107.004796 DOI: https://doi.org/10.2164/jandrol.107.004796
Davidoff MS, Middendorff R, Enikolopov G, Riethmacher D, Holstein AF, Dieter M. Progenitor cells. J Cell Biol 2004; 167: 935-44. http://dx.doi.org/10.1083/jcb.200409107 DOI: https://doi.org/10.1083/jcb.200409107
Weng Q, Medan MS, Ren L, Watanabe G, Tsubota T, Taya K. Immunolocalisation of steroidogenic enzymes. Exp Anim 2005; 54: 451-4. http://dx.doi.org/10.1538/expanim.54.451 DOI: https://doi.org/10.1538/expanim.54.451
Almeida J, Conley AJ, Mathewson L, Ball BA. Expression of steroidogenic enzymes. Reproduction 2011; 141: 841-48. http://dx.doi.org/10.1530/REP-10-0499 DOI: https://doi.org/10.1530/REP-10-0499
Bernier M, Gibb W, Haour F, Collu R, Saez JM, Ducharme JR. Studies with purified immature porcine Leydig cells in primary culture. Biol Reprod 1983; 29: 1172-78. http://dx.doi.org/10.1095/biolreprod29.5.1172 DOI: https://doi.org/10.1095/biolreprod29.5.1172
Geiger TL, Khan M, Whisnant CS, Prien SD, Khan SA. Regulation of DNA synthesis. Domest Anim Endocrinol 1999; 17: 65-75. http://dx.doi.org/10.1016/S0739-7240(99)00024-7 DOI: https://doi.org/10.1016/S0739-7240(99)00024-7
El-Sherbiny AM, Amin SO, Hernandez C, Carreau S. The immature rabbit testis: presence of two distinct populations of Leydig cells. World Rabbit Sci 1994; 2: 141-46. DOI: https://doi.org/10.4995/wrs.1994.230
Hedger MP, Risbridger GP, de Kretser DM. Autoradiographical localization. Aust J Biol Sci 1985; 38: 435-44. DOI: https://doi.org/10.1071/BI9850435
Machluf M, Orsola A, Boorjian S, Kershen R, Atala A. Microencapsulation of Leydig Cells. Endocrinology 2003; 144: 4975-79. http://dx.doi.org/10.1210/en.2003-0411 DOI: https://doi.org/10.1210/en.2003-0411
Anbalagan M, Sriraman V, Jagannadha Rao A. Isolation and characterization. J Endocrinol 2003; 179: 175-82. http://dx.doi.org/10.1677/joe.0.1790175 DOI: https://doi.org/10.1677/joe.0.1790175
Sun J, Zhong L, Zhu Y, Liu G. Research on isolation. J Reprod Dev 2011; 57: 433-6. http://dx.doi.org/10.1262/jrd.10-123N DOI: https://doi.org/10.1262/jrd.10-123N
Hess MF, Roser JF. A comparison of the effects. Anim Reprod Sci 2005; 89: 7-19. http://dx.doi.org/10.1016/j.anireprosci.2005.06.014 DOI: https://doi.org/10.1016/j.anireprosci.2005.06.014
Yoon MJ, Roser JF. Insulin-like growth factor-I. Anim Reprod Sci 2010; 122: 353-8. http://dx.doi.org/10.1016/j.anireprosci.2010.10.001 DOI: https://doi.org/10.1016/j.anireprosci.2010.10.001
Yoon MJ, Roser JF. A synergistic effect. Anim Reprod Sci 2011; 126: 195-99. http://dx.doi.org/10.1016/j.anireprosci.2011.05.008 DOI: https://doi.org/10.1016/j.anireprosci.2011.05.008
Ascoli M, Fanelli F, Segaloff DL. The lutropin/ choriogonadotropin receptor, a 2002 perspective. Endocr Rev 2002; 23: 141-74. http://dx.doi.org/10.1210/er.23.2.141 DOI: https://doi.org/10.1210/edrv.23.2.0462
Stocco DM, Wang X, Jo Y, Manna PR. Multiple signaling pathways. Mol Endocrinol 2005; 19: 2647-59. http://dx.doi.org/10.1210/me.2004-0532 DOI: https://doi.org/10.1210/me.2004-0532
Teerds KJ, Rijntjes E, Veldhuizen-Tsoerkan MB, Rommerts FF, de Boer-Brouwer M. The development of rat Leydig cell. J Endocrinol 2007; 194: 579-93. http://dx.doi.org/10.1677/JOE-06-0084 DOI: https://doi.org/10.1677/JOE-06-0084
Dufau ML. Endocrine regulation. Annu Rev Physiol 1988; 50: 483-508. http://dx.doi.org/10.1146/annurev.ph.50.030188.002411 DOI: https://doi.org/10.1146/annurev.ph.50.030188.002411
Brun HP, Leonard JF, Moronvalle V, Caillaud JM, Melcion C, Cordier A. Pig Leydig cell culture. Toxicol Appl Pharmacol 1991; 108: 307-20. http://dx.doi.org/10.1016/0041-008X(91)90120-4 DOI: https://doi.org/10.1016/0041-008X(91)90120-4
Krockova JZ, Massanyi P, Sirotkin AV, Pivko J, Makarevich AV, Lukac N, et al. Nickel induced structural. J Trace Elem Med Biol 2011; 25: 14-18. DOI: https://doi.org/10.1016/j.jtemb.2010.11.003
Wu L, Zhang A, Sun Y, Zhu X, Fan W, Lu X, et al. Sirt1 exerts anti-inflammatory effects. Fertil Steril 2012; 98: 194-9. http://dx.doi.org/10.1016/j.fertnstert.2012.04.008 DOI: https://doi.org/10.1016/j.fertnstert.2012.04.008
Lai KP, Wong MH, Wong CK. Inhibition of CYP450scc expression. J Endocrinol 2005; 185: 519-27. http://dx.doi.org/10.1677/joe.1.06054 DOI: https://doi.org/10.1677/joe.1.06054
Mondillo C, Falus A, Pignataro O, Pap E. Prolonged histamine deficiency. J Androl 2007; 28: 86-91.http://dx.doi.org/10.2164/jandrol.106.000257 DOI: https://doi.org/10.2164/jandrol.106.000257
Gunarajasingam D, Rajamahendran R, Downey BR, Lague PC. Testosterone secretion. Theriogenology 1985; 24: 185-95. http://dx.doi.org/10.1016/0093-691X(85)90182-7 DOI: https://doi.org/10.1016/0093-691X(85)90182-7
Downloads
Published
How to Cite
Issue
Section
License
Policy for Journals/Articles with Open Access
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgment of the work's authorship and initial publication in this journal.
- Authors are permitted and encouraged to post links to their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work