Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture A., Bhuyan D., Das P.P., Koushik S., Das B., Phookan A., Kharche S.D., Singh S.P., Chauhan M.S. (2020): Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture. Czech J. Anim. Sci., 65: 66-76.
supplementary materialdownload PDF

The present study was conducted to compare the stemness and morphobiometry of spermatogonial stem cells (SSCs) from the Doom pig on different days of culture (9th, 30th and 65th day) for the development of long-term culture method. The testes from 7–15-day old piglets were collected and two-step enzymatic digestion was used to isolate SSCs. Before in vitro culture of SSC-like cells on the Sertoli cell feeder layer, the cells were enriched by differential plating and Percoll density gradient centrifugation. The isolated SSCs were characterised by alkaline phosphatase and immunofluorescence staining and qPCR analysis of SSC specific marker genes. Stemness was compared based on the expression of different SSC specific marker genes. The putative spermatogonial stem cells (PSSCs) from all the days of culture were found to be positive for alkaline phosphatase and immunofluorescence staining. The results from qPCR analysis showed that PSSCs were positive for SSC marker genes, though their expression decreased gradually from day 9 to day 65 of culture. The shape of the cells was found to change from compact round or oval to amorphous shape on day 65 of culture. Colony diameter ranged from 68.92 ± 1.20 µm (day 9) to 213.53 ± 12.52 µm (day 65) and differed significantly from each other. The number of colonies on day 65 of culture was significantly lower than on days 9 and 30. These results suggest that the enriched SSCs from Doom pigs can be maintained up to two months in vitro in the present culture system.

Bai Y, Feng M, Liu S, Wei H, Li L, Zhang X, Shen C, Zhang S, Ma N. Differential gene expression in mouse spermatogonial stem cells and embryonic stem cells. Int. J. Mol. Med. 2016;38:423-432.
Beaulah JV, Ushakumary S, Kannan TA, William BJ, Ramesh G. Isolation and characterization of adipose derived mesenchymal stem cells (ADMSCs) in Madras Red Sheep (Ovis aries). Adv. Biomed. Pharma. 2016;3:156-160.
Brevini TA, Antonini S, Cillo F, Crestan M, Gandolfi F. Porcine embryonic stem cells: Facts, challenges and hopes. Theriogenology, 2007;68:206-213.
Cheng G, Feng ST. Studies on spermatogonial stem cells cultured in vitro of Wuzhishan mini porcine. Sheng Wu Gong Cheng Xue Bao, 2006;22:689-693.
de Rooij DG. The spermatogonial stem cell niche. Microsc. Res. Tech. 2009;72:580-585.
Dym M, Kokkinaki M, He Z. Spermatogonial stem cells: Mouse and human comparisons. Birth Defects Res. C Embryo Today, 2009;87:27-34.
Guaus SJB, Kim S, Mulero L, Vaquero JM, Morera C, Milanes RA, Veiga A, Raya A. Molecular markers of putative spermatogonial stem cells in the domestic cat. Reprod. Domest. Anim. 2017;52:177-186.
Hamidabadi HG, Bojnordi MN. Co-culture of mouse spermatogonial stem cells with Sertoli cells as a feeder layer, stimulates the proliferation and spermatogonial stemness profile. Middle East Fertil. Soc. J. 2018;23:107-111.
Han S, Gupta M, Uhm S, Lee H. Isolation and in vitro culture of pig spermatogonial stem cell. Asian-Australas. J. Anim. Sci. 2009;22:187-193.
Hernandez M, Ekwall H, Roca J, Vazquez JM, Martinez E, Rodriguez-Martinez H. Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars. Cryobiology, 2007;54:63-70.
Herrid M, McFarlane JR. Application of testis germ cell transplantation in breeding systems of food producing species: a review. Anim. Biotechnol. 2013;24:293-306.
Ju JY, Park CY, Gupta MK, Uhm SJ, Paik EC, Ryoo ZY, Cho YH, Chung KS, Lee HT. Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning. Fertil. Steril. 2008;89:1314-1323.
Kala S, Kaushik R, Singh KP, Kadam PH, Singh MK, Manik RS, Singla SK, Palta P, Chauhan MS. In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell. J. Assist. Reprod. Genet. 2012;29:1335-1342.
Kanatsu-Shinohara M, Takehashi M, Shinohara T. Brief history, pitfalls, and prospects of mammalian spermatogonial stem cell research. Cold Spring Harb. Symp. Quant. Biol. 2008;73:17-23.
Kokkinaki M, Lee TL, He Z, Jiang J, Golestaneh N, Hofmann MC, Chan WY, Dym M. The molecular signature of spermatogonial stem/progenitor cells in the 6-day-old mouse testis. Biol. Reprod. 2009;80:707-717.
Kokkinaki M, Djourabtchi A, Golestaneh N. Long-term culture of human SSEA-4 positive spermatogonial stem cells (SSCs). J. Stem Cell Res. Ther. 2011;s2:003.
Kon Y, Endoh D, Iwanaga T. Expression of protein gene product 9.5, a neuronal ubiquitin C-terminal hydrolase, and its developing change in sertoli cells of mouse testis. Mol. Reprod. Dev. 1999;54:333-341.
Lim JJ, Seol DW, Choi KH, Shin DH, Kim HJ, Song SH, Lee DR. Spermatogonial stem cell enrichment using simple grafting of testis and in vitro cultivation. Sci. Rep. 2014;4:5923.
Luo J, Megee S, Rathi R, Dobrinski I. Protein gene product 9.5 is a spermatogonia-specific marker in the pig testis: Application to enrichment and culture of porcine spermatogonia. Mol. Reprod. Dev. 2006;73:1531-1540.
Mutoji K, Singh A, Nguyen T, Gildersleeve H, Kaucher AV, Oatley MJ, Oatley JM, Velte EK, Geyer CB, Cheng K, McCarrey JR, Hermann BP. SPAN8 expression distinguishes spermatogonial stem cells in the prepubertal mouse testis. Biol. Reprod. 2016;95:1-14.
Nasiri Z, Hosseini SM, Hajian M, Abedi P, Bahadorani M, Baharvand H, Nasr-Esfahani MH. Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro. Theriogenology. 2012;77:1519-1528.
Pramod RK, Mitra A. In vitro culture and characterization of spermatogonial stem cells on sertoli cell feeder layer in goat (Capra hircus). J. Assist. Reprod. Genet. 2014;31:993-1001.
Sahare M, Kim SM, Otomo A, Komatsu K, Minami N, Yamada M, Imai H. Factors supporting long-term culture of bovine male germ cells. Reprod. Fertil. Dev. 2016;28:2039-2050.
Scarpino S, Morena AR, Petersen C, Froysa B, Soder O, Boitani CA. Rapid method of Sertoli cells isolation by DSA lectin, allowing mitotic analyses. Mol. Cell Endocrinol. 1998;146:121-127.
Shi R, Bai Y, Li S, Wei H, Zhang X, Li L, Tian XC, Jiang Q, Wang C, Qin L, Cai J, Zhang S. Characteristics of spermatogonial stem cells derived from neonatal porcine testis. Andrologia. 2015;47:765-778.
Sun YZ, Liu ST, Li XM, Zou LK. Progress in in vitro culture and gene editing of porcine spermatogonial stem cells. Zool. Res. 2019;20:1-6.
Takagi Y, Talbot NC, Rexroad Jr. CE, Pursel VG. Identification of pig primordial germ cells by immunocytochemistry and lectin binding. Mol. Reprod. Dev. 1997;46:567-580.
Zhang L, Tang J, Haines CJ, Feng LH, Lai L, Teng X, Han Y. c-kit and its related genes in spermatogonial differentiation. Spermatogenesis. 2011;1:186-194.
Zhang P, Chen X, Zheng Y, Zhu J, Qin Y, Lv Y, Zeng W. Long-term propagation of porcine undifferentiated spermatogonia. Stem Cells Dev. 2017;26:1121-1131.
Zheng Y, Tian XE, Zhang YQ, Qin JZ, An JH, Zeng WX. In vitro propagation of male germline stem cells from piglets. J. Assist. Reprod. Genet. 2013;30:945-952.
supplementary materialdownload PDF

© 2020 Czech Academy of Agricultural Sciences