Fetal bovine serum (FBS) enhances proliferation and colonization of caprine spermatogonial stem cells

MANISHA PATHAK; S D KHARCHE; S P SINGH; D JENA; JUHI PATHAK; DEEKSHA GUPTA; A K S SIKARWAR; M S CHAUHAN

doi:10.56093/ijans.v90i5.104609

Authors

MANISHA PATHAK ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India
S D KHARCHE ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India
S P SINGH ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India
D JENA ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India
JUHI PATHAK ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India
DEEKSHA GUPTA ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India
A K S SIKARWAR ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India
M S CHAUHAN ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India

https://doi.org/10.56093/ijans.v90i5.104609

Keywords:

Capra hircus, Differential plating, Fetal bovine serum, Pre pubertal testis, Spermatogonial stem cells

Abstract

Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. This study was undertaken to investigate the effect of serum concentration on colony formation and development of different types of SSC colonies with respect to passage number. Cells were isolated from pre-pubertal buck testes by two step enzymatic digestion method. The filtered cells were enriched by differential adherence selection method. Cells were then randomly divided into 8 groups, depending on concentration of FBS in culture medium ranging from 0% to 35%. In experiment 1, effect of different concentrations of FBS on total number pSSCs with reference to differential plating was observed while in experiment 2, effect of different concentrations of FBS on types of pSSC colonies with respect to passage number was observed. No colony formation was observed in control group (0% FBS) while significantly higher number of single, paired, cluster and rosette colonies observed were with 20% FBS group in differential 2 (D2) as compared to other groups. Alkaline phosphatase staining and immunocytochemistry staining (PGP9.5 and OCT4) were positive in SSCs colonies. The growth rate of the culture was significantly and consistently higher with 20% FBS.

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References

Adetunji P, Fayomi and Kyle E O. 2018. Spermatogonial stem cells and spermatogenesis in mice, monkeys and men. Stem Cell Research 29: 207–14. DOI: https://doi.org/10.1016/j.scr.2018.04.009

Aponte P M, Van Bragt M P, De Rooij D G and Van Pelt A M. 2005. Spermatogonial stem cells: characteristics and experimental possibilities. APMIS 113: 727–42. DOI: https://doi.org/10.1111/j.1600-0463.2005.apm_302.x

Baker H, DeAngelis B and Frank O. 1988. Vitamins and other metabolites in various sera commonly used for cell culturing. Experientia 44: 1007–1010. DOI: https://doi.org/10.1007/BF01939904

Heideri B, Rahmati-Ahmadabadi M, Akhondi M M, Zarnani A H, Jeddi-Tehrani M, Shirazi A, Naderi M M and Behzadi B. 2012. Isolation, identification, and culture of goat sscsusing c-kit and pgp9.5 markers. Journal of Assisted Reproduction and Genetics 29: 1029–38. DOI: https://doi.org/10.1007/s10815-012-9828-5

Jabarpour M and Tajik. 2018. Evaluating the behaviour of cultured sertoli cells in the presence and absence of spermatogonial stem cell. Stem Cell Investigation 5: 1. DOI: https://doi.org/10.21037/sci.2018.01.01

Kanatsu-Shinohara M, Muneto T, Lee J, Takenaka M and Chuma S. 2008. Long-term culture of male germline stem cells from hamster testes. Biology of Reproduction 78: 611–17. DOI: https://doi.org/10.1095/biolreprod.107.065615

Pramod R K and Mitra A. 2014. In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus). Journal of Assisted Reproduction and Genetics 31: 993–1001. DOI: https://doi.org/10.1007/s10815-014-0277-1

Reed S A and Johnson S E. 2008. Equine umbilical cord blood contains a population of stem cells that express Oct4 and differentiate into mesodermal and endodermal cell types. Journal of Cellular Physiology 215: 329–36. DOI: https://doi.org/10.1002/jcp.21312

Stefkova K, Prochazkova J and Pachernik J. 2015. Alkaline phosphatise in stem cells. Stem Cells International 2015: 628368. DOI: https://doi.org/10.1155/2015/628368

Takashima S and Shinohara T. 2018. Culture and transplantation of spermatogonial stem cells. Stem Cell Research 29: 46–55. DOI: https://doi.org/10.1016/j.scr.2018.03.006

Tegelenbosch R A and de Rooij D G. 1993. A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mutation Research 290: 193–200. DOI: https://doi.org/10.1016/0027-5107(93)90159-D

Van Der Valk J, Bieback K, Buta C, Cochrane B, Dirks W G, Fu J, Hickman J J, Hohensee C, Kolar R, Liebsch M, Pisto, Atp F, Schulz M, Thieme D, Weber T, Wiest J, Winkler S and G straunthalar G. 2018. Fetal bovine serum (FBS): Past-presentfuture. Alternatives to Animal Experimentation 35(1): 99–118. DOI: https://doi.org/10.14573/altex.1705101

Van Der Wee K S, Johnson E W, Dirami G, Dym M and Hofmann M. 2001. Immunomagnetic isolation and long-term culture of mouse Type A spermatogonia. Journal of Andrology 22(4): 696–704.