ISSN 1062-3604, Russian Journal of Developmental Biology, 2016, Vol. 47, No. 5, pp. 269–277. © Pleiades Publishing, Inc., 2016.
Effect of Different Serum Concentrations on Short Term
in vitro Culture of Goat Testicular Cells
*, Morteza S. Hosseini
, Hassan Abbasi
, and Mohammad H. Nasr-Esfahani
Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
Department of Reproductive Biotechnology at Reproductive Biomedicine Research Center,
Royan Institute for Biotechnology, ACECR, Isfahan, Iran
Department of Biology, Faculty of Science, Nour Danesh Institute of Higher Education, Meymeh, Isfahan, Iran
*e-mail: email@example.com, firstname.lastname@example.org
Received March 21, 2016; in final form, April 25, 2016
Abstract—Proper in vitro culture and proliferation of spermatogonial stem cells (SSCs) are key tools to
achieve functional potentials of SSCs. The fresh serum extracted has been successfully used to stimulate
growth of spermatogonial stem cells in vitro. However, there is no report on the optimal concentration of
serum for in vitro culture of goat SSCs. This study investigated this issue.
Material and methods: Retrieved testicular cells were cultured in DMEM in presence of different 0 to 15%
serum for 7 days and examined on 3
day of culture for colony formation, cell viability, immunocy-
tochemistry and quantitative gene expression analyses.
Results: The maximum numbers of colonies were observed in presence of 1% serum on days 3 and 7 of cul-
ture. Differential viable staining revealed that most cells were non-viable in complete absence of serum (0%).
Immunocytochemistry showed that cells positive for pan cytokeratin were observed in colonies developed in
presence of 1, 5, 10 and 15% serum but not 0%, suggesting the presence of epithelial cells within the develop-
ing colonies. The colonies developed in the presence of 1 and 5% serum had remarkable alkaline phosphatase
activity. The maximum expressions of Plzf, Bcl6b and Vasa were observed in colonies developed in presence
of 1% serum, with a significant difference to all the other groups. No significant difference was observed for
the expression of Vimentin between different treatment groups.
Conclusion: The results of this study indicate that for maintenance of SSCs, 1% serum required for maintain-
ing viability while allowing proliferation and maintenance of SSCs like colonies.
Keywords: Goat, SSCs, Serum, Culture
Spermatogonial stem cells (SSCs) are the founda-
tion of spermatogenesis in the testes (Tegelenbosch
and de Rooij, 1993). Although the total number of
SSCs in adult testes in as low as 0.02–0.03% of total
germ cells, they have a unique ability to both self-
renew (to maintain their initial number) and to differ-
entiate into mature sperm through the spermatogene-
sis process (Oatley and Brister, 2008).
SSCs are located at the base of seminiferous
tubules in a niche provided by supporting Sertoli cells.
Sertoli cells construct the blood-testis barrier by their
tight junctions and sustain all differentiating classes of
spermatogonial stem cells during the spermatogenesis
process (Cheng and Mruk, 2002). Seminiferous
tubules in turn are surrounded by myoid cells, a kind
of fibroblast cells contain contractile elements to con-
dense tubules for sperm propulsion. Leydig cells, (a
polyhedral epithelioid cell) release testosterone hor-
mone and are out of the tubules.
In vitro culture of SSCs provides promising oppor-
tunities for the study of spermatogenesis, restoring of
fertility and for production of transgenic animals.
Nagano et al. cultured murine SSCs for the first time
(Nagano et al., 1998). Since then, this technique of
SSCs culture was gradually developed (Nagano, 2011)
in different animal species, including human. How-
ever, proper in vitro culture of SSCs with the capacity
to restore fertility and contribution to the next genera-
tion has been so far achieved only in the mice, rats,
goats, chickens (Wyns, 2010) and to a certain extent in
The article is published in the original.
Abbreviations: SSC, spermatogonial stem cell; PBS, phosphate
buffer saline; DMEM, Dulbecco’s modified eagle medium;
FBS, fetal bovine serum.
DEVELOPMENTAL BIOLOGY OF MAMMALS