E. M. Kaiin et al. / Open Journal of Animal Sciences 3 (2013) 299-304 303
produced by isolated adult rat Leydig cells in vitro varies
from 0.5 ng up to 150 ng/106 Leydig cells per hour under
LH/hCG—stimulated condition [16]. Similar results oc-
curred in Leydig cell piglets cultures [15]. In this ex-
periment, Leydig cells were cultured in DMEM + ITS
which had the testosterone levels of 3.19 ng/ml. Pointis
et al. [14] stated that the addition of insulin in the culture
medium can increase the accumulation of testosterone in
fetal mouse Leydig cell cultures. Hormone that stimu-
lates steroidogenesis in Leydig cells is Luiteinizing
Hormone (LH) which is secreted by the pituitary.
Changes in LH levels in vivo can reflect variations of
physiological conditions that can induce morphological
changes and the ability of Leydig cells to synthesize and
secrete testosterone [22]. The use of hCG which is an
analog of LH that can bind to the same receptor with LH
receptors on Leydig cells [23] resulted in the secretion of
testosterone. The addition of hCG to the DMEM in-
creased the secretion of testosterone, whereas ITS in-
creased the cell proliferation that can increase the content
of testosterone in the culture medium.
The addition of 10% NBCS hormone in DMEM me-
dium produced the testosterone content of 1.29 ng/ml.
Sedelaar and Isaacs [24] reported that hormone testos-
terone concentrations between 1.2 and 7.5 ng/ml were
found in bovine serum from newborn to one year old
assayed using the RIA method. Serum contains several
nutritional components such as fatty acids, cholesterol,
T3, insulin, IGF, EGF and androgen [25]. Moreover, the
addition of serum is made to provide the hormones that
stimulate cell growth and function. Serum provides bio-
matriks that help the process of attachment and spreading
of cells, as well as carrier transport protein hormones,
minerals and lipids [26].
It can be concluded that the addition of ITS to the cul-
ture medium increased Leydig cell proliferation, whereas
addition of hCG increased concentrations of testosterone
hormone in the Leydig cell culture medium. The combi-
nation of ITS and hCG increased cell proliferation and
the concentration of testosterone in Leydig cell culture
medium. Further research is needed to increase the num-
ber of living cells in the production of Leydig cell line.
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