Development of <i>in vitro</i> culture of rat Leydig cells after purification with Nycodenz gradient

doi:10.4236/ojas.2013.34044

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Vol.3, No.4, 299-304 (2013) Open Journal of Animal Sciences

http://dx.doi.org/10.4236/ojas.2013.34044

Development of in vitro culture of rat Leydig cells

after purification with Nycodenz gradient

Ekayanti Mulyawati Kaiin1*, Ita Djuwita2, Tuty Laswardi Yusuf3, Mohamad Agus Setiadi3

1Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia;

*Corresponding Author: [email protected]

2Department of Anatomy, Physiology and Pharmacology, Faculty of Veterinary Medicine, Bogor Agriculture University (IPB),

Jl. Agatis Kampus Darmaga, Bogor, Indonesia

3Department of Veterinary Clinic, Reproduction and Pathology, Faculty of Veterinary Medicine, Bogor Agriculture University (IPB),

Jl. Agatis Kampus Darmaga, Bogor, Indonesia

Received 11 July 2013; revised 23 August 2013; accepted 12 September 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

The study examined the effect of human Chori-

onic Gonadotrophin (hCG) and/or Insulin Trans-

ferring Sodium Selenite (ITS) on the proliferation

and development of rat Leydig cells purified by

Nycodenz gradient. Leydig cells purity, viability

and proliferation after purification and 3 days of

cultured were evaluated. Leydig cells 1 × 106

cells/ml were cultured in DMEM containing 10%

Newborn Calf Serum (NBCS) and divided into

four kinds of treatments 1) as a control, 2) control

+ 2.5 IU/ml hCG, 3) control + ITS (5 μg/ml insulin,

10 μg/ml transferrin, 5 μg/ml Se), and 4) control +

hCG + ITS. Leydig cells purification results

showed 91.40% of purity, viability was 98.17%

and concentration 7.03 × 106 cells/ml. The addi-

tion of ITS and hCG + ITS in DMEM produced

Leydig cell proliferation by 88.35% and 90.64%

higher than in controls (86.82%) (p < 0.05). The

addition of hCG did not increase Leydig cell

proliferation (86.99%). In the primary culture of

Leydig cells, the population doubling time (PDT)

was 1.03 days, similar with the addition of hCG

treatment (1.02 day). Declined in value of PDT

significantly (p < 0.05) occurred in DMEM sup-

plemented with ITS (0.97 day) and hCG + ITS

(0.88 day). This result was also seen in the first

and second Leydig cell lines. DMEM added with

hCG or hCG + ITS resulted in a higher amount of

testosterone (5.06 ng/ml; 5.25 ng/ml)) than the

culture medium without hCG (2.46 ng/ml) (p <

0.05). It can be concluded that the combination

of hCG + ITS can increase the number of Leydig

cells and testosterone levels in the culture me-

dium.

Keywords: Leydig; In Vitro; Rat; Nycodenz; hCG;

ITS; Testosterone

1. INTRODUCTION

Androgen hormonal therapy for hypogonadism in men

has been done to maintain testosterone level physiologi-

cally. Bhasin & Bremer stated that this therapy can in-

crease muscle strength, improve osteoporosis, stabilized

bone density and restore the secondary sexual characters

[1]. However, provision of these hormones in the long

term can cause an increase in blood viscosity, red blood

cell formation abnormalities, hypertension, stroke, bone

density changes and emotional changes [1-3]. Therefore,

it is necessary to attempt an alternative therapy such as

Leydig cells transplantation which naturally produced

testosterone that can be used to replace the use of syn-

thetic testosterone hormone [1]. This cellular therapy has

limitations in terms of availability of tissue and cells.

Therefore, Leydig cell culture is required to obtain a

source of cells that will be used in cellular therapy. Tes-

ticular tissue consists of somatic cells like Sertoli cells,

Leydig cells, fibroblasts, and spermatogenic cells (male

gamet cells) in various stages of development, and other

cells so it requires to be isolated and purified with ap-

propriated methods like Nycodenz gradient [4]. Ny-

codenz or Iohexol has several advantages compared to

Percoll including lower osmolality and viscosity but

higher density. It was able to separate macromolecules

and cells effectively with a broader range of sizes for

more varied cell types. Nycodenz are stable, soluble in

water and can be sterilized with a syringe filter that is

easy to use in the laboratory and non cytotoxic against

mammalian cells. Additionally, Nycodenz can be easily

E. M. Kaiin et al. / Open Journal of Animal Sciences 3 (2013) 299-304

300

removed from the cell suspension by centrifugation com-

pared with gradients of other materials because the re-

sidual gradient can lead to cell death. Nycodenz can be

used easily by a simple method without any limitations

in temperature, pH and stability [5]. Nycodenz gradient

was used to separate blood cells, liver cells, the primor-

dial germ cell (PGC), spermatogonia and Sertoli cells

[6-8].

To obtain an optimum condition in supporting prolife-

ration of Leydig cell culture in vitro, medium culture

requires some bioactive materials such as human Chorio-

genic Gonadotrophin (hCG) and Insulin Transfferin So-

dium Selenite (ITS). Leydig cell may secrete several

bioactive materials such as peptide, growth hormone,

Interleukin-1 (IL-1) and Interleukin-6 (IL-6), testoster-

one and others to support their growth [9-11]. Luiteniz-

ing Hormone (LH) or hCG is necessary for the prolifera-

tion and differentiation of Leydig cells so that the cells

are able to produce testosterone [12]. The concentration

of testosterone in human Leydig cell culture medium has

increased after the addition of 1 IU hCG/ml [9] whereas

the addition of 5 IU hCG on Leydig cell culture medium

of young rabbit produce a large amount of testosterone

[13]. From several studies before, it seems that addition

of ITS is required as a bioactive material for the Leydig

cell proliferation. Chemes et al. [9] added transferrin 10

μg/ml and insulin that can enhance the production of

testosterone by the Leydig cells of fetal mice in the cul-

ture medium for 24 and 48 hours [14]. Bernier et al. [15]

added insulin and transferrin each 5 μg/ml and 1 IU

hCG/ml into the culture of Leydig cells piglets which

causes an increase in the synthesis of testosterone com-

pared to those without addition of hCG. The purpose of

this study was to test the influence of the addition of hCG,

ITS or in combination in DMEM medium on prolifera-

tion and development of Leydig cells and to get an opti-

mum condition of adult rat Leydig cells in vitro.

2. MATERIALS AND METHODS

2.1. Isolation and Purification of Leydig

Cells

Testes were collected from the rats (Sprague Dawley)

adult males aged 8 - 10 weeks after anesthetized and

sacrificed by cervical dislocation. Testicular tissue was

placed in a petri dish contains Dulbecco’s Phosphate

Buffer Saline (DPBS) without Ca and Mg (Gibco, 21600

- 010, Invitrogen, NY, USA). The tissue was then washed

three times using medium DPBS supplemented with

0.1% Newborn Calf Serum (NBCS, Gibco, 16010 - 159,

Invitrogen, New Zealand) (DBPS). Testicular tissue were

then put into tubes containing one ml of 0.04% colla-

genase type I (Sigma, C0130, St. Louis, MO, USA) and

10 μg/ml trypsin inhibitor (Sigma, T9003, St. Louis, MO,

USA) in DPBS and incubated in a water bath at a tem-

perature of 34˚C for 40 min. Cell suspension diluted four

times to initial volume with DPBS then allowed to pre-

cipitate. Supernatant was collected and centrifuged at

200 g for three minutes. Cell pellet then was washed

twice by using DPBS and diluted with 0.5 ml DPBS.

Isolation and purification of Leydig cells were conducted

by using Nycodenz gradient. The cell suspension were

put into Nycodenz gradient 4%, 8%, 10%, 12%, 15% and

then were centrifuge using rotor swing (KOKUSAN

H-26F) at 1500 g for 10 min. Cell layer formed were

collected and washed four times with DPBS and DMEM

(Sigma, D5532, St. Louis, MO, USA) supplemented 10%

NBCS and centrifuged at 200 g for three minutes. Cell

pellet was diluted with 0.5 ml DMEM and the cell con-

centration was calculated using Neubauer haemocytom-

eter.

2.2. In Vitro Culture and the Production of

Leydig Cells

Leydig cells as 1 × 106 cells/ml were placed in a petri

dish (Corning, 430 165, NY USA) 35 × 10 mm contain-

ing DMEM medium supplemented with 10% NBCS as a

control (group 1), with 2.5 IU/ml hCG (Chorulon, In-

tervet, EU) (group 2), with 5 mg/ml insulin, 10 mg/ml

transferrin, 5 μg/ml Se (ITS, Sigma I3146, St Louis, MO,

USA) (group 3) and hCG + ITS (group 4). All petri dish

then cultured in a 5% CO2 incubator (Sanyo, MCO-95,

Japan) at 37˚C. After cultured for three days, Leydig

cells were calculated for their concentration, viability

and purity. Leydig cells were stained by Trypan Blue and

specific staining 3-β HSD. Primary cultures from the

each treatment were passage on day 3 and after washing

with DPBS medium then the cells were calculated using

a Neubauer haemositometer. Cell was then cultured

again until reaching a confluent stage. Passage was per-

formed twice and was counted Population Doubling

Time (PDT) with the formula:







PDT day

log final cellnumberlog initial cellnumber3.32

time



2.3. Testosterone Content from Leydig Cell

Culture Medium

Leydig cell culture medium from each treatment were

collected on day 3 and tested by using Testosterone

ELISA kit (DRG Diagnostics EIA 1559). Tests were car-

ried out at the Laboratory of Hormones, Reproduction

Rehabilitation Unit, Faculty of Veterinary Medicine, Bo-

gor Agriculture University (IPB).

E. M. Kaiin et al. / Open Journal of Animal Sciences 3 (2013) 299-304 301

2.4. Experimental Design and Data Analysis

Leydig cell cultures performed three replications for

each treatment. The parameters measured were viability,

purity, PDT and number of Leydig cells alive. Leydig

cell lines made up to second passage and repeated three

times. The content of testosterone in the culture medium

were conducted three replications in each treatment. Data

were analyzed using one way ANOVA and if there is a

difference between treatments followed by Duncan’s. All

cases significance was set at p < 0.05.

3. RESULTS

The results showed that a percentage of Leydig cells

after purified with Nycodenz gradients were 91.40%,

while the viability was 98.17% and the cell concentration

was 6.30 × 106 cells/ml (Table 1).

The effect of hCG, ITS or in combination on Leydig

cell proliferation in vitro were presented at Table 2. Ley-

dig cells proliferation were cultured in DMEM + ITS and

DMEM + hCG + ITS significantly (p < 0.05) higher at

88.35% and 90.64% when compared to control (86.82%).

The addition of hCG to the Leydig cell culture medium

did not increase the proliferation of Leydig cells (86.99%).

The percentage of Leydig cells tend to decline at the end

of treatment by hCG, ITS and the combination.

The result of the passage of Leydig cells increased in

cell number at the end of the primary culture and also at

first and second cell lines (Table 3). In between treat-

ments shows that the role of hCG and ITS increased the

number of Leydig cells compared with the other treat-

ments.

Table 1. Purity, viability and concentration of Leydig cell after

purification with Nycodenz gradient.

Parameter Leydig Cells

Purity (%) 91.40 ± 5.02

Viability (%) 98.17 ± 0.51

Cell concentration (106 cells/ml) 7.03 ± 1.04

Number of living cells (106 cells/ml) 6.30

Table 2. Primary cultures of Leydig cells after purified with

Nycodenz gradient.

Medium treatment

Parameter DMEM

DMEM

+ hCG

DMEM

+ ITS

DMEM +

hCG + ITS

Proliferation

rate (%) 86.82a 86.99a 88.35b 90.64c

Leydig cells (%)

-Initial culture 90.75 90.75 90.75 90.75

-End of culture 88.75 88.25 88.00 88.50

Note: Different superscript letters indicate significant differences (p < 0.05)

by Duncan test on the same line.

Table 3. Number of cells and PDT from cell suspense in vitro.

Cell line in several

medium treatment

Initial cell

number

(106/ml)

End of cell

number

(106/ml)

PDT (day)

Primary cultures

DMEM 1 7.60aA 1.03a

DMEM + hCG 1 7.69a 1.02a

DMEM + ITS 1 8.63b 0.97b

DMEM + hCG + ITS1 10.69c 0.88c

1st cell line

DMEM 1 6.71aAB 1.09a

DMEM + hCG 1 7.60a 1.03a

DMEM + ITS 1 8.30a 0.99a

DMEM + hCG + ITS1 13.56b 0.82b

2nd cell line

DMEM 1 6.28aB 1.14a

DMEM + hCG 1 7.27ab 1.05a

DMEM + ITS 1 8.76b 0.96b

DMEM + hCG + ITS1 9.17c 0.94b

Note: Different small superscript letters indicate significant differences (p <

0.05) with Duncan test on the same cell lines. Different capital superscript

letters indicate significant differences (p < 0.05) with Duncan test in the

same treatment with the different cell lines.

The percentage of Leydig cells and the viability was

seen in all treatments (80% - 91%). The ability of Leydig

cells to proliferate seen from the highest number of Ley-

dig cells in the treatment DMEM + hCG + ITS in pri-

mary culture, first and second cell lines compared to

other treatments (Table 4).

The addition of ITS, hCG + ITS in DMEM increasing

the number of live Leydig cells (p < 0.05) in primary

cultures (6.68 × 106/ml and 8.31 × 106/ml cells). Number

of live Leydig cells in first and secondary cell lines on all

treatments were also increased (p < 0.05) compared with

controls. Treatment of the same medium in first and sec-

ond cell lines reduced the number of live Leydig cells (p

< 0.05) compared to primary culture except the treatment

of hCG + ITS on the first cell line, the Leydig cells alive

increased in number.

Table 5 shows the content of testosterone increased (p

< 0.05) in medium with hCG and hCG + ITS to 5.06

ng/ml and 5.25 ng/ml than without hCG (2.46 ng/ml).

4. DISCUSSION

Several different methods of purification of Leydig

cells have been reported by other researchers. The percen-

tage of Leydig cells purified by Nycodenz gradient in

this experiment was higher than Risbridger and Hedger

[16] which was 87%. The same result by Yang et al. (2003)

was 95% and also by Kaiin et al. [4] was 92.22% using

Percoll gradient. However, the concentration of Leydig

cells purified by Percoll was higher at 15.42 × 106 cells/ml

[4] A higher proliferation of Leydig cells was found when .

E. M. Kaiin et al. / Open Journal of Animal Sciences 3 (2013) 299-304

302

Table 4. The purity, viability and number of living cells of Leydig cell lines.

Cell line in several

medium treatment Cell concentration

(×106/ml) Leydig cells (%) Number of Leydig

cells (×106/ml) Cell Viability (%) Number of live

cells (×106/ml)

Primary culture

DMEM 7.60aA 85.17 6.47aA 88.67A 5.74aA

DMEM + hCG 7.69a 85.50A 6.58a 89.00C 5.85aD

DMEM + ITS 8.63b 86.67 7.48b 89.33E 6.68bG

DMEM + hCG + ITS 10.69c 87.00 9.30c 89.33G 8.31cI

1st cell line

DMEM 6.71aAB 85.67 5.74aAB 86.33A 4.96aB

DMEM + hCG 7.60a 85.33A 6.49a 84.00D 5.45bE

DMEM + ITS 8.30a 86.67 7.19a 87.00E 6.26cH

DMEM + hCG + ITS 13.56b 87.33 11.84b 88.33G 10.46dJ

2nd cell line

DMEM 6.28aB 86.67 5.44aB 80.67B 4.39aC

DMEM + hCG 7.27ab 89.17B 6.49ab 81.67D 5.30bF

DMEM + ITS 8.76b 90.67 7.94b 82.67F 6.57cH

DMEM + hCG + ITS 9.17c 91.17 8.36c 83.33H 6.97cK

Note: Different small superscript letters indicate significant differences (p < 0.05) with Duncan test in the same cell line. Different capital superscript letters

indicate significant differences (p < 0.05) with Duncan test in the same treatment with different cell lines.

Table 5. Testosterone content in Leydig cells culture medium.

Treatment n Testosterone (ng/ml)

DMEM + 10% NBCS (DMEM) 2 1.29a

Leydig cells + DMEM 3 2.46ab

Leydig cells + DMEM + hCG 3 5.06c

Leydig cells + DMEM + ITS 3 3.19b

Leydig cells + DMEM + hCG + ITS 3 5.25d

Note: The different superscript letters indicate significant difference (p <

0.05) in column.

cultured in vitro with ITS. Insulin Transferrin Sodium

Selenite (ITS) is the supplement that used to increase cell

proliferation. Insulin is a polypeptide hormone that helps

the absorption of glucose and amino acids, whereas

transferrin is an iron-carrying protein aims to help cell

nutrient absorption. Selenium is an essential trace ele-

ment present in the serum.

Doubling time is the time period required by the cell

to make twice of size or the number from the original

[18]. The faster of proliferation of the cell made the

value of PDT lower. The value of PDT from primary cul-

tures of Leydig cells was 1.03 days and that was equiva-

lent to DMEM + hCG treatment (1.02 day). The value of

PDT was significantly (p < 0.05) lower in DMEM + ITS

treatment (0.97 day) and DMEM + hCG + ITS (0.88

day). Similar results occurred in the first and second cell

lines. The results showed that the addition of ITS, as well

as hCG + ITS makes Leydig cells require shorter time to

achieve a cell number twice. It supports the ITS func-

tions as a bioactive material that can increase cell prolif-

eration. Butler [19] states that the time required for the

process of cell division in vivo occurs approximately 18 -

24 hours. After passage, the cell is more homogeneous

and able to adapt to the environmental conditions in vitro

[20]. Leydig cells in primary culture have a fairly high

percentage of purity (>90%) and because those in culture

were homogeneous, they made the value of PDT near to

the time of proliferation in vivo. Leydig cell number on

treatment with DMEM decreased in the first and second

cell lines (p < 0.05), therefore, resulted in a higher PDT

from primary culture. Similarly occurred in the other

treatment, except in the first cell line, that an increase in

the number of cells was occurred in hCG + ITS. The

addition of ITS in DMEM led to an increased in the

number of Leydig cells at the second cell lines compared

to the first lines. In general, a decline in the ability of

Leydig cell proliferation occurred after passage twice.

According to Hebert et al. [21], the stimulation of pro-

liferation of rat Leydig cell precursors is influenced by

growth factors such as Transforming Growth Factor

(TGF-α) and Insulin-like Growth Factor (IGF-I). In addi-

tion, Platelet-derived Growth Factor (PDGF-A) which is

secreted by Sertoli cells is also required for proliferation

and differentiation of adult Leydig cells. The decrease in

the number of live Leydig cells in the first and second

cell line is probably influenced by the availability of

these growth factors in the culture medium.

Leydig cells in culture have the capacity to secrete

testosterone up to 72 hours [22]. Level of testosterone

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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