Establishment of Primary Sertoli Cell Culture
from Immature Shiba Goat Testes

(KULTUR SEL SERTOLI PRIMER DARI TESTIS KAMBING SHIBA MUDA)
By
B. B. Andriana1, 4), A. A. A. Mirah Adi3), Y. Kanai1), Y. Hayashi2) and
M. Kurohmaru1).
1)Department of Veterinary Anatomy, 2)Department of Global Animal Resource Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. 3)Department of Veterinary Pathology, Udayana University, Bali, Indonesia. 4)Department of Animal Science, Bogor Agriculture University, Kampus Darmaga Bogor 16680, Indonesia. Email address: [email protected].

ABSTRACT
In order to evaluate the toxicity of xenobiotics, the application of in vitro model system has been increased in recent years. In this study, we attempted to establish the primary Sertoli cell culture from immature Shiba goat testes as such model. Two heads of immature (presumably 6-10 day-old) Shiba goats were sacrificed under anesthesia. The testes were excised, decapsulated, and cut into smaller pieces. Then, the sequential collagenase and trypsin-EDTA treatments were carried out. The cell culture with the cell density of 3.1×106 cells.cm-2 was incubated in a humidified atmosphere consisting of 95% air and 5% CO2 at 32oC, and maintained for 18 days. The culture was replaced, reseeded and incubated for 3 days to obtain the monolayer of Sertoli cell culture. Based on light and transmission electron microscopic observations, it became obvious that the intact primary Sertoli cell culture could be established through these processes. In the primary Sertoli cell culture, discernible intermediate filaments surrounding the nucleus of Sertoli cells could be apparently detected.

Key words: Primary Sertoli cell culture, Shiba goat

ABSTRAK
Dewasa ini, dalam rangka mengevaluasi xenobiotic toksikan, penggunaan model in vitro telah meningkat. Dalam penelitian ini, telah diusahakan pembentukan kultur sel Sertoli primer dari testis kambing Shiba muda sebagai hewan percobaannya. Dua ekor kambing Shiba muda (berumur kisaran 6 sampai 10 hari) dikorbankan dengan cara pembiusan (anastesi). Testis diambil, dikeluarkan dari pelapis pembungkusnya, dan dipotong-potong membentuk potongan kecil. Kemudian, perlakuan dengan pemberian collagenase dan trypsin-EDTA. Kultur sel dengan tingkat kepadatan 3.1×106 sel.cm-2 yang diperoleh kemudian diinkubasi selama 18 hari dalam tingkat kelembaban 95%, kandungan CO2 5%, dan pada tingkat suhu 32oC. Untuk memperoleh monolayer (sehelai lapisan kultur) dari sel Sertoli maka pada hari ke delapan belas perlu dilakukan pemindahan ulang kultur sel tersebut dari wadah, dan penanaman kembali, kemudian dilanjutkan inkubasi ulang sel selama tiga hari. Berdasarkan observasi mikroskop cahaya dan electronik, dapat dipastikan bahwa melalui metode tersebut diatas dapat diperoleh secara utuh kultur sel Sertoli primer. Dalam kultur sel Sertoli primer, keberadaan intermediate filaments sekitar inti sel Sertoli dapat terdeteksi.
Kata kunci: Kultur sel Sertoli primer, Kambing Shiba

INTRODUCTION
The in vitro model system to evaluate the toxicity of chemicals and drugs has become important. There are hundreds of compounds synthesized each year, and the cost of animal testing may exceed several million dollars. From a scientific perspective, there are several reasons for the popularity of in vitro model systems, while from a public perspective, in vitro model systems increase the popularity because their application may allow a reduction in the number of live animals employed in toxicity testing (Davila et al., 1998).

It has been clear that in vitro methods are usually used to answer question at the cellular or molecular level, whereas whole animals are used to answer questions at the organ and interorgan levels (Straughan et al., 1997). Characteristically, in vitro methods are usually easy to set up, cheaper, and simple to run and automate; yield quick result; and good quantification and permit replication (Davila et al., 1998). Since the primary cell cultures do not proliferate and therefore limit their widespread, they are convenient for application to study of toxic mechanism (Sorenson and Brabec, 2003).
Until present, some researchers have conducted to use the primary Sertoli cell culture of mouse (Imai et al., 2004), hamster (Davenvord et al., 1987), rat (Rich et al., 1983; Heindel et al., 1989; Clough et al., 1990; Chapin et al., 1991; Awal et al., 2002), and wall-lizard (Khan and Rai, 2004). But, no information is available on the establishment of the primary Sertoli cell culture from testes in domestic animals. Not only humans but also domestic animals have been threatened by chemicals. Therefore, it should be necessary to establish the primary Sertoli cell culture from domestic animals. In the present study, we attempted to establish the primary Sertoli cell culture from immature (presumably 6 – 10 day-old) Shiba goat (ruminant) testes for the risk assessment of chemicals.

MATERIALS AND METHODS
Animals
Two heads of 6 - 10 days old male Shiba goats were provided from the stock farm of the University of Tokyo, Ibaraki, Japan.

Sertoli cell isolation for primary Sertoli cell culture
Under chloroform anesthesia, immature Shiba goats were sacrificed. Then, the testes were excised, decapsulated, cut into smaller pieces (approximately 1 mm3), and enzymatically digested for 10 min. Digestive solution was composed of Dulbecco’s Modified Eagle’s Medium (DMEM; SIGMA) and 0.1% collagenase (SIGMA) with ratio 3:1. The dissociated seminiferous tubules were filtered, washed with DMEM, and conducted by the second enzymatic digestion (shaking in water bath at 37oC for 15 min [120 oscillations.min-1]). In the third enzymatic digestion, the samples with digestive solution were shaked in water bath at 37oC for 30 min. To obtain single cells, trypsin-EDTA solution (SIGMA) was dropped to the cell suspension. Then, they were gently pipetted for 2 min, and centrifuged. The sediment was washed with fetal bovine serum-trypsin inhibitor solution (SIGMA). The provided-single cells were washed once with DMEM [treated with antibiotics (200 units penicillin 100 IU.ml-1 and streptomycin 100 g.ml-1), and supplemented with 10% fetal bovine serum (32 VICTORIA AVENUE, CANTERBURY-3126 VICTORIA, AUSTRALIA)], and then seeded in the same medium with the cell density of 3.1×106 cells.cm-2. The seeding (in the dish with 10 cm in diameter) and incubation (in a humidified atmosphere consisting of 95% air and 5% CO2 at 32oC) were carried out. After 36 hr incubation, the medium was changed with the antibiotics-treated DMEM, and then the medium change was done twice per one week. After 18 days, Sertoli cell cultures were replaced to the eight-chamber (for light microscopy) and/or multichamber (for transmission electron microscopy). Seeding was done for 3 days to provide the monolayer of Sertoli cells. Based on the observation, this replacement could limit the spermatogenic cell number within the Sertoli cell culture. The Sertoli cell culture was fixed in 10% formalin for 10 min, washed again with PBS, dehydrated in a serial grade of ethanol (50%, 70%, 80%, 90%, and 100%), and embedded in paraffin for light microscopic observation.
In order to distinguish Sertoli cells from spermatogenic cells, DAPI (4′,6-diamidino-2-phenylindole; provided from the Tokyo Metropolitan Institute, JAPAN) staining was adopted. Morphologically, it was not difficult to distinguish Sertoli cells from spermatogenic cells by using DAPI staining. Paraffin sections were used for this observation. After deparaffinization and washing with phosphate buffered saline (PBS; magnesium and calcium-free), incubation with DAPI was done for 30 min. Then, the sections were washed again with PBS, mounted, and observed by light microscopy.
For transmission electron microscopy, the primary Sertoli cell cultures, seeded in the multichamber (Millicell-HA: Millipore Corp), were immediately washed with PBS, fixed in 2.5% glutaraldehyde-0.05M cacodylate buffer (pH 7.4) at 4oC for 2 hr, and washed 3 times with the same buffer. They were postfixed in 1% OsO4 for 2 hr, dehydrated through a graded series of ethanol (50%, 70%, 80%, 90%, 95%, and 100% ethanol), and embedded in Araldite M. Semithin sections of 1 μm were stained with 0.5% toluidine blue for light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined in a JEM-1200 EX transmission electron microscope at 60 kV.

RESULTS
Light microscopy
In the present study, the primary Sertoli cell culture could be established from immature Shiba goat testes. The testes of young goats are suitable for the primary Sertoli cell culture, because they contain only a few spermatogenic cells.
During the first week, the cell culture, seeded on plastic surfaces, contained a small cluster of Sertoli cells and some spermatogenic cells. After being cultured in the dish (10 cm in diameter) for 18 days, it was possible to provide the Sertoli cell culture. But, the culture still contained a small amount of spermatogenic cells. Therefore, replacement was conducted after 18 days incubation. At 3 days after replacement, the primary Sertoli cell culture could be established (Fig. 1) by elimination of spermatogenic and myoid cells. The primary Sertoli cell culture appeared as a monolayer on the eight-chamber (solid plastic support). When plated on solid plastic support, the Sertoli cells formed an epithelia-like monolayer.
DAPI staining for the primary Sertoli cell culture was also conducted to clarify its purity (Fig. 2). A characteristic DNA pattern in Sertoli cells can be detected by DAPI staining. As a result, it might have the purity of 95%. Fig. 2 shows the in vivo (Fig. 2a) and in vitro (Fig. 2b) Sertoli cells by DAPI staining.

Fig. 1. Light micrographs of the primary Sertoli cell culture. Toluidine blue staining. (a) Low magnification. Arrow indicates the nucleus of monolayered Sertoli cells. (b) Higher magnification. Scn: Sertoli cell nucleus

Fig. 2. Light micrographs of Sertoli cell from in vivo and in vitro at higher magnification. (a) Sertoli cell in vivo. DAPI staining. (b) Sertoli cell in vitro. DAPI staining. Arrows indicate the nucleolus of Sertoli cell. Scn: Sertoli cell nucleus.

Transmission electron microscopy
In the primary Sertoli cell culture, an ultrastructural study was carried out (Fig. 3a, b) to assure the purity of Sertoli cells. Sertoli cells, spermatogenic cells and peritubular myoid cells in immature Shiba goats could be easily distinguished at the electron microscopic level. Numerous intermediate filaments surrounding the nucleus are characteristic only in Sertoli cells. They were clearly visible around the nucleus of the cell in culture (Fig. 3b). The cell culture obtained here contained only Sertoli cells, but did not contain spermatogenic and peritubular myoid cells.

Fig. 3. Transmission electron micrographs of the primary Sertoli cell culture. (a) Low magnification. (b) Higher magnification. Numerous intermediate filaments (arrow) are discernible around the Sertoli cell nucleus. Scn: Sertoli cell nucleus.
DISCUSSION
Sertoli cell culture
The present research was the first attempt to establish the primary Sertoli cell culture from goat testes, though Merhi et al. (2001) conducted to establish the ovine (lamb) Sertoli cell lines. The Shiba goats at less than 1-month-old were adequate for the primary Sertoli cell culture. As Steinberger and Jakubowiak (1993) mentioned that the purity of isolating Sertoli cells from sexually mature animals was generally lower compared to that from immature animals, probably due to a greater degree of cell differentiation, presence of many germ cells at various stages of differentiation, and greater complexity of Sertoli-germ cells intercellular junction. In addition, the in vitro model may also improve the understanding of the mechanism of chemical-induced toxicity, because the in vivo model was complicated by the presence of structural and functional heterogeneities, and did not allow for mechanisms to be clearly defined (Davila et al., 1998).
In this study, in order to assure the purity of the primary Sertoli cell culture, we adopted 2 criteria obtained by transmission electron microscopy and DAPI staining. These steps were in well agreement with the previous reports by Tung and Frizt (1977), Steinberger (1979), and Griswold et al. (1988). They indicated that for clarification the successfulness of cell isolation and the purity of Sertoli cell cultures, the identity of cells must be verified using various morphological and functional criteria. For example, Sertoli cells exhibit a characteristic DNA pattern when stained with Hoeschst 33342 dye.
As mentioned before, during the first week, the cell culture, seeded on plastic surfaces, contained a small cluster of Sertoli cells and some spermatogenic cells. We used DMEM with serum-free to eliminate the proliferation of a small cluster of Sertoli cells. This step is consistent with the report of Rich et al. (1983) and Janecki et al. (1987). They recommended using serum-free, chemically defined media (e.g. DMEM/F12) supplemented with vitamins and hormones to eliminate a cluster of Sertoli cells in vitro. Regarding to eliminate spermatogenic cells in the primary Sertoli cell culture, we replaced and reseeded the cell cultures in the serum-free DMEM. As a result, spermatogenic and myoid cells could be eliminated. And, as an unexpected result, the Sertoli cells formed an epithelia-like monolayer. These steps are in well agreement with the report by Steinberger and Jakubowiak (1993). They stated that germ cell contamination of Sertoli cell cultures was relatively easy to eliminate, whereas peritubular myoid cells and fibroblasts were more difficult to detect and eliminate. They also suggested that peritubular myoid cells and fibroblasts tended to spread under a Sertoli cell monolayer, and when serum was added to the medium, actively proliferated. Thus, as soon as possible (just 3 days), the primary Sertoli cell culture had to be harvested, or if we intended to use them for the target of toxicity, they should be treated at 3 days after replacement.
Davila et al. (1998) stated that the advantages of cell culture models were as follows: (a) morphological characteristics of individual cells can be seen under a microscope; (b) nutritional and hormonal status, oxygen supply and concentration, and time exposure to test compounds are better controlled; (c) blood flow, heterogeneity of cell type, and nervous and humoral factors are eliminated; (d) cells have time to recover from trauma sustained during the isolation procedure; (e) monolayer cultures permit the study of the effects of toxicants on functions requiring organization of cells; and (f) monolayer cultures can be maintained for long periods of time.
Thus, in this study, the primary Sertoli cell culture from immature Shiba goat testes could be established, and it should be advantageous for screening the testicular toxicity of chemicals.

ACKNOWLEDGEMENT
The skilful technical assistance of Mr. I. Tsugiyama (Department of veterinary Anatomy, The University of Tokyo) is gratefully acknowledged.

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