Presentation Authors: Satoshi Nozaki*, Shoiciro Iwatsuki, Tomoki Takeda, Hiroki Kubota, Hiroyuki Kamiya, Shoichi Sasaki, Yukihiro Umemoto, Takahiro Yasui, Nagoya, Japan
Introduction: Leydig and Sertoli cells play important roles in spermatogenesis. To investigate the role of testicular somatic cells such as Leydig and Sertoli cells in spermatogenesis, they need to be isolated. However, little is known about effective isolation of these cells. In this study, we established a simple technique to isolate testicular somatic cells using Datura Stramonium (DSA) lectin and Percoll.
Methods: C57BL/6J mice were used for this experiment. The testes were collected and seminiferous tubules were dissociated using collagenase and trypsin. Tubules were layered over 5% Percoll and allowed to settle. The upper gradient of the Percoll mixture was centrifuged at 20,000 Ã— g to isolate the Leydig cell fraction, whereas the lower gradient was transferred to DSA lectin coated plates and incubated. Subsequently the Sertoli cell fraction was isolated from the cells that attached to the plate. We counted the number of cells and compared the positive ratio in immunostaining in each fraction. We used STAR antibody as Leydig cell-specific marker and SOX9 antibody as Sertoli cell-specific marker. The quantification of gene transcripts was carried out by quantitative PCR. We used 3bHSD and Star as Leydig cell-specific markers and Sox9 as Sertoli cell-specific markers.
Results: The numbers of cells in the Leydig and Sertoli cell fractions were 3.7 Ã— 106 and 4.4 Ã— 106, respectively. STAR positive ratio (STAR positive cells / total cells) in the unisolated, Leydig, and Sertoli cell fractions were 2.9%, 57.6%, and 2.3%, respectively. The STAR positive ratio in the Leydig cell fraction was higher than that in the others. Furthermore, SOX9 positive ratio (SOX9 positive cells / total cells) in the unisolated, Leydig, and Sertoli cell fractions were 10.5%, 7.7%, and 15.6%, respectively. The expression levels of 3bHSD and Star in the Leydig cell fraction were significantly higher than in the Sertoli cell fraction. The expression levels of Sox9 was not significantly different between Sertoli and Leydig cell fraction.
Conclusions: Although it was difficult to isolate Sertoli cells from the testes using DSA lectin and Percoll, we found that Leydig cells could be isolated efficiently. Our method provides a platform to expand the investigations related to testicular somatic cells.