ARTICLES

Cell Research (2004); 14(6):507-512

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

Gang Liang^1,4, Xiao dong Zhang¹, Lu jing Wang¹, Yu shen Sha², Jian chao Zhang², Shi ying Miao¹, Shu dong Zong², Lin fang Wang^1,*, S.S. Koide³

¹National Laboratory Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 5 Dong Dan San Tiao, 100005 Beijing, China
²National Research Institute for Family Planning, WHO Collaboration Center for Research in Human Reproduction, Beijing, 12 Da Hui Si, 100081 Beijing, China
³Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021, USA
⁴Chinese National Human Genome Center, Beijing, 3-707 North Yong Chang Road BDA, Beijing 100176, China

Received, Jun 30, 2003    Revised, Aug 4, 2004    Accpeted, Sep 16, 2004

Correspondence: Lin Fang Wang +86-10-65296418 (phone) +86-10-65240529 (fax) wanglf@ms.imicams.ac.cn

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Abstract

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.

Keywords: laser capture microdissection, suppressive subtractive hybridization, spermatogenesis, cytochrome c oxidase II, humanin.