Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique.
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.
Liang
Gang
G
National Laboratory Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 100005 Beijing, China.
Zhang
Xiao Dong
XD
Wang
Lu Jing
LJ
Sha
Yu Shen
YS
Zhang
Jian Chao
JC
Miao
Shi Ying
SY
Zong
Shu Dong
SD
Wang
Lin Fang
LF
Koide
S S
SS
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D003576:Electron Transport Complex IV / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D006652:Histological Techniques; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D013087:Spermatids / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D013090:Spermatocytes / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D013091:Spermatogenesis / Q000235:genetics; D013737:Testis / Q000166:cytology*
D018076:DNA, Complementary; D012313:RNA; C117108:cytochrome C oxidase subunit II; D003576:Electron Transport Complex IV