Transfer of spermatogenesis-related cDNAs into eel testis germ-somatic cell coculture pellets by electroporation: Methods for analysis of gene function

• Published in: Molecular reproduction and development 2007-04, Vol.74 (4), p.420-427
• Main Authors: Miura, C., Kuwahara, R., Miura, T.
• Format: Article
• Language: English
• Subjects: androgen; Anguilla - genetics; Animals; Biological and medical sciences; Biotechnology; Coculture Techniques; DNA, Complementary - isolation & purification; Electroporation; Fundamental and applied biological sciences. Psychology; Gene transfer; Gene Transfer Techniques; Genetic engineering; Genetic technics; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; in vitro spermatogenesis; Male; Methods. Procedures. Technologies; Spermatogenesis - genetics; Spermatogenesis - physiology; Spermatogonia - cytology; spermatogonial proliferation; Synthetic digonucleotides and genes. Sequencing; Testis - cytology
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/mrd.20653

Genes encoding spermatogenesis‐related substance (eSRSs) show unique expression patterns during spermatogenesis. To analyze their function, we developed a new assay system using gene transfer techniques combined with coculture of the eel germ‐somatic cells. First, we investigated the efficacy of in vitro electroporation transfer of gene into germ‐somatic cell pellets using green fluorescent protein (GFP) gene. Second, in order to define the function of the eSRSs, we electrophoretically transferred eel spermatogonial stem cell renewal factor (eSRS34) and eel spermatogenesis‐preventing substance (eSRS21) genes into germ‐somatic cell pellets. Presence of the transferred cDNA was examined by reverse transcription‐polymerase chain reaction (RT‐PCR). Furthermore, proliferating cells were detected histologically, after labeling with BrdU. Transfer of the eSRS34 gene induced spermatogonial stem cell renewal in the pellets. Moreover, 11‐ketotestosterone (11‐KT) treatment stimulated the proliferation of spermatogonia, which resulted in the appearance of late type B spermatogonia in the pellets. The proliferation of spermatogonia by 11‐KT stimulation was suppressed by transfer of the eSRS21 gene. These results indicate that the transferred eSRS34 and 21genes were functional in the pellets. Thus, an efficient in vitro gene transfer technique for coculture system of germ and somatic cell of Japanese eel was established. Mol. Reprod. Dev. 74: 420–427, 2007. © 2006 Wiley‐Liss, Inc.