Expression of Testis Specific Genes of Male Germ Cells in Pre-pubertal Mouse using in vitro Culture System / 대한남성과학회지

ABSTRACT

PURPOSE: To establish cultures of premeiotic mouse spermatogenic cells that could allow study of the meiotic process in vitro and to investigate expression of sperm-specific genes in premeiotic and postmeiotic division. MATERIALS AND METHODS: Male ICR mice were used. To obtain the premeiotic spermatogenic cells, we examined the histologic appearance of the germ cells in hematoxylin-eosin-stained testis sections during prepubertal development. To confirm the identity of the premeiotic cells, we preformed reverse transcriptase-polymerase chain reaction (RT-PCR) for the genes for LDH-C4, acrosin, protamine-2 (P-2), and SP-10, which are testis-specific marker proteins. In order to establish the culture system, the premeiotic spermatogenic cells were cocultured with mouse Sertoli cells in DMEM containing 10% fetal bovine serum (FBS), amino acids, FSH, and testosterone at 32 degrees C for 6 days, After 2 days of culture, RT-PCR was done to detect the sperm-specific genes. RESULTS: The four genes was not expressed in 10-day-old mice, but expression was detectable in 17-day-old mice. Thus, mice of this age have initiated the meiotic process, and round spermatids were seen in the testis. Premeiotic germ cells isolated from 15-day-old mice expressed LDH-C4 and acrosin but not P-2 and SP-10, which are post-meiotic marker proteins. Beginning after 2 days of culture, expression of the P-2 and SP-10 genes was detected in cultured premeiotic germ cells as in 15-day-old mice. CONCLUSIONS: P-2 and SP-10 may be marker genes for the premeiotic stage of spermatogenesis. Premeiotic male germ cells are able to differentiate into postmeiotic forms during coculture with Sertoli cells.