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

Lee, Ho Joon; Kim, Myo Kyung; Ko, Duck Sung; Kang, Hee Gyoo; Kim, Dong Hoon

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.