Differentiation of Mouse Germ Cells to Mature Sperm in Three-Dimensional Methylcellulose Culture System (MCS). Academic Article uri icon

abstract

  • Establishment of an in vitro system that enable development, proliferation and differentiation of testicular germ cells to sperm will be valuable in the study of spermatogenesis and in future treatment of male infertility. In our previous study using a soft agar culture system (SACS) with three-dimensional (3-D) character, we could induce undifferentiated spermatogonial stem cells (SSC) to meiotic and post-meiotic stages but not to sperm. The aim of the present study was to establish an in vitro culture conditions, using the 3-D methylcellulose system (MCS), with capacity to induce testicular germ cells to final stages of spermatogenesis and to produce sperm. The advantage of MCS is that it allows isolation and purification of cells from the culture dish that can then be subjected to detailed analysis by FACS and FISH. Due to residual agar, cells cultured with SACS cannot be processed for FISH and SACS. Methods: For achieving this aim, testes from 7-day-old mice were decapsulated and seminiferous tubules were enzymatically dissociated. Dissociated cells (106 cells/1 ml) were cultured in MCS containing RPMI and FCS (25%) alone or in the presence or absence of FSH (7.5 IU/ml) and testosterone (0.01 μM) (hormones; H), or mouse embryo fibroblast conditioned media (MEF). Developed colonies were picked after 28-30 days of culture. All colonies in a dish were collected and examined for markers representing different spermatogenic stages by immunofluorescence analysis: markers for 1) undifferentiated and differentiating spermatogonia: C-kit, Gfr-a-1, Cd9, a-6-integrin; 2) meiotic genes (boule and CREM-1) and 3) postmeiotic markers (Ldh, Protamine and acrosin). Marker genes for Sertoli cells (Abp) or peritubular cells (aSm) were also examined by RT-PCR. Colonies were subjected to FACS and FISH for determination of DNA content to determine absence or presence of haploid cells. The presence of sperm was examined histologically. Acrosome reacted sperm were evaluated by using PNA-FITC staining in the presence or absence of ionomycin. Results: We isolated distinct colonies which under any or all culture conditions did not express Abp or aSm. Marker related to early germ cells, meiotic and post meiotic stages were detected. MEF or hormones did not show significant effect on germ cell differentiation, as examined by immunostaining analysis. Flow cytometry revealed that the colonies contained subfractions of haploid cells (around 6%) after 28 days of culture in MCS. The presence of haploid cells was confirmed by FISH analysis. Sperm-like cells with complete characteristic morphology of head, neck and tail were detected in our culture. The generated sperm could undergo ionomycin-induced acrosome reaction. Conclusions: Our data show that MCS can be used as a novel in vitro approach to grow mouse male germ cells to mature sperm. The generation of sperm from germ cells in the absence of hormones may suggest that hormones are not crucial for spermatogenesis in vitro and/or that in immature mice spermatogenesis is suppressed in vivo and this suppression is removed under in vitro culture. This novel approach could be useful for understanding the cellular and biomolecular mechanisms of spermatogenesis under physiological and pathological conditions, and might be used for developing future strategies in treatment of male infertility. (platform)

publication date

  • January 1, 2009