315 AN ATTEMPT AT INDUCING DIFFERENTIATION INTO ROUND SPERMATIDS OF RAT SPERMATOGONIA BY CO-CULTURING WITH SERTOLI CELLS

Abstract

Mammalian spermatogenesis is a complex process of germ cell development at the seminiferous tubules whereby diploid spermatogonia proliferate and differentiate into haploid spermatozoa via round and elongating spermatids in close association with somatic Sertoli cells. In the present study, the potential of rat spermatogonia to undergo meiosis during co-culture with Sertoli cells was assessed. The type-A spermatogonia and Sertoli cells were prepared from Day 7 heterozygous transgenic male rats carrying EGFP DNA, and co-cultured on the dishes (coated; BD Falcon™ 35-3801, or non-coated: BD Falcon™ 35-1008, 4 × 10⁶ cells/4-mL dish) at 37°C for 3 days and at 34°C for a subsequent 7 days in 5% CO₂ in air. The culture medium was DMEM medium supplemented with 10% fetal bovine serum, growth factors (10 ng/mL EGF and 10 ng/mL IGF) and various hormones (500 ng/mL FSH, 133 μIU/mL hGH, 5 μg/mL insulin, 0.1 μM testosterone and 0.1 μM dihydrotestosterone). During culture, appearance of round spermatid-like cells (ca. 15 μm in cellular diameter and 7–8 μm in nuclear diameter) was traced. The ploidy of the cells was also analyzed by flow cytometry (FCM). At the end of culture, the proportion of EGFP DNA-bearing cells in the total cultured cell population was examined under UV light at 365 nm. Thereafter, continuation of the spermatid-like cells to full-term development was examined by ooplasmic microinjection (Kato et al. 2004 Contemp. Top. Lab. Anim. Sci. 43/2, 13). Briefly, oocytes from the Sprague-Dawley rats were denuded, activated with two direct-current pulses at 100 V/mm for 99 μs and held in 2 mM 6-dimethylaminopurine for 20 min. The nuclei of spermatid-like cells were microinjected into the oocytes by using a piezo impact driving unit, and the injected oocytes after 24 h culture were transferred into recipients. Round spermatid-like cells were first observed at the 5th day of culture on both dishes, but the proportion of spermatid-like cells on the coated dish was higher than that on the non-coated dish. The FCM analysis showed that a single peak of haploid cells was detected in the cell population cultured on the coated dish at the 5th day of culture, while no haploid peak was detected on the non-coated dish. The cultured cells exhibited two distinct patterns of EGFP fluorescence, with a proportion of EGFP-positive cells at 53.5% (total 1,000 counts). The microinsemination into 263 oocytes resulted in the production of 27 oocytes with two pronuclei (10.3%) and 15 cleaved oocytes (5.7%). However, the oviductal transfer of 143 microinseminated oocytes resulted in only 8 implantation sites without viable offspring (5.6%). These results indicated that rat type-A spermatogonial cells seemed to undergo meiosis, but the potential of the cultured spermatid-like cells to participate into full-term development was questionable.