The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells

The aim of the present study was to assess whether the whole meiotic proces s of spermatogenic cells is able to take place in vitro. Fragments of semin iferous tubules from 20- to 22- or 28-day-old rats were seeded in medium co ntaining 0.2% fetal calf serum in bicameral chambers and then cultured for 4 weeks in a chemically defined medium. The differentiation of meiotic germ inal cells was followed by four criteria: (i) ultramicroscopic examination of the different types of germ cells present in the cell layer throughout t he culture period; (ii) determination of the changes in DNA content per nuc leus of the cell population seeded with time in culture; (iii) assessment o f the ability of germinal cells to transcribe genes expressed after complet ion of meiosis; and (iv) monitoring the fate of BrdU-labeled leptotene sper matocytes. The ultrastructural study showed that the overall organization o f the cells in the culture well recalls that of the seminiferous epithelium throughout the culture period. Moreover the identification of young round spermatids 21 days after seeding suggested that these spermatids had been f ormed very recently in culture. Determination of DNA content per nucleus sh owed that a 1C cell population could be observed after several days of cult ures reaching 6 to 10% of total cells. An exponential-like increase in the amounts of the mRNAs encoding for TP1 or TP2 occurred from the time when 1C cells appeared in the culture until the end of the experiment, Finally, Br dU-labeled leptotene spermatocytes differentiated into pachytene spermatocy tes and then into secondary spermatocytes, and BdrU-labeled round spermatid s were observed from Day 21 of culture onward. Taken together these results indicate that the whole meiotic process from leptotene spermatocyte to rou nd spermatid can indeed occur in vitro under the present culture conditions , (C) 2000 Academic Press.