Stimulation of Ribonucleic Acid and Deoxyribonucleic Acid Synthesis in Spermatogenic Cells by Their Coculture with Sertoli Cells*

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Abstract
Germ cells were isolated from 30-day-old rat testes and purified by a Percoll gradient. Germ cells were cultured for 24 h to eliminate contaminant cells by utilizing the property of many nongerm cells of attaching to plastic surfaces. Germ cells were subsequently seeded on top of 7-day-old Serotoli cell monolayers. RNA and DNA synthetic activities were estimated by the rate of incorporation of [3H]uridine and [3H] thymidine into trichloroacetic acid-precipitable material. Germ cells increased RNA synthesis from 54,053 .+-. 22,824 to 168,019 .+-. 57,137 dpm/2 .cntdot. 106 cells (mean .+-. SD) between 4 and 24 h of coculure, respectively (P < 0.01), while they decreased this activity from 32,150 .+-. 68,00 to 6,014 .+-. 5,243 dpm when they were cultured alone for the same periods. Coculture of germ cells with either a cell derived from the adult bovine aortic endothelial cells (ABAE), or rat fibroblasts in primary culture prevented the fall in RNA synthesis but did not stimulate it. Addition of high concentrations of lactate to the culture medium did not have any affect in germ cells cultured alone but produced a slight stimulation in germ cells cocultured with ABAE cells, which was much smaller than the effect observed during coculture with Serotili cells. Germ cells in coculture with Serotoli cells also increased their DNA synthesis from 2,186 .+-. 765 at 4 h to 9,679 .+-. 4,057 dpm [3H]thymidine/2 h .cntdot. 106 cells at 24 h (P < 0.05). When Serotoli cells were treated with FSH (1 .mu.g/ml), the synthesis of both RNA and DNA of germ cells cocultured with these cells was significantly higher than for germ cells cocultured with nontreated Sertoli cells. The system of coculture of Sertoli cells and germ cells consisted of the reassociation of the two previously isolated cells in a coculture, followed by their separation which permitted the effect of the coculture on the individual cell type to be assessed. By using this technique it was possible to obtain evidence suggesting that a chemical messenger other than lactate might be involved in the stimulatory effect of Sertoli cells on germ cells and that FSH increased this putative chemical messenger.