INVITRO DNA-SYNTHESIS IN LEYDIG AND OTHER INTERSTITIAL-CELLS OF THE RAT TESTIS

Replicative DNA syntheSiS (I-125-labelled iododeoxyuridine incorporation) was measured in interstitial cells prepared from rat testes and separated by Percoll density gradient centrifugation. Leydig cells were identified by I-125-labelled human chorionic gonadotrophin (hCG) binding and 3-beta-hydroxysteroid dehydrogenase histochemistry. Continuous density gradients indicated that interstitial cell DNA synthesis was not associated with Leydig cells, and was greater in cells from the immature than from the mature rat testis. Fractionation of cells by discontinuous density gradients into Leydig cell-rich and -depleted pools did not result in a similar enrichment of DNA synthesis. Treatment of the adult rat with hCG increased DNA synthesis into both fractions but oestrogen had no effect. DNA synthesis was greater in cells from the immature rat but, in contrast to the adult, in-vivo hCG treatment had no effect, whilst oestrogen decreased synthesis. To characterize the cells synthesizing DNA further, interstitial cells were prepared from testes in which the Leydig cells were depleted by in-vivo treatment with ethane dimethanesulphonate (EDS) or depleted in their germ cells by treatment in utero with busulphan. EDS treatment had no effect on DNA synthesis by the interstitial cells in spite of the I-125-labelled hCG binding being markedly reduced. Similarly, busulphan treatment was also without effects upon DNA synthesis. Fluorescence-activated cell cycle analysis of cells from both fractions from germ cell-depleted testes indicated that only a small proportion (3%) of the interstitial cells were actively dividing and this was almost doubled in cells from the germ cell-depleted immature rat testes. The experiments showed that the majority of cells in the interstitium of the rat testes do not synthesize DNA and are not undergoing cell division. The small proportion of cells that are dividing are probably not Leydig cells. The experiments have identified a dividing interstitial cell population and, in consideration of the changes in the immature and mature rat as well as the effects of hormone treatment, they may be regarded as putative Leydig cell precursors.