Analysis of the progression of meiosis in dispersed rat testicular cells by flow cytofluorometry.
Initiation and progression of meiosis was followed in dispersed rat testicular cells by flow cytofluorometry and cytology. The DNA content of dissociated testicular cells of rats 6--30 days old, killed at daily intervals, was analysed by flow cytofluorometry using propidium iodide as a DNA-specific and quantitative fluorochrome. Testicular cells of a 6-day-old rat showed one peak of fluorescence. A second peak, at twice the modal channel number, appeared in testicular cells of 9-day-old animals. The number of cells under this peak increased progressively with age. A third peak, at half the channel number of the original one, appeared at 20 days and accounted for an increasing proportion of cells in testes taken from older rats. Cytological examination of the testicular tissue used for flow cytofluorometric analysis showed that preleptotene spermatocytes first appeared at 8 days after birth. Spermatids were first observed cytologically at 20 days after birth. The close temporal appearance of the fluorescence peaks with that of spermatocytes and spermatids, and the close association of the frequency of diploid and tetraploid cells as derived by flow cytofluorometry and cytology, indicated that the fluorescence peaks correspond--in order of increasing fluorescence--to spermatids, spermatogonia and somatic cells, and to spermatocytes. This conclusion was re-examined by analysing the ploidy levels of testicular cells of hypophysectomized or estradiol-treated by flow cytofluorodmetry. There was a loss of the haploid and tetraploid peaks subsequent to hypophysectomy. Estradiol dipropionate-treated rats, given weekly injections starting at 7 days of age, showed no appearance of the haploid peak and the regression of the tetraploid peak after an initial and transitory appearance. These results indicate that changes in ploidy levels that accompany the progression of meiosis in the testis were reflected in the sequential appearance of three fluorescence peaks as detected by flow cytofluorometry. The close correlation between the frequency of cell types as obtained by cytology and flow cytofluorometry indicates that the latter is a sensitive method for studying selected aspects of spermatogenesis in dissociated testicular cells.
D000818:Animals; D003584:Cytological Techniques; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D005470:Fluorometry; D006065:Gonadotropins, Pituitary / Q000502:physiology; D007016:Hypophysectomy; D008297:Male; D008540:Meiosis; D051381:Rats; D013087:Spermatids / Q000166:cytology; D013090:Spermatocytes / Q000166:cytology; D013091:Spermatogenesis / Q000187:drug effects; D013737:Testis / Q000166:cytology*
D006065:Gonadotropins, Pituitary; D004958:Estradiol