Differential Binding of Tritiated Actinomycin to the Nuclei of Mammalian Spermatogenic Cells in Vivo

Abstract

Chinese hamsters, Cricetulus griseus, were given a single injection of 2\m=.\0 \g=m\g[3H]actinomycin D directly into the left testis. For 24 hr after injection, the distribution of the radioisotope in the reproductive organs, blood plasma and liver was followed, using liquid scintillation counting techniques. After 24 hr, nearly 50% of the injected dose remained in the left testis while insignificant amounts of label were associated with the right testis. The left epididymis gradually accumulated radioisotope probably by intratubular transport from the left testis. The right epididymis possessed negligible amounts of label after 24 hr. Levels of the antibiotic in blood plasma and liver steadily decreased during the experimental period.Localization of [3H]actinomycin D within the spermatogenic cells of the seminiferous epithelium was determined by autoradiography. The results indicated that the antibiotic was differentially bound by various types of spermatogenic cells. Of the spermatocytes, Stage VIII leptotene nuclei (31\m=.\6grains/nucleus) were much more heavily labelled compared to early Stage V pachytene nuclei (6\m=.\2grains/nucleus). Actinomycin D was lightly bound to nuclei of Step 5 and Step 8 round spermatids (3\m=.\6 and 3\m=.\8 grains/nucleus, respectively). Elongating spermatid nuclei (Steps 9 to 14) were lightly labelled at best, whereas elongated spermatid nuclei (Steps 15 to 18) were very heavily grained. The label intensity decreased in Step-19 spermatids (spermiation step). Labelling variations among spermatogenic cells may be attributable to the physicochemical state of deoxyribonucleoprotein complexes in these cells.

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis

“…Similar observations in the testis of the hamster, bull and ram have been reported (Barcellona et al, 1974;Loir & Hochereau de Reviers, 1972). The stage-4 germ cells (elongated spermatids and testicular spermatozoa) therefore represent an interesting transitional state in the process of spermatogenesis.…”

“…The genomic inactivation during spermiogenesis has been attributed to the masking of free sites on DNA by chromosomal basic proteins (Brachet & Hulin, 1969;Darzynkiewicz et al, 1969b;Marushige & Dixon, 1969). However, the presence of unmasked DNA during spermiogenesis without concomitant synthesis of RNA in vivo (Loir & Hochereau de Reviers, 1972;Barcellona et al, 1974) indicates that other control mechanisms are essential for genomic inactivation.…”

7‐aminoactinomycin D binding and the final stages of sperm chromatin processing in the mouse

“…Actinomycin D binding to chromatin has been used to monitor changes in DNA condensation or accessibility in a variety of different cell types [Darzynkiewics et al, 1969;Tumanishvili et al, 1977;Kleiman and Huang, 1971;Wong et al, 1971;Calvin and Bedford, 1974;Barcellona et al, 1974;Tubbert and Berlowitz, 1974;Aczel and Enesco, 1975;Kushch et al, 1978;Borodina et al, 19791. Darzynluewicz et a1 [1969] were among the first to demonstrate that actinomycin-D binding was reduced drastically in the late-step spermatids and epididymal sperm of the bull.…”

Culture of pachytene spermatocytes for analysis of meiosis

“…AMD inhibits transcription by intercalating into DNA, thus presumably hindering the transcription complex. Interestingly, AMD, as a DNA intercalator, has also been used to assess whether chromatin is accessible to AMD; some of these studies have analyzed spermatogenic cells of different stages and species le.g., Barcellona et al, 1974;Darzynkiewicz et al, 19691. In the present study, when cultured pachytene cells were treated with AMD in concentrations sufficient to inhibit RNA synthesis, the chromatin was dramatically affected.…”