Abstract
The technique of velocity sedimentation at unit gravity has been used to separate rat testis cell suspensions into fractions enriched in particular cell types. Changes in the nuclear proteins from the various fractions have been characterized by polyacrylamide gel electrophoresis, and correlated with the changing morphology of the nucleus during spermatogenesis. The most striking alterations in both protein composition and nuclear morphology occur during spermatid maturation as both histone and non-histone proteins are replaced by highly basic, low molecular weight, spermatidal proteins. This replacement process is accompanied by a quantitative reduction in both histone and non-histone proteins. The synthesis of at least three basic proteins has been identified with late stage spermatids. One of these proteins is a highly basic sperm-specific protein containing high levels of cyst(e)ine and arginine. A second protein synthesized in late stage spermatids is lysine rich, while the third protein contains cyst(e)ine and co-migrates with histone F2a1 on acid-urea polyacrylamide gels. The changes in protein composition of rat testis nuclei after irradiation or hypophysectomy reflect the resulting changes in the cellular composition of the testis. After selective elimination of the germinal cells by irradiation, the electrophoretic pattern of acid-soluble proteins from the testis is very similar to that of somatic tissue. Thus, the cellular specificity of nuclear proteins demonstrated here using cell separation techniques is also apparent following treatments which selectively alter the cellular composition of the testis.
Highlights
Our results indicate that at least three acid-soluble proteins are actively synthesized in late stage spermatids
Nuclei from fractions enriched in late spermatids, are generally contaminated by the acrosome, as well as by the ribonucleoprotein aggregates derived from the residual bodies, and by the flagella of spermatids. These contaminants can be eliminated from late stage spermatids by sonication2 As a further check for possible cytoplasmic contamination, [Wlarginine-labeled nuclei were isolated in the presence of cytoplasm (10,000 x g supernatant) from [3H]arginine-labeled testis and vice versa
The cross-contamination of nuclear proteins by cytoplasmic proteins in these experiments was less than 2%
Summary
Sprague-Dawley rats (250 g) were used throughout obtained from Hormone this study. Hypophysectomized Assay, Chicago, and maintained rats were for 28 or 55 days before use. The testes of normal rats were locally irradiated using cobalt 60, with two exposures of 1300 rads each, given 1 week apart. EDTA-trypsin method described previously [16] for obtaining high yields of spermatogonia and young primary spermatocytes from mouse testes was used with some modifications. Suspensions were prepared using two minced testes in 40 ml of calcium-magnesium-free phosphatebuffered saline containing 5 mM EDTA and 0.17o glucose at pH 7.4. After 10 min of incubation at 31”, purified trypsin
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
