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Biology of Reproduction, Vol 25, 1065-1077, Copyright © 1981 by Society for the Study of Reproduction
1 Department of Experimental Radiotherapy,
The University of Texas System Cancer Center,
M. D. Anderson Hospital and Tumor Institute
Houston, Texas 77030 A method for obtaining highly purified fractions of rat testicular cells is described. Single cell
suspensions from adult rat testes were separated by centrifugal elutriation. Fractions enriched in
pachytene primary spermatocytes, early spermatids, and cytoplasts detached from late spermatids
were obtained. These fractions were further separated by equilibrium density centrifugation on
gradients of Percoll. In this manner fractions of 3 x 107 pachytene spermatocytes (98% purity),
1.1 x 108 early spermatids (93% purity), and 1.1 x 108 cytoplasts (98% purity) were obtained
within 6 h after sacrificing the rats. The cells appeared to be morphologically intact and to have
retained their biochemical integrity. Analysis of acid-soluble nuclear proteins by polyacrylamide gel electrophoresis showed that
histone 4 is synthesized during the pachytene stage, and confirmed that testis-specific histones are
also synthesized during this stage. Analysis of a microsomal RNA preparation from purified pachytene spermatocytes and purified early spermatids by sucrose gradients indicated that intact ribosomal RNA (rRNA) can be obtained from purified cells. Both cell types are active in synthesizing
presumptive messenger RNA (mRNA) with a wide range of sedimentation values, but no appreciable rRNA synthesis was detected.
2 General Medical Research,
Veterans Administration Hospital,
Shreveport, Louisiana 71130
3 Department of Pathology,
Baylor College of Medicine,
Houston, Texas 77030
Note:
ACKNOWLEDGMENTS
We thank Patricia K. Trostle and Nancy R. Hunter
for excellent technical assistance and Elsa Ramos for
sectioning samples for electron microscopy. We also
wish to acknowledge the excellent typing assistance
of Rozanne Goddard and Ann McCarver. This investigation was supported by grants PCM 78-06097 from
NSF and HD-11796, CA-17364, and CA-06294 from
NIH.
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