Single-strand nuclease action on heat-denatured spermiogenic chromatin
The sensitivity of chromatin was compared from representative cellular stages of spermiogenesis to a single-stranded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S/sub 1/ nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90/sup 0/C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60/sup 0/C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80/sup 0/C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of roundspermatids labeled with /sup 3/H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot trichloracetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgenstainable material occurred in these cells and may be due to depurination and elimination of Fuelgren-reactant aldehyde groups.
- Research Organization:
- Univ. of California, Livermore, CA (United States)
- OSTI ID:
- 7319246
- Journal Information:
- J. Histochem. Cytochem.; (United States), Vol. 24:8
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CHROMATIN
BIOCHEMICAL REACTION KINETICS
THERMAL DEGRADATION
DNA
HEAT
BIOLOGICAL EFFECTS
NUCLEASES
SPERMATOGENESIS
SPERMATOZOA
TEMPERATURE EFFECTS
ENERGY
ENZYMES
GAMETES
GAMETOGENESIS
GERM CELLS
KINETICS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PHOSPHOTRANSFERASES
REACTION KINETICS
TRANSFERASES
550200* - Biochemistry