IDENTIFICATION OF A DEOXYRIBONUCLEASE I-LIKE ENDONUCLEASE IN RAT GRANULOSA AND LUTEAL CELL-NUCLEI

Apoptosis, a process recently implicated as the cellular mechanism und erlying ovarian follicular atresia and luteal regression, is character ized by the internucleosomal degradation of DNA by a Ca2+/Mg2+-depende nt endonuclease. Although hormones and growth factors have been demons trated to modulate the DNA degradation associated with ovarian follicu lar apoptosis, the nature and identity of the endonuclease involved is not known, Ca2+/Mg2+-dependent endonuclease activity has a developmen tal pattern of expression in rat granulosa and luteal cell nuclei. Thu s, the present study was conducted to establish the presence of an end onuclease in the nuclei of ovarian granulosa and luteal cells and to e xamine the biochemical properties of the enzyme relevant to apoptosis. Nuclei from diethylstilbestrol (DES)-, eCG-, and hCG-primed rat ovari es were isolated and exposed to Ca2+ and Mg2+ in vitro. Nuclei from ra t ovaries primed with eCG and hCG, but not DES, substantially degraded their DNA in an apoptotic fashion, and this DNA degradation was Ca2+/ Mg2+-dependent and inhibited by Zn2+. Protein extracts from the nuclei of DES-, eCG-, and hCG-treated rat ovaries were tested for endonuclea se activity by a plasmid degradation assay. The extracts were found to contain endonuclease activity with the same developmental pattern and cation dependency as found in intact nuclei. These protein extracts w ere assessed for nuclease activity by zymography, and three nuclease a ctivities were identified depending on the type of DNA used in the gel and the electrophoresis conditions used for protein separation, A dou blet of 32/34 kDa was found to have the same developmental pattern and cation dependency as that demonstrated with the plasmid degradation a ssay and apoptotic DNA production in intact nuclei. A nuclease activit y of 27 kDa was also present but was active only on single-stranded DN A and was Mg2+-, but not Ca2+-, dependent. True nuclease activity was distinguished from false positive histone effects by silver staining t he DNA in the zymographic gel, a new technique that avoids the use of isotopically labeled DNA. The 32/34-kDa activity had molecular size, c ation dependency, and optimal substrate and electrophoresis requiremen ts similar to those of DNase I from bovine pancreas. These experiments suggest that the capacity of rat ovarian cells to undergo apoptosis ( as measured by DNA degradation) is developmentally regulated such that differentiated granulosa and luteal cell nuclei, but not undifferenti ated granulosa cell nuclei, possess an endogenous DNase I-like endonuc lease activity. This Dnase I-like endonuclease may be responsible for the characteristic DNA degradation observed during follicular atresia and luteal regression.