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Identification and localization of deoxyribonuclease I in the rat ovary
1Department of Obstetrics & Gynaecology and Cellular & Molecular Medicine, University of Ottawa, Ontario, Canada.
Abstract:
Our objective was to identify the endonuclease responsible for DNA degradation in the ovary and determine its localization relative to the developmental state of ovarian follicles. Immature rats were treated with diethylstilbestrol (DES; DES group), DES + eCG (eCG group) or DES + eCG + hCG (hCG group). Nuclei of the eCG and hCG but not the DES group contained a 32/34-kDA DNase I-like endonuclease activity that was Ca2+/Mg(2+)-dependent, stimulated by Mn2+, optimal at pH 7, and identified by anti-DNase I antibody. G-actin, Zn2+, dithiothreitol, aurintricarboxylic acid, and sodium aurothiomalate, but not iodoacetic acid, inhibited the activity. Addition of eCG nuclear protein extracts to nuclei from the DES group induced oligonucleosomal DNA fragmentation, which could be prevented by pretreatment of the extracts with anti-DNase I antibody. DNase I was immunolocalized in nuclei of healthy luteal cells, antral follicle granulosa cells, oocytes of preantral follicles, atretic preantral follicles, and testicular spermatogonia, but was not observed in granulosa cells of preantral follicles, theca cells, antral follicle oocytes, or testicular spermatocytes. Nuclear extracts of rat kidney, liver, and spleen, and bovine, chicken, and human ovaries displayed DNase I-like activity. These results suggest that an endonuclease indistinguishable from DNase I is responsible for ovarian apoptotic DNA degradation.
Insights
Researchers identified a DNase I-like endonuclease responsible for ovarian DNA degradation during follicular development. This enzyme
Area of Science:
- Reproductive Biology
- Molecular Biology
- Biochemistry
Background:
- Ovarian function involves complex cellular processes, including programmed cell death (apoptosis).
- DNA fragmentation is a hallmark of apoptosis, necessitating the identification of specific endonucleases involved.
Purpose of the Study:
- To identify the endonuclease responsible for DNA degradation in rat ovaries.
- To determine the localization of this endonuclease in relation to ovarian follicle development.
Main Methods:
- Immature rats were treated with diethylstilbestrol (DES), DES + eCG, or DES + eCG + hCG.
- Nuclear endonuclease activity was assayed, and characterization included dependence on divalent cations (Ca2+/Mg2+), stimulation by Mn2+, pH optimum, and inhibition by specific agents.
- Immunolocalization studies using anti-DNase I antibody were performed.
- DNase I-like activity was also assessed in nuclear extracts from various rat tissues and ovaries from different species.
Main Results:
- A 32/34-kDa DNase I-like endonuclease activity was detected in nuclei of eCG and hCG treated groups, but not in the DES group.
- This activity exhibited Ca2+/Mg2+-dependence, Mn2+ stimulation, pH optimum of 7, and was inhibited by G-actin, Zn2+, and other agents, but not iodoacetic acid.
- Addition of eCG nuclear extracts induced DNA fragmentation in DES group nuclei, an effect blocked by anti-DNase I antibody.
- DNase I was immunolocalized in nuclei of luteal cells, granulosa cells of antral follicles, oocytes of preantral follicles, atretic follicles, and testicular spermatogonia.
- DNase I-like activity was present in nuclear extracts of rat kidney, liver, spleen, and ovaries of bovine, chicken, and human.
Conclusions:
- An endonuclease indistinguishable from DNase I is responsible for apoptotic DNA degradation in the rat ovary.
- The localization of DNase I correlates with specific stages of ovarian follicle development and cellular processes, including atresia.
- DNase I-like activity is conserved across species and present in various somatic tissues, suggesting a fundamental role in DNA metabolism.
