392
IN SITU MOLECULAR IMMUNOLABELING OF DNA AND CYTOCHEMICAL LABELING OF HYALURONIC ACID DURING NUCLEAR CHANGES IN APOPTOTIC GRANULOSA AND CUMULUS CELLS.
Kan, Frederick1, Dejager, Peter1, 1
ABSTRACT- In the present study, we examined the sequential changes that occurred in the nucleus of apoptotic granulosa and cumulus cells in the hamster ovarian follicles and the oocyte-cumulus complexes (OCCs), respectively. Our study focused on the localization and distribution of DNA and hyaluronic acid and their fate during apoptosis in the granulosa and cumulus cells. Apoptotic cells in the ovarian follicles and OCCs were detected on paraffin sections by use of the terminal deoxynucleotidyl-transferase enzymatic reaction. To further characterize this nuclear apoptotic activity, an immunocytochemical study was carried out to examine the nuclear organization of DNA throughout the apoptotic process. A study of high resolution localization of hyaluronic acid in the nucleus was also made. Our results showed that the breakdown of the nucleus in apoptotic granulosa and cumulus cells followed a highly ordered manner. During the process, the nucleus is broken up into several apoptotic nuclear bodies each of which consists of two distinct subcompartments, namely, heterochromatin and euchromatin. Using the in situ molecular DNA labeling technique and the Hyaluronidase VI-gold method, we localized both DNA and hyaluronic acid to the same subcompartment in the apoptotic nuclear bodies. Results from our present study have added new information to our current understanding of cellular changes that are associated with programed cell death by showing the stepwise formation of nuclear bodies in relation to the mobilization and subsequent segregation of DNA and hyaluronic acid in apoptotic granulosa and cumulus cells. The localization of both DNA and hyaluronic acid to the same region of the nucleus suggests the possibility that hyaluronic acid may play a role in nuclear function through the interaction with DNA-protein complex in the nucleosome.
KEY WORDS: Apoptosis, Oocyte-cumulus-complex, Granulosa cells, Nuclear DNA and hyaluronic acid