Expression and distribution of AP-1 transcription factors in the porcine ovary.

The activator protein-1 (AP-1) transcription factors are important regulators of cell proliferation and differentiation. The developmental distribution of AP-1 family members in porcine ovary has not been previously investigated. We examined the expression of AP-1 factors in porcine ovarian follicles, granulosa cells, and corpora lutea at different stages of development. Immunoblot analyses confirmed that c-Jun, JunD, JunB, c-Fos, Fra-1, Fra-2, and FosB immunoreactive proteins were present in whole-cell extracts (WCE) of all antral follicles and midluteal phase corpora lutea (CL) as well as granulosa cells (GC) isolated from different-sized antral follicles. The intensities of c-Jun and c-Fos protein bands were decreased in CL WCE compared to antral follicles. In granulosa cells from preovulatory follicles (8-10 mm), Fra-2 exhibited a shift from 43 kDa to 46 kDa when compared to granulosa cells from smaller antral follicles. Separation of cytoplasmic and nuclear extracts was performed to determine if developmental differences between these fractions existed. Most AP-1 factors predominated in the nuclear fraction with notable exceptions. c-Fos predominated in the nucleus in GC and follicles but predominated in the cytoplasmic fraction of CL. With the exception of GC from 1-2-mm follicles, in which expression was similar between fractions, Fos-B was found predominantly in the cytoplasmic fraction. Fra-1 exhibited similar expression between cytoplasmic and nuclear fractions for all tissues. Immunohistochemical (IHC) analyses of porcine ovary sections were performed to determine the cellular distribution of these factors at different follicular stages, and immunopositive nuclei were evaluated. In primordial and primary unilaminar follicles, all AP-1 factors studied except for FosB were detected in granulosa nuclei. Granulosa cell nuclei of multilaminar preantral follicles were immunopositive for all factors, with lower expression of FosB. Antral follicles exhibited GC and thecal cell nuclear staining for all factors with the exception of FosB in theca. Luteal cells exhibited the most intense nuclear staining for JunD and Fra-2, whereas all other factors were present in luteal cell nuclei although to a lesser extent. IHC with FosB antibodies yielded mostly cytoplasmic staining but only weak luteal nuclear staining. In corpora albicantia, low levels of staining were seen for all AP-1 factors. The DNA-binding abilities of these factors in granulosa cells and CL were evaluated by EMSA. Nuclear extracts from granulosa cells from 1-2-mm or 8-10-mm antral follicles bound an AP-1 DNA consensus sequence and complexes consisted predominantly of c-Jun, JunD, JunB, c-Fos, and Fra-2. In CL, c-Jun, JunD, JunB, and Fra-2 were present in DNA-binding complexes, and c-Fos binding was not detected. In conclusion, our results suggest that expression and DNA-binding activity of AP-1 factors in follicular structures changes with luteinization. Differentiation to the luteal phenotype involves a reduction in nuclear c-Jun and c-Fos and a predominance of JunD and Fra-2.