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The physiological effects of estrogen on the pituitary, including cellular proliferation and regulation of hormone synthesis, are mediated by the nuclear estrogen receptor (ER). The purpose of this study was to determine ontogenetic expression of two types of ERs (ERalpha and ERbeta) in the pituitary using specific antibodies, monoclonal antibody (1D5) for ERalpha and polyclonal antibody generated against ERbeta. First, we confirmed the detection of 66- and 55-kDa bands for ERalpha and ERbeta, respectively, in the rat pituitary extract by Western blotting. Then immunostaining with these antibodies was performed using fetal and adult Wistar rat tissues, combined with PRL or LHbeta immunohistochemistry. Intense ERbeta signal was detected throughout the pituitary from day 12 of gestation. However, staining for ERalpha only became detectable from day 17 of gestation. In contrast with the fetal period, nuclei stained for ERalpha were widely distributed in the anterior lobe in the adult rat, whereas ERbeta-positive cells were restricted in the anterior lobe. LHbeta, but not PRL, was colocalized in ERbeta-positive cells. Our results indicated that the major population of ER subtypes in the rat pituitary gland has changed around the day of birth and that the expression of ERbeta may be involved in the differentiation of pituitary cell function to synthesize a specific hormone.

Apoptosis, a physiological process of cell death, may modulate the mass of the thyroid gland. We investigated the role of apoptosis and the possible involvement of Fas/Fas ligand (FasL) system in apoptosis during goiter formation and involution in a rat model of goiter. Rats were fed a low iodine diet and a goitrogen, 6-propyl-2-thiouracil, to induce goiter. Rats with goiter were then fed a high iodine diet to study the phase of involution. We examined the presence of apoptosis by electron microscopy (EM) and terminal deoxy-UTP nick end labeling (TUNEL). We also investigated the association between Fas and FasL expression and thyrocyte apoptosis using immunohistochemistry and Western blotting. To evaluate the proliferation of thyrocytes, proliferating cell nuclear antigen was examined immunohistochemically. The number of apoptotic cells increased during goiter formation and the early stage of involution, which were also associated with increased number of Fas-positive thyrocytes, and some of these cells c...

Current studies have shown that atresia of ovarian follicles is induced through apoptosis in granulosa cells. Several articles have been devoted to study of the molecular mechanisms responsible for APO-1/CD95 (Fas) is a cell surface protein that can mediate apoptosis in lymphoid cells, and Fas ligand was recently identified in a cytotoxic T cell line. To clarify the involvement of the Fas-Fas ligand system in granulosa cell apoptosis, we investigated the expression of Fas and Fas ligand at an individual cell level. For this purpose, we raised specific polyclonal antibodies against Fas and Fas ligand. Western blotting confirmed that our anti-Fas antibodies (anti-P2 and anti-P4) detect a specific band with a mol wt of 45 kDa in the lysate of ovaries from immature PMSG-treated rats or adult cyclic rats. In immature PMSG-treated rats, immunohistochemical analysis with these antibodies revealed specific staining of granulosa cells in secondary and tertiary follicles at an early stage of atresia, but not in healthy follicles. Fas messenger RNA was also found in granulosa cells of early atretic follicles using in situ hybridization. On the other hand, the anti-Fas ligand antibody (anti-P5) detected a specific 31-kDa band on a Western blot of the oocytes lysate, and the staining with the serum was localized to oocytes in most of developing follicles. Colocalization of Fas and Fas ligand in certain follicles intimately correlated with granulosa cell apoptosis, which was revealed by terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling staining of DNA strand breaks. Finally, we found that interferon-gamma increased Fas expression on granulosa cells in vitro. Coculturing interferon-gamma-pretreated granulosa cells with zona-free oocytes induced granulosa cell apoptosis, which was confirmed by Hoechst 33342 dye staining and terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling, and the killing effect of oocytes was abolished by the addition of anti-P2, which was expected to interrupt the interaction between Fas and Fas ligand. These results demonstrate that activation between Fas and Fas ligand. These results demonstrate that activation of the Fas-Fas ligand system is capable of initiating apoptosis in the ovary, as are a number of other stimuli, outside the immune system.

We have found that microwave (MW) stabilization greatly improves detection of the estrogen receptor (ER) in frozen sections of rhesus monkey oviduct by immunocytochemistry (ICC). Fresh samples of fimbriae were MW-irradiated, frozen, and then cryosectioned. The frozen sections were also MW-treated and then fixed in a paraformaldehyde-based fixative before ICC processing. A parallel set of samples from each monkey were frozen, sectioned and processed for ICC without any MW treatment. MW stabilization clearly increased immunostaining intensity with either of two ER-specific monoclonal antibodies, namely, H222 and 1D5. The greatest increase was noted in tissues collected from spayed or progesterone-treated animals. An antibody dilution series indicated that MW stabilization increased the sensitivity approximately 20- to 40-fold. In addition, we incubated spayed macaque fimbriae at 4 C in the presence of 10 nM [3H]Moxestrol and then either froze the tissues immediately (non-MW) or treated them with MW. Slide-mounted cryosections of non-MW and MW-treated tissue were then incubated with either a Tris-EDTA buffer (low salt) or the same buffer containing 4 M KCl (high salt). The quantity of [3H]Moxestrol-occupied ER extracted from the frozen sections by each buffer was determined by a sucrose gradient shift assay. The low salt buffer extracted significantly more radiolabeled ER from non-MW sections than from MW-treated sections (P < 0.01), whereas the high salt buffer extracted equal amounts of ER from both the MW-treated and non-MW sections. MW-irradiation enhanced ICC detectability of ER in frozen sections by greatly reducing the amount of ER extracted during the various washes used during normal ICC processing.