Hormonal Regulation Of Apoptosis Research Articles

Hormonal regulation of apoptosis in the endometrium of common marmosets ( Callithrix jacchus)

Phase-dependent apoptotic changes in the human endometrium during an ovarian cycle imply a potential role of steroids in the regulation of apoptosis. The present study was undertaken to determine the direct role of hormones in endometrial apoptosis in marmosets ( Callithrix jacchus), a primate species which shows similarity to humans in terms of the cycle length and pattern. Endometrial apoptosis was detected by 3′-end labeling (TUNEL) in various phases of ovarian cycle in naturally cycling healthy marmosets ( n = 14) and also in ovariectomized marmosets ( n = 13) treated with either estradiol alone (E) or progesterone alone (P) or estradiol followed by progesterone (E + P). Expressions of apoptosis associated genes such as Bcl-2 family members (Bax and Bcl-2), proliferating cell nuclear antigen (PCNA)—a proliferation marker and steroid receptors, ERα and PR A were analysed by immunohistochemical methods. Apoptosis was intense in the glandular epithelial cells of endometrium during the mid-luteal phase as compared to other phases in naturally cycling animals; in the E + P group as compared to other groups of ovariectomized animals ( P < 0.05). Pronounced apoptosis in the mid-luteal phase was accompanied by the increased expression of Bax in glandular epithelial cells; while Bcl-2 immunoreactivity remained unchanged. PCNA expression was higher in the naturally cycling animals in the follicular phase and in the E group of the ovariectomized animals as compared those in the other groups. Immunoreactive ERα and PR A in glandular epithelial cells were most abundant during early follicular phase in naturally cycling animals and in both E and E + P groups among the ovariectomized animals. The present study highlights the importance of apoptosis in endometrial remodeling during the ovarian cycle and secondly, the role of both estradiol and progesterone in the regulation of apoptosis.

Hormonal regulation of apoptosis and the Fas and Fas ligand system in human endometrial cells.

The process of apoptosis is responsible for normal cellular turnover in numerous tissues throughout the body. The endometrial layer of the uterus shows steroid-dependent cyclic changes in structure and function. After a proliferative and secretory phase, steroid support is withdrawn and the uterine epithelium is shed. We hypothesize that the apoptosis observed in endometrial cells following hormonal withdrawal is mediated by the Fas/Fas ligand (FasL) system. Normal endometrial cells and endometrial cancer cells were cultured in the presence of estrogen and progesterone. In order to mimic physiological hormonal changes, estrogen and progesterone were removed from the media. Apoptosis was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay and propidium iodide staining, while Fas and FasL expression were evaluated by Western blot analysis. The endometrial cells expressed Fas and low levels of FasL. Withdrawal of estrogen and/or progesterone from the culture induced apoptosis causing an approximately 50% decrease in cell viability. This coincided with increased Fas and FasL expression. Treatment of the cells with anti-FasL antibody prevented cell death following hormonal withdrawal. Estrogen and progesterone therefore represent survival factors which hamper cell death by impeding the expression of apoptotic factors. Our results indicate that Fas-mediated apoptosis is important for endometrial cycling and suggest that dysregulation of the Fas/FasL interactions may have an important role in the development of endometrial cancer.

Hormonal regulation of apoptosis in breast cells and tissues

Few studies have referred to the implication of apoptotic processes following hormonal treatment. No data are available on the effects of progesterone in breast cells. In order to gain insights on the effects of the gonadal steroids and antiestrogens in breast cells, we have carried out studies on apoptosis in different breast materials. We have developed a model of normal breast cells in cultures that remain hormone-dependent. On these cells and in some hormone-dependent breast cancer cell lines (T-47-D, ZR75–1, MCF-7) we have observed an antiapoptotic effect of estradiol (E 2) and a potent proapoptotic effect of some antiestrogens. Progestins were also proapoptotic in normal as well as in hormone-dependent breast cancer cells. In order to understand the mechanisms of these hormones on apoptosis, we studied the bcl-2 family proteins. We demonstrated that E 2 increased the antiapoptotic proteins, bcl-2 and bclx L, whereas, the progestins drastically decreased bcl-2 expression and weakly bclx L levels. We investigated the mechanisms by which E 2 increased bcl-2 expression. Our results using quantitative RT-PCR showed that E 2 increased bcl-2 mRNA levels at 48 h of treatment via a transcriptional mechanism. None of the hormone treatments altered the proapoptotic protein levels, bax and bak. We also studied the in vivo expression of bcl-2 and other members of its family in biopsies of normal breast tissues according to the menstrual cycle. Bcl-2 displayed a strong cyclical variation and seemed to be the most hormone-dependent member of the family.

Hormonal regulation of apoptosis in early antral follicles: follicle- stimulating hormone as a major survival factor

Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.

Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor.

Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.