Marta Tesone

Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis

OBJECTIVE To evaluate and compare spontaneous apoptosis and Bcl-2 and Bax expression in eutopic endometrium from women with and without endometriosis. DESIGN Apoptosis and Bcl-2 and Bax expression were examined in eutopic endometrium from women with and without endometriosis. SETTING Instituto de Biología y Medicina Experimental-CONICET, Department of Gynecology and Department of Gynecological Pathology, Clínicas University Hospital, Buenos Aires, Argentina. PATIENT(S) Women with untreated endometriosis (n = 14) and controls (n = 16). INTERVENTION(S) Collection of endometrial samples during diagnostic or therapeutic laparoscopy. MAIN OUTCOME MEASURE(S) Apoptotic cells were detected with use of the dUTP nick-end labeling (TUNEL) assay; Bcl-2 and Bax expressions were assessed with use of immunohistochemical techniques. RESULT(S) Spontaneous apoptosis was significantly lower in eutopic endometrium from patients with endometriosis, compared with healthy controls (2.26 +/- 0.53 and 9.37 +/- 1.69 apoptotic cells/field, respectively) and was independent of cycle phase. An increased expression of Bcl-2 protein was found in proliferative eutopic endometrium from patients with endometriosis. Bax expression was absent in proliferative endometrium, whereas there was an increase in its expression in secretory endometrium from both patients and controls. CONCLUSION(S) Women with endometriosis show decreased number of apoptotic cells in eutopic endometrium. The abnormal survival of endometrial cells may result in their continuing growth into ectopic locations.

Oral contraceptives suppress cell proliferation and enhance apoptosis of eutopic endometrial tissue from patients with endometriosis

OBJECTIVE To evaluate the effects of administering combination oral contraceptives (COCs) to patients with endometriosis on the regulation of cell growth in the eutopic endometrium. DESIGN Prospective study. SETTING Research institute and clinical fertility center. PATIENT(S) Thirteen women with untreated endometriosis and 13 controls. INTERVENTION(S) Biopsy specimens of the eutopic endometrium were obtained from all subjects. Apoptosis, cell proliferation, and Bcl-2 and Bax expression were examined at the epithelial and stromal levels in the eutopic endometrium from patients with endometriosis before and after 30 days of daily exposure to COCs and from controls. MAIN OUTCOME MEASURE(S) Apoptotic cells were detected by using the dUTP nick-end labeling assay; Ki-67, Bcl-2, and Bax expressions were assessed by using immunohistochemical techniques. RESULT(S) After exposure to COCs, apoptosis was significantly increased in the eutopic endometrium compared with before COC administration, both at epithelial and stromal levels. Cell proliferation was significantly lowered by COCs. CONCLUSION(S) COCs showed a positive effect on patients with endometriosis by down-regulating cell proliferation and enhancing apoptosis in the eutopic endometrium.

Effect of a gonadotropin-releasing hormone agonist on luteinizing hormone receptors and steroidogenesis in ovarian cells *

OBJECTIVE To examine the effect of a gonadotropin-releasing hormone agonist (GnRH-a), leuprolide acetate (LA), on human chorionic gonadotropin/luteinizing hormone (LH) receptors content and progesterone (P) and estradiol (E2) production in cultured granulosa or luteal cells. DESIGN Prospective. SETTING Private Fertility Clinic and National Research Institute. PATIENTS Twenty patients undergoing in vitro fertilization or gamete intrafallopian transfer programs. RESULTS Human chorionic gonadotropin/LH receptors in human granulosa cells increased after 48 hours of culture, and LA inhibited such effect. Leuprolide acetate, 1 ng/mL, in the cultures produced an increase in P production. On the contrary, LA inhibited E2 production. Additionally, the in vivo effect of LA (2 micrograms/rat per 7 days) was studied in corpus luteum of superovulated rats. Luteal cells from LA-treated rats in culture produced lower P than the controls but showed an increase in aromatase activity. Luteal LH receptors declined after 48 hours of culture with LA. CONCLUSION The high doses of gonadotropin necessary to induce ovarian hyperstimulation when GnRH-a is administered could be related with an inhibitory effect of these agonists on LH receptors and aromatase activity.

Regulation of follicular luteinization by a gonadotropin-releasing hormone agonist: Relationship between steroidogenesis and apoptosis

The purpose of this study was to evaluate the effects of GnRH‐analog (Leuprolide acetate, LA) administration on follicular luteinization in equine chorionic gonadotropin plus human chorionic gonadotropin (eCG + hCG)‐superovulated prepubertal treated rats. Results indicate that LA treatment decreases circulating levels of progesterone (P) and P accumulation in collagenase‐dispersed ovarian cell cultures, though estradiol(E2) production is increased. These data suggest that cells from the LA group may be less luteinized following gonadotropin treatment. Studies performed on histological ovarian sections after different times of eCG administration showed that LA injections produce lower amounts of corpora lutea and antral follicles, and a greater number of atretic and preantral follicles. The basal and LH‐stimulated P and progestagen accumulations are decreased in incubations of corpora lutea isolated from the LA group. In addition, the mitochondrial cholesterol side‐chain cleavage (P450SCC) levels in corpora lutea from LA‐treated rats are reduced, indicating that the decrease in P production observed is due in part to an alteration in the steroidogenic luteal capability. Immunocytochemical localization of nuclei exhibiting DNA fragmentation by the technique of terminal deoxynucleotidyl transferase end‐labeling showed that LA treatment causes an increase in the number of apoptotic cells in preantral and antral follicles at all times studied (1, 2, 4, or 7 days of LA administration). A similar effect, though less pronounced, was observed in corpora lutea. It is concluded that LA treatment produces a failure in the steroidogenic luteal capability and an increase of apoptotic mechanisms in the ovary, producing as a consequence an interference in the follicular recruitment, growth, and luteinization induced by gonadotropins. Mol. Reprod. Dev. 51:287–294, 1998.

Gonadotropin-releasing hormone agonist induces apoptosis and reduces cell proliferation in eutopic endometrial cultures from women with endometriosis

OBJECTIVE There is growing evidence that suggests a direct action of gonadotropin-releasing hormone agonist (GnRH-a) on endometrial growth. Consequently, our purpose was to evaluate the effect of GnRH-a on in vitro eutopic endometrial cell growth and apoptosis. DESIGN Prospective study. SETTING Research institute and clinical fertility center. PATIENT(S) Sixteen women with untreated endometriosis and 14 controls. INTERVENTION(S) Biopsy specimens of eutopic endometrium were obtained from all subjects. Apoptosis and cell proliferation were examined in epithelial endometrial cell cultures after incubation with leuprolide acetate (LA), antide, and a combination of both. MAIN OUTCOME MEASURE(S) The percentage of apoptotic cells was evaluated by the acridine orange-ethidium bromide technique; cell proliferation was assessed by (3)H-thymidine incorporation. RESULT(S) Leuprolide acetate (LA) (100 ng/mL) enhanced apoptosis in endometrial cultures from patients with endometriosis and controls, and this effect was reversed by antide 10(-7)M. Cell proliferation was down-regulated by LA at 1, 10, and 100 ng/mL in cultures from women without and with endometriosis. The addition of antide 10(-7)M reversed this inhibition. CONCLUSION(S) GnRH-a appears to have a direct effect by enhancing the apoptotic index and decreasing the cell proliferation in endometrial cells.

Effects of a Gonadotropin-Releasing Hormone Agonist on Rat Ovarian Follicle Apoptosis: Regulation by Epidermal Growth Factor and the Expression of Bcl-2-Related Genes

Abstract The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuprolide acetate (LA) on follicular development and apoptosis-related mechanisms in preovulatory ovarian follicles (POF) obtained from prepubertal eCG-treated rats. Serum progesterone and estradiol levels were measured, and a significant decrease in circulating estradiol levels was observed in the LA group, whereas serum progesterone levels remained unchanged. Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG. After 48 h of LA treatment, the numbers of atretic and preantral follicles were increased as compared with controls, whereas the number of antral follicles had decreased. Cells undergoing DNA fragmentation were quantified by performing in situ 3′ end labeling of DNA with digoxygenin-dUTP on ovarian sections. LA treatment caused an increase in the percentage of apoptotic cells in preantral and antral follicles. DNA isolated from these POF incubated 24 h in serum-free medium exhibited the typical apoptotic DNA degradation pattern. Treatment of follicles with epidermal growth factor (EGF) suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA follicles. POF obtained from LA-treated rats showed no changes in Bcl-2 or Bax protein levels. However, a reduction in the Bcl-xL:Bcl-xS ratio was observed, with a greater decrease in Bcl-xL compared with Bcl-xS during the incubation, suggesting a lower stability of the Bcl-xL isoform in the LA group. These results indicate that in vivo GnRH agonist treatment produces an increase in the apoptosis process in POF from eCG-treated rats, and this effect is reversed in vitro by EGF. This GnRH analogue also reduced the stability of the Bcl-xL protein, thus interfering with follicular development by an as yet unknown mechanism.

Effects of a selective cyclooxygenase-2 inhibitor on endometrial epithelial cells from patients with endometriosis

BACKGROUND Celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, also has anti-proliferative properties and pro-apoptotic effects on different in vivo and in vitro models, two actions that may be efficacious in therapy for endometriosis. We evaluated the effects of celecoxib on apoptosis and proliferation, and vascular endothelial growth factor (VEGF) production and COX-2 expression and activity in endometrial epithelial cells (EECs). METHODS AND RESULTS Thirty-two endometriosis and 13 control women were included in the study. EECs from eutopic endometrium and control biopsies were cultured with different doses of celecoxib. Celecoxib at 50, 75 and 100 microM (versus vehicle control) inhibited EEC proliferation in cultures from controls (P < 0.05, P < 0.01 and P < 0.01, respectively) and patients with endometriosis (P < 0.05, P < 0.01 and P < 0.01), as assessed by (3)H-thymidine uptake. Celecoxib at 50, 75 and 100 microM induced apoptosis in EEC from controls (P < 0.05, P < 0.001 and P < 0.001) and patients with endometriosis (P < 0.001, P < 0.001 and P < 0.01), as revealed by the Acridine Orange-Ethidium Bromide technique. Western blot analysis showed that celecoxib was effective at increasing COX-2 protein at 100 microM in EEC from endometriosis patients (P < 0.05). In EEC from endometriosis patients, celecoxib at 25, 50 and 100 microM was also effective in reducing COX-2 activity, reflected in the reduction of prostaglandin E(2) (PGE(2)) synthesis (P < 0.001), and VEGF secretion (P < 0.001; P < 0.05 and P < 0.001), assessed by enzyme-linked immunosorbent assay. Exogenous PGE(2) did not reverse celecoxib-induced growth inhibition. CONCLUSIONS This study suggests a direct effect of celecoxib on reduction of endometrial growth and supports further research on selective COX-2 inhibition as a novel therapeutic modality in endometriosis.

Effect of a vascular endothelial growth factor (VEGF) inhibitory treatment on the folliculogenesis and ovarian apoptosis in gonadotropin-treated prepubertal rats.

Abstract In the present study, we investigated whether vascular endothelial growth factor A (VEGFA) plays a critical intraovarian survival role in gonadotropin-dependent folliculogenesis. The effect of an intrabursal administration of a VEGFA antagonist on follicular development, apoptosis, and levels of pro- and antiapoptotic proteins of BCL2 family members (BAX, BCL2, and BCL2L1), as well as of TNFRSF6 (also known as FAS) and FAS ligand (FASLG), was examined. To inhibit VEGFA, a soluble FLT1/Fc Chimera (Trap) was administered to prepubertal eCG-treated rats. Injection of 0.5 μg of Trap per ovary did not change the number of preantral follicles (PFs) or early antral follicles (EAFs); however, it significantly decreased the number of periovulatory follicles 48 h after surgery and significantly increased the number of atretic follicles. No significant differences were found in any stage of the follicles either 12 or 24 h after injection. Cells undergoing DNA fragmentation were quantified by performing TUNEL on ovarian sections. Trap treatment caused a twofold increase in the number of apoptotic cells in EAFs. DNA isolated from antral follicles incubated for 24 h exhibited the typical apoptotic DNA pattern. Follicles obtained from Trap-treated ovaries showed a significant increase in the spontaneous onset of apoptotic DNA fragmentation. The injection of Trap significantly increased the levels of BAX and decreased the levels of BCL2 protein. The ratio of BCL2L1L:BCL2L1s was significantly diminished in follicles obtained from ovaries treated with Trap. No changes in the levels of TNFRSF6 or FASLG were observed after treatment. We concluded that the local inhibition of VEGFA activity appears to produce an increase in ovarian apoptosis through an imbalance among the BCL2 family members, thus leading a larger number of follicles to atresia.

Gonadotropin-Releasing Hormone Antagonist Antide Inhibits Apoptosis of Preovulatory Follicle Cells in Rat Ovary

Abstract Analogs of GnRH, including agonists (GnRH-a) and antagonists (GnRH-ant), have been widely used to inhibit gonadotropin pituitary release. Aside from the effect of GnRH analogs on the pituitary-gonadal axis, studies have shown that GnRH has extrapituitary effects, particularly on rat and human ovaries. In the present study, we evaluated the direct in vivo effects of the GnRH-a, leuprolide acetate (LA), or the GnRH-ant, Antide (Ant), either singly or together, on ovarian follicular development in prepubertal eCG-treated rats. LA significantly decreased ovarian weight, whereas Ant increased ovarian weight compared with controls; however, coinjection of both compounds had no effect. In addition, LA increased the number of preantral follicles (PFs) and atretic follicles, and decreased the number of early antral follicles (EAFs) and preovulatory follicles (POFs). Coinjection of Ant interfered with this LA effect. Ant alone increased the number of POFs compared with that of controls. Analysis of apoptosis has shown that LA increases the percentage of apoptotic cells in PFs, EAFs, and POFs; however, Ant prevented this effect. In addition, Ant alone decreased the percentage of apoptotic cells in EAFs and POFs. Data have shown that Ant per se inhibited BAX translocation from cytosol to mitochondria and retained cytochrome C in the mitochondria, whereas LA induced cytochrome C release. We conclude that Ant inhibits apoptosis in preovulatory follicles through a decrease of BAX translocation to mitochondria, suggesting that GnRH may act as a physiological intraovarian modulator factor that is able to interfere with follicular development through an increase in apoptotic events mediated by an imbalance among the BCL-2 family members.

Steroidogenic Acute Regulatory Protein in Ovarian Follicles of Gonadotropin-Stimulated Rats Is Regulated by a Gonadotropin-Releasing Hormone Agonist

Abstract The aim of the present study was to examine the acute and chronic effects of the gonadotropin-releasing hormone agonist (GnRH-a) leuprolide acetate (LA) on the expression of the steroidogenic acute regulatory protein (StAR), the cytochrome P450 side-chain cleavage enzyme (P450scc), and steroid production in antral ovarian follicles obtained from prepubertal equine choriogonadotropin (eCG)-treated rats. Follicular contents of StAR and P450scc proteins were measured by Western blotting following in vivo injection of eCG (control) and eCG+LA (LA) to prepubertal rats. Treatment with eCG for 2 h resulted in no change in StAR protein content, but it was markedly increased at 4 and 8 h after hormone treatment. However, coadministration of eCG+LA produced a significant increase (P < 0.05) in StAR protein levels at 2, 4, and 8 h when compared with eCG treatment. Acute and chronic treatment with either eCG or eCG+LA did not alter the P450scc protein levels in freshly isolated follicles. The increase in StAR protein expression following LA treatment was qualitatively similar to StAR mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, administration of eCG demonstrated a time-dependent increase (2–8 h) in the levels of StAR mRNA, and these levels were markedly increased by eCG+LA. However, the temporal response pattern of StAR mRNA was much greater at 2 h following LA administration when compared with controls. In addition, 48 h of LA treatment in eCG-treated rats resulted in a significant increase (P < 0.05) in follicular progesterone levels, whereas significant decreases in androgen (testosterone and androsterone) and estradiol levels were observed. Similar results were obtained when serum androgens and estradiol were measured, but serum progesterone levels were unchanged. Collectively, these findings demonstrate that the inhibitory effect of LA on ovarian androgen and estradiol levels is related to changes in the follicular levels of StAR protein and steroid production.