No abstract

Uterine leiomyoma is the most common benign smooth muscle cell tumor of the myometrium. Although Bcl-2 protein is known to be an apoptosis-inhibiting gene product and to prevent apoptotic cell death in a variety of cells, there are no published data regarding whether human leiomyomas express Bcl-2 protein. In the present study, we examined the expression of Bcl-2 protein in leiomyomas in comparison with that in the normal myometrium using an immunohistochemical method and immunoblot analysis with a monoclonal antibody to human Bcl-2 protein. Furthermore, we investigated whether sex steroid hormones could influence the levels of Bcl-2 protein expression in leiomyoma cells cultured in vitro under serum-free, phenol red-free conditions. Immunohistochemical staining for Bcl-2 protein was prominent in leiomyoma cells, but was scarcely present in normal myometrial smooth muscle cells. The expression of Bcl-2 protein in leiomyoma cells was most abundant in the secretory, progesterone-dominated, phase of the menstrual cycle, but was less abundant in the proliferative phase of the menstrual cycle. Western blot analyses of leiomyoma and myometrium tissue extracts revealed that Bcl-2 protein, with a molecular mass estimated at approximately 26 kDa, was abundantly present in leiomyoma tissue extracts, but was undetectable in normal myometrial tissue extracts. In monolayer cultures of uterine leiomyoma cells under a serum-free condition, the addition of progesterone (100 ng/mL) resulted in a striking increase in Bcl-2 protein expression in the cultured leiomyoma cells relative to that in control cultures, whereas the addition of 17 beta-estradiol (10 ng/mL) resulted in a reduction in Bcl-2 protein expression in the cells. The concentrations of sex steroids used were within the physiological tissue concentrations found in leiomyomas and myometrium. The present results suggest that the abundant expression of Bcl-2 protein may have a molecular basis characteristic of leiomyomas in the human uterus and that progesterone may play a vital role in the enhanced expression of Bcl-2 protein in human uterine leiomyoma cells.

No abstract

It is well known that T(3) plays a crucial role in the maintenance of early pregnancy through the induction of endocrine function in villous trophoblasts. The effects of T(3) on extravillous trophoblast (EVT) function, however, remain to be elucidated. To investigate the possible role of T(3) in the regulation of EVT invasion to the decidua, we have examined whether T(3) affects EVT invasive potential and the expression of matrix metalloproteinase-2 (MMP-2), MMP-3, tissue inhibitor metalloproteinase-1, fetal fibronectin (FN), and integrin alpha(5)beta(1) in cultured early placental EVTs. Isolation and purification of trophoblasts differentiating into EVTs were performed by the enzymatic digestion of the anchoring chorionic villi, with the use of human FN-precoated culture dishes and FN-precoated Matrigel Transwells. The cells attached to the dishes were subcultured in DMEM supplemented with 10% fetal bovine serum for 48 h and were characterized by RT-PCR analysis after 24-h subculture and immunocytochemical analysis after 48-h subculture for specific EVT markers. Thereafter, the cultured cells were stepped down to a 4% fetal bovine serum condition and cultured in the presence or absence of T(3) (10(-8) m) for the subsequent 72 h. Matrigel invasion assay demonstrated that the treatment with T(3) significantly increased the number of cell projections of subsequent 24-, 48-, and 72-h cultured EVTs. RT-PCR analysis revealed that the treatment with T(3) increased the expression of MMP-2, MMP-3, fetal FN, and integrin alpha(5)beta(1) mRNA in subsequent 24-h cultured EVTs compared with those in control cultures. Immunocytochemical and Western immunoblot analyses revealed that treatment with T(3) increased the expression of MMP-2 and MMP-3 in subsequent 48-h cultured EVTs compared with those in control cultures. The present results suggest that T(3) (10(-8) m) may play a vital role in up-regulating the invasive potential of EVTs into the decidua.

To characterize thyroid hormone action on the ovary, the direct effects of T4 or T3 were investigated in vitro using a monolayer culture system of porcine granulosa cells. Monolayer cultures were maintained for 6 days in 4% serum-supplemented medium in the absence or presence of porcine FSH (20 ng/ml), with or without graded doses of T4 or T3. Combined treatment with FSH and T4 (10(-7) M) induced morphological alternation resembling epithelioid cells, while FSH alone or T4 alone failed to bring about the epithelioid morphology. Concomitant treatment with FSH and T4 (10(-7) M) markedly increased FSH-stimulated induction of [125I]iodo-human CG binding to cultured granulosa cells obtained from small follicles. The combined treatment with FSH and T4 (10(-7) M) also resulted in a significant increase in progesterone and estrogen secretion by the cultured cells relative to treatment with FSH alone. Increases in progesterone, 17 beta-estradiol, and estrone secretion caused by the combined treatment with FSH and T4 (10(-7) M) were further augmented in response to the addition of exogenously provided substrate pregnenolone, testosterone, and androstenedione, respectively. Furthermore, aromatase activity assessed by the release of [3H]water from [1 beta-3H, 4-14C]androstenedione was significantly higher in cells treated concomitantly with FSH and T4 (10(-7) M) than that in cells treated with FSH alone. All the stimulatory effects of T4 (10(-7) M) on the morphological and functional differentiation of cultured granulosa cells were also found in combined treatment with FSH and T3 (10(-9) M). Either treatment with higher or lower concentrations of T4 or T3 gave attenuated effects, and T4 or T3 alone without FSH was incapable of exhibiting these stimulatory effects. These findings suggest that thyroid hormones synergize with FSH to exert direct stimulatory effects on granulosa cell functions, including morphological differentiation, LH/human CG receptor formation and steroidogenic enzyme (3 beta-hydroxysteroid dehydrogenase and aromatase) induction. Hence, decreases in ovarian functions during the states of hypo- or hyperthyroidism may account for diminished responsiveness of the granulosa cells to FSH.