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Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. In order to characterize the molecular mechanism of sex steroidal regulation of leiomyoma growth, we examined whether sex steroids could influence the proliferation of leiomyoma cells. As epidermal growth factor (EGF) has been shown to mediate estrogen action and play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on EGF and EGF receptor (EGF-R) expression in leiomyoma cells. In cultures of leiomyoma cells, the addition of either estradiol (E₂; 10 ng/ml) or progesterone (P₄; 100 ng/ml) resulted in an increase in proliferating cell nuclear antigen (PCNA) expression in the cells, whereas in cultures of normal myometrial cells, the addition of E₂ augmented PCNA expression in the cells, but P₄ did not. Immunoblot analysis revealed that leiomyoma cells contained immunoreactive EGF and that P₄ treatment resulted in an increase in EGF expression in the cells, whereas E₂ treatment resulted in a lower EGF expression in the cells. By contrast, E₂ treatment augmented EGF-R expression in cultured leiomyoma cells, but P₄ did not. These results indicate that P₄ upregulates the expression of PCNA and EGF in leiomyoma cells, whereas E₂ upregulates the expression of PCNA and EGF-R in those cells. It is, therefore, conceivable that P₄ and E₂ act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression. We also found that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to that in normal myometrium and that Bcl-2 protein expression in leiomyoma cells was upregulated by P₄, but downregulated by E₂. It seems, therefore, likely that P₄ may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. The abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of a leiomyoma relative to that of normal myometrium in the uterus.

Abstract.In order to evaluate placental trophoblast proliferation and apoptosis during pregnancy, we investigated proliferating cell nuclear antigen (PCNA) expression, apoptosis and Bcl-2 protein expression in the human placenta using avidin/biotin immunoperoxidase method to examine PCNA and Bcl-2 protein expression, and TUNEL method to assess apoptosis. The appearance of apoptotic cells in very early term placental trophoblasts was also examined by transmission electron microscopy.PCNA was immunolocalized in the nuclei of cytotrophoblasts (C-cells). Determination of the mean percentage of PCNA-positive nuclei of C-cells revealed that PCNA expression in C-cells was highest in very early term (4th to 5th wk) placentas and significantly decreased with the advance of pregnancy. Bcl-2 protein was immunolocalized in the cytoplasm of syncytiotrophoblast (S-cell), being least abundant in very early term placentas, less abundant in early term and midterm placentas, and most abundant in term placentas.On the basis of TUNEL method, apoptosis was apparent in the nuclei of both C-cells and S-cell. The apoptosis positive rate of C-cell nuclei was highest in very early term 4th to 5th wk placentas, and significantly decreased in early term 7th to 9th wk and midterm placentas, but somewhat increased in term placentas compared to that in midterm placentas. On the other hand, apoptosis positive rate of S-cell nuclei was remarkably higher only in very early term 4th to 5th wk placentas compared to that in early term, midterm and term placentas.Transmission electron microscopy revealed the appearance of apoptotic nucleus in very early term placental trophoblasts.These results demonstrate for the first time that apoptosis in the human normal placenta predominates in both C-cells and S-cell in very early term 4th to 5th wk pregnancy and drastically diminished after 7th wk of pregnancy.An apparent increase in apoptosis in C-cells in term placentas compared to that in midterm placentas may reflect aging of the placenta or parturitionassociated biological change. The abundant expression of Bcl-2 protein in S-cell in term placentas may be responsible for the diminished occurrence of apoptosis in S-cell in term placentas.

Abstract. The cytologic localization and cellular levels of insulin receptors in the human ovary during follicular growth, regression and atresia were examined by the avidin/biotin immunoperoxidase techniques with a monoclonal antibody to insulin receptor. In primordial follicles, only the oocyte showed a weak immunostaining for insulin receptor, whereas the stromal cells surrounding primordial follicles were moderately immunostained. The earliest stage of follicular growth at which immunostaining for insulin receptor in granulosa cells and theca interna cells became apparent was the preantral stage. With the increase in the size of the follicles, the immunostaining of the oocyte and follicular elements intensified, whereas the staining intensity of the stromal cells surrounding growing follicles was reduced compared to those surrounding primordial follicles. The immunostaining in granulosa and theca interna cells persisted in the corpus luteum, and further intensified during the midluteal phase. In the regressing corpus luteum, the immunostaining was present only in the peripheral lutein cells adjacent to the central scar tissue. The corpus albicans was negative for the immunostaining, but the surrounding stromal cells exhibited predominant staining. In atretic follicles, the theca interna cells exhibited intense staining for insulin receptor without appreciable staining in the scattered granulosa cells, whereas the surrounding stromal cells were moderately immunostained. This is the first study to demonstrate notable changes in insulin receptor expression in the oocyte, granulosa cells, theca cells, lutein cells and surrounding stromal cells during follicular growth, regression and atresia. The results obtained indicate insulin participation in oocyte maturation, follicular growth and stromal cell function. The increased expression of insulin receptors in theca interna cells of atretic follicles and in stromal cells surrounding the corpora albicans raises the intriguing possibility of insulin involvement in the transformation of theca interna cells into stromal cells. This implies that insulin may participate in remodelling ovarian local tissues following follicular atresia and luteolysis in the human ovary.