Takashi Samoto

Effects of progesterone on uterine leiomyoma growth and apoptosis

Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. Recently we have found that the use of levonorgestrel-releasing intrauterine system (IUS) is effective in the long-term contraception and management of menorrhagic women with uterine myomas because of a striking reduction in menorrhagia. These clinical experiences prompted us to characterize the effects of progestin on the proliferation and apoptosis of leiomyoma cells cultured in vitro. 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 E(2) (10 ng/ml) or P(4) (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 E(2) augmented PCNA expression in the cells, but P(4) did not. Immunoblot analysis revealed that leiomyoma cells contained immunoreactive EGF and that P(4) treatment resulted in an increase in EGF expression in the cells. In contrast, E(2) treatment augmented EGF-R expression in cultured leiomyoma cells, but P(4) did not. These results indicate that P(4) up-regulates the expression of PCNA and EGF in leiomyoma cells, whereas E(2) up-regulates the expression of PCNA and EGF-R in those cells. It is, therefore, conceivable that P(4) and E(2) 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, suggesting that the abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Furthermore, Bcl-2 protein expression in leiomyoma cells was up-regulated by P(4), but down-regulated by E(2). Therefore, it seems likely that P(4) may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells.

Molecular Bases for the Actions of Ovarian Sex Steroids in the Regulation of Proliferation and Apoptosis of Human Uterine Leiomyoma

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.

Comparative Analysis of the Effects of Gonadotropin-Releasing Hormone Agonist on the Proliferative Activity, Apoptosis, and Steroidogenesis in Cultured Porcine Granulosa Cells at Varying Stages of Follicular Growth

This study was conducted to analyze comparative effects of gonadropin-releasing hormone (GnRH) agonist on the proliferation, apoptosis, and differentiated function of cultured porcine granulosa cells from varying follicle stages. Comparative analyses of porcine granulosa cells from varying follicle stages to respond to GnRH agonist were performed in terms of proliferating cell nuclear antigen (PCNA) expression, occurrence of apoptosis, and 17β-estradiol (E2) and progesterone (P) secretion. PCNA expression was examined by the avidin/biotin immunoperoxidase method with a monoclonal antibody to PCNA, and apoptosis was assessed by in situ DNA 3′-end labeling method and DNA fragmentation analysis. E2 and P were measured by radioimmunoassays. The PCNA positive rate of granulosa cells cultured in the presence of GnRH agonist (10−9M) was lower compared with that of cells cultured in the absence of GnRH agonist. However, the apoptosis positive rate was higher, and E2 and P secretion by cultured granulosa cells was lower in the presence of GnRH agonist (10−9M) compared with that in the absence of GnRH agonist. The inhibitory effect of GnRH agonist on PCNA positive rate of cultured cells was prominent in granulosa cells from small and medium but not from large follicles. By contrast, the inhibitory effect of GnRH agonist on E2 and P secretion by cultured cells was prominent in granulosa cells from large but not small and medium follicles. The stimulatory effect of GnRH agonist on apoptosis positive rate of cultured cells was, however, uniform regardless of the stages of follicular growth. These results demonstrate that GnRH agonist exerts diverse actions on granulosa cells over the course of follicular growth. One downregulates granulosa proliferation in immature follicles as well as steroidogenesis in mature follicles, and the other upregulates apoptosis of granulosa cells regardless of the stages of follicular growth.

Transient postpartum diabetes insipidus in twin pregnancy associated with HELLP syndrome

Abstract Diabetes insipidus during pregnancy is an uncommon medical problem, and its cause is not entirely clear. We present a woman with twin pregnancy associated with HELLP syndrome, who developed diabetes insipidus during postpartum period. A hypertonic saline infusion study with measurement of plasma arginine vasopressin concentrations confirmed the diagnosis. She had mild response to 1-desamino-8-d-arginine-vasopressin (dDAVP) during the immediate postpartum period. On the 3rd postpartum day two doses of 100μl of dDAVP were administered, and her urinary volume gradually decreased. We could stop dDAVP on the 30th postpartum day. This exacerbation may result from increased vasopressinase activity caused by the excessive production in the placenta due to twin pregnancy, together with the insufficient degradation in the liver due to HELLP syndrome.

Biology of human trophoblast

In order to elucidate the regulation of placental growth, we have characterized the expression of proliferating cell nuclear antigen (PCNA), apoptotic DNA fragmentation and bc1‐2 protein in human placenta during pregnancy. PCNA and bc1‐2 protein expression were examined by immunohistochemical techniques, while the occurrence of apoptotic DNA fragmentation was assessed by in situ analysis of DNA 3′‐end labeling method. Both PCNA expression and apoptotic DNA fragmentation were found in cytotrophoblasts (C‐cells), being most abundant in early placenta, less abundant in midterm placenta and least abundant in term placenta. In contrast, bc1‐2 protein expression was found in syncytiotrophoblasts (S‐cells), being least abundant in early placenta, less abundant in midterm placenta and most abundant in term placenta. These data indicate that early placenta is characterized by the highly proliferative activity of C‐cells associated with the increased occurrence of apoptosis, whereas term placenta is characterized by the abundant expression of bc1‐2 protein in S‐cells.

Factors Regulating SCC Antigen Expression in Squamous Cell Carcinoma of the Uterine Cervix

Expression of squamous cell carcinoma (SCC) antigen emerged concurrently with squamous formation of the uterine cervix and increased during the neoplastic transformation of the cervical squamous epithelium. SCC antigen expression differed considerably among the histomorphologic cell types of cervical carcinoma. Large cell nonkeratinizing carcinoma contained high levels of the antigen. In contrast, no appreciable expression of SCC antigen was observed in small cell nonkeratinizing carcinoma. The pattern of SCC antigen expression closely coincided with EGF receptor (EGF-R) expression in cervical squamous neoplasia. This suggests that the expression of SCC and EGF-R in cervical carcinoma is related to the differentiation or dedifferentiation processes of the tumor cells. SCC production by CaSki cervical epidermoid carcinoma cells was stimulated by EGF. It seems likely that an autocrine system, in which EGF serves as the signal, may exist in cervical squamous carcinoma. 17β-estradiol and L-triiodothyronine were found to upregulate EGF-R expression, proliferative potential and SCC production in the CaSki cervical carcinoma cells.

Estradiol/progesterone-releasing vaginal rings for hormone replacement therapy in postmenopausal women

In order to investigate whether vaginal rings delivering estradiol and progesterone could prevent endometrial hyperplasia and relieve climacteric symptoms ,two variants of rings were used in 20 postmenopausal women with intact uteri for 4 months. One ring designated as PI-002 (n = 8) delivered in vitro estradiol 160 μg/day and progesterone 20 mg/day ,while the other (PI-003; n = 12) delivered the same dosage of estradiol but only half the progesterone (10 mg/day). Serum estrone ,estradiol and progesterone were measured at pretreatment ,weekly for 4 weeks ,and then monthly for 4 months. The incidence of hot flushes ,frequency of night sweats ,mood scores ,vaginal discharge and bleeding profiles were recorded. Endometrial thickness was monitored by ultrasonography. The mean estrone level was 50 pg/ml for 16 weeks. The mean serum estradiol level was 75 pg/ml for the first 4 weeks and gradually decreased to 50 pg/ml at 16 weeks. The mean progesterone level with the PI-002 ring was 5 ng/ml for the first 4 weeks and decreased to 3.5 ng/ml at 16 weeks. With the PI-003 ring ,the mean progesterone level was initially 3.5 ng/ml and then decreased to 2.5 ng/ml thereafter. Significant decreases in the incidence of hot flushes and night sweats as well as a striking improvement in mood scores were noted as early as 2 weeks after insertion. Three of the 20 women discontinued the treatment ,owing to ring expulsion. Increased vaginal discharge was observed with both rings in the first 6 weeks. Vaginal bleeding was more frequently apparent among users of the PI-002 ring ,although bleeding and spotting were confined to the first 6 weeks. Ultrasonographic monitoring of the endometrium constantly revealed a thickness of < 3 mm for both variants throughout use for 16 weeks. An estradiol/progesterone-releasing vaginal ring is a potential alternative to long-term hormone replacement therapy with minimum attention required. It provides effective protection against endometrial hyperplasia.