Taisen Iguchi

Polyovular follicles in the ovary of immature mice exposed prenatally to diethylstilbestrol

SummaryPolyovular follicles (PF) occur in the ovary of 30-day-old offspring of ICR/JCL mice given 4 daily subcutaneous injections of 20–2,000 μg diethylstilbestrol (DES)/day from days 15 to 18 of gestation. PF containing 2–9 oocytes per follicle in the prenatally DES-exposed mice are increased 33- to 112-fold as compared to controls. In 5- to 25-day-old offspring of mothers given injections of 2,000 μg DES/day, PF are observed 17–65 times more frequent than in controls.

Development of Vaginal Adenosis-like Lesions and Uterine Epithelial Stratification in Mice Exposed Perinatally to Diethylstilbestrol

Abstract Pregnant ICR/JCL mice were given either four daily subcutaneous injections of 20-2000 μg diethylstilbestrol (DES)/day or a 4-day intravenous infusion of 20-200 μg DES/day starting on Day 15 of gestation. Female mice were injected with 0.01-50 μg DES/day for 5 days starting on the day of birth. Females given oil injections during the neonatal period and offspring of mothers given injections of an infusion of respective vehicles alone served as controls when corresponding ages were attained. Incidence of adenosis-like lesions (ADL) in the vaginal epithelium and stratification of the uterine epithelium (UST) were determined in both offspring and neonatally exposed mice at 30 days of age. Injections of 0.1-50 μg DES/day during the neonatal period induced ADL in the fornical epithelium of vagina (36-70%), but not UST. A high incidence of ADL (80-88%) was found in the fornical and upper-vaginal epithelia in offspring of mothers given injections of 200-2000 μg DES/day, and UST was encountered in 38-70% of these mice. Offspring of mothers given an infusion of 20 μg DES/day had low incidences of ADL (11%) and UST (22%), whereas offspring of mothers infused with 200 μg DES/day exhibited high incidences of ADL (71%) and UST (86%). In offspring of mothers given injections of 2000 μg DES/day, ADL appeared in the fornical and upper-vaginal epithelia as early as 15 days of age. Thus, the present study revealed that ADL and UST occur at a high incidence in infantile mice exposed prenatally to high doses of DES. Previous studies failed to detect such effects perhaps due to the strain of mice, methods, duration, and/or dose of DES used.

Postnatal Development of Uterine Abnormalities in Mice Exposed to DES in Utero

Postnatal development of uterine abnormalities was examined in the offspring of mice given 4 daily subcutaneous injections of 2,000 micrograms diethylstilbestrol (DES) and of oil vehicle alone from days 15 to 18 of gestation. The prenatally DES-exposed mice (DES mice) exhibited a disorganization of the inner circular musculature of the uterus as early as at 5 days of age. Invaginations of the luminal epithelium into the stroma which began to form in 5-day-old control and 10-day-old DES mice developed into uterine glands at 10 and 15 days, respectively. The number of glands per uterine section was significantly reduced in DES mice as compared with the controls at 10-30 days. Stratification with or without squamous metaplasia (ST) was found in the luminal epithelium of DES mice at 20 days. Five groups each of DES mice and the controls ovariectomized (OVX) at 10 days were given 5 daily subcutaneous injections of 10(-4), 10(-3), 10(-2), 10(-1), and 1 microgram 17 beta-estradiol (E2) starting at 25 days. ST was never observed in OVX mice given prepubertal injections of any E2 dose, whereas in DES OVX mice aged 30 days, ST was produced by prepubertal injections of E2 doses higher than 10(-2) micrograms/day. Uterine responsiveness, weight gain and gland formation were significantly reduced in DES mice given prepubertal E2 injections compared to the controls.

Growth of mouse vaginal epithelial cells in culture: Functional integrity of the estrogen receptor system and failure of estrogen to induce proliferation

Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 40-day-old BALB/cCrg1 mice were purified by Percoll density gradient centrifugation and grown in primary culture using a collagen gel matrix and a serum-free complete medium. During the 9-day culture period, a 6-fold increase in cell number was observed. Addition of estrogen to the medium did not enhance epithelial cell proliferation. In fact, all doses of estrogen (180 fM to 18 nM) were inhibitory, resulting in only a 3- to 4-fold increase in cell number by day 9 of culture. Continuous exposure to estradiol (1.8 nM) for 9 days in the serum-free complete medium resulted in a decrease in cytosolic estrogen receptors with associated nuclear accumulation of estrogen receptors. A corresponding increase of cytosolic progestin receptors was also observed, indicating that no qualitative modification of the estrogen receptor system had occurred. Thus, despite its effectiveness in specific product synthesis (progestin receptors), estrogen does not stimulate proliferation of vaginal cells in this culture system, but rather inhibits epithelial cell proliferation.

Abnormal Development of the Os Penis in Male Mice Treated Neonatally with Tamoxifen

The os penis of male C57BL/Tw mice given 5 daily injections of 100 micrograms tamoxifen (Tx) starting on the day of birth (day 0) was examined at ages of 5-60 days; the bones of males given Tx injections for 5 days starting at 0-10 days and of those given neonatal injections of 100 micrograms clomiphene or nafoxidine were examined at 60 days. In the control males given the vehicle alone, the proximal segment of the os penis, composed of a compact cell mass found at day 0, developed at 5 days into the membrane bone with bone marrow and hyaline cartilage; the distal segment, composed of mesenchymatous cells until 10 days, developed at 30 days into fibrocartilage characterized by a distribution of type I collagen. By contrast, in Tx mice, fibrocartilage in the distal segment, and hyaline cartilage characterized by a distribution of type II collagen, and bone marrow in the proximal segment disappeared by 30 days. The maximum area of the proximal and distal segments gradually increased with age in control mice, whereas the proximal segment area remained unchanged in Tx mice. In clomiphene and nafoxidine mice at 60 days, the proximal segment was composed of hyaline cartilage; however, the distal segment lacked fibrocartilage. Hyaline cartilage in the proximal segment and fibrocartilage in the distal segment disappeared in all 60-day-old mice given Tx starting within 5 days. Neonatal castration did not suppress the formation of bone marrow and fibrocartilage in the os penis, though the bone size was smaller than in the intact controls. Formation of spines on the glans penis skin was suppressed by Tx given within 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth of mouse vaginal epithelial cells in culture: Effect of sera and supplemented serum-free media

SummaryNormal mouse vaginal epithelial cells isolated from ovariectomized ca. 35-d-old BALB/cCrgl mice were grown in primary culture using collagen gel metrix and a serum-free medium composed of a 1∶1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 (D:H) medium supplemented with insulin (IN), epidermal growth factor (EGF), cholera toxin, transferrin, and bovine serum albumin V (BSA). Three-dimensional cellular outgrowths occurred inside the collagen gel matrix. The contribution of each factor to cell growth was examined by individual addition to the basic D:H medium and by individual deletion from the complete serum-free medium. When added individually, only IN promoted growth. Deletion of IN from the complete serum-free medium markedly, diminished growth; deletion of EGF or BSA slightly diminished growth. When horse, fetal bovine, or chicken serum was added to the basal D:H medium, only with increasing doses of horse serum was there enhanced cell growth. The effect of 17ß-estradiol and diethylstilbestrol on the growth of cells was also tested, using a suboptimal medium of D:H supplemented with BSA and IN, or a minimal medium supplemented with IN alone. During the 8-d time period, addition of estrogen did not enhance cell growth in either medium. To date, we have been unable to demonstrate a mitogenic effect of estrogen; rather a dose-dependent inhibition of proliferation is seen.

Changes in the uterus and vagina of mice treated neonatally with antiestrogens

Adenosis-like lesions (ADL) were found in the upper vagina including the cervical region of ICR mice as early as 1 day after 5 daily injections of 200 micrograms tamoxifen (Tx) starting on the day of birth (200-micrograms Tx mice). ADL developed progressively from 5 to 30 days of age. Involution of the musculature and suppression of the gland genesis occurred in the uterus of 5- to 30-day-old, 200-micrograms Tx mice. The uterine abnormality resulted in a loss of type III collagen and laminin, and in an increase in fibronectin and type I collagen in the mesenchymal stroma of 15-day-old, 200-microgram Tx mice. Vaginal ADL and uterine myometrial involution were also encountered in 35-day-old mice given neonatal injections of 2-200 micrograms Tx, 200 micrograms clomiphene (Clm) and 200 micrograms nafoxidine (Naf), respectively. These changes were never observed in control and 0.2-microgram Tx mice. Ovariectomy performed at 10 days resulted in a reduction in the weight of uteri and the extent of ADL occupancy in Tx, Clm and Naf mice. Five daily injections of 0.1 microgram 17 beta-estradiol (E2) beginning at 30 days of age increased uterine weight and ADL occupancy extent in Tx (2-200 micrograms), Clm and Naf mice. The present findings suggest, therefore, that the antiestrogens act as an estrogen agonist to mouse uterus and vagina and that the induced ADL develops progressively depending on ovarian estrogen.

Estrogen participation in induction of cervicovaginal adenosis-like lesions in immature mice exposed prenatally to diethylstilbestrol.

A high incidence of vaginal adenosis-like lesions (ADL, 67%) was found in 30-day-old offspring of ICR/JCL mice given 4 daily subcutaneous injections of 2,000 micrograms diethylstilbestrol (DES) on days 15-18 of gestation. The prenatally DES-exposed mice and the controls ovariectomized at 10 days of age were given 5 daily subcutaneous injections of 10(-4), 10(-3), 10(-2), 10(-1), or 1 micrograms 17 beta-estradiol (E2), respectively, starting at 25 days of age. ADL was never observed in the age-matched controls given prepubertal injections of any E2 dose. In contrast, high incidences of ADL (63-100%) were found in the vaginal fornix and upper vagina of 30-day-old DES mice receiving prepubertal injections of 10(-3)-1 micrograms E2/day, whereas in DES mice given prepubertal injections of the vehicle alone and a lower daily dose of 10(-4) micrograms E2, incidences were nil and 25%, respectively. Mitotic figures were found in the columnar epithelium with ADL, although the rate was lower than in the ADL-freed stratified epithelium. It is suggested, therefore, that an ovarian hormone (probably estrogen) participates in the prepubertal induction of ADL in DES mice.