Yugo Fukazawa

Apoptotic cell death during the estrous cycle in the rat uterus and vagina

Rodent uterus and vagina show marked histological changes during the estrous cycle. Apoptotic cell death has been demonstrated in hamster and rat uterine epithelium during the estrous cycle by electron microscopy: Numerous epithelial cells undergo apoptosis at estrus. We examined cell death and cell proliferation in rat uterus and vagina during estrous cycle.

Effects of estrogenic hormones on early development of Xenopus laevis

Many chemicals released into the environment have estrogenic activity and can disrupt animal development and the function of endocrine systems. In order to study the effects of estrogens on aquatic animals, we examined the effects of certain estrogens on early development in Xenopus laevis. X. laevis embryos were kept in water containing 10(-10), 10(-9), 10(-7), 10(-6), and 10(-5) M 17 beta-estradiol (E2); 17 alpha-estradiol; diethylstilbestrol (DES); 10(-5) M progesterone (P); or dihydrotestosterone (DHT) beginning at developmental stage 3. Survival rates of the embryos developed in water containing 10(-10)-10(-6) M E2 or DES, all concentrations of 17 alpha-estradiol, and 10(-5) M P or DHT, which were over 70% after stage 48, whereas the rates of the embryos treated with 10(-5) M E2 and DES decreased remarkably after stage 27 and all embryos were dead by stages 42 and 32, respectively. Embryos treated with 10(-5) M E2 showed malformations of the head and abdomen and suppressed organogenesis, including crooked vertebrae at stage 38; the head was smaller and the abdomen was larger than in the controls. Similar effects were observed in embryos developed in 10(-5) M DES but not in 10(-5) M 17 alpha-estradiol, P, or DHT. After 10(-5) M E2 treatment, abnormalities were induced only when the treatment was started before stage 39. However, on day 30 after fertilization, the stage of the embryos treated with 10(-6) M E2 was more progressed than that of the controls. Estrogen receptor (ER 4) mRNA was examined in eggs, embryos, and adult female liver by reverse-transcription polymerase chain reaction. ER4 mRNA was expressed in adult liver, unfertilized and fertilized eggs, and embryos, but ER3 mRNA was not expressed. ER4 mRNA in 10(-6) and 10(-5) M E2-treated embryos showed different expression patterns, which may result from the diverse developmental effects of E2. The present results demonstrate that 10(-5) M E2 and DES induced embryo death and malformations and that ER may be involved in the induction of various developmental defects in X. laevis embryos.

Effect of Estrogen on Ontogenic Expression of Progesterone and Estrogen Receptors in Rat Uterus

Abstract The ontogenic expression of progesterone and estrogen receptors (PR and ER) and effect of estrogen on these receptors were investigated immunohistochemically in rat uterus from the day of birth (=0 day) to 30 days of age. Uterine epithelial and stromal cells showed a negative PR immunoreaction at 0 day. The PR in the epithelial cell nuclei appeared by 5 days, while the stromal cells showed a negative PR reaction until 12 days. The staining of the stromal cells appeared from 12 to 15 days. In both the epithelial and stromal cells, the initiation of the PR appearance was not affected by ovariectomy performed at 0 day or 5 days prior to the appearance of PR in the epithelial and stromal cells. Estrogen injections from 0 day failed to initiate the appearance of PR in the epithelial cells, regardless of doses of estradiol-17&bgr;(0.1, 1 and 10&mgr;g daily), but induced PR in the stromal cells. The staining of ER appeared at 5 days in the epithelial cells and at 1 day in the stromal cells, respectively. ER appeared after 2–3 daily injections of estrogen from 0 day depending upon the doses. These results suggest that steroid hormones secreted from neonatal ovary do not play any important role in ontogenic expression of PR during the postnatal uterine maturation.

Effect of Neonatal Exposure to Diethylstilbestrol and Tamoxifen on Pelvis and Femur in Male Mice

Permanent abnormalities have been reported in reproductive and non‐reproductive organs of mice and humans exposed perinatally to a synthetic estrogen, diethylstilbestrol (DES). Recent studies demonstrated that sex hormones affected the shape of the innominate bone in mice. Therefore, we analyzed the long‐term effects of neonatal exposure of DES and tamoxifen, an anti‐estrogen, in mouse bones.

Effects of Steroid Hormones and Growth Factors on the Development of the Male Mouse Reproductive Tract In Vitro

Abstract In order to determine what growth-promoting factors may be involved in androgen-induced male reproductive tract development, the newborn mouse urogenital sinus region with seminal vesicles attached was cultured for 5 days on collagen gel matrix in a serum-free medium composed of DMEM and Hams F-12 (1:1) supplemented with BSA, insulin, cholera toxin and transferrin. Testosterone and 5 α-dihydrotestosterone (DHT) stimulated development of seminal vesicle (SV), coagulating gland (CG), prostate (P) and bulbourethral gland (BG). Epidermal growth factor (EGF) stimulated development of CG, P and BG, but inhibited that of SV. Transforming growth factor-α (TGF-α) inhibited SV development, but had stimulatory effects on both CG and P. Addition of anti-EGF antibody significantly inhibited the DHT-induced development of CG and BG, but not of SV and P. These findings suggest that both EGF and TGF-α have organspecific regulatory actions on male reproductive tract development and that EGF may mediate the action of DHT in the development of CG and BG.

Involvement of the TNF-α system and the Fas system in the induction of apoptosis of mouse mammary glands after weaning

Mammary gland involution after cessation of milk production is associated with extensive loss of secretory epithelial cells. In order to study the mechanism of mammary gland involution, litters were removed on day 20 of lactation and morphological and biochemical changes were examined in GR/A mice andlprcg mice lacking functional Fas. DNA fragmentation occurred 24 h after weaning both in GR/A mice andlprcg mice indicating that apoptotic cell death occurs during involution of mammary glands.In situ 3′-end labelling method revealed apoptotic cells in epithelial cells lining alveolar lumens and in cells shed into the alveolar lumen. The number of apoptotic cells plateaued on day 2 of weaning in mammary glands of GR/A mice. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that expression of bcl-2, Fas ligand, keratinocyte growth factor (KGF) and transforming growth factor-β1 (TGF-β1) increased on day 1 of weaning in the process of involution of mammary glands in GR/A mice. In mammary glands oflprcg mice, RT-PCR showed that expression of bcl-2, Fas ligand, KGF, tumour necrosis factor-α(TNF-α) and TGF-βs increased in the process of involution. These results suggest that the Fas ligand, TGF-β1 and TNF-α are involved in the involution of mammary glands after weaning. TNF-α and anti-Fas antibody directly killed more than 80% of mammary cells from p53 knockout micein vitro within 24 h in the presence of actinomycin D, supporting the hypothesis that Fas and/or TNF-α are involved in the induction of apoptosis of mouse mammary glands.