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T. Sawada


Differences in the secretion of pregnane compounds by follicular polycystic ovaries of androgen-sterilized rats and by normal preovulatory ovaries of early prooestrous rats were compared. Some rats were injected i.v. with LH 30 min before bleeding, in order to stimulate the secretion of steroids. This injection of LH greatly increased the secretion of progesterone, 5α-pregnane-3,20-dione and 3α-hydroxy-5α-pregnan-20-one by both types of ovaries. The response of the two progesterone metabolites in the polycystic ovaries was low, suggesting low 5α-reductase activity.

Because it is known that the preovulatory LH surge is absent in androgen-sterilized rats, a classical approach was taken to circumvent the probable deficit in cyclic release of LH by giving an i.v. injection of LH (25 μg) every 4 days for 16 days. Ovarian venous blood was collected 4 days after the last injection. The mean secretion of 5α-pregnane-3,20-dione and 3α-hydroxy-5α-pregnan-20-one from the ovaries of such androgen-sterilized rats became much (P <0·01) higher than that of multiple saline-treated controls. These results suggest that low 5α-reductase activity of polycystic ovaries in androgen-sterilized rats may be due to the absence of cyclic release of LH from the pituitary gland.

J. Endocr. (1986) 110, 507–510

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T Sawada, K Oofusa, and K Yoshizato

Gene (Rmmp1) of matrix metalloproteinase 1 (MMP1) of the bullfrog, Rana catesbeiana, has been shown previously to contain a thyroid hormone response element (TRE)-like sequence in its 5'-upstream region. The present study aimed to determine whether this TRE-like sequence is functional in vivo as a true TRE, and to characterize the sequences of the 5'-upstream region with respect to the regulation of the activity of the TRE when the TRE-like sequence was proved to be a true TRE. With this aim, various sequences of TRE-like sequence-containing 5'-upstream region were constructed and fused to the enhanced green fluorescent protein gene as a reporter gene. The fusion constructs were bombarded to the skin of bullfrog tadpoles and the activity of the TRE was quantitatively determined by measuring increased intensities of fluorescence when the animals were exposed to thyroid hormone. The present study clearly demonstrated that the sequence of Rmmp1 is a biologically active TRE in vivo. In addition, a unique 36 bp long sequence directly flanked to the 3'-end of the TRE was identified which worked co-operatively with TRE to regulate the transcriptional promoter activity. It should be emphasized that the presence of TRE in the Rmmp1 gene is unique, because its presence has not been reported in the known promoter region of vertebrate MMPs.

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H Tamada, Y Shimizu, T Inaba, N Kawate, and T Sawada

It is well known that progesterone and estrogen are essential hormones for maintaining pregnancy in most mammals. Some specific roles of progesterone for the maintenance of pregnancy have been clarified, but the role of estrogen is not well known. This study examines the effects of the aromatase inhibitor, fadrozole hydrochloride (Fad), on fetuses, uterine physical properties and the mRNA expression of the uterine enzymes that are related to collagen metabolism during late pregnancy in rats. Continuous s.c. infusion with 300 micro g/day Fad from day 14 of pregnancy (day 1=the day of sperm detection) reduced the concentration of plasma estradiol-17beta (E(2)), and did not change that of plasma progesterone, compared with controls. The treatment increased the intrauterine pressure and reduced the size and compliance of the uterine tissue framework. It also caused injuries (hematomata on the extremities) in about one-quarter of fetuses by day 20. The collagen content of the uterine ampullae was not changed by the treatment. Uterine mRNA expressions of matrix metalloproteinase-1 (MMP-1), which degrades collagens, and of lysyl oxidase (LO), which is necessary for the formation of intra- and inter-molecular cross-links of collagen, were examined by quantitative RT-PCR. The treatment with Fad had no effect on the expression of MMP-1 mRNA and increased that of LO mRNA. Daily s.c. injection with 0.2 micro g E(2) restored the changes in uterine physical properties and the mRNA expression of LO caused by the Fad treatment, and prevented fetal injury, indicating that the influences of Fad treatment are due to estrogen deficiency but not to toxicological effects of Fad. These results imply that estrogen deficiency during late pregnancy in rats obstructs development of the uterine tissue framework so as to cause fetal injury. It is possible that an increase in the uterine expression of LO gene may be involved in this obstruction.

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M Yamaguchi, L Ogren, R Barnard, T Imai, T Sawada, A Miyake, and F Talamantes


The placental members of the prolactin-GH-placental lactogen (PL) gene family of the mouse include mPL-I, mPL-II, proliferin (PLF) and proliferin-related protein (PRP). The aim of the present study was to assess the effects of tumour necrosis factor-α (TNF-α) on the secretion of these proteins in primary cultures of placental cells from days 7, 9 and 12 of pregnancy. The effects of epidermal growth factor (EGF) on the secretion of PLF and PRP were also determined. EGF has previously been shown to stimulate mPL-I and inhibit mPL-II secretion. Incubation of placental cells from day 7 of pregnancy for 5 days with 10 nmol human (h)TNF-α/1 did not affect the mPL-II concentration of the medium, but similar treatment of cells from days 9 or 12 of pregnancy resulted in a significant reduction in the mPL-II concentration of the medium by the second or third day of culture. The intracellular concentration of mPL-II, the number of cells that released mPL-II as assessed by reverse haemolytic plaque assay, and steady-state levels of mPL-II mRNA as assessed by Northern analysis were also reduced by hTNF-α treatment. The lowest concentration of hTNF-α that significantly inhibited mPL-II secretion by cells from day 12 of pregnancy was 0·01 nmol/l. hTNF-α treatment did not affect the secretion of mPL-I, PLF or PRP, as assessed by the concentrations of these proteins in the medium during a 5-day incubation. Incubation of the cells with 20 ng EGF/ml also did not affect the PLF or PRP concentration of the medium during 5 days of culture. To determine whether the effect of hTNF-α on mPL-II secretion was mediated by interleukin-6 (IL-6), the IL-6 concentration of the medium of control and hTNF-α-treated cells was determined. Bioactive and immuno-reactive IL-6 could not be detected in medium from either treatment group. The presence of binding sites for hTNF-α was assessed in cells from day 12 of pregnancy. Scatchard analysis detected a single class of binding sites having a Kd of 1·61±0·34 nmol/l, with about 1350 sites per cell. The results of this study demonstrate that hTNF-α inhibits the secretion of mPL-II by placental cells from days 9 and 12 of pregnancy, suggesting that TNF-α may be one of the factors that regulate the production of this hormone in vivo.

Journal of Endocrinology (1994) 143, 95–105