Yoshito Ibuki

CLONING AND SEQUENCING OF HUMAN LH/HCG RECEPTOR CDNA

We have isolated and sequenced a cDNA encoding the human luteinizing hormone--choriogonadotropin (LH/hCG) receptor. The deduced amino acid sequence (699 residues) containing seven putative transmembrane segments displays sequence similarity to G protein-coupled receptors. The receptor consists of 335 residue extracellular domain which contains six N-linked glycosylation sites. While the protein is 85 and 87% identical overall with the previously cloned rat and porcine LH/hCG receptor respectively, the most highly conserved regions are the putative transmembrane segments (91 and 94% similarity, respectively).

Cloning and sequencing of human FSH receptor cDNA

We have isolated and sequenced a cDNA encoding the follicle stimulating hormone (FSH) receptor. The deduced amino acid sequence (678 residues) containing seven putative transmembrane segments which displays sequence similarity to G protein-coupled receptors. The receptor consists of 359 residue extracellular domain which contains four N-linked glycosylation sites. While the protein is 89% identical overall with the previously cloned rat FSH receptor, the most highly conserved regions are the putative transmembrane segments (95% similarity).

A Comparative Study on Transforming Growth Factor-β and Activin A for Preantral Follicles from Adult, Immature, and Diethylstilbestrol-Primed Immature Mice

Both transformation growth factor-β (TGFβ) and activin belong to the TGFβ superfamily, and each receptor is structurally related. We have shown that the action of activin A on folliculogenesis is different in immature and adult mice, so it is of interest to study whether TGFβ has such an action on follicular development. The effect of TGFβ on folliculogenesis was studied in isolated preantral follicles from immature, adult, and diethylstilbestrol (DES)-primed immature mice and was compared with that of activin A. TGFβ caused a significant increase in follicular diameter and estradiol and immunoreactive inhibin secretion in adult mice in a dose-related manner, but did not affect the size of preantral follicles from immature mice. Activin A, on the other hand, caused a significant increase in the size of follicles from immature mice, but did not change the size of preantral follicles from adult mice. TGFβ enhanced the effect of FSH, whereas activin A completely blocked the action of FSH on preantral follicl...

Regulation of inhibin production by rat granulosa cells

Inhibin production by cultured granulosa cells from immature diethylstilbestrol (DES)-primed rats was studied in relation to estradiol and progesterone production. The inhibin content in culture media was assayed with a specific radioimmunoassay (RIA) using an antibody to porcine 32 kDa inhibin that recognizes rat inhibin as well. Inhibin production was about 10 ng/ml/2 X 10(4) cells/72 h at the basal levels and was maximally stimulated with 25 ng/ml of follicle stimulating hormone (FSH) to 45 ng/ml which was 4.5 times the basal levels, with an ED50 value of 2.0 ng/ml. A cyclic AMP analog (dibutyryl cyclic AMP) or reagents that promote cAMP production were also effective in inhibin production, indicating that FSH stimulates inhibin production through a cAMP-dependent pathway. Luteinizing hormone (LH) was not effective in producing inhibin from freshly prepared granulosa cells, whereas granulosa cells pre-incubated with FSH for 48 h because responsive to LH regarding inhibin production. Testosterone sensitized the granulosa cells to the FSH stimulation, whereas hydrocortisone (4 ng/ml) decreased the sensitivity of granulosa cells by increasing the ED50 value for inhibin production by FSH about 10 times. A similar effect was observed regarding estradiol production, while progesterone production due to stimulation by FSH was enhanced by the hydrocortisone treatment. Insulin and platelet extract both stimulated inhibin production and enhanced the maximal response of inhibin production due to stimulation by FSH without altering, or even increasing the ED50 values. Epidermal growth factor (EGF), (D-Leu6)Des-Gly10-LHRH N-ethylamide (GnRH agonist) and 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator, inhibited both inhibin production and estradiol or progesterone production. Consequently, the regulation of inhibin production was similar to that of estradiol production, but markedly different from that of progesterone. However, inhibin and estradiol production were modulated differently by various growth factors and hormones. These phenomena might account for possible discrete changes in the plasma levels of inhibin and estradiol in vivo.

Hormonal regulation of gonadotropin receptor mRNA in rat ovary during follicular growth and luteinization

To investigate the regulation of the follicle-stimulating hormone (FSH) and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor genes by gonadotropins, we examined the effect of pregnant mares serum gonadotropin (PMSG) or PMSG-hCG on the expression of FSH and LH/hCG receptors in rat ovaries. After administration of PMSG, Northern blot analysis using the FSH receptor cDNA probe revealed that a major band of 2400 nucleotides was detected which reached the maximal level on day 3. On the other hand, the level of LH/hCG receptor mRNA, a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300 and 1200 nucleotides, increased progressively during 4 days. Treatment with hCG resulted in a decrease of FSH and LH/hCG receptor mRNA levels, and the level of FSH receptor mRNA was completely suppressed. Although the level of LH/hCG receptor mRNA was also suppressed from 3 h to an almost undetectable level at 24 h after hCG injection, it recovered to the control level by 48 h and exceeded this level several fold by 72 h. The reappearance of LH/hCG receptors following desensitization was preceded by an increase in mRNA levels. These studies demonstrate that hormonal regulation of gonadotropin receptor mRNAs on rat ovary reflects the changes in gonadotropin receptor levels.

Prevention of postmenopausal bone loss with minimal uterine bleeding using low dose continuous estrogen/progestin therapy: a 2-year prospective study

OBJECTIVE To re-examine the minimal effective dose of conjugated estrogen (CEE)-progestin hormone replacement on postmenopausal bone loss. DESIGN A 2-year, prospective, open label, randomized study. SETTING Department of Obstetrics and Gynecology of a university hospital. PARTICIPANTS Fifty-two postmenopausal or oophorectomized women. INTERVENTION One of the following regimens was continuously administered for 2 years: (1) CEE 0.625 mg/day, (2) CEE 0.625 mg + medroxyprogesterone (MPA) 2.5 mg/day, (3) CEE 0.31 mg + MPA 2.5 mg/day and (4) control. MEASUREMENTS Lumbar spine and femoral BMD by dual energy X-ray absorptiometry (DXA), a monthly based incidence of bleeding, serum lipids, PTH, calcitonin. A1-p, and osteocalcin. RESULTS Of the 52 patients enrolled in this study, 49 patients completed the 1 year of therapy and 36 completed the 2- year study. The control group showed a significant decrease in lumbar BMD over the 2 years (P < 0.05). The % changes in lumbar BMD at 2 years of CEE alone, CEE 0.625 + MPA and CEE 0.31 + MPA were 8.52% (95% confidence intervals; 4.61 approximately 12.4%), 7.4% (0.60 approximately 14.2%) and 3.20% (0.61 approximately 5.84%), respectively, and were significantly higher than pretreatment values. The incidence of bleeding was significantly lower in women taking CEE 0.31 mg + MPA. HDL cholesterol increased in women taking CEE 0.625 mg alone or with MPA. No significant changes in lipid profiles were seen in the control or in the group of women taking CEE 0.31 mg + MPA. CONCLUSIONS Continuous hormone replacement therapy (HRT) using 0.31 mg of CEE and 2.5 mg of MPA is effective in increasing lumbar BMD in postmenopausal or oophorectomized women and can be an appropriate option for women with a normal lipid profile or those women wishing to eliminate unscheduled bleeding.

Do assisted reproductive technologies have effects on the demography of monozygotic twinning

This study examined the incidence of monozygotic twinning of one embryo with the use of cycles in which only one embryo, treated by various assisted reproductive technologies (ART), was transferred. It also examined the possibility of demographic alterations in the overall incidence of monozygotic twinning in Japan induced by ART. A total of 134 clinics or institutes participated in this study. Overall, it is noted that the incidence of monozygotic twinning in Japan has been almost constant (0.402%). In ART treatments, the monozygotic twinning rate was significantly higher than that of natural pregnancies (P = 0.0204). For in vitro fertilization (IVF) treatment cycles, the monozygotic twinning rate was also higher than that of natural pregnancies (P = 0.6285). However, this difference was not statistically significant because of the small number of IVF pregnancies. Moreover, in the context of microinsemination cycles, the monozygotic twinning rate was significantly higher than that of natural pregnancies (P = 0.0285) or IVF (P = 0.0006). In terms of the possible impact of ART on the demography of monozygotic twinning, adoption of mechanically assisted hatching for all embryos in Japan may alter the demography of monozygotic twinning.

Mode and site of action of clomiphene

Abstract Direct action of clomiphene citrate upon the hypothalamus, especially on the median eminence and the anterior hypothalamus was demonstrated by the increase of plasma and pituitary LH following the stereotaxic implantation of clomiphene into the rat brain. Subcutaneous 5 day injection of clomiphene also increased both LH release and synthesis in rats. FSH release from the pituitary increased transiently, but FSH synthesis was not simulated following both the subcutaneous injection and the intra-hypothalamic implantation of clomiphene. These results indicate the mode and site of action of clomiphene citrate in the induction of human ovulation.

Effect of Estriol on Bone Loss in Postmenopausal Japanese Women: A Multicenter Prospective Open Study

Objectives: To assess the effects of oral estriol on the bone mineral density (BMD) and bone metabolism in postmenopausal women.

Expression of follicle-stimulating hormone receptor in human ovary

The gonadotropins, follicle‐stimulating hormone (FSH) and luteinizing hormone (LH), are key hormones in the regulation of ovarian function. The acquisition of FSH receptors during folliculogenesis is believed to be a key event in the subsequent development of the follicle. However, the binding and biochemical properties of the human FSH receptor are not well‐characterized owing to the low abundance of these receptors and the limited availability of human tissue. The binding experiments show that, while the affinity of the FSH receptor does not change through the menstrual cycle, the total number of FSH receptors in the leading follicles increases by about two‐fold at mid‐follicular phase compared with those at other periods. Northern blot analysis was used to measure relative levels of FSH receptor mRNA, and in situ hybridization was used to localize FSH receptor transcripts. Northern blot analysis of human ovaries detected two transcripts (4.1 and 2.4 kb) for the FSH receptor. The FSH receptor mRNA was abundant in the preovulatory follicle, with expression decreased by about 50% in the corpus luteum. Using in situ hybridization, FSH receptor mRNA was found to be confined to the granulosa cells of developing follicles. A reverse transcription–polymerase chain reaction amplification was used to detect the expression of different isoforms of the FSH receptor mRNA in human corpus luteum and placenta.