Kazuko Abe

A Role of Insulin-Like Growth Factor I for Follicle-Stimulating Hormone Receptor Expression in Rat Granulosa Cells

Abstract The present study was undertaken to identify the mechanisms underlying the effect of insulin-like growth factor I (IGF-I) on FSH receptor (FSHR) in rat granulosa cells. Treatment with FSH produced a substantial increase in FSHR mRNA level, as was expected, while concurrent treatment with increasing concentrations of IGF-I brought about dose-dependent increases in FSH-induced FSHR mRNA, with a maximal response 2.8-fold greater than that induced by FSH alone. IGF-I, either alone or in combination with FSH, did not affect intracellular cAMP levels, whereas it enhanced the effect of 8-bromo (Br)-cAMP on FSHR mRNA production. Taken together, these findings suggest that the ability of IGF-I to enhance FSH action concerning the induction of FSHR is exerted at sites distal to cAMP generation. We then investigated whether the effect of IGF-I and FSH on FSHR mRNA levels was the result of increased transcription and/or altered mRNA stability. The rates of FSHR mRNA gene transcription, assessed by nuclear run-on transcription assay, were not increased by the addition of IGF-I. On the other hand, the decay curves for the 2.4-kilobase (kb) FSHR mRNA transcript in primary granulosa cells significantly altered the slope of the FSHR mRNA decay curve in the presence of IGF-I and increased the half-life of the FSHR mRNA transcript. These data suggest a possible role for changes in FSHR mRNA stability in the IGF-I-induced regulation of FSHR in rat granulosa cells. Treatment with activin produced a substantial increase in FSHR mRNA level, as was expected, and concurrent treatment with IGF-I did not affect activin-induced FSHR mRNA. Our data suggest that the IGF-I effect on FSHR expression is related to cAMP production induced by FSH and may maintain FSHR mRNA level because of prolonged FSHR mRNA stability.

The mechanisms of retinoic acid-induced regulation on the follicle-stimulating hormone receptor in rat granulosa cells

The present study was undertaken to identify the mechanisms underlying the effect of retinoic acid (RA) on follicle-stimulating hormone receptor (FSH-R) in rat granulosa cells. Treatment with FSH produced a substantial increase in FSH-R mRNA level, as was expected, while concurrent treatment with increasing concentrations of RA brought about dose-dependent decreases in FSH-induced FSH-R mRNA, with a maximal inhibition one-third lower than that induced by FSH alone. RA, either alone or in combination with FSH, did not affect intracellular cAMP levels, while it inhibited the effect of 8-Br-cAMP on FSH-R mRNA production. These results suggested that RA diminished the action of FSH on FSH-R expression at sites distal to cAMP generation in the granulosa cells. Whether the effect of RA and FSH on FSH-R mRNA levels was the result of decreased transcription and/or altered mRNA stability was also investigated. The rate of FSH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, was found to decrease by the addition of RA. On the other hand, the decay curves for the 2.4 kb FSH-R mRNA transcript in primary granulosa cells did not alter the slope of the FSH-R mRNA decay curve in the presence of RA. Our data suggests for the first time that the effect of RA on FSH-R expression is possibly mediated by the reduction of the FSH-R mRNA level due to a negative regulation of the FSH-R gene in the presence of FSH. These findings assist in understanding the molecular mechanism underlying the effect of RA on reproductive function in rat granulosa cells.

Effect of Transforming Growth Factor β on the Expression of Luteinizing Hormone Receptor in Cultured Rat Granulosa Cells

Abstract The present study was undertaken in order to identify the mechanism underlying the effect of transforming growth factor-β (TGFβ) on LH receptor (LH-R) expression in rat granulosa cells. Treatment with FSH produced a substantial increase in LH-R mRNA level, and concurrent treatment with increasing concentrations of TGFβ brought about dose-dependent increases in FSH-induced LH receptor mRNA. TGFβ, either alone or in combination with FSH, did not affect intracellular cAMP levels. We then investigated whether the effect of TGFβ and FSH on LH-R mRNA levels results in increased transcription and/or altered mRNA stability. To determine whether the LH receptor 5′-flanking region plays a role in directing LH receptor mRNA expression, the proximal area of the LH receptor 5′-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. FSH (30 ng/ml) significantly enhanced the activity of 1389 base pairs of the LH receptor 5′-flanking region, but treatment with TGFβ did not significantly influence the activity induced by FSH. On the other hand, the decay curves for LH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFβ.

Expression of adrenomedullin in the endometrium of the human uterus.

OBJECTIVE Our purpose was to determine whether adrenomedullin is present in the human uterus. METHODS We obtained specimens from five patients with leiomyoma and four patients with endometrial adenocarcinoma who were undergoing abdominal hysterectomy. Tissue sections were stained with antibody against adrenomedullin using an avidin-biotin-peroxidase complex technique. Total RNA from normal and cancerous endometrium, myometrium, and leiomyoma was prepared and analyzed by northern blot methodology. RESULTS Sections stained with an antibody to adrenomedullin showed prominent expression of adrenomedullin in the normal and cancerous endometrial epithelial cells. Northern blot analysis showed that normal and cancerous endometrium expressed the messenger RNA (mRNA) for adrenomedullin and that the size of the mRNA transcript was 1.6 kilobases (kb). No significant difference was observed between the abundance of mRNA in normal and cancerous endometrium. CONCLUSION The findings in this study demonstrated that both adrenomedullin protein and mRNA are present in normal and carcinomatous endometrium of the human uterus.

Expression of adrenomedullin in the human corpus luteum

OBJECTIVE Adrenomedullin (AM) is a potent hypotensive peptide found in human pheochromocytoma tissue. In the present study, the expression of AM mRNA in the human ovary was examined. DESIGN Ovarian mRNA was analyzed in the follicle, the corpus luteum of mid-luteal phase, and early pregnancy. SETTING Gunma University School of Medicine, Gunma, Japan. PATIENT(S) Premenopausal women with histologically normal ovary who were undergoing salpingoophorectomy. INTERVENTION(S) The dominant follicle and corpora lutea were isolated and total RNA was extracted from these tissues. MAIN OUTCOME MEASURE(S) Northern blot analysis of AM, receptor activity-modifying protein 2 (RAMP2), and LH/hCG receptor mRNA in human samples. RESULT(S) An AM mRNA transcript of 1.6 kilobases (kb) was detected in corpus luteum tissue; this transcript was identical to that which has been detected in placenta and fetal membrane. The AM and LH/hCG receptor mRNA levels were low in the mature follicle but increased in the corpus luteum of the mid-luteal phase and were maintained during early pregnancy. A single transcript of 0.8 kb for RAMP2 was also seen in the follicle and corpus luteum, the level of RAMP2 mRNA was relatively high in the preovulatory follicle and RAMP2 was present in the corpus luteum. CONCLUSION(S) The expression of AM, its receptor, and LH/hCG receptor may be an important component in the process of development and differentiation of the corpus luteum.

Mechanisms of Action of Transforming Growth Factor β on the Expression of Follicle-Stimulating Hormone Receptor Messenger Ribonucleic Acid Levels in Rat Granulosa Cells

Abstract The present study was undertaken to identify the mechanisms underlying the effect of transforming growth factor (TGF) β on FSH receptor (FSH-R) in rat granulosa cells. Compared to the control, the treatment of granulosa cells with TGFβ (10 ng/ml) increased FSH-R mRNA transcripts (5.5 and 2.4 kilobases) in a time-dependent manner, with a maximum increase of approximately 2-fold at 48 h. We then investigated whether the effect of TGFβ on FSH-R mRNA levels was the result of increased transcription and/or altered mRNA stability. To determine whether the FSH-R 5′-flanking region plays a role in directing FSH-R mRNA expression, the proximal area of the FSH-R 5′-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. The FSH (30 ng/ml) significantly enhanced the activity of 1862 base pairs of the FSH-R 5′-flanking region, but treatment with TGFβ did not significantly influence the activity induced by FSH. On the other hand, the decay curves for FSH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFβ. Transforming growth factor β stimulates the expression of follistatin mRNA accumulation in a dose- and time-dependent manner. Treatment with activin produced a substantial increase in FSH-R mRNA level. Concurrent treatment with follistatin neutralized this activin effect on FSH-R mRNA, as reported, although concurrent treatment with follistatin did not affect TGFβ-induced FSH-R mRNA. Therefore, the profile of the TGFβ effect on FSH-R mRNA granulosa cells may be caused by the increased stability of FSH-R mRNA and insensitivity to the follistatin.

Expression of steroidogenic acute regulatory protein (StAR) in rat granulosa cells

Steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein that is indispensable for the synthesis of steroids. To study the mechanisms of regulation of StAR in rat granulosa cells, we used granulosa cells obtained from diethylstilbestrol-treated immature rats. Northern blot analysis revealed two major transcripts of about 3.6 kb and 1.6 kb of rat StAR mRNA. Rat StAR mRNA had strongly increased within 2 h due to the treatment of FSH or 8-Br-cAMP in this culture, a parallel increase of transcripts of both sizes was observed. Compared to the control, StAR mRNA levels increased in a dose-dependent manner in the presence of increasing concentrations of FSH (1-100 ng/ ml) and 8-Br-cAMP (0.25-5 mM). Although co-treatment of rat granulosa cells with FSH and TGF-beta did not change FSH-induced StAR mRNA levels, these levels in granulosa cells were markedly increased by pretreatment with TGF-beta before being acutely (2 h) stimulated with an effective dose of FSH. The stimulatory effect of TGF-beta was time- and concentration-dependent (1-30 ng/ml).

Effect of IGF-1 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of LH receptors during cell differentiation in cultured granulosa cells

Ovarian granulosa cells undergo a complex differentiation process during the growth and maturation of ovarian follicle. This process includes the acquisition of cell surface LH receptor, which mediates the granulosa cells ability to respond to circulating LH. The results of the actions of LH on the mature granulosa cell include steroidogenesis, luteinization, and ovulation. As such, induction of the LH receptor in granulosa cells is a critical step in reproductive physiology. In the present study, we attempted to assess the effects of IGF-1 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on FSH-induced LH receptor expression in rat granulosa cells to understand the actions of these factors on normal reproductive function. Treatment with FSH, as expected, produced a substantial increase in LH receptor mRNA level, and concurrent treatment with increasing concentrations of IGF-1 brought about dose-dependent increases in FSH-induced LH receptor mRNA. On the other hand, the concurrent treatment of TCDD (10 pM) resulted in a significant decrease in LH receptor after 24 h. The decay curves for LH receptor mRNA transcript showed a significant increase in the half-life after the addition of IGF-1 and a significant decrease after addition of TCDD. These data suggests a possible role for changes in LH receptor mRNA stability in the IGF-1 and TCDD induced regulation of LH receptor in rat granulosa cells. The rates of LH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, were not increased by the addition of IGF-1, but decreased by the addition of TCDD. The data of IGF-1 present that the interface between circulating hormones and paracrine/autocrine systems could provide an important mechanism to amplify the effects of gonadotropin hormones at the local level. In addition, the endocrine-disrupting effects of TCDD are, at least in part, caused by direct action on the expression of LH receptor expression in granulosa cells.