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Translational Medical Center for Stem Cell Therapy & Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China
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Translational Medical Center for Stem Cell Therapy & Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China
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. 1971 b , Godinho et al . 2015 ). Extracellular cAMP mediates glucagon actions such as endocrine inhibition of sodium and phosphate reabsorption in the renal proximal tubule ( Ahloulay et al . 1996 ). Liver-borne extracellular cAMP is also able to
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Pituitary equine luteinizing hormone (eLH) and fetal chorionic gonadotrophin (eCG) have identical polypeptidic chains, but different linked carbohydrates. In equine tissues, eCG and eLH bind only to the LH/CG receptor (eLH/CG-R) and have no FSH activity. However, radio-receptor assays on equine luteal or testicular tissues have shown that eCG binds to the eLH/CG-R with only 2–4% of the binding activity of eLH. In order to study the structure–function relationship of eLH and eCG in a homologous sytem, we undertook the cloning and functional expression of the eLH/CG-R.
Based on sequence homologies among mammalian sequences for the LH/CG-R, overlapping partial fragments of LH/CG-R cDNAs were obtained from mare luteal RNA using reverse transcription-PCR and 5′-rapid amplification of cDNA ends. Ligations of the partial cDNA fragments encoded a part of the signal peptide followed by a putative 672 amino acid eLH/CG-R mature protein. The mature eLH/CG-R displayed 88.2–92.8% overall sequence homology with the other mammalian LH/CG-Rs and contained one unique seventh N-glycosylation site in its extracellular domain.
COS-7 cells were transiently transfected with a cDNA construct encoding an engineered complete signal peptide and the mature eLH/CG-R. Membrane preparations from transfected COS-7 cells bound 125I-eLH with high affinity (K d 3.8 × 10−10 M). On a molar basis, eCG competed with 125I-eLH on membrane preparations with only 12.4% of the eLH binding activity. In transfected COS-7, both eLH and eCG increased the extracellular cAMP concentration in a dose-dependent manner, whereas eFSH did not. Furthermore, on a molar basis, eCG stimulated cAMP production with only 13.9% of the eLH stimulating activity.
We conclude that the cloned cDNA encodes a The differences functional eLH/CG-R. between eLH and eCG activities towards this receptor will be useful in studies of the influence of carbohydrates on gonadotrophin receptor binding and activation.
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ABSTRACT
The effects of recombinant rat interferon-γ (rRaIFN-γ) and rat IFN (RaIFN, a mixture of IFN-γ and -α) on basal and FSH-induced ovarian granulosa cell function were studied. Granulosa cells were harvested from diethylstilboestrol-treated immature rats and cultured (2 × 105 viable cells/well per 0·5 ml) in serumfree medium with or without treatment for 48 h. In the presence of FSH (20 ng/ml), rRaIFN-γ (10–1000 U/ml) significantly inhibited FSH-stimulated aromatase activity (76·4 ± 2·3% maximum inhibition compared with FSH treatment alone), inhibin (40·4 ± 3·7%), progesterone (47·7 ± 8·6%) and 20α-hydroxypregn-4-en-3-one (20α-OHP) (51·8±1·7%) production in a dose-dependent manner. Furthermore, rRaIFN-γ inhibited FSH- and forskolin (FSK; 30 μmol/l)-induced extracellular cAMP accumulation (46·0 ± 6·6% and 29·1 ± 7·3% respectively). The inhibitory effect of rRaIFN-γ on FSK-induced cAMP was accompanied by decreased FSK-induced aromatase activity, inhibin, progesterone and 20α-OHP production. rRaIFN-γ had no detectable effect on aromatase activity, progesterone production and 20α-OHP production in the absence of FSH, but significantly stimulated basal inhibin production by 1·5-fold. rRaIFN-γ alone also caused a small but significant increase in basal levels of cAMP. The timecourse studies showed that FSH-induced aromatase activity and inhibin production were consistently suppressed by rRaIFN-γ, FSH-induced progesterone and 20α-OHP were inhibited at 1 and 2 days and then stimulated on days 3, 4 and 5 relative to FSH alone. There was no difference in DNA content between treatment and non-treatment wells during 5 days of culture. RaIFN had similar effects to rRaIFN-γ. We conclude that IFN-γ can inhibit FSH-induced granulosa cell differentiation and that, in the absence of FSH, IFN-γ stimulated undifferentiated granulosa cells to produce more inhibin. The mechanism of its action is likely to involve changes in cAMP production.
Journal of Endocrinology (1992) 133, 131–139
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). After 24-h treatment, extracellular cAMP levels in the culture medium containing 0.1 mM IBMX were determined by enzyme immunoassay. Results are shown as mean± s.e.m. of data from at least three separate experiments, each performed with triplicate
Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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Department of Endocrinology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyouku, Tokyo, Japan
Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo, Japan
Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama 5-1, Myondaiji-cho, Okazaki, Japan
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presence of 1 mM 1,3-dimethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor. After collecting culture medium for measurement of extracellular cAMP, cells were treated with 5% trichloroacetic acid and boiled for 5 min, for determination of the
