LH induces de novo cholesterol biosynthesis via SREBP activation in granulosa cells during ovulation in female mice

Endocrinology ◽  
2021 ◽  
Author(s):  
Tomoya Nakanishi ◽  
Risa Tanaka ◽  
Shingo Tonai ◽  
Joo Yeon Lee ◽  
Manami Yamaoka ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Granulosa Cells ◽  
De Novo ◽  
Cholesterol Biosynthesis ◽  
Active Form ◽  
Control Group ◽  
Element Binding Protein ◽  
Matured Oocyte

Abstract In the liver, the sterol response element binding protein (SREBP) and the SREBP cleavage-activated protein (SCAP) complex upregulates cholesterol biosynthesis by gene induction of de novo cholesterol synthetic enzymes (Hmgcr, Cyp51, and Dhcr7). Insulin induced gene 1 (INSIG1) negatively regulates cholesterol biosynthesis by the inhibition of de novo cholesterol biosynthetic gene expression. In the ovary, cholesterol is de novo synthesized; however, the roles of SREBP and its regulators (SCAP and INSIG1) are not well understood. In this study, when immature mice were treated with gonadotropins (eCG followed by hCG), eCG induced and hCG maintained the expression of SREBP-1a, -2, and SCAP granulosa cells, whereas INSIG1 expression was dramatically downregulated after hCG injection. Downregulation of INSIG1 led to generate the SREBPs active form and translocate the SREBPs active form to nuclei. Inhibition of generation of the SREBPs active form by fatostatin or Scap siRNA in both in vivo and in vitro significantly decreased the expressions of de novo cholesterol biosynthetic enzymes, cholesterol accumulation, and progesterone (P4) production compared to control group. Fatostatin treatment inhibited the ovulation and increased the formation of abnormal corpus luteum which trapped the matured oocyte in the corpus luteum, however, the phenomenon was abolished by P4 administration. The results showed that decreasing INSIG1 level after hCG stimulation activated SREBP-induced de novo cholesterol biosynthesis in granulosa cells of preovulatory follicles, which is essential for P4 production and the rupture of matured oocyte during ovulation process.

dSLIM Immunomodulators Induce Anti-Tumor Responses Both In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1727-1727
Author(s):  
Manuel Schmidt ◽  
Javier de Cristobal ◽  
Astrid Sander ◽  
Bernadette Brzezicha ◽  
Sven A. König Merediz ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Tumor Cell Line ◽  
Control Group ◽  
Γδt Cells ◽  
Ifn Γ

Abstract Cytosine-guanine (CpG) motifs containing oligonucleotides (ODN) are commonly used for immunomodulatory purpose in cancer therapy and for the treatment of allergic diseases since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response with secretion of IL-12 and IFN-γ, In addition their broad potential includes activation of B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6, and stimulation of plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. Usually phosphorothioate (PS) modifications are to enhance the stability, but these are leading to several side-effects, like severe organ enlargements, morphological changes and immunosuppression in mice. We designed immunomodulatory molecules based on short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. To evaluate the anti-tumor effect of the dSLIM molecules we developed an in vitro anti-tumor assay. This assay uses supernatant from dSLIM-activated human PBMCs for incubation with tumor cells in vitro. We observed increased apoptosis and necrosis of the HT-29 tumor cell line after incubation with supernatant from dSLIM-treated PBMC which was significantly higher than the effect of supernatant from non-treated PBMC. In addition, supernatant from dSLIM-treated PBMC increased the expression of HLA-ABC on the tumor cells, a pre-requisite for tumor cell recognition by the immune system. These effects were confirmed with human HEK293 and murine Renca cell lines. Analyzing the effect with neutralizing antibodies to various apoptosis-related cytokines, we observed a crucial role of IFN-γ but not IFN-α or TNFα. To investigate the anti-tumor effects of dSLIM in vivo, we employed a SKH1 murine model which is prone to spontaneous development of papillomas. Using chemicals for initiation and weekly promotion of de novo papilloma development we compared groups of weekly s.c. or i.p. dSLIM injections, respectively, with the PBS control group. The number of papilloma developing mice was significantly lower in the dSLIM groups and the total number of papillomas on all mice was reduced by approximately 50%. In conclusion, we showed that dSLIM immunomodulators exhibit potent anti-tumor effects in vitro and in vivo.

scholarly journals Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine

1999 ◽  
Vol 339 (2) ◽  
pp. 291-298 ◽  
Author(s):  
Annette L. HENNEBERRY ◽  
Christopher R. McMASTER
Keyword(s):  
De Novo ◽  
Active Form ◽  
Fatty Acyl ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Kennedy Pathway ◽  
Membrane Spanning

Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.

Oxytocin determination in steroid producing tissues and in vitro production in ovarian follicles

1985 ◽  
Vol 109 (4) ◽  
pp. 530-536 ◽  
Author(s):  
D. Schams ◽  
Th. A. M. Kruip ◽  
R. Koll
Keyword(s):  
Corpus Luteum ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
De Novo ◽  
Prostate Gland ◽  
Seminal Vesicles ◽  
Corpora Lutea ◽  
In Vitro Production ◽  
Wet Weight

Abstract Immunoreactive oxytocin was measured in ovaries (corpus luteum and follicular fluid) and adrenals of cows, and in testes, seminal vesicles, prostate gland and adrenals of bulls. Secretion of oxytocin was further measured after culture of whole follicles, granulosa cells and theca tissue. Concentrations of oxytocin increased in corpora lutea of cycling cattle until mid-luteal phase (447 ± 93 ng/g wet weight) and decreased afterwards. Low concentrations were found in corpora lutea of pregnant animals (6 ± 3 ng/g wet weight). Follicular fluid contains some oxytocin (on average 42–108 pg/ml) but concentrations were significantly higher in the fluid of ovarian cysts (190 pg/ml). After culture of follicles the amount of oxytocin released into the medium increased indicating de novo synthesis. The granulosa cells were the main source of follicular oxytocin. Production increased during luteinization indicating that luteinization is an important step for the production of oxytocin in ovaries. Tissues of testes (65 ± 10 pg/g wet weight) and adrenals from cows (122 ± 39 pg/g wet weight) and bulls (111 ±2 pg/g wet weight) contained oxytocin but at much lower concentrations compared to corpus luteum tissue. About 10 times higher concentrations of oxytocin were measured in the adrenal medulla (717 ± 96 pg/g wet weight) compared to the cortex (72 ± 11 pg/g wet weight). Seminal vesicles and prostate gland contained no measurable amounts of oxytocin (< 5 pg/g wet weight).

scholarly journals Coordinated Formation of IMPDH2 Cytoophidium in Mouse Oocytes and Granulosa Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Shiwen Ni ◽  
Teng Zhang ◽  
Chenmin Zhou ◽  
Min Long ◽  
Xuan Hou ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
De Novo ◽  
Biological Significance ◽  
Preimplantation Embryos ◽  
Inosine Monophosphate Dehydrogenase ◽  
Cell Stage ◽  
Simultaneous Formation

Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme catalyzing de novo biosynthesis of guanine nucleotides, aggregates under certain circumstances into a type of non-membranous filamentous macrostructure termed “cytoophidium” or “rod and ring” in several types of cells. However, the biological significance and underlying mechanism of IMPDH assembling into cytoophidium remain elusive. In mouse ovaries, IMPDH is reported to be crucial for the maintenance of oocyte–follicle developmental synchrony by providing GTP substrate for granulosa cell natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system to produce cGMP for sustaining oocyte meiotic arrest. Oocytes and the associated somatic cells in the ovary hence render an exciting model system for exploring the functional significance of formation of IMPDH cytoophidium within the cell. We report here that IMPDH2 cytoophidium forms in vivo in the growing oocytes naturally and in vitro in the cumulus-enclosed oocytes treated with IMPDH inhibitor mycophenolic acid (MPA). Inhibition of IMPDH activity in oocytes and preimplantation embryos compromises oocyte meiotic and developmental competences and the development of embryos beyond the 4-cell stage, respectively. IMPDH cytoopidium also forms in vivo in the granulosa cells of the preovulatory follicles after the surge of luteinizing hormone (LH), which coincides with the resumption of oocyte meiosis and the reduction of IMPDH2 protein expression. In cultured COCs, MPA-treatment causes the simultaneous formation of IMPDH cytoopidium in cumulus cells and the resumption of meiosis in oocytes, which is mediated by the MTOR pathway and is prevented by guanosine supplementation. Therefore, our results indicate that cytoophidia do form in the oocytes and granulosa cells at particular stages of development, which may contribute to the oocyte acquisition of meiotic and developmental competences and the induction of meiosis re-initiation by the LH surge, respectively.

scholarly journals Maprotiline Suppresses Cholesterol Biosynthesis and Hepatocellular Carcinoma Progression Through Direct Targeting of CRABP1

2021 ◽  
Vol 12 ◽  
Author(s):  
Cancan Zheng ◽  
Yidong Zhu ◽  
Qinwen Liu ◽  
Tingting Luo ◽  
Wenwen Xu
Keyword(s):  
Hepatocellular Carcinoma ◽  
Signaling Pathway ◽  
Binding Protein ◽  
Regulatory Element ◽  
Cholesterol Biosynthesis ◽  
Antitumor Effects ◽  
Element Binding Protein ◽  
Hcc Cells

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related death and has a poor prognosis worldwide, thus, more effective drugs are urgently needed. In this article, a small molecule drug library composed of 1,056 approved medicines from the FDA was used to screen for anticancer drugs. The tetracyclic compound maprotiline, a highly selective noradrenergic reuptake blocker, has strong antidepressant efficacy. However, the anticancer effect of maprotiline remains unclear. Here, we investigated the anticancer potential of maprotiline in the HCC cell lines Huh7 and HepG2. We found that maprotiline not only significantly restrained cell proliferation, colony formation and metastasis in vitro but also exerted antitumor effects in vivo. In addition to the antitumor effect alone, maprotiline could also enhance the sensitivity of HCC cells to sorafenib. The depth studies revealed that maprotiline substantially decreased the phosphorylation of sterol regulatory element-binding protein 2 (SREBP2) through the ERK signaling pathway, which resulted in decreased cholesterol biosynthesis and eventually impeded HCC cell growth. Furthermore, we identified cellular retinoic acid binding protein 1 (CRABP1) as a direct target of maprotiline. In conclusion, our study provided the first evidence showing that maprotiline could attenuate cholesterol biosynthesis to inhibit the proliferation and metastasis of HCC cells through the ERK-SREBP2 signaling pathway by directly binding to CRABP1, which supports the strategy of repurposing maprotiline in the treatment of HCC.

Prior exposure to gonadotrophins prevents the subsequent antigonadotrophic actions of cloprostenol by a cyclic AMP-dependent mechanism in cultured human granulosa cells

1991 ◽  
Vol 131 (2) ◽  
pp. 319-325 ◽  
Author(s):  
A. E. Michael ◽  
G. E. Webley
Keyword(s):  
Cyclic Amp ◽  
Corpus Luteum ◽  
Granulosa Cells ◽  
Early Pregnancy ◽  
Prior Exposure ◽  
Vitro Fertilization ◽  
Human Granulosa Cells ◽  
Dependent Mechanism

ABSTRACT The antigonadotrophic action of a prostaglandin F2α analogue, cloprostenol, has been investigated in human granulosa cells obtained from cycles stimulated for in-vitro fertilization and induced to secrete luteal quantities of progesterone by culture in serum-supplemented medium. Cells were exposed to conditions which may mimic those occurring in early pregnancy to establish the roles of human chorionic gonadotrophin (hCG) versus LH and that of cyclic AMP (cAMP) in the anti-gonadotrophic action of cloprostenol. When human granulosa cells were cultured in the absence of treatment for 3 days, exposure to cloprostenol had no effect on basal progesterone production but inhibited hCG-stimulated progesterone (60% decrease; P<0·01), hCG-stimulated cAMP (40% decrease; P < 0·05) and the progesterone response to dibutyryl cAMP (dbcAMP; 70% decrease; P < 0·01), suggesting pre- and post-cAMP sites of cloprostenol action. The inhibitory actions of cloprostenol were prevented when the granulosa cells were either continuously exposed to treatment from the start of culture or pre-exposed for 3 days to maximum concentrations of LH, hCG, dbcAMP or 8-bromo-cAMP. We conclude that prior exposure either in vivo or in vitro to LH or hCG prevents the subsequent antigonadotrophic action of cloprostenol via a cAMP-dependent mechanism. Prevention of the antigonadotrophic action of cloprostenol after exposure to hCG may be a mechanism through which CG prevents regression of the corpus luteum in early pregnancy, while the suppressive effect of LH pretreatment may account for the refractory response of the early corpus luteum to cloprostenol following the midcycle LH surge. Journal of Endocrinology (1991) 131, 319–325

scholarly journals Hepatic one-carbon metabolism in early folate deficiency in rats

1993 ◽  
Vol 291 (1) ◽  
pp. 145-149 ◽  
Author(s):  
M Balaghi ◽  
D W Horne ◽  
C Wagner
Keyword(s):  
Enzyme Activity ◽  
De Novo ◽  
Folate Deficiency ◽  
Male Rats ◽  
Control Group ◽  
Deficient Diet ◽  
Ad Libitum ◽  
Deficient Group

Glycine N-methyltransferase (GNMT) is inhibited by 5-methyltetrahydrofolate polyglutamate in vitro. It is believed to play a regulatory role in the synthesis de novo of methyl groups. We have used the amino-acid-defined diet of Walzem and Clifford [(1988) J. Nutr. 118, 1089-1096] to determine whether folate deficiency in vivo would affect GNMT activity, as predicted by the studies in vitro. Weanling male rats were fed on the folate-deficient diet or a folate-supplemented diet pair-fed to the deficient group. A third group was fed on the folate-supplemented diet ad libitum. Development of folate deficiency rapidly resulted in decreased levels of S-adenosylmethionine (SAM) and elevation of S-adenosylhomocysteine (SAH). The ratios of SAM to SAH were 1.8, 2.7 and 1.5 in the deficient group for weeks 2, 3 and 4 of the experiment, and the values were 9.7, 7.1 and 8.9 for the pair-fed control group and 10.3, 8.8 and 8.0 for the control group ad libitum fed. The activity of GNMT was significantly higher in the deficient group than in either of the two control groups at each time period. This was not due to increased amounts of GNMT protein, but reflected an increase in specific enzyme activity. Levels of folate in both the cytosol and mitochondria were severely lowered after only 2 weeks on the diet. The distribution of folate coenzymes was also affected by the deficiency, which resulted in a marked increase in the percentage of tetrahydrofolate polyglutamates in both cytosol and mitochondria and a very large decrease in cytosolic 5-methyltetrahydrofolate. The increased GNMT activity is therefore consistent with decreased folate levels and decreased inhibition of enzyme activity.

dSLIM Immunomodulators Induce Anti-Tumor Responses Both In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5527-5527
Author(s):  
Manuel Schmidt ◽  
Javier de Cristobal ◽  
Astrid Sander ◽  
Bernadette Brzezicha ◽  
Sven A. Koenig-Merediz ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Tumor Cell Line ◽  
Control Group ◽  
Γδt Cells ◽  
Ifn Γ

Abstract Cytosine-guanine (CpG) motifs containing oligonucleotides (ODN) are commonly used for immunomodulatory purpose in cancer therapy and for the treatment of allergic diseases since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response with secretion of IL-12 and IFN-γ, In addition their broad potential includes activation of B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6, and stimulation of plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. Usually phosphorothioate (PS) modifications are to enhance the stability, but these are leading to several side-effects, like severe organ enlargements, morphological changes and immunosuppression in mice. We designed immunomodulatory molecules based on short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. To evaluate the anti-tumor effect of the dSLIM molecules we developed an in vitro anti-tumor assay. This assay uses supernatant from dSLIM-activated human PBMCs for incubation with tumor cells in vitro. We observed increased apoptosis and necrosis by the HT-29 tumor cell line after incubation with supernatant from dSLIM-treated PBMC which was significantly higher than the effect of supernatant from non-treated PBMC. In addition, supernatant from dSLIM-treated PBMC increased the expression of HLA-ABC on the tumor cells, a pre-requisite for tumor cell recognition by the immune system. These effects were confirmed with human HEK293 and murine Renca cell lines. Analyzing the effect with neutralizing antibodies to various apoptosis-related cytokines, we observed a crucial role of IFN-γ but not IFN-α or TNFα. To investigate the anti-tumor effects of dSLIM in vivo, we employed a SKH1 murine model which is prone to spontaneous development of papillomas. Using chemicals for initiation and weekly promotion of de novo papilloma development we compared groups of weekly s.c. or i.p. dSLIM injections, respectively, with the PBS control group. The number of papilloma developing mice was significantly lower in the dSLIM groups and the total number of papillomas on all mice was reduced by approximately 50%. In conclusion, we showed that dSLIM immunomodulators exhibit potent anti-tumor effects in vitro and in vivo.

scholarly journals Hydroxysteroid (17β) Dehydrogenase 7 Activity Is Essential for Fetal de Novo Cholesterol Synthesis and for Neuroectodermal Survival and Cardiovascular Differentiation in Early Mouse Embryos

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1884-1892 ◽  
Author(s):  
Heli Jokela ◽  
Pia Rantakari ◽  
Tarja Lamminen ◽  
Leena Strauss ◽  
Roxana Ola ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Heart Development ◽  
De Novo ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Yolk Sac ◽  
Mouse Embryos ◽  
Metabolic Role

Hydroxysteroid (17β) dehydrogenase 7 (HSD17B7) has been shown to catalyze the conversion of both estrone to estradiol (17-ketosteroid reductase activity) and zymosterone to zymosterol (3-ketosteroid reductase activity involved in cholesterol biosynthesis) in vitro. To define the metabolic role of the enzyme in vivo, we generated knockout mice deficient in the enzyme activity (HSD17B7KO). The data showed that the lack of HSD17B7 results in a blockage in the de novo cholesterol biosynthesis in mouse embryos in vivo, and HSD17BKO embryos die at embryonic day (E) 10.5. Analysis of neural structures revealed a defect in the development of hemispheres of the front brain with an increased apoptosis in the neuronal tissues. Morphological defects in the cardiovascular system were also observed from E9.5 onward. Mesodermal, endodermal, and hematopoietic cells were all detected by the histological analysis of the visceral yolk sac, whereas no organized vessels were observed in the knockout yolk sac. Immunohistological staining for platelet endothelial cell adhesion molecule-1 indicated that the complexity of the vasculature also was reduced in the HSD17B7KO embryos, particularly in the head capillary plexus and branchial arches. At E8.5–9.5, the heart development and the looping of the heart appeared to be normal in the HSD17B7KO embryos. However, at E10.5 the heart was dilated, and the thickness of the cardiac muscle and pericardium in the HSD17B7KO embryos was markedly reduced, and immunohistochemical staining for GATA-4 revealed that HSD17B7KO embryos had a reduced number of myocardial cells. The septum of the atrium was also defected in the knockout mice.

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