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1.
Diabetologia ; 46(2): 276-83, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12627327

ABSTRACT

AIMS/HYPOTHESIS: Previous studies have shown that diabetic placentas are characterized by structural and biochemical anomalies, including defects in the differentiation of trophoblasts. In this study, the Rcho-1 cell line was used to investigate the impact of high glucose concentrations on different markers of differentiation of rat trophoblast cells in giant cells (endoreduplication, invasive phenotype and endocrine phenotype). MATERIALS: Rcho-1 cells were incubated for 12 days in medium supplemented with different concentrations of glucose and 10% horse serum to optimize differentiation. The cells were examined for the proportion of nuclei showing signs of apoptosis. The effect of high glucose was investigated on the endoreduplication process, on invasive phenotype (secretion of gelatinase B) and on endocrine phenotype (expression of placental lactogen I (PL-I) and II (PL-II) and progesterone secretion). RESULTS: Apoptosis was not induced by high glucose in Rcho-1. The number of cells was higher in the cultures exposed to high glucose (p<0.05) and their nuclei contained more DNA compared with control cells (p<0.001), while their nuclear size was smaller (p<0.001). Gelatinase B secretion increased during differentiation but no difference was found when gelatinase B secretion from trophoblasts exposed to high glucose was compared with the control cells. Rcho-1 cell cultures showed an increase in PL-I and PL-II mRNA expressions during differentiation and which was not affected by high glucose. Progesterone secretion increased during differentiation in control cultures. However, this increase was abolished when trophoblasts were cultured in high glucose. CONCLUSIONS/INTERPRETATION: Our data suggest that high glucose influences the endoreduplication process and the steroidogenesis during differentiation of rattrophoblasts.


Subject(s)
Glucose/administration & dosage , Trophoblasts/cytology , Animals , Apoptosis , Cell Count , Cell Differentiation/drug effects , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Dose-Response Relationship, Drug , Matrix Metalloproteinase 9/metabolism , Mitotic Index , Osmolar Concentration , Placental Lactogen/metabolism , Progesterone/metabolism , Rats , Trophoblasts/drug effects , Trophoblasts/metabolism , Trophoblasts/physiology
2.
Biol Reprod ; 68(5): 1808-12, 2003 May.
Article in English | MEDLINE | ID: mdl-12606487

ABSTRACT

Previous investigations have shown that maternal diabetes impairs rodent embryo development during the earliest phase of gestation. Exposure to high concentrations of glucose before implantation results in a decrease in the number of cells per embryo and in a concomitant increase in two nuclear markers of apoptosis: chromatin degradation and nuclear fragmentation. In the present study, we show that caspase-6 is expressed in rat blastocysts, using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Caspase-6 is detected in all cells of the blastocyst and is excluded from the nucleus. To assess the role of caspase-6 in the glucose-induced apoptosis, rat blastocysts were incubated for 24 h in either 6 or 28 mM glucose in the presence or absence of a specific inhibitor of caspase-6 (VEID-CHO, 100 nM). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation and nuclear fragmentation. Addition of VEID-CHO was found to inhibit nuclear fragmentation, but did not prevent the increase in chromatin degradation triggered by excess glucose. Our data indicate that chromatin degradation and nuclear fragmentation are two nuclear damages that are induced separately by high glucose in rat blastocysts. Furthermore, nuclear fragmentation in rat blastocysts is apparently mediated by the activation of caspase-6.


Subject(s)
Apoptosis/drug effects , Blastocyst/enzymology , Caspases/metabolism , Glucose/toxicity , Animals , Blastocyst/drug effects , Caspase 6 , Caspase Inhibitors , Cell Count , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Chromatin/genetics , Embryo, Mammalian/enzymology , Enzyme Inhibitors/pharmacology , Female , Immunohistochemistry , In Situ Nick-End Labeling , In Vitro Techniques , Microscopy, Confocal , Pregnancy , Rats , Reverse Transcriptase Polymerase Chain Reaction
3.
Diabetologia ; 45(4): 571-9, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12032635

ABSTRACT

AIMS/HYPOTHESIS: Signs of apoptosis have been observed in rodent blastocysts exposed to high d-glucose concentrations in vitro. The mechanism underlying the detrimental influence of glucose remains to be identified. It has been postulated that high d-glucose concentrations induced oxidative stress in rat post-implantation embryos in vitro. A decreased glucose uptake has also been implicated in the embryotoxicity of glucose in pre-implantation mouse embryos. We examined whether the high incidence of cell death in high d-glucose-treated embryos was associated with a disrupted redox status and with alterations in glucose transport and metabolism. METHODS: After blastocysts were incubated in different concentrations of d-glucose for 24 h, they were examined for the proportion of nuclei showing signs of chromatin degradation using the TUNEL technique, for the generation of reactive oxygen species and for the mitochondrial membrane potential using specific fluoroprobes and the confocal microscopy. Glucose transport and metabolism were assessed using radiolabelled 3-O-methylglucose and glucose, respectively. RESULTS: Compared to the control blastocysts, high d-glucose-treated embryos showed a higher incidence of TUNEL-positive nuclei and reactive oxygen species generation principally in the inner cell mass cells. Decreased glucose transport and glycolytic activity but unmodified pentose phosphate pathway activity were detected in these embryos. CONCLUSION/INTERPRETATION: Incubation in high d-glucose concentrations in vitro increased cell death, induced oxidative stress and decreased glucose transport and metabolism in mouse blastocysts. As only glycolysis was affected, however, we suggest that metabolic inhibition occurred downstream glucose transport and glucose-6-phosphate formation.


Subject(s)
Blastocyst/cytology , Cell Death/drug effects , Glucose/metabolism , Glucose/pharmacology , Animals , Apoptosis/drug effects , Blastocyst/drug effects , Blastocyst/physiology , Dose-Response Relationship, Drug , Kinetics , Mice , Oxidative Stress/drug effects , Oxidative Stress/physiology , Reactive Oxygen Species/metabolism
4.
Diabetologia ; 44(10): 1318-25, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11692181

ABSTRACT

AIMS/HYPOTHESIS: Paracrine interactions are thought to operate between the inner cell mass and trophectoderm cell lineages to co-ordinate their expansion and differentiation during embryo implantation. We aimed to determine whether hyperglycaemia could interfere with this regulatory process. METHODS: Mouse blastocysts were pre-exposed to either control or high concentrations of d-glucose for 24 h in vitro and tested for their ability to attach and spread over a fibronectin-coated culture substrate. Quantitative immunocytochemistry was done on blastocysts to assess the protein expression of Fibroblast Growth Factor-4, a growth factor preferentially produced by the inner cell mass and thought to restrict trophectoderm differentiation into giant trophoblasts. Experiments were then done combining the pre-exposure to high D-glucose with the addition of recombinant fibroblast growth factor-4. RESULTS: Compared with control blastocysts, high D-glucose pre-treated embryos were found to form 18 % larger trophoblast outgrowths. These blastocysts also showed a 30 % reduction in the expression of fibroblast growth factor-4 protein by inner cell mass cells as they were outgrowing. The trophoblast surface area per outgrowth and the trophoblast nuclear area were corrected when the addition of recombinant fibroblast growth factor-4 was combined with the pre-exposure to high D-glucose. CONCLUSION/INTERPRETATION: The data suggests that trophectoderm differentiation is impaired by high D-glucose and that this effect is secondary to a deficiency in fibroblast growth factor-4 protein in the inner cell mass. These observations add a novel perspective to the study of the peri-implantation embryopathy associated with maternal diabetes


Subject(s)
Blastocyst/drug effects , Fibroblast Growth Factors/analysis , Glucose/administration & dosage , Trophoblasts/cytology , Animals , Apoptosis , Blastocyst/chemistry , Blastocyst/cytology , Cell Count , Cell Differentiation/drug effects , Culture Techniques , Female , Fibroblast Growth Factors/pharmacology , Male , Mice , Pregnancy , Recombinant Proteins/pharmacology , Trophoblasts/chemistry
5.
Mol Reprod Dev ; 60(1): 38-46, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11550266

ABSTRACT

Previous studies have suggested that fibroblast growth factor-4 (FGF-4) may be a paracrine signal used by inner cell mass (ICM) cells to maintain adjacent trophectoderm (TE) cells in an undifferentiated state. In the present work, immunocytochemical analysis of mouse blastocysts confirmed that FGF-4 was predominantly detected in the ICM before and after spreading over a fibronectin-coated culture substrate. Addition of human recombinant FGF-4 did not influence morphological progression, cell allocation and proliferation in ICM and TE lineages or mitosis and karyorhexis frequencies during blastocyst expansion. Addition of FGF-4 to outgrowing blastocysts, in contrast, induced a significant decrease in the surface of the trophoblast outgrowths formed by the TE cells and in the proportion of giant trophoblasts per outgrowth. The fact that blastocysts display excessive trophoblast expansion and spreading over their culture substrate upon pre-exposure to high concentrations of glucose in vitro was used to further assess the regulatory effect of FGF-4. Addition of FGF-4 was indeed found to fully neutralize the disruptive impact of high glucose on trophoblast outgrowths. Altogether, our data indicate that ICM-derived FGF-4 participates actively in the regulation of trophoblast development.


Subject(s)
Cell Differentiation , Fibroblast Growth Factors/metabolism , Glucose/pharmacology , Proto-Oncogene Proteins/metabolism , Trophoblasts/cytology , Trophoblasts/metabolism , Animals , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Nucleus/drug effects , Cell Size/drug effects , Female , Fibroblast Growth Factor 4 , Fibroblast Growth Factors/pharmacology , Glucose/metabolism , Humans , Immunohistochemistry , Male , Mice , Proto-Oncogene Proteins/pharmacology , Recombinant Proteins/pharmacology , Trophoblasts/drug effects
6.
Diabetes Metab ; 27(3): 329-36, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11431598

ABSTRACT

OBJECTIVE: To analyse the clinical characteristics and relevant hormonal profile in type 1 diabetic patients with and without ED. MATERIAL AND METHODS: Fifty one type 1 diabetic patients were studied. ED was assessed by direct interview. Chronic diabetic complications, smoking and alcohol status as well as current use of medications were recorded. Hormonal profile consisted of plasma LH, FSH, prolactin, androstenedione (Delta(4)), dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEA-S), free testosterone (FT), estradiol (E(2)), sex hormone binding globulin (SHBG), dihydrotestosterone (DHT), cortisol, TSH and free thyroxine (FT(4)). RESULTS: ED was present in 24 patients (47%) (group 1), who were older (P<0.001), had a longer diabetes duration (P<0.001) and a higher systolic blood pressure (P=0.017) when compared to the subjects who did not complain (group 2). ED was positively correlated to all diabetes-related complications (P<0.02). Antidepressive drug(s) were more frequent in group 1 (P=0.007), as well as prokinetics (P=0.043) and ACE-inhibitors (P=0.010). HbA(1)c was comparable. Patients with ED had lower levels of Delta(4) (P=0.003), DHEA (P<0.001), DHEA-S (P=0.002), FT (P=0.08) while SHBG (P=0.010) and LH (P=0.022) were higher compared to group 2. Multiple logistic regression analysis showed an independent association of ED with Delta(4) (P=0.016), DHEA-S (P=0.037), SHBG (P=0.001) and insulin dose (P=0.025). There was no significant difference for all other measured hormones. CONCLUSION: ED is impressively prevalent in type 1 diabetes and is associated with age, diabetes duration, chronic complications and decreased androgens.


Subject(s)
Androgens/blood , Diabetes Mellitus, Type 1/physiopathology , Erectile Dysfunction/blood , Erectile Dysfunction/physiopathology , Age Factors , Alcohol Drinking , Blood Pressure , C-Peptide/blood , Cohort Studies , Diabetes Mellitus, Type 1/blood , Diabetic Angiopathies/epidemiology , Diabetic Angiopathies/physiopathology , Diabetic Neuropathies/epidemiology , Diabetic Neuropathies/physiopathology , Diabetic Retinopathy/epidemiology , Diabetic Retinopathy/physiopathology , Estradiol/blood , Follicle Stimulating Hormone/blood , Humans , Hydrocortisone/blood , Hypertension/epidemiology , Hypertension/physiopathology , Luteinizing Hormone/blood , Male , Middle Aged , Prolactin/blood , Smoking
7.
Biol Reprod ; 64(2): 555-62, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11159358

ABSTRACT

Previous investigations have shown that maternal diabetes impairs rodent embryo development during the earliest phase of gestation. Exposure to high concentrations of glucose before implantation results in a decrease in the number of cells per embryo and in a concomitant increase in two nuclear markers of apoptosis, chromatin degradation and nuclear fragmentation. In the present study, we show that two intracellular effectors of apoptosis, caspase-3 and caspase-activated deoxyribonuclease (CAD), are involved in the embryotoxicity of high glucose. Using reverse transcription-polymerase chain reaction and immunocytochemistry, we first demonstrated that these two effectors were expressed in rat blastocysts. The two effectors were detected in all the cells of the blastocysts and the immuno-signals were excluded from the nuclei. Rat blastocysts were incubated for 24 h in either 6 mM or 28 mM glucose in the presence or absence of specific inhibitors (DEVD-CHO [10 microM] against caspase-3 and aurin [1 microM] against CAD). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation or nuclear fragmentation. Addition of DEVD-CHO or aurin was found to inhibit the increase in chromatin degradation induced by high glucose. None of these two inhibitors prevented the increase in nuclear fragmentation triggered by excess glucose. Our data indicate that chromatin degradation and nuclear fragmentation are two nuclear damages that are induced separately by high glucose in rat blastocysts. Chromatin degradation is apparently mediated by the activation of caspase-3 and CAD.


Subject(s)
Blastocyst/enzymology , Caspases/analysis , Chromatin/metabolism , Deoxyribonucleases/analysis , Glucose/pharmacology , Animals , Apoptosis/drug effects , Base Sequence , Caspase 3 , Caspase Inhibitors , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Chromatin/drug effects , Deoxyribonucleases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Female , Immunohistochemistry , Male , Microscopy, Confocal , Molecular Sequence Data , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction
8.
Diabetes ; 50(1): 143-9, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11147780

ABSTRACT

Bcl-2 mRNA expression was detected in rat blastocysts by in situ hybridization. The distribution of mRNA expression was rather heterogenous, with approximately 2% of high-expressing cells. In vitro exposure to 28 mmol/l D-glucose for 24 h resulted in a significant increase in the proportion of these cells compared with control embryos in either 6 mmol/l D-glucose or 28 mmol/l D+L-glucose. Heterogeneity in the expression of Bcl-2 was also observed at the protein level by immunocytochemistry. Exposure to 28 mmol/l D-glucose significantly increased the incidence of chromatin degradation (karyolysis) and nuclear fragmentation (karyorhexis), two nuclear markers of apoptosis in rat blastocysts. When two different antisense oligodeoxynucleotides designed to block Bcl-2 expression were added to 28 mmol/1 D-glucose, the incidence of karyolysis (but not karyorhexis) was increased compared with embryos in 28 mmol/l D-glucose alone. These data suggest that Bcl-2 is involved in the protective response against the induction of karyolysis in blastocysts on their exposure to high concentrations of D-glucose in vitro, whereas karyorhexis appears to result from the activation of an intracellular pathway that is independent of Bcl-2.


Subject(s)
Blastocyst/drug effects , Blastocyst/physiology , Glucose/pharmacology , Proto-Oncogene Proteins c-bcl-2/physiology , Animals , Blastocyst/metabolism , Culture Techniques , Dose-Response Relationship, Drug , Female , Oligonucleotides, Antisense/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar
9.
Cytokine ; 11(7): 500-9, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10419651

ABSTRACT

Previous observations have shown that tumour necrosis factor alpha (TNF-alpha) synthesis is increased in the uterus of diabetic rats and that the epithelial layer lining the uterine lumen is the major site of TNF-alpha over-production. In the present study, TNF-alpha secretion was found to be stimulated by high D-glucose levels in primary cultures of mouse uterine luminal cells but not in cultures of the mouse uterine epithelial WEG-1 cell line. Experiments were performed to investigate the possibility that non-epithelial cells may mediate the influence of high D-glucose on TNF-alpha production by uterine epithelial cells. Immunocytochemical analysis revealed the reproducible presence of a small proportion of macrophages in primary cultures. Macrophages of the RAW 264.7 cell line were found to secrete more interleukin (IL)-1beta (but not TNF-alpha) when cultured in high D-glucose. TNF-alpha production in WEG-1 cells was increased upon exposure to IL-1beta and both protein kinase-C and tyrosine kinase pathways appeared to be involved in TNF-alpha stimulation. Addition of IL-1 receptor antagonist to primary cultures partially abrogated the effect of high D-glucose. Since WEG-1 cells do not produce IL-1beta, the data lend support to the hypothesis that uterine epithelial cells synthesize high levels of TNF-alpha in response to hyperglycaemia via an increase in IL-1beta secretion by stromal macrophages.


Subject(s)
Epithelial Cells/metabolism , Glucose/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Uterus/cytology , Animals , Cells, Cultured/drug effects , Epithelial Cells/drug effects , Female , Hyperglycemia/physiopathology , Interleukin-1/pharmacology , Interleukin-1/physiology , Macrophages/drug effects , Mice , Tumor Necrosis Factor-alpha/biosynthesis , Uterus/metabolism
10.
Biol Reprod ; 60(2): 479-83, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9916017

ABSTRACT

Mouse blastocysts were exposed for 24 h to various concentrations of recombinant mouse tumor necrosis factor alpha (TNFalpha) and observed for their capacity to implant in vitro on a fibronectin-coated substrate or to develop in vivo after their transfer into surrogate females. Compared with findings in control blastocysts, exposure to TNFalpha resulted in a significant reduction in the average number of cells in the inner cell mass (ICM) lineage. This effect was associated with a significant increase in the frequency of cells identified as engaged in apoptosis by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling technique. No difference was found in the incidence of nuclear fragmentation between control and TNFalpha-exposed blastocysts. When TNFalpha-pretreated blastocysts were allowed to implant in vitro, significantly fewer embryos were able to maintain a structured ICM cluster at the center of the trophectoderm outgrowth. Although no difference was found in the average surface area of the outgrowths, implants derived from TNFalpha-treated blastocysts contained significantly fewer nuclei than implants from control embryos. After transfer into recipient mice, TNFalpha-pretreated blastocysts implanted at about the same rate as control embryos, but a significantly higher rate of resorption was found among fetuses after exposure to the cytokine. In addition, the weight of the surviving fetuses was significantly lower than for control fetuses. These data indicate that the impact of TNFalpha on blastocysts is specifically aimed at the ICM lineage and that TNFalpha decreases the ability of embryos to differentiate into fetuses after implantation.


Subject(s)
Blastocyst/physiology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Culture Media , Culture Techniques , Embryo Transfer , Embryonic Development , Embryonic and Fetal Development , Female , Fibronectins , Mice , Pregnancy , Pseudopregnancy , Recombinant Proteins/pharmacology
11.
Biol Reprod ; 58(6): 1416-24, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9623600

ABSTRACT

Previous studies have shown the adverse impact of the cytokine tumor necrosis factor alpha (TNFalpha) on the development of the inner cell mass in mouse blastocysts. In the present study, two embryonic stem (ES) cell lines were used to further investigate the action of TNFalpha. The expression of TNFalpha receptors in ES cells was tested by reverse transcription-polymerase chain reaction and Northern blot analysis. Transcripts encoding the two distinct receptor isoforms were detected in these cells. Using different approaches, our data showed a TNFalpha dose-dependent decrease in the number of ES cells after 24 h of exposure. Simultaneous blocking of the two receptors with antagonist antibodies was needed to completely abrogate the inhibitory effect of the cytokine. Extensive DNA nicks (visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling [TUNEL] method), but not nuclear fragmentation, was found with a higher incidence in ES cells exposed to TNFalpha. The possibility that TNFalpha may stimulate ES cell differentiation was investigated with a test based on the expression of alkaline phosphatase. The results indicated that TNFalpha cannot over-ride the negative control exerted by leukemia inhibitory factor on differentiation. The opposite possibility, that TNFalpha blocks differentiation, was tested in suspended medium drops. In this system, TNFalpha was found to decrease the ability of ES cells to differentiate into embryoid bodies. In addition, expression of Rex-1, a marker gene for undifferentiated ES cells, was increased in ES cells exposed to TNFalpha. Thus our data support the hypothesis that TNFalpha is a significant (negative) effector of proliferation and differentiation in inner cell mass-derived ES cells.


Subject(s)
Embryo, Mammalian , Stem Cells/cytology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Apoptosis , Cell Differentiation , Cell Division , Cell Line , DNA Fragmentation , Gene Expression , Mice , Polymerase Chain Reaction , Receptors, Tumor Necrosis Factor/genetics , Stem Cells/metabolism
12.
Diabetes ; 46(7): 1214-24, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9200658

ABSTRACT

The production of tumor necrosis factor-alpha (TNF-alpha) was investigated in uterine explants from normal, diabetic, or insulin-treated diabetic pregnant rats. Explants from diabetic rats released more soluble TNF-alpha than did those in the other groups. The extent of this secretion was correlated with blood glucose concentration at the time of explantation. The concentration of cell membrane-associated TNF-alpha in the explants was not altered by diabetes. Daily insulin administration failed to normalize uterine TNF-alpha secretion despite correction of glycemia in the diabetic rats. Explants from normal pregnant rats cultured in vitro with increasing concentrations of D-glucose showed a dose-dependent increase in TNF-alpha secretion. The production of TNF-alpha in high glucose was also tested in primary cultures of uterine cells isolated from either immature or adult rats. TNF-alpha secretion was increased in high D-glucose but not in iso-osmolar concentrations of L-glucose, D-raffinose, D-galactose, or mannitol. Cell membrane-associated TNF-alpha was not influenced by high D-glucose. Semiquantitative reverse transcription-amplification of RNA extracted from primary cultures of uterine cells showed that the steady-state level of TNF-alpha transcripts was increased by high D-glucose but not by high L-glucose. The results are consistent with the hypothesis that hyperglycemia is instrumental in the overexpression of TNF-alpha in the diabetic uterus. Because TNF-alpha has a demonstrated negative impact on embryonic growth, enhanced TNF-alpha synthesis in the pregnant uterus may contribute to the embryopathy associated with maternal diabetes.


Subject(s)
Diabetes Mellitus, Experimental/physiopathology , Glucose/pharmacology , Tumor Necrosis Factor-alpha/biosynthesis , Uterus/metabolism , Animals , Carbohydrates/pharmacology , Cell Death/drug effects , Cell Death/physiology , Cells, Cultured , Diabetes Mellitus, Experimental/chemically induced , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Glucose/chemistry , Organ Culture Techniques , Pregnancy , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/drug effects , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Uterus/cytology , Uterus/drug effects , Uterus/immunology
13.
Development ; 124(23): 4827-36, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9428419

ABSTRACT

The morphogenetic function of the transient phase of cell death that occurs during blastocyst maturation is not known but it is thought that its regulation results from a delicate balance between survival and lethal signals in the uterine milieu. In this paper, we show that blastocysts from diabetic rats have a higher incidence of dead cells than control embryos. Differential lineage staining indicated that increased nuclear fragmentation occurred mainly in the inner cell mass. In addition, terminal transferase-mediated dUTP nick end labeling (TUNEL) demonstrated an increase in the incidence of non-fragmented DNA-damaged nuclei in these blastocysts. Analysis of the expression of clusterin, a gene associated with apoptosis, by quantitative reverse transcription-polymerase chain reaction detected an increase in the steady-state level of its transcripts in blastocysts from diabetic rats. In situ hybridization revealed that about half the cells identified as expressing clusterin mRNA exhibited signs of nuclear fragmentation. In vitro experiments demonstrated that high D-glucose increased nuclear fragmentation, TUNEL labeling and clusterin transcription. Tumor necrosis factor-alpha (TNF-alpha), a cytokine whose synthesis is up-regulated in the diabetic uterus, did not induce nuclear fragmentation nor clusterin expression but increased the incidence of TUNEL-positive nuclei. The data suggest that excessive cell death in the blastocyst, most probably resulting from the overstimulation of a basal suicidal program by such inducers as glucose and TNF-alpha, may be a contributing factor of the early embryopathy associated with maternal diabetes.


Subject(s)
Blastocyst/cytology , Glucose/pharmacology , Molecular Chaperones , Pregnancy in Diabetics , Tumor Necrosis Factor-alpha/pharmacology , Animals , Blastocyst/drug effects , Cell Death/drug effects , Cell Nucleus/ultrastructure , Clusterin , DNA Fragmentation , Female , Glycoproteins/genetics , Male , Pregnancy , RNA, Messenger , Rats
15.
Clin Endocrinol (Oxf) ; 44(4): 429-33, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8706309

ABSTRACT

OBJECTIVE: Pregnancy induced hypertension has been shown to be associated with a normal or low activity of the maternal circulating renin-angiotensin system (RAS) but little is known of the local RAS in placenta and fetal membranes. The present study attempts to determine, at full term of human preeclamptic pregnancies, the activity of the chorioplacental renin-angiotensin system. PATIENTS AND MEASUREMENTS: We analysed the concentrations of active renin, prorenin, angiotensin converting enzyme (ACE) and angiotensin II in homogenates of human placenta and fetal membranes from preeclamptic patients at full term pregnancy. The values of renin, ACE and angiotensin II found in the patients with moderate preeclampsia (gestosis index 0-1) (n = 10) were compared with those of normal pregnant women (n = 8). RESULTS: Our experiments showed that in preeclamptic pregnancies, the chorion membrane contained the highest concentrations of active renin (2905 +/- 152 pg/g, mean +/- SD), prorenin (21,315 +/- 2849 pg/g) and ACE (1258 +/- 302 U/g) whereas the placenta had more angiotensin II than the chorion and amnion (741 +/- 45 vs 456 +/- 40 and 428 +/- 64 pg/g, respectively). In the placenta, as in the fetal membranes, no significant difference was found in the levels of active renin, ACE or angiotensin II between hypertensive patients and normal subjects but a slightly lower level of chorionic prorenin (P < 0.05) was observed in pregnancy induced hypertension. CONCLUSION: These findings indicate that in moderate preeclampsia (gestosis index 0-1), the activity of the renin-angiotensin system in term human placenta and fetal membranes remains essentially normal.


Subject(s)
Angiotensin II/analysis , Extraembryonic Membranes/chemistry , Peptidyl-Dipeptidase A/analysis , Placenta/chemistry , Pre-Eclampsia/metabolism , Renin/analysis , Amnion/chemistry , Chorion/chemistry , Extraembryonic Membranes/enzymology , Female , Humans , Placenta/enzymology , Pre-Eclampsia/enzymology , Pregnancy , Pregnancy Trimester, Third
16.
J Clin Endocrinol Metab ; 81(3): 998-1002, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8772564

ABSTRACT

Regulation of the angiotensin AT1 receptor in human placenta is poorly understood. In this study, we analyzed the time course of angiotensin AT1 receptor expression, internalization, and recycling by human trophoblast cells. We also studied the effects of estradiol, progesterone, and chloroquine on regulation of the angiotensin AT1 receptor in 48-h cell culture. The angiotensin II receptor expression increased with the time of incubation, reaching a level at 48 h of culture that was about 120% above the initial value. A large majority of angiotensin II receptors was of the AT1 subtype, as it was completely inhibited by losartan (1 mumol/L). The internalization of [125]angiotensin II binding and the angiotensin AT1 receptor recycling were also time dependent, with t1/2 values of 12 and 21 min, respectively. In human trophoblast cells exposed to progesterone (10 mumol/L) for 48 h, angiotensin AT1 receptor density was decreased by 49%, whereas estradiol (10 mumol/L) or chloroquine (100 mumol/L) treatment was ineffective. In the freshly isolated trophoblast cells initially treated with unlabeled angiotensin II (200 nmol/L) for 30 min, chloroquine was shown to decrease angiotensin AT1 receptor recycling by 73%, whereas estradiol and progesterone had no effect. These findings indicate that progesterone induces a down-regulation of the angiotensin AT1 receptor in human placenta and that the recycling of this receptor can be delayed by chloroquine.


Subject(s)
Angiotensin I/metabolism , Down-Regulation , Placenta/metabolism , Progesterone/pharmacology , Receptors, Angiotensin/drug effects , Receptors, Angiotensin/metabolism , Angiotensin II/drug effects , Angiotensin II/metabolism , Chloroquine/pharmacology , Female , Humans , Pregnancy , Trophoblasts/cytology , Trophoblasts/metabolism
17.
Placenta ; 17(2-3): 103-110, 1996.
Article in English | MEDLINE | ID: mdl-8730880

ABSTRACT

The recent discovery of a local renin-angiotensin system in trophoblastic tissues has raised many questions regarding its role in the physiology of normal gestation and its implications in the pathophysiology of hypertension during pregnancy. In this article, the authors first review the most interesting aspects of the chorioplacental renin-angiotensin system, dwelling on the tissue distribution of angiotensin II and its receptor subtypes in the placenta and fetal membranes of different species. The relationship between angiotensin II and other locally synthesized chorioplacental substances is also analysed and the therapeutic implications of phenomena observed in pregnancy-associated hypertension are discussed.


Subject(s)
Angiotensin II/physiology , Extraembryonic Membranes/physiology , Receptors, Angiotensin/physiology , Reproduction/physiology , Trophoblasts/physiology , Angiotensin II/classification , Animals , Female , Humans , Pregnancy , Pregnancy Complications/physiopathology , Pregnancy Complications/therapy , Species Specificity
18.
Biol Reprod ; 52(6): 1316-26, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7543296

ABSTRACT

Antisense oligodeoxyribonucleotide inhibition of gene expression was used to test whether the p60 form of the tumor necrosis factor alpha (TNF alpha) receptor (Rp60) is responsible for mediating the negative effect of TNF alpha on the development of rat blastocysts in vitro. The antisense oligonucleotide was designed to overlap the translation initiation codon of the TNF alpha Rp60 mRNA. Preliminary experiments showed that concentrations of oligonucleotides above 10 microM in the culture medium were embryotoxic over 24 h. When used at nontoxic concentrations (8 microM), antisense oligonucleotides specifically decreased the abundance of intact TNF alpha Rp60 transcripts by 80% within 3 h of exposure. In contrast to results with control embryos, mRNA for the second form of TNF alpha receptor, TNF alpha Rp80, was detected in blastocysts exposed to antisense oligonucleotides to TNF alpha Rp60. Antisense oligonucleotides to TNF alpha Rp60 blocked the 25-30% decrease in cell proliferation induced by 50 ng/ml TNF alpha added to a standard culture medium and by 5 ng/ml TNF alpha added to a medium that had been conditioned by rat uterine cells. Sense oligonucleotides had no such protective effect. Because uterine cells from diabetic rats secrete higher levels of TNF alpha than those from control rats, antisense oligonucleotides were also tested in a medium that had been conditioned by diabetic uterine cells (cell-secreted TNF alpha concentration was 50 pg/ml in this medium, and no exogenous TNF alpha was added). Addition of antisense oligonucleotides to TNF alpha Rp60 improved the quality of this medium with respect to cell proliferation but failed to correct the high frequency of dead cells observed in the blastocysts.


Subject(s)
Blastocyst/cytology , Cell Division/drug effects , Oligonucleotides, Antisense/pharmacology , Receptors, Tumor Necrosis Factor/genetics , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Base Sequence , Blastocyst/drug effects , DNA, Complementary/chemistry , Female , Molecular Sequence Data , Oligonucleotides, Antisense/toxicity , Polymerase Chain Reaction , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , RNA-Directed DNA Polymerase , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/pharmacology
19.
Diabetes ; 44(5): 531-6, 1995 May.
Article in English | MEDLINE | ID: mdl-7729611

ABSTRACT

Tumor necrosis factor (TNF) bioactivity was assessed in culture media conditioned with uterine cells collected from control or diabetic rats on days 5 and 8 of pregnancy. On both days, diabetic uterine cells released significantly more biologically active TNF than did control cells, and this activity was significantly decreased by the addition of anti-TNF-alpha antibodies but not by the addition of normal IgG when WEHI 164 cells were used as a target. When uterine tissues from day 5 or day 8 pregnant diabetic rats were tested by Northern blot analysis, TNF-alpha mRNAs were twofold more abundant than in control samples, but the difference was not statistically significant (P = 0.086 and 0.100, respectively). Immunohistochemical analysis of diabetic day 5 uterine sections revealed that most of the TNF-alpha synthesis occurs in the epithelium lining the uterine lumen. Finally, the growth of day-5 embryos in culture medium conditioned with day-5 diabetic uterine cells was significantly reduced when compared with that of embryos in medium conditioned with control cells. Embryonic development was markedly improved when anti-TNF-alpha antibodies were added to the diabetic-cell conditioned medium. Our data support the hypothesis that TNF-alpha may be implicated in the developmental deficiencies observed in preimplantation embryos from pregnant diabetic rats.


Subject(s)
Congenital Abnormalities/etiology , Diabetes Mellitus, Experimental/complications , Pregnancy in Diabetics , Tumor Necrosis Factor-alpha/physiology , Animals , Culture Media, Conditioned , Culture Techniques , Diabetes Mellitus, Experimental/physiopathology , Embryonic Development , Female , Immunohistochemistry , Pregnancy , Pregnancy in Diabetics/physiopathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Up-Regulation , Uterus/metabolism
20.
J Clin Endocrinol Metab ; 80(4): 1233-7, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7714093

ABSTRACT

A complete renin-angiotensin system has been shown to be present in human placenta, but its physiological role is poorly known. To investigate the implication of this system in the regulation of steroid hormone secretion, we studied the effect of angiotensin-II on the release of estradiol and progesterone from human placental explants. Our experiments showed that angiotensin-II stimulated estradiol secretion from term placental explants in a dose- and time-dependent fashion, although progesterone release was unaffected. Estradiol release induced by angiotensin-II (0.2 mumol/L) was blocked by angiotensin AT1 receptor antagonist losartan in a dose-dependent manner, suggesting the involvement of the AT1 receptor subtype in the process. On the contrary, the angiotensin AT2 receptor antagonist PD123319 (1 mumol/L) or the angiotensin AT2 receptor agonist CGP42112A (1 mumol/L) had no effect. Analysis of the amount of steroid hormones in the placental tissues incubated for 12 h showed that angiotensin-II increased estradiol production by 34% compared with the unstimulated explants, whereas the total levels of the estrogen precursor androstenedione and testosterone were decreased by 30-45% in the presence of the peptide, suggesting a stimulatory effect on the aromatization step. This hypothesis was reinforced by the absence of effect of angiotensin-II on both estradiol and testosterone concentrations in the placental explants pretreated with the aromatase inhibitor 4-hydroxyandrostenedione (25 mumol/L). Progesterone synthesis was not affected by angiotensin-II. The present study indicates that angiotensin-II induces the secretion of estradiol from human placenta through the angiotensin AT1 receptor subtype activation, and this effect seems to be linked to the stimulation of local androgen aromatization.


Subject(s)
Angiotensin II/pharmacology , Estradiol/metabolism , Placenta/metabolism , Receptors, Angiotensin/physiology , Culture Techniques , Dose-Response Relationship, Drug , Female , Humans , Pregnancy , Progesterone/metabolism , Receptors, Angiotensin/classification
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