ABSTRACT
OBJECTIVE: GnRH-agonist (GnRH-Ag) used in controlled ovarian hyperstimulation (COH) for IVF-ET has been known to affect directly on apoptosis of human ovarian cells, but its mechanism is not clearly understood. Therefore, the purpose of the present study was to investigate whether caspase-3 and -9 activation and poly-(ADP-ribose)-polymerase (PARP) cleavage are involved in the mechanism(s) by which GnRH-Ag induces apoptosis in human granulosa-luteal cells. METHODS: Human granulosa-luteal cells collected from IVF-ET patients were cultured and treated with 10(-6) M GnRH-Ag or saline as a control. To access apoptosis in the cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay and DNA fragmentation analysis were preformed 24 h after the treatment. Activity of caspase-3 and -9 in the cells was examined by using a fluorogenic substrate. Caspase-3 and -9 activation and poly (ADP-ribose) polymerase (PARP) cleavage were analyzed by Western blotting. RESULTS: DNA fragmentation in the cells increased in the higher concentration over 10(-6) M GnRH-Ag. In the result of TUNEL assay, the rate of apoptotic cells in GnRH-Ag treatment increased significantly compared with that of saline treatment (p<0.05). The activity of caspase-3 and -9 investigated by using a fluorogenic substrate increased only in the apoptotic cells. In Western blot analysis, the cells treated with GnRH-Ag revealed an increase in active forms of caspase-3 and -9 compared with those of the saline treatment. In addition, cleavage of PARP also increased in the cells treated with GnRH-Ag. CONCLUSION: These results suggest that activation of caspase-3 and -9 and cleavage of PARP might be involved in apoptosis of human granulosa-luteal cells induced by GnRH-Ag.
Subject(s)
Female , Humans , Apoptosis , Blotting, Western , Caspase 3 , DNA Fragmentation , DNA Nucleotidylexotransferase , Fluorescent Dyes , In Situ Nick-End Labeling , Luteal CellsABSTRACT
OBJECTIVE: To investigate whether GnRH-agonist (GnRH-Ag) using in IVF-ET affects apoptosis of human granulosa-luteal cells and expression of peripheral benzodiazepine receptor (PBR) protein involved in the apoptosis of the cells. METHODS: Granulosa-luteal cells obtained during oocyte retrieval were cultured and treated with 10(-5) M GnRH-Ag. Apoptosis of the cells by the treatment was confirmed using DNA fragmentation analysis 24 h after culture. The presence of PBR protein within the cells was examined by immunofluorescence staining and the expression of the protein was analyzed by Western blotting. In addition, it was measured for progesterone and nitric oxide (NO) produced by granulosa-luteal cells after GnRH-Ag treatment. To evaluate the relationship between NO production and PBR expression, sodium nitroprusside (SNP) as a NO donor was added in media and investigated the expression of PBR protein by Western blotting. RESULTS: Apoptosis increased in the granulosa-luteal cells 24 h after GnRH-Ag treatment, whereas the expression of PBR protein significantly decreased. Furthermore, the production of progesterone and nitric oxide (NO) by the cells significantly fell from 12 h after the treatment. In the results of Western blotting after SNP treatment, the expression of PBR protein increased in the treatment with SNP alone to the granulosa-luteal cells, but was suppressed in the treatment with GnRH-Ag and SNP. Additionally, the staining result of PBR protein in the cells showed the even distribution of it through the cell. CONCLUSION: These results demonstrate that GnRH-Ag treatment induces apoptosis, decreasing expression of PBR protein and NO production in human granulosa-luteal cells. The present study suggests that one of the apoptosis mechanism of human granulosa-luteal cells by GnRH-Ag might be a signal transduction pathway via NO and PBR.
Subject(s)
Female , Humans , Apoptosis , Blotting, Western , DNA Fragmentation , Fluorescent Antibody Technique , Luteal Cells , Nitric Oxide , Nitroprusside , Oocyte Retrieval , Progesterone , Receptors, GABA-A , Signal Transduction , Tissue DonorsABSTRACT
To examine the electrophysiological properties of luteal cells and the relationship between membrane potential and luteal steroidogenic capacity, the membrane potential of luteal cells and the luteal steroidogenesis were measured under different ionic conditions following treatment with various drugs and gonadotropins. The membrane potential of luteal cells did not vary throughout the estrous cycle and was -55 +/- 1 mV. The membrane potential was highly dependent upon the external K+ concentration and was depolarized by the deprivation of external Ca2+, however) there seemed to be a lower K+ permeability in luteal membranes as the presence of 10-9 M valinomycin, a K+ ionophore Caused hyperpolarization from -55 to -91 mV. Luteal progestin production was increased in a high K+ solution but not m a Ca2+-free solution indicating that Ca2+ may be essential for steroid synthesis and/or secretion by luteal cells. Gonadotropins and ouabain induced a depolarization of the membrane potential and stimulated luteal steroidogenesis; however; prostaglandin F2alpha stimulated only steroidogenesis without any changes in membrane potential. These results suggest that the relationship between steroidogenesis and the changes in membrane potential by drugs and gonadotropins is still obscure and remains to be eluridated. The relationship between membrane potential and steroidogenesis in the luteal cell may be dependent upon the availability of intracelluar Ca2+.
Subject(s)
Female , Rats , Animals , Corpus Luteum/metabolism , Estrus/metabolism , Ions , Luteal Cells/metabolism , Membrane Potentials , Rats, Inbred Strains , Steroids/biosynthesisABSTRACT
Ten pregnant rats randomly chosen from the total twenty rats on day 9 ofprognancy were injected a high dosage of LRH-A.The other ten were used ascontrol group.After one day and two days the ultrastructure of the luteal cell wasobserved by transmission electron microscope.Compared with the control group,theexperimental group showed an increase of lipid droplets,and a lot of large lipiddroplets appeared.The smooth endoplasmic reticulum decreased,dissolved and lostits regular array.Mitochondria degenerated,Autophagocytic vacuole,lysosome,myelin figure,multivesicular body as well as residual body increased.Golgi appa-ratus swelled in the first day after injection,and then shrank.Microvilli on thesurface of the cell decreased.The above results all suggest that with the injection of high dosage of LRH-Ainto the pregnant rats,luteal cell shows morphologic regression and decrease insecretive function.