Synaptotagmin 1 regulates cortical granule exocytosis during mouse oocyte activation.

Synaptotagmin 1 (Syt1) is an abundant and important presynaptic vesicle protein that binds Ca2+ for the regulation of synaptic vesicle exocytosis. Our previous study reported its localization and function on spindle assembly in mouse oocyte meiotic maturation. The present study was designed to investigate the function of Syt1 during mouse oocyte activation and subsequent cortical granule exocytosis (CGE) using confocal microscopy, morpholinol-based knockdown and time-lapse live cell imaging. By employing live cell imaging, we first studied the dynamic process of CGE and calculated the time interval between [Ca2+]i rise and CGE after oocyte activation. We further showed that Syt1 was co-localized to cortical granules (CGs) at the oocyte cortex. After oocyte activation with SrCl2, the Syt1 distribution pattern was altered significantly, similar to the changes seen for the CGs. Knockdown of Syt1 inhibited [Ca2+]i oscillations, disrupted the F-actin distribution pattern and delayed the time of cortical reaction. In summary, as a synaptic vesicle protein and calcium sensor for exocytosis, Syt1 acts as an essential regulator in mouse oocyte activation events including the generation of Ca2+ signals and CGE.

Role of mammalian target of rapamycin signaling pathway in regulation of fatty acid oxidation in a preeclampsia-like mouse model treated with pravastatin.

BACKGROUND: Fatty acid oxidation (FAO) disorder is involved in the pathogenesis of some cases of preeclampsia (PE). Several studies show that mammalian target of rapamycin (mTOR) signaling pathway is related to FAO. Pravastatin (Pra) can promote FAO in Nω-nitro-L-arginine methyl ester (L-NAME) PE-like mouse model in our previous study. This study aimed to investigate the effect of mTOR signaling pathway in PE-like model treated with Pra. METHODS: Pregnant mice were randomly injected with L-NAME as PE-like model group or saline as control group respectively, from gestational 7th to 18th day. Giving Pra (L-NAME + Pra, Control + Pra, n = 8) or normal saline (NS; L-NAME + NS, Control + NS, n = 8) from gestational 8th to 18th day, the mice were sacrificed on day 18 and their liver and placental tissues were collected. Then the activation of mTOR and its substrates in the liver and placenta were detected. And the association between mTOR activation and serum free fatty acid (FFA) levels and the expression of long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) were evaluated using Pearson correlation test. Differences between groups were analyzed using independent t-test or one-way analysis of variance (ANOVA). RESULTS: Both in the maternal liver and placenta, the activation of mTOR protein and its effect on substrates increased significantly in the L-NAME + NS group and decreased significantly in the L-NAME + Pra group. The p-mTOR/mTOR protein ratio decreased in the L-NAME + Pra group significantly than that in the L-NAME + NS group both in liver and placenta (liver: 0.74 ±â€Š0.08 vs. 0.85 ±â€Š0.06, t = 2.95, P < 0.05; placenta: 0.63 ±â€Š0.06 vs. 0.77 ±â€Š0.06, t = 4.64, P < 0.05). The activation of mTOR protein in the liver and placenta negatively correlated with the expression of LCHAD in the L-NAME + NS group (liver: r = -0.745, P < 0.05; placenta: r = -0.833, P < 0.05) and that in the maternal liver negatively correlated with the expression of LCHAD (r = -0.733, P < 0.05) and positively with the serum FFA levels (r = 0.841, P < 0.05) in the L-NAME + Pra group. CONCLUSION: The inhibition of mTOR signaling pathway might be involved in the regulation of FAO in mouse model treated with Pra.

Different Effects of Pravastatin on Preeclampsia-like Symptoms in Different Mouse Models.

BACKGROUND: Pravastatin (Pra) exerts protective effects on preeclampsia. Preeclampsia is a multifactorial and pathogenic pathway syndrome. The present study compared the effects of Pra on clinical manifestations of preeclampsia in different pathogenic pathways. METHODS: Two different preeclampsia-like mouse models used in this study were generated with Nω-nitro-L-arginine methyl ester (L-NAME) and used lipopolysaccharide (LPS) from day 7 of gestation, respectively. Pra treatment was administered on day 2 after the models were established in each group (L-NAME + Pra, LPS + Pra, and Control + Pra, n = 8) or normal saline (NS) for the control group (L-NAME + NS, LPS + NS, and Control + NS, n = 8). Maternal weight, serum lipids, the histopathological changes, and lipid deposition in the liver and placenta were observed. The pregnancy outcomes were compared. The blood pressure analysis was carried out on repeated measurements of variance. Student's t-test was used for comparing the two groups. The enumeration data were compared by Chi-square test. RESULTS: The mean arterial pressure (MAP) and 24-h urinary protein in the L-NAME + NS and LPS + NS groups were significantly higher than the Control + NS group (F = 211.05 and 309.92 for MAP, t = 6.63 and 8.63 for 24-h urinary protein; all P < 0.05) and reduced in the L-NAME + Pra group as compared to the L-NAME + NS group (F = 208.60 for MAP, t = 6.77 for urinary protein; both P < 0.05). Urinary protein was decreased in the LPS + Pra group as compared to the LPS + NS group (t = 5.33; P < 0.05), whereas MAP had no statistical significance (F = 3.37; P > 0.05). Compared to the Control + NS group, the placental efficiency in the L-NAME + NS and LPS + NS groups decreased significantly (t = 3.09 and 2.89, respectively; both P < 0.05); however, no significant difference was observed in L-NAME + Pra and LPS + Pra groups (t = 1.37 and 0.58, respectively; both P > 0.05). Free fatty acid was elevated in the L-NAME + NS group as compared to the Control + NS group (t = 3.99; P < 0.05) at day 18 of pregnancy and decreased in the L-NAME + Pra group as compared to the L-NAME + NS group (t = 3.28; P < 0.05); however, no significant change was observed in the LPS model (F = 0.32; P > 0.05). CONCLUSION: This study suggested that Pra affected the clinical manifestations differently in preeclampsia-like mouse models generated in various pathogenic pathways.

Effects of Rapamycin on Clinical Manifestations and Blood Lipid Parameters in Different Preeclampsia-like Mouse Models.

BACKGROUND: The pathogenesis of some types of preeclampsia is related to fatty acid oxidation disorders. Rapamycin can regulate fatty acid metabolism. This study aimed to investigate the effects of rapamycin on the clinical manifestations and blood lipid parameters in different preeclampsia-like mouse models. METHODS: Two preeclampsia-like mouse models and a control group were established: L-NA (injected with Nω-nitro-L-arginine methyl ester), LPS (injected with lipopolysaccharide), and the control group with normal saline (NS). The mouse models were established at preimplantation (PI), early- and late-pregnancy (EP, LP) according to the time of pregnancy. The administration of rapamycin (RA; L-NA+RA, LPS+RA, and NS+RA) or vehicle as controls (C; L-NA+C, LPS+C, NS+C) were followed on the 2nd day after the mouse models' establishment. Each subgroup consisted of eight pregnant mice. The mean arterial pressure (MAP), 24-h urinary protein, blood lipid, fetus, and placental weight were measured. The histopathological changes and lipid deposition of the liver and placenta were observed. Student's t-test was used for comparing two groups. Repeated measures analysis of variance was used for blood pressure analysis. Qualitative data were compared by Chi-square test. RESULTS: The MAP and 24-h urinary protein in the PI, EP, and LP subgroups of the L-NA+C and LPS+C groups were significantly higher compared with the respective variables in the NS+C group (P < 0.05). The preeclampsia-like mouse models were established successfully. There was no significant difference in the MAP between the PI, EP, and LP subgroups of the L-NA+RA and L-NA+C groups and the LPS+RA and LPS+C groups. The 24-h urine protein levels in the PI and EP subgroups of the L-NA+RA group were significantly lower compared with the respective levels in the L-NA+C groups (1037 ± 63 vs. 2127 ± 593 µg; 976 ± 42 vs. 1238 ± 72 µg; bothP < 0.05), also this effect appeared similar in the PI and EP subgroups of the LPS+RA and LPS+C groups (1022 ± 246 vs. 2141 ± 432 µg; 951 ± 41 vs. 1308 ± 30 µg; bothP < 0.05). The levels of serum-free fatty acid (FFA) in the PI and EP subgroups of the L-NA+RA groups were significantly lower compared with the respective levels in the L-NA+C group (2.49 ± 0.44 vs. 3.30 ± 0.18 mEq/L; 2.23 ± 0.29 vs. 2.84 ± 0.14 mEq/L; bothP < 0.05). The levels of triglycerides (TG) and total cholesterol in the PI subgroup of the L-NA+RA group were significantly lower compared with the respective levels in the L-NA+C (1.51 ± 0.16 vs. 2.41 ± 0.37 mmol/L; 2.11 ± 0.17 vs. 2.47 ± 0.26 mmol/L; bothP < 0.05), whereas high-density lipoprotein serum concentration was significantly higher (1.22 ± 0.19 vs. 0.87 ± 0.15 mmol/L;P < 0.05) and low-density lipoprotein serum concentration did not exhibit a significant difference. There were no significant differences in the FFA of the PI, EP, and LP subgroups between the LPS+RA and the LPS+C groups. The levels of TG in the PI subgroup of the LPS+RA group were significantly lower compared with the respective levels in the LPS+C group (0.97 ± 0.05 vs. 1.22 ± 0.08 mmol/L;P < 0.05). CONCLUSION: Rapamycin can improve clinical manifestations and blood lipid profile in part of the preeclampsia-like mouse models.