ABSTRACT
ObjectiveThe mechanism of histone phosphorylation modification in oocyte meiosis is less studied. This study is designed to investigate the pattern of histone H3 phophorylation and regulation of maturation process in the porcine oocytes.MethodsThe histone H3Ser10 (H3S10) phosphorylation expression was examined on the porcine oocyte meiotic process. The porcine cumulus oocyte complexes (COCs) were divided into four groups, one group was cultured as control group, and the other 3 groups were supplemented with 5, 10, and 30 μmol/L ZM447439, and cultured in vitro for 27 h, respectively, 5, 10, and 30 μmol/L ZM4474349 treatment group. The proportion of each meiotic stage was counted. The phosphorylation pattern of histone H3S10 and the expression level of protein kinase Aurora B were detected at the porcine oocytes.ResultsCompared with histone H3S10 phosphorylation level of oocyte GVBD phase, the MI and AI phases were significantly increased (P<0.05), and H3S10 phosphorylation level of AI phase was remarkedly higher than that of MII phase (P<0.05). Compared with the control group, the proportion of oocytes at the GVBD phase in the 10 and 30 μmol/L ZM4447439 treatment group [(32.14±0.51)%, (95.34±0.59)%]was higher than that of the control group [(2.56±0.03)%, P<0.05], the proportion of oocytes at the MI phase [(66.88±0.13)%, (4.66±0.04)%] significantly decreased than that of the control group [(87.42±0.14)%, P<0.05], and the proportion of oocytes at the AI stage [(1.01±0.03)%, (0.000±0.00)%] significantly decreased compared with the control group[(10.02 ± 0.21)%, P<0.05]. Compared with the control group (0), oocytes H3S10 dephosphorylation modification ratio in the 10 μmol/L and 30 μmol/L ZM4474349 treatment group [(35.2±0.39)%, (95.4±0.65)%]significantly increased (P<0.05). Compared with the control group, the relative expression level of Aurora B in the 10 and 30 μmol/L ZM4447439 treatment group was significantly reduced (P<0.05).ConclusionHstone H3S10 phosphorylation plays arolein the maturation of mammalian oocytes. AuroraB kinase inhibitors (ZM447439) treatment can reduce H3S10 phosphorylation and Aurora B expression level and lead to oocytesmaturation disorder.
ABSTRACT
Background At present,a new drug,platelet-derived growth factor-BB (PDGF-BB),as a drug knife for the treatment of glaucoma is under study to expect it directly working on the trabecular meshwork without disrupting the normal physiological structure of anterior chamber angle,clearing the trabecular meshwork aqueous outflow channel so as to achieve the purpose of lowering intraocular pressure.Objective This study aimed to investigate the effect of PDGF-BB on matrix metalloproteinase-2 (MMP-2) expression in cultured bovine trabecular meshwork cells.Methods Trabcular tissue was obtained from fresh bovine eyes,and trabcular meshwork cells were cultured and passaged using explant method.Cultured cells were identified by morphological evaluation and neuronspecific enolase (NSE) staining.The third generation of cells were inoculated to 6-well plate,and different concentrations (0,5.0,12.5,25.0 μg/L) of PDGF-BB was added into the medium for 2 hours.Expression levels (A value) of MMP-2 mRNA (MMP-2 mRNA/β-actin) and protein in the cells were assayed by reverse transcription polymerase chain reaction (RT-PCR) and immunochemistry,respectively.Results Trabcular meshwork cells appeared 5-9 days after cultured.The third generation of cells presented with many process and showed the green influence in cytoplasm.MMP-2 mRNA/β-actin value (A) was 0.127 ± 0.026,0.147 ± 0.045,0.178 ± 0.053 and 0.222±0.062 in the O,5.0,12.5,25.0 μg/L PDGF-BB group,respectively,showing a significant difference among them (F =56.71,P＜0.05),and the MMP-2 mRNA/β3-actin value in the 5.0,12.5,25.0 μg/L PDGF-BB group was elevated in comparison with that of the 0 μg/L PDGF-BB group (all P＜0.05).The expression value (A value) of PDGF-BB protein in the cells was 446.12±13.81,1444.65±54.64,2086.18±73.18,3488.65±25.98 in the 0,5.0,12.5,25.0 μg/L PDGF-BB group,respectively,with a significant difference among the four groups (F=213.12,P＜0.01),and the expression value (A value) of PDGF-BB protein was gradually increased with the ascend of concentration of PDGF-BB(all P＜0.05).Conclusions PDGF-BB can promote the expression of MMP-2 in bovine trabcular meshwork cells in vitro in concentration-dependent manner.