| Lysosome is an important organelle that exists in the cells of all protozoa and multicellular animals,to isolate macromolecules submitted through endocytosis and autophagy for degradation and recycling.Large-scale macromolecular renewal is very important for the growth and development of mammalian oocytes.However,the functional role of lysosomes in meiotic maturation of mammalian oocytes is still unclear.In this study,we studied the role and possible mechanisms of lysosomes on in vitro maturation of porcine oocytes,by treating porcine cumulusoocyte complex(COC)with chloroquine(CQ),a specific lysosome inhibitor,providing novel insights intorelated studies.The main results of this study are as follows:(1)Detection of lysosomes by fluorescence staining showed that lysosomes existed during every stages of maturing oocytes and mainly distributed in the cortical and peripheral cytoplasmic regions of ooplasm.Quantitative analysis showed that the relative fluorescence intensity of lysosomes remained s Tab.from GV to GVBD,but then significantly decreased from GVBD to MII(1.12 vs.0.83,P <0.001).These data indicate that lysosomes existed and changed dynamically during porcine oocyte maturation.(2)Adding different concentrations of specific lysosome inhibitor CQ(0,25,35,45μM)to in vitro mature COCs did not change the rate of oocytes with the first polar body extrusion,but significantly increased the rate of MII oocytes with abnormal status of nuclear chromosomes(6.2% of control vs.34.7%,39.9%,71.2%,P < 0.05),These results suggest that the inhibition of lysosomes by CQ during in vitro maturation of porcine oocytes induces nuclear abnormalities in porcine oocytes in a dose-dependent manner.(3)Detection of the spindle assembly,oxidative stress level and early apoptosis of oocytesusing fluorescence staining showed that 35μM CQ treatment induced abnormal microtubule morphology(34.7% of control vs.57.9%,P < 0.001)and destroyed the normal distribution of microfilaments on the cell membrane(1.00 of control vs.0.60,P < 0.001),but did not affect the distribution of microfilaments within the cytoplasm and p-ERK1/2 level.At the same time,inhibition of lysosome could significantly increase the percentage of oocytes with early apoptotic signals(9.1% of control vs.19.2%,P < 0.01)and reduce the m RNA level of anti-apoptosis gene Bcl2(1.00 vs.0.16,P < 0.01),but there was no significant change for ROS level.In summary,inhibition of lysosomes during oocyte maturation in vitro caused abnormal cytoskeleton assembly and increased the proportion of oocytes with early apoptosis.(4)CQ treatments to inhibit lysosomes during in vitro maturation of oocytes followed by parthenogenetic activation and in vitro fertilization of mature oocytes showed that for parthenogenetic embryos,the dealth rates in CQ treatment groups significantly increased(Control vs.35 μM vs.45 μM: 2.75% vs.24.50% vs.50.59%,P < 0.01),the cleavage rates decreased significantly(Control vs.35 μM vs.45 μM: 89.90% vs.76.60% vs.75.51%,P < 0.05),the blastocyst rates decreased significantly(Control vs.35 μM vs.45 μM: 61.05% vs.33.70% vs.33.92%,P < 0.001),and the cell number per blastocyst decreased significantly(Control vs.35μM vs.45 μM: 35.7 vs.25.0 vs.21.2).At the same time,the cleavage rates of IVF embryos decreased significantly(Control vs.35 μM vs.45 μM: 62.31% vs.44.21% vs.32.74%,P < 0.05),the fragmentation rates of embryos increased significantly(Controlvs.35 μM vs.45 μM: 4.64% vs.16.49% vs.23.83%,P < 0.05),and the blastocyst rates decreased significantly(Control vs.25 μM vs.35 μM vs.45 μM: 23.52% vs.16.41% vs.11.93% vs.5.54%,P <0.05),and the cell number per blastocyst decreased significantly(Control vs.35 μM vs.45 μM: 34.8 vs.21.3 vs.16.5,P < 0.001).In summary,inhibition of lysosomes in oocytes may affect their subsequent development potential after parthenogenetic activation or in vitro fertilization by causing embryo death or inducing embryo fragmentation.(5)Detection of the expression levels of autophagy-related m RNA and proteins showed that the level of LC3 protein in oocytes treated with 35 μM CQ increased significantly(0.60 of control vs.5.8,P < 0.001),and the m RNA levels of lysosome membrane markers Lamp1 and 2 decreased significantly(1.00 vs.0.46,P < 0.05;1.00 vs.0.14,P < 0.001).In summary,inhibition of lysosomes during in vitro maturation of porcine oocytes could affect the autophagy function.Taken together,lysosomes exist and change dynamically during in vitro maturation of porcine oocytes,which play an important role for oocyte meiotic maturation.Lysosomes affect the nuclear maturation and subsequent development of pig oocytes mainly by regulating cytoskeleton assembly and autophagy pathway. |
