Background Atresia and degeneration,a follicular developmental fate that reduces female fertility and is triggered by granulosa cell(GC)apoptosis,have been induced by dozens of miRNAs.Here,we report a miRNA,miR-423,th...Background Atresia and degeneration,a follicular developmental fate that reduces female fertility and is triggered by granulosa cell(GC)apoptosis,have been induced by dozens of miRNAs.Here,we report a miRNA,miR-423,that inhibits the initiation of follicular atresia(FA),and early apoptosis of GCs.Results We showed that miR-423 was down-regulated during sow FA,and its levels in follicles were negatively correlated with the GC density and the P4/E2 ratio in the follicular fluid in vivo.The in vitro gain-of-function experiments revealed that miR-423 suppresses cell apoptosis,especially early apoptosis in GCs.Mechanically speaking,the miR-423 targets and interacts with the 3’-UTR of the porcine SMAD7 gene,which encodes an apoptosis-inducing factor in GCs,and represses its expression and pro-apoptotic function.Interestingly,FA and the GC apoptosis-related lncRNA NORHA was demonstrated as a ceRNA of miR-423.Additionally,we showed that a single base deletion/insertion in the miR-423 promoter is significantly associated with the number of stillbirths(NSB)trait of sows.Conclusion These results demonstrate that miR-423 is a small molecule for inhibiting FA initiation and GC early apoptosis,suggesting that treating with miR-423 may be a novel approach for inhibiting FA initiation and improving female fertility.展开更多
Ovarian follicle development is associated with the physiological functions of granulosa cells(GCs),including proliferation and apoptosis.The level of miR-24-3p in ovarian tissue of high-yielding Yorkshire×Landra...Ovarian follicle development is associated with the physiological functions of granulosa cells(GCs),including proliferation and apoptosis.The level of miR-24-3p in ovarian tissue of high-yielding Yorkshire×Landrace sows was significantly higher than that of low-yielding sows.However,the functions of miR-24-3p on GCs are unclear.In this study,using flow cytometry,5-ethynyl-2′-de-oxyuridine(EdU)staining,and cell count,we showed that miR-24-3p promoted the proliferation of GCs increasing the proportion of cells in the S phase and upregulating the expression of cell cycle genes,moreover,miR-24-3p inhibited GC apoptosis.Mechanistically,on-line prediction,bioinformatics analysis,a luciferase reporter assay,RT-qPCR,and Western blot results showed that the target gene of miR-24-3p in proliferation and apoptosis is cyclin-dependent kinase inhibitor 1B(P27/CDKN1B).Furthermore,the effect of miR-24-3p on GC proliferation and apoptosis was attenuated by P27 overexpression.These findings suggest that miR-24-3p regulates the physiological functions of GCs.展开更多
Background:SMAD family proteins(SMADs)are crucial transcription factors downstream of transforming growth factor beta(TGF-ß)/SMAD signaling pathways that have been reported to play a pivotal role in mammalian rep...Background:SMAD family proteins(SMADs)are crucial transcription factors downstream of transforming growth factor beta(TGF-ß)/SMAD signaling pathways that have been reported to play a pivotal role in mammalian reproduction.However,the role of SMAD family member 8(SMAD8,also known as SMAD9),a member of the SMAD family,in mammalian reproduction remains unclear.Methods:We employed RNA interference techniques to knock down Smad8 expression in mouse granulosa cells(GCs)to investigate the effects of Smad8 on GC growth and steroidogenesis.Results:Our findings revealed a significant decrease in the proliferative capacity and a substantial increase in the apoptosis rate of GCs after transfection with Smad8-siRNA for 48 h.Subsequent hormone assays demonstrated a significant decrease in estradiol(E2)levels,whereas progesterone(P4)remained unchanged.Further mechanistic analysis showed that the mRNA expression of proliferating cell nuclear antigen(Pcna),Cyclin D2,cell cycle-dependent kinase 4(Cdk4),B-cell lymphoma-2(Bcl-2),estrogen receptor(Er),luteinizing hormone receptor(Lhr)and cytochrome P450 family 19 subfamily A member 1(Cyp19a1)significantly decreased.Conversely,the mRNA of cysteine aspartate proteinase 3(Caspase 3)significantly increased,wheras Bcl2-associated X(Bax),folliclestimulating hormone receptor(Fshr)and cytochrome P450 family 11 subfamily A member 1(Cyp11a1)remained unchanged compared to the controls.Conclusion:This study indicates that Smad8 knockdown inhibits cell proliferation,promotes apoptosis,reduces Er and Lhr transcription,and decreases E2 production in mouse GCs.These findings suggest that Smad8 may serve as a novel genetic marker for mammalian reproduction.展开更多
Background Clock circadian regulator(CLOCK)is a core factor of the mammalian biological clock system in regulat-ing female fertility and ovarian physiology.However,CLOCK’s specific function and molecular mechanism in...Background Clock circadian regulator(CLOCK)is a core factor of the mammalian biological clock system in regulat-ing female fertility and ovarian physiology.However,CLOCK’s specific function and molecular mechanism in porcine granulosa cells(GCs)remain unclear.In this study,we focused on CLOCK’s effects on GC proliferation.Results CLOCK significantly inhibited cell proliferation in porcine GCs.CLOCK decreased the expression of cell cycle-related genes,including CCNB1,CCNE1,and CDK4 at the mRNA and protein levels.CDKN1A levels were upregulated by CLOCK.ASB9 is a newly-identified target of CLOCK that inhibits GC proliferation;CLOCK binds to the E-box element in the ASB9 promoter.Conclusions These findings suggest that CLOCK inhibits the proliferation of porcine ovarian GCs by increasing ASB9 level.展开更多
Background During the transition period,the insufficient dry matter intake and a sharply increased in energy consumption to produce large quantities of milk,high yielding cows would enter a negative energy balance(NEB...Background During the transition period,the insufficient dry matter intake and a sharply increased in energy consumption to produce large quantities of milk,high yielding cows would enter a negative energy balance(NEB)that causes an increase in ketone bodies(KBs)and decrease in reproduction efficiency.The excess concentrations of circulating KBs,represented byβ-hydroxybutyric acid(BHBA),could lead to oxidative damage,which potentially cause injury to follicular granulosa cells(fGCs)and delayed follicular development.Sirtuin 3(Sirt3)regulates mitochondria reactive oxygen species(mitoROS)homeostasis in a beneficial manner;however,the molecular mechanisms underlying its involvement in the BHBA-induced injury of fGCs is poorly understood.The aim of this study was to explore the protection effects and underlying mechanisms of Sirt3 against BHBA overload-induced damage of fGCs.Results Our findings demonstrated that 2.4 mmol/L of BHBA stress increased the levels of mitoROS in bovine fGCs.Further investigations identified the subsequent mitochondrial dysfunction,including an increased abnormal rate of mitochondrial architecture,mitochondrial permeability transition pore(MPTP)opening,reductions in mitochondrial membrane potential(MMP)and Ca^(2+)release;these dysfunctions then triggered the caspase cascade reaction of apoptosis in fGCs.Notably,the overexpression of Sirt3 prior to treatment enhanced mitochondrial autophagy by increasing the expression levels of Beclin-1,thus preventing BHBA-induced mitochondrial oxidative stress and mitochondrial dysfunction in fGCs.Furthermore,our data suggested that the AMPK-mTOR-Beclin-1 pathway may be involved in the protective mechanism of Sirt3 against cellular injury triggered by BHBA stimulation.Conclusions These findings indicate that Sirt3 protects fGCs from BHBA-triggered injury by enhancing autophagy,attenuating oxidative stress and mitochondrial damage.This study provides new strategies to mitigate the fGCs injury caused by excessive BHBA stress in dairy cows with ketosis.展开更多
Objective:To investigate the effect of abnormal ovarian granulosa cell metabolism on in vitro fertilization and embryo transfer(IVF-ET)outcomes in obese polycystic ovary syndrome(PCOS)patients.Methods:Patients with PC...Objective:To investigate the effect of abnormal ovarian granulosa cell metabolism on in vitro fertilization and embryo transfer(IVF-ET)outcomes in obese polycystic ovary syndrome(PCOS)patients.Methods:Patients with PCOS who met the study criteria were screened according to the inclusion criteria.A total of 32 patients with obese PCOS were recruited into the study group,and 39 patients with non-obese PCOS were recruited into the control group.The general data(age,body mass index,and years of infertility),insulin resistance index(HOMA-IR),follicle-stimulating hormone(FSH),luteinizing hormone(LH),granulosa cell mitochondrial function,and IVF-ET outcome of patients in the study group and control group were retrospectively analyzed.Results:The differences in age and years of infertility between the study group and the control group were insignificant(P>0.05),and the body mass index(BMI)of the study group and control group was 30.5±1.24 kg/m2 and 22.3±1.12 kg/m2,respectively,in which the difference was statistically significant(P<0.05);the HOMA-IR of the study group was significantly higher than that of the control group(P<0.05);the reactive oxygen species(ROS)in the study group was significantly higher than that in the control group(P<0.05),and the ATP content in the study group was significantly lower than that in the control group(P<0.05);comparing the FSH and LH levels between the two groups,the difference was not statistically significant(P>0.05);the rate of IVF-ET failure was significantly higher in the study group than in the control group.Conclusion:PCOS is a complex endocrine disorder,and obesity is one of the independent risk factors for the development of PCOS.展开更多
基金supported by the National Key R&D Program of China(2022YFD1600903)the National Natural Science Foundation of China(32072693)the College Students’Innovative Entrepreneurial Training Plan Program(202110307028).
文摘Background Atresia and degeneration,a follicular developmental fate that reduces female fertility and is triggered by granulosa cell(GC)apoptosis,have been induced by dozens of miRNAs.Here,we report a miRNA,miR-423,that inhibits the initiation of follicular atresia(FA),and early apoptosis of GCs.Results We showed that miR-423 was down-regulated during sow FA,and its levels in follicles were negatively correlated with the GC density and the P4/E2 ratio in the follicular fluid in vivo.The in vitro gain-of-function experiments revealed that miR-423 suppresses cell apoptosis,especially early apoptosis in GCs.Mechanically speaking,the miR-423 targets and interacts with the 3’-UTR of the porcine SMAD7 gene,which encodes an apoptosis-inducing factor in GCs,and represses its expression and pro-apoptotic function.Interestingly,FA and the GC apoptosis-related lncRNA NORHA was demonstrated as a ceRNA of miR-423.Additionally,we showed that a single base deletion/insertion in the miR-423 promoter is significantly associated with the number of stillbirths(NSB)trait of sows.Conclusion These results demonstrate that miR-423 is a small molecule for inhibiting FA initiation and GC early apoptosis,suggesting that treating with miR-423 may be a novel approach for inhibiting FA initiation and improving female fertility.
基金supported by the National Natural Science Foundation of China(32272849)the National Key R&D Program of China(2021YFF1000602)the earmarked fund for CARS-35-PIG。
文摘Ovarian follicle development is associated with the physiological functions of granulosa cells(GCs),including proliferation and apoptosis.The level of miR-24-3p in ovarian tissue of high-yielding Yorkshire×Landrace sows was significantly higher than that of low-yielding sows.However,the functions of miR-24-3p on GCs are unclear.In this study,using flow cytometry,5-ethynyl-2′-de-oxyuridine(EdU)staining,and cell count,we showed that miR-24-3p promoted the proliferation of GCs increasing the proportion of cells in the S phase and upregulating the expression of cell cycle genes,moreover,miR-24-3p inhibited GC apoptosis.Mechanistically,on-line prediction,bioinformatics analysis,a luciferase reporter assay,RT-qPCR,and Western blot results showed that the target gene of miR-24-3p in proliferation and apoptosis is cyclin-dependent kinase inhibitor 1B(P27/CDKN1B).Furthermore,the effect of miR-24-3p on GC proliferation and apoptosis was attenuated by P27 overexpression.These findings suggest that miR-24-3p regulates the physiological functions of GCs.
基金supported by the High-Level Talent Research Start-Up Funds of West Anhui University(No.WGKQ2021031)Key Project of Natural Science Foundation of Anhui Province of China(No.2108085QC136)+2 种基金Key Project of Quality Engineering in Higher Education Institutions of Anhui Province(No.2020jyxm2128)National College Student Innovation and Entrepreneurship Training Program(No.202110370093)Innovation and Entrepreneurship Training Program for College Students of Anhui Province(No.S202010376114).
文摘Background:SMAD family proteins(SMADs)are crucial transcription factors downstream of transforming growth factor beta(TGF-ß)/SMAD signaling pathways that have been reported to play a pivotal role in mammalian reproduction.However,the role of SMAD family member 8(SMAD8,also known as SMAD9),a member of the SMAD family,in mammalian reproduction remains unclear.Methods:We employed RNA interference techniques to knock down Smad8 expression in mouse granulosa cells(GCs)to investigate the effects of Smad8 on GC growth and steroidogenesis.Results:Our findings revealed a significant decrease in the proliferative capacity and a substantial increase in the apoptosis rate of GCs after transfection with Smad8-siRNA for 48 h.Subsequent hormone assays demonstrated a significant decrease in estradiol(E2)levels,whereas progesterone(P4)remained unchanged.Further mechanistic analysis showed that the mRNA expression of proliferating cell nuclear antigen(Pcna),Cyclin D2,cell cycle-dependent kinase 4(Cdk4),B-cell lymphoma-2(Bcl-2),estrogen receptor(Er),luteinizing hormone receptor(Lhr)and cytochrome P450 family 19 subfamily A member 1(Cyp19a1)significantly decreased.Conversely,the mRNA of cysteine aspartate proteinase 3(Caspase 3)significantly increased,wheras Bcl2-associated X(Bax),folliclestimulating hormone receptor(Fshr)and cytochrome P450 family 11 subfamily A member 1(Cyp11a1)remained unchanged compared to the controls.Conclusion:This study indicates that Smad8 knockdown inhibits cell proliferation,promotes apoptosis,reduces Er and Lhr transcription,and decreases E2 production in mouse GCs.These findings suggest that Smad8 may serve as a novel genetic marker for mammalian reproduction.
基金supported by National Natural Science Foundation of China (32272849)China Agriculture Research System of MOF and MARA
文摘Background Clock circadian regulator(CLOCK)is a core factor of the mammalian biological clock system in regulat-ing female fertility and ovarian physiology.However,CLOCK’s specific function and molecular mechanism in porcine granulosa cells(GCs)remain unclear.In this study,we focused on CLOCK’s effects on GC proliferation.Results CLOCK significantly inhibited cell proliferation in porcine GCs.CLOCK decreased the expression of cell cycle-related genes,including CCNB1,CCNE1,and CDK4 at the mRNA and protein levels.CDKN1A levels were upregulated by CLOCK.ASB9 is a newly-identified target of CLOCK that inhibits GC proliferation;CLOCK binds to the E-box element in the ASB9 promoter.Conclusions These findings suggest that CLOCK inhibits the proliferation of porcine ovarian GCs by increasing ASB9 level.
基金supported by the National Natural Science Foundation of China(32102549)the National Key R&D Program of Ningxia(2021BEF02023)+2 种基金the earmarked fund for CARS(CARS-36)the Agricultural Science and Technology Innovation Program(ASTIP-IAS06)the National Key R&D Program of Gansu(21YF5NJ196)。
文摘Background During the transition period,the insufficient dry matter intake and a sharply increased in energy consumption to produce large quantities of milk,high yielding cows would enter a negative energy balance(NEB)that causes an increase in ketone bodies(KBs)and decrease in reproduction efficiency.The excess concentrations of circulating KBs,represented byβ-hydroxybutyric acid(BHBA),could lead to oxidative damage,which potentially cause injury to follicular granulosa cells(fGCs)and delayed follicular development.Sirtuin 3(Sirt3)regulates mitochondria reactive oxygen species(mitoROS)homeostasis in a beneficial manner;however,the molecular mechanisms underlying its involvement in the BHBA-induced injury of fGCs is poorly understood.The aim of this study was to explore the protection effects and underlying mechanisms of Sirt3 against BHBA overload-induced damage of fGCs.Results Our findings demonstrated that 2.4 mmol/L of BHBA stress increased the levels of mitoROS in bovine fGCs.Further investigations identified the subsequent mitochondrial dysfunction,including an increased abnormal rate of mitochondrial architecture,mitochondrial permeability transition pore(MPTP)opening,reductions in mitochondrial membrane potential(MMP)and Ca^(2+)release;these dysfunctions then triggered the caspase cascade reaction of apoptosis in fGCs.Notably,the overexpression of Sirt3 prior to treatment enhanced mitochondrial autophagy by increasing the expression levels of Beclin-1,thus preventing BHBA-induced mitochondrial oxidative stress and mitochondrial dysfunction in fGCs.Furthermore,our data suggested that the AMPK-mTOR-Beclin-1 pathway may be involved in the protective mechanism of Sirt3 against cellular injury triggered by BHBA stimulation.Conclusions These findings indicate that Sirt3 protects fGCs from BHBA-triggered injury by enhancing autophagy,attenuating oxidative stress and mitochondrial damage.This study provides new strategies to mitigate the fGCs injury caused by excessive BHBA stress in dairy cows with ketosis.
基金Baoding Science and Technology Program Project(Grant No.2241ZF120)Hebei Health Care Commission Scientific Research Funding Project(Grant No.20170827)+1 种基金Funding Project of Affiliated Hospital of Hebei University(Grant No.2016Q016)Funding Project of Affiliated Hospital of Hebei University(No.2022QC66).
文摘Objective:To investigate the effect of abnormal ovarian granulosa cell metabolism on in vitro fertilization and embryo transfer(IVF-ET)outcomes in obese polycystic ovary syndrome(PCOS)patients.Methods:Patients with PCOS who met the study criteria were screened according to the inclusion criteria.A total of 32 patients with obese PCOS were recruited into the study group,and 39 patients with non-obese PCOS were recruited into the control group.The general data(age,body mass index,and years of infertility),insulin resistance index(HOMA-IR),follicle-stimulating hormone(FSH),luteinizing hormone(LH),granulosa cell mitochondrial function,and IVF-ET outcome of patients in the study group and control group were retrospectively analyzed.Results:The differences in age and years of infertility between the study group and the control group were insignificant(P>0.05),and the body mass index(BMI)of the study group and control group was 30.5±1.24 kg/m2 and 22.3±1.12 kg/m2,respectively,in which the difference was statistically significant(P<0.05);the HOMA-IR of the study group was significantly higher than that of the control group(P<0.05);the reactive oxygen species(ROS)in the study group was significantly higher than that in the control group(P<0.05),and the ATP content in the study group was significantly lower than that in the control group(P<0.05);comparing the FSH and LH levels between the two groups,the difference was not statistically significant(P>0.05);the rate of IVF-ET failure was significantly higher in the study group than in the control group.Conclusion:PCOS is a complex endocrine disorder,and obesity is one of the independent risk factors for the development of PCOS.
