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
Polycystic ovary syndrome (PCOS) is a major cause of anovulatory infertility. Laparoscopic ovarian drilling (LOD) is a treatment for PCOS that allows the laparoscopic identification of other intra-abdominal lesions an...Polycystic ovary syndrome (PCOS) is a major cause of anovulatory infertility. Laparoscopic ovarian drilling (LOD) is a treatment for PCOS that allows the laparoscopic identification of other intra-abdominal lesions and the provision of diagnostic treatment. This study reports a case of PCOS with an ovarian mass in which LOD was aggressively used and a granulosa cell tumor (GCT) was found. A 34-year-old woman with secondary amenorrhea and irregular menstrual cycles presented to the emergency department with abdominal pain of unknown etiology. Imaging studies revealed a 6-cm left ovarian mass with an internal appearance suggestive of a hemorrhage. The patient’s secondary amenorrhea was subsequently diagnosed as PCOS, and LOD was performed to preserve her fertility. Simultaneously, a cystectomy was performed to evaluate the tumor in the left ovary;the diagnosis was adult-type GCT. Although concomitant GCT and PCOS are extremely rare, the two conditions have similar clinical manifestations. In women of reproductive age, the impact of surgery on future fertility should be considered, and the initial surgical technique should be chosen carefully.展开更多
The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL)...The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the con- trol rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regu- lating the apoptosis of granulosa cells in PCOS rats.展开更多
BACKGROUND High humidity and temperature in Taiwan have significant effects on the reproductivity of Holstein cattle,resulting in the occurrence of bovine ovarian follicular cyst(OFC).Because of economic loss from OFC...BACKGROUND High humidity and temperature in Taiwan have significant effects on the reproductivity of Holstein cattle,resulting in the occurrence of bovine ovarian follicular cyst(OFC).Because of economic loss from OFC,manual rupture and hormone injection have been advocated for the management of OFC.However,these incomplete treatments increase hormone resistance in cattle.Mesenchymal stem cells(MSCs)derived from placental stem cells(PSCs)demonstrate potential properties for the treatment of several diseases via promoting angiogenesis and immune modulation.AIM To establish the possibility of cattle placental stem cells(CPSCs)as a treatment modality for OFC of cows in Taiwan.METHODS The cows with OFC were divided into three groups:control(BC1 and BC2),hormone(H1 and H2),and CPSC(PS1 and PS2)treatment groups.In the hormone treatment group,the cows were given gonadotrophin-releasing hormone(GnRH)-prostaglandin-GnRH intramuscular injection with or without drainage of follicular fluid.In the CPSC treatment group,CPSCs were isolated from the placenta after labor.With the identification of surface antigen on stem cells,the cows were administered ovarian injection of 1×106 or 6×106 CPSCs with drainage.In all groups,OFC was scanned by ultrasound once a week for a total of seven times.The concentrations of estradiol and progesterone in serum were tested in the same period.The estrus cycle was analyzed by food intake and activity.If estrus was detected,artificial insemination was conducted.Then the cow was monitored by ultrasound for confirmation of pregnancy.RESULTS After 7 d of culture,CPSCs were successfully isolated from placental pieces.CPSCs significantly proliferated every 24 h and had high expression of MSC markers such as cluster of differentiation 44,as determined by flow cytometry.Ultrasound showed lower numbers of OFCs with drainage of follicular fluid.We achieved recovery rates of 0%,50%,50%,75%,75%and 75%in BC1,BC2,H1,H2,PS1,and PS2,respectively.Higher concentrations of progesterone were detected in the CPSC treatment groups.However,both hormone and CPSC treatment groups had no significant difference in the concentration of estradiol.The estrus rate was 0%,100%,25%,75%,75%and 75%in BC1,BC2,H1,H2,PS1,and PS2,respectively.The two fetuses were born in H2 and PS1.In brief,cows with CPSC injection achieved higher recovery,estrus,and inseminated conception rates.CONCLUSION CPSCs have efficacy in treating cows with OFC,and thus,may serve as an alternative treatment for reproductive disorders.展开更多
Background The ovaries are one of the first organs that undergo degenerative changes earlier in the aging process,and ovarian aging is shown by a decrease in the number and quality of oocytes.However,little is known a...Background The ovaries are one of the first organs that undergo degenerative changes earlier in the aging process,and ovarian aging is shown by a decrease in the number and quality of oocytes.However,little is known about the molecular mechanisms of female age-related fertility decline in different types of ovarian cells during aging,especially in goats.Therefore,the aim of this study was to reveal the mechanisms driving ovarian aging in goats at single-cell resolution.Results For the first time,we surveyed the single-cell transcriptomic landscape of over 27,000 ovarian cells from newborn,young and aging goats,and identified nine ovarian cell types with distinct gene-expression signatures.Functional enrichment analysis showed that ovarian cell types were involved in their own unique biological processes,such as Wnt beta-catenin signalling was enriched in germ cells,whereas ovarian steroidogenesis was enriched in granulosa cells(GCs).Further analysis showed that ovarian aging was linked to GCs-specific changes in the antioxidant system,oxidative phosphorylation,and apoptosis.Subsequently,we identified a series of dynamic genes,such as AMH,CRABP2,THBS1 and TIMP1,which determined the fate of GCs.Additionally,FOXO1,SOX4,and HIF1A were identified as significant regulons that instructed the differentiation of GCs in a distinct manner during ovarian aging.Conclusions This study revealed a comprehensive aging-associated transcriptomic atlas characterizing the cell typespecific mechanisms during ovarian aging at the single-cell level and offers new diagnostic biomarkers and potential therapeutic targets for age-related goat ovarian diseases.展开更多
Polycystic ovary syndrome(PCOS)is a complex disorder.Genome-wide association studies(GWAS)have identified several genes associated with this condition,including DENND1A.DENND1A encodes a clathrin-binding protein that ...Polycystic ovary syndrome(PCOS)is a complex disorder.Genome-wide association studies(GWAS)have identified several genes associated with this condition,including DENND1A.DENND1A encodes a clathrin-binding protein that functions as a guanine nucleotide exchange factor involved in vesicular transport.However,the specific role of DENND1A in reproductive hormone abnormalities and follicle development disorders in PCOS remain poorly understood.In this study,we investigated DENND1A expression in ovarian granulosa cells(GCs)from PCOS patients and its correlation with hormones.Our results revealed an upregulation of DENND1A expression in GCs from PCOS cases,which was positively correlated with testosterone levels.To further explore the functional implications of DENND1A,we generated a transgenic mouse model overexpressing Dennd1a(TG mice).These TG mice exhibited subfertility,irregular estrous cycles,and increased testosterone production following PMSG stimulation.Additionally,the TG mice displayed diminished responsiveness to FSH,characterized by smaller ovary size,less well-developed follicles,and abnormal expressions of FSH-priming genes.Mechanistically,we found that Dennd1a overexpression disrupted the intracellular trafficking of follicle stimulating hormone receptor(FSHR),promoting its internalization and inhibiting recycling.These findings shed light on the reproductive role of DENND1A and uncover the underlying mechanisms,thereby contributing valuable insights into the pathogenesis of PCOS and providing potential avenues for drug design in PCOS treatment.展开更多
Background:Follicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility,which are regulated by many factors such as microRNAs(miRNAs),which constitute a class...Background:Follicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility,which are regulated by many factors such as microRNAs(miRNAs),which constitute a class of noncoding RNAs(ncRNAs).However,little is known about long noncoding RNAs(lncRNAs),which constitute another ncRNA family that regulate follicular atresia.Results:A total of 77 differentially expressed lncRNAs,including 67 upregulated and 10 downregulated lncRNAs,were identified in early atretic follicles compared to healthy follicles by RNA-Sequencing.We characterized a noncoding RNA that was highly expressed in atretic follicles(NORHA).As an intergenic lncRNA,NORHA was one of the upregulated lncRNAs identified in the atretic follicles.To determine NORHA function,RT-PCR,flow cytometry and western blotting were performed,and the results showed that NORHA was involved in follicular atresia by influencing GC apoptosis with or without oxidative stress.To determine the mechanism of action,bioinformatics analysis,luciferase reporter assay and RNA immunoprecipitation assay were performed,and the results showed that NORHA acted as a‘sponge’,that directly bound to the miR-183-96-182 cluster,and thus prevented its targeted inhibition of FoxO1,a major sensor and effector of oxidative stress.Conclusions:We provide a comprehensive perspective of lncRNA regulation of follicular atresia,and demonstrate that NORHA,a novel lncRNA related to follicular atresia,induces GC apoptosis by influencing the activities of the miR-183-96-182 cluster and FoxO1 axis.展开更多
Background The mechanism by which Meishan(MS)sows are superior to white crossbred sows in ovarian follicle development remains unclear.Given gut microbiota could regulate female ovarian function and reproductive capac...Background The mechanism by which Meishan(MS)sows are superior to white crossbred sows in ovarian follicle development remains unclear.Given gut microbiota could regulate female ovarian function and reproductive capacity,this study aimed to determine the role of gut microbiota-ovary axis on follicular development in sows.Methods We compared the ovarian follicular development,gut microbiota,plasma metabolome,and follicular fluid metabolome between MS and Landrace×Yorkshire(L×Y)sows.A H_(2)O_(2)-induced cell apoptosis model was used to evaluate the effects of multi-omics identified metabolites on the apoptosis of porcine ovarian granulosa cells in vitro.Results Compared with L×Y sows,MS sows have greater ovary weight and improved follicular development,including the greater counts of large follicles of diameter≥5 mm,secondary follicles,and antral follicles,but lesser atretic follicles.The ovarian granulosa cells in MS sows had alleviated apoptosis,which was indicated by the increased BCL-2,decreased caspases-3,and decreased cleaved caspases-3 than in L×Y sows.The ovarian follicular fluid of MS sows had higher concentrations of estradiol,progesterone,follicle-stimulating hormone,luteinizing hormone,and insulin like growth factor 1 than L×Y sows.Gut microbiota of MS sows formed a distinct cluster and had improved alpha diversity,including increased Shannon and decreased Simpson than those of L×Y sows.Corresponding to the enhanced function of carbohydrate metabolism and elevated short-chain fatty acids(SCFAs)in feces,the differential metabolites in plasma between MS and L×Y sows are also mainly enriched in pathways of fatty acid metabolism.There were significant correlations among SCFAs with follicular development,ovarian granulosa cells apoptosis,and follicular fluid hormones,respectively.Noteworthily,compared with L×Y sows,MS sows had higher follicular fluid SCFAs concentrations which could ameliorate H_(2)O_(2)-induced porcine granulosa cells apoptosis in vitro.Conclusion MS sows have more secondary and antral follicles,but fewer atretic follicles and apoptotic ovarian granulosa cells,as well as harbored a distinctive gut microbiota than L×Y sows.Gut microbiota may participate in regulating ovarian follicular development via SCFAs affecting granulosa cells apoptosis in sows.展开更多
Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no...Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no established, reliable method for assessing the number of viable GC within an isolated follicle. Methods: Centrifugation conditions and the Trypan Blue (TB) Exclusion assay were optimised for low cell densities compatible with the numbers of GC in follicles. Mouse ovarian follicles were disaggregated to produce a single cell suspension of GC which were examined by TB (n = 4), but also by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3). GC viability in vitro was characterised further by using enzyme-linked immunoassays to quantify GC production of anti-Mullerian hormone (AMH) and estrogen. Results: The centrifugation and low cell density TB protocol could accurately measure the viability of 78 GC in 10 μL, with an intra-assay coefficient of variation (CoV) 22%, and inter-assay CoV 7%. The best follicle disaggregation method (30 min 37°C exposure to 2 mg/mL collagenase prior to 30 min exposure to 0.025% hyaluronidase) yielded (656 ±87) GC per antral follicle of which 82% ±5% were viable. Culturing 312 - 20,000 GC per well for 24 hours and assessing viability by crystal violet assay generated a linear correlation between OD value and viable GC number (R2 = 0.98) and estrogen concentration per well (R2 = 0.92). 20,000 GC per well produced 143 ±16 pg/mL estrogen during 24 hours in vitro, but no detectable AMH. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents.展开更多
In mammals,the growth and maturation of oocytes within growing follicles largely depends on ovarian granulosa cells(GCs)in response to gonadotropin stimulation.Many signals have been shown to regulate GC proliferation...In mammals,the growth and maturation of oocytes within growing follicles largely depends on ovarian granulosa cells(GCs)in response to gonadotropin stimulation.Many signals have been shown to regulate GC proliferation and apoptosis.However,whether the tyrosine phosphatase SHP2 is involved remains unclear.In this study,we identified the crucial roles of SHP2 in modulating GC proliferation and apoptosis.The production of both mature oocytes and pups was increased in mice with Shp2 specifically deleted in ovarian GCs via Fshr-Cre.Shp2 deletion simultaneously promoted GC proliferation and inhibited GC apoptosis.Furthermore,Shp2 deficiency promoted,while Shp2 overexpression inhibited,the proliferation of cultured primary mouse ovarian GCs and the human ovarian granulosa-like tumor cell line KGN in vitro.Shp2 deficiency promoted follicule-stimulating hormone(FSH)-activated phosphorylation of AKT in vivo.SHP2 deficiency reversed the inhibitory effect of hydrogen peroxide(H_(2)O_(2))on AKT activation in KGN cells.H_(2)O_(2) treatment promoted the interaction between SHP2 and the p85 subunit of PI3K in KGN cells.Therefore,SHP2 in GCs may act as a negative modulator to balance follicular development by suppressing PI3K/AKT signaling.The novel function of SHP2 in modulating proliferation and apoptosis of GCs provides a potential therapeutic target for the clinical treatment of follicle developmental dysfunction.展开更多
OBJECTIVE:To investigate the protective efficacy of Bushen Culuan decoction补肾促卵方,BCD)on ovarian follicle and follicular granulosa cells in mice with premature ovarian insufficiency(POI)induced by tripterygium wil...OBJECTIVE:To investigate the protective efficacy of Bushen Culuan decoction补肾促卵方,BCD)on ovarian follicle and follicular granulosa cells in mice with premature ovarian insufficiency(POI)induced by tripterygium wilfordii polyglycoside,and to study the potential mechanism underlying the action.METHODS:Eighty female Balb/c mice were randomly divided into 4 groups(n=20 each):blank group,model group,Bushen Culuan decoction intervening group(BCD group)and estradiol valerate intervening group(EV group).In the first 14 model establishing d,mice in model group,BCD group and EV group were under Tripterygium wilfordii polyglycoside(TWP)gavage to establish POI models.In the 14-day therapeutic stage,mice in BCD group were taken BCD 18.35 mg·kg^(-1)·d^(-1),mice in EV group were taken EV solution 0.15 mg·kg^(-1)·d^(-1),while mice in blank group and model group were taken normal saline.When the mice accomplished therapy,whole blood was collected for serum hormone including follicle stimulating hormone(FSH),luteal hormone(LH),estradiol(E2),antimullerian hormone(AMH)levels and vascular endothelial growth factor(VEGF),bone morphogenetic protein-7(BMP-7)measurement.Ovarian tissues were harvested for morphologic observation,follicle counting,ovarian follicular graulosa cell apoptosis test and testing BMP-7 and caspase-3 expressions.RESULTS:The body weights of the mice kept growing stably in the process expect in TWP intervening stage.Compared with model group,BCD group had significantly higher ovarian index,serum E_(2),AMH,VEGF,BMP-7 levels and significantly lower FSH level(P<0.05).Meanwhile the VEGF level in BCD group was higher than in EV group(P<0.05).Compared with model group,the histopathological damage and GCs apoptosis were mitigated;developing follicle counting,BMP-7 expression were up-regulated,and caspase-3 expression was downregulated in BCD groups(P<0.05).CONCLUSION:BCD treatment could attenuate pathological process in POI ovaries,suppress GC apoptosis,probably through promoting BMP-7 expression and following inhibiting caspase-3 activation.展开更多
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.
文摘Polycystic ovary syndrome (PCOS) is a major cause of anovulatory infertility. Laparoscopic ovarian drilling (LOD) is a treatment for PCOS that allows the laparoscopic identification of other intra-abdominal lesions and the provision of diagnostic treatment. This study reports a case of PCOS with an ovarian mass in which LOD was aggressively used and a granulosa cell tumor (GCT) was found. A 34-year-old woman with secondary amenorrhea and irregular menstrual cycles presented to the emergency department with abdominal pain of unknown etiology. Imaging studies revealed a 6-cm left ovarian mass with an internal appearance suggestive of a hemorrhage. The patient’s secondary amenorrhea was subsequently diagnosed as PCOS, and LOD was performed to preserve her fertility. Simultaneously, a cystectomy was performed to evaluate the tumor in the left ovary;the diagnosis was adult-type GCT. Although concomitant GCT and PCOS are extremely rare, the two conditions have similar clinical manifestations. In women of reproductive age, the impact of surgery on future fertility should be considered, and the initial surgical technique should be chosen carefully.
文摘The relationship between apoptosis of granulosa cells and follicle development arrest in polycystic ovarian syndrome (PCOS) rats, and the contribution of tumor necrosis factor related apoptosis inducing ligand (TRAIL) in apoptosis of granulosa cells were explored. By using sodium prasterone sulfate rat PCOS model was induced. The apoptosis of granulosa cells in ovaries of rats was observed by TdT-mediated dUTP-biotin nick end-labeling (TUNEL), and the expression of TRAIL protein and mRNA in granulosa cells was detected by using immunhistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) respectively. The apoptotic rate and the expression of protein TRAIL in granulosa cells were significantly higher in antral follicles from the PCOS rats than in those from the control rats (P<0.01, P<0.05). There was no significant difference in apoptotic rate and the expression of TRAIL protein in granulosa cells of preantral follicles between the PCOS rats and the control rats (P>0.05). No apoptosis and the expression of TRAIL protein in granulosa cells of primordial follicles were found in the two groups. The expression of TRAIL mRNA was significantly stronger in granulosa cells from the PCOS rats than in those from the con- trol rats (P<0.01). It was suggested that the apoptotic rate in granulosa cells was significantly higher in antral follicle from the PCOS rats than in those from the control rats. TRAIL played a role in regu- lating the apoptosis of granulosa cells in PCOS rats.
文摘BACKGROUND High humidity and temperature in Taiwan have significant effects on the reproductivity of Holstein cattle,resulting in the occurrence of bovine ovarian follicular cyst(OFC).Because of economic loss from OFC,manual rupture and hormone injection have been advocated for the management of OFC.However,these incomplete treatments increase hormone resistance in cattle.Mesenchymal stem cells(MSCs)derived from placental stem cells(PSCs)demonstrate potential properties for the treatment of several diseases via promoting angiogenesis and immune modulation.AIM To establish the possibility of cattle placental stem cells(CPSCs)as a treatment modality for OFC of cows in Taiwan.METHODS The cows with OFC were divided into three groups:control(BC1 and BC2),hormone(H1 and H2),and CPSC(PS1 and PS2)treatment groups.In the hormone treatment group,the cows were given gonadotrophin-releasing hormone(GnRH)-prostaglandin-GnRH intramuscular injection with or without drainage of follicular fluid.In the CPSC treatment group,CPSCs were isolated from the placenta after labor.With the identification of surface antigen on stem cells,the cows were administered ovarian injection of 1×106 or 6×106 CPSCs with drainage.In all groups,OFC was scanned by ultrasound once a week for a total of seven times.The concentrations of estradiol and progesterone in serum were tested in the same period.The estrus cycle was analyzed by food intake and activity.If estrus was detected,artificial insemination was conducted.Then the cow was monitored by ultrasound for confirmation of pregnancy.RESULTS After 7 d of culture,CPSCs were successfully isolated from placental pieces.CPSCs significantly proliferated every 24 h and had high expression of MSC markers such as cluster of differentiation 44,as determined by flow cytometry.Ultrasound showed lower numbers of OFCs with drainage of follicular fluid.We achieved recovery rates of 0%,50%,50%,75%,75%and 75%in BC1,BC2,H1,H2,PS1,and PS2,respectively.Higher concentrations of progesterone were detected in the CPSC treatment groups.However,both hormone and CPSC treatment groups had no significant difference in the concentration of estradiol.The estrus rate was 0%,100%,25%,75%,75%and 75%in BC1,BC2,H1,H2,PS1,and PS2,respectively.The two fetuses were born in H2 and PS1.In brief,cows with CPSC injection achieved higher recovery,estrus,and inseminated conception rates.CONCLUSION CPSCs have efficacy in treating cows with OFC,and thus,may serve as an alternative treatment for reproductive disorders.
基金supported by the National Key Research and Development Program of China(2022YFD1300202)the Technology Innovation and Application Development Special Project of Chongqing(cstc2021jscx-gksb X0008)+2 种基金the National Natural Science Foundation of China(32102623)the National Natural Science Foundation of Chongqing(cstc2021jcyj-msxm X0875)the Ph D Train Scientific Research Project of Chongqing(CSTB2022BSXM-JCX0002)。
文摘Background The ovaries are one of the first organs that undergo degenerative changes earlier in the aging process,and ovarian aging is shown by a decrease in the number and quality of oocytes.However,little is known about the molecular mechanisms of female age-related fertility decline in different types of ovarian cells during aging,especially in goats.Therefore,the aim of this study was to reveal the mechanisms driving ovarian aging in goats at single-cell resolution.Results For the first time,we surveyed the single-cell transcriptomic landscape of over 27,000 ovarian cells from newborn,young and aging goats,and identified nine ovarian cell types with distinct gene-expression signatures.Functional enrichment analysis showed that ovarian cell types were involved in their own unique biological processes,such as Wnt beta-catenin signalling was enriched in germ cells,whereas ovarian steroidogenesis was enriched in granulosa cells(GCs).Further analysis showed that ovarian aging was linked to GCs-specific changes in the antioxidant system,oxidative phosphorylation,and apoptosis.Subsequently,we identified a series of dynamic genes,such as AMH,CRABP2,THBS1 and TIMP1,which determined the fate of GCs.Additionally,FOXO1,SOX4,and HIF1A were identified as significant regulons that instructed the differentiation of GCs in a distinct manner during ovarian aging.Conclusions This study revealed a comprehensive aging-associated transcriptomic atlas characterizing the cell typespecific mechanisms during ovarian aging at the single-cell level and offers new diagnostic biomarkers and potential therapeutic targets for age-related goat ovarian diseases.
基金supported by the National Key Research and Development Program of China(2021YFC2700400)the Basic Science Center Program(31988101)+2 种基金the National Natural Science Foundation of China(82071606,31871509,82201800,82071610)the Shandong Provincial Key Research and Development Program(2020ZLYS02)Ferring Institute of Reproductive Medicine(FIRMA180310).
文摘Polycystic ovary syndrome(PCOS)is a complex disorder.Genome-wide association studies(GWAS)have identified several genes associated with this condition,including DENND1A.DENND1A encodes a clathrin-binding protein that functions as a guanine nucleotide exchange factor involved in vesicular transport.However,the specific role of DENND1A in reproductive hormone abnormalities and follicle development disorders in PCOS remain poorly understood.In this study,we investigated DENND1A expression in ovarian granulosa cells(GCs)from PCOS patients and its correlation with hormones.Our results revealed an upregulation of DENND1A expression in GCs from PCOS cases,which was positively correlated with testosterone levels.To further explore the functional implications of DENND1A,we generated a transgenic mouse model overexpressing Dennd1a(TG mice).These TG mice exhibited subfertility,irregular estrous cycles,and increased testosterone production following PMSG stimulation.Additionally,the TG mice displayed diminished responsiveness to FSH,characterized by smaller ovary size,less well-developed follicles,and abnormal expressions of FSH-priming genes.Mechanistically,we found that Dennd1a overexpression disrupted the intracellular trafficking of follicle stimulating hormone receptor(FSHR),promoting its internalization and inhibiting recycling.These findings shed light on the reproductive role of DENND1A and uncover the underlying mechanisms,thereby contributing valuable insights into the pathogenesis of PCOS and providing potential avenues for drug design in PCOS treatment.
基金This work was supported by the National Natural Science Foundation of China(32072693 and 31630072)the Qing Lan Project of Jiangsu Province(2020).
文摘Background:Follicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility,which are regulated by many factors such as microRNAs(miRNAs),which constitute a class of noncoding RNAs(ncRNAs).However,little is known about long noncoding RNAs(lncRNAs),which constitute another ncRNA family that regulate follicular atresia.Results:A total of 77 differentially expressed lncRNAs,including 67 upregulated and 10 downregulated lncRNAs,were identified in early atretic follicles compared to healthy follicles by RNA-Sequencing.We characterized a noncoding RNA that was highly expressed in atretic follicles(NORHA).As an intergenic lncRNA,NORHA was one of the upregulated lncRNAs identified in the atretic follicles.To determine NORHA function,RT-PCR,flow cytometry and western blotting were performed,and the results showed that NORHA was involved in follicular atresia by influencing GC apoptosis with or without oxidative stress.To determine the mechanism of action,bioinformatics analysis,luciferase reporter assay and RNA immunoprecipitation assay were performed,and the results showed that NORHA acted as a‘sponge’,that directly bound to the miR-183-96-182 cluster,and thus prevented its targeted inhibition of FoxO1,a major sensor and effector of oxidative stress.Conclusions:We provide a comprehensive perspective of lncRNA regulation of follicular atresia,and demonstrate that NORHA,a novel lncRNA related to follicular atresia,induces GC apoptosis by influencing the activities of the miR-183-96-182 cluster and FoxO1 axis.
基金supported by the National Natural Science Foundation of China(32230099 and 31925037)Hubei Hongshan Laboratory(2021hszd018).
文摘Background The mechanism by which Meishan(MS)sows are superior to white crossbred sows in ovarian follicle development remains unclear.Given gut microbiota could regulate female ovarian function and reproductive capacity,this study aimed to determine the role of gut microbiota-ovary axis on follicular development in sows.Methods We compared the ovarian follicular development,gut microbiota,plasma metabolome,and follicular fluid metabolome between MS and Landrace×Yorkshire(L×Y)sows.A H_(2)O_(2)-induced cell apoptosis model was used to evaluate the effects of multi-omics identified metabolites on the apoptosis of porcine ovarian granulosa cells in vitro.Results Compared with L×Y sows,MS sows have greater ovary weight and improved follicular development,including the greater counts of large follicles of diameter≥5 mm,secondary follicles,and antral follicles,but lesser atretic follicles.The ovarian granulosa cells in MS sows had alleviated apoptosis,which was indicated by the increased BCL-2,decreased caspases-3,and decreased cleaved caspases-3 than in L×Y sows.The ovarian follicular fluid of MS sows had higher concentrations of estradiol,progesterone,follicle-stimulating hormone,luteinizing hormone,and insulin like growth factor 1 than L×Y sows.Gut microbiota of MS sows formed a distinct cluster and had improved alpha diversity,including increased Shannon and decreased Simpson than those of L×Y sows.Corresponding to the enhanced function of carbohydrate metabolism and elevated short-chain fatty acids(SCFAs)in feces,the differential metabolites in plasma between MS and L×Y sows are also mainly enriched in pathways of fatty acid metabolism.There were significant correlations among SCFAs with follicular development,ovarian granulosa cells apoptosis,and follicular fluid hormones,respectively.Noteworthily,compared with L×Y sows,MS sows had higher follicular fluid SCFAs concentrations which could ameliorate H_(2)O_(2)-induced porcine granulosa cells apoptosis in vitro.Conclusion MS sows have more secondary and antral follicles,but fewer atretic follicles and apoptotic ovarian granulosa cells,as well as harbored a distinctive gut microbiota than L×Y sows.Gut microbiota may participate in regulating ovarian follicular development via SCFAs affecting granulosa cells apoptosis in sows.
文摘Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no established, reliable method for assessing the number of viable GC within an isolated follicle. Methods: Centrifugation conditions and the Trypan Blue (TB) Exclusion assay were optimised for low cell densities compatible with the numbers of GC in follicles. Mouse ovarian follicles were disaggregated to produce a single cell suspension of GC which were examined by TB (n = 4), but also by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3). GC viability in vitro was characterised further by using enzyme-linked immunoassays to quantify GC production of anti-Mullerian hormone (AMH) and estrogen. Results: The centrifugation and low cell density TB protocol could accurately measure the viability of 78 GC in 10 μL, with an intra-assay coefficient of variation (CoV) 22%, and inter-assay CoV 7%. The best follicle disaggregation method (30 min 37°C exposure to 2 mg/mL collagenase prior to 30 min exposure to 0.025% hyaluronidase) yielded (656 ±87) GC per antral follicle of which 82% ±5% were viable. Culturing 312 - 20,000 GC per well for 24 hours and assessing viability by crystal violet assay generated a linear correlation between OD value and viable GC number (R2 = 0.98) and estrogen concentration per well (R2 = 0.92). 20,000 GC per well produced 143 ±16 pg/mL estrogen during 24 hours in vitro, but no detectable AMH. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents.
基金National Key Research and Development Program of China(2018YFC1003701 and 2017YFC1001402)National Natural Science Foundation of China(31970797 and 31671564).
文摘In mammals,the growth and maturation of oocytes within growing follicles largely depends on ovarian granulosa cells(GCs)in response to gonadotropin stimulation.Many signals have been shown to regulate GC proliferation and apoptosis.However,whether the tyrosine phosphatase SHP2 is involved remains unclear.In this study,we identified the crucial roles of SHP2 in modulating GC proliferation and apoptosis.The production of both mature oocytes and pups was increased in mice with Shp2 specifically deleted in ovarian GCs via Fshr-Cre.Shp2 deletion simultaneously promoted GC proliferation and inhibited GC apoptosis.Furthermore,Shp2 deficiency promoted,while Shp2 overexpression inhibited,the proliferation of cultured primary mouse ovarian GCs and the human ovarian granulosa-like tumor cell line KGN in vitro.Shp2 deficiency promoted follicule-stimulating hormone(FSH)-activated phosphorylation of AKT in vivo.SHP2 deficiency reversed the inhibitory effect of hydrogen peroxide(H_(2)O_(2))on AKT activation in KGN cells.H_(2)O_(2) treatment promoted the interaction between SHP2 and the p85 subunit of PI3K in KGN cells.Therefore,SHP2 in GCs may act as a negative modulator to balance follicular development by suppressing PI3K/AKT signaling.The novel function of SHP2 in modulating proliferation and apoptosis of GCs provides a potential therapeutic target for the clinical treatment of follicle developmental dysfunction.
基金Supported by Chinese Traditional Medicine“One Belt One Road”International Cooperation Project of China Academy of Chinese Medical Science(No.GH-2017-02-02)。
文摘OBJECTIVE:To investigate the protective efficacy of Bushen Culuan decoction补肾促卵方,BCD)on ovarian follicle and follicular granulosa cells in mice with premature ovarian insufficiency(POI)induced by tripterygium wilfordii polyglycoside,and to study the potential mechanism underlying the action.METHODS:Eighty female Balb/c mice were randomly divided into 4 groups(n=20 each):blank group,model group,Bushen Culuan decoction intervening group(BCD group)and estradiol valerate intervening group(EV group).In the first 14 model establishing d,mice in model group,BCD group and EV group were under Tripterygium wilfordii polyglycoside(TWP)gavage to establish POI models.In the 14-day therapeutic stage,mice in BCD group were taken BCD 18.35 mg·kg^(-1)·d^(-1),mice in EV group were taken EV solution 0.15 mg·kg^(-1)·d^(-1),while mice in blank group and model group were taken normal saline.When the mice accomplished therapy,whole blood was collected for serum hormone including follicle stimulating hormone(FSH),luteal hormone(LH),estradiol(E2),antimullerian hormone(AMH)levels and vascular endothelial growth factor(VEGF),bone morphogenetic protein-7(BMP-7)measurement.Ovarian tissues were harvested for morphologic observation,follicle counting,ovarian follicular graulosa cell apoptosis test and testing BMP-7 and caspase-3 expressions.RESULTS:The body weights of the mice kept growing stably in the process expect in TWP intervening stage.Compared with model group,BCD group had significantly higher ovarian index,serum E_(2),AMH,VEGF,BMP-7 levels and significantly lower FSH level(P<0.05).Meanwhile the VEGF level in BCD group was higher than in EV group(P<0.05).Compared with model group,the histopathological damage and GCs apoptosis were mitigated;developing follicle counting,BMP-7 expression were up-regulated,and caspase-3 expression was downregulated in BCD groups(P<0.05).CONCLUSION:BCD treatment could attenuate pathological process in POI ovaries,suppress GC apoptosis,probably through promoting BMP-7 expression and following inhibiting caspase-3 activation.
