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 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
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
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
Background: Heat stress is known to alter follicular dynamics and granulosa cell function and may contribute to the diminished reproductive efficiency commonly observed in mammals during the summer. Although several i...Background: Heat stress is known to alter follicular dynamics and granulosa cell function and may contribute to the diminished reproductive efficiency commonly observed in mammals during the summer. Although several investigators have studied heat-induced ovarian injury, few reports have focused on the effects of chronic heat stress on ovarian function and the molecular mechanisms through which it induces ovarian injury.Methods: In Exp. 1, 48 female mice were assigned to a control or heat-stressed treatment. After exposure to a constant temperature of 25 °C for 7, 14, 21 or 28 d(n = 6) or to 42 °C for 3 h per d for 7, 14, 21 or 28 d(n = 6), the mice were euthanized and their ovaries were analyzed for follicular atresia, granulosa cell apoptosis, changes in the abundance of HSP70 protein and serum concentrations of estradiol. In Exp. 2, the expression of HSP70 and aromatase was quantified in antral follicles cultured in vitro at 37 or 42 °C for 24 h. In Exp. 3, granulosa cells from ovaries maintained at 37 or 41 °C for 2 h were analyzed for their expression of HSP70, Bim, caspase-3 and cleaved caspase-3.Results: In Exp. 1, body weight and food intake of heat-stressed mice decreased(P < 0.05) compared with control mice while the concentration of estradiol in serum was lower(P < 0.05) in heat-stressed mice than in control mice. Compared with control mice, the percentage of atretic follicles and the number of antral follicles with severe apoptotic signals were increased(P < 0.05) after 21 d of heat-stressed treatment. HSP70 protein was more abundant(P < 0.05) in heat-stressed mice than control mice. In Exp. 2, heat stress increased HSP70 and decreased aromatase proteins(P < 0.05) in antral follicles. In Exp. 3, TUNEL-positive granulosa cells from heat-stressed ovaries were observed concomitant with a significant increase in HSP70, Bim and cleaved caspase-3 protein.Conclusion: Heat-stress in mice decrease estradiol in serum and aromatase in antral follicles but increased number of atretic follicles and granulosa cell undergoing apoptosis which may explain the decreased fertility commonly observed in heat-stressed animals.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
Follicle-stimulating hormone(FSH),an important hypothalamic-pituitary-gonadal axis(HPG)hormone,is secreted by the pituitary gland.This study confirms that FSH is expressed in chicken follicles at different stages,and ...Follicle-stimulating hormone(FSH),an important hypothalamic-pituitary-gonadal axis(HPG)hormone,is secreted by the pituitary gland.This study confirms that FSH is expressed in chicken follicles at different stages,and positive FSHβ mRNA signals were stronger(P<0.05)in granulosa cells than in oocytes.The 369 bp coding sequence of FSHβ in ovaries is 100%identical to that in the pituitary gland.The experiment in vitro revealed that the ovary possessed FSH secretory capacity.Further,FSHβ mRNA was significantly upregulated(P<0.05)in follicles and significantly higher(P<0.05)than that in the pituitary gland by approximately 2–23 times with the development.The number of granulosa cells decreased significantly(P<0.05)in the cells with siRNA treatment,confirming that the ovarian FSH could promote granulosa cell proliferation.This view was supported by cell cycle analysis and CCND2 and CCNE2 expression.Further research indicated that no difference(P>0.05)was observed between the number of granulosa cells treated with FSHβ siRNA and in exogenous FSH.However,the number of granulosa cells without FSHβ siRNA transfection was significantly higher(P<0.05)for exogenous FSH.This finding suggests that the proliferative effect of exogenous FSH on ovarian granulosa cells depend on endogenous FSH.This study demonstrated that the FSH gene was expressed in chicken follicles and promoted ovarian granulosa cell proliferation,which enriched the theory on HPG axis.展开更多
Objective: To study the interaction between granulosa and thecal cel1s in steroidogenesis in an am-nion dual chamber in comparison with the cellulose dual chamber.Method: A dual chamber culture system was designed and...Objective: To study the interaction between granulosa and thecal cel1s in steroidogenesis in an am-nion dual chamber in comparison with the cellulose dual chamber.Method: A dual chamber culture system was designed and prepared with amnion membrane from human term placenta. The isolated porcine granulosa and thecal cells were grown on both sides of the amnion membrane, with granulosa cells in the inner chamber and thecal cells in the outer chamber. The concentrations of estradiol (E,), progesterone (P) and testosterone (T) in the culture media were mea-sured by radioimmunoassay.Results: The growth of both cells and their steroidogenic function were more active in amnion dual chamber system than in cellulose dual chamber system: (1) more T produced by thecal cells in the outer chamber, passing into inner chamber through the amnion membrane. T was used by granulosa cells as the substrate of aromatization, so that granulosa cells produced more E2 (up to 2 435 pmol/L); (2) the production of P (52. 5 μmol/L) and T (10. 2μmol/L) by granulosa cells cultured in the amnion mem-brane dual chamber system were also higher.Conclusions:The dual chamber system made of amnion mpmbrane showed better effect in studying steroidogenesis than with cellulose dual chamber system, and can be used as a model for studying paracrine regulatory interaction between granulosa and thecal cells in vitro.展开更多
Background: Granulosa cells(GCs) proliferation and estradiol synthesis significantly affect follicular development.The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yiel...Background: Granulosa cells(GCs) proliferation and estradiol synthesis significantly affect follicular development.The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yielding sows,indicating that miR-214-3p may be involved in sow fertility. However, the functions and mechanisms of miR-214-3p on GCs are unclear. This study focuses on miR-214-3p in terms of the effects on GCs proliferation and estradiol synthesis.Results: Our findings revealed that miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine GCs. MiR-214-3p can increase the percentage of S-phase cells, the number of EdU labeled positive cells, and cell viability. However, E2 concentration was reduced after miR-214-3p agomir treatment. We also found that miR-214-3p up-regulates the expression of cell cycle genes including cell cycle protein B(Cyclin B), cell cycle protein D(Cyclin D), cell cycle protein E(Cyclin E), and cyclin-dependent kinase 4(CDK4) at the transcription and translation levels, but down-regulates the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and steroidogenic acute regulatory protein(StAR)(i.e., the key enzymes in estradiol synthesis). On-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target genes of miR-214-3p in proliferation and estradiol synthesis are Mfn2 and NR5A1, respectively.Conclusions: Our findings suggest that miR-214-3 p plays an important role in the functional regulation of porcine GCs and therefore may be a target gene for regulating follicular development.展开更多
Background:MiRNAs act as pivotal post-transcriptional gene mediators in the regulation of diverse biological processes,including proliferation,development and apoptosis.Our previous study has showed that miR-101-3p is...Background:MiRNAs act as pivotal post-transcriptional gene mediators in the regulation of diverse biological processes,including proliferation,development and apoptosis.Our previous study has showed that miR-101-3p is differentially expressed in dairy goat ovaries compared single with multiple litters.The objective of this research was to explore the potential function and molecular mechanism of miR-101-3p via its target STC1 in goat ovarian growth and development.Results:cDNA libraries were constructed using goat granulosa cells transfected with miR-101-3p mimics and negative control by RNA-sequencing.In total,142 differentially expressed unigenes(DEGs)were detected between two libraries,including 78 down-regulated and 64 up-regulated genes.GO and KEGG enrichment analysis showed the potential impacts of DEGs on ovarian development.STC1 was singled out from DEGs for further research owing to it regulates reproductive-related processes.In vitro,bioinformatics analysis and 3′-UTR assays confirmed that STC1 was a target of miR-101-3p.ELISA was performed to detect the estrogen(E2)and progesterone(P4)levels.CCK8,EdU and flow cytometry assays were performed to detect the proliferation and apoptosis of granulosa cells.Results showed that miR-101-3p regulated STAR,CYP19A1,CYP11A1 and 3β-HSD steroid hormone synthesis-associated genes by STC1 depletion,thus promoted E2 and P4 secretions.MiR-101-3p also affected the key protein PI3K,PTEN,AKT and mTOR in PI3K-AKT pathway by STC1,thereby suppressing proliferation and promoting apoptosis of granulosa cells.In vivo,the distribution and expression levels of miR-101-3p in mouse ovaries were determined through fluorescence in situ hybridisation(FISH).Immunohistochemistry results showed that STC1 expression was suppressed in mouse ovaries in miR-101-3p-agonist and siRNA-STC1 groups.Small and stunted ovarian fragments,decreased numbers of follicles at diverse stages were observed using Hematoxylin-eosin(HE)staining,thereby showing unusual ovarian development after miR-101-3p overexpression or STC1 depletion.Inhibition of miR-101-3p manifested opposite results.Conclusions:Taken together,our results demonstrated a regulatory mechanism of miR-101-3p via STC1 in goat granulosa cells,and offered the first in vivo example of miR-101-3p and STC1 functions required for ovarian development.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: Granulosa cell tumors (GCTs) are rare neoplasms with a relatively favorable prognosis. They are characterized by a prolonged history and a tendency to late recurrences. It is the most common type of sex co...Background: Granulosa cell tumors (GCTs) are rare neoplasms with a relatively favorable prognosis. They are characterized by a prolonged history and a tendency to late recurrences. It is the most common type of sex cord-stromal tumors. Aims: To analyze, to report and to better understand the clinico-pathologic features and results of treatment, and prognostic factors of these tumors. Materials and Methods: A retrospective single-institutional review 17 cases of GCTs were treated in National Cancer Institute—Cairo University from January 2010 till December 2014. The clinical and pathological characteristics, treatment, and outcomes of patients with ovarian GCTs were analyzed. Results: Data from 17 patients were obtained. The median age was 54 years (range;14 - 72). Abdominal pain was the most common presentation (64.7%). The mean tumor size was 14 cm (range;7 - 23 cm). The majority of our patients were stage I (n = 11;64.7%), while (n = 3;17.6%) had stage III and (n = 2, 11.8%) were stage IV. Only one case (5.9%) had an unknown stage (explored outside NCI). The majority of cases were of adult type disease (n = 14) and low grade pathology (n = 10). In follow-up period (median = 42 months;ranging 9 - 60) three patients relapsed;the median overall survival time was not reached yet, however, the estimated 3-year survival was 72.5%. Conclusion: Granulosa cell tumors are rare neoplasms of the ovaries. They progress slowly and often are diagnosed in an early stage. Surgery is the main line of treatment. Prolonged post-therapeutic follow-up is necessary. Definition of proper prognostic factors is mandatory.展开更多
Background:Oleic acid is an abundant free fatty acid present in livestock that are in a negative energy-balance state,and it may have detrimental effects on female reproduction and fertility.Oleic acid induces lipid a...Background:Oleic acid is an abundant free fatty acid present in livestock that are in a negative energy-balance state,and it may have detrimental effects on female reproduction and fertility.Oleic acid induces lipid accumulation in bovine granulosa cells,which leads to a foam cell-like morphology and reduced steroidogenesis.However,why oleic acid increases lipid accumulation but decreases steroidogenesis remains unclear.This study focused on oleic acid’s effects on lipid type and steroidogenesis.Results:Oleic acid increased the lipid accumulation in a concentration-dependent manner and mainly increased the triglyceride level and decreased the cholesterol ester level.Oleic acid also led to a decline in estradiol and progesterone production in porcine granulosa cells in vitro.In addition,oleic acid up-regulated the expression of CD36 and diacylglycerol acyltransferase 2,but down-regulated the expression of 3-hydroxy-3-methylglutarylcoenzyme A reductase,scavenger receptor class B member 1 and acetyl-Coenzyme A acetyltransferase 2,as well as steroidogenesis-related genes,including cytochrome P450 family 11 subfamily A member 1,cytochrome P450family 19 subfamily A member 1 and 3 as well as steroidogenic acute regulatory protein at the mRNA and protein levels.An oleic acid-rich diet also enhanced the triglyceride levels and reduced the cholesterol levels in ovarian tissues of female mice,which resulted in lower estradiol levels than in control-fed mice.Compared with the control,decreases in estrus days and the numbers of antral follicles and corpora lutea,as well as an increase in the numbers of the atretic follicles,were found in the oleic acid-fed female mice.Conclusions:Oleic acid changed the lipid type stored in lipid droplets of ovarian granulosa cells,and led to a decrease in steroidogenesis.These results improve our understanding of fertility decline in livestock that are in a negative energy-balance state.展开更多
BACKGROUND Meigs’syndrome is regarded as a benign ovarian tumor accompanied by pleural effusion and ascites,both of which resolve after removal of the tumor.Patients often seek treatment in the Department of Respirat...BACKGROUND Meigs’syndrome is regarded as a benign ovarian tumor accompanied by pleural effusion and ascites,both of which resolve after removal of the tumor.Patients often seek treatment in the Department of Respiratory and Critical Care Medicine or other internal medicine departments due to symptoms caused by ascites or hydrothorax.Here,we report a rare case of Meigs'syndrome caused by granulosa cell tumor accompanied with intrathoracic lesions.CASE SUMMARY A 52-year-old women was admitted to the Department of Respiratory and Critical Care Medicine due to coughing and expectoration accompanied with shortness of breath.Chest X-ray and chest computed tomography showed a modest volume of pleural fluid with pleural thickening in the right lung.The carbohydrate antigen 125(CA125)concentration was 150.8 U/mL(normal,0-35 U/mL)and no tumor cells were observed in pleural fluid.Nodules and a neoplasm with a fish meat-like appearance in the parietal pleura and nodules with a‘string of beads’-like appearance in the diaphragm were found by thoracoscopic examination.Furthermore,pelvic magnetic resonance revealed a pelvic mass measuring about 11.6 cm×10.0 cm×12.4 cm with heterogeneous signal intensity and multiple hypointense separations.Total abdominal hysterectomy,bilateral adnexectomy,and separation of pelvic adhesion were performed under general anesthesia.The pathology results showed granulosa cell tumor.At the 2-mo follow-up after the surgery,the hydrothorax subsided,and the CA125 level returned to normal.CONCLUSION For postmenopausal women with unexplained hydrothorax and elevated CA125,in addition to being suspected of having gynecological malignancy,Meigs’syndrome should be considered.展开更多
Introduction: Extraovarian granulosa cell tumor (GCT) is a very uncommon tumor, assumed to arise from the ectopic gonadal tissue along the embryonal route of the genital ridge. Case report: We report the case of a 25 ...Introduction: Extraovarian granulosa cell tumor (GCT) is a very uncommon tumor, assumed to arise from the ectopic gonadal tissue along the embryonal route of the genital ridge. Case report: We report the case of a 25 year-old woman who was presented at the emergency unit with a severe abdominal pain focused on the left iliac fossa. The patient had delivered normally 2 months before. An ovarian mass of 79 × 67 × 89 mm was shown in vaginal ultrasound as well as at the abdominal scan. Exploratory laparoscopy was performed and a torsion of the fallopian tube and an hemato-salpinges was visualized. Re-vascularisation was not achieved and a left salpingectomy took place. The immuno-histopathology report revealed an extra-ovarian GCT deriving from the fimbriae of the tube. Follow-up surgery was discussed. The case is presented for its rarity.展开更多
Based on published sequences for chicken Bcl-2,three siRNAs（small interfering RNA）were designed,and expression vectors were constructed and transfected into goose granulosa cells cultured in vitro.Bcl-2 protein,apop...Based on published sequences for chicken Bcl-2,three siRNAs（small interfering RNA）were designed,and expression vectors were constructed and transfected into goose granulosa cells cultured in vitro.Bcl-2 protein,apoptosis and proliferation of granulosa cells,48 h after the transf ection,were analyzed by flow cytometry,and progesterone（P）secreted into the culture medium was measured by radioimmunoassay.In addition,apoptosis and Bcl-2 protein level were assessed in untreated granulosa cells from the four largest preovulatory follicles（F<sub>1</sub><sup>F</sup><sub>4</sub>）,the smallest preovulatory follicles（SPF）,small yellow follicles（SYF）and atretic follicles.The highest level of Bcl-2 protein was observed in granulosa cells from SPF,and levels in cells from healthy follicles were significantly higher than those of atretic follicles（P【0.05）.Bcl-2 protein levels in cells subjected to RNAi were significantly lower than those of controls（P【0.05）,while apoptosis indices（AI）,proliferation indices（PI）and P secretion in the RNAi treatments were higher than those of controls（P【0.05）.展开更多
Background: Long non-coding RNAs(lncRNAs) may regulate gene expression in numerous biological processes including cellular response to xenobiotics.The exposure of living organisms to 2,3,7,8-tetrachlorodibenzo-p-dioxi...Background: Long non-coding RNAs(lncRNAs) may regulate gene expression in numerous biological processes including cellular response to xenobiotics.The exposure of living organisms to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD),a persistent environmental contaminant,results in reproductive defects in many species including pigs.The aims of the study were to identify and characterize lncRNAs in porcine granulosa cells as well as to examine the effects of TCDD on the lncRNA expression profile in the cells.Results: One thousand six hundred sixty-six lncRNAs were identified and characterized in porcine granulosa cells.The identified lncRNAs were found to be shorter than mRNAs.In addition,the number of exons was lower in lncRNAs than in m RNAs and their exons were longer.TCDD affected the expression of 22 lncRNAs(differentially expressed lncRNAs [DELs]; log2 fold change ≥ 1,P-adjusted < 0.05) in the examined cells.Potential functions of DELs were indirectly predicted via searching their target cis-and trans-regulated protein-coding genes.The coexpression analysis revealed that DELs may influence the expression of numerous genes,including those involved in cellular response to xenobiotics,dioxin metabolism,endoplasmic reticulum stress and cell proliferation.Aryl hydrocarbon receptor(AhR) and cytochrome P450 1 A1(CYP1 A1) were found among the trans-regulated genes.Conclusions: These findings indicate that the identified lncRNAs may constitute a part of the regulatory mechanism of TCDD action in granulosa cells.To our knowledge,this is the first study describing lncRNAs in porcine granulosa cells as well as TCDD effects on the lncRNA expression profile.These results may trigger new research directions leading to better understanding of molecular processes induced by xenobiotics in the ovary.展开更多
MicroRNAs(miRNAs) are endogenous 18-24 nucleotide(nt) non-coding RNAs,some of which have been indicated to play key roles in granulosa cells(GCs) function.However,little is known about how the miRNA gene expression it...MicroRNAs(miRNAs) are endogenous 18-24 nucleotide(nt) non-coding RNAs,some of which have been indicated to play key roles in granulosa cells(GCs) function.However,little is known about how the miRNA gene expression itself is regulated in the GCs.Our previous study showed that miR-34 c,identified to be a pro-apoptotic and anti-proliferative factor in many cell types,exerted the same effects in porcine GCs.Here,the transcriptional regulation of miR-34 c expression in GCs was further investigated.5' rapid amplification of cDNA ends(RACE) assay indicated that the pri-miR-34 c transcription start site was located in 1 556 bp upstream of pre-miR-34 c.With dual-luciferase reporter assay,we confirmed a 69 bp core promoter region(-1 799 bp/-1 730 bp) was indispensable for the transcription of miR-34 c.Chromatin immunoprecipitation(ChIP) assay demonstrated that p53,p50,and p65 could bind to the transcription factor binding sites within the 69 bp core promoter region.In addition,deletion of transcripition factor binding sites resulted in obvious change of the miR-34 c promoter activity.Finally,using overexpression and knockdown of p53,p50,and p65 strategies,we showed that p53 and p50 could positively regulated miR-34 c expression,whereas p65 neletively regulated miR-34 c expression in GCs.Our results provide new data about the transcription regulatory mechanism of miRNA genes in GCs.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 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.
基金The design of the study and collection,analysis,and interpretation of data and in writing the manuscript were supported by the Specialized Research Fund for the Doctoral Program of Higher Education(20130008130001)
文摘Background: Heat stress is known to alter follicular dynamics and granulosa cell function and may contribute to the diminished reproductive efficiency commonly observed in mammals during the summer. Although several investigators have studied heat-induced ovarian injury, few reports have focused on the effects of chronic heat stress on ovarian function and the molecular mechanisms through which it induces ovarian injury.Methods: In Exp. 1, 48 female mice were assigned to a control or heat-stressed treatment. After exposure to a constant temperature of 25 °C for 7, 14, 21 or 28 d(n = 6) or to 42 °C for 3 h per d for 7, 14, 21 or 28 d(n = 6), the mice were euthanized and their ovaries were analyzed for follicular atresia, granulosa cell apoptosis, changes in the abundance of HSP70 protein and serum concentrations of estradiol. In Exp. 2, the expression of HSP70 and aromatase was quantified in antral follicles cultured in vitro at 37 or 42 °C for 24 h. In Exp. 3, granulosa cells from ovaries maintained at 37 or 41 °C for 2 h were analyzed for their expression of HSP70, Bim, caspase-3 and cleaved caspase-3.Results: In Exp. 1, body weight and food intake of heat-stressed mice decreased(P < 0.05) compared with control mice while the concentration of estradiol in serum was lower(P < 0.05) in heat-stressed mice than in control mice. Compared with control mice, the percentage of atretic follicles and the number of antral follicles with severe apoptotic signals were increased(P < 0.05) after 21 d of heat-stressed treatment. HSP70 protein was more abundant(P < 0.05) in heat-stressed mice than control mice. In Exp. 2, heat stress increased HSP70 and decreased aromatase proteins(P < 0.05) in antral follicles. In Exp. 3, TUNEL-positive granulosa cells from heat-stressed ovaries were observed concomitant with a significant increase in HSP70, Bim and cleaved caspase-3 protein.Conclusion: Heat-stress in mice decrease estradiol in serum and aromatase in antral follicles but increased number of atretic follicles and granulosa cell undergoing apoptosis which may explain the decreased fertility commonly observed in heat-stressed animals.
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.
基金This work was funded by the National Natural Science Foundation of China(31802057)the Second Batch of Special Grant from China Postdoctoral Science Foundation(2020TQ0252)+1 种基金the Postdoctoral Research Funding Project of Jiangsu Province,China in 2020(2020Z213)the Open Project Program of Joint International Research Laboratory of Agriculture and Agri-Product Safety,the Ministry of Education of China(JILAR-KF202017).
文摘Follicle-stimulating hormone(FSH),an important hypothalamic-pituitary-gonadal axis(HPG)hormone,is secreted by the pituitary gland.This study confirms that FSH is expressed in chicken follicles at different stages,and positive FSHβ mRNA signals were stronger(P<0.05)in granulosa cells than in oocytes.The 369 bp coding sequence of FSHβ in ovaries is 100%identical to that in the pituitary gland.The experiment in vitro revealed that the ovary possessed FSH secretory capacity.Further,FSHβ mRNA was significantly upregulated(P<0.05)in follicles and significantly higher(P<0.05)than that in the pituitary gland by approximately 2–23 times with the development.The number of granulosa cells decreased significantly(P<0.05)in the cells with siRNA treatment,confirming that the ovarian FSH could promote granulosa cell proliferation.This view was supported by cell cycle analysis and CCND2 and CCNE2 expression.Further research indicated that no difference(P>0.05)was observed between the number of granulosa cells treated with FSHβ siRNA and in exogenous FSH.However,the number of granulosa cells without FSHβ siRNA transfection was significantly higher(P<0.05)for exogenous FSH.This finding suggests that the proliferative effect of exogenous FSH on ovarian granulosa cells depend on endogenous FSH.This study demonstrated that the FSH gene was expressed in chicken follicles and promoted ovarian granulosa cell proliferation,which enriched the theory on HPG axis.
文摘Objective: To study the interaction between granulosa and thecal cel1s in steroidogenesis in an am-nion dual chamber in comparison with the cellulose dual chamber.Method: A dual chamber culture system was designed and prepared with amnion membrane from human term placenta. The isolated porcine granulosa and thecal cells were grown on both sides of the amnion membrane, with granulosa cells in the inner chamber and thecal cells in the outer chamber. The concentrations of estradiol (E,), progesterone (P) and testosterone (T) in the culture media were mea-sured by radioimmunoassay.Results: The growth of both cells and their steroidogenic function were more active in amnion dual chamber system than in cellulose dual chamber system: (1) more T produced by thecal cells in the outer chamber, passing into inner chamber through the amnion membrane. T was used by granulosa cells as the substrate of aromatization, so that granulosa cells produced more E2 (up to 2 435 pmol/L); (2) the production of P (52. 5 μmol/L) and T (10. 2μmol/L) by granulosa cells cultured in the amnion mem-brane dual chamber system were also higher.Conclusions:The dual chamber system made of amnion mpmbrane showed better effect in studying steroidogenesis than with cellulose dual chamber system, and can be used as a model for studying paracrine regulatory interaction between granulosa and thecal cells in vitro.
基金supported by grants from the National Natural Science Foundation (No.31802047)the National Science and Technology Major Project of China (No. 2016ZX08006003)Shaanxi Provincial Key Research and Development Project (CN)(No. 2018ZDXM-NY-035)。
文摘Background: Granulosa cells(GCs) proliferation and estradiol synthesis significantly affect follicular development.The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yielding sows,indicating that miR-214-3p may be involved in sow fertility. However, the functions and mechanisms of miR-214-3p on GCs are unclear. This study focuses on miR-214-3p in terms of the effects on GCs proliferation and estradiol synthesis.Results: Our findings revealed that miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine GCs. MiR-214-3p can increase the percentage of S-phase cells, the number of EdU labeled positive cells, and cell viability. However, E2 concentration was reduced after miR-214-3p agomir treatment. We also found that miR-214-3p up-regulates the expression of cell cycle genes including cell cycle protein B(Cyclin B), cell cycle protein D(Cyclin D), cell cycle protein E(Cyclin E), and cyclin-dependent kinase 4(CDK4) at the transcription and translation levels, but down-regulates the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and steroidogenic acute regulatory protein(StAR)(i.e., the key enzymes in estradiol synthesis). On-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target genes of miR-214-3p in proliferation and estradiol synthesis are Mfn2 and NR5A1, respectively.Conclusions: Our findings suggest that miR-214-3 p plays an important role in the functional regulation of porcine GCs and therefore may be a target gene for regulating follicular development.
基金supported by the National Natural Science Foundation of China(31601925)Shaanxi Science and Technology Innovation Project Plan(2020ZDLNY02–01,2020ZDLNY02–02,2018ZDCXL-NY-01-04,2018ZDCXL-NY-01-02 and 2017ZDXM-NY-081)Natural Science Foundation of Shaanxi Province(2020JQ-868)。
文摘Background:MiRNAs act as pivotal post-transcriptional gene mediators in the regulation of diverse biological processes,including proliferation,development and apoptosis.Our previous study has showed that miR-101-3p is differentially expressed in dairy goat ovaries compared single with multiple litters.The objective of this research was to explore the potential function and molecular mechanism of miR-101-3p via its target STC1 in goat ovarian growth and development.Results:cDNA libraries were constructed using goat granulosa cells transfected with miR-101-3p mimics and negative control by RNA-sequencing.In total,142 differentially expressed unigenes(DEGs)were detected between two libraries,including 78 down-regulated and 64 up-regulated genes.GO and KEGG enrichment analysis showed the potential impacts of DEGs on ovarian development.STC1 was singled out from DEGs for further research owing to it regulates reproductive-related processes.In vitro,bioinformatics analysis and 3′-UTR assays confirmed that STC1 was a target of miR-101-3p.ELISA was performed to detect the estrogen(E2)and progesterone(P4)levels.CCK8,EdU and flow cytometry assays were performed to detect the proliferation and apoptosis of granulosa cells.Results showed that miR-101-3p regulated STAR,CYP19A1,CYP11A1 and 3β-HSD steroid hormone synthesis-associated genes by STC1 depletion,thus promoted E2 and P4 secretions.MiR-101-3p also affected the key protein PI3K,PTEN,AKT and mTOR in PI3K-AKT pathway by STC1,thereby suppressing proliferation and promoting apoptosis of granulosa cells.In vivo,the distribution and expression levels of miR-101-3p in mouse ovaries were determined through fluorescence in situ hybridisation(FISH).Immunohistochemistry results showed that STC1 expression was suppressed in mouse ovaries in miR-101-3p-agonist and siRNA-STC1 groups.Small and stunted ovarian fragments,decreased numbers of follicles at diverse stages were observed using Hematoxylin-eosin(HE)staining,thereby showing unusual ovarian development after miR-101-3p overexpression or STC1 depletion.Inhibition of miR-101-3p manifested opposite results.Conclusions:Taken together,our results demonstrated a regulatory mechanism of miR-101-3p via STC1 in goat granulosa cells,and offered the first in vivo example of miR-101-3p and STC1 functions required for ovarian development.
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.
文摘Background: Granulosa cell tumors (GCTs) are rare neoplasms with a relatively favorable prognosis. They are characterized by a prolonged history and a tendency to late recurrences. It is the most common type of sex cord-stromal tumors. Aims: To analyze, to report and to better understand the clinico-pathologic features and results of treatment, and prognostic factors of these tumors. Materials and Methods: A retrospective single-institutional review 17 cases of GCTs were treated in National Cancer Institute—Cairo University from January 2010 till December 2014. The clinical and pathological characteristics, treatment, and outcomes of patients with ovarian GCTs were analyzed. Results: Data from 17 patients were obtained. The median age was 54 years (range;14 - 72). Abdominal pain was the most common presentation (64.7%). The mean tumor size was 14 cm (range;7 - 23 cm). The majority of our patients were stage I (n = 11;64.7%), while (n = 3;17.6%) had stage III and (n = 2, 11.8%) were stage IV. Only one case (5.9%) had an unknown stage (explored outside NCI). The majority of cases were of adult type disease (n = 14) and low grade pathology (n = 10). In follow-up period (median = 42 months;ranging 9 - 60) three patients relapsed;the median overall survival time was not reached yet, however, the estimated 3-year survival was 72.5%. Conclusion: Granulosa cell tumors are rare neoplasms of the ovaries. They progress slowly and often are diagnosed in an early stage. Surgery is the main line of treatment. Prolonged post-therapeutic follow-up is necessary. Definition of proper prognostic factors is mandatory.
基金supported by grants from the National Natural Science Foundation(No.31802047)the National Science and Technology Major Project of China(No.2016ZX08006003)。
文摘Background:Oleic acid is an abundant free fatty acid present in livestock that are in a negative energy-balance state,and it may have detrimental effects on female reproduction and fertility.Oleic acid induces lipid accumulation in bovine granulosa cells,which leads to a foam cell-like morphology and reduced steroidogenesis.However,why oleic acid increases lipid accumulation but decreases steroidogenesis remains unclear.This study focused on oleic acid’s effects on lipid type and steroidogenesis.Results:Oleic acid increased the lipid accumulation in a concentration-dependent manner and mainly increased the triglyceride level and decreased the cholesterol ester level.Oleic acid also led to a decline in estradiol and progesterone production in porcine granulosa cells in vitro.In addition,oleic acid up-regulated the expression of CD36 and diacylglycerol acyltransferase 2,but down-regulated the expression of 3-hydroxy-3-methylglutarylcoenzyme A reductase,scavenger receptor class B member 1 and acetyl-Coenzyme A acetyltransferase 2,as well as steroidogenesis-related genes,including cytochrome P450 family 11 subfamily A member 1,cytochrome P450family 19 subfamily A member 1 and 3 as well as steroidogenic acute regulatory protein at the mRNA and protein levels.An oleic acid-rich diet also enhanced the triglyceride levels and reduced the cholesterol levels in ovarian tissues of female mice,which resulted in lower estradiol levels than in control-fed mice.Compared with the control,decreases in estrus days and the numbers of antral follicles and corpora lutea,as well as an increase in the numbers of the atretic follicles,were found in the oleic acid-fed female mice.Conclusions:Oleic acid changed the lipid type stored in lipid droplets of ovarian granulosa cells,and led to a decrease in steroidogenesis.These results improve our understanding of fertility decline in livestock that are in a negative energy-balance state.
基金Supported by the Scientific Research Project of Sichuan Provincial Health and Family Planning Commission,No.18PJ409.
文摘BACKGROUND Meigs’syndrome is regarded as a benign ovarian tumor accompanied by pleural effusion and ascites,both of which resolve after removal of the tumor.Patients often seek treatment in the Department of Respiratory and Critical Care Medicine or other internal medicine departments due to symptoms caused by ascites or hydrothorax.Here,we report a rare case of Meigs'syndrome caused by granulosa cell tumor accompanied with intrathoracic lesions.CASE SUMMARY A 52-year-old women was admitted to the Department of Respiratory and Critical Care Medicine due to coughing and expectoration accompanied with shortness of breath.Chest X-ray and chest computed tomography showed a modest volume of pleural fluid with pleural thickening in the right lung.The carbohydrate antigen 125(CA125)concentration was 150.8 U/mL(normal,0-35 U/mL)and no tumor cells were observed in pleural fluid.Nodules and a neoplasm with a fish meat-like appearance in the parietal pleura and nodules with a‘string of beads’-like appearance in the diaphragm were found by thoracoscopic examination.Furthermore,pelvic magnetic resonance revealed a pelvic mass measuring about 11.6 cm×10.0 cm×12.4 cm with heterogeneous signal intensity and multiple hypointense separations.Total abdominal hysterectomy,bilateral adnexectomy,and separation of pelvic adhesion were performed under general anesthesia.The pathology results showed granulosa cell tumor.At the 2-mo follow-up after the surgery,the hydrothorax subsided,and the CA125 level returned to normal.CONCLUSION For postmenopausal women with unexplained hydrothorax and elevated CA125,in addition to being suspected of having gynecological malignancy,Meigs’syndrome should be considered.
文摘Introduction: Extraovarian granulosa cell tumor (GCT) is a very uncommon tumor, assumed to arise from the ectopic gonadal tissue along the embryonal route of the genital ridge. Case report: We report the case of a 25 year-old woman who was presented at the emergency unit with a severe abdominal pain focused on the left iliac fossa. The patient had delivered normally 2 months before. An ovarian mass of 79 × 67 × 89 mm was shown in vaginal ultrasound as well as at the abdominal scan. Exploratory laparoscopy was performed and a torsion of the fallopian tube and an hemato-salpinges was visualized. Re-vascularisation was not achieved and a left salpingectomy took place. The immuno-histopathology report revealed an extra-ovarian GCT deriving from the fimbriae of the tube. Follow-up surgery was discussed. The case is presented for its rarity.
基金supported by National Natural Science Foundation of China(30300253)Chen guang youth technology program of Wuhan(20065004116-25)
文摘Based on published sequences for chicken Bcl-2,three siRNAs（small interfering RNA）were designed,and expression vectors were constructed and transfected into goose granulosa cells cultured in vitro.Bcl-2 protein,apoptosis and proliferation of granulosa cells,48 h after the transf ection,were analyzed by flow cytometry,and progesterone（P）secreted into the culture medium was measured by radioimmunoassay.In addition,apoptosis and Bcl-2 protein level were assessed in untreated granulosa cells from the four largest preovulatory follicles（F<sub>1</sub><sup>F</sup><sub>4</sub>）,the smallest preovulatory follicles（SPF）,small yellow follicles（SYF）and atretic follicles.The highest level of Bcl-2 protein was observed in granulosa cells from SPF,and levels in cells from healthy follicles were significantly higher than those of atretic follicles（P【0.05）.Bcl-2 protein levels in cells subjected to RNAi were significantly lower than those of controls（P【0.05）,while apoptosis indices（AI）,proliferation indices（PI）and P secretion in the RNAi treatments were higher than those of controls（P【0.05）.
基金supported by National Science Centre(2012/05/B/NZ9/03333)The Ministry of Science and Higher Education in Poland(UWM No.528.0206.0806)
文摘Background: Long non-coding RNAs(lncRNAs) may regulate gene expression in numerous biological processes including cellular response to xenobiotics.The exposure of living organisms to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD),a persistent environmental contaminant,results in reproductive defects in many species including pigs.The aims of the study were to identify and characterize lncRNAs in porcine granulosa cells as well as to examine the effects of TCDD on the lncRNA expression profile in the cells.Results: One thousand six hundred sixty-six lncRNAs were identified and characterized in porcine granulosa cells.The identified lncRNAs were found to be shorter than mRNAs.In addition,the number of exons was lower in lncRNAs than in m RNAs and their exons were longer.TCDD affected the expression of 22 lncRNAs(differentially expressed lncRNAs [DELs]; log2 fold change ≥ 1,P-adjusted < 0.05) in the examined cells.Potential functions of DELs were indirectly predicted via searching their target cis-and trans-regulated protein-coding genes.The coexpression analysis revealed that DELs may influence the expression of numerous genes,including those involved in cellular response to xenobiotics,dioxin metabolism,endoplasmic reticulum stress and cell proliferation.Aryl hydrocarbon receptor(AhR) and cytochrome P450 1 A1(CYP1 A1) were found among the trans-regulated genes.Conclusions: These findings indicate that the identified lncRNAs may constitute a part of the regulatory mechanism of TCDD action in granulosa cells.To our knowledge,this is the first study describing lncRNAs in porcine granulosa cells as well as TCDD effects on the lncRNA expression profile.These results may trigger new research directions leading to better understanding of molecular processes induced by xenobiotics in the ovary.
基金supported by the National Natural Science Foundation of China (31201771)the earmarked fund for the China Agriculture Research System (CARS-36)
文摘MicroRNAs(miRNAs) are endogenous 18-24 nucleotide(nt) non-coding RNAs,some of which have been indicated to play key roles in granulosa cells(GCs) function.However,little is known about how the miRNA gene expression itself is regulated in the GCs.Our previous study showed that miR-34 c,identified to be a pro-apoptotic and anti-proliferative factor in many cell types,exerted the same effects in porcine GCs.Here,the transcriptional regulation of miR-34 c expression in GCs was further investigated.5' rapid amplification of cDNA ends(RACE) assay indicated that the pri-miR-34 c transcription start site was located in 1 556 bp upstream of pre-miR-34 c.With dual-luciferase reporter assay,we confirmed a 69 bp core promoter region(-1 799 bp/-1 730 bp) was indispensable for the transcription of miR-34 c.Chromatin immunoprecipitation(ChIP) assay demonstrated that p53,p50,and p65 could bind to the transcription factor binding sites within the 69 bp core promoter region.In addition,deletion of transcripition factor binding sites resulted in obvious change of the miR-34 c promoter activity.Finally,using overexpression and knockdown of p53,p50,and p65 strategies,we showed that p53 and p50 could positively regulated miR-34 c expression,whereas p65 neletively regulated miR-34 c expression in GCs.Our results provide new data about the transcription regulatory mechanism of miRNA genes in GCs.
