miR‑423 sponged by lncRNA NORHA inhibits granulosa cell apoptosis

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.

miR-24-3p promotes proliferation and inhibits apoptosis of porcine granulosa cells by targeting P27

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.

Smad8 is involvement in follicular development via the regulation of granulosa cell growth and steroidogenesis in mice

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.

CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9

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.

Sirtuin 3 regulation:a target to alleviateβ-hydroxybutyric acid-induced mitochondrial dysfunction in bovine granulosa cells

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.

Effect of Mitochondrial Function of Ovarian Granulosa Cells on In Vitro Fertilization and Embryo Transfer Outcomes in Obese Polycystic Ovary Syndrome Patients

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.

Expression of Leptin Long-form Receptor mRNA in Luteinized Granulosa Cells of Obese Women with Polycystic Ovary Syndrome

To investigate the expression of mRNA of leptin long-form receptor （OB-Rb） in luteinized granulosa ceils of obese women with polycystic ovary syndrome （PCOS）, and to determine the role of leptin in the physiopathology of PCOS, luteinized granulosa cells were collected from the follicle fluid of 10 obese women who met the diagnostic criteria for PCOS and their BMI was equal to or greater than 25 kg/m^2, and at the same time, granulosa cells were collected from 10 normal women undergoing IVF-ET who served as the control group. Some luteinized granulosa cells were taken from normal women for in-vitro culture, into which human leptin of different concentrations was added （0, 10, 100 and 1000 ng/mL）. After stimulation with leptin for 48 h, RT-PCR was employed for the detection of the expression of OB-RLInRNA in the luteinized granulosa cells. Our results showed that the level of OB-RLmRNA in luteinized granulosa cells of obese PCOS women was higher than those in the control （P〈0.05）. In luteinized granulosa cells cultured in vitro and stimulated by human leptin for 48 h, the level of OB-RLmRNA was higher than those without leptin stimulation （P〈0.01）, and when leptin concentration was at 100 ng/mL, and the level of OB-RLmRNA reached a peak, It is concluded that in obese PCOS women, the level of serum leptin is increased, which promotes the expression of OB-RL in luteinized granulosa cells and increases the sensitivity of the granulosa cells to leptin. Leptin may contribute to anovulation in obese women with PCOS.

Apoptosis and Expression of Protein TRAIL in Granulosa Cells of Rats with Polycystic Ovarian Syndrome

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.

Follicle-stimulating hormone is expressed in ovarian follicles of chickens and promotes ovarian granulosa cell proliferation

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.

MiR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells

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.

Interaction between porcine granulosa and thecal cells in steroidogenesis in an amnion dual chamber culture system

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.

Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1

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.

NORHA,a novel follicular atresia-related lncRNA,promotes porcine granulosa cell apoptosis via the miR-183-96-182 cluster and FoxO1 axis

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.

Effects of chronic heat stress on granulosa cell apoptosis and follicular atresia in mouse ovary

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 ℃ for 7, 14, 21 or 28 d（n = 6） or to 42 ℃ 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 ℃ for 24 h. In Exp. 3, granulosa cells from ovaries maintained at 37 or 41 ℃ 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.

Effect of vitamin D3 on production of progesterone in porcine granulosa cells by regulation of steroidogenic enzymes

1,25-dihydroxyvitamin D3 （VD3）, an active form of Vitamin D, is photosynthesized in the skin of vertebrates in response to solar ultraviolet B radiation （UV-B）. VD3 deficiency can cause health problems such as immune disease, metabolic disease, and bone disorders. It has also been demonstrated that VD3 is involved in reproductive functions. Female sex hormones such as estrogen and progesterone are biosynthesized mainly in ovarian granulosa cells as the ovarian follicle develops. The functions of sex hormones include regulation of the estrus cycle and puberty as well as maintenance of pregnancy in females. In this study, we isolated granulosa cells from porcine ovaries and cultured them for experiments. To examine the effects of VD3 on ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by Real-time PCR and Western blotting assay. Production of progesterone from granulosa cells was also measured by ELISA assay. As a result, transcriptional and translational regulation of progesterone biosynthesis-related genes in granulosa cells was significantly altered by VD3. Furthermore, progesterone concen- trations in porcine granulosa cell-cultured media decreased in response to VD3. These results show that VD3 was a strong regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.

Granulosa Cell Tumors of the Ovary: Retrospective Analysis of 17 Cases

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.

MicroRNA-34c regulates porcine granulosa cell function by targeting forkhead box O3a

Granulosa cells（GCs） are somatic cells of ovary, the behaviors of GCs are important for ovarian function. MicroRNAs（miRNAs） are a class of endogenous 18–24 nucleotide（nt） non-coding RNAs, some of which have been shown to be important regulators of GCs function. miR-34c involved in the regulation of various biological processes and was identified to be a pro-apoptotic and anti-proliferative factor in many cell types. However, the roles of miR-34c in GCs function remain unknown. In this study, we used Annexin V-FITC and Ed U assays to demonstrate that miR-34c exerted pro-apoptotic and anti-proliferative effects in porcine GCs. Dual-luciferase reporter assays, quantitative real-time PCR（q RT-PCR） and Western blotting identified Forkhead box O3a（Fox O3a） as a direct target gene of miR-34c. The overexpression of FoxO3a rescued the phenotypic change caused by miR-34c in porcine GCs. In conclusion, miR-34c regulate the function of porcine GCs by targeting FoxO3a.

Oleic acid reduces steroidogenesis by changing the lipid type stored in lipid droplets of ovarian granulosa cells

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.

miRNA sequencing analysis of healthy and atretic follicles of chickens revealed that miR-30a-5p inhibits granulosa cell death via targeting Beclin1

Background:The egg production performance of chickens is affected by many factors,including genetics,nutrition and environmental conditions.These factors all play a role in egg production by affecting the development of follicles.MicroRNAs(miRNAs)are important non-coding RNAs that regulate biological processes by targeting genes or other non-coding RNAs after transcription.In the animal reproduction process,miRNA is known to affect the development and atresia of follicles by regulating apoptosis and autophagy of granulosa cells(GCs).Results:In this study,we identified potential miRNAs in the atretic follicles of broody chickens and unatretic follicles of healthy chickens.We identified gga-miR-30a-5p in 50 differentially expressed miRNAs and found that gga-miR-30a-5p played a regulatory role in the development of chicken follicles.The function of miR-30a-5p was explored through the transfection test of miR-30a-5p inhibitor and miR-30a-5p mimics.In the study,we used qPCR,western blot and flow cytometry to detect granulosa cell apoptosis,autophagy and steroid hormone synthesis.Confocal microscopy and transmission electron microscopy are used for the observation of autophagolysosomes.The levels of estradiol(E2),progesterone(P4),malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by ELISA.The results showed that miR-30a-5p showed a negative effect on autophagy and apoptosis of granulosa cells,and also contributed in steroid hormones and reactive oxygen species(ROS)production.In addition,the results obtained from the biosynthesis and dual luciferase experiments showed that Beclin1 was the target gene of miR-30a-5p.The rescue experiment conducted further confirmed that Beclin1 belongs to the miR-30a-5p regulatory pathway.Conclusions:In summary,after deep miRNA sequencing on healthy and atretic follicles,the results indicated that miR-30a-5p inhibits granulosa cell death by inhibiting Beclin1.