UCHL1 promotes the proliferation of porcine granulosa cells by stabilizing CCNB1

Background The proliferation of porcine ovarian granulosa cells(GCs)is essential to follicular development and the ubiquitin–proteasome system is necessary for maintaining cell cycle homeostasis.Previous studies found that the deubiquitinase ubiquitin carboxyl-terminal hydrolase 1(UCHL1)regulates female reproduction,especially in ovarian development.However,the mechanism by which UCHL1 regulates porcine GC proliferation remains unclear.Results UCHL1 overexpression promoted GC proliferation,and knockdown had the opposite effect.UCHL1 is directly bound to cyclin B1(CCNB1),prolonging the half-life of CCNB1 and inhibiting its degradation,thereby promoting GC proliferation.What's more,a flavonoid compound-isovitexin improved the enzyme activity of UCHL1 and promoted the proliferation of porcine GCs.Conclusions UCHL1 promoted the proliferation of porcine GCs by stabilizing CCNB1,and isovitexin enhanced the enzyme activity of UCHL1.These findings reveal the role of UCHL1 and the potential of isovitexin in regulating proliferation and provide insights into identifying molecular markers and nutrients that affect follicle development.

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.

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.

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.

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.