Mechanism Investigation Of Ovarian Insufficiency Caused By The Absence Of METTL3 In Theca Cells

Premature ovarian insufficiency（POI）is a common reproductive disorder that can lead to decreased ovarian function in livestock,mainly manifested as disruption of the estrous cycle in livestock,livestock do not ovulate or delay ovulation.At the same time,due to ovarian insufficiency,the ovarian function of livestock will be in a static state,without periodic activity,and the ovarian function will decline for a long time,causing tissue atrophy and sclerosis.Studies have shown that the occurrence of POI is related to estrogen deficiency,but its specific pathogenesis is complex,the pathogenesis is unclear,and the lack of effective prevention and treatment methods has become one of the main reasons affecting the reproductive efficiency of female animals.M⁶A is the most important modification method in RNA methylation.m⁶A modification refers to methylation at the N6 position of the adenosine base,and its biological function is mediated by specific binding proteins that recognize this modification.m⁶A is involved in regulating a variety of biological processes,including nuclear processing,splicing,transportation,as well as cytoplasmic translation and degradation.Methyltransferase like 3（METTL3）is one of the important components of m⁶A methyltransferase,which is crucial for various cell,tissue development,and disease occurrence processes,such as DNA repair,circadian rhythm regulation,obesity occurrence,immune regulation,spermatogenesis,and embryonic development.However,the role of METTL3 in follicular development remains unclear.In this study,we first constructed theca cells specific knockout METTL3 mice,and in-depth studied the role of METTL3 in follicular development.The results showed that after the deletion of METTL3 from theca cells,the mice fertility decreased and showed a POI like phenotype.In order to further explore the mechanism of METTL3 in follicular development,theca cells（TCs）and granulosa cells（GCs）were used as cell models to explore the mechanism of METTL3 regulating follicular development.The main research contents and results are as follows:1.Effect of METTL3 deficiency on ovarian development in mice:In this study,we successfully constructed TCs METTL3 specific knockout mice using the Cre/Lox P system.The results showed that the absence of METTL3 in the theca cells can lead to impaired fertility in mice,mainly manifested by the disturbance of the mice estrus cycle,a decrease in the number of corpus luteum in the ovary,and a decrease in the number of litter size.In order to further study the causes of reproductive damage caused by the deletion of METTL3 in the follicular membrane of mice,this experiment conducted RNA sequencing on the mice ovaries.Through GO and KEGG enrichment analysis,it was found that these differential genes are mainly closely related to inflammation and steroid synthesis.Detection of related gene expression showed that the expression of steroid synthesis genes STAR,and inflammatory gene IL-1β（interleukin 1β）in the ovary was significantly upregulated（p<0.05）after theca cells deletion of METTL3,and promoted testosterone（T）synthesis and IL-1βsecretion（p<0.05）,while the expression of CYP19A1,FSHR were downregulated（p<0.05）,and estradiol（E₂）secretion was decreased（p<0.05）.Combining the above detection indicators,it was found that the theca cells METTL3 specific knockout mice exhibited a POI-like phenotype.2.Effect of si METTL3 on the secretion of IL-1βin theca cells and study of its mechanismTo further investigate the molecular mechanism by which follicular theca cells METTL3-specific knockout mice develop impaired reproduction,this study used mouse theca cells and porcine theca cells as an in vitro cellular model,knocked down the expression of endogenous METTL3 by using si RNA interference,and detected the secretion of IL-1βby mouse theca cells and porcine follicular theca cells.The results of the study revealed that si METTL3 resulted in reduced synthesis of METTL3（p<0.05）in theca cells following the reduction of endogenous METTL3,which promoted the expression of testosterone synthesis-associated genes（STAR）and IL-1βgenes（p<0.05）,as well as the secretion of testosterone and IL-1β（p<0.05）.In order to further explore the theca cells the molecular mechanisms underlying the reduction of METTL3 and upregulation of IL-1β.In this study,mi RNA expression in porcine theca cells after si METTL3 was detected by mi RNA sequencing.Among the mi RNAs expressed in high abundance,30 mi RNAs were reduced,indicating that METTL3 affects mi RNA synthesis in porcine theca cells.The down-regulated mi RNAs were subjected to interaction network analysis and dual luciferase assay with IL-1β.mi R-21-5p was shown to target IL-1β.An in-depth study revealed that reduced synthesis of METTL3in porcine theca cells reduced the level of m⁶A modification of pri-mi R-21,which further reduced the recognition and binding of the RNA-binding protein DGCR8 to pri-mi R-21,leading to a reduction in the synthesis of mature mi R-21-5p.Inhibition of mi R-21-5p in porcine theca cells increased IL-1βexpression（p<0.05）and promoted IL-1βsecretion（p<0.05）,suggesting that mi R-21-5p is involved in regulating IL-1βgene expression.3.Effects of IL-1βaccumulation in TCs on ovarian development in miceThis study used mice as an animal model to study the effect of IL-1βaccumulation in theca cells on mouse ovarian development.Overexpression of IL-1βin mouse theca cells were achieved through adenoviruses.The results showed that the accumulation of IL-1βin theca cells can lead to impaired fertility in mice,mainly manifested by the disorder of the mouse estrus cycle and the reduction of litter size.To further investigate the cause of reproductive damage caused by IL-1βaccumulation in mice,the expression levels of steroid synthesis genes CYP19A1 and FSHR genes in ovarian tissue were detected,and the results showed that CYP19A1 and FSHR m RNA and protein levels were significantly down-regulated（p<0.05）.The levels of IL-1βand E₂ in serum were further tested,and it was found that the accumulation of IL-1βin theca cells promoted the secretion of IL-1β（p<0.05）,while the E₂ level had a downward trend（p=0.069）.4.IL-1βcauses GCs apoptosis by increasing intracellular ROS-Ca²⁺levels leading to mitochondrial damageTo further explore the effects of increased IL-1βsecretion on follicular development due to METTL3 deletion/reduction in the TCs,the studies in this chapter explored the effects of IL-1βon GCs apoptosis using porcine follicular GCs,mouse GCs and KGN cells as cell models.GCs viability were measured by CCK8,apoptosis level,cellular reactive oxygen species level,intracellular Ca²⁺level,mitochondrial membrane permeability and mitochondrial membrane potential by flow cytometry.The findings revealed that IL-1βinduced a decrease in cell viability and an increase in intracellular reactive oxygen species levels in GCs,which disrupted intracellular Ca²⁺homeostasis,increased the proportion of apoptotic cells,and an increase in mitochondrial membrane permeability further leading to a decrease in the mitochondrial membrane potential.5.Effect of IL-1βon GCs apoptosis via the cAMP-PKA-NFκB signalling pathwayTo further explore the mechanism of action of IL-1βin inducing apoptosis in GCs.The following studies were performed.The expression of apoptosis-related genes and estradiol synthesis-related genes were detected after IL-1βtreatment of GCs.The results revealed that m RNA levels and protein levels of BAX/BCL2 and TNFαwere significantly increased（p<0.05）,and m RNA levels and protein levels of CYP19A1 and FSHR were significantly decreased（p<0.05）after IL-1βtreatment of GCs.Meanwhile,the results of the study found that autophagy levels and p-PKA levels were significantly reduced（p<0.05）and NFκB pathway p-P65 levels were significantly increased（p<0.05）after IL-1βtreatment of GCs.To further validate that IL-1βinduces apoptosis in GCs through the cAMP-PKA-NFκB signalling pathway,GCs were treated with the cAMP-PKA pathway inhibitor,H89,and the activator,8-Br-cAMP,in this study.The results revealed that the m RNA level and protein level of BAX/BCL2 and TNFαgenes were significantly increased after H89 treatment of GCs（p<0.05）,and the m RNA level and protein level of CYP19A1 gene were significantly decreased（p<0.05）,meanwhile,the level of autophagy was significantly decreased after H89 treatment of GCs（p<0.05）,and the level of the NFκB pathway p-P65 was were significantly increased（p<0.05）.While the addition of activator 8-Br-cAMP significantly decreased the m RNA level and protein level of BAX/BCL2 and TNFαgenes（p<0.05）and significantly increased the m RNA level and protein level of CYP19A1 gene（p<0.05）compared with the IL-1βgroup,at the same time,the level of autophagy was significantly increased by the treatment of GCs with 8-Br-cAMP（p<0.05）,and the level of NFκB pathway p-P65 was significantly reduced（p<0.05）.The results indicated that IL-1βinduced apoptosis in GCs by inhibiting the cAMP-PKA signalling pathway and activating the NFκB pathway.Conclusively,this study report the first observation that METTL3 knockout in TCs caused a POI-like phenotype.Further,a METTL3 reduction resulted in decreased pri-mi R-21 m⁶A modifications,thus suppressing DGCR8 binding and recognition,leading to a decrease in the synthesis of mature mi R-21-5p,which then promoted IL-1βsecretion from TCs.Acting in a paracrine manner,IL-1βinhibited the cAMP–PKA pathway and activated the NFκB pathway in follicular GCs.This increased ROS levels in GCs,causing disturbances in the intracellular Ca²⁺balance and mitochondrial damage.These cellular events ultimately led to GCs apoptosis and decreased E₂synthesis,resulting in POI development.This is the first study reporting a mechanism of POI occurrence from the perspective of TCs and offers a theoretical foundation to investigate the involvement of m⁶A in ovary functions and a new theory for the development of POI disease.