Mevalonate metabolites boost aged oocyte quality through small GTPases prenylation

Mevalonate metabolites boost aged oocyte quality through small GTPases prenylation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mevalonate metabolites boost aged oocyte quality through small GTPases prenylation Lijun Ding, Chuanming Liu, Huidan Zhang, Jialian Mao, Sainan Zhang, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4762298/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Declined oocyte quality is the major contributor to female subfertility in aged mammals. Currently, there are no effective interventions to ameliorate aged oocyte quality. We found that oocytes from aged mice exhibited lower levels of mevalonate (MVA) pathway metabolites, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) and reduced cortical F-actin. We further demonstrated that MVA supplementation improved the FPP level, the cortical F-actin and the quality of aged oocytes. Mechanistically, we found that MVA supplementation induced granulosa cells to synthesize FPP, which was subsequently transferred to aged oocytes. Transported FPP increased small GTPases prenylation, including CDC42 and RAC1, and promoted membrane localization of CDC42-N-WASP-Arp2/3 and RAC1-WAVE2-Arp2/3 complexes, promoting cortical F-actin re-assembly and reducing aneuploidy of aged oocytes. We also identified an oral drug 8-isopentenyl flavone, as an isoprenoid donor from Epimedium brevicornu Maxim, which could increase CDC42 and RAC1 prenylation, improving the cortical F-actin and the competence of aged oocytes, ameliorating reproductive outcomes in aged female mice. Collectively, increasing small GTPases prenylation via MVA metabolites or 8-IPF provide a therapeutic approach for boosting fertility in women of advanced maternal age. Biological sciences/Developmental biology/Ageing Health sciences/Endocrinology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Human ovaries are one of the earliest organs to exhibit aging-associated dysfunctions, with a significant functional decline in women over 35 1,2 . Ovarian aging mainly manifests as declines in oocyte quality and quantity, resulting in female subfertility and infertility 3 . Abnormal F-actin assembly and aneuploidy are important contributors to age-related decline in oocyte quality 4 – 6 . Arp2/3-dependent assembly of the cortical F-actin affect oocyte contractions, cytoplasmic organization and developmental potential 7 . However, whether cortical F-actin of oocytes is involved in ovarian aging remains largely unknown. Mevalonate (MVA) pathway is crucial for cholesterol synthesis and regulates multiple cellular processes through protein prenylation, a lipidic modification that facilitates the anchoring of proteins to cell membranes 8 . Small GTPases could be prenylated and regulate cell division and morphogenesis by promoting cytoskeleton organization and cortex formation 9 . Recently, we reported that abnormal metabolism of the MVA pathway in granulosa cells (GCs) is an important contributing factor to meiotic defects and aneuploidy and MVA supplementation increases normal meiosis in aged oocyte 10 . How MVA pathway acts on actin assembly of aged oocytes within cumulus-oocyte complexes (COCs) .needs to be clarified Recent studies have reported several therapeutic strategies for improving the quality of aged oocyte, including the use of antioxidants, growth hormones, melatonin, nicotinamide mononucleotide and the polyamine metabolite spermidine, their effectiveness is yet to be tested in clinic 11 – 15 . MVA pathway is a pivotal water-soluble upstream for cholesterol and more than 50,000 terpenoids synthesis and is required for cell cycle progression and cell proliferation 8 , 16 , 17 . Previous studies have shown that MVA pathway is important for the coordination of regulatory T-cell proliferation by enhancing transforming growth factor (TGF)-β signalling and mediation of training immunity via IGF1-R and mTOR activation 18 , 19 . MVA supplementation has also been demonstrated to counteract adverse developmental effects of statins on blastocyst formation 20 , 21 . In this study, we found that Arp2/3-dependent assembly of the cortical F-actin was decreased in aged oocyte, accompanied by reduced MVA pathway metabolites (farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP)) levels. MVA supplementation improved the quality of aged oocytes in COCs by promoting cortical F-actin assembly, ameliorating meiotic errors and promoting embryonic development both in vitro and in vivo . Mechanistically, MVA activated the FPP metabolic pathway in GCs and promoted cortical F-actin assembly by increasing CDC42 and RAC1 prenylation and Arp2/3 complex formation in aged oocytes. In addition, 8-isopentenyl flavone (8-IPF), a natural chemical that is being used as an oral drug, also ameliorated the decline in ovarian reserve and significantly increased oocyte quality in aged mice by enhancing the prenylation of CDC42 and RAC1 on the oocyte membrane to assemble the cortical F-actin. Results Decreased MVA pathway metabolites and cortical F-actin distribution in aged oocytes. Our recent study indicates that downregulation of the MVA pathway in aged ovaries and FPP- and GGPP-mediated protein prenylation may be involved in the decrease of quality in aged oocytes 10 . To comprehensively study the change of FPP and GGPP metabolites in oocytes and surrounding GCs, we established a targeted metabolomics approach to detect FPP and GGPP using low cell inputs. We collected 10 oocytes at metaphase I (MI) stage and 10 4 surrounding GCs from young (6-week-old) and aged (10-month-old) mice with seven biological replicates for the MVA metabolite test (Fig. 1 a). Remarkably, aged oocyte contained significantly fewer FPP (2.9 ± 0.2 ng/ml versus 7.1 ± 0.7 ng/ml, P = 0.0001) and GGPP (7.0 ± 0.5 ng/ml versus 16.0 ± 2.2 ng/ml, P = 0.002) compared with young oocyte (Fig. 1 b,d,e). In addition, the FPP (0.6 ± 0.05 ng/ml versus 1.1 ± 0.05 ng/ml, P < 0.0001) and GGPP (1.0 ± 0.04 ng/ml versus 2.1 ± 0.05 ng/ml, P < 0.0001) metabolites levels were also significantly decreased in aged GC (Fig. 1 c,d,e). It was reported that FPP and GGPP are necessary for protein localization on the cell membrane for cytoskeletal organization 8 . Actin filaments become denser at the oocyte periphery and assemble cortical F-actin with the recruitment of proteins from the cytoplasm to membranes 22 . To explore the changes of cortical F-actin during ovarian aging, oocytes were collected from young and aged COCs after cultured for 9 h, and cortical F-actin distribution of oocytes at MI was examined. The fluorescence intensity of cortical F-actin of aged oocytes were much weaker than that of young oocytes (0.2 ± 0.03 versus 1.0 ± 0.09, P < 0.0001) (Fig. 1 f,g). The assembly of cortical F-actin requires the activity of the Arp2/3 complex, which functions as an actin nucleator 7 , 23 . Aged oocytes showed decreased Arp3 expression compared with the young oocytes (0.6 ± 0.09 versus 2.8 ± 0.2, P < 0.0001) (Fig. 1 h,i). These results indicate that the decreased MVA downstream metabolites and defective nucleation of cortical F-actin may be involved in abnormal assembly of the cortical F-actin during ovarian aging. MVA promotes cortical F-actin assembly and aged oocyte quality via the surrounding GCs. Since the levels of FPP and GGPP were significantly reduced in aged oocytes and MVA is the precursor for terpenoids, including FPP and GGPP 16 , 17 , we next investigated the effects of MVA supplementation on aged oocyte quality. We isolated COCs from 10-month-old female mice and cultured them in in vitro maturation (IVM) medium supplemented with 50 µM MVA (Fig. 2 a). We first compared the cortical F-actin between the two groups treated with or without MVA and found that MVA supplementation markedly promoted cortical F-actin assembly of MI ooctyes from aged COCs (6.3 ± 0.4 versus 1.0 ± 0.2, P < 0.0001) (Fig. 2 b,c). In addition, the decreased Arp3 expression in aged MI oocyte cortex was rescued after MVA supplementation (1.5 ± 0.08 versus 1.0 ± 0.04, P < 0.0001) (Fig. 2 d,e). Since actin is responsible for various essential function, including spindle migration, accurate chromosome segregation, and PBE during oocyte meiosis, we next compared meiotic progression in oocytes from the two groups 23 . The PBE rate was significantly higher in the group treated with MVA than in the control group (95.1% ± 3.3% versus 81.2% ± 4.0%, P = 0.02) although the germinal vesicle breakdown (GVBD) rates of oocytes collected from COCs were not different between the groups with or without MVA supplementation (Fig. 2 f and Extended Data Fig. 1 a,b). We further evaluated meiotic defects and developmental potential of MII oocytes collected from the two groups. The incidence of meiotic defects (18.2% ± 5.9% versus 38.2% ± 4.1%, P = 0.03) and aneuploidy (16.7% ± 1.9% versus 38.9% ± 2.0%, P = 0.001) were markedly reduced after MVA supplementation (Fig. 2 g,h and Extended Data Fig. 1 c,d). Moreover, the MII oocytes derived from MVA-treated COCs were more competent to develop into 2-cell embryos (79.7% ± 4.2% versus 54.1% ± 5.1%, P = 0.005) and blastocysts (28.2% ± 3.6% versus 13.1% ± 2.4%, P = 0.008) than those derived from the control group (Fig. 2 i-k). To further clarify whether MVA acted on oocytes or GCs, we also treated denuded oocytes (DOs) with 50 µM MVA. Notably, the incidence of GVBD, PBE and meiotic defects were not improved in DOs after MVA supplementation (Extended Data Fig. 1 e-i), indicating that the effects of MVA on aged oocytes were executed via GCs. To further evaluate the effects of MVA on aged COCs in vivo , 9-month-old female mice were intraperitoneally injected with 5 mg kg − 1 MVA for 30 days (Fig. 3 a). The ovarian index of aged mice was considerably increased (1.4 ± 0.07 versus 1.1 ± 0.08, P = 0.03) without affecting body weight after MVA supplementation (Fig. 3 b and Extended Data Fig. 2 a). MVA supplementation also ameliorated age-related depletion in ovarian reserve and increased the number of follicles for all developmental stages, especially the antral stage (Extended Data Fig. 2 b-d). Moreover, the number of ovulated MII oocytes doubled after MVA injection (9.8 ± 1.2 versus 4.5 ± 1.1, P = 0.003) (Fig. 3 c). MVA treatment significantly increased the cortical F-actin assembly (2.8 ± 0.2 versus 1.0 ± 0.1, P < 0.0001) and Arp3 expression (1.7 ± 0.3 versus 1.0 ± 0.2, P = 0.05) of MI oocytes from aged mice (Fig. 3 d-g). As shown in Extended Data Fig. 2 e,f, the percentage of abnormal spindles and misaligned chromosomes were considerably lower in oocytes from MVA-treated mice than those from control mice (26.3% ± 2.3% versus 46.3% ± 6.5%, P = 0.04). Chromosome spread experiments further confirmed a markedly lower frequency of aneuploidy in oocytes after MVA injection (25.1% ± 4.3% versus 50.5% ± 2.9%, P = 0.001) (Fig. 3 h,i). Moreover, more 2-cell embryos (63.8% ± 2.4% versus 43.7% ± 3.8%, P = 0.02) and blastocysts (53.8% ± 2.3% versus 39.3% ± 4.1%, P = 0.04) could be obtained from oocytes from MVA-treated mice (Fig. 3 j-l). Taken together, these results suggest that MVA ameliorates cortical F-actin assembly, meiotic defects, and aneuploidy in aged oocytes via GCs both in vitro and in vivo . MVA activates the FPP metabolic pathway in aged GCs. To further investigate how MVA supplementation improved the quality of aged oocytes, we performed RNA sequencing analysis of oocytes and neighbouring GCs from aged COCs treated with or without MVA (Fig. 4 a). PCA and comparison between the two groups revealed a highly dynamic change in gene expression after MVA supplementation. A total of 2116 downregulated and 1287 upregulated differentially expressed genes (DEGs) were identified in oocytes after MVA supplementation (Extended Data Fig. 3 a,b). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs revealed that genes associated with the cell cycle and oocyte meiosis were upregulated, whereas genes associated with ribosomes and thermogenesis were downregulated in oocytes from MVA-treated COCs (Extended Data Fig. 3 b,c). Many meiosis-associated genes such as Cdc20 , Bub3 , Ccnb1 and Mos play important roles during oocyte meiosis, and the downregulation of these genes results in meiotic defects and aneuploidy 24 – 27 . As shown in Fig. 4 C, the expression of Ccna2, Cdc20, Bub3, Ccnb1, Ppp2r1a, Mos, Mad2l1, Pk1, Map2K1, Cdc42ep2 , and Cdc42se2 was markedly increased in oocytes from MVA-treated COCs. To investigate how MVA improved cortical F-actin assembly and meiosis in aged oocyte via GCs, we further analyzed the transcriptome of GCs. PCA and volcano plotting revealed differences in the transcriptome profile of the MVA-treated GCs and control GCs, with 9317 downregulated and 1279 upregulated DEGs (Fig. 4 d and Extended Data Fig. 3 d). KEGG analysis revealed that genes associated with ribosome, oxidative phosphorylation, and proteasome were upregulated, while genes associated with neuroactive ligand‒receptor interaction and calcium signalling pathway were downregulated after MVA supplementation (Extended Data Fig. 3 e,f). Because MVA is an early rate metabolite in the cholesterol synthesis pathway 8 , we next analysed the expression of metabolic genes related to the MVA pathway (Extended Data Fig. 4 a). The expression of genes involved in the metabolism of MVA to FPP, including MVK , PMVK , MVD , IDI1 , and FDPS , was increased 1.8- to 4.0-fold in GCs after MVA supplementation (Fig. 4 e and Extended Data Fig. 4 a). However, genes encoding metabolic enzymes at biosynthetic steps from acetyl-CoA to MVA and FPP to cholesterol did not exhibit obvious changes in expression (Extended Data Fig. 4 a-c). qRT‒PCR and Western blotting analysis also confirmed that both the mRNA and protein levels of MVK and FDPS were significantly elevated after MVA supplementation (Fig. 4 f,g and Extended Data Fig. 4 d). Moreover, the MVA (896.8 ± 7.6 ng/ml versus 506.6 ± 42.5 ng/ml, P = 0.001) and FPP (0.17 ± 0.005 ng/ml versus 0.14 ± 0.007 ng/ml, P = 0.03) metabolites levels and protein prenylation level were significantly increased after MVA supplementation (Fig. 4 h-j). Taken together, our results suggest that MVA supplementation activates the FPP synthesis pathway and increases the protein prenylation levels in aged COCs. MVA promotes cortical F-actin assembly via prenylation by FPP from GCs. To clarify whether the effects of MVA on oocyte cortical F-actin assembly and meiosis was mediated by prenylation via FPP synthesis from GCs, COCs were isolated from 10-month-old female mice and subsequently cultured in IVM medium supplemented with FOH, which can be catabolized to FPP in cells 28 . FOH supplementation promoted cortical F-actin assembly (0.6 ± 0.009 versus 0.2 ± 0.03, P < 0.0001) in MI ooctyes, increased the rate of PBE (89.5% ± 2.5% versus 70.2% ± 1.9%, P = 0.004), and reduced the incidence of meiotic defects (23.9% ± 2.5% versus 43.3% ± 1.7%, P = 0.003) in oocytes from aged COCs, similar to MVA treatment (Fig. 5 a-d and Extended Data Fig. 5 a,b). To further explore the relationships between prenylation and oocyte quality in aged COCs, FTI-277, an inhibitor of protein prenylation, was added to IVM medium supplemented with MVA. Indeed, the addition of FTI-277 reduced cortical F-actin assembly (0.5 ± 0.03 versus 1.0 ± 0.07, P < 0.0001), decreased PBE rate (70.0% ± 1.8% versus 86.8% ± 3.4%, P = 0.01), and increased the incidence of meiotic defects (46.0% ± 0.2% versus 20.5% ± 1.5%, P < 0.0001) in oocytes from aged COCs in the presence of MVA (Fig. 5 a-d and Extended Data Fig. 5 a,b). To investigate whether FPP can be transferred from GCs to oocytes, an alkynyl-farnesol chemical reporter based on farnesol (alk-FOH) was used (Fig. 5 e). To study the incorporation of alk-FOH, human granulosa-like tumor cell line, KGN cells were first incubated with different concentrations of alk-FOH for 24 h, then the total cellular protein was ligated to azide-biotin via CuAAC (Extended Data Fig. 5 c). Western blotting analysis revealed that the alk-FOH was incorporated at 20 µM and increased considerably with 100 µM (Fig. 5 f). Moreover, alk-FOH incorporation was sensitive to competition by the natural metabolite FOH, suggesting that addition of the alkyne group did not compromise the activity of FOH (Extended Data Fig. 5 d). Next, we isolated DOs and COCs from 3-week-old female mice and cultured with alk-FOH for 14 h and they were visualized using fluorescence imaging (Fig. 5 g). No signals were detected in DOs, indicating that oocytes could not directly uptake FOH (Extended Data Fig. 5 e). Interestingly, obvious fluorescence signals were detected in both GCs and oocytes from COCs (Fig. 5 h). Moreover, alk-FOH signals also exhibited in the transzonal projections between GCs and oocyte (Fig. 5 i and Extended Data Fig. 5 f). Collectively, these results indicate that MVA activates the synthesis of FPP in GCs, and that the transfer of FPP from GCs to oocytes ameliorates aged oocyte cortical F-actin assembly and quality through prenylation. Prenylation increases CDC42 and RAC1 cortical localization and F-actin assembly. Prenylation involves the covalent addition of either farnesyl or geranylgeranyl isoprenoids to conserved cysteine (Cys) residues at the carboxyl-terminal CaaX or C(X)C motifs (Fig. 6 a). Immunofluorescence analysis revealed that the KGN cells also uptake FOH (Extended Data Fig. 6 a). To identify potential prenylated proteins involved in cortical F-actin assembly and oocyte meiosis, we performed large-scale profiling of prenylated proteins in KGN cells using alk-FOH. After incubation with azide-biotin, affinity enrichment, selective elution, liquid chromatography-tandem mass spectrometry (LC‒MS/MS) was used to identify prenylated proteins (Extended Data Fig. 6 a). Western blotting analysis revealed that a diverse range of proteins were labelled by alk-FOH (Fig. 6 b). A total of 140 putative prenylated proteins were identified by alk-FOH supplementation when compared to the control group; 39 of these proteins had a carboxyl-terminal CaaX or Rab motif (Extended Data Fig. 6 b). In addition, fifty-two percent (73/140) of the detected prenylated proteins were membrane-associated proteins (Extended Data Fig. 6 c). Prenylation can facilitate the anchoring of proteins to cell membranes, mediating protein‒protein interactions and signal transduction 16 . A Venn diagram showed that 24 proteins labelled by alk-FOH, which included CDC42 and RAC1, had a carboxyl-terminal CaaX or Rab motif and localized to the cell membrane (Extended Data Fig. 6 d,e). KEGG analysis revealed that 12 detected prenylated proteins were involved in the regulation of actin cytoskeleton, also including CDC42 and RAC1 (Fig. 6 c). CDC42 and RAC1 are small GTP binding proteins of the Rho subfamily and play critical roles in the establishment of oocyte polarity and subsequent meiosis 29 , 30 . To further validate whether CDC42 and RAC1 could be prenylated, we conducted immunoblot analysis using affinity enrichment of the labelled proteins. Both of these proteins could be labelled by alk-FOH, and the labelling was sensitive to competition with natural FOH, indicating that CDC42 and RAC1 were indeed prenylated (Fig. 6 d). Next, we examined whether MVA affected CDC42 and RAC1 membrane partitioning and cellular localization via prenylation. As shown in Fig. 6 e,f, MVA supplementation considerably increased the localization of CDC42 and RAC1 to the cell cortex, similar to the effect observed following treatment with FOH. As expected, supplementation with exogenous FTI-277 markedly reduced the MVA-induced anchoring of CDC42 and RAC1 to cell membranes, suggesting that MVA induces CDC42 and RAC1 prenylation and promotes their localization to the cell cortex. CDC42 and RAC1 are enriched in the oocyte cortex after chromosome migration to the periphery and regulate actin dynamics during oocyte meiosis 29 , 30 . To investigate the relationship between CDC42 and RAC1 prenylation and cortical F-actin assembly, we examined CDC42 and RAC1 localization in oocytes at MI by immunofluorescence. CDC42 and RAC1 failed to cortical localization in oocytes from aged COCs, and MVA supplementation induced CDC42 and RAC1 relocation to the cortex in aged oocytes, exhibiting a distribution pattern similar to that of F-actin, while the ameliorative effect of MVA was disrupted by FTI-277 (Extended Data Fig. 7 a-d). Next, we perturbed the CDC42/RAC1 signaling by overexpressing a CDC42/RAC1 dominant-negative mutant (CDC42 C188Y / RAC1 C188Y ; which is conserved position to the cysteine 188 at CVLL/CLLL motif to tyrosine respectively; previously demonstrated to inhibit CDC42/RAC1 prenylation) 31 , 32 . Expressing constitutively active CDC42 and RAC1 mutant failed to locate on the oocyte cortical F-actin, suggesting that CDC42 and RAC1 prenylation is essential for cortical F-actin assembly (Extended Data Fig. 7 e,f). The Arp2/3 complex can be activated by the CDC42/N-WASP or RAC1-WAVE2 signalling pathways to promote the cortical F-actin assembly in oocytes 9 , 33 . Reciprocal coimmunoprecipitation analysis further confirmed the endogenous interaction between CDC42 with N-WASP and the Arp2/3 complex, as well as the interaction between endogenous RAC1, WAVE2 and the Arp2/3 complex. In addition, FOH supplementation significantly enhanced the formation of CDC42-N-WASP-Arp2/3 and RAC1-WAVE2-Arp2/3 complexes (Fig. 6 g-j). Moreover, the Arp3 expression (2.9 ± 0.4 versus 1.0 ± 0.2, P = 0.0004) at the cortex of MI oocytes were significantly increased, while the addition of exogenous FTI-277 reduced the cortical Arp3 expression (1.1 ± 0.2 versus 2.6 ± 0.3, P = 0.0003) (Fig. 6 k,l). Together, these results suggest that MVA or FOH promotes the formation of CDC42-N-WASP-Arp2/3 and RAC1-WAVE2-Arp2/3 complexes for cortical F-actin assembly. For the first time, we found that prenylation of CDC42 and RAC1 by FPP, facilitates their localization to the membrane and contributes to cortical F-actin assembly in oocytes. The isopentenyl compounds 8-IPF facilitates cortical F-actin assembly in aged oocytes. Although MVA can effectively ameliorate ovarian reserve and oocyte quality in aged mice, currently there are no drugs containing MVA in the clinic. In order to improve the fertility of women with advanced age, approved and preclinical drugs were screened for their efficacy in increasing the expression of MVK and FDPS and protein prenylation. Among them, we identified 8-IPF, a traditional natural chemical used in China with an isopentenyl side chain 34 . Treatment with 50 µg L − 1 8-IPF considerably increased the prenylation level and the expression of MVK and FDPS in KGN cells (Fig. 7 a and Extended Data Fig. 8a,b). In addition, 8-IPF supplementation increased CDC42 and RAC1 cortical localization, similar to the results observed following MVA treatment in KGN cells (Fig. 7 b,c). We next analysed whether 8-IPF has therapeutic effects on ovarian aging in vivo using an aged mouse model. Briefly, 5 mg kg − 1 8-IPF was intragastrically administered to 9.5-month-old female mice for 14 days via gavage (Fig. 7 d). The ovarian index (2.0 ± 0.4 versus 1.4 ± 0.4, P = 0.0001) and the numbers of follicles considerably increased for all developmental stages after 8-IPF supplementation. Improvement effects were especially evident in growing follicles (secondary follicles (235.7 ± 28.4 versus 87.1 ± 21.7, P = 0.002) and antral follicles (76.4 ± 6.5 versus 22.9 ± 5.2, P = 0.0001)) (Fig. 7 e and Extended Data Fig. 8c-e). Moreover, the number of ovulated oocytes increased significantly after 8-IPF treatment (10.4 ± 1.3 versus 3.7 ± 0.3, P = 0.0002) (Extended Data Fig. 8f). More importantly, cortical F-actin staining of MI oocytes from aged COCs isolated from 8-IPF-treated mice were stronger than those from control oocytes (1.3 ± 0.1 versus 1.0 ± 0.1, P = 0.05) (Fig. 7 f,g). In addition, the decreased Arp3 expression in aged MI oocyte cortex was rescued after 8-IPF supplementation (2.6 ± 0.4 versus 1.0 ± 0.1, P = 0.001) (Fig. 7 h,i). When compared to the control, 8-IPF supplementation markedly reduced the incidence of meiotic defects (32.4% ± 3.1% versus 54.6% ± 2.4%, P = 0.005) and aneuploidy (28.3% ± 5.1% versus 53.9% ± 2.1%, P = 0.01) (Fig. 7 j,k and Extended Data Fig. 8g,h). Moreover, more 2-cell embryos (63.7% ± 4.7% versus 46.6% ± 1.7%, P = 0.03) and blastocysts (52.2% ± 4.7% versus 24.3% ± 7.3%, P = 0.03) could be obtained after 8-IPF treatment (Extended Data Fig. 8i-k). Excitingly, after natural mating with 8-week-old male mice, the pregnancy rate (85.7% versus 57.1%) and litter size (6.3 ± 0.3 versus 2.0 ± 0.6, P = 0.0001) were significantly higher in female mice subjected to 8-IPF treatment than in control mice (Fig. 7 l-n). Overall, 8-IPF ameliorates ovarian reserve depletion and oocyte meiotic defects, improving fertility outcomes in aged female mice by increasing CDC42 and RAC1 prenylation and cortical F-actin assembly in aged oocytes. Discussion The identification of effective methods to improve the quality of aged oocytes is a grand challenge in reproductive medicine. In this study, we found that MVA supplementation promoted cortical F-actin assembly and reduced meiotic defects in oocytes, improving the quality of oocytes from COCs in aged mice. Furthermore, MVA activated the FPP synthesis pathway in surrounding GCs, and GCs subsequently transferred the metabolite to aged oocytes through gap junction. FPP prenylated CDC42 and RAC1 in oocytes and promotes cortical F-actin assembly, which was critical for decreasing meiotic defects in oocytes. In addition, we found that natural herb compound 8-IPF could ameliorate ovarian reserve depletion and improved oocyte quality in aged mice by increasing CDC42 and RAC1 cortical localization for F-actin assembly (Extended Data Fig. 9). Oocytes and surrounding GCs form follicles, which constitute the fundamental reproductive units in the ovary. In addition, GCs establish a metabolic community with oocytes and transmit metabolites such as pyruvate, amino acids, and cholesterol for oocyte development and maturation 35 – 37 . Our recent studies and others reported that GCs exhibit a robust increase in MVA pathway gene expression during oocyte meiotic resumption, whereas oocytes show low expression of these regulatory genes 9 , 38 . This finding indicates that active MVA pathway metabolic synthesis occurs in GCs, in which MVA pathway metabolites are subsequently supplied to oocytes for meiosis. The present study further confirmed the metabolic coupling between GCs and oocytes using alk-FOH chemical reporters. Fluorescent labelling of FOH metabolism showed that active uptake of FOH occured in GCs within 14 h and FOH metabolites were transferred to oocytes through gap junctions in COCs, affecting oocyte meiosis, whereas denuded oocytes were unable to directly uptake FOH. Mammalian oocyte meiosis represents a distinctive form of asymmetric cell division, in which a mature egg and a diminutive polar body are produced. The cortical F-actin assembly in the oocyte is essential for oocyte polarization and the completion of chromosome segregation 23 . The Arp2/3 complex comprises actin nucleators that promote actin formation and can be activated through the CDC42-N-WASP or RAC1-WAVE2 signalling pathways 33 . CDC42 and RAC1 are members of the Rho GTPases family, a small guanosine triphosphatase (GTPase) subfamily of proteins that oscillate between the GTP-bound (active) and GDP-bound (inactive) states 39 . CDC42 and RAC1 are enriched at the cortex after spindle migration and cortical F-actin assembly are necessary for the extrusion of the first polar body during oocyte meiosis 40 – 45 . We confirmed that CDC42 and RAC1 have CaaX motifs at the carboxyl terminus and can be prenylated by FPP in oocytes. Prenylation facilitates the membrane localization of CDC42 and RAC1, promoting the assembly of the CDC42-N-WASP-Arp2/3 and RAC1-WAVE2-Arp2/3 complexes and subsequent assemble cortical F-actin at the MI stage. Decreased CDC42 and RAC1 membrane localization and abnormal cortical F-actin assembly led to meiotic defects and aneuploidy in aged oocytes. More importantly, supplementation with MVA or FOH rescued aged oocyte quality by increasing prenylation and cortical F-actin assembly. These findings reveals a novel mechanism for quality decline in aged oocytes during ovarian aging. For several decades, many studies have focused on developing therapeutic strategies to improve the quality of aged oocyte. However, there are still no effective clinical methods for reducing meiotic defects and aneuploidy in aged oocytes. Recent studies have shown that the MVA pathway participates in the aging of multiple organs, such as the testis, muscle, and ovary 9 , 46 , 47 . Our present study is the first to present initial evidence for the reduction of abnormal F-actin distribution, meiotic defects and aneuploidy in aged oocytes by supplementation with MVA. Therefore, we are carrying a single-center clinical study that maturation media for aged COCs supplemented with MVA metabolite, a water-soluble chemical, to improve aged oocyte euploidy and quality (Clinical trial: NCT05788822). Interestingly, we found that 8-IPF, a chemical from the traditional Chinese medicine Epimedium brevicornu Maxim, could activate the FPP synthesis pathway and promote the localization of CDC42 and RAC1 to the oocyte membrane for cortical F-actin assembly. Indeed, 8-IPF supplementation improved the quality of oocytes from aged ovaries and increased female mouse fertility. Furthermore, 8-IPF can be orally administered, does not cause direct damage to the skin or mucous membranes, and has a relatively short half-life 34 . Therefore, with further clinical study, 8-IPF may be suitable for clinical use via oral administration to improve fertility in females with advanced maternal age. In conclusion, the present study revealed that decreased prenylation of CDC42 and RAC1 in oocytes affects cortical F-actin assembly during oocyte meiosis, consequently contributing to meiotic defects and aneuploidy in aged oocytes. MVA and 8-IPF supplementation is instrumental in facilitating the synthesis of FPP from neighbouring GCs, enhancing the cortical localization of CDC42 and RAC1 to activate Arp2/3 complex for normal cortical F-actin assembly in oocytes. Thus, our study provides unprecedented insights into the mechanisms of age-related ooctye cortex abnormality, aneuploidy and clinical directions for improving the reproductive outcomes or extending the reproductive lifespan in women with advanced maternal age. Methods Animals and drug administration. Six-week-old and 8-month-old female C57BL/6 mice and 3-week-old female ICR mice were purchased from SPF Biotechnology Co., Ltd (Beijing, China). The mice were raised until experimental time in the Animal Laboratory Center of Nanjing Drum Tower Hospital under a 12-h light/dark cycle at a constant temperature (20–23°C) and had free access to food and water according to the institutional guidelines. Nine-month-old female C57BL/6 mice were intraperitoneally injected with 5 mg kg − 1 MVA (Sigma, St. Louis, MO, USA; M4667) every day at 18:00 hours for 30 consecutive days. 9.5-month-old female C57BL/6 mice were intragastrically gavaged with 5 mg kg − 1 8-IPF (Yuanye, Shanghai, China; B21576) every day at 18:00 hours for 14 consecutive days. Approval for the procedures involving mouse protocols and experiments was obtained from the Experimental Animal and Welfare Ethics Committee of Nanjing Drum Tower Hospital (2023AE01053). COC and DO IVM in MEMα maturation medium. Female mice were intraperitoneally injected with 10 IU pregnant mare serum gonadotropin (PMSG) (Sansheng Pharmaceuticals, Ningbo, China) and sacrificed 48 h later. COCs and DOs were isolated from the ovarian antral follicles using a disposable syringe with a 20-gauge needle and subsequently cultured in MEMα (Gibco, Waltham, MA, USA; 32561037) maturation medium covered with liquid paraffin oil in an incubator at 37 ℃ with 5% CO 2 . The MEMα maturation medium contained 10% foetal bovine serum (FBS) (Gibco, Waltham, MA, USA; 10270106), 10 ng/mL epidermal growth factor (EGF) (Gibco, Waltham, MA, USA; 53003-018), and 1.5 IU/mL human chorionic gonadotropin (hCG) (Sansheng Pharmaceutical, Ningbo, China). After culturing in IVM for 4 h and 14 h, respectively, the GVBD and PBE rate were analysed. 50 µM MVA, 10 µM FOH (Sigma, St. Louis, MO, USA; F203), and 10 µM FTI-277 (Aladdin, China; F331609) were administered by addition to the MEMα maturation medium in this study. Oocyte immunofluorescence. Oocytes were fixed in 4% paraformaldehyde (PFA) (Sigma, St. Louis, MO, USA; 158127) for 30 min before permeabilizing in 0.5% Triton X-100 (Sigma, St. Louis, MO, USA; T9284) for 20 min. Then, blocking was performed using 1% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA; 10711454001) for 1 h. Oocytes were incubated with mouse monoclonal anti-α-tubulin-FITC (Sigma, St. Louis, MO, USA; F2168; 1:200), 594-phalloidin (US Everbright® Inc., Suzhou, China; YP0052L, 1:200), mouse monoclonal anti-Arp3 (Santa Cruz Biotechnology, California, USA; sc-48344, 1:100), mouse monoclonal anti-CDC42 antibodies (Santa Cruz Biotechnology, California, USA; sc-8401; 1:200) and mouse monoclonal anti-RAC1 antibodies (Santa Cruz Biotechnology, California, USA; sc-514583; 1:200) at 4°C overnight. The oocytes were then treated with a secondary antibody for 1 h at room temperature. After three 5 min washes in phosphate-buffered saline with 0.05% Tween 20 (PBST), the oocytes were incubated with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA; H3570) for 10 min at room temperature. Then, the oocytes were mounted on glass slides and observed under a fluorescence microscope (Leica, Germany; DM3000). Chromosome spread. MII oocytes were first treated with Tyrode’s buffer (Sigma, St. Louis, MO, USA; T2397) for 3 min at 37°C to remove the zona pellucida. Then, the cells were cultured in M2 medium (Sigma, St. Louis, MO, USA; M7167) for 10 min and fixed in 1% PFA with 0.15% Triton X-100 (pH 9.2) on a glass slide. After drying, the samples were incubated with a human anti-centromere antibody (Incorporated, Davis, CA, USA; CA95617; 1:100) at 4°C overnight. After three 5 min washes in PBST, the samples were incubated with Hoechst 33342 for 10 min at room temperature. Finally, the number of spread chromosomes was counted under a DM3000 LED microscope (Leica, Germany). In vitro fertilization (IVF) and embryo culture. The sperm from the epididymides of 12-week-old C57BL/6 male mice were collected and capacitated for 1 h in human tubal fluid (HTF) medium (Merck Millipore, St. Louis, MO, USA; MR-070). COCs after IVM for 14 h or obtained from oviductal ampullae of C57BL/6 female mice were added to be fertilized by the addition of capacitated sperm for 6 h in a 37°C incubator with 5% CO 2 . The fertilized oocytes were subsequently transferred to KSOM (Merck Millipore, St. Louis, MO, USA; MR-106-D) for subsequent culture. The 2-cell embryos and blastocysts formation rates were subsequently calculated. Metabolite extraction and targeted metabolomics analysis. Metabolites were extracted using a method described previously with some modifications 48 . Mice oocytes and GCs were briefly washed with PBS after isolation before quenching. To quench cells and stop metabolism, oocytes and GCs were harvested in tubes and quick-frozen in liquid nitrogen immediately after washing. Oocytes or cells were then incubated in an extraction buffer containing 80% MS grade methanol and 2 µg/ml 4-CL-phenylalanine (Sigma, St. Louis, MO, USA; C6506) as an internal standard which was precooled at -80°C freezer. Metabolites were extracted by 3 rounds of bead-beating. After two rounds of centrifugation by 15,000 g for 10 minutes at 4°C, the supernatant fraction containing soluble metabolites was harvested and dried using a CentriVap Concentrator system (Labconco). For running samples, a dried metabolite extract sample was re-suspended in 50% MS grade acetonitrile for injection. MVA pathway metabolites such as FPP and GGPP levels were quantitatively analyzed in a negative ion mode using multiple reaction monitoring (MRM) acquisition with a triple quadrupole mass spectrometer (Triple Quad 6500+, AB SCIEX) that is coupled to a high performance liquid chromatography. A mix standard containing FPP (Sigma, St. Louis, MO, USA; F6892) and GGPP (Sigma, St. Louis, MO, USA; G6025) with series dilution for 20 ng/ml, 40 ng/ml, 200 ng/ml, 500 ng/ml, 2 µg/ml, 5 µg/ml was used to make standard curves. Metabolites were separated chromatographically on a UPLC HSS T3 column (ACQUITY 1.8 µm 150 x 2.1 mm, Waters). Flow rate was set to 0.25 mL/min using the following method: Buffer A: 10 mM ammonium carbonate, Buffer B: 100% acetonitrile. T = 0 min, 10% B; T = 1 min, 10% B, T = 4 min, 65% B; T = 6 min, 65% B; T = 6.5 min, 95% B; T = 8.5 min, 95% B; T = 9 min, 10% B; T = 12 min, 10% B stop. The retention time for each MRM peak was compared to an appropriate standard. The area under each peak was then quantitated by using SCIEX OS software and re-inspected for accuracy. RNA-seq library construction and analysis. Oocytes and GCs isolated from COCs were lysed to obtain cDNA using a Discover-sc WTA Kit V2 (Vazyme Biotech, China; N712). A TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Biotech, China; TD503) was used for library construction. The Illumina HiSeq X platform (Nanjing Genemap Co., Ltd., China) was used to perform sequencing. High-quality reads were aligned to the Mus musculus UCSC mm9 reference genome, and the FPKM value of each gene was calculated. Highly variable genes (coefficient of variation > 1) were selected for PCA, and the resulting PCA plot was generated using the ggplot2 package in R studio. The DESeq2 package was used to identify DEGs. KEGG enrichment analysis was performed with KEGG Mapper. qRT‒PCR. All primers were mixed in nuclease-free water to a final concentration of 0.1 µM as a primer assay pool. For each mixture, 5 µL of solution was prepared as follows: 2.5 µL of reaction mixture, 0.5 µL of primer mixture, 0.1 µL of RT-Taq mixture, and 1.9 µL of nuclease-free water (Vazyme Biotech, China; P621). GCs isolated from COCs were added to the mixture and placed in a -80°C freezer for 2 min. The mixture was centrifuged at 1000 g for 2 min, after which PCR was performed. For qRT‒PCR, 5 µL of SYBR-Green (Vazyme Biotech, China; Q121), 0.5 µL of Primer-forward (10 µM), 0.5 µL of Primer-reverse (10 µM), 2 µL of cDNA, and 2 µL of ddH 2 O were mixed. The following primer sequences were used: MVK (mouse): forward, 5’-AGCGTCAATTTACCCAACATCG-3’, reverse, 5’-GAGACATCACCTTGCTCAAGAAA-3’; FDPS (mouse): forward, 5’-GGGTTTGACCGTGGTACAAG-3’, reverse, 5’-AAGCCTGGAGCAGTTCTACAC-3’; GGPPS (mouse): forward, 5’-TTTTGCATACACTCGACACACT-3’, reverse, 5’-GGCCTCAATTTGTTTGTAGGCT-3’; SQLE (mouse): forward, 5’-GACCTCGTTCGTGACGGAC-3’, reverse, 5’-CTCCCCAACTATCCTGTCGG-3’; and 18S (mouse): forward, 5’-ATGGCCGTTCTTAGTTGGTG-3’, reverse, 5’-CGGACATCTAAGGGCATCAC-3’. All data were normalized to the expression of 18S using the comparative 2 −ΔΔCt method. Cell culture and metabolic labelling. Human KGN cells were cultured in DMEM/F12 (Gibco, Waltham, MA, USA; C11330500BT) supplemented with 10% FBS (Gibco) and 1% penicillin‒streptomycin (Gibco, Waltham, MA, USA; 15140122) in a humidified 37°C incubator with 5% CO 2 . Cells were grown in 10 cm plates to approximately 70% confluence and treated with alk-FOH (20, 50, or 100 µM; from 100 mM stock solution in DMSO) or DMSO only for 48 h. For antagonistic coincubation, both 50 µM alk-FOH and 50 µM FOH (Sigma, St. Louis, MO, USA; F203) were added to KGN cells for 48 h. Then, the cells were washed three times with cold phosphate-buffered saline. The cells were lysed for western blotting and pull-down assays. Western blotting. The proteins were quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA; 23227) following the manufacturer’s instructions. Next, the equivalent proteins were separated via 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, St. Louis, MO, USA; 03010040001). The membranes were incubated with primary antibodies, rabbit polyclonal anti-Farnesyl (Invitrogen, Waltham, MA, USA; PA1-12554; 1:1500), FDPS (Abcam, MA, USA; ab153805; 1:1000), MVK (Proteintech, China; 12228-1-AP; 1:1000), rabbit monoclonal anti-CDC42 (Abcam, MA, USA; ab187643; 1:1000), rabbit monoclonal anti-RAC1 (Cell signaling Technology, Danvers, MA, USA; 4651S; 1:1000), rabbit monoclonal anti-Arp2 (Abcam, MA, USA; ab128934; 1:1000), rabbit monoclonal anti-Arp3 (Abcam, MA, USA; ab181164; 1:1000), rabbit monoclonal anti-N-WASP (Abclonal, Wuhan, China; A2270; 1:1500), mouse monoclonal anti-WAVE2 (Abclonal, Wuhan, China; A19601; 1:1500), rabbit monoclonal anti-β-actin (Bioworld, Beijing, China; AP0060; 1:100000), and rabbit polyclonal anti-Calnexin (Proteintech, China; 10427-2-AP; 1:10000) at 4℃ overnight after blocking with 5% nonfat milk in PBST for 1 h at room temperature. The next day, the PVDF membranes were washed three times with PBST, incubated with HRP-conjugated goat anti-rabbit IgG (Abcam, MA, USA; ab97051; 1:10000) at room temperature for 1 h, washed three times with PBST, and then developed using enhanced chemiluminescence reagents (GE, Piscataway, NJ, USA); the mean grey value was estimated with ImageJ software (NIH, USA, Version 1.0). Cu-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction. Metabolic labelling and protein extraction were performed as described above. Protein concentrations were adjusted to 2.2 mg/mL. For each, 3000 µL of protein lysate click reagent mixture was prepared as follows: 2520 µL of protein (2.2 mg/mL), 60 µL of azide biotin (Confluore, China; 908007-17-0) (0.1 mM, 5 mM stock solution in DMSO), 120 µL of BTTAA-CuSO4 (2:1, 1 mM:0.5 mM), and 300 µL of fresh sodium ascorbate (2.5 mM, 25 mM stock solution in PBS), which were added last. Then, the reaction mixture was incubated on a shaker at room temperature for 3 h. After the CuAAC reaction, the metabolic labeled proteins were precipitated overnight in methanol at -40°C. The next day, the samples were centrifuged at 5,000 × g for 15 min at 4°C and subsequently washed twice with 7.5 mL of precooled methanol. The precipitated proteins were subsequently completely dissolved in 1.67 mL of buffer (1.2% SDS in PBS) after the methanol was discarded, after which the mixture was left at room temperature for 20 min of volatilization. One hundred microlitres of Pierce High Capacity Streptavidin Agarose (Thermo Fisher Scientific, Waltham, MA, USA; 20359) was washed with PBS three times and resuspended in 8.33 mL of PBS in a 15 mL tube. The redissolved proteins were added to 15 mL tubes filled with the agarose and incubated gently with rotation at room temperature for 4 h. The nonspecific binding proteins were then washed with 0.2% SDS in PBS (10 mL, once), PBS (10 mL, three times), and ddH 2 O (10 mL, three times) and centrifuged at 500 g for 1 min, after which the beads were transferred to 1.5 mL centrifuge tubes with ddH 2 O. Then, 2× loading buffer (Beyotime, Shanghai, China; P0015L) was added to the precipitated beads, which were subsequently heated at 95°C for 12 min. Next, the samples were centrifuged, after which approximately 40 µL of 2× loading buffer was transferred to a new 1.5 mL tube to obtain the protein released from the beads. The proteins were subsequently separated via 10% SDS-PAGE for Western blotting or were subjected to LC‒MS/MS. LC‒MS/MS analysis. The enriched protein samples were analysed using an Easy-nLC 1000 (Thermo Fisher) (Buffer A: 0.1% formic acid solution; Buffer B: 0.1% formic acid + 80% acetonitrile solution; Thermo Fisher, USA). After the column was equilibrated with 95% Buffer A, the sample was loaded onto a Trap column. Then, the samples were separated by chromatography and analysed via mass spectrometry (MS). MS analysis was performed with a Q Exactive mass spectrometer (Thermo Fisher, USA). The raw MS files were searched using MaxQuant 1.6.14 and compared to the Homo sapiens database downloaded from UniProt and finally the identified protein results were obtained. CuAAC reaction for fluorescence imaging. COCs were incubated with 50 µM alk-FOH for 9 h. COCs were fixed with 4% PFA for 30 min. Then, the COCs were incubated with 50 µM azide AZDye 488, the BTTAA-CuSO 4 complex (BTTAA/CuSO 4 6:1), and 2.5 mM sodium ascorbate in PBS at room temperature for 30 min. After the click reaction, COCs were incubated with 594-Phalloidin and Hochest 333342 at room temperature for 30 min. Fluorescence imaging was subsequently performed under LSM 900 confocal laser scanning microscope (Zeiss). Total, membrane and cytoplasmic protein isolation. Total proteins were isolated by lysing cells with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China; P0013B) supplemented with protease inhibitors at 4°C for 30 min. The supernatant of the lysates was collected after centrifugation at 12,000 rpm for 15 min. Cell membrane and cytoplasmic proteins were isolated using a Membrane and Cytosol Protein Extraction Kit with protease inhibitors according to the manufacturer’s instructions (Beyotime, Shanghai, China; P0033). In brief, approximately 3 × 10 7 cells were homogenized using 1 mL of membrane protein extraction reagent A supplemented with PMSF and protease inhibitors for 30 min at 4°C. The nuclei and unbroken cells were removed by centrifuging at 700 g for 10 min. The supernatant was then collected for further centrifugation at 12,000 rpm for 30 min to obtain the plasma proteins. The remaining sediment was fully vortexed in 200 µl of membrane protein extraction reagent B on ice for 10 min and subsequently centrifuged at 12,000 rpm for 30 min to obtain the membrane proteins. Coi mmunoprecipitation (co-IP) assay. KGN cells were treated with 10 µM FOH for 48 h, total protein was extracted with RIPA buffer supplemented with protease inhibitor, and total protein was quantified with a BCA protein assay kit. Next, 500 g of total protein was incubated with 1 g of anti-mouse immunoglobulin G (IgG), anti-CDC42 (Santa Cruz Biotechnology, California, USA; sc-8401), or anti-RAC1 (Santa Cruz Biotechnology, California, USA; sc-514583) at 4°C overnight. Then, 30 L of agarose beads was added to the cell lysates, which were incubated at 4°C for 4 h. Subsequently, the cell lysates were incubated with 2 × loading buffer at 95°C for 10 min. Finally, Western blotting was performed to analyse the protein samples. Expression Constructs, mRNA Synthesis and microinjection. The mcherry coding sequences and CDC42 or RAC1 coding sequences were fused and inserted into pGEMHE plasmids to obtain pGMHE-mCherry-CDC42 and pGMHE-mCherry-RAC1. pGMHE-mCherry-CDC42 C188Y (M-CDC42) and pGMHE-mCherry-RAC1 C188Y (M-RAC1) were obtained using a site-directed mutagenesis kit (NEB, E0554) for in vitro transcription. After linearization of the template with AscI (NEB, R0558V), capped mRNA was synthesized using HiScribe T7 High yield RNA Synthesis Kit (NEB, E2040S), and dephosphorylated for uncapped RNA using Antarctic phosphatase (NEB,M0289). Finally, purified for phenol/chloroform and dissolve in 11 µl nuclease-free H 2 O for mRNA. mRNA concentrations were determined by NanoDrop (Thermo Fisher Scientific). Mouse oocytes were microinjected with 3.5 pl of mRNA. mRNA was microinjected at a needle concentration (final concentration in the microinjection needle) of 100 ng/µl. Oocytes were allowed to express the mRNAs for 3 h before release in M2 medium containing 250 µM dbcAMP (Sigma-Aldrich). After 3 h, the oocytes were maintained in dbcAMP-free M2 medium and performed using LSM 900 confocal laser scanning microscope (Zeiss). Body weight and ovarian index. The mice in each group were euthanized after weighing. A U-shaped incision in the hypogastrium was made to explore the main organs. The bilateral ovaries connected to the bicorned uterus via the fallopian tubes were separated, and the peri-ovarian adipose tissue was removed under a stereoscope (Leica, Germany). The ovaries were subsequently weighed on an electronic analytical balance when no liquid remained. A representative picture was taken on dry sterile gauze, and the ovarian index was determined based on the ovarian wet weight (mg) / body weight (g). Histological analysis and follicle counting. The oestrous cycles of the mice were detected by daily examination of vaginal smears, and the mice were sacrificed in the dioestrus phase to collect ovaries. Ovaries were fixed with 4% paraformaldehyde in PBS overnight, dehydrated in 70%, 80%, 90%, 95%, and 100% ethanol, cleared with xylene and embedded in paraffin. The ovarian tissues were serially sectioned at 5 µm, sequentially deparaffinized in xylene, rehydrated in a descending series of graded ethanol solutions, and stained with haematoxylin and eosin (HE). One of every five slices was used to count the follicles. The follicles at the primordial, primary, secondary, and antral stages in the histological sections of the ovaries were observed and classified based on the histological morphology under a microscope at 200× magnification. Only follicles containing a visible nucleus were counted independently by two researchers. The result was calculated as a five-fold counting value. Fertility test. After two weeks of gavage, to evaluate the fertility function after 8-IPF administration, female mice were mated with 10-week-old C57BL/6 male mice with proven fertility. At 8:00 AM the next day, vaginal plugs were observed to confirm whether the female mice were pregnant. Female mice were considered infertile if, after continuous mating for two-weeks, no vaginal plugs were observed. The pregnancy was terminated by caesarean section at 18.5 days gestation, and after routine anaesthesia, the uterus was exposed through a U-shaped incision in the lower abdomen. The pregnancy and embryo implanted conditions of the mice were observed and photographed, after which the embryos and placenta were separated, weighed and recorded. The number of viable offspring in each litter was recorded. Statistical analysis. Normally distributed data are presented as the mean ± standard error of the mean (SEM) and were compared by unpaired two-tailed Student’s t test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Continuous variables without a normal distribution are presented as medians and interquartile ranges and were analysed by the Mann‒Whitney U test. All the statistical comparisons were performed with GraphPad Prism 8.0. Schematics. Schematic cartoons in Fig. 1 a, 2 a, 3 a, 4 a, 5 g, 6 a, 7 d and Extended Data Fig. 6 c, 7 e were created with BioRender.com. Declarations Data availability The mouse oocyte and GC RNA-seq data can be found under accession numbers CRA017269 and CRA017270. Competing interests The authors declare no competing interests. Author contributions L.J.D., H.X.S., and C.S. designed the research. C.M.L., H.D.Z., J.L.M., S.N.Z., L.J.D., X.T., Y.B.Z., C.J.W., H.J.P., N.N.K., J.S.F., Y.Z., J.D.Z., and X.Z. performed the experiments. 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Mol Cell 66:180–193e8. https://doi.org/10.1016/j.molcel.2017.02.026 Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedDataFig.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4762298","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":330283732,"identity":"0ce45d6e-74a4-4e37-b455-ae78fc00f714","order_by":0,"name":"Lijun 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Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Chuanming","middleName":"","lastName":"Liu","suffix":""},{"id":330283734,"identity":"918cdb78-569d-439f-878a-15f81f6f882d","order_by":2,"name":"Huidan Zhang","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huidan","middleName":"","lastName":"Zhang","suffix":""},{"id":330283735,"identity":"14d0d111-dd23-4e74-af67-35810697f7a2","order_by":3,"name":"Jialian Mao","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jialian","middleName":"","lastName":"Mao","suffix":""},{"id":330283736,"identity":"5b9313b7-1828-4888-815e-6d489452c2dc","order_by":4,"name":"Sainan Zhang","email":"","orcid":"","institution":"Nanjing Drum Tower 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University","correspondingAuthor":false,"prefix":"","firstName":"Chaojun","middleName":"","lastName":"Li","suffix":""},{"id":330283757,"identity":"0f7c903e-f188-467d-b579-665e10e6e76a","order_by":16,"name":"Yali Hu","email":"","orcid":"","institution":"Nanjing Drum Tower Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yali","middleName":"","lastName":"Hu","suffix":""},{"id":330283758,"identity":"abd116bd-7e43-4bcc-886a-5be6da5e7a0f","order_by":17,"name":"Cunqi Ye","email":"","orcid":"https://orcid.org/0000-0003-3117-8680","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Cunqi","middleName":"","lastName":"Ye","suffix":""},{"id":330283759,"identity":"5019c1b5-6790-42ac-8ad2-565a3dfb2d22","order_by":18,"name":"Ran Xie","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Ran","middleName":"","lastName":"Xie","suffix":""},{"id":330283760,"identity":"b5275294-1d12-45d2-84d6-090142d053fc","order_by":19,"name":"Chun So","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"So","suffix":""},{"id":330283761,"identity":"2cbc243c-b8a2-43df-9685-59fe0c95b187","order_by":20,"name":"Haixiang Sun","email":"","orcid":"","institution":"Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Haixiang","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-07-18 11:35:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4762298/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4762298/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61280573,"identity":"23ab943f-9232-43e6-8e22-33a8946ed875","added_by":"auto","created_at":"2024-07-29 04:58:37","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDecreased MVA pathway metabolites and cortical F-actin distribution in aged oocytes. a, \u003c/strong\u003eSchematic illustration showing the collection of MI oocytes and GCs from 6-week-old mice and 10-month-old mice for targeted metabolomics. \u003cstrong\u003eb-c,\u003c/strong\u003e Heatmaps showing the abundance of FPP and GGPP in oocytes and GCs by LC-MS/MS analysis. Scaled value bar indicates the relative concentration. \u003cstrong\u003ed-e,\u003c/strong\u003e Box plot showing the levels of FPP and GGPP in MI oocyte and MI GC from young and aged mice. The data are shown as the mean ± SEM of seven independent experiments. \u003cstrong\u003ef,\u003c/strong\u003e Fluorescence imaging showing F-actin expression at the oocyte cortex from the Young and Old groups. Young group: Young COCs cultured in MEMα maturation medium; Old group: Old COCs cultured in MEMα maturation medium. Scale bar, 25 µm. \u003cstrong\u003eg,\u003c/strong\u003e F-actin fluorescence intensity in the Young (n=17) and Old (n=25) groups. \u003cstrong\u003eh,\u003c/strong\u003e Fluorescence imaging showing Arp3 expression at the oocyte cortex from the Young and Old groups. Scale bar, 25 µm. \u003cstrong\u003ei,\u003c/strong\u003e Arp3 fluorescence intensity in the Young (n=18) and Old (n=18) groups. An unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e test was used for statistical analysis. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, or \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4762298/v1/93bcafe0025958f9ee92027b.jpeg"},{"id":61282663,"identity":"3fa299cd-e212-486b-971a-57003c5813fb","added_by":"auto","created_at":"2024-07-29 05:30:37","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":306753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMVA promotes cortical F-actin assembly and aged oocyte quality via the surrounding GCs \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a,\u003c/strong\u003e Schematic illustration of the experimental protocol used to analyse the effect of MVA supplementation on the mouse oocyte meiotic process. \u003cstrong\u003eb,\u003c/strong\u003e Fluorescence imaging showing F-actin expression at the oocyte cortex in the CTL and MVA groups. CTL group: cumulus-oocyte complexes (COCs) cultured in MEMα maturation medium; MVA group: COCs cultured in MEMα maturation medium with 50 µM MVA. Scale bar, 25 µm. \u003cstrong\u003ec,\u003c/strong\u003e F-actin fluorescence intensity in the CTL (n=25) and MVA (n=28) groups. d, Fluorescence imaging showing Arp3 expression at the oocyte cortex in the CTL and MVA groups. Scale bar, 25 µm. \u003cstrong\u003ee,\u003c/strong\u003e Arp3 fluorescence intensity in the CTL (n=13) and MVA (n=13) groups. \u003cstrong\u003ef,\u003c/strong\u003e Rate of polar body extrusion (PBE) in the CTL and MVA groups. The data are shown as the mean ± SEM of six independent experiments. \u003cstrong\u003eg,\u003c/strong\u003e Chromosome spread analysis showing representative images of aneuploid and euploid MII oocytes from the CTL and MVA groups. \u003cstrong\u003eh,\u003c/strong\u003e Rate of aneuploidy in the CTL and MVA groups. The data are shown as the mean ± SEM of three independent experiments. \u003cstrong\u003ei,\u003c/strong\u003e Images of 2-cell embryos and blastocysts from the CTL and MVA groups. The arrowheads denote blastocysts. Scale bar, 100 µm. \u003cstrong\u003ej-k,\u003c/strong\u003e Rates of 2-cell embryos and blastocysts in the CTL and MVA groups. The data are shown as the mean ± SEM of five independent experiments. An unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e test was used for statistical analysis. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, or \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4762298/v1/ce6204d0c53f0d74c3327cff.jpeg"},{"id":61280574,"identity":"9ca5dfe5-9868-4434-8453-08ab049719be","added_by":"auto","created_at":"2024-07-29 04:58:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":301645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMVA promotes cortical F-actin assembly and aged oocyte quality \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ea,\u003c/strong\u003e Schematic illustration of \u003cem\u003ein vivo\u003c/em\u003e injection of MVA into 9-month-old mice. NS: normal saline; MVA: mevalonate. \u003cstrong\u003eb,\u003c/strong\u003e Ovary index of mice in the CTL and MVA injected groups. CTL group: Nine-month-old female mice intraperitoneally injected with normal saline. MVA injected group, 9-month-old female mice intraperitoneally injected with 5 mg kg\u003csup\u003e-1\u003c/sup\u003e MVA every day for 30 days. The data are shown as the mean ± SEM of ten independent experiments. \u003cstrong\u003ec,\u003c/strong\u003e The number of MII oocytes in the CTL and MVA injected groups. The data are shown as the mean ± SEM of twelve independent experiments. \u003cstrong\u003ed,\u003c/strong\u003e Fluorescence imaging showing F-actin expression at the oocyte cortex in the CTL and MVA injected groups. Scale bar, 25 µm. \u003cstrong\u003ee,\u003c/strong\u003e F-actin fluorescence intensity in the CTL (n=15) and MVA (n=21) injected groups. \u003cstrong\u003ef,\u003c/strong\u003e Fluorescence imaging showing Arp3 expression at the oocyte cortex in the CTL and MVA injected groups. Scale bar, 25 µm. \u003cstrong\u003eg,\u003c/strong\u003e Arp3 fluorescence intensity in the CTL (n=9) and MVA (n=8) injected groups. \u003cstrong\u003eh,\u003c/strong\u003e Chromosome spread showing representative images of aneuploid and euploid MII oocytes from the CTL and MVA injected groups. \u003cstrong\u003ei, \u003c/strong\u003eAneuploidy rate was measured in the CTL and MVA injected groups. The data are shown as the mean ± SEM of five independent experiments. \u003cstrong\u003ej,\u003c/strong\u003e Images of 2-cell embryos and blastocysts from the CTL and MVA injected groups. The arrowheads indicate blastocysts. Scale bar, 200 µm. \u003cstrong\u003ek-l,\u003c/strong\u003e Two-cell embryos and blastocysts rate were measured in the CTL and MVA injected groups. The data are shown as the mean ± SEM of at least three independent experiments.\u003c/p\u003e\n\u003cp\u003eAn unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e test was used for statistical analysis. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, or \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4762298/v1/8fa8efbeb8a6d3a59f1383a9.jpeg"},{"id":61280581,"identity":"ee9a0dd0-c517-4b70-be02-dab83564c0b9","added_by":"auto","created_at":"2024-07-29 04:58:37","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":392701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMVA promotes the expression of meiosis-associated genes in oocytes from aged COCs and activates the FPP pathway in GCs. a,\u003c/strong\u003e Schematic showing the collection of aged oocytes and GCs for RNA-seq. \u003cstrong\u003eb,\u003c/strong\u003e Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the upregulated differentially expressed genes (DEGs) in oocytes. \u003cstrong\u003ec,\u003c/strong\u003e Violin plot showing the expression levels of meiosis-associated genes in CTL and oocytes from 50 µM MVA-treated aged COCs. \u003cstrong\u003ed,\u003c/strong\u003e PCA plot of GCs in the CTL- and MVA- treated groups based on gene expression patterns separated by PC1 and PC2. \u003cstrong\u003ee, \u003c/strong\u003eViolin plot showing the expression levels of \u003cem\u003eMVK\u003c/em\u003e, \u003cem\u003ePMVK\u003c/em\u003e, \u003cem\u003eMVD\u003c/em\u003e, \u003cem\u003eIDI1\u003c/em\u003e, and \u003cem\u003eFDPS\u003c/em\u003e in CTL- and MVA-treated aged GCs based on the RNA-seq results. \u003cstrong\u003ef,\u003c/strong\u003e mRNA levels of the \u003cem\u003eMVK\u003c/em\u003e, \u003cem\u003eFDPS\u003c/em\u003e, \u003cem\u003eGGPPS\u003c/em\u003e, and \u003cem\u003eSQLE\u003c/em\u003e genes in aged GCs from the CTL and MVA- treated groups. NS, not significant. The data are shown as the mean ± SEM of ten independent experiments. \u003cstrong\u003eg,\u003c/strong\u003e Western blotting analysis of MVK and FDPS expression in KGN cells treated with 50 µM MVA. \u003cstrong\u003eh-i,\u003c/strong\u003e Box plot showing the levels of MVA and FPP in primary GCs treated with or without 50 µM MVA. The data are shown as the mean ± SEM of four independent experiments. \u003cstrong\u003ej,\u003c/strong\u003e Prenylation levels in KGN cells treated with 50 µM MVA. An unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e test was used for statistical analysis. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, or \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4762298/v1/7e80cdb81e04e580e8164ce1.jpeg"},{"id":61280575,"identity":"5c049113-8048-4ffc-811a-82ff3983dde7","added_by":"auto","created_at":"2024-07-29 04:58:37","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":432941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMVA promotes aged oocyte cortical F-actin assembly via prenylation by FPP from GCs.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Fluorescence imaging showing F-actin expression at the oocyte cortex in the CTL, MVA, MVA+FTI, and FOH groups. CTL group: Old COCs cultured in MEMα maturation medium; MVA group: Old COCs cultured in MEMα maturation medium supplemented with 50 µM MVA; MVA+FTI group: Old COCs were cultured in MEMα maturation medium supplemented with 50 µM MVA and 10 µM FTI-277; FOH group: Old COCs cultured in MEMα maturation medium supplemented with 10 µM FOH. Scale bar, 25 µm. \u003cstrong\u003eb,\u003c/strong\u003e F-actin fluorescence intensity in the CTL (n=25), MVA (n=28), MVA+FTI (n=22), and FOH (n=43) groups. \u003cstrong\u003ec,\u003c/strong\u003e Images of oocytes isolated from aged COCs of 10-month-old mice after 14 h of maturation in the MVA, MVA+FTI, and FOH groups. Scale bar, 100 µm. \u003cstrong\u003ed,\u003c/strong\u003e Rate of PBE in the CTL, MVA, MVA+FTI, and FOH groups. The data are shown as the mean ± SEM of three independent experiments. \u003cstrong\u003ee, \u003c/strong\u003eSynthesis of the prenylation reporter, alk-FOH. \u003cstrong\u003ef,\u003c/strong\u003e Western blotting analysis showing prenylated proteins in KGN cells treated with 0, 20, 50, and 100 µM alk-FOH. \u003cstrong\u003eg,\u003c/strong\u003e Schematic of mouse DO and COC labelling. DOs and COCs were incubated with 50 µM alk-FOH for 14 h. \u003cstrong\u003eh, \u003c/strong\u003eFluorescence imaging of COCs after incubation with 50 µM alk-FOH for 14 h. O: oocyte. GC: granulosa cells. Scale bar, 5 µm. \u003cstrong\u003ei, \u003c/strong\u003eFluorescence imaging showing the alk-FOH signals in the gap junction from COCs after incubation with 50 µM alk-FOH for 14 h. Scale bar, 5 µm. An unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e test was used for statistical analysis.\u003csup\u003e *\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, or \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4762298/v1/7971a5e21bcee9881dd996be.jpeg"},{"id":61280578,"identity":"32ee2282-52fb-4262-adfd-efe578a33aeb","added_by":"auto","created_at":"2024-07-29 04:58:37","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":231643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrenylation increases CDC42 and RAC1 cortical localization and F-actin assembly. a,\u003c/strong\u003e Schematic showing prenylated proteins with a CaaX motif. n=2, farnesylated proteins; n=3, geranylgeranylated proteins. \u003cstrong\u003eb,\u003c/strong\u003e Western blotting analysis of proteins after pull-down experiments. \u003cstrong\u003ec,\u003c/strong\u003e KEGG analysis of the 140 prenylated proteins. \u003cstrong\u003ed,\u003c/strong\u003e Western blotting analysis validated the labelling of the prenylated proteins CDC42 and RAC1. PD: pull-down, TL: total. \u003cstrong\u003ee,\u003c/strong\u003e CDC42 and RAC1 protein expression in the membrane fractions of CTL, MVA, MVA+FTI, and FOH groups. \u003cstrong\u003ef, \u003c/strong\u003eWestern blotting analysis of CDC42 and RAC1 expression in the membrane fractions of CTL, MVA, MVA+FTI, and FOH groups. The data are shown as the mean ± SEM of three independent experiments. \u003cstrong\u003eg-h,\u003c/strong\u003e Endogenous CDC42-N-WASP, CDC42-Arp2, and CDC42-Arp3 interactions were detected by immunoprecipitation analysis in KGN cells with or without FOH treatment. The data are shown as the mean ± SEM of three independent experiments. \u003cstrong\u003ei-j,\u003c/strong\u003e Endogenous RAC1-WAVE2, RAC1-Arp2, and RAC1-Arp3 interactions were detected by immunoprecipitation analysis in KGN cells with or without FOH treatment. The data are shown as the mean ± SEM of three independent experiments. \u003cstrong\u003ek,\u003c/strong\u003e Fluorescence imaging showing Arp3 expression at the oocyte cortex in the CTL, MVA, MVA+FTI, and FOH groups. CTL group: Old COCs cultured in MEMα maturation medium; MVA group: Old COCs cultured in MEMα maturation medium supplemented with 50 µM MVA; MVA+FTI group: Old COCs were cultured in MEMα maturation medium supplemented with 50 µM MVA and 10 µM FTI-277; FOH group: Old COCs cultured in MEMα maturation medium supplemented with 10 µM FOH. Scale bar, 25 µm. \u003cstrong\u003el,\u003c/strong\u003e Arp3 fluorescence intensity in the CTL (n=15), MVA (n=15), MVA+FTI (n=15), and FOH groups (n=15). An unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e test was used for statistical analysis. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, or \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4762298/v1/a42a1a4bc481b38f36514799.jpeg"},{"id":61281339,"identity":"1336bd6d-2933-4a9a-9bbb-423a1e9ed7e4","added_by":"auto","created_at":"2024-07-29 05:06:37","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":409862,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe natural isopentenyl compounds 8-IPF facilitates cortical F-actin assembly in aged oocytes. a,\u003c/strong\u003e Prenylation levels in the CTL and 8-IPF groups. 8-IPF group: KGN cells treated with 50 µg L\u003csup\u003e-1 \u003c/sup\u003e8-IPF. \u003cstrong\u003eb,\u003c/strong\u003e CDC42 and RAC1 protein expression in the membrane fractions of the CTL and 8-IPF groups. \u003cstrong\u003ec,\u003c/strong\u003e Western blotting analysis of CDC42 and RAC1 expression in the membrane fractions of the CTL and 8-IPF groups. The data are shown as the mean ± SEM of three independent experiments. \u003cstrong\u003ed,\u003c/strong\u003e Schematic of 8-IPF supplementation \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003ee, \u003c/strong\u003eFollicle counts of CTL (n=7) and 8-IPF-treated (n=7) mouse ovaries. The data are shown as the mean ± SEM. \u003cstrong\u003ef,\u003c/strong\u003e Oocyte immunofluorescence showing the expression of actin at oocytes cortex from the CTL and 8-IPF-treated groups. Scale bar, 25 µm. \u003cstrong\u003eg,\u003c/strong\u003e Oocyte immunofluorescence intensity of F-actin in the CTL (n=23) and 8-IPF-treated (n=15) groups. The data are shown as the mean ± SEM. \u003cstrong\u003eh,\u003c/strong\u003e\u0026nbsp; Fluorescence imaging showing Arp3 expression at the oocyte cortex in the CTL and 8-IPF-treated groups. Scale bar, 25 µm. \u003cstrong\u003ei,\u003c/strong\u003e Arp3 fluorescence intensity in the CTL (n=13) and 8-IPF-treated (n=12) groups. The data are shown as the mean ± SEM. \u003cstrong\u003ej,\u003c/strong\u003e Confocal images showing aneuploidy in MII oocytes from control and euploid MII oocytes from 8-IPF-treated aged mice. \u003cstrong\u003ek,\u003c/strong\u003e Histogram showing the incidence of aneuploidy in MII oocytes from the CTL- and 8-IPF-treated groups. The data are shown as the mean ± SEM of three independent experiments. \u003cstrong\u003el, \u003c/strong\u003eFertility of mice treated with normal saline or 8-IPF. \u003cstrong\u003em,\u003c/strong\u003e Pregnancy rates in the CTL (n=7) and 8-IPF-treated (n=7) mice. \u003cstrong\u003en,\u003c/strong\u003e Litter sizes in CTL (n=4) and 8-IPF-treated (n=6) mice. The data are shown as the mean ± SEM. An unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e test was used for statistical analysis. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, or \u003csup\u003e****\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4762298/v1/e30365412a40dfc5b171c11b.jpeg"},{"id":61282665,"identity":"fbf720e9-284d-4d8e-a9f6-e7c3430d125d","added_by":"auto","created_at":"2024-07-29 05:30:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3392922,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4762298/v1/6ca6e046-4248-4772-b82f-bb7d3eff0b76.pdf"},{"id":61282207,"identity":"39ae0a08-551f-479d-a858-ac45f9400575","added_by":"auto","created_at":"2024-07-29 05:22:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2514701,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-4762298/v1/d549caacc1f8faddb65f4129.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mevalonate metabolites boost aged oocyte quality through small GTPases prenylation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman ovaries are one of the earliest organs to exhibit aging-associated dysfunctions, with a significant functional decline in women over 35\u003csup\u003e1,2\u003c/sup\u003e. Ovarian aging mainly manifests as declines in oocyte quality and quantity, resulting in female subfertility and infertility\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Abnormal F-actin assembly and aneuploidy are important contributors to age-related decline in oocyte quality\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Arp2/3-dependent assembly of the cortical F-actin affect oocyte contractions, cytoplasmic organization and developmental potential\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, whether cortical F-actin of oocytes is involved in ovarian aging remains largely unknown.\u003c/p\u003e \u003cp\u003eMevalonate (MVA) pathway is crucial for cholesterol synthesis and regulates multiple cellular processes through protein prenylation, a lipidic modification that facilitates the anchoring of proteins to cell membranes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Small GTPases could be prenylated and regulate cell division and morphogenesis by promoting cytoskeleton organization and cortex formation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Recently, we reported that abnormal metabolism of the MVA pathway in granulosa cells (GCs) is an important contributing factor to meiotic defects and aneuploidy and MVA supplementation increases normal meiosis in aged oocyte\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. How MVA pathway acts on actin assembly of aged oocytes within cumulus-oocyte complexes (COCs) .needs to be clarified\u003c/p\u003e \u003cp\u003eRecent studies have reported several therapeutic strategies for improving the quality of aged oocyte, including the use of antioxidants, growth hormones, melatonin, nicotinamide mononucleotide and the polyamine metabolite spermidine, their effectiveness is yet to be tested in clinic\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. MVA pathway is a pivotal water-soluble upstream for cholesterol and more than 50,000 terpenoids synthesis and is required for cell cycle progression and cell proliferation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that MVA pathway is important for the coordination of regulatory T-cell proliferation by enhancing transforming growth factor (TGF)-β signalling and mediation of training immunity via IGF1-R and mTOR activation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. MVA supplementation has also been demonstrated to counteract adverse developmental effects of statins on blastocyst formation\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we found that Arp2/3-dependent assembly of the cortical F-actin was decreased in aged oocyte, accompanied by reduced MVA pathway metabolites (farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP)) levels. MVA supplementation improved the quality of aged oocytes in COCs by promoting cortical F-actin assembly, ameliorating meiotic errors and promoting embryonic development both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, MVA activated the FPP metabolic pathway in GCs and promoted cortical F-actin assembly by increasing CDC42 and RAC1 prenylation and Arp2/3 complex formation in aged oocytes. In addition, 8-isopentenyl flavone (8-IPF), a natural chemical that is being used as an oral drug, also ameliorated the decline in ovarian reserve and significantly increased oocyte quality in aged mice by enhancing the prenylation of CDC42 and RAC1 on the oocyte membrane to assemble the cortical F-actin.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDecreased MVA pathway metabolites and cortical F-actin distribution in aged oocytes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur recent study indicates that downregulation of the MVA pathway in aged ovaries and FPP- and GGPP-mediated protein prenylation may be involved in the decrease of quality in aged oocytes\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To comprehensively study the change of FPP and GGPP metabolites in oocytes and surrounding GCs, we established a targeted metabolomics approach to detect FPP and GGPP using low cell inputs. We collected 10 oocytes at metaphase I (MI) stage and 10\u003csup\u003e4\u003c/sup\u003e surrounding GCs from young (6-week-old) and aged (10-month-old) mice with seven biological replicates for the MVA metabolite test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Remarkably, aged oocyte contained significantly fewer FPP (2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 ng/ml versus 7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 ng/ml, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) and GGPP (7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 ng/ml versus 16.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 ng/ml, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) compared with young oocyte (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb,d,e). In addition, the FPP (0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ng/ml versus 1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ng/ml, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and GGPP (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 ng/ml versus 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ng/ml, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) metabolites levels were also significantly decreased in aged GC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d,e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt was reported that FPP and GGPP are necessary for protein localization on the cell membrane for cytoskeletal organization\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Actin filaments become denser at the oocyte periphery and assemble cortical F-actin with the recruitment of proteins from the cytoplasm to membranes\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. To explore the changes of cortical F-actin during ovarian aging, oocytes were collected from young and aged COCs after cultured for 9 h, and cortical F-actin distribution of oocytes at MI was examined. The fluorescence intensity of cortical F-actin of aged oocytes were much weaker than that of young oocytes (0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 versus 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef,g). The assembly of cortical F-actin requires the activity of the Arp2/3 complex, which functions as an actin nucleator\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Aged oocytes showed decreased Arp3 expression compared with the young oocytes (0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 versus 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh,i). These results indicate that the decreased MVA downstream metabolites and defective nucleation of cortical F-actin may be involved in abnormal assembly of the cortical F-actin during ovarian aging.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMVA promotes cortical F-actin assembly and aged oocyte quality via the surrounding GCs.\u003c/b\u003e Since the levels of FPP and GGPP were significantly reduced in aged oocytes and MVA is the precursor for terpenoids, including FPP and GGPP\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, we next investigated the effects of MVA supplementation on aged oocyte quality. We isolated COCs from 10-month-old female mice and cultured them in \u003cem\u003ein vitro\u003c/em\u003e maturation (IVM) medium supplemented with 50 \u0026micro;M MVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We first compared the cortical F-actin between the two groups treated with or without MVA and found that MVA supplementation markedly promoted cortical F-actin assembly of MI ooctyes from aged COCs (6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 versus 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c). In addition, the decreased Arp3 expression in aged MI oocyte cortex was rescued after MVA supplementation (1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 versus 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed,e). Since actin is responsible for various essential function, including spindle migration, accurate chromosome segregation, and PBE during oocyte meiosis, we next compared meiotic progression in oocytes from the two groups\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The PBE rate was significantly higher in the group treated with MVA than in the control group (95.1% \u0026plusmn; 3.3% versus 81.2% \u0026plusmn; 4.0%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) although the germinal vesicle breakdown (GVBD) rates of oocytes collected from COCs were not different between the groups with or without MVA supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). We further evaluated meiotic defects and developmental potential of MII oocytes collected from the two groups. The incidence of meiotic defects (18.2% \u0026plusmn; 5.9% versus 38.2% \u0026plusmn; 4.1%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) and aneuploidy (16.7% \u0026plusmn; 1.9% versus 38.9% \u0026plusmn; 2.0%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) were markedly reduced after MVA supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg,h and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d). Moreover, the MII oocytes derived from MVA-treated COCs were more competent to develop into 2-cell embryos (79.7% \u0026plusmn; 4.2% versus 54.1% \u0026plusmn; 5.1%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) and blastocysts (28.2% \u0026plusmn; 3.6% versus 13.1% \u0026plusmn; 2.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008) than those derived from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei-k). To further clarify whether MVA acted on oocytes or GCs, we also treated denuded oocytes (DOs) with 50 \u0026micro;M MVA. Notably, the incidence of GVBD, PBE and meiotic defects were not improved in DOs after MVA supplementation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-i), indicating that the effects of MVA on aged oocytes were executed via GCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate the effects of MVA on aged COCs \u003cem\u003ein vivo\u003c/em\u003e, 9-month-old female mice were intraperitoneally injected with 5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MVA for 30 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The ovarian index of aged mice was considerably increased (1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 versus 1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) without affecting body weight after MVA supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). MVA supplementation also ameliorated age-related depletion in ovarian reserve and increased the number of follicles for all developmental stages, especially the antral stage (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). Moreover, the number of ovulated MII oocytes doubled after MVA injection (9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 versus 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). MVA treatment significantly increased the cortical F-actin assembly (2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 versus 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and Arp3 expression (1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 versus 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05) of MI oocytes from aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-g). As shown in Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee,f, the percentage of abnormal spindles and misaligned chromosomes were considerably lower in oocytes from MVA-treated mice than those from control mice (26.3% \u0026plusmn; 2.3% versus 46.3% \u0026plusmn; 6.5%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). Chromosome spread experiments further confirmed a markedly lower frequency of aneuploidy in oocytes after MVA injection (25.1% \u0026plusmn; 4.3% versus 50.5% \u0026plusmn; 2.9%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh,i). Moreover, more 2-cell embryos (63.8% \u0026plusmn; 2.4% versus 43.7% \u0026plusmn; 3.8%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) and blastocysts (53.8% \u0026plusmn; 2.3% versus 39.3% \u0026plusmn; 4.1%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04) could be obtained from oocytes from MVA-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej-l). Taken together, these results suggest that MVA ameliorates cortical F-actin assembly, meiotic defects, and aneuploidy in aged oocytes via GCs both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMVA activates the FPP metabolic pathway in aged GCs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate how MVA supplementation improved the quality of aged oocytes, we performed RNA sequencing analysis of oocytes and neighbouring GCs from aged COCs treated with or without MVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). PCA and comparison between the two groups revealed a highly dynamic change in gene expression after MVA supplementation. A total of 2116 downregulated and 1287 upregulated differentially expressed genes (DEGs) were identified in oocytes after MVA supplementation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs revealed that genes associated with the cell cycle and oocyte meiosis were upregulated, whereas genes associated with ribosomes and thermogenesis were downregulated in oocytes from MVA-treated COCs (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,c). Many meiosis-associated genes such as \u003cem\u003eCdc20\u003c/em\u003e, \u003cem\u003eBub3\u003c/em\u003e, \u003cem\u003eCcnb1\u003c/em\u003e and \u003cem\u003eMos\u003c/em\u003e play important roles during oocyte meiosis, and the downregulation of these genes results in meiotic defects and aneuploidy\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, the expression of \u003cem\u003eCcna2, Cdc20, Bub3, Ccnb1, Ppp2r1a, Mos, Mad2l1, Pk1, Map2K1, Cdc42ep2\u003c/em\u003e, and \u003cem\u003eCdc42se2\u003c/em\u003e was markedly increased in oocytes from MVA-treated COCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate how MVA improved cortical F-actin assembly and meiosis in aged oocyte via GCs, we further analyzed the transcriptome of GCs. PCA and volcano plotting revealed differences in the transcriptome profile of the MVA-treated GCs and control GCs, with 9317 downregulated and 1279 upregulated DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). KEGG analysis revealed that genes associated with ribosome, oxidative phosphorylation, and proteasome were upregulated, while genes associated with neuroactive ligand‒receptor interaction and calcium signalling pathway were downregulated after MVA supplementation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee,f). Because MVA is an early rate metabolite in the cholesterol synthesis pathway\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, we next analysed the expression of metabolic genes related to the MVA pathway (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The expression of genes involved in the metabolism of MVA to FPP, including \u003cem\u003eMVK\u003c/em\u003e, \u003cem\u003ePMVK\u003c/em\u003e, \u003cem\u003eMVD\u003c/em\u003e, \u003cem\u003eIDI1\u003c/em\u003e, and \u003cem\u003eFDPS\u003c/em\u003e, was increased 1.8- to 4.0-fold in GCs after MVA supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). However, genes encoding metabolic enzymes at biosynthetic steps from acetyl-CoA to MVA and FPP to cholesterol did not exhibit obvious changes in expression (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). qRT‒PCR and Western blotting analysis also confirmed that both the mRNA and protein levels of MVK and FDPS were significantly elevated after MVA supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef,g and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Moreover, the MVA (896.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6 ng/ml versus 506.6\u0026thinsp;\u0026plusmn;\u0026thinsp;42.5 ng/ml, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and FPP (0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 ng/ml versus 0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007 ng/ml, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) metabolites levels and protein prenylation level were significantly increased after MVA supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh-j). Taken together, our results suggest that MVA supplementation activates the FPP synthesis pathway and increases the protein prenylation levels in aged COCs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMVA promotes cortical F-actin assembly via prenylation by FPP from GCs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo clarify whether the effects of MVA on oocyte cortical F-actin assembly and meiosis was mediated by prenylation via FPP synthesis from GCs, COCs were isolated from 10-month-old female mice and subsequently cultured in IVM medium supplemented with FOH, which can be catabolized to FPP in cells\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. FOH supplementation promoted cortical F-actin assembly (0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009 versus 0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in MI ooctyes, increased the rate of PBE (89.5% \u0026plusmn; 2.5% versus 70.2% \u0026plusmn; 1.9%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004), and reduced the incidence of meiotic defects (23.9% \u0026plusmn; 2.5% versus 43.3% \u0026plusmn; 1.7%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) in oocytes from aged COCs, similar to MVA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). To further explore the relationships between prenylation and oocyte quality in aged COCs, FTI-277, an inhibitor of protein prenylation, was added to IVM medium supplemented with MVA. Indeed, the addition of FTI-277 reduced cortical F-actin assembly (0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 versus 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), decreased PBE rate (70.0% \u0026plusmn; 1.8% versus 86.8% \u0026plusmn; 3.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01), and increased the incidence of meiotic defects (46.0% \u0026plusmn; 0.2% versus 20.5% \u0026plusmn; 1.5%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in oocytes from aged COCs in the presence of MVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether FPP can be transferred from GCs to oocytes, an alkynyl-farnesol chemical reporter based on farnesol (alk-FOH) was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). To study the incorporation of alk-FOH, human granulosa-like tumor cell line, KGN cells were first incubated with different concentrations of alk-FOH for 24 h, then the total cellular protein was ligated to azide-biotin via CuAAC (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Western blotting analysis revealed that the alk-FOH was incorporated at 20 \u0026micro;M and increased considerably with 100 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Moreover, alk-FOH incorporation was sensitive to competition by the natural metabolite FOH, suggesting that addition of the alkyne group did not compromise the activity of FOH (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Next, we isolated DOs and COCs from 3-week-old female mice and cultured with alk-FOH for 14 h and they were visualized using fluorescence imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). No signals were detected in DOs, indicating that oocytes could not directly uptake FOH (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Interestingly, obvious fluorescence signals were detected in both GCs and oocytes from COCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). Moreover, alk-FOH signals also exhibited in the transzonal projections between GCs and oocyte (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Collectively, these results indicate that MVA activates the synthesis of FPP in GCs, and that the transfer of FPP from GCs to oocytes ameliorates aged oocyte cortical F-actin assembly and quality through prenylation.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrenylation increases CDC42 and RAC1 cortical localization and F-actin assembly.\u003c/b\u003e Prenylation involves the covalent addition of either farnesyl or geranylgeranyl isoprenoids to conserved cysteine (Cys) residues at the carboxyl-terminal CaaX or C(X)C motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Immunofluorescence analysis revealed that the KGN cells also uptake FOH (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). To identify potential prenylated proteins involved in cortical F-actin assembly and oocyte meiosis, we performed large-scale profiling of prenylated proteins in KGN cells using alk-FOH. After incubation with azide-biotin, affinity enrichment, selective elution, liquid chromatography-tandem mass spectrometry (LC‒MS/MS) was used to identify prenylated proteins (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Western blotting analysis revealed that a diverse range of proteins were labelled by alk-FOH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). A total of 140 putative prenylated proteins were identified by alk-FOH supplementation when compared to the control group; 39 of these proteins had a carboxyl-terminal CaaX or Rab motif (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). In addition, fifty-two percent (73/140) of the detected prenylated proteins were membrane-associated proteins (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Prenylation can facilitate the anchoring of proteins to cell membranes, mediating protein‒protein interactions and signal transduction\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. A Venn diagram showed that 24 proteins labelled by alk-FOH, which included CDC42 and RAC1, had a carboxyl-terminal CaaX or Rab motif and localized to the cell membrane (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed,e). KEGG analysis revealed that 12 detected prenylated proteins were involved in the regulation of actin cytoskeleton, also including CDC42 and RAC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCDC42 and RAC1 are small GTP binding proteins of the Rho subfamily and play critical roles in the establishment of oocyte polarity and subsequent meiosis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To further validate whether CDC42 and RAC1 could be prenylated, we conducted immunoblot analysis using affinity enrichment of the labelled proteins. Both of these proteins could be labelled by alk-FOH, and the labelling was sensitive to competition with natural FOH, indicating that CDC42 and RAC1 were indeed prenylated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Next, we examined whether MVA affected CDC42 and RAC1 membrane partitioning and cellular localization via prenylation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee,f, MVA supplementation considerably increased the localization of CDC42 and RAC1 to the cell cortex, similar to the effect observed following treatment with FOH. As expected, supplementation with exogenous FTI-277 markedly reduced the MVA-induced anchoring of CDC42 and RAC1 to cell membranes, suggesting that MVA induces CDC42 and RAC1 prenylation and promotes their localization to the cell cortex.\u003c/p\u003e \u003cp\u003eCDC42 and RAC1 are enriched in the oocyte cortex after chromosome migration to the periphery and regulate actin dynamics during oocyte meiosis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To investigate the relationship between CDC42 and RAC1 prenylation and cortical F-actin assembly, we examined CDC42 and RAC1 localization in oocytes at MI by immunofluorescence. CDC42 and RAC1 failed to cortical localization in oocytes from aged COCs, and MVA supplementation induced CDC42 and RAC1 relocation to the cortex in aged oocytes, exhibiting a distribution pattern similar to that of F-actin, while the ameliorative effect of MVA was disrupted by FTI-277 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-d). Next, we perturbed the CDC42/RAC1 signaling by overexpressing a CDC42/RAC1 dominant-negative mutant (CDC42\u003csup\u003eC188Y\u003c/sup\u003e/ RAC1\u003csup\u003eC188Y\u003c/sup\u003e; which is conserved position to the cysteine\u003csup\u003e188\u003c/sup\u003e at CVLL/CLLL motif to tyrosine respectively; previously demonstrated to inhibit CDC42/RAC1 prenylation) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Expressing constitutively active CDC42 and RAC1 mutant failed to locate on the oocyte cortical F-actin, suggesting that CDC42 and RAC1 prenylation is essential for cortical F-actin assembly (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee,f). The Arp2/3 complex can be activated by the CDC42/N-WASP or RAC1-WAVE2 signalling pathways to promote the cortical F-actin assembly in oocytes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Reciprocal coimmunoprecipitation analysis further confirmed the endogenous interaction between CDC42 with N-WASP and the Arp2/3 complex, as well as the interaction between endogenous RAC1, WAVE2 and the Arp2/3 complex. In addition, FOH supplementation significantly enhanced the formation of CDC42-N-WASP-Arp2/3 and RAC1-WAVE2-Arp2/3 complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-j). Moreover, the Arp3 expression (2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 versus 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004) at the cortex of MI oocytes were significantly increased, while the addition of exogenous FTI-277 reduced the cortical Arp3 expression (1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 versus 2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek,l). Together, these results suggest that MVA or FOH promotes the formation of CDC42-N-WASP-Arp2/3 and RAC1-WAVE2-Arp2/3 complexes for cortical F-actin assembly. For the first time, we found that prenylation of CDC42 and RAC1 by FPP, facilitates their localization to the membrane and contributes to cortical F-actin assembly in oocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe isopentenyl compounds 8-IPF facilitates cortical F-actin assembly in aged oocytes.\u003c/b\u003e Although MVA can effectively ameliorate ovarian reserve and oocyte quality in aged mice, currently there are no drugs containing MVA in the clinic. In order to improve the fertility of women with advanced age, approved and preclinical drugs were screened for their efficacy in increasing the expression of MVK and FDPS and protein prenylation. Among them, we identified 8-IPF, a traditional natural chemical used in China with an isopentenyl side chain\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Treatment with 50 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 8-IPF considerably increased the prenylation level and the expression of MVK and FDPS in KGN cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;8a,b). In addition, 8-IPF supplementation increased CDC42 and RAC1 cortical localization, similar to the results observed following MVA treatment in KGN cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb,c).\u003c/p\u003e \u003cp\u003eWe next analysed whether 8-IPF has therapeutic effects on ovarian aging \u003cem\u003ein vivo\u003c/em\u003e using an aged mouse model. Briefly, 5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 8-IPF was intragastrically administered to 9.5-month-old female mice for 14 days via gavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). The ovarian index (2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 versus 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) and the numbers of follicles considerably increased for all developmental stages after 8-IPF supplementation. Improvement effects were especially evident in growing follicles (secondary follicles (235.7\u0026thinsp;\u0026plusmn;\u0026thinsp;28.4 versus 87.1\u0026thinsp;\u0026plusmn;\u0026thinsp;21.7, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) and antral follicles (76.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 versus 22.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee and Extended Data Fig.\u0026nbsp;8c-e). Moreover, the number of ovulated oocytes increased significantly after 8-IPF treatment (10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 versus 3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002) (Extended Data Fig.\u0026nbsp;8f). More importantly, cortical F-actin staining of MI oocytes from aged COCs isolated from 8-IPF-treated mice were stronger than those from control oocytes (1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 versus 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef,g). In addition, the decreased Arp3 expression in aged MI oocyte cortex was rescued after 8-IPF supplementation (2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 versus 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh,i). When compared to the control, 8-IPF supplementation markedly reduced the incidence of meiotic defects (32.4% \u0026plusmn; 3.1% versus 54.6% \u0026plusmn; 2.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) and aneuploidy (28.3% \u0026plusmn; 5.1% versus 53.9% \u0026plusmn; 2.1%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej,k and Extended Data Fig.\u0026nbsp;8g,h). Moreover, more 2-cell embryos (63.7% \u0026plusmn; 4.7% versus 46.6% \u0026plusmn; 1.7%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) and blastocysts (52.2% \u0026plusmn; 4.7% versus 24.3% \u0026plusmn; 7.3%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) could be obtained after 8-IPF treatment (Extended Data Fig.\u0026nbsp;8i-k). Excitingly, after natural mating with 8-week-old male mice, the pregnancy rate (85.7% versus 57.1%) and litter size (6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 versus 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) were significantly higher in female mice subjected to 8-IPF treatment than in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003el-n). Overall, 8-IPF ameliorates ovarian reserve depletion and oocyte meiotic defects, improving fertility outcomes in aged female mice by increasing CDC42 and RAC1 prenylation and cortical F-actin assembly in aged oocytes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe identification of effective methods to improve the quality of aged oocytes is a grand challenge in reproductive medicine. In this study, we found that MVA supplementation promoted cortical F-actin assembly and reduced meiotic defects in oocytes, improving the quality of oocytes from COCs in aged mice. Furthermore, MVA activated the FPP synthesis pathway in surrounding GCs, and GCs subsequently transferred the metabolite to aged oocytes through gap junction. FPP prenylated CDC42 and RAC1 in oocytes and promotes cortical F-actin assembly, which was critical for decreasing meiotic defects in oocytes. In addition, we found that natural herb compound 8-IPF could ameliorate ovarian reserve depletion and improved oocyte quality in aged mice by increasing CDC42 and RAC1 cortical localization for F-actin assembly (Extended Data Fig.\u0026nbsp;9).\u003c/p\u003e \u003cp\u003eOocytes and surrounding GCs form follicles, which constitute the fundamental reproductive units in the ovary. In addition, GCs establish a metabolic community with oocytes and transmit metabolites such as pyruvate, amino acids, and cholesterol for oocyte development and maturation\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Our recent studies and others reported that GCs exhibit a robust increase in MVA pathway gene expression during oocyte meiotic resumption, whereas oocytes show low expression of these regulatory genes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This finding indicates that active MVA pathway metabolic synthesis occurs in GCs, in which MVA pathway metabolites are subsequently supplied to oocytes for meiosis. The present study further confirmed the metabolic coupling between GCs and oocytes using alk-FOH chemical reporters. Fluorescent labelling of FOH metabolism showed that active uptake of FOH occured in GCs within 14 h and FOH metabolites were transferred to oocytes through gap junctions in COCs, affecting oocyte meiosis, whereas denuded oocytes were unable to directly uptake FOH.\u003c/p\u003e \u003cp\u003eMammalian oocyte meiosis represents a distinctive form of asymmetric cell division, in which a mature egg and a diminutive polar body are produced. The cortical F-actin assembly in the oocyte is essential for oocyte polarization and the completion of chromosome segregation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The Arp2/3 complex comprises actin nucleators that promote actin formation and can be activated through the CDC42-N-WASP or RAC1-WAVE2 signalling pathways\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. CDC42 and RAC1 are members of the Rho GTPases family, a small guanosine triphosphatase (GTPase) subfamily of proteins that oscillate between the GTP-bound (active) and GDP-bound (inactive) states\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. CDC42 and RAC1 are enriched at the cortex after spindle migration and cortical F-actin assembly are necessary for the extrusion of the first polar body during oocyte meiosis\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42 CR43 CR44\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We confirmed that CDC42 and RAC1 have CaaX motifs at the carboxyl terminus and can be prenylated by FPP in oocytes. Prenylation facilitates the membrane localization of CDC42 and RAC1, promoting the assembly of the CDC42-N-WASP-Arp2/3 and RAC1-WAVE2-Arp2/3 complexes and subsequent assemble cortical F-actin at the MI stage. Decreased CDC42 and RAC1 membrane localization and abnormal cortical F-actin assembly led to meiotic defects and aneuploidy in aged oocytes. More importantly, supplementation with MVA or FOH rescued aged oocyte quality by increasing prenylation and cortical F-actin assembly. These findings reveals a novel mechanism for quality decline in aged oocytes during ovarian aging.\u003c/p\u003e \u003cp\u003eFor several decades, many studies have focused on developing therapeutic strategies to improve the quality of aged oocyte. However, there are still no effective clinical methods for reducing meiotic defects and aneuploidy in aged oocytes. Recent studies have shown that the MVA pathway participates in the aging of multiple organs, such as the testis, muscle, and ovary\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Our present study is the first to present initial evidence for the reduction of abnormal F-actin distribution, meiotic defects and aneuploidy in aged oocytes by supplementation with MVA. Therefore, we are carrying a single-center clinical study that maturation media for aged COCs supplemented with MVA metabolite, a water-soluble chemical, to improve aged oocyte euploidy and quality (Clinical trial: NCT05788822). Interestingly, we found that 8-IPF, a chemical from the traditional Chinese medicine \u003cem\u003eEpimedium brevicornu\u003c/em\u003e Maxim, could activate the FPP synthesis pathway and promote the localization of CDC42 and RAC1 to the oocyte membrane for cortical F-actin assembly. Indeed, 8-IPF supplementation improved the quality of oocytes from aged ovaries and increased female mouse fertility. Furthermore, 8-IPF can be orally administered, does not cause direct damage to the skin or mucous membranes, and has a relatively short half-life\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Therefore, with further clinical study, 8-IPF may be suitable for clinical use via oral administration to improve fertility in females with advanced maternal age.\u003c/p\u003e \u003cp\u003eIn conclusion, the present study revealed that decreased prenylation of CDC42 and RAC1 in oocytes affects cortical F-actin assembly during oocyte meiosis, consequently contributing to meiotic defects and aneuploidy in aged oocytes. MVA and 8-IPF supplementation is instrumental in facilitating the synthesis of FPP from neighbouring GCs, enhancing the cortical localization of CDC42 and RAC1 to activate Arp2/3 complex for normal cortical F-actin assembly in oocytes. Thus, our study provides unprecedented insights into the mechanisms of age-related ooctye cortex abnormality, aneuploidy and clinical directions for improving the reproductive outcomes or extending the reproductive lifespan in women with advanced maternal age.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eAnimals and drug administration.\u003c/b\u003e Six-week-old and 8-month-old female C57BL/6 mice and 3-week-old female ICR mice were purchased from SPF Biotechnology Co., Ltd (Beijing, China). The mice were raised until experimental time in the Animal Laboratory Center of Nanjing Drum Tower Hospital under a 12-h light/dark cycle at a constant temperature (20\u0026ndash;23\u0026deg;C) and had free access to food and water according to the institutional guidelines. Nine-month-old female C57BL/6 mice were intraperitoneally injected with 5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MVA (Sigma, St. Louis, MO, USA; M4667) every day at 18:00 hours for 30 consecutive days. 9.5-month-old female C57BL/6 mice were intragastrically gavaged with 5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 8-IPF (Yuanye, Shanghai, China; B21576) every day at 18:00 hours for 14 consecutive days. Approval for the procedures involving mouse protocols and experiments was obtained from the Experimental Animal and Welfare Ethics Committee of Nanjing Drum Tower Hospital (2023AE01053).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCOC and DO IVM in MEMα maturation medium.\u003c/b\u003e Female mice were intraperitoneally injected with 10 IU pregnant mare serum gonadotropin (PMSG) (Sansheng Pharmaceuticals, Ningbo, China) and sacrificed 48 h later. COCs and DOs were isolated from the ovarian antral follicles using a disposable syringe with a 20-gauge needle and subsequently cultured in MEMα (Gibco, Waltham, MA, USA; 32561037) maturation medium covered with liquid paraffin oil in an incubator at 37 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. The MEMα maturation medium contained 10% foetal bovine serum (FBS) (Gibco, Waltham, MA, USA; 10270106), 10 ng/mL epidermal growth factor (EGF) (Gibco, Waltham, MA, USA; 53003-018), and 1.5 IU/mL human chorionic gonadotropin (hCG) (Sansheng Pharmaceutical, Ningbo, China). After culturing in IVM for 4 h and 14 h, respectively, the GVBD and PBE rate were analysed. 50 \u0026micro;M MVA, 10 \u0026micro;M FOH (Sigma, St. Louis, MO, USA; F203), and 10 \u0026micro;M FTI-277 (Aladdin, China; F331609) were administered by addition to the MEMα maturation medium in this study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOocyte immunofluorescence.\u003c/b\u003e Oocytes were fixed in 4% paraformaldehyde (PFA) (Sigma, St. Louis, MO, USA; 158127) for 30 min before permeabilizing in 0.5% Triton X-100 (Sigma, St. Louis, MO, USA; T9284) for 20 min. Then, blocking was performed using 1% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA; 10711454001) for 1 h. Oocytes were incubated with mouse monoclonal anti-α-tubulin-FITC (Sigma, St. Louis, MO, USA; F2168; 1:200), 594-phalloidin (US Everbright\u0026reg; Inc., Suzhou, China; YP0052L, 1:200), mouse monoclonal anti-Arp3 (Santa Cruz Biotechnology, California, USA; sc-48344, 1:100), mouse monoclonal anti-CDC42 antibodies (Santa Cruz Biotechnology, California, USA; sc-8401; 1:200) and mouse monoclonal anti-RAC1 antibodies (Santa Cruz Biotechnology, California, USA; sc-514583; 1:200) at 4\u0026deg;C overnight. The oocytes were then treated with a secondary antibody for 1 h at room temperature. After three 5 min washes in phosphate-buffered saline with 0.05% Tween 20 (PBST), the oocytes were incubated with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA; H3570) for 10 min at room temperature. Then, the oocytes were mounted on glass slides and observed under a fluorescence microscope (Leica, Germany; DM3000).\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosome spread.\u003c/b\u003e MII oocytes were first treated with Tyrode\u0026rsquo;s buffer (Sigma, St. Louis, MO, USA; T2397) for 3 min at 37\u0026deg;C to remove the zona pellucida. Then, the cells were cultured in M2 medium (Sigma, St. Louis, MO, USA; M7167) for 10 min and fixed in 1% PFA with 0.15% Triton X-100 (pH 9.2) on a glass slide. After drying, the samples were incubated with a human anti-centromere antibody (Incorporated, Davis, CA, USA; CA95617; 1:100) at 4\u0026deg;C overnight. After three 5 min washes in PBST, the samples were incubated with Hoechst 33342 for 10 min at room temperature. Finally, the number of spread chromosomes was counted under a DM3000 LED microscope (Leica, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003efertilization (IVF) and embryo culture.\u003c/b\u003e The sperm from the epididymides of 12-week-old C57BL/6 male mice were collected and capacitated for 1 h in human tubal fluid (HTF) medium (Merck Millipore, St. Louis, MO, USA; MR-070). COCs after IVM for 14 h or obtained from oviductal ampullae of C57BL/6 female mice were added to be fertilized by the addition of capacitated sperm for 6 h in a 37\u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. The fertilized oocytes were subsequently transferred to KSOM (Merck Millipore, St. Louis, MO, USA; MR-106-D) for subsequent culture. The 2-cell embryos and blastocysts formation rates were subsequently calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetabolite extraction and targeted metabolomics analysis.\u003c/b\u003e Metabolites were extracted using a method described previously with some modifications\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Mice oocytes and GCs were briefly washed with PBS after isolation before quenching. To quench cells and stop metabolism, oocytes and GCs were harvested in tubes and quick-frozen in liquid nitrogen immediately after washing. Oocytes or cells were then incubated in an extraction buffer containing 80% MS grade methanol and 2 \u0026micro;g/ml 4-CL-phenylalanine (Sigma, St. Louis, MO, USA; C6506) as an internal standard which was precooled at -80\u0026deg;C freezer. Metabolites were extracted by 3 rounds of bead-beating. After two rounds of centrifugation by 15,000 g for 10 minutes at 4\u0026deg;C, the supernatant fraction containing soluble metabolites was harvested and dried using a CentriVap Concentrator system (Labconco).\u003c/p\u003e \u003cp\u003eFor running samples, a dried metabolite extract sample was re-suspended in 50% MS grade acetonitrile for injection. MVA pathway metabolites such as FPP and GGPP levels were quantitatively analyzed in a negative ion mode using multiple reaction monitoring (MRM) acquisition with a triple quadrupole mass spectrometer (Triple Quad 6500+, AB SCIEX) that is coupled to a high performance liquid chromatography. A mix standard containing FPP (Sigma, St. Louis, MO, USA; F6892) and GGPP (Sigma, St. Louis, MO, USA; G6025) with series dilution for 20 ng/ml, 40 ng/ml, 200 ng/ml, 500 ng/ml, 2 \u0026micro;g/ml, 5 \u0026micro;g/ml was used to make standard curves. Metabolites were separated chromatographically on a UPLC HSS T3 column (ACQUITY 1.8 \u0026micro;m 150 x 2.1 mm, Waters). Flow rate was set to 0.25 mL/min using the following method: Buffer A: 10 mM ammonium carbonate, Buffer B: 100% acetonitrile. T\u0026thinsp;=\u0026thinsp;0 min, 10% B; T\u0026thinsp;=\u0026thinsp;1 min, 10% B, T\u0026thinsp;=\u0026thinsp;4 min, 65% B; T\u0026thinsp;=\u0026thinsp;6 min, 65% B; T\u0026thinsp;=\u0026thinsp;6.5 min, 95% B; T\u0026thinsp;=\u0026thinsp;8.5 min, 95% B; T\u0026thinsp;=\u0026thinsp;9 min, 10% B; T\u0026thinsp;=\u0026thinsp;12 min, 10% B stop. The retention time for each MRM peak was compared to an appropriate standard. The area under each peak was then quantitated by using SCIEX OS software and re-inspected for accuracy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-seq library construction and analysis.\u003c/b\u003e Oocytes and GCs isolated from COCs were lysed to obtain cDNA using a Discover-sc WTA Kit V2 (Vazyme Biotech, China; N712). A TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Biotech, China; TD503) was used for library construction. The Illumina HiSeq X platform (Nanjing Genemap Co., Ltd., China) was used to perform sequencing. High-quality reads were aligned to the \u003cem\u003eMus musculus\u003c/em\u003e UCSC mm9 reference genome, and the FPKM value of each gene was calculated. Highly variable genes (coefficient of variation\u0026thinsp;\u0026gt;\u0026thinsp;1) were selected for PCA, and the resulting PCA plot was generated using the ggplot2 package in R studio. The DESeq2 package was used to identify DEGs. KEGG enrichment analysis was performed with KEGG Mapper.\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT‒PCR.\u003c/b\u003e All primers were mixed in nuclease-free water to a final concentration of 0.1 \u0026micro;M as a primer assay pool. For each mixture, 5 \u0026micro;L of solution was prepared as follows: 2.5 \u0026micro;L of reaction mixture, 0.5 \u0026micro;L of primer mixture, 0.1 \u0026micro;L of RT-Taq mixture, and 1.9 \u0026micro;L of nuclease-free water (Vazyme Biotech, China; P621). GCs isolated from COCs were added to the mixture and placed in a -80\u0026deg;C freezer for 2 min. The mixture was centrifuged at 1000 g for 2 min, after which PCR was performed. For qRT‒PCR, 5 \u0026micro;L of SYBR-Green (Vazyme Biotech, China; Q121), 0.5 \u0026micro;L of Primer-forward (10 \u0026micro;M), 0.5 \u0026micro;L of Primer-reverse (10 \u0026micro;M), 2 \u0026micro;L of cDNA, and 2 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO were mixed. The following primer sequences were used: \u003cem\u003eMVK\u003c/em\u003e (mouse): forward, 5\u0026rsquo;-AGCGTCAATTTACCCAACATCG-3\u0026rsquo;, reverse, 5\u0026rsquo;-GAGACATCACCTTGCTCAAGAAA-3\u0026rsquo;; \u003cem\u003eFDPS\u003c/em\u003e (mouse): forward, 5\u0026rsquo;-GGGTTTGACCGTGGTACAAG-3\u0026rsquo;, reverse, 5\u0026rsquo;-AAGCCTGGAGCAGTTCTACAC-3\u0026rsquo;; \u003cem\u003eGGPPS\u003c/em\u003e (mouse): forward, 5\u0026rsquo;-TTTTGCATACACTCGACACACT-3\u0026rsquo;, reverse, 5\u0026rsquo;-GGCCTCAATTTGTTTGTAGGCT-3\u0026rsquo;; \u003cem\u003eSQLE\u003c/em\u003e (mouse): forward, 5\u0026rsquo;-GACCTCGTTCGTGACGGAC-3\u0026rsquo;, reverse, 5\u0026rsquo;-CTCCCCAACTATCCTGTCGG-3\u0026rsquo;; \u003cem\u003eand 18S\u003c/em\u003e (mouse): forward, 5\u0026rsquo;-ATGGCCGTTCTTAGTTGGTG-3\u0026rsquo;, reverse, 5\u0026rsquo;-CGGACATCTAAGGGCATCAC-3\u0026rsquo;. All data were normalized to the expression of \u003cem\u003e18S\u003c/em\u003e using the comparative 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture and metabolic labelling.\u003c/b\u003e Human KGN cells were cultured in DMEM/F12 (Gibco, Waltham, MA, USA; C11330500BT) supplemented with 10% FBS (Gibco) and 1% penicillin‒streptomycin (Gibco, Waltham, MA, USA; 15140122) in a humidified 37\u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were grown in 10 cm plates to approximately 70% confluence and treated with alk-FOH (20, 50, or 100 \u0026micro;M; from 100 mM stock solution in DMSO) or DMSO only for 48 h. For antagonistic coincubation, both 50 \u0026micro;M alk-FOH and 50 \u0026micro;M FOH (Sigma, St. Louis, MO, USA; F203) were added to KGN cells for 48 h. Then, the cells were washed three times with cold phosphate-buffered saline. The cells were lysed for western blotting and pull-down assays.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting.\u003c/b\u003e The proteins were quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA; 23227) following the manufacturer\u0026rsquo;s instructions. Next, the equivalent proteins were separated via 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, St. Louis, MO, USA; 03010040001). The membranes were incubated with primary antibodies, rabbit polyclonal anti-Farnesyl (Invitrogen, Waltham, MA, USA; PA1-12554; 1:1500), FDPS (Abcam, MA, USA; ab153805; 1:1000), MVK (Proteintech, China; 12228-1-AP; 1:1000), rabbit monoclonal anti-CDC42 (Abcam, MA, USA; ab187643; 1:1000), rabbit monoclonal anti-RAC1 (Cell signaling Technology, Danvers, MA, USA; 4651S; 1:1000), rabbit monoclonal anti-Arp2 (Abcam, MA, USA; ab128934; 1:1000), rabbit monoclonal anti-Arp3 (Abcam, MA, USA; ab181164; 1:1000), rabbit monoclonal anti-N-WASP (Abclonal, Wuhan, China; A2270; 1:1500), mouse monoclonal anti-WAVE2 (Abclonal, Wuhan, China; A19601; 1:1500), rabbit monoclonal anti-β-actin (Bioworld, Beijing, China; AP0060; 1:100000), and rabbit polyclonal anti-Calnexin (Proteintech, China; 10427-2-AP; 1:10000) at 4℃ overnight after blocking with 5% nonfat milk in PBST for 1 h at room temperature. The next day, the PVDF membranes were washed three times with PBST, incubated with HRP-conjugated goat anti-rabbit IgG (Abcam, MA, USA; ab97051; 1:10000) at room temperature for 1 h, washed three times with PBST, and then developed using enhanced chemiluminescence reagents (GE, Piscataway, NJ, USA); the mean grey value was estimated with ImageJ software (NIH, USA, Version 1.0).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCu-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction.\u003c/b\u003e Metabolic labelling and protein extraction were performed as described above. Protein concentrations were adjusted to 2.2 mg/mL. For each, 3000 \u0026micro;L of protein lysate click reagent mixture was prepared as follows: 2520 \u0026micro;L of protein (2.2 mg/mL), 60 \u0026micro;L of azide biotin (Confluore, China; 908007-17-0) (0.1 mM, 5 mM stock solution in DMSO), 120 \u0026micro;L of BTTAA-CuSO4 (2:1, 1 mM:0.5 mM), and 300 \u0026micro;L of fresh sodium ascorbate (2.5 mM, 25 mM stock solution in PBS), which were added last. Then, the reaction mixture was incubated on a shaker at room temperature for 3 h. After the CuAAC reaction, the metabolic labeled proteins were precipitated overnight in methanol at -40\u0026deg;C. The next day, the samples were centrifuged at 5,000 \u0026times; g for 15 min at 4\u0026deg;C and subsequently washed twice with 7.5 mL of precooled methanol. The precipitated proteins were subsequently completely dissolved in 1.67 mL of buffer (1.2% SDS in PBS) after the methanol was discarded, after which the mixture was left at room temperature for 20 min of volatilization. One hundred microlitres of Pierce High Capacity Streptavidin Agarose (Thermo Fisher Scientific, Waltham, MA, USA; 20359) was washed with PBS three times and resuspended in 8.33 mL of PBS in a 15 mL tube. The redissolved proteins were added to 15 mL tubes filled with the agarose and incubated gently with rotation at room temperature for 4 h. The nonspecific binding proteins were then washed with 0.2% SDS in PBS (10 mL, once), PBS (10 mL, three times), and ddH\u003csub\u003e2\u003c/sub\u003eO (10 mL, three times) and centrifuged at 500 g for 1 min, after which the beads were transferred to 1.5 mL centrifuge tubes with ddH\u003csub\u003e2\u003c/sub\u003eO. Then, 2\u0026times; loading buffer (Beyotime, Shanghai, China; P0015L) was added to the precipitated beads, which were subsequently heated at 95\u0026deg;C for 12 min. Next, the samples were centrifuged, after which approximately 40 \u0026micro;L of 2\u0026times; loading buffer was transferred to a new 1.5 mL tube to obtain the protein released from the beads. The proteins were subsequently separated via 10% SDS-PAGE for Western blotting or were subjected to LC‒MS/MS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLC‒MS/MS analysis.\u003c/b\u003e The enriched protein samples were analysed using an Easy-nLC 1000 (Thermo Fisher) (Buffer A: 0.1% formic acid solution; Buffer B: 0.1% formic acid\u0026thinsp;+\u0026thinsp;80% acetonitrile solution; Thermo Fisher, USA). After the column was equilibrated with 95% Buffer A, the sample was loaded onto a Trap column. Then, the samples were separated by chromatography and analysed via mass spectrometry (MS). MS analysis was performed with a Q Exactive mass spectrometer (Thermo Fisher, USA). The raw MS files were searched using MaxQuant 1.6.14 and compared to the \u003cem\u003eHomo sapiens\u003c/em\u003e database downloaded from UniProt and finally the identified protein results were obtained.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCuAAC reaction for fluorescence imaging.\u003c/b\u003e COCs were incubated with 50 \u0026micro;M alk-FOH for 9 h. COCs were fixed with 4% PFA for 30 min. Then, the COCs were incubated with 50 \u0026micro;M azide AZDye 488, the BTTAA-CuSO\u003csub\u003e4\u003c/sub\u003e complex (BTTAA/CuSO\u003csub\u003e4\u003c/sub\u003e 6:1), and 2.5 mM sodium ascorbate in PBS at room temperature for 30 min. After the click reaction, COCs were incubated with 594-Phalloidin and Hochest 333342 at room temperature for 30 min. Fluorescence imaging was subsequently performed under LSM 900 confocal laser scanning microscope (Zeiss).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTotal, membrane and cytoplasmic protein isolation.\u003c/b\u003e Total proteins were isolated by lysing cells with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China; P0013B) supplemented with protease inhibitors at 4\u0026deg;C for 30 min. The supernatant of the lysates was collected after centrifugation at 12,000 rpm for 15 min.\u003c/p\u003e \u003cp\u003eCell membrane and cytoplasmic proteins were isolated using a Membrane and Cytosol Protein Extraction Kit with protease inhibitors according to the manufacturer\u0026rsquo;s instructions (Beyotime, Shanghai, China; P0033). In brief, approximately 3 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells were homogenized using 1 mL of membrane protein extraction reagent A supplemented with PMSF and protease inhibitors for 30 min at 4\u0026deg;C. The nuclei and unbroken cells were removed by centrifuging at 700 g for 10 min. The supernatant was then collected for further centrifugation at 12,000 rpm for 30 min to obtain the plasma proteins. The remaining sediment was fully vortexed in 200 \u0026micro;l of membrane protein extraction reagent B on ice for 10 min and subsequently centrifuged at 12,000 rpm for 30 min to obtain the membrane proteins.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCoi\u003c/strong\u003e \u003cp\u003e \u003cb\u003emmunoprecipitation (co-IP) assay.\u003c/b\u003e KGN cells were treated with 10 \u0026micro;M FOH for 48 h, total protein was extracted with RIPA buffer supplemented with protease inhibitor, and total protein was quantified with a BCA protein assay kit. Next, 500 g of total protein was incubated with 1 g of anti-mouse immunoglobulin G (IgG), anti-CDC42 (Santa Cruz Biotechnology, California, USA; sc-8401), or anti-RAC1 (Santa Cruz Biotechnology, California, USA; sc-514583) at 4\u0026deg;C overnight. Then, 30 L of agarose beads was added to the cell lysates, which were incubated at 4\u0026deg;C for 4 h. Subsequently, the cell lysates were incubated with 2 \u0026times; loading buffer at 95\u0026deg;C for 10 min. Finally, Western blotting was performed to analyse the protein samples.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression Constructs, mRNA Synthesis and microinjection.\u003c/b\u003e The mcherry coding sequences and CDC42 or RAC1 coding sequences were fused and inserted into pGEMHE plasmids to obtain pGMHE-mCherry-CDC42 and pGMHE-mCherry-RAC1. pGMHE-mCherry-CDC42\u003csup\u003eC188Y\u003c/sup\u003e(M-CDC42) and pGMHE-mCherry-RAC1\u003csup\u003eC188Y\u003c/sup\u003e(M-RAC1) were obtained using a site-directed mutagenesis kit (NEB, E0554) for \u003cem\u003ein vitro\u003c/em\u003e transcription. After linearization of the template with AscI (NEB, R0558V), capped mRNA was synthesized using HiScribe T7 High yield RNA Synthesis Kit (NEB, E2040S), and dephosphorylated for uncapped RNA using Antarctic phosphatase (NEB,M0289). Finally, purified for phenol/chloroform and dissolve in 11 \u0026micro;l nuclease-free H\u003csub\u003e2\u003c/sub\u003eO for mRNA. mRNA concentrations were determined by NanoDrop (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eMouse oocytes were microinjected with 3.5 pl of mRNA. mRNA was microinjected at a needle concentration (final concentration in the microinjection needle) of 100 ng/\u0026micro;l. Oocytes were allowed to express the mRNAs for 3 h before release in M2 medium containing 250 \u0026micro;M dbcAMP (Sigma-Aldrich). After 3 h, the oocytes were maintained in dbcAMP-free M2 medium and performed using LSM 900 confocal laser scanning microscope (Zeiss).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBody weight and ovarian index.\u003c/b\u003e The mice in each group were euthanized after weighing. A U-shaped incision in the hypogastrium was made to explore the main organs. The bilateral ovaries connected to the bicorned uterus via the fallopian tubes were separated, and the peri-ovarian adipose tissue was removed under a stereoscope (Leica, Germany). The ovaries were subsequently weighed on an electronic analytical balance when no liquid remained. A representative picture was taken on dry sterile gauze, and the ovarian index was determined based on the ovarian wet weight (mg) / body weight (g).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological analysis and follicle counting.\u003c/b\u003e The oestrous cycles of the mice were detected by daily examination of vaginal smears, and the mice were sacrificed in the dioestrus phase to collect ovaries. Ovaries were fixed with 4% paraformaldehyde in PBS overnight, dehydrated in 70%, 80%, 90%, 95%, and 100% ethanol, cleared with xylene and embedded in paraffin. The ovarian tissues were serially sectioned at 5 \u0026micro;m, sequentially deparaffinized in xylene, rehydrated in a descending series of graded ethanol solutions, and stained with haematoxylin and eosin (HE).\u003c/p\u003e \u003cp\u003eOne of every five slices was used to count the follicles. The follicles at the primordial, primary, secondary, and antral stages in the histological sections of the ovaries were observed and classified based on the histological morphology under a microscope at 200\u0026times; magnification. Only follicles containing a visible nucleus were counted independently by two researchers. The result was calculated as a five-fold counting value.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFertility test.\u003c/b\u003e After two weeks of gavage, to evaluate the fertility function after 8-IPF administration, female mice were mated with 10-week-old C57BL/6 male mice with proven fertility. At 8:00 AM the next day, vaginal plugs were observed to confirm whether the female mice were pregnant. Female mice were considered infertile if, after continuous mating for two-weeks, no vaginal plugs were observed. The pregnancy was terminated by caesarean section at 18.5 days gestation, and after routine anaesthesia, the uterus was exposed through a U-shaped incision in the lower abdomen. The pregnancy and embryo implanted conditions of the mice were observed and photographed, after which the embryos and placenta were separated, weighed and recorded. The number of viable offspring in each litter was recorded.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Normally distributed data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) and were compared by unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Continuous variables without a normal distribution are presented as medians and interquartile ranges and were analysed by the Mann‒Whitney U test. All the statistical comparisons were performed with GraphPad Prism 8.0.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSchematics.\u003c/b\u003e Schematic cartoons in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee were created with BioRender.com.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe mouse oocyte and GC RNA-seq data can be found under accession numbers CRA017269 and CRA017270.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eL.J.D., H.X.S., and C.S. designed the research. C.M.L., H.D.Z., J.L.M., S.N.Z., L.J.D., X.T., Y.B.Z., C.J.W., H.J.P., N.N.K., J.S.F., Y.Z., J.D.Z., and X.Z. performed the experiments. C.M.L., H.D.Z., J.L.M., S.N.Z., C.S., and L.J.D. analyzed the data and wrote the manuscript. L.J.D., H.X.S., C.S., R.X., C.Q.Y., Y.L.H., C.J.L, and G.J.Y. revised the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (82271671), National Key Research and Development Program of China (2018YFC1004701) to Lijun Ding; a grant from the National Natural Science Foundation of China (82201830) to Chuanming Liu; a grant from the self-research project of State Key Laboratory of Reproductive Medicine (SKLRM-2022D2) and research project of Changzhou Medical Center of Nanjing Medical University (CMCM202203) to Haixiang Sun.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBroekmans FJ, Soules MR, Fauser BC (2009) Ovarian aging: mechanisms and clinical consequences. 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Mol Cell 66:180\u0026ndash;193e8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.molcel.2017.02.026\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2017.02.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4762298/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4762298/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDeclined oocyte quality is the major contributor to female subfertility in aged mammals. Currently, there are no effective interventions to ameliorate aged oocyte quality. We found that oocytes from aged mice exhibited lower levels of mevalonate (MVA) pathway metabolites, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) and reduced cortical F-actin. We further demonstrated that MVA supplementation improved the FPP level, the cortical F-actin and the quality of aged oocytes. Mechanistically, we found that MVA supplementation induced granulosa cells to synthesize FPP, which was subsequently transferred to aged oocytes. Transported FPP increased small GTPases prenylation, including CDC42 and RAC1, and promoted membrane localization of CDC42-N-WASP-Arp2/3 and RAC1-WAVE2-Arp2/3 complexes, promoting cortical F-actin re-assembly and reducing aneuploidy of aged oocytes. We also identified an oral drug 8-isopentenyl flavone, as an isoprenoid donor from \u003cem\u003eEpimedium brevicornu\u003c/em\u003e Maxim, which could increase CDC42 and RAC1 prenylation, improving the cortical F-actin and the competence of aged oocytes, ameliorating reproductive outcomes in aged female mice. Collectively, increasing small GTPases prenylation via MVA metabolites or 8-IPF provide a therapeutic approach for boosting fertility in women of advanced maternal age.\u003c/p\u003e","manuscriptTitle":"Mevalonate metabolites boost aged oocyte quality through small GTPases prenylation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 04:58:32","doi":"10.21203/rs.3.rs-4762298/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"776b45a3-ed08-4b25-a64e-c89e0eea4edb","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":34961862,"name":"Biological sciences/Developmental biology/Ageing"},{"id":34961863,"name":"Health sciences/Endocrinology"}],"tags":[],"updatedAt":"2024-11-06T16:41:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 04:58:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4762298","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4762298","identity":"rs-4762298","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}