{"paper_id":"e0ab3218-3377-47ee-bd29-0307eff185ae","body_text":"Oocyte quality plays an important role in the success of assisted reproductive\ntechnology (ART) procedures ( Li  et\nal. , 2008 ;  Assou  et\nal. , 2010 ). Communication between the oocyte and the\nsurrounding cumulus cells (CCs) via gap junctions and paracrine signals is essential\nfor oocyte competence and influences fertilization outcomes ( Molinari  et al. , 2016 ).\nCCs support oocyte nuclear and cytoplasmic maturation by transferring required\nmolecules for oocytes such as amino acids and protecting the oocytes against harmful\nfactors such as oxidative stress ( Eppig  et\nal. , 2005 ;  Uhde  et\nal. , 2018 ;  Martinez\n et al. , 2023 ). They also sustain energy within the\ncumulus-oocyte complex (COC) by providing required molecules for ATP synthesis, like\npyruvate, lactate, and cholesterol precursors ( Da\nBroi  et al. , 2018 ). On the other hand, oocytes regulate\nCCs differentiation, proliferation and apoptosis through the secretion of paracrine\nfactors ( Hourvitz  et al. ,\n2010 ;  Huang & Wells, 2010 ).\nAny disruption of this cross-talk might impair the oocyte competence and\nfertilization outcomes ( Da Broi  et\nal. , 2018 ). Despite technological progress, molecular\nmarkers of oocyte competence within CCs are not completely understood. Recent\nstudies have suggested that CC-derived transcription factors (TFs) are linked to\nembryo quality ( Wathlet  et al. ,\n2011 ;  Burnik Papler  et\nal. , 2015 ;  Li  et\nal. , 2015a ;  2015b ;\n Shen  et al. , 2020 ).\nHowever, the specific role of TFs in CCs performance is still poorly characterized.\nTFs such as Zinc Finger 83 (ZNF83), and related genes including structural\nmaintenance of chromosomes 5 (SMC5), and Aminoacylase 1 (ACY-1) have been suggested\nas candidates for CCs performance based on their involvement in critical pathways.\nFor example ZNF83 regulates RNA polymerase II activity, potentially modulating RNA\nsupply to the oocyte ( Lambert  et\nal. , 2018 ); while animal studies have shown that SMC5\nmaintains chromosomal stability during meiosis ( Hwang  et al. , 2017 ;  Pradhan  et al. , 2023 ); and ACY-1 facilitates amino acid\nrecycling and oxidative stress defense ( Al-Helaly\n& Mahmood, 2021 ;  Fernández-Hernández  et al. , 2021 ). These\ngenes selected based on transcriptomic data from a prior screen ( Govahi  et al. , 2022 ) and it\nseems to be effective on the function of CCs and the two-waycommunication between\nCCs and oocytes by affecting processes such as cell division, DNA damage repair\n( Pradhan  et al. , 2023 ),\nprotection against oxidative stress ( Al-Helaly &\nMahmood, 2021 ), and providing the oocyte with the necessary materials\n( Fernández-Hernández  et\nal. , 2021 ).\nWe hypothesized that dysregulation of these TFs in CCs would correlate with oocyte\nfertilization failure. Our study aims to compare the expression of  ZNF83,\nACY-1 , and  SMC5  in CCs by fertilized oocytes producing\nhigh-quality embryos versus non-fertilized oocytes following intracytoplasmic sperm\ninjection (ICSI).\n\nThe study began after approval by the Ethics Committee of Iran University of\nMedical Sciences (Approval ID: IR.IUMS.REC.1400.509) and obtaining informed\nconsent from all participants. Eighteen oocyte donors meeting the following\ninclusion criteria were enrolled:\nProven fertility (≥1 live birth from natural conception)\nNo reproductive tract abnormalities or underlying diseases\nAge <35 years and BMI 18-28\nThe partner of the couples receiving the donated oocyte was aged <45\nyears\nTo minimize confounding effects of paternal factors on fertilization and embryo\nquality, sperm samples with abnormal semen parameters (outside WHO standards) or\nDNA Fragmentation Index (DFI) >20% were excluded ( Kandil  et al. , 2021 ). Antagonist protocol\nwas used for stimulation. Cumulus-oocyte complexes (COCs) were collected via\ntransvaginal ultrasound. After a 2-hour incubation, CCs were mechanically\ndenuded from Metaphase II (MII) oocytes. MII oocytes were placed in labeled\nculture droplets for intracytoplasmic sperm injection (ICSI). Injected oocytes\nwere maintained in G1 medium until day 3 of development. CCs from each oocyte\nwere centrifuged and cryopreserved in liquid nitrogen (-196°C). Two samples were\ntaken from everyone. CC from fertilized oocyte that formed high-quality embryos\n(6-8 cell, no fragmentation, evenly sized blastomeres) ( Sakkas & Gardner, 2017 ) were considered as the\nfertilized group and CC from oocyte failing to fertilize were considered as the\nnon-fertilized group.\nSperm DNA fragmentation (SDF) assay was performed by a DNA fragmentation assay\nkit (Dianbio Assay Co.). Assessment was done according to the manufacturer’s\nprotocol. Briefly, dilution of semen to achieve concentration of 5-10 ×\n10 6  sperm/mL was performed by phosphate-buffered saline (PBS; pH\n7.4). Diluted semen was embedded in low-melting-point agarose gel. Subsequently,\nsamples mounted on pre-coated slides. Cellular denaturation and lysis were\nperformed using acid and lysis solutions, respectively, followed by dehydration\nthrough a graded ethanol series (70%, 90%, and 100%). Two hundred sperms per\nsample were analyzed, with DNA fragmentation classified based on the presence or\nabsence of characteristic halo patterns: spermatozoa exhibiting minimal or no\nhalos were classified as containing fragmented DNA. DFI was calculated as the\npercentage of spermatozoa with abnormal halo formation. In alignment with\nclinical thresholds, a DFI value ≤ 20% was deemed within the normal range\n( Mirsanei  et al. ,\n2023 ).\nTotal RNA extraction was performed using the Trizol reagent (Sigma, Poole, UK).\nSpectrophotometry (NanoDrop) was used for RNA concentration and purity\nassessment. Then, Complementary DNA (cDNA) was synthesized by reverse\nTranscription Kit (Bio-Rad, #1725037), according to the manufacturer’s\ninstructions. Quantitative PCR (qPCR) was conducted in triplicate to evaluate\nthe expression levels of target genes ( ZNF83, ACY-1 ,\nand SMC5 ). The relative gene expression was calculated using\nthe 2-∆∆Ct method. Primer sequences used in the assay are listed in  Table 1 .\nPrimer sequences used in the assay\nWe used CCs from two groups and performed Western blot analyses as previously\ndescribed, with some modifications ( Jabarpour\n et al. , 2018 ;  Gholipour  et al. , 2023 ). Briefly, the cells were\nlysed with ripa buffer. The lysates were removed by centrifugation. Protein\nconcentration was determined by the Bradford Protein Quantification kit\n(DNAbioTech, Iran). The tissue lysates were mixed with a Laemmlisample buffer.\nLysates were then subjected to SDS-PAGE and subsequently transferred to a 0.2\nµm Immune-Blot™ polyvinylidene difluoride (PVDF) membrane (Bio-Rad\nLaboratories, CA, USA). The membranes were then blocked with 5% BSA (Sigma\nAldrich) in 0.1% Tween 20. Then, the membranes were incubated with Anti-ACY-1\n(abcam), Anti-SMC5 (abcam), Anti-ZNF83 (abcam), and anti-β actin-loading\ncontrol antibodies (Abcam). After washing and incubation of the membranes, goat\nanti-rabbit IgG H&L (HRP) (Abcam) secondary antibody was used. The membranes\nwere then incubated with enhanced chemiluminescence (ECL). β-actin was\nused for normalization.\nData was presented as mean ± standard deviation (SD). Statistical\ncomparisons were performed using independent sample-test. All analyses were\nconducted using GraphPad Prism version 8.0 , with\n p -value <0.05  considered statistically\nsignificant.\n\nAs described in  Figure 1 , Real-time results\nshowed that the expression of ZNF83  was significantly\ndownregulated in the non-fertilized group than in the fertilized group\n( p <0.0001). Similarly, the expression of genes\n ACY-1  and SMC5  was also significantly\nreduced in the non-fertilized group compared to the fertilized group\n( p <0.0001).\nFigure 1 Real-time PCR analysis of ZNF83 (zinc Finger 83), ACY-1\n(aminoacylase-1), and SMC5 (structural maintenance of chromosomes 5)\ngenes expression in non-fertilized groups versus fertilized group.\nExpression level of all three genes (ACY-1, SMC5, ZNF83) were\nsignificantly downregulated in the non-fertilized group compared to\nthe fertilized group ( p <0.0001****).\nβ-actin was used for normalization. Number of biological\nreplicates (n): 3.\nReal-time PCR analysis of ZNF83 (zinc Finger 83), ACY-1\n(aminoacylase-1), and SMC5 (structural maintenance of chromosomes 5)\ngenes expression in non-fertilized groups versus fertilized group.\nExpression level of all three genes (ACY-1, SMC5, ZNF83) were\nsignificantly downregulated in the non-fertilized group compared to\nthe fertilized group ( p <0.0001****).\nβ-actin was used for normalization. Number of biological\nreplicates (n): 3.\nAs described in  Figure 2 , Western blot\nresults revealed that the protein expression of  ZNF83  was\nsignificantly downregulated in the non-fertilized group than in the fertilized\ngroup ( p <0.01). Similarly, the protein expression\nof ACY-1  and  SMC5  was also significantly\nreduced in the non-fertilized group compared to the fertilized group\n( p <0.01,  p <0.05).\nFigure 2 Western blot analysis ofZNF83 (zinc Finger 83), ACY-1\n(aminoacylase-1), and SMC5 (structural maintenance of chromosomes 5)\nexpression. Protein levels of three target (ACY-1, SMC5, ZNF83)\nshowed significant downregulation in the non-fertilized group\ncompared to the fertilized group (* p <0.05/\n** p <0.01). Molecular Weight: ZNF83: 60kda,\nACY-1:46kda, SMC5:129kda, β-actin:42kda. β-actin was\nused for normalization. Number of biological replicates (n):2.\nWestern blot analysis ofZNF83 (zinc Finger 83), ACY-1\n(aminoacylase-1), and SMC5 (structural maintenance of chromosomes 5)\nexpression. Protein levels of three target (ACY-1, SMC5, ZNF83)\nshowed significant downregulation in the non-fertilized group\ncompared to the fertilized group (* p <0.05/\n** p <0.01). Molecular Weight: ZNF83: 60kda,\nACY-1:46kda, SMC5:129kda, β-actin:42kda. β-actin was\nused for normalization. Number of biological replicates (n):2.\n\nThis study compared the gene and protein expression levels of  ZNF83,\nACY-1 , and  SMC5  in CCs from oocytes that failed to\nfertilize (non-fertilized group) and CCs from oocytes that successfully fertilized\nand developed into high-quality embryos (fertilized group) following ICSI. A\nparallel decrease in both mRNA and protein expression of  ZNF83,\nACY-1 , and  SMC5  in the non-fertilized group was\nobserved in this study. These findings suggest that these molecules may play a\nfunctional role in oocyte developmental competence and could serve as novel\nbiomarkers for predicting fertilization potential.\nDue to the well-established role of CCs in oocyte maturation, fertilization, and\nearly embryo development, functional impairments of these cells may contribute to\nreduced fertilization rates ( Turathum  et\nal. , 2021 ).  McReynolds\n et al.  (2012)  demonstrated that alterations in the\nproteomic profile of CCs is associated with follicular senescence.  Li  et al.  (2015a)  have\nreported increased mRNA levels of PRSS35 in CCs from fertilized oocytes compared to\nnon-fertilized oocytes. Reduced HLA-G expression in CCs has been shown to be\nassociated with poor oocyte quality and impaired embryo development ( Aftabsavad  et al. , 2021 ).\nMany studies have reported that, in addition to small molecules, large molecules such\nas long RNA are transferred from CCs to the oocyte ( Macaulay  et al. , 2014 ). One of the key functions of CCs\nis to supply the RNA required by the oocyte to provide maternal reserves ( Russell  et al. , 2016 ). Until\nembryonic genome activation, the developing embryo relies on the oocytes’ RNA for\nproper development ( Hand  et al. ,\n2017 ). Maternal RNA reserves in the oocyte are stored as\nribonucleoprotein complexes. Since CC-derived transcripts do not require the\ndissociation of these protein complexes for translation, their translation within\nthe oocyte is more efficient and accessible. These transcripts are primarily\ninvolved in critical processes such as meiosis regulation, transcription, and\ntranslation. According to ( Macaulay  et\nal. , 2016 ), this transfer follows a precise timeline,\noccurring just before meiosis resumption. Notably, some of these transferred\ntranscripts increase when oocyte transcription is silenced, suggesting their\nfunctional importance for the oocyte’s function ( Macaulay  et al. , 2014 ; 2016).\nIn our study,  ZNF83  expression was significantly lower in the\ninfertile group. Considering its role as a TF, a reduction in  ZNF83 \nexpression among CCs may impair the synthesis of essential RNAs and their transfer\nto the oocyte, leading to reduced oocyte competence. Also,  ZNF83 \nplays a critical role in cell cycle regulation via its interaction with RNA\npolymerase II, so its downregulation could disrupt the cell cycle, leading to\naberrant cell division and increased apoptosis rates.\nWe also observed that SMC5, a component of the chromosomal maintenance complex, was\nnotably downregulated in the non-fertilized group. SMC5 plays a critical role in\ngene expression regulation, chromosome segregation, and DNA damage repair ( Pradhan  et al. , 2023 ).\nConsequently, defects in this protein may impair DNA repair mechanisms. If DNA\ndamage remains unrepaired due to a compromised repair system, it often leads to\napoptosis ( De Zio  et al. ,\n2013 ) and increased apoptosis in CCs can diminish their ability to\nsupport the oocyte, potentially contributing to abnormal embryonic cleavage\ndivisions ( Burrows  et al. ,\n2006 ). Notably, gametes and embryos derived from COCs with minimal or no\napoptosis exhibit a higher developmental competence, reaching the blastocyst stage\nmore efficiently ( Corn  et al. ,\n2005 ). Additionally, research in mouse oocytes has shown that SMC5 levels\ndecline with maternal age, and this protein is essential for the correct segregation\nof homologous chromosomes during meiosis I ( Hwang\n et al. , 2017 ). Based on these functional properties\nof SMC5, the reduced expression of SMC5 in the CCs of the non-fertilized group could\ncontribute to lower oocyte fertilization potential.\nWe also observed reduced expression of the ACY-1 gene and protein in CCs of\nnon-fertilized oocytes compared with fertilized oocytes. This enzyme hydrolyzes\nacetylated proteins, leading to protein recycling into amino acids ( Fernández-Hernández  et\nal. , 2021 ). CCs supply vital amino acids to the oocyte, and\ntheir absence impairs oocyte metabolism, fertilization and early embryo development\n( Martinez  et al. , 2023 ).\nPrior studies have reported that embryo cleavage divisions depend on amino acids\nprovided by CCs. In the absence of CCs, only exogenous amino acid supplementation\nsupports proper division ( Juetten & Bavister,\n1983 ). Thus, the observed ACY-1 downregulation in non-fertilized oocytes\nmay impair amino acid supply, reducing the oocytes’ fertilization potential.\nACY-1 also can protect against oxidative stress by increasing glutathione peroxidase\nand reducing malondialdehyde ( Al-Helaly &\nMahmood, 2021 ). Elevated reactive oxygen species (ROS) in CCs is linked\nto follicular senescence and infertility ( Lin\n et al. , 2020 ). Therefore, lower ACY-1 levels in\nnon-fertilized oocytes could heighten oxidative stress, thus weakening the CCs’\nability to support oocyte fertilization and embryo development. Significantly\nreduced expression of ZNF83, ACY-1, and SMC5 in CCs from non-fertilized oocytes\nhighlights their critical role in supporting oocyte fertilization. The consistency\nbetween gene and protein expression patterns reinforces their functional relevance\nin CC-mediated oocyte support.\nGiven the reproductive health of the study groups, it is not possible to say for sure\nwhat factors may have affected the quality of CCs and the reduction expression of\nthese genes. The observed decrease in the expression of these genes in the\nnon-fertilized group could indicate a disruption in the communication between the\noocyte and the CCs. Because in addition to supporting the CCs from the oocyte, the\noocyte also affects the proliferation and activity of these cells by secreting a\nnumber of factors such as GDF9/BMP15 ( Su  et\nal. , 2008 ). Therefore, one possibility could be a decrease\nin the signaling of the oocyte itself. Epigenetic changes (such as Histone\nmodifications and DNA methylation) cannot be ignored either. These changes, which\ncan be the result of intrinsic (endogenous) and extrinsic (environmental) factors,\ncan affect the expression of the genes ( Nalvarte\n et al. , 2018 ). Considering the limitations of direct\nexamination of the human oocyte, designing studies with the aim of controlling\nclinical variables and functional assays of these genes in CCs can help to identify\nthe responsible factors in this field”.\nThese findings show the way for novel biomarkers and therapeutic strategies to\nimprove IVF outcomes. More functional studies are needed to support the hypothesis\nof this study and explore the clinical applications of these markers in ART.","source_license":"public-domain-us","license_restricted":false}