{"paper_id":"f0a209a6-e9e8-4efa-b75f-485a941165fc","body_text":"In vitro  maturation (IVM) of human oocytes is a valuable technique in\nassisted reproductive technology (ART). During the IVM procedure, immature oocytes are\nretrieved from small antral follicles and then meiosis progression occurs in the laboratory\n( 1 ).\nIVM could be an appropriate alternative in various conditions such as patients who have\npoor ovarian response to gonadotropin stimulation, high numbers of antral follicles,\npolycystic ovarian syndrome, egg factor problems with only germinal vesicle (GV) oocytes in\ntheir stimulation cycles, and those who suffer from cancer who intend to cryopreserve their\noocytes prior to the onset of cancer treatment ( 2 ). However, the developmental potential of\noocytes reduces after IVM compared to  in vivo  matured oocytes ( 2 ,  3 ).\nThe IVM process differs from natural ovulation, in which an oocyte resumes meiosis after\nthe luteinizing hormone (LH) surge. Therefore, IVM changes the usual timeline of cytoplasmic\nand nuclear maturation processes ( 2 ). Many events occur during oocyte maturation, such as\ncytoplasmic reorganization, cytoskeletal dynamics, and meiotic resumption, which are\nessential for regular fertilization and embryonic development ( 1 ). The preovulatory LH surge\nby the activation of a signaling cascade leads to meiosis resumption in the oocyte\n in vivo  ( 4 ).\nOocyte meiotic progression involves protein phosphorylation pathways that are regulated via\ncyclindependent kinases (CDKs) ( 5 ). Maturation-promoting factor (MPF) consists of two\nsubunits, CDK1 and cyclin B, and is a key factor in meiotic resumption ( 6 ). MPF activity\ndepends on the availability of cyclin B and phosphorylation status of CDK1 ( 7 ).\nMitogen-activated protein kinase (MAPK) signalling is involved in the oocyte maturation\nprocess ( 5 ). In human oocytes, MAPK is inactive in the GV stage oocytes, reaches its highest\nactivity in the metaphase II (MII) stage, and has reduced activity after pronucleus\nformation ( 8 ). MAPK signalling regulates MPF activation, and the MAPK inhibitors block\ngerminal vesicle breakdown (GVBD) in oocytes ( 6 ). High cyclic adenosine monophosphate (cAMP)\nin oocytes promote meiotic arrest until ovulation ( 7 ). In the somatic follicular cells, cAMP\nsynthesis is catalysed from ATP by the adenylyl cyclase enzyme and is transferred into the\noocyte via gap junctions ( 5 ). During oocyte meiotic arrest, high levels of protein kinase A\n(PKA), a cAMP cell cycle mediator, inhibit the CDK1 subunit of MPF ( 5 ,  7 ). In the  in\nvivo  study, a preovulatory LH surge starts the meiosis resumption by destructing\noocyte-somatic cell communication, preventing cAMP transfer, and it also activates the MAPK\ncascade in cumulus cells ( 5 ). The MAPK signalling cascade, which is known as extracellular\nsignal-regulated kinases (ERK1/2) elevates phosphodiesterase, a cAMP degrading-endzyme\nactivity in the oocyte, and thereby decreases cAMP in the oocyte. cAMP-degrading in the\noocyte reduces PKA activity ( 7 ). A decrease in PKA activity leads to dephosphorylation of\nthe inhibitory sites of CDK1 and results in MPF activation. Activated MPF phosphorylates\nhistones, lamins, and other cellular components. Subsequently, meiosis is resumed ( 9 ).\nGlutathione (γ-glutamyl-cysteinyl glycine, GSH)\nis an intracellular free thiol, which is an essential nonenzymatic antioxidant within cells. GSH levels in the\noocyte are an excellent cytoplasmic maturation marker\nafter IVM ( 10 ). A critical role of GSH, as an antioxidant,\nis defending the oocyte against oxidative injuries through\ndecreasing reactive oxygen species (ROS) production in\nmitochondrial metabolism ( 11 ). The oxidative damage\nstatus in oocytes is one of the essential markers to assess\nthe health of the oocyte. ROS damage in the oocyte can\nlead to unexpected apoptosis and subsequent arrest in\nembryonic development ( 12 ).\nReduction in anti-apoptotic factors, such as BCL-2,\nleads to an elevation in pro-apoptotic factors, including\nBAX. These apoptotic factors cause oocyte apoptosis\n( 13 ). The BAX/BCL-2 ratio regulates a cascade of\nmolecular events that determine the cell’s fate (survival or\napoptosis). Increased BAX/BCL-2 alters mitochondrial\nmembrane polarization and results in cytochrome C\ninflux from mitochondria into the cytosol, which involves\ninactivation of the initiator (8 and 9) and effector ( 3 ,  6 ,\nand 7) caspases in oocytes ( 14 ). The BCL-2 protein kinase\nsites are phosphorylated during the G2 to M transition of\nthe cell cycle. Phosphorylation of Thr-56, Thr-74, and\nSer-87 BCL-2 residues inhibit proteasome function and\nprevent apoptosis. It has been suggested that MAPK and\nMPF have an essential role in this process ( 15 ).\nCalcium ionophore (CI) is a fat-soluble molecule that increases cytosolic calcium\n(Ca 2+ ) by transferring Ca 2+  from the plasma membrane to the\ncytoplasm ( 16 ). Ca 2+  signaling is a key factor in the physiology of oocytes from\noogenesis to maturation and fertilization. The passage through the meiosis phase to another\nphase is controlled by cell checkpoints, which act in many species by increasing\nintracellular Ca 2+  levels ( 17 ). During fertilization, sperm-induced elevation in\nintracellular Ca 2+  is necessary for oocyte activation, which is a trigger for\ntransforming an oocyte into an embryo ( 1 ). Also, Ca 2+  changes the activity of\nspecific transcription factors in the nucleus, and these factors affect chromatin structure\nand, as a result, gene expression ( 18 ).\nPrevious studies have shown the relationship between Ca 2+  and GVBD. Increasing\nCa 2+  during GVBD can indicate a correlation between intracellular\nCa 2+  and oocyte maturation in different species of mammals ( 17 ). Furthermore,\nit is reported that the duration of Ca 2+  oscillation increases during oocyte\nmaturation. Oocytes with increases levels of cytosolic Ca 2+  have higher\nspontaneous parthenogenetic activation ( 19 ).\nIVM conditions may influence the oocyte’s developmental\ncompetence. In general, there is no accepted procedure in\ninfertility clinics for the IVM of oocytes. On the other hand,\nIVM, as a clinical approach, should be optimized for the\nfuture ( 2 ). The current study was carried out to clarify the\nrole of CI on IVM of human oocytes.\n\nEthics approval for the current randomized clinical\ntrial study was given by the Ethics Committee at Shahid\nBeheshti Medical University, Tehran, Iran (IR.SBMU.\nMSP.REC.1396.416). Participants gave verbal and\nwritten consent for study participation. All procedures\nin this research were in accordance with the ethical\nguidelines of responsible institutional and national\ncommittees that involve human experimentation\n(IRCT20140707018381N4).\nThe oocytes were donated for the current study by the\npatients of the Genetics and In Vitro Assisted Reproductive\n(GIVAR) Center at Erfan and Taleghani Hospitals (Tehran,\nIran) between October, 2017-November, 2018.\nA total of 552 GV oocytes from 216 intracytoplasmic\nsperm injection (ICSI) procedures were included in the\ncurrent study. These oocytes were not suitable for the ICSI\nprocedure. All women participants were ≤40 years of age\n(mean: 32.13 ± 4.96 years). Cycles diagnosed as male factor infertility (n=135), tubal factor infertility (n=70),\nuterine factor (n=4), and unexplained infertility (n=7)\nwere included in the current study. Women who suffered\nfrom polycystic ovarian syndrome, endometriosis, and\ngenetic disorders were excluded from this study.\nOvarian stimulation was carried out using the long\nprotocol. Briefly, gonadotropin-releasing hormone\n(GnRH) agonist (Superfact, Aventis Pharma, Germany)\nwas adminstered on day 21 of the menstrual cycle.\nrFSH (Gonal-F, Merck Serono, Germany) was injected\nsubcutaneously each day (150–300 IU/day) after the third\nday of menstrual bleeding for a duration of five days.\nFor triggering ovulation, intramuscular administration\nof 10000 IU units of human chorionic gonadotropin (hCG)\n(Ovitrelle, Merck Serono Europe; Pregnyl, Organon) was\nperformed when one of the follicles reached >18 mm in\nsize as viewed by ultrasound. Transvaginal oocyte pickup via ultrasound guidance was carried out 36–38 hours\nfollowing the hCG injection.\nAfter oocyte retrieval, the oocytes were denuded by\nbrief exposure to hyaluronidase (LifeGlobal) and frequent\npipetting. Then, oocytes were evaluated under an inverted\nmicroscope for nuclear maturation assessment: i. GV\nstage showed a germinal vesicle in the cytoplasm, ii.\nmeiosis I (MI) stage did not show any germinal vesicle in\nthe ooplasm and first polar body (PB) in the perivitelline\nspace, and iii. MII stage showed the presence of the first\nPB in the perivitelline space.\nA total of 552 GV stage oocytes were obtained from women who had an adequate number of\nMII oocytes after oocyte retrieval (>80%). Dimethyl sulphoxide (DMSO) was used to dissolve\nthe CI A23187 (Sigma Aldrich; St. Louis, MO, USA) according to the manufacturer’s\nprotocol. Just before IVM, individual oocytes were transferred to 50 μL droplets that\ncontained 10 µM CI of a stock solution diluted in culture medium (Global R, Life Global)\nfor 15 minutes based on an artificial oocyte activation protocol ( 20 ). Then, the oocytes\nwere washed in two, 50 μL droplets of culture medium. In the control group, GV oocytes\nwere not exposed to CI. Oocytes from the treated and control groups were transferred to 50\nμL droplets of culture medium (Global R, LifeGlobal) under mineral oil (LifeGlobal) and\nincubated in 6% CO 2  air atmosphere at 37˚C. After 24-28 hours, oocyte\nmaturation was assessed. Oocytes with the first PB (MII stage) were used for this study.\nMAPK3, CDK1, CCNB1, cyclin D1 (CCND1), BCL2, BAX, Caspase-3 , and \nβ-actin  gene expressions were assessed using real-time reverse transcription\npolymerase chain reaction (RT-PCR) in the IVM oocytes at the MII stage. Reverse\ntranscriptions of samples were carried out as explained previously ( 21 ). In summary, a\ntotal of 78 oocytes (39 oocytes in each group) were washed in phosphate-buffered saline\n(PBS, Invitrogen Corp.) + 1% polyvinyl alcohol (PVA), and pooled into six Eppendorf tubes\n(13 oocytes in each microtube) with 1.5 µL of lysis buffer to isolate the RNA from the\noocytes. The Eppendorf tubes were stored at -80˚C. Next, we added 5 μL nucleasefree water\nand 3 µL random hexamer to the Eppendorf tubes and placed them in a Bio-Rad\nthermocycler.\nComplementary DNA (cDNA) synthesis was performed\nwith 10 mmol/L dNTP, 200 U RT enzyme, 10 U RNase\ninhibitor, and 5× RT buffer in a total reaction volume of\n21 µL for 10 minutes at 25˚C, 15 minutes at 37˚C, 45\nminutes at 42˚C, and 10 minutes at 72˚C followed by\novernight incubation at 4˚C.\nThe investigated genes ( MAPK3, CDK1, CCNB1, CCND1, BAX, BCL-2 , and\n Caspase-3 ) and the internal control ( β-actin ) were\namplified as follows. We added 1 μg cDNA, 3 μL nuclease-free water, 5 μL Master Mix\n(Amplicon, Denmark), and 10 nmol specific forward and reverse primers ( Table 1 ) to the PCR\nEppendorf tubes and processed them for 5 minutes at 94˚C, 30 seconds at 94˚C, 30 seconds\nat 60˚C, and 45 seconds at 72˚C and 40 extension cycles. The amount of RNA was visualized\nafter loading the samples. The amplification products were visualized on agarose gel\nelectrophoresis under short UV.\nIn order to quantify  MAPK3, CDK1, CCNB1, CCND1, BAX, BCL-2 , and\n Caspase-3  gene expressions, realtime RT-PCR was performed in 13 μL of\nreaction buffer that contained synthesized cDNA, forward and reverse specific primers (1\nmmol/L for each gene), and DNA Master SYBR Green I mix. The gene amplification program\nincluded 2 minutes at 95˚C, 5 seconds at 95˚C, 30 seconds at 60˚C, 10 seconds at 72˚C, and\n40 extension cycles. The experiment for each sample was carried out in three replicates.\nRelative Expression Software Tool (REST, version 2009) was applied to calculate the\nexpression of each of the investigated genes.\nThe IVM-MII oocytes were collected from each group\nto determine their intracellular GSH (20 oocytes in each\ngroup) and ROS (23 oocytes in each group) levels by\npreviously described methods ( 22 ). Briefly, the GSH and\nROS content of the oocytes were detected using Cell\nTracker Blue (CMF2HC; 4-chloromethyl-6,  8 -difluoro7-hydroxycoumarin; Invitrogen), and H2DCFDA\n(2’,7’-dichlorodihydrofluorescein diacetate; Invitrogen)\nfluorescent dyes. Oocytes were transferred to a 30 µL PBS\ndroplet that consisted of 10 µM Cell Tracker Blue, 10 µM\nH2DCFDA, and 1 mg/mL PVA in the dark at 37˚C for\n45 minutes followed by three washes in PBS + 1% PVA.\nThe samples’ intracellular GSH and ROS concentrations\nwere observed as blue and green fluorescence under\na fluorescence microscope (Labomed Lx 400; Labo America). The GSH and ROS contents were detected\nby 370 nm and 460 nm ultraviolet filters, respectively.\nFluorescence images of oocytes were recorded as TIFF\nformat graphics files and evaluated by ImageJ software\n(NIH, Bethesda, MD, USA), version 1.41.\nImmunocytologic staining of the spindle structure\nand chromosome arrangement in the IVM-MII oocytes\n(10 oocytes in each group) was carried out using a\npreviously described method ( 23 ). Briefly, MII stage\noocytes were treated for about 30 seconds by Tyrode’s\nacidic solution (pH=2.5) at room temperature to remove\nthe zona pellucida. Next, 4% paraformaldehyde in PBS\n(pH=7.4) was applied for 30 minutes at 4˚C to fix the\noocytes. Following three washes in PBS + 0.02% Tween\n20, oocyte membrane permeabilization was induced by\n0.25% Triton X-100 for 60 minutes at room temperature.\nThen, the oocytes were exposed to 4N HCl for 30 minutes\nat room temperature, followed by 0.1 M Tris-HCl for\nneutralization. The oocytes were transferred to a blocking\nsolution that contained 2% bovine serum albumin (BSA,\nSigma Aldrich; St. Louis, MO, USA) + 0.02% Tween 20 in\nPBS for 60 minutes at room temperature. Subsequently, the\noocytes were placed in mouse monoclonal anti-β-tubulin\nantibody (1/100 dilution, Sigma Aldrich; St. Louis, MO,\nUSA) in the blocking solution overnight in a humidified\nchamber at 4˚C. After several washes, meiotic spindle\nstaining was carried out following 30 minutes incubation\nat room temperature of the oocytes with conjugated goat\nanti-mouse (IgG) fluorescein isothiocyanate (FITC) at\n1/100 dilution (Sigma Aldrich; St. Louis, MO, USA) in\nthe dark. After several washes, the oocytes were placed\nin 10 mg of propidium iodide (PI; Sigma Aldrich; St.\nLouis, MO, USA) for chromatin staining for 20 minutes.\nThe samples were individually mounted on microscope\nslides and a coverslip and etched rings were applied to\nprevent the samples from being ruptured by the coverslip.\nThe slide was observed under a fluorescent microscope\n(Labomed Lx 400; Labo America) and the chromosomes,\nand spindle configurations were defined as normal\n(aligned chromosomes at the metaphase plate with barrelshaped spindles) or abnormal (misaligned chromosomes\nin the metaphase plate with non-barrel-shaped spindles).\nThe t test and chi-square test using SPSS (SPSS,\nChicago, IL, USA) software (version 16.0) was applied\nto analyse differences between the two groups. Mean ±\nstandard deviation (SD) and percentages were used to the\nexpress data. A p-value <0.05 was considered statistically\nsignificant.\nPrimer sequences used in real-time RT-PCR\nRT-PCR; Reverse transcription polymerase chain reaction, GC; Guanine-cytosine, and Tm; Melting temperature\n\nOverall, 216 couples participated in this study ( Table\n2 ). Out of 552 GV oocytes, 390 (70.65%) reached the\nMII stage and 96 (17.39%) arrested in the MI stage.\nThere were 50 (9.05%) oocytes that arrested in the\nGV stage and 16 (2.89%) oocytes were degenerated.\nAlthough the MII oocyte rate was higher in CI‐\ntreated oocytes (73.53%) compared to the control\ngroup (67.43%), this difference was not statistically\nsignificant (P=0.13). The GV arrested oocyte rate (CI‐\ntreated oocytes: 8.24% and control: 9.96%, P=0.06),\noocyte degeneration rates (CI‐treated oocytes: 2.40%\nand control: 3.44%, P=0.46), and arrested MI oocyte\nrates (CI‐treated oocytes: 15.80% and control: 19.15%,\nP=0.87) after IVM was not statistically significant\nbetween CI‐treated oocytes and the control group\n( Table 3 ). This finding suggested that CI treatment\nsignificantly affected the first PB extrusion in human\noocytes.\nBaseline characteristics of the study population\nThe t test was applied for statistical analysis. There was no statistically significant difference in any parameter between the CI-treated and control groups.\nCI; Calcium ionophore, Ns; Not significant, GV; Germinal vesicle, MI; Metaphase I, MII; Metaphase II, and SD; Standard deviation.\nMeiotic maturation of human oocytes after 24-28 hours of culture\nThe t test was applied for statistical analysis. There was no significant difference in the meiotic maturation rate between the two groups. CI; Calcium\nionophore, GV; Germinal vesicle, MI; Metaphase I, MII; Metaphase II, and SD; Standard deviation.\nIn the present study, the transcript profiles of several oocyte maturation-related\ngenes ( MAPK3, CCNB1, CDK1, and CCND1 ) were evaluated by real-time RTPCR.\nThe results showed that  MAPK3, CCNB1, CDK1 , and  CCND1\n mRNA expression levels compared with the housekeeping gene\n( β-actin ) were up-regulated significantly in CI-treated oocytes\n(P<0.05; Fig.1). These findings led to the hypothesis that exposure of CI to human\noocytes resulted in an apparent up-regulation in  MAPK3, CCNB1, CDK1 , and\n CCND1  mRNA expressions.\nA molecular mechanism that modulates human oocyte apoptosis might be induced by CI\ntreatment. Therefore, we evaluated the  BCL-2, BAX , and  Caspase-3\n relative expression levels by real-time RT-PCR. The results of real-time RT-PCR\ndemonstrated that the expression of anti-apoptotic  BCL-2  was remarkably\nup-regulated after treatment with CI (P=0.001; Fig.1), whereas the expression of\npro-apoptotic  BAX  did not change significantly (P=0.76). Thus, the\n BAX/BCL-2  ratio decreased (13.60%). Also, real-time RT-PCR revealed\nthat the expression level of Caspase-3 mRNA did not change significantly in human oocytes\nafter exposure to CI (P=0.81; Fig.1).\nNuclear maturation and apoptosis-related gene mRNA expressions of human oocytes. The relative\nexpression levels of mitogen-activated protein kinase 3  (MAPK3), CCNB1,\nCDK1 , and cyclin  D1 (CCND1)  were significantly higher and\n BCL-2  was significantly lower in calcium ionophore (CI)-treated\nhuman  in vitro  maturation-meiosis II (IVM-MII) oocytes\n(*P<0.05, **P<0.001).\nThe human oocyte GSH content was evaluated in the CI\ntreatment and control groups. Analyses with ImageJ software\nindicated that CI treatment induced a statistically remarkable\nincrease in oocyte intracellular GSH concentration (P=0.005,\n Fig .2A, B ). A comparison of the intracellular ROS content\nof human oocytes (23 oocytes in each group) revealed\nsignificantly diminished ROS content in CI‐treated oocytes\ncompared with the control group (P=0.04; Fig.2C, D).\nGlutathione (GSH) and reactive oxygen species (ROS) content in human  in vitro\n maturation-meiosis II (IVM-MII) oocytes evaluated by fluorescent staining.\n A.  Oocytes were stained with Cell Tracker Blue to assess the level of\nintracellular GSH and  B.  2-7-dichlorodihydrofluorescein diacetate\n(H2DCFDA) to determine ROS (bar: 100 μm).  C.  GSH and  D.  ROS\ncontent in calcium ionophore (CI)-treated human oocyte and control groups. The data\nwere analysed using the t test. As the graph depicts, CI‐treated oocyte cytoplasm had\nsignificantly higher GSH and lower ROS content (**P<0.01, *P<0.05).\nMeiotic spindle configuration and chromosome alignment in human  in vitro\n maturation-meiosis II (IVM-MII) oocytes evaluated using immunocytochemistry.\nIVM-MII oocytes in calcium ionophore (CI)-treated human oocyte and control groups were\nfixed and stained for β-tubulin (green) and chromosomes (propidium iodide [PI], red),\nand analysed for meiotic spindle configuration and chromosome alignment. Meiotic\nspindles were classified as  A.  Normal with aligned chromosomes at the\nmetaphase plate with barrel-shaped spindles and  B.  Abnormal with\nmisaligned chromosomes in the metaphase plate with non-barrel-shaped spindles (bar: 50\nμm).\nIn order to find out whether the CI treatment could affect\nchromosome and spindle structure in human oocytes after\nIVM, we stained IVM-MII oocytes for β-tubulin to assess\nspindle configuration and PI to detect chromosomes. A total\nof 20 oocytes (10 oocytes in each group) were examined\nfor meiotic spindle structure and chromosome alignment.\nFollowing the evaluation of β-tubulin positive spindles by\nfluorescent microscopy, one abnormal chromosome and\nspindle structure were observed in each group. There was\nno significant difference in normal spindle configuration\nand chromosome alignment rate (normal oocytes/\nexamined oocytes) between the CI‐treated group (90%)\nand control group (90%,  Fig .3 ). This result showed that\nmeiotic spindle bipolarity and chromosome alignment of\nhuman IVM-MII oocytes was not significantly affected\nby CI treatment.\n\nDue to the absence of ovarian niches, human oocyte maturation following IVM is suboptimal.\nSome studies have reported morphological and structural differences after IVM of human\noocytes in comparison with  in vivo  oocytes ( 2 ,  3 ).\nAlthough in previous animal and human studies the influence of Ca 2+  on oocyte\nmaturation has been identified, its central role in human IVM as a mediator of MAPK, MPF,\nand apoptosis signalling cascade has not been proven.\nIn this study, in order to demonstrate the effect of CI\nin oocyte maturation, we used CI before oocyte meiosis\nresumption during 24-28 hours of IVM. The results\nshowed beneficial effects of CI on increasing nuclear\nmaturation and anti-apoptotic gene expressions and\ncytoplasmic maturation.\nThe effects of CI on human artificial oocyte activation\nhave been shown before ( 20 ,  24 ). To our knowledge, this\nstudy is the first to identify the effects of CI on IVM of\nthe human oocyte.\nPromotive effects of CI on the IVM of human oocyte\ncan be through several pathways.\nThe results of the present study demonstrated that CI up-regulates  MAPK, Cyclin\nB , and  CDK1  gene expressions. These findings support the report\nof Liu et al., which stated that the cortical distribution of the calcium-sensing receptor\nregulated by gonadotropins in porcine oocytes improved oocyte IVM through the MAPK-dependent\nsignalling cascade ( 25 ). The current study demonstrated that CI up-regulated\n MAPK , which then improved human oocyte maturation. This process might\noccur via the MAPK-related pathway. Protein kinase C (PKC) is the Ca 2+  target\ndownstream molecule ( 26 ). Cell cycle regulation by PKC cascades is involved in the\nactivation of MAPK and MPF. CDK1 and cyclin B1 are PKC substrates. PKC inhibitor decreases\nMPF activity in the oocyte and PKC regulates MAPK signalling ( 6 ). It has been shown that\nMAPK is activated in cumulus cells by PKC activators ( 4 ). The present study findings\ncontradict a previous observation by Ito et al. in which porcine oocytes were\nparthenogenetically activated by CI. They reported that  MAPK  activity\ndecreased after pronucleus formation ( 27 ). It should be considered while we evaluated the\nMAPK levels in MII stage oocytes; the latter study reported the MAPK levels decreased after\nfertilization. Zhang et al. reported that MAPK levels increased during oocyte maturation\nuntil the MII stage, but the levels decreased after fertilization ( 28 ).\nThe  CCNB1  expression level in the oocyte is a marker for cytoplasmic\nmaturation ( 25 ). Liang et al. showed that stored mRNA of  CCNB1  in the\ncytoplasm of the oocyte could influence MAPK and the MPF pathway ( 29 ). These results\nindicated that CI could increase cytoplasmic maturation in IVM-MII oocytes by enhancement of\nMAPK activity. The finding of the present study supported their views.\nThe relative expression level of  CCND1 , a cell cycle regulator gene, is a\nproliferative marker. Increasing expression of  CCND 1 has been reported\nduring meiosis progression in mouse oocytes ( 31 ). In mammalian oocytes,\n CCND1  was expressed both in the oocyte and granulosa cells during\nfollicular growth ( 30 ) and has a crucial role in follicles and granulosa cell proliferation,\nsurvival, and early embryonic transition ( 30 ). Gatius et al. ( 32 ) showed that MAPK\nsignalling promotes cell proliferation by activation of CCND1. Up-regulation of\n MAPK  in the present study might be the result of the activation of\n CCND1 .\nIn general, oxidative stress induced by overloading of Ca 2+  is an apoptotic\nsignal that can increase BAX/BCL-2 and increase apoptosis in the oocyte ( 14 ). The findings\nof the present study show that CI could upregulate antiapoptotic BCL-2. It does not\nup-regulate pro-apoptotic  BAX  and effector  Caspase-3  gene\nexpression in IVM-MII human oocytes. In agreement with our findings, several studies have\nshown that decreased levels of MAPK and MPF in oocytes also lead to increased BCL-2 protein\ndegradation and activation of the apoptotic pathway in mice ( 15 ), rat ( 13 ), and canine ( 33 )\noocytes. Also, it has been reported that inhibition of CDK1 activity by reducing the MPF\nheterodimer prevents meiotic cell cycle progression and induces apoptosis ( 13 ,  15 ).\nDecreased CDK1 phosphorylation as well as increased degradation of cyclin B1 lead to MPF\ninstability and result in fas ligand-induced apoptosis in oocytes ( 14 ). Thus, the increased\nexpression levels of MAPK and MPF genes in our study might be responsible for an increased\nsurvivalpromoting signalling in IVM-MII oocytes. Tripathi and Chaube added different\nconcentrations of CI (0.5,  1 ,  2 ,  3 , 4 μM) to rat MII oocyte culture medium for 3 hours and\nshowed that high concentrations (3 and 4 μM) of CI led to increased ROS production and\napoptosis in oocytes ( 34 ). Moreover, Chaube et al. reported that the addition of CI (1.6 μM)\nto the culture medium of rat MII stage oocytes for 3 hours induced hydrogen peroxide\nformation and apoptosis in these oocytes ( 35 ). In both of these studies, the oocyte\ndevelopmental stage, CI concentrations, and exposure duration were not similar to our\nwork.\nIn the current study, we observed higher GSH and lowered ROS content in CI‐treated oocyte\ncytoplasm. Intracellular GSH concentration is an oocyte cytoplasmic maturation marker.\nIncreasing GSH synthesis in oocytes starts from meiosis resumption in the GVBD stage and\nreaches its highest concentration at the MII stage ( 19 ). GSH regulates many processes in the\noocyte, including modulating the intracellular redox balance, defending oocytes from ROS\ndamage, influencing sperm nuclear decondensation, and male pronucleus formation, DNA\nsynthesis, and amino acid and protein transport ( 11 ). BCL-2 prevents the intrinsic apoptotic\npathway in mitochondria. Besides its anti-apoptotic function, BCL2 has an antioxidant-like\nproperty that has been related to the regulation of the intracellular concentration of\n GSH . Previous studies have reported that increased BCL2 expression causes\nan increase in intracellular GSH content by enhanced GSH synthesis and reduced cellular GSH\nefflux ( 36 ). In our research, overexpression of BCL2 induced by CI treatment might be the\nreason for the increase in GSH content and, subsequently, reduced ROS status in oocytes\nafter IVM.\nIn the present study, we showed that CI did not disturb\nthe meiotic spindle structure and chromosome alignment.\nAbnormal spindle assembly and chromosome segregation\ncause aneuploidy in oocytes, which leads to the embryo\ndevelopment arrest and spontaneous abortion ( 37 ). Our\nfinding might be due to the MAPK and MPF pathway that\nhas a significant role in the remodeling of actin filaments\nand microtubule organization ( 4 ,  25 ). In agreement with\nour findings, Luo et al. ( 38 ) showed that inhibition of the\nactivation of MAPK during porcine oocyte maturation\nresulted in prevention spindle microtubules assembly and\nfirst PB extrusion. Choi et al. ( 39 ) reported that increased\noxidative stress and a decreased intracellular concentration\nof GSH led to the spindle structure defect in IVM mouse\noocytes. Nevertheless, the normal spindle morphology\nwas reported in IVM-MII macaque oocytes, which GSH\nethyl ester was added to the IVM culture medium ( 10 ).\nConsidering the protective effect of GSH on the meiotic\nspindle structure and cytoplasmic microtubules, CI might\nprevent the meiotic spindle disruption and chromosome\nmisalignment in IVM-MII human oocytes through\nincreased levels of the intracellular GSH level.\nWe did not find any effect of CI on the first PB extrusion\nin human oocytes. In contrast to our finding, Makki et al.\n( 40 ) reported that addition of 15 μg/ml selenium, 10 μg/\nml calcium, and 5 μg/ml CI to the IVM medium for 24\nhours improved IVM and fertilization of oocytes, and the embryo cleavage rate. The differences between the\nfindings of this study and our work might be due to the\nvarious times of exposure and compounds which were\nadded to the culture medium.\nIn the current study, we showed that CI could improve oocyte cytoplasmic and nuclear\nmaturation during IVM of human oocytes, but it could not alter the extrusion of the first PB\nof the oocytes. It should be mentioned that we evaluated the expression of genes related to\nmaturation in oocytes at the RNA level, whereas the first PB extrusion was regulated when\nthese RNAs were translated into protein. Hence, it seemed that the prolonged  in\nvitro  culture of the oocyte might lead to the conversion of maturation related\nRNA genes to proteins and improve the first PB extrusion of the oocytes. Therefore, further\nclarification of the impact of the CI on maturation related proteins is required.\n\nThe finding of the current study seems to supports the\nbeneficial effect of CI on the developmental competence\nof human oocytes, including nuclear and cytoplasmic\nmaturation, and apoptosis of human oocytes. We suggest\nthat the CI may optimize the human IVM procedure in the\nART clinic.","source_license":"CC-BY-4.0","license_restricted":false}