Mouse Germinal Vesicle Oocytes Research Articles

P-501 Superovulation alters telomere length and telomerase expression in mouse oocytes, possibly by changing estradiol and progesterone levels

Abstract Study question How does the single and repeated superovulation affect the telomerase components expression and telomere length in mouse GV and MII oocytes? Summary answer Superovulation significantly altered the expression of the telomerase components Tert and Terc genes, TERT protein levels, and telomere length in the GV and MII oocytes. What is known already Assisted reproductive technologies (ARTs) are commonly used to treat infertility problems. One of the basic ART steps is controlled ovarian stimulation, also known as superovulation. Superovulation can lead to several adverse effects, such as increased oxidative stress, decreased oocyte and embryo quality, and mitochondrial dysfunction. These effects of superovulation largely overlap with those that causing telomere shortening. Study design, size, duration The effect of superovulation on the telomerase expression and telomere length of GV and MII oocytes is not fully addressed yet. In this study, we investigated the potential effects of single and repeated superovulation on telomerase expression and telomere length in mouse oocytes at germinal vesicle (GV) and metaphase II (MII) stages. In addition, we measured the estradiol and progesterone as well as oxidant and antioxidant capacity in serum samples of the oocytes-collected mice. Participants/materials, setting, methods Four groups of female Balb/C mice were formed: Control (C; n = 12), superovulated once (1S; n = 12), superovulated three times (3S; n = 12), and superovulated five times (5S; n = 12) at one-week intervals. The Tert and Terc gene expression, TERT protein levels, and telomere length in the mouse oocytes were assessed by RT-qPCR, immunofluorescence, and q-PCR, respectively. The hormone levels were analyzed by ELISA, and oxidant-antioxidant capacities were measured. All the ovarian follicles were counted in the H&E stained-sections. Main results and the role of chance We found that superovulation (1S, 3S, and 5S) significantly altered expression of the telomerase components Tert and Terc genes, TERT protein levels, and telomere length in the GV and MII oocytes (P<0.05). While estradiol levels in GV oocyte-collected and progesterone levels of GV and MII oocyte-collected mice were higher in the 1S, 3S, and 5S groups compared with the C group (P<0.01), the estradiol levels in MII oocyte-collected mice decreased progressively from C to 5S groups (P<0.01). However, no significant changes were found in the total oxidant status (TOS), antioxidant status (TAS), and oxidative stress index (OSI) between the groups. Also, primordial and atretic follicle numbers in the ovaries of 3S and 5S groups were significantly decreased compared to the control and 1S groups (P<0.05). Limitations, reasons for caution The limitation of this study is that we could not evaluate telomerase activity and oxidative stress levels in the mouse oocytes. Wider implications of the findings All these results suggest that altered telomerase expression and telomere length in GV and MII oocytes, possibly due to changed estradiol and progesterone levels, may be associated with adverse effects of superovulation on oocyte maturation and even preimplantation embryo development. Trial registration number not applicaple

The role of SRPK1-mediated phosphorylation of SR proteins in the chromatin configuration transition of mouse germinal vesicle oocytes.

Meiotic resumption in mammalian oocytes involves nucleus and organelle structural changes, notably chromatin configuration transitioning from non-surrounding nucleolus (NSN) to surrounding nucleolus (SN) in germinal vesicle (GV) oocytes. Our study found that nuclear speckles, a subnuclear structure mainly composed of serine-arginine (SR) proteins, changed from a diffuse spotted distribution in mouse NSN oocytes to an aggregation pattern in SN oocytes. We further discovered that SRPK1, an enzyme phosphorylating SR proteins, co-localized with NS at SN stage and NSN oocytes failed to convert into SN oocytes after inhibiting the activity of SRPK1. Furthermore, the typical structure of chromatin ring around the nucleolus in SN oocytes collapsed after inhibitor treatment. To explore the underlying mechanism, phosphorylated SR proteins were confirmed to be associated with chromatin by salt extraction experiment, and in situ DNase I assay showed that the accessibility of chromatin enhanced in SN oocytes with SRPK1 inhibited, accompanied by decreased repressive modification on histone and abnormal recurrence of transcriptional signal. In conclusion, our results indicated that SRPK1-regulated phosphorylation on SR proteins was involved in the NSN to SN transition and played an important role in maintaining the condensation nucleus of SN oocytes via interacting with chromatin.

Comparing the effects of vitrification, before and after mouse oocyte in vitro maturation on developmental competence, changes in epigenetic regulators and stress oxidative response

Since the developmental stage of oocyte is a challenging issue in the success of vitrification, this study investigated the effects of vitrification, before and after in vitro maturation, on the survival and maturation rates, developmental competence and the expression levels of genes involved in apoptosis, oxidative stress and epigenetic modifications. Mouse germinal vesicle (GV) oocytes were divided into four groups: fresh in vitro matured oocytes without vitrification (fIVM), in vitro matured oocytes after vitrification (vIVM), in vitro matured oocytes before vitrification (IVMv). In addition, in vivo matured oocytes (MII) were used as control. After oocytes collection, maturation and survival rates as well as the intracellular reactive oxygen species (ROS) level were evaluated. Also, the expression level of various genes was analyzed by qRT-PCR. In addition, following artificial activation (parthenogenesis), the developmental competence of oocytes to the blastocyst stage was evaluated. A significant decrease in maturation rate and survival of vIVM oocytes was observed compared to fIVM and IVMv oocytes. Intracellular ROS levels were significantly increased in both vitrified groups compared to the fIVM group, and no significant difference between vitrified groups. Pro-apoptotic genes; BAX and Bcl2 as well as genes related to oxidative stress response Hsp1a, Hsp1b and SOD1were significantly increased in the vIVM group compared to the IVMv group. Interestingly, epigenetic regulators genes DNMT1, DNMT3a and DNMT3b were highly expressed in IVMv oocytes along with a decrease in the artificial activation rate compared to the vIVM oocytes. Our results indicated that despite observing more negative effects of vitrification before IVM on the survival rate and maturation as well as apoptosis status, less epigenetic changes in vIVM oocytes can make this process a better option in the treatment of infertility than IVM of oocytes followed by vitrification, a hypothesis that needs to be investigation in human oocytes.

Chromatin Configuration in Diplotene Mouse and Human Oocytes during the Period of Transcriptional Activity Extinction.

In the oocyte nucleus, called the germinal vesicle (GV) at the prolonged diplotene stage of the meiotic prophase, chromatin undergoes a global rearrangement, which is often accompanied by the cessation of its transcriptional activity. In many mammals, including mice and humans, chromatin condenses around a special nuclear organelle called the atypical nucleolus or formerly nucleolus-like body. Chromatin configuration is an important indicator of the quality of GV oocytes and largely predicts their ability to resume meiosis and successful embryonic development. In mice, GV oocytes are traditionally divided into the NSN (non-surrounded nucleolus) and SN (surrounded nucleolus) based on the specific chromatin configuration. The NSN-SN transition is a key event in mouse oogenesis and the main prerequisite for the normal development of the embryo. As for humans, there is no single nomenclature for the chromatin configuration at the GV stage. This often leads to discrepancies and misunderstandings, the overcoming of which should expand the scope of the application of mouse oocytes as a model for developing new methods for assessing and improving the quality of human oocytes. As a first approximation and with a certain proviso, the mouse NSN/SN classification can be used for the primary characterization of human GV oocytes. The task of this review is to analyze and discuss the existing classifications of chromatin configuration in mouse and human GV oocytes with an emphasis on transcriptional activity extinction at the end of oocyte growth.

Expression pattern and subcellular localization of p62/SQSTM 1 during mouse oocyte maturation

Objective: Autophagy is a survival mechanism, and it is initiated by several factors such as oxidative stress. Cells give autophagy response against oxidative stress through Nrf2-Keap1-p62 pathway. p62 or Sequestosome 1 (SQSTM 1) which takes place during autophagy, and it is showed that p62 plays critical role during primordial follicle formation. During oocyte maturation, Germinal Vesicle (GV) oocytes resume meiosis with the beginning of puberty and progress through Metaphase I and Metaphase II stages. Metaphase II (MII) oocytes are ready to be fertilized and until fertilization they are arrested at metaphase II stages. It is known that oxidative stress is increasing during oocyte maturation. However, it is not shown that spatial and temporal expression of p62 throughout oocyte maturation. Thus, the aim of the study was to reveal expression pattern and the subcellular localization of the p62 protein in mouse GV, MI and MII oocytes. Methods: GV oocytes were received from female Balb/C mice at 4 weeks aged and GV oocytes were cultured for 8h to develop into MI oocytes and for 14h to mature into MII oocytes. Then, expression of p62 protein was observed in oocytes at GV, MI and MII maturation stages by using immunofluorescence method. Results: Our results showed that there is a significant increasing p62 expression when GV oocytes are compared to MI and MII oocytes, but there is no difference between MI and MII oocytes. Moreover, we revealed that p62 is localized in cytoplasm of GV oocytes, while it is localized around chromosomes in MI and MII oocytes. Conclusion: In conclusion, our results indicate that further study is mandatory to understand the participation of p62 during meiosis, and the impact of p62 during oocyte maturation.

Comprehensive analysis of nonsurrounded nucleolus and surrounded nucleolus oocytes on chromatin accessibility using ATAC-seq.

Mouse germinal vesicle (GV) oocytes are divided into surrounded nucleolus (SN) and nonsurrounded nucleolus (NSN) oocytes based on chromatin morphology. NSN oocytes spontaneously transform into SN oocytes after accumulating enough maternal transcripts. SN oocytes show transcriptional silencing. When oocyte maturation is abnormal or takes place in vitro, NSNoocytes do not go through SN stage before proceeding to MII. Nontransitive oocytes show developmental retardation, a low fertilization rate, and arrest at the two-cell embryo stage in mice. Here, chromatin-binding ribonucleic acid polymerase II (RNAP II) activity, newly synthesized RNA, and chromatin accessibility in GV oocytes were examined. In SN oocytes, RNAP II did not bind to DNA, neo-RNA was not generated in nuclei, and the phosphorylation state of RNAP II did not affect the chromatin-binding activity. The number of accessible genes in SN oocytes was remarkably lower than that in NSN oocytes. The accessibility of different functional genes was also different between the two types of oocytes. Thus, low chromatin accessibility leads to transcriptional silencing in SN oocytes.

Granulosa Cell Conditioned Medium Enhances The Rate of Mouse Oocyte In Vitro Maturation and Embryo Formation.

In vitro maturation (IVM) and cryopreservation of oocytes are two important parts of assisted reproductive technology (ART), but their efficacy is low. This study aimed to improve the quality of in vitro vitrified-warmed maturated oocytes using granulosa cell conditioned medium (GCCM). In the experimental study, fresh/non-vitrified and vitrified-warmed mouse germinal vesicle (GV) oocytes (as F and V) were In vitro maturated using basal medium (BM) and also BM supplemented with 50% GCCM as treated groups (GM), and categorized as FBM, FGM, VBM and VGM groups, respectively. The rate of successful IVM (MII oocyte formation), mitochondrial membrane potential and the viability of MII oocytes were determined using inverted microscopy, JC-1 and trypan blue staining. Then, the rate of In vitro fertilization (IVF) and subsequent two-cell embryo formation was calculated. Finally, the expression levels of Oct4, Sox2, Cdk-2, Gdf9, Integrin beta1 and Igf2 were analyzed using real-time polymerase chain reaction (PCR) in MII oocytes and two-cell embryos. These analyses showed that GCCM significantly increased the IVM rate, oocyte meiotic resumption and mitochondrial membrane potential (P<0.05). In addition, the rate of IVF and two-cell embryo formation was significantly higher in FGM and VGM compared to FBM and VBM (P<0.05). Interestingly, GCCM significantly affected the expression of the studied genes. Our findings suggest that GCCM might be useful for improving the efficiency of IVM and the subsequent IVF outcomes.

O-289 Effects of Taxol on the developmental potency of human and mouse GV oocytes

Abstract Study question Is it possible to improve the quality of oocytes with delayed maturation or aging? Summary answer Yes, modification of oocyte microtubules at the germinal vesicle (GV) stage inhibits abnormal chromosome separation in in-vitro maturation and enhances early cleavage. What is known already Oocyte aging is characterized by an increase of aneuploidy and a decrease of the developmental potency with maternal ages, which will results in low frequencies of the blastocyte formation and implantation. In addition to the 1st meiotic division, it has become clear that the 2nd meiotic division also significantly contributes to aneuploidy production which is followed by pre-implantation embryo loss. However, no method for overcoming the oocyte aging has been established. Study design, size, duration Human GV oocytes with delayed maturation obtained from consented female patients were used. Mouse GV oocytes collected from 9-15 month old ICR mice were also used. After exposure to Taxol for 1hr, the oocytes were matured in vitro and examined cytogenetically and cytologically at the GV stage, the MII stage and pronuclear stage. Participants/materials, setting, methods RNA transcripts of the GV stage oocytes were compared before and after Taxol exposure with microarray in both species. Chromosome aberrations at the MII stage and blastocyst formation rate were examined in human IVM oocytes. Mouse IVM oocytes were evaluated on 2nd polar body (PB) extrusion and O2 consumption (CRAS3.0, Crino Co Inc.) at the pronuclear stage after parthenogenetic activation with electro-stimulation. Main results and the role of chance After Taxol treatment, premature chromosome separation was significantly reduced from 96% to 7% and the blastocyst formation rate increased from 3% to 16% in human. In mice, normal PB extrusion rate increased from 29% to 92%, and oxygen consumption was higher in some pronuclear oocytes. In human GV oocytes, taxol treatment altered gene expression only in a few factors related to chromosome attachment and segregation. On the other hand, in mouse GV oocytes, gene expression was significantly altered by oocyte aging and Taxol treatment. Limitations, reasons for caution The present results were obtained in human GV oocytes with delayed maturation which were collected after PMS and HCG administrations. It is not clear whether Taxol is effective on the GV oocytes collected before HCG administration. Wider implications of the findings Our results indicate that the combination of biochemical modification and IVM in the GV stage may be able to restore the aging of human oocytes. Trial registration number not applicable

Mettl3 downregulation in germinal vesicle oocytes inhibits mRNA decay and the first polar body extrusion during maturation†.

In oocytes, mRNA decay is essential for maturation and subsequent events, such as maternal-zygotic transition, zygotic genomic activation, and embryo development. Reversible N6-methyladenosine RNA methylation directly regulates transcription, pre-mRNA splicing, mRNA export, mRNA stability, and translation. Here, we identified that downregulation of N6-methyladenosine modification by microinjecting a methyltransferase-like 3 (Mettl3)-specific small interfering RNA into mouse germinal vesicle oocytes led to defects in meiotic spindles and the first polar body extrusion during maturation in vitro. By further quantitative real-time polymerase chain reaction and Poly(A)-tail assay analysis, we found that N6-methyladenosine methylation mainly acts by reducing deadenylation of mRNAs mediated by the carbon catabolite repression 4-negative on TATA less system, thereby causing mRNA accumulation in oocytes. Meanwhile, transcriptome analysis of germinal vesicle oocytes revealed the downregulation of transcripts of several genes encoding ribosomal subunits proteins in the Mettl3 small interfering RNA-treated group, suggesting that N6-methyladenosine modification might affect translation. Together, our results indicate that RNA methylation accelerates mRNA decay, confirming the critical role of RNA clearance in oocyte maturation.

Ribosomal DNA methylation in human and mouse oocytes increases with age.

An age-dependent increase in ribosomal DNA (rDNA) methylation has been observed across a broad spectrum of somatic tissues and the male mammalian germline. Bisulfite pyrosequencing (BPS) was used to determine the methylation levels of the rDNA core promoter and the rDNA upstream control element (UCE) along with two oppositely genomically imprinted control genes (PEG3 and GTL2) in individual human germinal vesicle (GV) oocytes from 90 consenting women undergoing fertility treatment because of male infertility. Apart from a few (4%) oocytes with single imprinting defects (in either PEG3 or GTL2), the analyzed GV oocytes displayed correct imprinting patterns. In 95 GV oocytes from 42 younger women (26-32 years), the mean methylation levels of the rDNA core promoter and UCE were 7.4±4.0% and 9.3±6.1%, respectively. In 79 GV oocytes from 48 older women (33-39 years), methylation levels increased to 9.3±5.3% (P = 0.014) and 11.6±7.4% (P = 0.039), respectively. An age-related increase in oocyte rDNA methylation was also observed in 123 mouse GV oocytes from 29 4-16-months-old animals. Similar to the continuously mitotically dividing male germline, ovarian aging is associated with a gain of rDNA methylation in meiotically arrested oocytes. Oocytes from the same woman can exhibit varying rDNA methylation levels and, by extrapolation, different epigenetic ages.

Effects of melatonin and human follicular fluid supplementation of in vitro maturation medium on mouse vitrified germinal vesicle oocytes: A laboratory study.

Background Vitrification as the most efficient method of cryopreservation, enables successful storage of oocytes for couples who undergo specific procedures including surgery and chemotherapy. However, the efficacy of in vitro maturation (IVM) methods with vitrified germinal vesicle (GV) oocytes could be improved.Objective As melatonin and follicular fluid (FF) might enhance IVM conditions, we used these supplements to assess the maturation rate of vitrified GV oocytes and their artificial fertilization rate.Materials and MethodsFour hundred mouse GV oocytes were harvested, vitrified, and assigned into control (C-Vit-GV) and treatment groups of melatonin (M-Vit-GV), human follicular fluid (HFF-Vit-GV), and a combination (M + HFF-Vit-GV). A non-vitrified group of GV oocytes (non-Vit-GV) and a group of in vivo matured metaphase II (Vivo-MII) oocytes served as control groups to evaluate the vitrification and IVM conditions, respectively. Maturation of GV oocytes to MII and further development to two-cell-stage embryos were determined in the different groups.ResultsDevelopment to two-cell embryos was comparable between the Vivo-MII and non-Vit-GV groups. IVM and in vitro fertilization (IVF) results in the non-Vit-GV group were also comparable with the C-Vit-GV oocytes. In addition, the IVM and IVF outcomes were similar across the different treatment groups including the M-Vit-GV, HFF-Vit-GV, M + HFF-Vit-GV, and C-Vit-GV oocytes.ConclusionEmploying an appropriate technique of vitrification followed by suitable IVM conditions can lead to reasonable IVF outcomes which may not benefit from extra supplementations. However, whether utilizing other supplementation formulas could improve the outcome requires further investigation.

Acrylamide impairs the developmental potential of germinal vesicle oocytes by inducing mitochondrial dysfunction and autophagy/apoptosis in mice

Background: Acrylamide (ACR), an important endogenous contaminant in carbohydrate-rich foods, has been involved in various negative effects on multiple organ networks, including the reproductive system. Previous studies have reported that ACR affects oocyte quality and fertility.Purpose: This study aimed to explore the toxic effects and regulatory mechanisms of ACR on mouse germinal vesicle (GV) oocytes. Research Design: In this study, adult female mice were exposed to ACR at 10mg/kg/day/body weight through their drinking water continuously for 4weeks. Study Sample and Data Analysis: The mitochondrial function, autophagy/apoptosis, and development potential of GV oocytes were investigated. Results: The results showed that ACR reduced the oocyte diameter, sperm-binding ability, parthenogenetic activation and in vitro fertilization (IVF) rate, and development potential of pre-implantation embryos. We also found that ACR exposure disrupted chromatin configuration, mitochondrial distribution, and membrane potential (Δφm) of oocytes. Actin filament expression was significantly reduced in both the membrane and cytoplasm of mouse oocytes. Moreover, ACR exposure increased LC3-positive signals, early apoptosis rate, aberrant ATG3, ATG5, LC3, Beclin1, and mTOR mRNA expression. Conclusions: These results suggest that ACR exposure can affect the developmental potential of GV oocytes by inducing mitochondrial dysfunction, actin filament assembly, and autophagy/apoptosis.

Melatonin Promotes in vitro Development of Vitrified-Warmed Mouse GV Oocytes, Potentially by Modulating Phosphorylation of Drp1

Previous studies have shown that melatonin can mitigate cryopreservation-induced mitochondrial dysfunction in oocytes; however, the underlying molecular mechanism remains unclear. The objective of the present study was to investigate whether melatonin can improve the mitochondrial function during in vitro maturation of vitrified-warmed mouse germinal vesicle (GV) oocytes by modulating phosphorylation of dynamin related protein 1 (Drp1). Vitrification/warming procedures resulted in the following: (1) After cryopreservation of mouse GV oocytes, the phosphorylation level of Drp1 at Ser616 (p-Drp1 Ser616) in metaphase II (MII) oocytes was increased (P < 0.05). Furthermore, the rates of in vitro maturation, cleavage and blastocyst formation after parthenogenetic activation were decreased (P < 0.05). (2) In MII oocytes, the expression levels of translocase of the mitochondrial outer membrane 20 (TOMM20), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, and mRNA levels of mitochondrial biogenesis-related genes (Sirt1, Pgc-1α, Tfam) were all decreased (P < 0.05), and (3) Reactive oxygen species (ROS) level, early apoptosis level, Cytochrome C release and mRNA levels of pro-apoptotic related genes (Bax, Caspase9, Caspase3) in MII oocytes were all increased (P < 0.05). The results of this study further revealed that negative impacts of GV oocyte cryopreservation were mitigated by supplementation of warming and in vitro maturation media with 10−7mol /L melatonin or 2 x 10−5mol/L Mdivi-1 (Drp1 inhibitor). Therefore, we concluded that 10−7mol/L melatonin improved mitochondrial function, reduced oxidative stress and inhibited apoptosis by regulating phosphorylation of Drp1, thereby enhancing in vitro development of vitrified-warmed mouse GV oocytes.

Melatonin Promotes In Vitro Maturation of Vitrified-Warmed Mouse Germinal Vesicle Oocytes, Potentially by Reducing Oxidative Stress through the Nrf2 Pathway.

Simple SummaryCryopreservation of oocytes can cause high oxidative stress, reduce the quality of vitrified-warmed oocytes, and seriously hinder the application of oocyte cryopreservation technology in production and medicine. In this work, we found for the first time that melatonin can exert antioxidant effects through receptors and regulate the Nrf2 antioxidant pathway to respond to oxidative stress of vitrified-warmed oocytes, thereby improving both oocyte quality and the potential for subsequent development. The results illustrated the molecular mechanism of melatonin’s antioxidant effect in vitrified-warmed oocytes and provided a theoretical basis for the application of melatonin in the cryopreservation of oocytes. These findings are of great significance for the further application of oocyte cryopreservation technology to production and assisted reproduction in the future.Previously it was reported that melatonin could mitigate oxidative stress caused by oocyte cryopreservation; however, the underlying molecular mechanisms which cause this remain unclear. The objective was to explore whether melatonin could reduce oxidative stress during in vitro maturation of vitrified-warmed mouse germinal vesicle (GV) oocytes through the Nrf2 signaling pathway or its receptors. During in vitro maturation of vitrified-warmed mouse GV oocytes, there were decreases (p < 0.05) in the development rates of metaphase I (MI) oocytes and metaphase II (MII) and spindle morphology grades; increases (p < 0.05) in the reactive oxygen species (ROS) levels; and decreases (p < 0.05) in expressions of Nrf2 signaling pathway-related genes (Nrf2, SOD1) and proteins (Nrf2, HO-1). However, adding 10−7 mol/L melatonin to both the warming solution and maturation solutions improved (p < 0.05) these indicators. When the Nrf2 protein was specifically inhibited by Brusatol, melatonin did not increase development rates, spindle morphology grades, genes, or protein expressions, nor did it reduce vitrification-induced intracellular oxidative stress in GV oocytes during in vitro maturation. In addition, when melatonin receptors were inhibited by luzindole, the ability of melatonin to scavenge intracellular ROS was decreased, and the expressions of genes (Nrf2, SOD1) and proteins (Nrf2, HO-1) were not restored to control levels. Therefore, we concluded that 10−7 mol/L melatonin acted on the Nrf2 signaling pathway through its receptors to regulate the expression of genes (Nrf2, SOD1) and proteins (Nrf2, HO-1), and mitigate intracellular oxidative stress, thereby enhancing in vitro development of vitrified-warmed mouse GV oocytes.

Melatonin promotes in vitro maturation of vitrified-warmed mouse GV oocytes potentially by modulating MAD2 protein expression of SAC component through MTRs

Previous studies have shown that melatonin (MT) can ameliorate vitrification-inflicted damage in mouse germinal vesicle (GV) oocytes, however, the key mechanistic basis of this improvement still remains poorly understood. This study was conducted to investigate whether MT can improve in vitro developmental potential of vitrified-warmed GV oocytes through its receptors. The fresh oocytes were randomly divided into four groups: untreated (control group, F), vitrified by open-pulled straw method (vitrification group, V), vitrification group with 100 nmol/L MT supplementation (vitrification + MT group, VM), and with 100 nmol/L MT plus 100 nmol/L luzindole administration (vitrification + MT + luzindole group, VML) or with 50 nmol/L ramelteon addition (vitrification + ramelteon group; VR). After warming, oocytes were cultured in vitro, and MT receptors (MTRs), MAD2 (mitotic arrest deficient 2), Securin and CyclinB1 protein levels and spindle morphology were evaluated. The ratio of oocytes developed to the metaphase I (MI) and metaphase II (MII) stages was also assessed. The results showed that after vitrification-warming, the in vitro maturation rate of GV oocytes was significantly lower compared to the control (F) group. Vitrification also significantly impaired the spindle morphology, decreased the protein level of MTRs and Securin, and decreased MAD2 levels in MI oocytes. However, when MT or ramelteon (MTRs agonist) were added (group wise) to warming and maturation media, the maturation rate of GV oocytes was significantly increased, the normal proportion of the spindle morphology increased, and the expression level of MAD2 increased in their resulting MI oocytes compared to the vitrification group. However, following addition of both MT and ramelteon, the maturation rate of GV oocyte showed no significant difference between VML and vitrification groups. The spindle morphology and MAD2 levels in MI oocytes were comparable to the vitrification group but differed significantly from the VM group. Taken together, finding of the present study shows that MT (100 nmol/L) can ameliorate the in vitro maturation of vitrified-warmed mouse GV oocytes, potentially by improving the spindle morphology, modulating MAD2 protein level and promoting the development of MI stage oocytes through MTRs.

The Effect of Sodium Selenite on Expression of Mitochondrial Transcription Factor A during In Vitro Maturation of Mouse Oocyte.

Background:The aim of the present study was to investigate the effect of Sodium Selenite (SS) supplemented media on oocyte maturation, expression of mitochondrial transcription factor A (TFAM) and embryo quality.Methods:Mouse Germinal Vesicle (GV) oocytes were collected after administration of Pregnant Mare Serum Gonadotropin (PMSG); in experimental group 1, oocytes were cultured and then subjected for in vitro maturation in the absence of SS, and in experimental group 2, they were matured in vitro in the presence of 10 ng/ml of SS up to 16 hr. The control group included MII oocytes obtained from the fallopian tubes after ovarian stimulation with PMSG, followed by human chorionic gonadotropin. Then, the expression of TFAM in MII oocytes in all three groups was investigated using real-time RT-PCR. The fertilization and embryo developmental rates were assessed, and finally the quality of the blastocysts was evaluated using propidium iodide staining.Results:The oocyte maturation rate to MII stage in SS treated group was significantly higher than non-treated oocytes (75.65 vs. 68.17%, p<0.05). Also, the rates of fertilization, embryo development to blastocyst stage as well as the cell number of blastocyst in SS supplemented group were higher than other experimental group (p<0.05). There was a significant decrease in TFAM gene expression in both in vitro groups compared to the group with in vivo obtained oocytes (p<0.05). Moreover, there was a significant increase in TFAM gene expression in oocytes that matured in the presence of SS compared to that of the group without SS (p<0.05).Conclusion:Supplementation of oocyte maturation culture media with SS improved the development rate of oocytes and embryo and also enhanced TFAM expression in MII oocytes which can affect the mitochondrial biogenesis of oocytes.

Enhanced Cryoprotective Effect of Melatonin and Resveratrol by Coencapsulation: Improved In Vitro Development of Vitrified-Warmed Mouse Germinal Vesicle Oocytes.

Oocyte vitrification, as a vital step in reproductive medicine, is strongly associated with lower development caused by cryodamaging factors, such as oxidative stress. In this study, we evaluated the antioxidative synergistic effects of Melatonin (Mel) and Resveratrol (RES) coencapsulated by solid lipid nanocarriers (SLNs) against the pure antioxidant combination (Mel+RES). In this research, the formation of Mel+RES-SLN was confirmed by Fourier-transformed infrared spectroscopy. The average mean diameter, size distribution, polydispersity index, and zeta potential of particles were measured by Zetasizer, and the morphology was evaluated by scanning electron microscopy. In addition, the encapsulation efficiency (EE%) or drug loading capacity (DL%) of the nanocapsule was determined by spectrophotometric methods. Germinal vesicle (GV)-stage oocytes harvested from 6- to 12-week-old female NMRI mice were randomly divided into seven groups for in vitro studies. In these groups, (0, 10-12 M + 0.5 μM, 10-9 M + 2 μM, or 10-6 M + 10 μM) of Mel+RES/Mel+RES-SLN were added into vitrification media. After thawing, oocytes were matured, fertilized, and cultured for 3 days. Extra/intracellular reactive oxygen species (ROS) levels were measured in in vitro maturation medium after 24 hours. Our results revealed a significant improvement in the normal morphology of warmed GV-stage oocytes, GV breakdown (GVBD) rate, Metaphase II (MII)-stage oocyte formation, fertilization rate, early embryo development, and a significant reduction in intra/extracellular ROS level when vitrification media was supplemented with the lowest Mel+RES-SLN concentration. In vitro studies also demonstrated that the highest concentration of Mel+RES-SLN was safe, without a detrimental effect on embryonic development upon treatment. In conclusion, the lowest concentration of Mel+RES-SLN supplementation in GV-stage oocyte vitrification media improved maturation, fertilization, and embryo development rate and decreased extra/intracellular ROS level through an enhanced/controlled intracellular penetration compared to the pure Mel+RES.

Proteomic-based identification of oocyte maturation-related proteins in mouse germinal vesicle oocytes.

Oocyte proteins play an important role in oocyte maturation, fertilization and embryonic development. However, the protein composition of mouse germinal vesicle (GV) oocytes is still unclear. Using one-dimensional Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) and Reverse-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS), we constructed a protein profile of mouse GV oocytes. First, our proteomics profile identified 1,405 different proteins from 11,000 mouse GV oocytes lacking zona pellucida. Second, with detailed bioinformatics analysis, a group of proteins that play an essential role in oocyte maturation was screened. In addition, the expression and localization of suppressor of G2 allele of skp1(SUGT1, also called SGT1), heterogeneous nuclear ribonucleoprotein K(Hnrpk), Seruin, Cullin1(Clu1) and nuclear distribution protein C (Nudc) in mouse ovaries and early embryos were also captured and investigated in this study. Moreover, the protein profile was submitted to the Proteomics Identifications Database (PRIDE) and is available via ProteomeXchange with the identifier PXD014314. Our research provides valuable resources for the study of oocyte proteins and oocyte maturation and helps to clarify the mechanisms of oocyte maturation.

Ceratonia siliqua (Carob) extract improved in vitro development of vitrified-warmed mouse germinal vesicle oocytes: assessment of possible mechanism.

Oocyte banking is a vital step for safekeeping and spreading genetic resources of animals. It is also used for fertility preservation of human. Oocyte vitrification is closely related to the lower developmental competence which includes the cryo-injury arisen during vitrification. The aim of the present study was to evaluate the maturation, embryonic development and production of reactive oxygen species (ROS) of mice oocytes following the supplementation vitrification media with different concentrations of Ceratonia siliqua (carob) extracts. In this experimental study, germinal vesicle oocytes collected from 8 to 10week-old female NMRI mice (30-40gr) were randomly divided into six groups of vitrification media supplemented with 0 (control), 5, 10, 20, 30 and 50µg/ml C. siliqua. After thawing, oocytes were put in an in vitro maturation medium (IVM) (α-MEM: Alpha Minimum Essential Medium). 3-4 and 24h (hr) later, the oocyte nuclear maturity was checked. Standard invitro fertilization was performed on the matured oocytes (MII), and embryonic development was followed. Extra- and intra-cellular ROS was measured in IVM medium after 24h of oocyte incubation. The addition of 20 and 30μg/ml C. siliqua extract to vitrification media improved normal morphology of warmed germinal vesicle (GV) oocytes, rate of germinal vesicle break down (GVBD), and metaphase 2 (MII) oocyte formation significantly (p < 0.05). Fertilization rate, (embryonic development to 2 cells stage, 4-8 cells stage, and > 8 cells stage increased in the 30μg/ml C. siliqua group significantly (p < 0.05). Furthermore, supplementation of 30μg/ml C. siliqua in vitrification media significantly decreased extra- and intra-cellular of ROS as well as embryonic fragmentation (p < 0.05). In conclusion, supplementation of GV oocyte vitrification media with carob extract improved maturation, fertilization, and embryonic development rate and decreased extra- and intra-cellular ROS levels.

Actinomycin D causes oocyte maturation failure by inhibiting chromosome separation and spindle assembly†.

Actinomycin D (ActD) has been considered as one of the most effective and safe chemotherapeutic medications for treating a number of cancers. Although ActD has been used in the treatment of gynecological tumors and pediatric tumors for more than 50years, the toxic effects of ActD on mammalian oocytes remain unknown. In this study, the influence of ActD on mouse and human oocyte maturation and the possible mechanisms were investigated. Notably, ActD inhibited oocyte maturation and arrested oocytes at the metaphase I (MI) stage in a dose-dependent manner. In addition, ActD arrested oocyte maturation when the oocytes were treated at different successive stages, including the germinal vesicle (GV), germinal vesicle breakdown, and MI stages. In ActD-treated oocytes, disordered chromosome condensation and irregular spindle assembly occurred, resulting in incomplete chromosome segregation and oocytes arresting at the MI phase; these results possibly occurred because ActD triggered the formation of reactive oxygen species, resulting in DNA damage and decreased ATP in mouse GV oocytes. Besides, in vivo treatment with ActD also inhibited mouse oocyte maturation. Similar effects were seen in human oocytes. Collectively, our results indicated that ActD exposure disrupted oocyte maturation by increasing DNA damage, which is a finding that might help with optimizing future methods for female fertility preservation before undergoing chemotherapy.