Study Of The Influence Of In-vitro Maturation On The Expression Of ESET And H3K9Me3 In Mouse Oocytes

IntroductionThe technology of in-vitro maturation (IVM) of mammalian oocytes combined with in-vitro fertilization (IVF) and in-vitro embryo culture (IVC) has been widely used in basic research studies as well as in the clinical treatment of human infertility.IVM is the process whereby oocytes are retrieved when they are still immature and matured in-vitro to metaphase stage of meiosis II (MII) in a specified culture system. There are several important reasons for the clinical application of oocyte IVM. First, the routine hormonal stimulation protocols used for induction of ovulation in human IVF are not suitable for some infertile patients at risk of ovarian hyper-stimulation syndrome (e.g., women suffering from polycystic ovarian syndrome). Second, IVM combined with ovarian or oocyte cryopreservation could provide the reproductive opportunities to the women who undergo chemotherapy or radiotherapy for cancer treatment. Third, clinical super-ovulation protocols still have many indirect problems, including the high cost of hormones and the side-effects of hormonal stimulations. Because of all these advantages, many infertility clinics have attempted to use IVM to treat patients. However, so far, the clinical pregnant rates of IVM are still much lower than those in routine IVF. A higher incidence of spindle abnormalities, chromosome defects and changes of epigenetic modification in IVM oocytes result in the worries for its safety.There is the crosstalk between oocytes and their surrounding somatic cells during oogenesis. It is well known that factors such as nutrition, hormones and environmental compounds can have a profound effect on oocyte quality and subsequently embryo development. The oocytes in IVM culture system develop in the micro-environment that imitates to the internal environment. Due to lack of the self-regulation and control system in-vitro, the imitated environment is not so stable as in in-vivo. Secondly, the process of in-vitro culture and manipulation will inevitably expose the oocytes to a changing environments, such as room temperature, visible light, and so they may influence the potential growth of the oocytes.The developmental program is controlled by genetic and epigenetic mechanisms. During development of mammalians, different cells and tissues acquire different programs of gene expression. It is thought that this is substantially regulated by epigenetic modifications such as DNA methylation, histone tail modifications and non-histone proteins that bind to chromatin. Epigenetic markers are heritable through mitosis and meiosis, and are not dependent on changes in DNA sequence. The precise timing of gene activation/inactivation is crucial for normal preimplantation development, and any delay or lack of expression leads to abnormalities. Thus, the reprogramming needs to attain the correct timing of activation and the well-orchestrated pattern of gene expression, in order to initiate and continue embryonic development after somatic nuclear transfer.The ability of the oocyte to operate an efficient block to polyspermy was markedly affected by maturation. The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation. Oocytes reorganize the penetrated sperm chromatin into male pronucleus and in parallel, the epigenetic processes of global chromatin methylation and acetylation are carried out. IVM oocytes are more likely to have abnormal chromosome configurations and disorganized meiotic spindle microtubules. IVM oocytes also have a reduced epigenetic competence. The fertilization rate was significantly lower in those in IVM than that in in-vivo oocytes.Eukaryotic DNA is packaged within the nucleus through its association with histones to form the fundamental repeating unit of chromatin, the nucleosome. The nucleosome consists of 146 bp of DNA wrapped around a histone core octamer composed of two each of H2A, H2B, H3, and H4. A striking feature of the histones is that they are subjected to a number of posttranslational covalent modifications such as acetylation, phosphorylation, methylation, ubiquitinylation and sumoylation. The nucleosomal diversity created by these modifications forms the basis of the 'histone code' hypothesis, which proposes that chromatin domains, defined by local combinatorial signatures of histone modifications, have distinct outcomes for chromatin structure and gene expression. In histone modifications related to gene silencing, the best studied are the methylation and acetylation. Methylation is one of the most complex process for histone modification, both in terms of the nature of the signal and its biological consequences. Histone methylation occurs on both arginines and lysines. Both the site and the degree of methylation affect the biological outcome of methylation. These methyl marks serve as binding sites for proteins that assemble complexes that in turn regulate chromatin structure and gene transcription.Histone methylation is catalyzed by members of the histone methyltransferases. So far, five mammalian H3-K9-specific histone methyltransferases (HMTs) have been identified. Histone H3 lysine 9 (H3K9) methylation plays a crucial role for heterochromatin formation and maintenance and for gene silencing.The mouse homolog of SETDB1, ESET [ERG (ets -related gene)-associated protein specifically methylates lysine-9 of histone H3 and is the only euchromatic HMT that can also catalyze H3-K9 triplemethylation. ESET/SETDB1 interacts with a number of enzymes and transcription factors, connecting transcriptional silencing of gene expression and localized heterochromatin formation.H3K9Me3 and ESET play crucial roles in gene transcription during oogenesis and the epigenetic reprogramming of oocytes and fertilization. The investigation of the expression of H3K9Me3 and ESET of IVM oocytes may offer us a chance to study the function of external factors which influence the ability of the epigenetic reprogramming in oocytes and experimental data for safety evaluation of IVM.The ESET mRNA level of these oocytes was detected by RT-PCR, and the expression and distribution of H3K9Me3 and ESET proteins in oocytes were investigated by fluorescent immunocytochemistry. All of these were compared with those in-vivo matured oocytes.ObjectiveThe aim of the present study is to investigate the effects of in-vitro maturation on the expression and distribution of H3K9Me3 and ESET in mouse oocytes, analyze the molecular mechanisms of the changes of the epigenetic reprogramming associated with the procedures of in-vitro maturation, and evaluate the safety of IVM.Material & methods1. Animals: ICR female mice at ages of 6～8 weeks.2. IVM groupFor the collection of GV oocytes, mice were each injected with 7.5 IU of pregnant mare's serum gonadotropin (PMSG) and killed by cervical dislocation 46～48h later. Ovaries were isolated and antral follicles were punctured by a sharp needle with a 1ml syringe. Cumulus-oocyte complexes (COCs) were released into MHTF medium. Only spherical oocytes with a distinct GV and attached cumulus cells were collected and rinsed one time in MHTF. COCs were cultured in the drops of 30ul culture medium with recombination human follicle stimulating hormone (r-FSH, 0.075IU/ml) and human choriongonadotropin (hCG, 0.5IU/ml) at 37℃, 100% humidity and 5% CO₂. About 14～16 hr later, oocytes were liberated from the surrounding cumulus cells by gentle pipetting through a narrow-bore glass pipette. The spherical oocytes with a distinct first polar body (MII oocyte) were selected for this group. The diameters and zona thickness of MII oocytes were measured.3. In-vivo groupFor the collection of in-vivo MII oocytes, mice were injected with 10IU PMSG followed by 10IU of hCG 46～48 h later. The mice were killed at 13～15 h post HCG injection. The swollen of oviducts were punctured in MHTF. COCs were briefly exposed to 80 IU/ml hyaluronidase at 37℃to remove attached cumulus granulosa cells. Only spherical oocytes with a distinct first polar body (MII oocyte) were selected for this group. The diameters and zona thickness of MII oocytes were measured.4. RT-PCRSome of MII oocytes in each group were collected for RNA extraction. RT-PCR was used to detect ESET mRNA level in the oocytes from IVM and the control group respectively.5. ImmunocytochemistryFluorescent immunocytochemistry was used to detect the expression and distribution of H3K9Me3 and ESET proteins in the oocytes from IVM and in-vivo group respectively.6. Statistical AnalysisAll statistical analyses were conducted using the SPSS 15.0 statistical software program (SPSS, Chicago, IL, USA). Statistical analyses were carried out by independent-one-sample t test. Data were expressed as mean±SD. The results were considered statistically significant when P values were less than 0.05. Results1. About 80% of oocytes in IVM group reached the MII stage. The mean of the diameters of the oocytes from IVM (75.03±2.98 um) was significantly lower than that in-vivo MII oocytes (95.77±4.03 um) (P<0.001).2. RT-PCRThe level of ESET mRNA was detected by measuring the gray value of the PCR product using KODAK EDAS 290 imagining system software. No statistical difference was found between IVM and in-vivo groups by RT-PCR for ESET mRNA (P>0.05).3. ImmunocytochemistryThe ESET protein was detectable in the MII oocytes from both groups. The distribution of ESET immuno-staining in MII oocytes was evenly in the cytoplasm and enhanced in the peripheral region. The fluorescent intensity of ESET signals in the oocytes of in-vivo group showed significantly higher than that in IVM oocytes (p<0.05). However, there were no differences of H3K9Me3 expression and distribution between the MII oocyte from the two groups. The signal of anti-trimethyl-H3-K9 was only observed in the spindle and first polar body, the region of DAPI stained. The H3K9Me3 signals in the first polar bodies were always stronger than those in spindles.Conclusions1. IVM process could decrease the diameters of oocyte, one of important parameters for oocyte growth.2. IVM process did not alter the ESET gene transcription but down-regulate the ESET protein expression in MII oocyte. ESET proteins are mainly distributed in the cytoplasm of MII oocytes.3. H3K9Me3, the methylation mode of H3K9, was only expressed in the region with chromatins or chromosomes. There was no obvious effect of IVM on H3K9Me3 expression. 4. IVM down-regulated ESET but no obvious effects on the expression of H3K9Me3, the only targets of ESET might result from the distribution differences of the two proteins, which suggested that the functions of ESET at MII stage of oocyte were maternal storages for histone modification in fertilization and further development of embryos.