Project description:Basonuclin, which is a zinc-finger protein found in abundance only in the keratinocytes of the stratified epithelium, male germ cells and oocytes, qualifies as a maternal-effect gene because the source of pre-implantation embryonic basonuclin is maternal. Using a transgenic-RNAi approach, we knocked-down basonuclin specifically in mouse oocytes, which led to female sub-fertility. Basonuclin deficiency in oocytes perturbed both RNA polymerase I- and II-mediated transcription and oocyte morphology was affected as evidenced by cytoplasmic and cell surface abnormalities. The affected oocytes, however, could still mature to and arrest at metaphase II and be ovulated, suggesting the impaired pathways were not essential for oocyte development and maturation. Nevertheless, an early embryonic failure in pre-implantation development was identified and likely accounted for the sub-fertility phenotype. These results suggest that basonuclin is a new member of the mammalian maternal-effect genes and interestingly, differs from the previously reported mammalian maternal-effect genes in that it also apparently perturbs oogenesis.
Project description:In this study, we applied 1D SDS-PAGE and RP-LC-MS/MS to investigate the proteins stored in GV mouse oocytes. This high-performance strategy allowed us to define a set of 1405 different mouse GV oocyte proteins. It is confirmed that this study will help us to understand the diverse biological processes occurring in mouse oocytes and during early embryo development. However, compared with proteomic analysis of other cells and tissues, such as embryonic stems from cells and liver, the proteins identified in mature mouse oocytes were limited. This was mainly due to the fact that oocytes obtained from each mouse were very limited. We believe that the catalog of maternal proteins presented in this article is a starting point and we anticipate that more researches on the oocyte proteome will deduce most of the maternal proteins.
Project description:In order to establish an obese mouse model, female mice were continuously fed with a high-fat diet (HFD) or a normal diet (control) for 16 weeks beginning at three weeks of age. In this paper, these mice are termed ‘HFD mice’ and ‘control mice’, respectively. Accordingly, we call their oocytes ‘HFD oocytes’ and ‘control oocytes’. Substantial evidence indicates that the effects of maternal obesity on embryo/offspring development can be attributed to factors within the oocyte (9). To identify such potential effectors, we performed a comparative proteomic analysis of ovulated MII oocytes from control and HFD mice.
Project description:The long-standing view of 'immortal germ line versus mortal soma' poses a fundamental question in biology concerning how oocytes age in molecular terms. A mainstream hypothesis is that maternal aging of oocytes has its roots in gene transcription. Investigating the proteins resulting from mRNA translation would reveal how far the levels of functionally available proteins correlate with mRNAs, and would offer novel insight into the changes oocytes undergo during maternal aging. Gene ontology semantic analysis reveals the high similarity of the detected proteome (2,324 proteins) to the transcriptome (22,334 mRNAs), though not all proteins have a cognate mRNA. Concerning their dynamics, 4-fold changes of abundance are more frequent in the proteome (3%) than the transcriptome (0.05%), with correlation. Whereas proteins associated with the nucleus (e.g. structural maintenance of chromosomes, spindle-assembly checkpoints) are largely represented among those that change in oocytes during maternal aging; proteins associated with oxidative stress/damage (e.g. superoxide dismutase) are infrequent. These quantitative alterations are either impoverishing or enriching. Using gene ontology analysis, these alterations do not relate in any simple way to the classic signature of aging known from somatic tissues. We conclude that proteome analysis of mouse oocytes may not be surrogated with transcriptome analysis, given the lack of correlation. Furthermore, we conclude that the classic features of aging may not be transposed from somatic tissues to oocytes in a one-to-one fashion. Overall, there is more to the maternal aging of oocytes than mere cellular deterioration exemplified by the notorious increase of meiotic aneuploidy. Three pools of 20 zona-enclosed B6C3F1 oocytes from each age group were subjected for experiment.
Project description:STUDY QUESTION: Does maternal age affect the maturated oocyte quality and the fol-lowing development after fertilization in human? SUMMARY ANSWER: Maternal age affects the quality of maturated oocytes by altering the stored mRNA levels in human, such as TOP2B. WHAT IS KNOWN ALREADY: Intracellular mRNAs in maturated oocytes are tran-scripted from the maternal genome during oogenesis and important for the zygotic genome activation (ZGA) after fertilization. Microarray data showed that maternal age affected polyadenylated transcript abundance in human oocytes. These genes are involved in in signaling pathway related to cell cycle regulation, chromosome alignment. However, which genes are the key genes affected by maternal age and important for the development after fertilization had not been reported. Therefore, single-cell RNA sequencing (scRNA-Seq) technology is employed in this study to screen the key genes affected by maternal age in human maturated oocytes. STUDY DESIGN, SIZE, DURATION: We isolated mRNA from maturated (MII) oo-cytes donated by IVF or ICSI patients (three oocytes from young (≤ 30 years) and three oocytes from advanced maternal age (≥ 40 years) patients) undergoing controlled ovarian stimulation. Thus, a total of six maturated oocytes were individually processed for scRNA-seq analysis. The key genes screened from scRNA-seq analysis are confirmed using mouse model. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients undergoing infertility treatment at the Yuhuangding Hospital of Yantai underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ul-trasound guided oocyte retrieval. We isolated RNA, generated single cell RNA-seq librar-ies (Smart-Seq2) and sequenced by Illumina Hiseq X-ten platform with 150 bp paired-end. Bioinformatics analysis of the sequencing data was done to find the biological processes and key genes that led to the decline in the quality of oocytes with advanced maternal age. To validate the findings, we used mouse model and validated candidate genes by RT-PCR and knockdown experiments. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 1439 genes differentially expressed between older and younger women's maturated oocytes (|foldchange|>2, P < 0.05). These genes are significantly enriched with annotations related to transporter activity, cytoskeleton, oxidative stress, catalytic activity, immune function, cellular senescence and biosynthesis. The key candidate gene TOP2B was found by protein interaction network analysis, and knockdown verification on young mouse maturated oocytes showed that TOP2B was a key gene affecting the oocyte quality and disturbing early embryo development. LARGE SCALE DATA Raw data from this study can be accessed through GSE. LIMITATIONS, REASONS FOR CAUTION: The human maturated oocytes used in this study were from patients with different causes of infertility and may affect oocyte gene expression. In addition, the study was based on a lim-ited number of patients, and there are possible natural biological variance existed in human samples. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, we used scRNA-seq to detect global gene transcriptome of maturated oocytes in young and older women. These results are useful to indicate the molecular mechanisms of female ovary aging and establishing a criterion to evaluate the quality of oocytes in women with advanced maternal age. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the National Key Research and Development Program of China (2018YFC1004304, 2016YFA0100203), Medical and Health Science Technology Development Plan Project of Shandong Province (Grant#. 2017WS566). There are no competing interests.
Project description:Basonuclin, which is a zinc-finger protein found in abundance only in the keratinocytes of the stratified epithelium, male germ cells and oocytes, qualifies as a maternal-effect gene because the source of pre-implantation embryonic basonuclin is maternal. Using a transgenic-RNAi approach, we knocked-down basonuclin specifically in mouse oocytes, which led to female sub-fertility. Basonuclin deficiency in oocytes perturbed both RNA polymerase I- and II-mediated transcription and oocyte morphology was affected as evidenced by cytoplasmic and cell surface abnormalities. The affected oocytes, however, could still mature to and arrest at metaphase II and be ovulated, suggesting the impaired pathways were not essential for oocyte development and maturation. Nevertheless, an early embryonic failure in pre-implantation development was identified and likely accounted for the sub-fertility phenotype. These results suggest that basonuclin is a new member of the mammalian maternal-effect genes and interestingly, differs from the previously reported mammalian maternal-effect genes in that it also apparently perturbs oogenesis. Keywords: RNAi knockdown
Project description:Here we asked if oocyte proteomes are representative of the transcriptomes, how the abundance of specific genes’ mRNA and protein responds to maternal aging, and if oocyte aging presents the features characteristic of somatic aging. To address these questions on the proteomic level, we employed stable isotope labeling of amino acids in cell culture (SILAC; Geiger et al. 2011) as the method of choice, and performed a SILAC screen of mouse metaphase II (MII) oocytes superovulated at 3, 8+-1 and 58+-10 weeks of maternal age, which correspond to pre-puberty, mature age and climacterium, respectively. We used heavy F9 embryonic carcinoma (EC) cells as internal or "Spike-in" standard for the quantification of oocytes proteins because in contrast to the oocytes they can easily be cultured feeder-free, have stem cell properties and should harbor the majority of all oocyte proteins (although with different relative abundances). The SILAC screen was conducted in parallel with conventional microarray analysis to compare the concordance of protein and transcript levels in these oocytes. Associated microarray data have been deposited to NCBI GEO with accession number GSE42959.
Project description:Chromosome aneuploidy increases in oocytes with maternal age, and is considered the leading cause for the increased incidence of infertility, miscarriage, and birth defects. Using mRNA-Sequencing of oocytes from 12 month old mouse versus 3 month young mouse, we identified a spindle assembly checkpoint gene, BubR1, whose expression was significantly decreased. We employed a mRNA microinjection based approach to increase BubR1 expression in aging oocytes. We find that increased expression of BubR1 protects against aneuploidy and chromosome misalignment in aging oocytes. After in vitro fertilization, the embryos derived from BubR1 increased expression aging oocytes exhibited chromosome stability as robust as those of the young ones. Furthermore, following embryo transfer, these embryos showed greatly improved developmental competency, with comparable levels of full-term development to those of the young ones. These results indicate that the decline in oocyte quality may be reversible and could lead to treatments that prolong female fertility. Examination of the effect of maternal aging on the mRNA expression in the mature oocytes of the female mice. Naturally ovulated mature oocytes (MII stage) were collected from 6 young (3 month) and 6 aging (12 month) female mice (3 oocytes per mice, 18 oocytes for each group).
Project description:Background: Early embryonic development is governed by maternal transcripts stored within the oocyte during oogenesis. Transcriptional activity of the oocyte ultimately dictates its developmental potential and may be influenced by maternal age, resulting in reduced competence of oocytes derived from women of advanced age, compared with the young. In the current study, RNA-Seq was used to perform transcriptome profiling of human GV and MII oocytes derived from young and advanced maternal age women. Participants/Materials and Methods: Cumulus dissection from donated oocytes was performed. GV and MII oocytes underwent deep RNA sequencing using the SMART-Seq v4 Ultra Low Input RNA protocol (Takara-Clontech, USA) and Nextera XT DNA library preparation kit (Illumina, USA). Data processing, quality assessment and bioinformatics analysis were performed using source-software, including FastQC, HISAT2, StringTie, edgeR and DAVID. Results: Following deep single-cell RNA-Seq on GV and MII oocytes, hundreds of transcripts were significantly differentially expressed between young maternal age (YMA) and advanced maternal age (AMA) groups, with the most significant biological processes relating to mitochondrial reserves. The GV to MII transition shares common biological processes between young and AMA groups, however, some genes involved in mitochondria function were altered during ageing. A decrease in mitochondrial-related transcripts was also observed during the GV to MII transition. However, there was a much greater reduction of mitochondrial-related transcripts in MII oocytes of AMA. This observation was confirmed when YMA MII oocytes were compared with the AMA MII group with mitochondrial-related transcripts being significantly higher expressed in the YMA group, including biological processes, such as mitochondrial electron transport and ATP biosynthetic process. These results indicate a higher energy potential in YMA MII oocytes that is decreased with age. Other significantly higher biological processes in the YMA MII group include transcripts involved in the regulation of ubiquitin-dependent degradation. Lack of these transcripts could lead to a non-appropriate removal of oogenesis remnants following fertilisation in the AMA MII group. Discussion: Understanding reproductive ageing effects at the RNA level in human oocytes may reveal differences in the mechanisms regulating the GV to MII transition that impact on oocyte quality in YMA and AMA patients. Further investigations of the up-/down-regulated transcripts during ageing could guide and improved IVF outcomes for AMA patients.
Project description:Sensitivity of murine offspring’s oocytes to a maternal high-fat/high-sugar (HF/HS) diet and how preconception care interventions (diet normalization (DN) or caloric restriction (CR)) might influence this. A proteomic insight.