Oocyte Quality Control Research Articles

ARHGAP26 deficiency drives the oocyte aneuploidy and early embryonic development failure.

Aneuploidy, the presence of a chromosomal anomaly, is a major cause of spontaneous abortions and recurrent pregnancy loss in humans. However, the underlying molecular mechanisms still remain poorly understood. Here, we report that ARHGAP26, a putative tumor suppressor gene, is a newly identified regulator of oocyte quality to maintain mitochondrial integrity and chromosome euploidy, thus ensuring normal embryonic development and fertility. Taking advantage of knockout mouse model, we revealed that genetic ablation of Arhgap26 caused the oocyte death at GV stage due to the mitochondrial dysfunction-induced ROS accumulation. Lack of Arhgap26 also impaired both in vitro and in vivo maturation of survived oocytes which results in maturation arrest and aneuploidy, and consequently leading to early embryonic development defects and subfertility. These observations were further verified by transcriptome analysis. Mechanistically, we discovered that Arhgap26 interacted with Cofilin1 to maintain the mitochondrial integrity by regulating Drp1 dynamics, and restoration of Arhgap26 protein level recovered the quality of Arhgap26-null oocytes. Importantly, we found an ARHGAP26 mutation in a patient with history of recurrent miscarriage by chromosomal microarray analysis. Altogether, our findings uncover a novel function of ARHGAP26 in the oocyte quality control and prevention of aneuploidy and provide a potential treatment strategy for infertile women caused by ARHGAP26 mutation.

Follicular Fluid Proteomic Analysis to Identify Predictive Markers of Normal Embryonic Development.

Ageing populations, mass "baby-free" policies and children born to mothers at the age at which they are biologically expected to become grandmothers are growing problems in most developed societies. Therefore, any opportunity to improve the quality of infertility treatments seems important for the survival of societies. The possibility of indirectly studying the quality of developing oocytes by examining their follicular fluids (hFFs) offers new opportunities for progress in our understanding the processes of final oocyte maturation and, consequently, for predicting the quality of the resulting embryos and personalising their culture. Using mass spectrometry, we studied follicular fluids collected individually during in vitro fertilisation and compared their composition with the quality of the resulting embryos. We analysed 110 follicular fluids from 50 oocyte donors, from which we obtained 44 high-quality, 39 medium-quality, and 27 low-quality embryos. We identified 2182 proteins by Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) using a TripleTOF 5600+ hybrid mass spectrometer, of which 484 were suitable for quantification. We were able to identify several proteins whose concentrations varied between the follicular fluids of different oocytes from the same patient and between patients. Among them, the most important appear to be immunoglobulin heavy constant alpha 1 (IgA1hc) and dickkopf-related protein 3. The first one is found at higher concentrations in hFFs from which oocytes develop into poor-quality embryos, the other one exhibits the opposite pattern. None of these have, so far, had any specific links to fertility disorders. In light of these findings, these proteins should be considered a primary target for research aimed at developing a diagnostic tool for oocyte quality control and pre-fertilisation screening. This is particularly important in cases where the fertilisation of each egg is not an option for ethical or other reasons, or in countries where it is prohibited by law.

Chii Jou Chan: The positives of being under "pressure".

Chii Jou Chan investigates how tissue hydraulics regulates mammalian development, with a special focus on folliculogenesis and oocyte quality control.

Maternal Gliadin Intake Reduces Oocyte Quality with Chromosomal Aberrations and Increases Embryonic Lethality through Oxidative Stress in a Caenorhabditis elegans Model.

Oocyte quality is essential for reproductive capacity, but it rapidly declines with age. In addition to aging, maternal nutrition is a major concern in maintaining oocyte quality. Gliadin, a major component of gluten, causes gluten toxicity, which has been reported in a variety of gluten-related disorders. The basis of gluten toxicity in reproduction is being understood using simple animal models such as Caenorhabditis elegans. In this study, we examined the effects of gliadin peptide (GP; amino acids 151-170) intake on oocyte quality control in C. elegans. We found that GP intake impaired oocyte quality through chromosomal aberrations and mitochondrial oxidative stress, which was suppressed by antioxidant treatment. The reduced oocyte quality by GP intake consequently increased embryonic lethality. Furthermore, the expression of oxidative stress-responding genes prdx-3 and gst-4 was significantly increased by GP intake. The increased DAF-16 activity by GP intake suggests that DAF-16 is a possible transactivator of these antioxidant genes. Taken together, GP intake reduced reproductive capacity in C. elegans by decreasing oocyte quality and increasing embryonic lethality through mitochondrial oxidative stress.

Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control.

p53 gene family members in humans and other organisms encode a large number of protein isoforms whose functions are largely undefined. Using Drosophila as a model, we find that a p53B isoform is expressed predominantly in the germline where it colocalizes with p53A into subnuclear bodies. It is only p53A, however, that mediates the apoptotic response to ionizing radiation in the germline and soma. In contrast, p53A and p53B are both required for the normal repair of meiotic DNA breaks, an activity that is more crucial when meiotic recombination is defective. We find that in oocytes with persistent DNA breaks p53A is also required to activate a meiotic pachytene checkpoint. Our findings indicate that Drosophila p53 isoforms have DNA lesion and cell type-specific functions, with parallels to the functions of mammalian p53 family members in the genotoxic stress response and oocyte quality control.

RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging

ABSTRACT There is increasing evidence that mitophagy, a specialized form of autophagy to degrade and clear long-lived or damaged mitochondria, is impaired in aging and age-related disease. Previous study has demonstrated the obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy. However, it remains unknown whether mitophagy functions in oocyte and what’s the regulatory mechanism in oocyte aging. In the study, when fully grown oocytes were treated with CCCP, an uncoupling agent to induce mitophagy, we found the activation of the PRKN-mediated mitophagy pathway accompanied the blockage of meiosis at metaphase I stage. Our result then demonstrated its association with the decreased activity of RAB7 and all the observed defects in CCCP treated oocytes could be effectively rescued by microinjection of mRNA encoding active RAB7Q67L or treatment with the RAB7 activator ML098. Further study indicated PRKN protein level as a rate-limiting factor to facilitate degradation of RAB7 and its GEF (guanine nucleotide exchange factor) complex CCZ1-MON1 through the ubiquitin-proteasome system. In GV oocytes collected during ovarian aging, we found the age-related increase of PINK1 and PRKN proteins and a significant decrease of RAB7 which resulted in defects of mitophagosome formation and the accumulation of damaged mitochondria. The age-related retardation of female fertility was improved after in vivo treatment of ML098. Thus, RAB7 activity is required to maintain the balance between mitophagy and chromosome stability and RAB7 activator is a good candidate to ameliorate age-related deterioration of oocyte quality. Abbreviations: ATG9: autophagy related 9A; ATP: adenosine triphosphate; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CCZ1: CCZ1 vacuolar protein trafficking and biogenesis associated; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAPs: GTPase-activating proteins; GEF: guanine nucleotide exchange factor; GV: germinal vesicle; GVBD: germinal vesicle breakdown; LAMP1: lysosomal-associated membrane protein 1; MI: metaphase I stage of meiosis; MII: metaphase II stage of meiosis; Mito: MitoTracker; mtDNA: mitochondrial DNA; MON1: MON1 homolog, secretory trafficking associated; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB7: RAB7, member RAS oncogene family; ROS: reactive oxygen species; TEM: transmission electron microscopy; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; UB: ubiquitin.

Impeding DNA Break Repair Enables Oocyte Quality Control

Oocyte quality control culls eggs with defects in meiosis. In mouse, oocyte death can be triggered by defects in chromosome synapsis and recombination, which involve repair of DNA double-strand breaks (DSBs) between homologous chromosomes. We show that RNF212, a SUMO ligase required for crossing over, also mediates oocyte quality control. Both physiological apoptosis and wholesale oocyte elimination in meiotic mutants require RNF212. RNF212 sensitizes oocytes to DSB-induced apoptosis within a narrow window as chromosomes desynapse and cells transition into quiescence. Analysis of DNA damage during this transition implies that RNF212 impedes DSB repair. Consistently, RNF212 is required for HORMAD1, a negative regulator of inter-sister recombination, to associate with desynapsing chromosomes. We infer that oocytes impede repair of residual DSBs to retain a "memory" of meiotic defects that enables quality-control processes. These results define the logic of oocyte quality control and suggest RNF212 variants may influence transmission of defective genomes.

CHK2 sets the stage for CK1 in oocyte quality control.

CHK2 sets the stage for CK1 in oocyte quality control.

Oocyte Quality Control: Causes, Mechanisms, and Consequences.

Oocyte quality and number are key determinants of reproductive life span and success. These variables are shaped in part by the elimination of oocytes that experience problems during the early stages of meiosis. Meiotic prophase-I marks an extended period of genome vulnerability in which epigenetic reprogramming unleashes retroelements and hundreds of DNA double-strand breaks (DSBs) are inflicted to initiate the programmed recombination required for accurate chromosome segregation at the first meiotic division. Expression of LINE-1 retroelements perturbs several aspects of meiotic prophase and is associated with oocyte death during the early stages of meiotic prophase I. Defects in chromosome synapsis and recombination also trigger oocyte loss, but typically at a later stage, as cells transition into quiescence and form primordial follicles. Interrelated pathways that signal defects in DSB repair and chromosome synapsis mediate this late oocyte attrition. Here, I review our current understanding of early and late oocyte attrition based on studies in mouse and describe how these processes appear to be both distinct and overlapping and how they help balance the quality and size of oocyte reserves to maximize fecundity.

Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level.

Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.

Analysis of the oligomeric state and transactivation potential of TAp73α

The proteins p73 and p63 are members of the p53 protein family and are involved in important developmental processes. Their high sequence identity with the tumor suppressor p53 has suggested that they act as tumor suppressors as well. While p63 has a crucial role in the maintenance of epithelial stem cells and in the quality control of oocytes without a clear role as a tumor suppressor, p73's tumor suppressor activity is well documented. In a recent study we have shown that the transcriptional activity of TAp63α, the isoform responsible for the quality control in oocytes, is regulated by its oligomeric state. The protein forms an inactive, dimeric and compact conformation in resting oocytes, while the detection of DNA damage leads to the formation of an active, tetrameric and open conformation. p73 shows a high sequence identity to p63, including those domains that are crucial in stabilizing its inactive state, thus suggesting that p73's activity might be regulated by its oligomeric state as well. Here, we have investigated the oligomeric state of TAp73α by size exclusion chromatography and detailed domain interaction mapping, and show that in contrast to p63, TAp73α is a constitutive open tetramer. However, its transactivation potential depends on the cellular background and the promoter context. These results imply that the regulation of p73's transcriptional activity might be more closely related to p53 than to p63.

What is the optimal condition for fertilization of IVM oocytes?

Application of in vitro maturation (IVM) is recently increasing for human infertility, especially to rescue patients of polycystic ovarian syndrome and ovarian hyperstimulation syndrome. To increase the application of IVM oocytes for embryo production and the efficiency of successful production of babies using IVM oocytes, quality control of oocytes and achievement of fertilization in the most suitable condition may be very important. In this paper, suitable conditions for fertilization of IVM oocytes will be discussed with recent knowledge about IVM and in vitro fertilization of oocytes in domestic animals. Currently, human oocytes are collected mainly from patients' ovaries 36 h following mild gonadotropin stimulation and used for IVM for 24-26 h. However, asynchronous progression of those oocytes to reach the metaphase-II stage may have occurred during the IVM culture. In the oocytes that have already progressed to the metaphase-II stage, sudden aging such as reduction in maturation promoting factor and MAP kinases will start to occur. Application of specific inhibitors of phosphodiesterase to control intracellular cAMP (cyclic adenosine monophosphate) level may be effective to synchronize timings of the germinal vesicle breakdown and consequently the meiotic progression of oocytes, and to improve the developmental competence. Furthermore, treatment of aging oocytes with caffeine appears to rescue them from reductions in maturation promoting factor and MAP kinases and to improve the developmental competence. Assessment methods to select oocytes with good quality may also be important to improve the successful rates.

DNA Damage in Oocytes Induces a Switch of the Quality Control Factor TAp63α from Dimer to Tetramer

SummaryTAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63α's activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ∼20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes.

Caenorhabditis elegans reproductive aging: Regulation and underlying mechanisms

Female reproductive decline is one of the first aging phenotypes in humans, manifested in increasing rates of infertility, miscarriage, and birth defects in children of mothers over 35. Recently, Caenorhabditis elegans (C. elegans) has been developed as a model to study reproductive aging, and several studies have advanced our knowledge of reproductive aging regulation in this organism. In this review, we describe our current understanding of reproductive cessation in C. elegans, including the relationship between oocyte quality, ovulation rate, progeny number, and reproductive span. We then discuss possible mechanisms of oocyte quality control, and provide an overview of the signaling pathways currently identified to be involved in reproductive span regulation in C. elegans. Finally, we extend the relevance of C. elegans reproductive aging studies to the issue of human female reproductive decline, and we discuss ideas concerning the relationship between reproductive aging and somatic longevity.

Two Regions in the N-terminal Domain of Ionotropic Glutamate Receptor 3 Form the Subunit Oligomerization Interfaces That Control Subtype-specific Receptor Assembly

The N-terminal domain (NTD) of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate glutamate receptors plays an important role in controlling subtype specific receptor assembly. To identify NTD subdomains involved in this process we generated AMPA glutamate receptor 3 (GluR3) mutants having intra-NTD substitutions with the corresponding regions of the kainate receptor GluR6 and tested their ability to form functional heteromers with wild-type subunits. The chimeric design was based on the homology of the NTD to the NTD of the metabotropic GluR1, shown to form two globular lobes and to assemble in dimers. Accordingly, the NTD was divided into four regions, termed here N1-N4, of which N1 and N3 correspond to the regions forming lobe-1 and N2 and N4 to those forming lobe-2. Substituting N1 or N3 impaired functional heteromerization but allowed protein-protein interactions. Conversely, exchanging N2 or N4 preserved functional heteromerization, although it significantly decreased homomeric activity, indicating a role in subunit folding. Moreover, a deletion in GluR3 corresponding to the hotfoot mouse mutation of the glutamate receptor delta2, covering part of N2, N3, and N4, impaired both homomeric and heteromeric oligomerization, thus explaining the null-like mouse phenotype. Finally, computer modeling suggested that the dimer interface, largely formed by N1, is highly hydrophobic in GluR3, whereas in GluR6 it contains electrostatic interactions, hence offering an explanation for the subtype assembly specificity conferred by this region. N3, however, is positioned perpendicular to the dimer interface and therefore may be involved in secondary interactions between dimers in the assembled tetrameric receptor.

Histochemistry and Cytochemistry of Glucose-6-Phosphate Dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)