Maintain Oocyte Quality Research Articles

The role of declining ataxia-telangiectasia-mutated (ATM) function in oocyte aging

Despite the advances in the understanding of reproductive physiology, the mechanisms underlying ovarian aging are still not deciphered. Recent research found an association between impaired ATM-mediated DNA double-strand break (DSB) repair mechanisms and oocyte aging. However, direct evidence connecting ATM-mediated pathway function decline and impaired oocyte quality is lacking. The objective of this study was to determine the role of ATM-mediated DNA DSB repair in the maintenance of oocyte quality in a mouse oocyte knockdown model. Gene interference, in vitro culture, parthenogenesis coupled with genotoxicity assay approaches, as well as molecular cytogenetic analyses based upon next-generation sequencing, were used to test the hypothesis that intact ATM function is critical in the maintenance of oocyte quality. We found that ATM knockdown impaired oocyte quality, resulting in poor embryo development. ATM knockdown significantly lowered or blocked the progression of meiosis in vitro, as well as retarding and reducing embryo cleavage after parthenogenesis. After ATM knockdown, all embryos were of poor quality, and none reached the blastocyst stage. ATM knockdown was also associated with an increased aneuploidy rate compared to controls. Finally, ATM knockdown increased the sensitivity of the oocytes to a genotoxic active metabolite of cyclophosphamide, with increased formation of DNA DSBs, reduced survival, and earlier apoptotic death compared to controls. These findings suggest a key role for ATM in maintaining oocyte quality and resistance to genotoxic stress, and that the previously observed age-induced decline in oocyte ATM function may be a prime factor contributing to oocyte aging.

Surfeit locus protein 4 modulates endoplasmic reticulum function and maintains oocyte quality

ABSTRACT Surfeit locus protein 4 is a cargo receptor mediating cargo transport from the endoplasmic reticulum lumen to the Golgi apparatus. Loss of Surf4 gene led to embryonic lethality in mice. However, the role of Surf4 during oocyte development remains unknown. In this study, we generated the mouse model with oocyte-specific knockout of Surf4 gene. We found that adult mice with deletion of Surf4 showed normal folliculogenesis, ovulation and fertility. However, loss of Surf4 slightly impaired oocyte quality, thus led to partial oocyte meiotic arrest and reduced ratio of blastocyst formation. Consistent with this, the distribution of endoplasmic reticulum was disturbed in Surf4-deficient oocytes in mice. These results demonstrated that although Surf4 is dispensable for female mouse fertility, Surf4 modulates endoplasmic reticulum arrangement and participates in regulation of developmental competence of oocytes.

Diminishing oocyte quality with advancing age is associated with deficiency of nitric oxide synthase cofactors, tetrahydrobiopterin, and zinc, in mouse oocytes

To study the implications of decreased zinc and tetrahydrobiopterin (H4B) associated with chronological aging on oocyte quality using a mouse model. H4B and zinc are essential cofactors for nitric oxide synthase (NOS), because they aid in electron transfer and dimeric stability, and their bioavailability is crucial in regulating NOS coupling. We have previously shown that sufficient levels of nitric oxide (NO) are essential for maintaining oocyte quality and activation, and NO levels decrease in the oocyte as a function of age. Thus, it is plausible that zinc and H4B may decrease as a function of age, resulting in NOS dysfunction with subsequent depletion of NO. Additionally, increased production of reactive oxygen species from the monomeric form can further disrupt oocyte quality and NO bioavailability. Experimental laboratory study. Laboratory. B6D2F1 mice. Sibling oocytes were retrieved from super-ovulated B6D2F1 mice from 3 age groups: 8-14 weeks (young breeders [YBs]), 48-52 weeks (retired breeders [RBs]), and 80-84 weeks (old animals [OAs]). Oocytes were scored for ooplasmic/spindle microtubule (MT) morphology, chromosomal alignment, and cortical granule (CG) intactness using immunofluorescence and confocal microscopy with 3 dimension image reconstruction and subjected to an high-performance liquid chromatography assay to measure the concentrations of H4B and its metabolites, as well as the zinc measurement using atomic absorption spectrophotometry. Oocyte scoring showed a reduction in "good" quality oocyte percentage as age increases, with YB having the highest percentage of quality oocytes followed by RB and OA. The high-performance liquid chromatography analysis showed a significant progressive decrease in total H4B in RB and OA (0.00098 picogram (pg)/oocyte and 0.00069 pg/oocyte, respectively) compared with YB (0.00125 pg/oocyte). Atomic absorbance spectrophotometry revealed a significant progressive decrease in zinc concentration in RB and OA compared with YB (8.45 pg/oocyte and 5.82 pg/oocyte vs. 10.05 pg/oocyte, respectively). Age-related diminution in oocyte quality is paralleled by a decline in the levels of H4B and zinc. The resultant deficiency in the oocytes can lead to the inability of NOS to maintain dimerization. Consequent uncoupling of NOS generates superoxide instead of NO, which participates in a multitude of reactions contributing to oxidative stress. Therefore, dysfunction of NOS secondary to zinc and H4B loss is a major mechanism involved in reactive oxygen species generation and oocyte quality deterioration related to the chronological age.

Zinc deficiency compromises the maturational competence of porcine oocyte by inducing mitophagy and apoptosis

Zinc, an essential trace mineral, plays a pivotal role in cell proliferation, maintenance of redox homeostasis, apoptosis, and aging. Serum zinc concentrations are reduced in patients with polycystic ovary syndrome (PCOS). However, the underlying mechanism of the effects of zinc deficiency on the female reproductive system, especially oocyte quality, has not been fully elucidated. Thus, we established an in vitro experimental model by adding N,N,N′,N′-Tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) into the culture medium, and to determine the potential regulatory function of zinc during porcine oocytes maturation. In the present study, we found that zinc deficiency caused aberrant meiotic progress, accompanied by the disrupted cytoskeleton structure in porcine oocytes. Zinc deficiency impaired mitochondrial function and dynamics, leading to the increase of reactive oxygen species (ROS) and acetylation level of the antioxidative enzyme superoxide dismutase 2 (SOD2), eventually induced the occurrence of oxidative stress and early apoptosis. Moreover, zinc deficiency perturbed cytosolic Ca2+ homeostasis, lipid droplets formation, demonstrating the aberrant mitochondrial function in porcine oocytes. Importantly, we found that zinc deficiency in porcine oocytes induced the occurrence of mitophagy by activating the PTEN-induced kinase 1/Parkin signaling pathway. Collectively, our findings demonstrated that zinc was a critical trace mineral for maintaining oocyte quality by regulating mitochondrial function and autophagy in porcine oocytes.

Vitellogenin accumulation leads to reproductive senescence by impairing lysosomal function.

The maintenance of proteostasis is essential for cellular and organism healthspan. How proteostasis collapse influences reproductive span remains largely unclear. In Caenorhabditis elegans, excess accumulation of vitellogenins, the major components in yolk proteins, is crucial for the development of the embryo and occurs throughout the whole body during the aging process. Here, we show that vitellogenin accumulation leads to reproduction cessation. Excess vitellogenin is accumulated in the intestine and transported into the germline, impairing lysosomal activity in these tissues. The lysosomal function in the germline is required for reproductive span by maintaining oocyte quality. In contrast, autophagy and sperm depletion are not involved in vitellogenin accumulation-induced reproductive aging. Our findings provide insights into how proteome imbalance has an impact on reproductive aging and imply that improvement of lysosomal function is an effective approach for mid-life intervention for maintaining reproductive health in mammals.

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes.

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Maternal contributions to pregnancy success: from gamete quality to uterine environment.

The establishment and maintenance of a pregnancy that goes to term is sine qua non for the long-term sustainability of dairy and beef cattle operations. The oocyte plays a critical role in providing the factors necessary for preimplantation embryonic development. Furthermore, the female, or maternal, environment where oocytes and embryos develop is crucial for the establishment and maintenance of a pregnancy to term. During folliculogenesis, the oocyte must sequentially acquire meiotic and developmental competence, which are the results of a series of molecular events preparing the highly specialized gamete to return to totipotency after fertilization. Given that folliculogenesis is a lengthy process in the cow, the occurrence of disease, metabolic imbalances, heat stress, or other adverse events can make it challenging to maintain oocyte quality. Following fertilization, the newly formed embryo must execute a tightly planned program that includes global DNA remodeling, activation of the embryonic genome, and cell fate decisions to form a blastocyst within a few days and cell divisions. The increasing use of assisted reproductive technologies creates an additional layer of complexity to ensure the highest oocyte and embryo quality given that in vitro systems do not faithfully recreate the physiological maternal environment. In this review, we discuss cellular and molecular factors and events known to be crucial for proper oocyte development and maturation, as well as adverse events that may negatively affect the oocyte; and the importance of the uterine environment, including signaling proteins in the maternal-embryonic interactions that ensure proper embryo development. We also discuss the impact of assisted reproductive technologies in oocyte and embryo quality and developmental potential, and considerations when looking into the prospects for developing systems that allow for in vitro gametogenesis as a tool for assisted reproduction in cattle.

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.

Mitochondrial E3 ubiquitin ligase MARCH5 is required for mouse oocyte meiotic maturation†.

As the most abundant organelles in oocytes, mitochondria play an important role in maintaining oocyte quality. Here, we report that March5, encoding a mitochondrial ubiquitin ligase that promotes mitochondrial elongation, plays a critical role in mouse oocyte meiotic maturation via regulating mitochondrial function. The subcellular localization of MARCH5 was similar to the mitochondrial distribution during mouse oocyte meiotic progression. Knockdown of March5 caused decreased ratios of the first polar body extrusion. March5-siRNA injection resulted in oocyte mitochondrial dysfunctions, manifested by increased reactive oxygen species, decreased ATP content as well as decreased mitochondrial membrane potential, leading to reduced ability of spindle formation and an increased ratio of kinetochore-microtubule detachment. Further study showed that the continuous activation of the spindle assembly checkpoint and the failure of Cyclin B1 degradation caused MI arrest and first polar body (PB1) extrusion failure in March5 knockdown oocytes. Taken together, our results demonstrated that March5 plays an essential role in mouse oocyte meiotic maturation, possibly via regulation of mitochondrial function and/or ubiquitination of microtubule dynamics- or cell cycle-regulating proteins.

Large RNP granules in Caenorhabditis elegans oocytes have distinct phases of RNA-binding proteins.

The germ line provides an excellent in vivo system to study the regulation and function of RNP granules. Germ granules are conserved germ line-specific RNP granules that are positioned in the Caenorhabditis elegans adult gonad to function in RNA maintenance, regulation, and surveillance. In Caenorhabditis elegans, when oogenesis undergoes extended meiotic arrest, germ granule proteins and other RNA-binding proteins assemble into much larger RNP granules whose hypothesized function is to regulate RNA metabolism and maintain oocyte quality. To gain insight into the function of oocyte RNP granules, in this report, we characterize distinct phases for four protein components of RNP granules in arrested oocytes. We find that the RNA-binding protein PGL-1 is dynamic and has liquid-like properties, while the intrinsically disordered protein MEG-3 has gel-like properties, similar to the properties of the two proteins in small germ granules of embryos. We find that MEX-3 exhibits several gel-like properties but is more dynamic than MEG-3, while CGH-1 is dynamic but does not consistently exhibit liquid-like characteristics and may be an intermediate phase within RNP granules. These distinct phases of RNA-binding proteins correspond to, and may underlie, differential responses to stress. Interestingly, in oocyte RNP granules, MEG-3 is not required for the condensation of PGL-1 or other RNA-binding proteins, which differs from the role of MEG-3 in small, embryonic germ granules. Lastly, we show that the PUF-5 translational repressor appears to promote MEX-3 and MEG-3 condensation into large RNP granules; however, this role may be associated with regulation of oogenesis.

3,3'-Diindolylmethane Supplementation Maintains Oocyte Quality by Reducing Oxidative Stress and CEP-1/p53-Mediated Regulation of Germ Cells in a Reproductively Aged Caenorhabditis elegans Model.

In recent decades, maternal age at first birth has increased, as has the risk of infertility due to rapidly declining oocyte quality with age. Therefore, an understanding of female reproductive aging and the development of potential modulators to control oocyte quality are required. In this study, we investigated the effects of 3,3′-diindolylmethane (DIM), a natural metabolite of indole-3-cabinol found in cruciferous vegetables, on fertility in a Caenorhabditis elegans model. C. elegans fed DIM showed decreased mitochondrial dysfunction, oxidative stress, and chromosomal aberrations in aged oocytes, and thus reduced embryonic lethality, suggesting that DIM, a dietary natural antioxidant, improves oocyte quality. Furthermore, DIM supplementation maintained germ cell apoptosis (GCA) and germ cell proliferation (GCP) in a CEP-1/p53-dependent manner in a reproductively aged C. elegans germ line. DIM-induced GCA was mediated by the CEP-1-EGL-1 pathway without HUS-1 activation, suggesting that DIM-induced GCA is different from DNA damage-induced GCA in the C. elegans germ line. Taken together, we propose that DIM supplementation delays the onset of reproductive aging by maintaining the levels of GCP and GCA and oocyte quality in a reproductively aged C. elegans.

Phosphoglycerate mutase family member 5 maintains oocyte quality via mitochondrial dynamic rearrangement during aging.

Decline in ovarian reserve with aging is associated with reduced fertility and the development of metabolic abnormalities. Once mitochondrial homeostasis is imbalanced, it may lead to poor reproductive cell quality and aging. However, Phosphoglycerate translocase 5 (PGAM5), located in the mitochondrial membrane, is associated with necroptosis, apoptosis, and mitophagy, although the underlying mechanisms associated with ovarian aging remain unknown. Therefore, we attempted to uncover whether the high phosphoglycerate mutant enzyme family member 5 (PGAM5) expression is associated with female infertility in cumulus cells, and aims to find out the underlying mechanism of action of PGAM5. We found that PGAM5 is highly expressed and positively associated with aging, and has the potential to help maintain and regulate mitochondrial dynamics and metabolic reprogramming in aging granulosa cells, ovaries of aged female mice, and elderly patients. PGAM5 undergoes activation in the aging group and translocated to the outer membrane of mitochondria, co‐regulating DRP1; thereby increasing mitochondrial fission. A significant reduction in the quality of mitochondria in the aging group, a serious imbalance, and a significant reduction in energy, causing metabolism shift toward glycolysis, were also reported. Since PGAM5 is eliminated, the mitochondrial function and metabolism of aging cells are partially reversed. A total of 70 patients undergoing in vitro fertilization (IVF) treatment were recruited in this clinical study. The high expression of PGAM5 in the cumulus cells is negatively correlated with the pregnancy rate of infertile patients. Hence, PGAM5 has immense potential to be used as a diagnostic marker.

Germ cell apoptosis is critical to maintain Caenorhabditis elegans offspring viability in stressful environments.

Maintaining reproduction in highly variable, often stressful, environments is an essential challenge for all organisms. Even transient exposure to mild environmental stress may directly damage germ cells or simply tax the physiology of an individual, making it difficult to produce quality gametes. In Caenorhabditis elegans, a large fraction of germ cells acts as nurse cells, supporting developing oocytes before eventually undergoing so-called physiological germ cell apoptosis. Although C. elegans apoptosis has been extensively studied, little is known about how germline apoptosis is influenced by ecologically relevant environmental stress. Moreover, it remains unclear to what extent germline apoptosis contributes to maintaining oocyte quality, and thus offspring viability, in such conditions. Here we show that exposure to diverse environmental stressors, likely occurring in the natural C. elegans habitat (starvation, ethanol, acid, and mild oxidative stress), increases germline apoptosis, consistent with previous reports on stress-induced apoptosis. Using loss-of-function mutant alleles of ced-3 and ced-4, we demonstrate that eliminating the core apoptotic machinery strongly reduces embryonic survival when mothers are exposed to such environmental stressors during early adult life. In contrast, mutations in ced-9 and egl-1 that primarily block apoptosis in the soma but not in the germline, did not exhibit such reduced embryonic survival under environmental stress. Therefore, C. elegans germ cell apoptosis plays an essential role in maintaining offspring fitness in adverse environments. Finally, we show that ced-3 and ced-4 mutants exhibit concomitant decreases in embryo size and changes in embryo shape when mothers are exposed to environmental stress. These observations may indicate inadequate oocyte provisioning due to the absence of germ cell apoptosis. Taken together, our results show that the central genes of the apoptosis pathway play a key role in maintaining gamete quality, and thus offspring fitness, under ecologically relevant environmental conditions.

Tet1 Deficiency Leads to Premature Ovarian Failure.

Tet enzymes participate in DNA demethylation and play critical roles in stem cell pluripotency and differentiation. DNA methylation alters with age. We find that Tet1 deficiency reduces fertility and leads to accelerated reproductive failure with age. Noticeably, Tet1-deficient mice at young age exhibit dramatically reduced follicle reserve and the follicle reserve further decreases with age, phenomenon consistent with premature ovarian failure (POF) syndrome. Consequently, Tet1-deficient mice become infertile by reproductive middle age, while age matched wild-type mice still robustly reproduce. Moreover, by single cell transcriptome analysis of oocytes, Tet1 deficiency elevates organelle fission, associated with defects in ubiquitination and declined autophagy, and also upregulates signaling pathways for Alzheimer’s diseases, but down-regulates X-chromosome linked genes, such as Fmr1, which is known to be implicated in POF. Additionally, Line1 is aberrantly upregulated and endogenous retroviruses also are altered in Tet1-deficient oocytes. These molecular changes are consistent with oocyte senescence and follicle atresia and depletion found in premature ovarian failure or insufficiency. Our data suggest that Tet1 enzyme plays roles in maintaining oocyte quality as well as oocyte number and follicle reserve and its deficiency can lead to POF.

Cellular and Molecular Events that Occur in the Oocyte during Prolonged Ovarian Storage in Sheep.

Simple SummaryEstablishing efficient in vitro embryo production (IVP) protocols in sheep usually requires prolonged transportation of post-mortem ovaries since adult animals are often slaughtered in abattoirs far from laboratories. In this study, different analyses were carried out to investigate important cellular and molecular aspects of hypoxic injury on excised ovaries over time in order to understand the factors jeopardizing the development of competent oocytes during prolonged transport times. We observed that, when ovaries were stored for more than 7 h, the quality and developmental potential of oocytes and cumulus cells were greatly reduced. Moreover, the use of medium TCM199 over saline solution also had deleterious effects. Beyond transport time, strategies aimed at reducing these damages may improve oocyte quality and developmental competence.For the past two decades, there has been a growing interest in the application of in vitro embryo production (IVP) in small ruminants such as sheep. To improve efficiency, a large number abattoir-derived ovaries must be used, and long distances from the laboratory are usually inevitable when adult animals are used. In that scenario, prolonged sheep ovary transportation may negatively affect oocyte developmental competence. Here, we evaluated the effect of ovary storage time (3, 5, 7, 9, 11 and 13 h) and the medium in which they were transported (TCM199 and saline solution) on oocyte quality. Thus, live/dead status, early apoptosis, DNA fragmentation, reduced glutathione (GSH) and reactive oxygen species (ROS) content, caspase-3 activity, mitochondrial membrane potential and distribution, and relative abundance of mRNA transcript levels were assessed in oocytes. After in vitro maturation (IVM), cumulus cell viability and quality, meiotic and fertilization competence, embryo rates and blastocyst quality were also evaluated. The results revealed that, after 7 h of storage, oocyte quality and developmental potential were significantly impaired since higher rates of dead oocytes and DNA fragmentation and lower rates of viable, matured and fertilized oocytes were observed. The percentage of cleavage, blastocyst rates and cumulus cell parameters (viability, active mitochondria and GSH/ROS ratio) were also decreased. Moreover, the preservation of ovaries in medium TCM199 had a detrimental effect on cumulus cells and oocyte competence. In conclusion, ovary transport times up to 5 h in saline solution are the most adequate storage conditions to maintain oocyte quality as well as developmental capacity in sheep. A strategy to rescue the poor developmental potential of stored oocytes will be necessary for successful production of high-quality embryos when longer ovarian preservation times are necessary.

Association of exosomal microRNAs in human ovarian follicular fluid with oocyte quality

The postponement of childbearing by women has led to an increase in infertility. The reproductive aging process leads to a decrease in both the quantity and quality of oocytes. The aim of this study was to investigate exosomal microRNAs in human ovarian follicular fluid and explore their potential association with oocyte quality. We collected ovarian follicle fluid from 68 patients and assigned the patients to A (superior oocyte quality) or B (poor oocyte quality) group according to their oocyte quality. Exosomal miRNAs were extracted, library constructed and sequenced using the Illumina HiSeq platform. Subsequently, we analyzed exosomal miRNA expression, predicted the miRNA target genes, and enriched Gene Ontology terms using GOSeq. Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed using miRanda. A total of 47 miRNAs were found to be significantly differentially expressed between group A and group B (p < 0.05). Among nine differentially expressed miRNAs that were previously known, seven were upregulated in group B. In silico analysis indicated that several of these exosomal miRNAs were involved in pathways implicated in oocyte quality. Analysis of the expression of exosomal miRNAs in human ovarian follicular fluid showed that they were critical for maintaining oocyte quality. Our findings provide the basis for further investigations of the functions of exosomal miRNAs in the ovarian microenvironment and suggest that these exosomal miRNAs may be potential biomarkers for evaluating oocyte quality. The findings are potentially important to maintain oocyte quality in clinical settings.

Mitochondrial Function in Modulating Human Granulosa Cell Steroidogenesis and Female Fertility.

Ovarian follicle steroidogenesis associated with embryo quality results in a successful pregnancy. Each follicle consists of an oocyte surrounded by granulosa cells, which secrete several steroid and peptide hormones. Follicles harvested from women who conceived after assisted reproductive therapy (ART) had significantly higher estradiol levels in follicular fluids than the follicles from women who failed to conceive after ART. The higher follicular estradiol levels correlate well with successful fertilization following ART. Mitochondria are the central sites for steroid hormone biosynthesis. The first and rate-limiting step in the biosynthesis of steroid hormones occurs in the mitochondria of granulosa cells. In the present study, we hypothesized that the mitochondria in granulosa cells are critical for maintaining oocyte quality and fertility capacity. This study aims to clarify the relationship between mitochondrial function and granulosa cell steroidogenesis, and the relationship between hormone levels and fertility capacity. Sera, follicular fluids and granulosa cells were obtained from individuals undergoing IVF-ET treatment. The oocyte numbers, oocyte quality, fertilization rate, and pregnancy rate were also recorded. The patients who provided the granulosa cells were further classified into four groups: endometriosis, ovarian endometrioma, endometriosis without ovarian endometrioma, and polycystic ovary syndrome (PCOS); patients with other female factor infertility and male factor infertility were used as controls. We measured the levels of estradiol (E2) by radioimmunoassay. Concurrently, we analyzed the mitochondrial mass and membrane potential, and apoptosis by flow cytometry using nonyl acridine orange, TMRE, Annexin V-FITC and PI. Mitochondrial morphology was visualized by transfection with pLV-mitoDsRed. In addition, we assessed the protein levels of steroidogenic enzymes, steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) by Western blot. The results showed significantly decreased serum E2 and follicular E2 levels, and decreased IVF outcomes, in the patients with endometriosis. Reduced mitochondrial mass and decreased mitochondrial membrane potential were correlated with lower E2. Furthermore, a significant decrease in StAR and 3β-HSD was found in patients with ovarian endometrioma. The enzyme levels of StAR and 3β-HSD were highly correlated with E2 levels. Finally, elevated cumulus cell apoptosis was found in the patient group with ovarian endometrioma and PCOS. In conclusion, mitochondrial dysfunction of human granulosa cells may contribute to the decline of steroidogenesis, decreased fertilization rate, oocyte maturation rate, and oocyte quality, and it can ultimately jeopardize fertility.

Molecular analysis of lipid uptake- and necroptosis-associated factor expression in vitrified-warmed mouse oocytes

BackgroundWe had previously demonstrated that vitrification reduces the levels of certain phospholipid classes, and that oocytes from aged mice show a similar lipidome alteration, even without vitrification. In the current investigation, we examined if vitrification-warming of mouse oocytes from young and aged mice causes any changes in molecular aspects of lipid-associated features.MethodsMetaphase II (MII) stage oocytes were harvested from young (10–14-week-old) and aged (45–54-week-old) mice by a superovulation regime with PMSG followed by hCG. We examined the status of the intracellular lipid pool and the integrity of the plasma membrane by staining oocytes with BODIPY 500/510 and CellMask live dyes. Expression of lipid uptake- and necroptosis-associated genes was assessed by quantitative PCR analyses, in oocytes from young and old mice, before and after vitrification. Localization patterns of two crucial necroptosis proteins, phosphorylated MLKL (pMLKL) and phosphorylated RIPK1 (pRIPK1) were examined in mouse oocytes by immunofluorescence staining. Necrostain-1 (Nec1), an inhibitor of RIPK1, was used to examine if RIPK1 activity is required to maintain oocyte quality during vitrification.ResultsWe confirmed that vitrified-warmed oocytes from aged mice showed noticeable decrease in both CellMask and BODIPY 500/510 dyes. Among the lipid uptake-associated genes, Cd36 expression was higher in oocytes from aged mice. Necroptosis is a type of programmed cell death that involves damage to the plasma membrane, eventually resulting in cell rupture. The expression of necroptosis-associated genes did not significantly differ among groups. We observed that localization patterns of pMLKL and pRIPK1 were unique in mouse oocytes, showing association with microtubule organizing centers (MTOCs) and spindle poles. pMLKL was also localized on kinetochores of MII chromosomes. Oocytes treated with Nec1 during vitrification showed a decreased survival rate, indicating the importance of RIPK1 activity in oocyte vitrification.ConclusionsWe report that oocytes from aged mice show differential expression of CD36, which suggests that CD36-mediated lipid uptake may be influenced by age. We also show for the first time that pMLKL and pRIPK1 exhibit unique localization pattern in mouse oocytes and this may suggest role(s) for these factors in non-necroptosis-associated cellular processes.

Resveratrol treatment increases mitochondrial biogenesis and improves viability of porcine germinal-vesicle stage vitrified-warmed oocytes

Vitrification induces mitochondrial dysfunction in warmed oocytes, and degeneration and biogenesis of mitochondria are crucial for maintaining oocyte quality. The present study addresses a hypothesis that treatment of vitrified-warmed oocytes with resveratrol could improve the viability of oocytes by activating mitochondrial biosynthesis. Cumulus oocyte complexes (COCs) collected from gilt ovaries were vitrified, warmed, and cultured in a medium containing vehicle or 1 μM resveratrol. Resveratrol treatment improved survival, maturation, and mitochondrial membrane potential of vitrified-warmed oocytes, but did not improve the development into blastocysts after parthenogenetic activation. Resveratrol treatment increased mitochondrial synthesis, as determined by the expression levels of TOMM20 and mitochondrial DNA copy number, in vitrified-warmed oocytes, but not in non-vitrified oocytes. In addition, the effect of resveratrol treatment on mitochondrial synthesis was reduced by EX527, a SIRT1 inhibitor. Resveratrol treatment of vitrified-warmed oocytes increased the expression levels of genes involved in mitochondrial synthesis (TFAM, POLG, and PGC1α) and increased nuclear translocation of NRF2. Furthermore, vitrification induced mitophagy, as confirmed by a reduction in TOMM20 expression and decreased p62 aggregation, whereas resveratrol treatment did not affect these mitophagic features. In conclusion, vitrification induced mitochondrial clearance in oocytes, whereas activation of SIRT1 by resveratrol treatment facilitated the recovery of vitrified-warmed oocytes through the activation of mitochondrial synthesis.

The ubiquitin ligase KBTBD8 regulates PKM1 levels via Erk1/2 and Aurora A to ensure oocyte quality.

Tight control of energy metabolism is essential for normal cell function and organism survival. PKM (pyruvate kinase, muscle) isoforms 1 and 2 originate from alternative splicing of PKM pre-mRNA. They are key enzymes in oxidative phosphorylation and aerobic glycolysis, respectively, and are essential for ATP generation. The PKM1:PKM2 expression ratio changes with development and differentiation, and may also vary under metabolic stress and other conditions. Until now, there have been no reports about the function and regulation of PKM isozymes in oocytes. Here, we demonstrate that PKM1 or PKM2 depletion significantly disrupts ATP levels and mitochondrial integrity, and exacerbates free-radical generation and apoptosis in mouse oocytes. We also show that KBTBD8, a female fertility factor in the KBTBD ubiquitin ligase family, selectively regulates PKM1 levels through a signaling cascade that includes Erk1/2 and Aurora A kinases as intermediates. Finally, using RNA sequencing and protein network analysis, we identify several regulatory proteins that may be govern generation of mature PKM1 mRNA. These results suggest KBTBD8 affects PKM1 levels in oocytes via a KBTBD8→Erk1/2→Aurora A axis, and may also affect other essential processes involved in maintaining oocyte quality.