Ectopic expression of human TUBB8 leads to increased aneuploidy in mouse oocytes.

Aneuploidy seriously compromises female fertility and increases incidence of birth defects. Rates of aneuploidy in human eggs from even young women are significantly higher than those in other mammals. However, intrinsic genetic factors underlying this high incidence of aneuploidy in human eggs remain largely unknown. Here, we found that ectopic expression of human TUBB8 in mouse oocytes increases rates of aneuploidy by causing kinetochore-microtubule (K-MT) attachment defects. Stretched bivalents in mouse oocytes expressing TUBB8 are under less tension, resulting in continuous phosphorylation status of HEC1 by AURKB/C at late metaphase I that impairs the established correct K-MT attachments. This reduced tension in stretched bivalents likely correlates with decreased recruitment of KIF11 on meiotic spindles. We also found that ectopic expression of TUBB8 without its C-terminal tail decreases aneuploidy rates by reducing erroneous K-MT attachments. Importantly, variants in the C-terminal tail of TUBB8 were identified in patients with recurrent miscarriages. Ectopic expression of an identified TUBB8 variant in mouse oocytes also compromises K-MT attachments and increases aneuploidy rates. In conclusion, our study provides novel understanding for physiological mechanisms of aneuploidy in human eggs as well as for pathophysiological mechanisms involved in recurrent miscarriages.

Antioxidant procyanidin B2 protects oocytes against cryoinjuries via mitochondria regulated cortical tension.

BACKGROUND: Irreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation. Antioxidants were always used to antagonist the oxidative stress caused by vitrification. However, the comprehensive mechanism underlying the protective role of antioxidants has not been studied. Procyanidin B2 (PCB2) is a potent natural antioxidant and its functions in response to vitrification are still unknown. In this study, the effects of PCB2 on vitrified-thawed oocytes and subsequent embryo development were explored, and the mechanisms underlying the protective role of PCB2 were systematically elucidated. RESULTS: Vitrification induced a marked decline in oocyte quality, while PCB2 could improve oocyte viability and further development after parthenogenetic activation. A subsequent study indicated that PCB2 effectively attenuated vitrification-induced oxidative stress, rescued mitochondrial dysfunction, and improved cell viability. Moreover, PCB2 also acts as a cortical tension regulator apart from strong antioxidant properties. Increased cortical tension caused by PCB2 would maintain normal spindle morphology and promote migration, ensure correct meiosis progression and finally reduce the aneuploidy rate in vitrified oocytes. Further study reveals that ATP biosynthesis plays a crucial role in cortical tension regulation, and PCB2 effectively increased the cortical tension through the electron transfer chain pathway. Additionally, PCB2 would elevate the cortical tension in embryo cells at morula and blastocyst stages and further improve blastocyst quality. What's more, targeted metabolomics shows that PCB2 has a beneficial effect on blastocyst formation by mediating saccharides and amino acids metabolism. CONCLUSIONS: Antioxidant PCB2 exhibits multi-protective roles in response to vitrification stimuli through mitochondria-mediated cortical tension regulation.

Mito-Q promotes porcine oocytes maturation by maintaining mitochondrial thermogenesis via UCP2 downregulation.

Mitochondrial thermogenesis is an adaptive response of cells to their surrounding stress. Oxidative stress is one of the common stresses during in vitro maturation (IVM) of oocytes, which leads to mitochondrial dysfunction. This study aimed to probe the effects of the mitochondria-targeted antioxidant Mito-Q on oocyte development and unravel the role of Mito-Q in mitochondrial ATP production and thermogenesis regulation. Our results showed that Mito-Q had a positive effect on porcine oocytes maturation and subsequent embryo development. During oocytes IVM, Mito-Q could reduce ATP levels and ROS, increase lipid droplets accumulation, induce autophagy, and maintain mitochondrial temperature stability. Moreover, in metaphase II (MII) oocytes, Mito-Q would induce mitochondrial uncoupling manifested by decreased ATP, attenuated mitochondrial membrane potential (MMP), and increased mitochondrial thermogenesis. Notably, the expression of mitochondrial uncoupling protein (UCP2) was significantly reduced in oocytes treated with Mito-Q. Further study indicated that specific depletion of UCP2 in oocytes also resulted in increased thermogenesis, decreased ATP and declined MMP, suggesting that UCP2 downregulation may participate in Mito-Q-induced mitochondrial uncoupling. In summary, our data demonstrate that Mito-Q promotes oocyte maturation in vitro and maintains the stability of mitochondrial thermogenesis by inhibiting UCP2 expression.

Proteomic profile of mouse oocytes after vitrification: A quantitative analysis based on 4D label-free technique.

Mature oocyte cryopreservation represents an important trend for future fertility preservation, however, the relatively low efficiency has hampered its clinical application. Proteomic profiling is a method of choice for the exploration of the molecular mechanism underlying cryoinjuries. Here, a systematic comparison of protein expression between fresh and vitrified oocytes was performed based on the 4D label-free technique, an informative method with high sensitivity. Our results indicated that the oocyte survival rate was significantly reduced after vitrification. Proteomic results showed that 32 proteins were up-regulated, while 77 proteins were down-regulated in vitrified oocytes compared with the fresh counterparts. Gene Ontology (GO) functional analysis revealed that differentially expressed proteins (DEPs) were involved in metabolism, mitochondrial function, cytoskeleton and other cell functions. Moreover, proteins that participated in signaling transduction mechanisms were the largest category based on Clusters of Orthologous Groups of protein/EuKaryotic Orthologous Groups (COG/KOG) functional classification. In addition, over-expressed DEPs were enriched for "nucleus", "protein binding", "membrane", "cytoplasm" as well as mitochondrial function. Furthermore, we discovered that the DEPs were clustered in pyruvate metabolism, citric acid (TCA) cycle and glucose metabolism by Protein-Protein Interaction (PPI) network evaluation. In conclusion, our data demonstrate that vitrification induces multi-level damages in oocytes, the dynamic proteomic profiling will provide systematic insights into uncovering the mechanism underlying cryoinjuries.

Procyanidin B2 Protects Aged Oocytes Against Meiotic Defects Through Cortical Tension Modulation.

Defects in meiotic process are the main factors responsible for the decreased developmental competence in aged oocytes. Our recent research indicated that natural antioxidant procyanidin B2 (PCB2) promoted maturation progress in oocytes from diabetic mice. However, the effect of PCB2 on aging-induced chromosome abnormalities and the underlying mechanism have not been explored. Here, we found that PCB2 recovered aging-caused developmental arrest during meiotic maturation, germinal vesicle breakdown (GVBD) rate was significantly higher in aged oocytes treated with PCB2 (P < 0.05). Furthermore, we discovered that cortical mechanics were altered during aging process, cortical tension-related proteins were aberrantly expressed in aged oocytes (P < 0.001). PCB2 supplementation efficaciously antagonized aging-induced decreased cortical tension (P < 0.001). Moreover, PCB2 restored spindle morphology (P < 0.01), maintained proper chromosome alignment (P < 0.05), and dramatically reduced reactive oxygen species (ROS) level (P < 0.05) in aged oocytes. Collectively, our results reveal that PCB2 supplementation is a feasible approach to protect oocytes from reproductive aging, contributing to the improvement of oocytes quality.

Artificially Increasing Cortical Tension Improves Mouse Oocytes Development by Attenuating Meiotic Defects During Vitrification.

Oocyte cryopreservation demonstrates great benefits in the conservation of animal germplasm resources and assisted reproductive technology. However, vitrification causes damages in oocytes, which would lead to the decrease of oocyte quality, and embryonic development post fertilization. Cytoskeleton plays an important role in regulating cell shape, organelle migration, cell division and mechanical signal transduction. Cortical tension is a reflection of the physiological state and contractile ability of cortical cytoskeleton. Appropriate cortical tension is prerequesite for normal oocyte meiosis. In the present study, oocyte cortical tension was examined by evaluating the levels of cortical tension-related protein pERM (Phospho-Ezrin/Radixin/Moesin) and pMRLC (Phospho-Myosin Light Chain 2). We found that the cortical tension of vitrified oocytes was decreased. Increasing cortical tension of vitrified oocytes by adding 10 µg/ml ConA during in vitro culture could significantly improve the polar body extrusion rate and embryo development. Furthermore, increasing the cortical tension could improve spindle positioning, maintain kinetochore-microtubule (KT-MT) attachment, strengthen spindle assembly checkpoint (SAC) activity, and reduce the aneuploidy rate in vitrified oocytes. In conclusion, vitrification induced a remarkable decrease in cortical tension, and increasing the cortical tension could rescue the meiosis defect and improve oocyte quality.

Nampt affects mitochondrial function in aged oocytes by mediating the downstream effector FoxO3a.

Maternal aging can impair the quality and decrease the developmental competence of ovulated oocytes. In this study, compromised germinal vesicle breakdown (GVBD) was found in aged mice oocytes. Furthermore, we observed increased reactive oxygen species (ROS) and mitochondrial Ca2+ levels, along with reduced mitochondrial temperature in aged oocytes. Maternal aging also changed the crotonylation level in oocytes. Forkhead box O3 (FoxO3a), a member of the forkhead protein family involved in the regulation of cell survival and life span reached a peak level in the metaphase II stage. Compared with a younger group, FoxO3a expression increased in aged oocytes. Intracellular localization of FoxO3a changed from the cytoplasm to chromatin in response to aging. The expression of the upstream regulator nicotinamide-phosphoribosyltransferase (Nampt) peaked in the GVBD stage. Moreover, Nampt expression was increased in aged oocytes, and more intense staining of Nampt was found in aged mice ovary. To further study the role of Nampt in mitochondrial function, specific agonist P7C3 and inhibitor FK866 were applied to aged oocytes, and FK866 significantly decreased adenosine triphosphate and mitochondrial membrane potential. In conclusion, mitochondrial dysfunction in aged oocytes was associated with elevated FoxO3a, and suppression of Nampt could further impair mitochondrial function.

Procyanidin B2 Improves Oocyte Maturation and Subsequent Development in Type 1 Diabetic Mice by Promoting Mitochondrial Function.

Type 1 diabetes (T1D) results in decreased oocyte quality and compromised early embryonic development. Procyanidin B2 (PB2) is a natural compound extracted from grape seeds and has strong antioxidant activity in vivo. This study evaluated the effect of PB2 on oocyte maturation in diabetic mice. Diabetic mice were induced by streptozotocin (STZ) injection. PB2 was supplemented in the in vitro maturation medium, and the ratio of germinal vesicle breakdown (GVBD) and polar body extrusion (PBE), reactive oxygen species (ROS) levels, mitochondrial function, developmental ability, as well as crotonylation at H4K5 were determined in oocytes. PB2 can promote the extrusion of PBE (88.34% vs. 75.02%, P < 0.05); reduce the generation of ROS (1.12 vs. 1.96, P < 0.05); and improve the level of mitochondrial membrane potential (0.87 vs. 0.79 Δφm, P < 0.05), ATP level (1.31 vs. 0.71 pmol, P < 0.05), and mitochondria temperature (618.25 vs. 697.39 pixels, P < 0.05). The addition of PB2 also improved the level of oocyte crotonylation at H4K5 (crH4K5) (47.26 vs. 59.68 pixels, P < 0.05) and increased the blastocyst rate (61.51% vs. 36.07%, P < 0.05) after parthenogenetic activation. Our results are the first to reveal a role for PB2 in promoting the viability of oocytes by regulating the mitochondrial function. Moreover, we uncover that PB2 can improve the level of crH4K5, which provides a new strategy to combat the decline in oocyte quality of diabetic.