Inheritance of perturbed methylation and metabolism caused by uterine malnutrition via oocytes.

BACKGROUND: Undernourishment in utero has deleterious effects on the metabolism of offspring, but the mechanism of the transgenerational transmission of metabolic disorders is not well known. In the present study, we found that undernourishment in utero resulted in metabolic disorders of female F1 and F2 in mouse model. RESULTS: Undernutrition in utero induced metabolic disorders of F1 females, which was transmitted to F2 females. The global methylation in oocytes of F1 exposed to undernutrition in utero was decreased compared with the control. KEGG analysis showed that genes with differential methylation regions (DMRs) in promoters were significantly enriched in metabolic pathways. The altered methylation of some DMRs in F1 oocytes located at the promoters of metabolic-related genes were partially observed in F2 tissues, and the expressions of these genes were also changed. Meanwhile, the abnormal DNA methylation of the validated DMRs in F1 oocytes was also observed in F2 oocytes. CONCLUSIONS: These results indicate that DNA methylation may mediate the transgenerational inheritance of metabolic disorders induced by undernourishment in utero via female germline.

Epigallocatechin gallate improves the quality of diabetic oocytes.

BACKGROUND: Maternal diabetes compromises the quality and developmental potential of oocytes. Therefore, it is important to study how to ameliorate the adverse effects of diabetes on oocyte quality. Epigallocatechin gallate (EGCG) has a variety of physiological activities, including anti-inflammatory, antioxidant, and anti-diabetes. In the present study, we evaluated the effect of EGCG on the maturation of diabetic oocytes in vitro. OBJECTIVE: Investigating the role of EGCG in restoring the adverse effects of diabetes on oocyte quality. METHODS: Diabetes mouse model was established by a single injection of streptozotocin (STZ). Oocytes were collected and matured in vitro with/without EGCG in M16 medium. RESULTS: Compared with control, diabetic oocytes have a higher frequency of spindle defects and chromosome misalignment, but EGCG effectively reduces the incidence of oocytes with abnormal spindle assembly and chromosome mismatches. Moreover, the abnormal mitochondrial membrane potential (MMP) of diabetic oocytes is significantly alleviated by EGCG, and the reduced expression of genes regulating mitochondrial fusion (Mfn1 and Mfn2) and fission (Drp1) in diabetic oocytes is significantly increased while EGCG is added. EGCG also decreases the higher level of reactive oxygen species (ROS) in diabetic oocytes that may be regulated by the increased expression of superoxide dismutase 1 (Sod1) and superoxide dismutase 2 (Sod2). EGCG can also reduce the DNA damage of diabetic oocytes. CONCLUSIONS: Our results suggest that EGCG, at least partially, improve the quality of diabetic oocytes.

Protocatechuic Acid Delays Postovulatory Oocyte Ageing in Mouse.

SCOPE: Tea is a popular beverage worldwide and has many health functions. Protocatechuic acid (PCA) is an important bioactive component of tea and has benefit to health. In some cases, oocytes after ovulation may miss the optimal fertilization time and enter a postovulatory ageing process. Therefore, to investigate the role of PCA in delaying oocyte ageing is aimed. METHODS AND RESULTS: Metaphase II (MII) oocytes aged in vitro are randomly divided into three groups: control, aged, and aged + PCA. PCA treatment (30 µM) reduces the fragmentation rate and the incidence of abnormal spindle morphology and chromosome misalignment of oocytes aged 24 h in vitro. The mitochondrial dysfunction of aged oocytes, such as decreased mitochondrial membrane potential and excessive accumulation of reactive oxygen (ROS), is also alleviated by PCA. PCA also delays apoptosis of aged oocytes, and improves the sperm binding capacity. Otherwise, aged oocytes treated with PCA have a higher fertilization rate and blastocyst rate compared with untreated aged oocytes in vitro. CONCLUSION: PCA is an important bioactive ingredient of tea that improves aged oocyte quality, suggesting that PCA is available to improve the quality of aged oocytes in vitro.

Epigallocatechin gallate improves meiosis maturation against Diazinon exposure in porcine oocytes.

Diazinon (DZN) is a refractory organophosphorus pesticide (OP) in the surrounding environment due to its overuse in agriculture. The antioxidant activity of Epigallocatechin gallate (EGCG) from green tea is at least 100 times greater than that of vitamin C. This study aimed to study the effects of DZN on the meiotic maturation of porcine oocytes, as well as the protective roles of EGCG. Firstly, the effects of DZN and EGCG on meiotic nuclear maturation of porcine oocytes were detected, and then embryonic development was investigated by chemical parthenogenetic activation. Next, the spindle assembly, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA damage, and finally the early apoptosis of oocytes were examined by immunofluorescence staining. The results revealed that DZN exposure significantly reduced the quality of porcine oocytes, such as failure of nuclear and cytoplasmic maturation, evidenced by abnormal spindle assembly, disordered chromosome alignment, low MMP, observably increased ROS, severe DNA damage, and early apoptosis. Appropriate EGCG could significantly reduce all these defects caused by DZN. In conclusion, EGCG can help prevent the harm that DZN exposure can do. These findings offer convincing support for enhancing the oocyte quality from EGCG through daily ordinary beverages.

Melatonin improves meiosis maturation against diazinon exposure in mouse oocytes.

AIMS: Organophosphorus pesticide diazinon (DZN) has adverse effects on animals and humans by direct contact or the spread of food chain. The antioxidant melatonin has protective effects on female reproduction. This study aimed to explore the effects of DZN on meiosis maturation in mouse cumulus oocyte complexes (COCs) and the effects of melatonin. MAIN METHODS: Different concentrations of DZN and melatonin were added during the in vitro maturation of COCs. Then we detected the extrusion rate of the first polar body, the number of sperms binding to oocyte, mitochondrial membrane potential, reactive oxygen species (ROS), early apoptosis. Subsequently, the expression of Juno, CX37, CX43 and ERK1/2 were detected by immunofluorescence staining and Western blotting. KEY FINDINGS: DZN exposure results in the failure of nuclear and cytoplasmic maturation of oocyte meiosis. Destruction of repositioning and function of mitochondria increases the levels of ROS and early apoptosis. The DZN-exposed oocytes express less Juno resulting to bind less sperms than normal. The loss of gap junctions and failure to activate ERK1/2 also contribute to the failure of cytoplasmic maturation. All these ultimately lead to the poor oocyte quality and low fertility. Appropriate melatonin can effectively restore all these defects. SIGNIFICANCE: Under DZN exposure, melatonin can significantly improve the quality of oocytes, and melatonin promotes oocyte maturation by protecting gap junction and restoring ERK1/2 pathway, which is a new breakthrough for improving female fertility.

Tea polyphenols alleviate the adverse effects of diabetes on oocyte quality.

Maternal diabetes mellitus reduces oocyte quality, such as abnormalities of spindle assembly and chromosome segregation, mitochondrial dysfunction, decrease of fertilization rate, increase of ROS, and so on. So, it is important to research how to restore the decreased oocyte quality induced by maternal diabetes mellitus. Polyphenols are the most abundant bioactive components of green tea. It is reported that tea polyphenols have many health functions, for instance anti-oxidation, anti-inflammation, anti-obesity, and anti-diabetes. Thus, we hypothesize that tea polyphenols may play a crucial role in alleviating adverse effects of diabetes on oocyte quality. In the present study, we researched the effects of tea polyphenols on diabetic oocyte maturation in vitro. Compared with the control, oocytes from diabetic mice displayed a lower maturation rate and a higher frequency of spindle defects and chromosome misalignment. However, tea polyphenols significantly increased the oocyte maturation rate, and reduced the incidence of abnormal spindle assembly and chromosome segregation. Tea polyphenols also obviously decreased the reactive oxygen species (ROS) levels in diabetic oocytes, and increased the expression of antioxidant genes (Sod1 and Sod2). Abnormal mitochondrial membrane potential was also alleviated in diabetic oocytes, and the expression of genes regulating mitochondrial fusion (Opa1, Mfn1 and Mfn2) and fission (Drp1) was significantly increased while tea polyphenols were added. Meanwhile, tea polyphenols reduced DNA damage in diabetic oocytes which may be mediated by the increased expression of Rad51, related to DNA damage repair. Our results suggest that tea polyphenols would, at least partially, restore the adverse effects of diabetes mellitus on oocyte quality.

Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy.

Mitochondrial replacement therapy (MRT) has been used to prevent maternal transmission of disease-causing mutations in mitochondrial DNA (mtDNA). However, because MRT requires nuclear transfer, it carries the risk of mtDNA carryover and hence of the reversion of mtDNA to pathogenic levels owing to selective replication and genetic drift. Here we show in HeLa cells, mouse embryos and human embryos that mtDNA heteroplasmy can be reduced by pre-labelling the mitochondrial outer membrane of a donor zygote via microinjection with an mRNA coding for a transmembrane peptide fused to an autophagy receptor, to induce the degradation of the labelled mitochondria via forced mitophagy. Forced mitophagy reduced mtDNA carryover in newly reconstructed embryos after MRT, and had negligible effects on the growth curve, reproduction, exercise capacity and other behavioural characteristics of the offspring mice. The induction of forced mitophagy to degrade undesired donor mtDNA may increase the clinical feasibility of MRT and could be extended to other nuclear transfer techniques.

Correction: Potential role of tea extract in oocyte development.

Correction for 'Potential role of tea extract in oocyte development' by Lei Zhao et al., Food Funct., 2021, 12, 10311-10323, DOI: 10.1039/D1FO01725J.

Repeated Superovulation Accelerates Primordial Follicle Activation and Atresia.

For humans, ARTs (assisted reproductive technologies) have become the most effective method to treat subfertility/infertility in clinic. To obtain enough oocytes during ART, ovarian stimulation is performed by exogenous hormones, and some patients undergo several ovarian stimulation cycles. Although some adverse effects of ARTs on women and offspring are reported, few studies are focused on the effects of multiple superovulation on ovarian reserve. In the present study, we found that repeated superovulation significantly reduced primordial follicle number and the serum AMH. Compared to the decreased antral follicle number, the expression of genes related to primordial follicle activation, such as Foxo3, Akt, and Rptor, and the atretic follicle number in ovaries were increased by superovulation times. We further found that repeated superovulation reduced the plasma level of FSH, LH, and estradiol, and increased the expression of genes related to apoptosis (Bax, Casp3 (caspase-3), Casp8, and Casp9) in granulosa cells, providing evidence that repeated superovulation disrupted the balance between survival and death in granulosa cells. In summary, our results suggest that repeated superovulation has adverse effects on folliculogenesis.

Potential role of tea extract in oocyte development.

Tea is the second most popular beverage in the world and beneficial to health. It has been demonstrated that tea polyphenols can reduce the risk of diseases, such as cancers, diabetes, obesity, Alzheimer's disease, etc. But the knowledge of tea extract on the female germline is limited. Folliculogenesis is a complicated process and prone to be affected by ROS. Tea polyphenols can reduce the accumulation of ROS in folliculogenesis and affect oocyte maturation. Tea extract also influences granulosa cell proliferation and expansion during oocyte growth and maturation. However, the studies about the benefits of tea extract on female germline are few, and the underlying mechanisms are obscure. In the present study, we will mainly discuss the effects of tea extract on ovarian function, oocyte maturation, and the underlying possible mechanisms, and according to the discussion, we suggest that tea extract may have benefits for oocytes at an appropriate dose.

Effects of intrauterine exposure to 2,3',4,4',5-pentachlorobiphenyl on the reproductive system and sperm epigenetic imprinting of male offspring.

Polychlorinated biphenyls (PCBs) are a class of persistent organic environmental pollutants with a total of 209 homologs. The homolog 2,3',4,4',5-pentachlorobiphenyl (PCB118) is one of the most important dioxin-like PCBs and is highly toxic. PCB118 can accumulate in human tissues, serum and breast milk, which leads to direct exposure of the fetus during development. In the present study, pregnant mice were exposed to 0, 20 and 100 µg/kg/day of PCB118 during the stage of fetal primordial germ cell migration. Compared with the control group, we found morphological alterations of the seminiferous tubules and a higher sperm deformity rate in the male offspring in the treatment groups. Furthermore, the methylation patterns in the treatment groups of the imprinted genes H19 and Gtl2 in the sperm were altered in the male offspring. We also characterized the disturbance of the expression levels of DNA methyltransferase 1 (Dnmt1), Dnmt3a, Dnmt3b, Dnmt3l, and Uhrf1. The results indicated that intrauterine exposure to low doses of PCB118 could significantly damage the reproductive health of the male offspring. Therefore, attention should be paid to the adverse effects of PCB118 exposure during pregnancy on the reproductive system of male offspring.

Paraquat Reduces the Female Fertility by Impairing the Oocyte Maturation in Mice.

Paraquat (PQ) is a widely used non-selective and oxidizing herbicide in farmland, orchards, flower nursery, and grassland. Overuse of PQ will accumulate in the body and affect the reproduction in mammals. In this study, we found that PQ could reduce the female fertility by oral administration for 21 days in mice. PQ exposure could impair the nuclear maturation by perturbing the spindle assembly and kinetochore-microtubule attachment to cause the misaligned chromosomes during meiosis. In the meantime, PQ exposure disturbed the mitochondrial distribution and enhanced the level of reactive oxygen species and early apoptosis, which thereby deteriorated the early embryo development. Also, PQ administration could cause some changes in epigenetic modifications such as the level of H3K9me2 and H3K27me3. Therefore, PQ administration reduces the female fertility by impairing the nuclear and cytoplasmic maturation of oocytes in mice.

Multiple superovulations alter histone modifications in mouse early embryos.

It is demonstrated that repeated superovulation has deleterious effects on mouse ovaries and cumulus cells. However, little is known about the effects of repeated superovulation on early embryos. Epigenetic reprogramming is an important event in early embryonic development and could be easily disrupted by the environment. Thus, we speculated that multiple superovulations may have adverse effects on histone modifications in the early embryos. Female CD1 mice were randomly divided into four groups: (a) spontaneous estrus cycle (R0); (b) with once superovulation (R1); (c) with three times superovulation at a 7-day interval (R3) and (d) with five times superovulation at a 7-day interval (R5). We found that repeated superovulation remarkably decreased the fertilization rate. With the increase of superovulation times, the rate of early embryo development was decreased. The expression of Oct4, Sox2 and Nanog was also affected by superovulation in blastocysts. The immunofluorescence results showed that the acetylation level of histone 4 at lysine 12 (H4K12ac) was significantly reduced by repeated superovulation in mouse early embryos (P < 0.01). Acetylation level of histone 4 at lysine 16 (H4K16ac) was also significantly reduced in pronuclei and blastocyst along with the increase of superovulation times (P < 0.01). H3K9me2 and H3K27me3 were significantly increased in four-cell embryos and blastocysts. We further found that repeated superovulation treatment increased the mRNA level of histone deacetylases Hdac1, Hdac2 and histone methyltransferase G9a, but decreased the expression level of histone demethylase-encoding genes Kdm6a and Kdm6b in early embryos. In a word, multiple superovulations alter histone modifications in early embryos.

Tributyltin oxide exposure impairs mouse oocyte maturation and its possible mechanisms.

Tributyltin oxide (TBTO) has been widely used as marine antifouling composition, preservative, biocide, and a stabilizer in plastic industry. Previous studies have indicated that TBTO can cause immunotoxicity as an environmental pollutant. However, little is known about its reproductive toxicity, especially on female oocyte maturation and the underlying mechanisms. In this study, mouse oocytes were cultured with different concentrations of TBTO in vitro, and several crucial events during meiotic maturation were evaluated. We found that the first polar body extrusion rate was significantly reduced, which reflected the disruption of meiotic maturation. The rate of abnormal spindle organization increased significantly, accompanied with a higher rate of chromosome misalignment. In addition, TBTO treatment increased reactive oxygen species generation markedly, which also accelerated the early-stage apoptosis. Moreover, heterogeneous mitochondrial distribution, mitochondrial dysfunction, and higher rate of aneuploidy were detected, which consequently disrupted in vitro fertilization. In conclusion, our results indicated that TBTO exposure could impair mouse oocyte maturation by affecting spindle organization, chromosome alignment, mitochondria functions, oxidative stress, and apoptosis.

Causative Mutations and Mechanism of Androgenetic Hydatidiform Moles.

Androgenetic complete hydatidiform moles are human pregnancies with no embryos and affect 1 in every 1,400 pregnancies. They have mostly androgenetic monospermic genomes with all the chromosomes originating from a haploid sperm and no maternal chromosomes. Androgenetic complete hydatidiform moles were described in 1977, but how they occur has remained an open question. We identified bi-allelic deleterious mutations in MEI1, TOP6BL/C11orf80, and REC114, with roles in meiotic double-strand breaks formation in women with recurrent androgenetic complete hydatidiform moles. We investigated the occurrence of androgenesis in Mei1-deficient female mice and discovered that 8% of their oocytes lose all their chromosomes by extruding them with the spindles into the first polar body. We demonstrate that Mei1-/- oocytes are capable of fertilization and 5% produce androgenetic zygotes. Thus, we uncover a meiotic abnormality in mammals and a mechanism for the genesis of androgenetic zygotes that is the extrusion of all maternal chromosomes and their spindles into the first polar body.

High-glucose concentrations change DNA methylation levels in human IVM oocytes.

STUDY QUESTION: What are the effects of high-glucose concentrations on DNA methylation of human oocytes? SUMMARY ANSWER: High-glucose concentrations altered DNA methylation levels of Peg3 and Adiponectin in human in vitro maturation oocytes. WHAT IS KNOWN ALREADY: Maternal diabetes has a detrimental influence on oocyte quality including epigenetic modifications, as shown in non-human mammalian species. STUDY DESIGN, SIZE, DURATION: Immature metaphase I (MI) stage oocytes of good quality were retrieved from patients who had normal ovarian potential and who underwent ICSI in the Reproductive Medicine Center of People's Hospital of Zhengzhou University. MI oocytes were cultured in medium with different glucose concentrations (control, 10 mM and 15 mM) in vitro and 48 h later, oocytes with first polar body extrusion were collected to check the DNA methylation levels. PARTICIPANTS/MATERIALS, SETTING, METHODS: MI oocytes underwent in vitro maturation (IVM) at 37°C with 5% mixed gas for 48 h. Then the mature oocytes were treated with bisulfite buffer. Target sequences were amplified using nested or half-nested PCR and the DNA methylation status was tested using combined bisulfite restriction analysis (COBRA) and bisulfite sequencing (BS). MAIN RESULTS AND THE ROLE OF CHANCE: High-glucose concentrations significantly decreased the first polar body extrusion rate. Compared to controls, the DNA methylation levels of Peg3 in human IVM oocytes were significantly higher in 10 mM (P < 0.001) and 15 mM (P < 0.001) concentrations of glucose. But the DNA methylation level of H19 was not affected by high-glucose concentrations in human IVM oocytes. We also found that there was a decrease in DNA methylation levels in the promoter of adiponectin in human IVM oocytes between controls and oocytes exposed to 10 mM glucose (P = 0.028). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: It is not clear whether the alterations are beneficial or not for the embryo development and offspring health. The effects of high-glucose concentrations on the whole process of oocyte maturation are still not elucidated. Another issue is that the number of oocytes used in this study was limited. WIDER IMPLICATIONS OF THE FINDINGS: This is the first time that the effects of high-glucose concentration on DNA methylation of human oocytes have been elucidated. Our result indicates that in humans, the high risk of chronic diseases in offspring from diabetic mothers may originate from abnormal DNA modifications in oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the fund of National Natural Science Foundation of China (81401198) and Doctor Foundation of Qingdao Agricultural University (1116008).The authors declare that there are no potential conflicts of interest relevant to this article.

Toxic effects and possible mechanisms of hydrogen sulfide and/or ammonia on porcine oocyte maturation in vitro.

Previous studies suggest that hydrogen sulfide (H2S) and ammonia (NH3) are two major air pollutants which can cause damage to porcine health. However, the mechanisms underlying toxic effects of these compounds on porcine oocyte maturation are not clear. To clarify the mechanism, we evaluated the oocyte quality by detecting some events during oocytes maturation. In our study, porcine oocytes were cultured with different concentrations of Na2S and/or NH4Cl in vitro and the rate of the first polar body extrusion decreased significantly. Also, actin filament was seriously disrupted to damage the cytoskeleton which resulted in reduced rate of oocyte maturation. We explored the reactive oxygen species (ROS) generation and found that the ROS level was increased significantly after Na2S treatment but not after NH4Cl treatment. Moreover, early stage apoptosis rate was significantly increased and autophagy protein LC3 B expression level was higher in oocytes treated with Na2S and/or NH4Cl, which might be caused by ROS elevation. Additionally, exposure to Na2S and/or NH4Cl also caused ROS generation and early apoptosis in cumulus cells, which might further affect oocyte maturation in vitro. In summary, our data suggested that exposure to H2S and/or NH3 decreased porcine oocyte maturation in vitro, which might be caused by actin disruption, ROS generation, early apoptosis and autophagy.

The role of L-type calcium channels in mouse oocyte maturation, activation and early embryonic development.

Calcium ion fluctuation is closely related to the transformation of cell cycle. However, little is known about the function of L-type calcium channel in mammalian oocyte and embryo development. We thus studied the roles of L-type calcium channel in mouse oocyte meiotic maturation, parthenogenetic activation and early embryonic development. We used the antagonist Amlodipine to block L-type calcium channel. Oocytes or zygotes were cultured to different time points with 0 µM, 10 µM, 30 µM and 50 µM Amlodipine. Then we checked the rate of first polar body extrusion, spindle formation, asymmetric division parthenogenetic activation and early embryo cleavage. The results showed that Amlodipine treatment did not affect germinal vesicle breakdown, but caused disruption of cytoskeleton organization, symmetric division, formation of mature oocytes with a large polar body, or reduced the first polar body extrusion, depending on its concentrations. Amlodipine treatment also resulted in decreased parthenogenetic activation and arrested early embryonic development. Overall, these data suggest that proper function of L-type calcium channel is critical for oocyte maturation, activation, and early embryonic development.