Widespread Enhancer Dememorization and Promoter Priming during Parental-to-Zygotic Transition.

The epigenome plays critical roles in controlling gene expression and development. However, how the parental epigenomes transit to the zygotic epigenome in early development remains elusive. Here we show that parental-to-zygotic transition in zebrafish involves extensive erasure of parental epigenetic memory, starting with methylating gametic enhancers. Surprisingly, this occurs even prior to fertilization for sperm. Both parental enhancers lose histone marks by the 4-cell stage, and zygotic enhancers are not activated until around zygotic genome activation (ZGA). By contrast, many promoters remain hypomethylated and, unexpectedly, acquire histone acetylation before ZGA at as early as the 4-cell stage. They then resolve into either activated or repressed promoters upon ZGA. Maternal depletion of histone acetyltransferases results in aberrant ZGA and early embryonic lethality. Finally, such reprogramming is largely driven by maternal factors, with zygotic products mainly contributing to embryonic enhancer activation. These data reveal widespread enhancer dememorization and promoter priming during parental-to-zygotic transition.

Regulation of the mammalian maternal-to-embryonic transition by eukaryotic translation initiation factor 4E.

Eukaryotic translation initiation factor 4E (eIF4E) mediates cap-dependent translation. Genetic and inhibitor studies show that eIF4E expression is required for the successful transition from maternal to embryonic control of mouse embryo development. eIF4E was present in the oocyte and in the cytoplasm soon after fertilization and during each stage of early development. Functional knockout (Eif4e-/-) by PiggyBac [Act-RFP] transposition resulted in peri-implantation embryonic lethality because of the failure of normal epiblast formation. Maternal stores of eIF4E supported development up to the two- to four-cell stage, after which new expression occurred from both maternal and paternal inherited alleles. Inhibition of the maternally acquired stores of eIF4E (using the inhibitor 4EGI-1) resulted in a block at the two-cell stage. eIF4E activity was required for new protein synthesis in the two-cell embryo and Eif4e-/- embryos had lower translational activity compared with wild-type embryos. eIF4E-binding protein 1 (4E-BP1) is a hypophosphorylation-dependent negative regulator of eIF4E. mTOR activity was required for 4E-BP1 phosphorylation and inhibiting mTOR retarded embryo development. Thus, this study shows that eIF4E activity is regulated at key embryonic transitions in the mammalian embryo and is essential for the successful transition from maternal to embryonic control of development.

Longitudinal profiling of human androgenotes through single-cell analysis unveils paternal gene expression dynamics in early embryo development.

STUDY QUESTION: How do transcriptomics vary in haploid human androgenote embryos at single cell level in the first four cell cycles of embryo development? SUMMARY ANSWER: Gene expression peaks at the fourth cell cycle, however some androcytes exhibit unique transcriptional behaviors. WHAT IS KNOWN ALREADY: The developmental potential of an embryo is determined by the competence of the oocyte and the sperm. However, studies of the contribution of the paternal genome using pure haploid androgenotes are very scarce. STUDY DESIGN, SIZE, DURATION: This study was performed analyzing the single-cell transcriptomic sequencing of 38 androcytes obtained from 10 androgenote bioconstructs previously produced in vitro (de Castro et al., 2023). These results were analyzed through different bioinformatics software such as g: Profiler, GSEA, Cytoscape, and Reactome. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single cell sequencing was used to obtain the transcriptomic profiles of the different androcytes. The results obtained were compared between the different cycles studied using the DESeq2 program and functional enrichment pathways using g: Profiler, Cytoscape, and Reactome. MAIN RESULTS AND THE ROLE OF CHANCE: A wave of paternally driven transcriptomic activation was found during the third-cell cycle, with 1128 upregulated and 225 downregulated genes and the fourth-cell cycle, with 1373 upregulated and 286 downregulated genes, compared to first-cell cycle androcytes. Differentially expressed routes related to cell differentiation, DNA-binding transcription, RNA biosynthesis and RNA polymerase II transcription regulatory complex, and cell death were found in the third and fourth with respect to the first-cell cycle. Conversely, in the fourth cell cycle, 153 downregulated and 332 upregulated genes were found compared with third cell cycle, associated with differentially expressed processes related to E-box binding and zinc finger protein 652 (ZNF652) transcription factor. Further, significant overexpression of LEUTX, PRAMEF1, DUXA, RFPL4A, TRIM43, and ZNF675 found in androgenotes, compared to biparental embryos, highlights the paternal contributions to zygote genome activation. LARGE SCALE DATA: All raw sequencing data are available through the Gene Expression Omnibus (GEO) under accessions number: GSE216501. LIMITATIONS, REASONS FOR CAUTION: Extrapolation of biological events from uniparental constructs to biparental embryos should be done with caution. Maternal and paternal genomes do not act independently of each other in a natural condition. The absence of one genome may affect gene transcription of the other. In this sense, the haploid condition of the bioconstructs could mask the transcriptomic patterns of the single cells. WIDER IMPLICATIONS OF THE FINDINGS: The results obtained demonstrated the level of involvement of the human paternal haploid genome in the early stages of embryo development as well as its evolution at the transcriptomic level, laying the groundwork for the use of these bioconstructs as reliable models to dispel doubts about the genetic role played by the paternal genome in the early cycles of embryo development. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Instituto de Salud Carlos III (ISCIII) through the project 'PI22/00924', co-funded by European Regional Development Fund (ERDF); 'A way to make Europe'. F.D. was supported by the Spanish Ministry of Economy and Competitiveness through the Miguel Servet program (CPII018/00002). M.J.E. was supported by Instituto de Salud Carlos III (PI19/00577 [M.J.E.]) and FI20/00086. P.dC. was supported by a predoctoral grant for training in research into health (PFIS PI19/00577) from the Instituto de Salud Carlos III. All authors declare having no conflict of interest with regard to this trial.

microRNAs associated with the quality of follicular fluids affect oocyte and early embryonic development.

Purpose: Oocyte and embryo quality differs significantly among individuals. Follicular fluid (FF) is a solo environment of oocyte maturation and may flux into the oviduct. Supplementation of in vitro maturation (IVM) and culture (IVC) medium with extracellular vesicles of FFs supports oocyte maturation and embryonic development. We addressed a hypothesis that miRNA profiles in FFs are crucial background of oocyte maturation and embryonic development. Methods: FFs were collected from the ovaries of individual cows, and the FFs were classified into Good or Poor FF based on the developmental rate to the blastocyst stage of enclosed oocytes. miRNAs associated with the Good FFs were explored using small RNA sequencing. In addition, FFs were classified using the concentration of Good-FF-associated miRNAs. These classified FFs or miRNA were added to the IVM or IVC mediums. Results: Supplementation of IVM and IVC medium with Good FF improved embryonic development. Good FFs contained miR-151-3p and miR-425-5p at a high concentration compared with those in Poor FFs. FFs selected by the concentration of miR-151-3p and miR-425-5p improved oocyte maturation and embryonic development. Supplementation of IVM or IVC medium with either miR-151-3p or miR-425-5p improved embryonic development to the blastocyst stage. Conclusion: miRNAs were associated with the Good FFs determined oocyte maturation and embryonic development.

Histone acetyltransferases and histone deacetyl transferases play crucial role during oogenesis and early embryo development.

Dynamic epigenetic regulation is critical for proper oogenesis and early embryo development. During oogenesis, fully grown germinal vesicle oocytes develop to mature Metaphase II oocytes which are ready for fertilization. Fertilized oocyte proliferates mitotically until blastocyst formation and the process is called early embryo development. Throughout oogenesis and early embryo development, spatio-temporal gene expression takes place, and this dynamic gene expression is controlled with the aid of epigenetics. Epigenetic means that gene expression can be altered without changing DNA itself. Epigenome is regulated through DNA methylation and histone modifications. While DNA methylation generally ends up with repression of gene expression, histone modifications can result in expression or repression depending on type of modification, type of histone protein and its specific residue. One of the modifications is histone acetylation which generally ends up with gene expression. Histone acetylation occurs through the addition of acetyl group onto amino terminal of the core histone proteins by histone acetyltransferases (HATs). Contrarily, histone deacetylation is associated with repression of gene expression, and it is catalyzed by histone deacetylases (HDACs). This review article focuses on what is known about alterations in the expression of HATs and HDACs and emphasizes importance of HATs and HDACs during oogenesis and early embryo development.

Metabolomics Identifies a Panel of Diagnostic Biomarkers for Early Human Embryonic Development Arrest.

Early embryonic development arrest (EEDA) is a unique form of early spontaneous abortion in pregnant women, which is previously suggested to be associated with metabolic abnormalities. Noninvasive biomarkers would significantly improve its diagnosis and clinical outcome. Here, we performed a targeted metabolomics study in plasma from EEDA patients (n = 27) and normal pregnant women (NPW, n = 27) using liquid chromatography coupled with mass spectrometry (LC-MS) to identify potential diagnostic marker metabolites. Our results showed significantly different plasma metabolic profiles between EEDA patients and NPW. Particularly, EEDA patients showed significant alterations in amino acid, carbohydrate, and vitamin metabolism, which were characterized by 21 significantly increased metabolites and five decreased metabolites in plasma. Further receiver operating characteristic analysis showed that an optimal combination of S-methyl-5'-thioadenosine, kynurenine, leucine, and malate could be used as a panel of metabolites for EEDA diagnosis. The area under the curve of the metabolite panel was 0.941, suggesting a better performance than any single metabolite for the diagnosis of EEDA. In summary, our study identifies a panel of differential metabolites in plasma that could act as potential biomarkers for the diagnosis of EEDA in clinical settings.

Single intraovarian dose of stem cell- and platelet-secreted factors mitigates age-related ovarian infertility in a murine model.

BACKGROUND: Systemic administration of soluble factors from bone marrow-derived stem cells combined with activated platelet-rich plasma (SC-PRP) restored ovarian function, mediated through paracrine signaling, in murine models of chemotherapy-induced ovarian damage and human tissue from poor responder patients. However, the effects against age-related infertility and the efficacy of local administration have not been evaluated yet. OBJECTIVE: This study aimed to assess whether a single intraovarian dose of stem cells combined with activated platelet-rich plasma can recover ovarian function, oocyte quality, and developmental competence in older mice. STUDY DESIGN: The effects of stem cells combined with activated platelet-rich plasma against age-related infertility were assessed following controlled ovarian stimulation in an aging murine model reproducing 3 physiological stages of women's reproductive life, namely young, advanced maternal age, and menopausal (n=12 animals per group). Female mice were randomized to receive a single intraovarian injection (10 µL/ovary) of either saline, activated platelet-rich plasma, or stem cells combined with activated platelet-rich plasma. Seven days later, the mice were stimulated, naturally mated, and sacrificed to harvest their ovaries for histologic assessment and molecular analysis and their oviducts to evaluate oocyte maturation and to assess early embryo development. RESULTS: A single intraovarian injection of stem cells combined with activated platelet-rich plasma promoted follicle activation and development in young, advanced maternal age, and old mice. Furthermore, stem cells combined with activated platelet-rich plasma rescued fertility in older mice by enhancing the quantity and quality of ovulated mature oocytes and supporting early embryo development to the blastocyst stage in all the evaluated ages. These fertility outcomes were positively associated with mitochondrial quality, treatment-increased mitochondrial DNA copy numbers, and reduced oxidative damage and apoptosis. Finally, the effects observed by histologic analysis were supported at the proteomic level. Functional proteomic analyses revealed molecular mechanisms involved in oocyte maturation and quality, mitochondrial function, and recovery of the ovarian stroma. CONCLUSION: Bone marrow-derived stem cells combined with activated platelet-rich plasma is a promising treatment with the potential to improve the reproductive outcomes of women with age-related infertility, exceeding the restorative effects of platelet-rich plasma alone. Although further research in human ovarian samples is still required, the autologous nature of stem cell factors collected by noninvasive mobilization, their combination with platelet-rich plasma, and the local administration route suggest that stem cells combined with activated platelet-rich plasma treatment could be a potentially effective and safe application for future clinical practice.

Nucleoporin37 may play a role in early embryo development in human and mice.

Maternal-effect genes (MEGs) play an important role in maintaining the survival and development of mammalian embryos at the cleavage stage after fertilization. Despite long-term efforts, the MEGs that regulate preimplantation embryo development remain largely unknown. Here, using whole-exome sequencing and homozygosity mapping, we identified a potential candidate gene associated with early embryo development: nucleoporin37 (NUP37), a nucleoporin gene that encodes a member of the nuclear pore complexes and regulates nuclear pore permeability and nucleocytoplasmic transport. Moreover, we determined the temporal and spatial expression patterns of Nup37 in mouse oocytes and early embryos, and explored the role of NUP37 in oocyte maturation and preimplantation embryo development. Immunoprecipitation assays confirmed that yes-associated protein-1 (YAP1) binds to TEA domain transcription factor 4 (TEAD4) and NUP37. Furthermore, Nup37 gene knockdown reduced the nuclear import of YAP1 and down-regulated the expression of YAP1-TEAD pathway downstream genes Rrm2 and Rpl13 in early embryos. Our study provides evidence that maternal NUP37 contributes to the nuclear import of YAP1 and then activates the YAP1-TEAD pathway, a signalling pathway essential for zygotic genome activation. Nup37 may be a key gene involved in preimplantation embryo development in mammals.

PABPN1L mediates cytoplasmic mRNA decay as a placeholder during the maternal-to-zygotic transition.

Maternal mRNA degradation is a critical event of the maternal-to-zygotic transition (MZT) that determines the developmental potential of early embryos. Nuclear Poly(A)-binding proteins (PABPNs) are extensively involved in mRNA post-transcriptional regulation, but their function in the MZT has not been investigated. In this study, we find that the maternally expressed PABPN1-like (PABPN1L), rather than its ubiquitously expressed homolog PABPN1, acts as an mRNA-binding adapter of the mammalian MZT licensing factor BTG4, which mediates maternal mRNA clearance. Female Pabpn1l null mice produce morphologically normal oocytes but are infertile owing to early developmental arrest of the resultant embryos at the 1- to 2-cell stage. Deletion of Pabpn1l impairs the deadenylation and degradation of a subset of BTG4-targeted maternal mRNAs during the MZT. In addition to recruiting BTG4 to the mRNA 3'-poly(A) tails, PABPN1L is also required for BTG4 protein accumulation in maturing oocytes by protecting BTG4 from SCF-ßTrCP1 E3 ubiquitin ligase-mediated polyubiquitination and degradation. This study highlights a noncanonical cytoplasmic function of nuclear poly(A)-binding protein in mRNA turnover, as well as its physiological importance during the MZT.

Repro-protective role of royal jelly in phenylhydrazine-induced hemolytic anemia in male mice: Histopathological, embryological, and biochemical evidence.

To estimate the repro-protective effect of royal jelly (RJ) on phenylhydrazine (PHZ)-induced anemia's detrimental effects, 24 mature mice were divided into control group (0.10 mL normal saline; intra-peritoneally), RJ group (100 mg/kg/day; orally), experimental anemia (EA) group that received only PHZ (6 mg/100 g/48 h; intra-peritoneally), and RJ + EA (according to the previous prescription) group. After 35 days, testicular histoarchitecture, RNA damage in germinal cells, sperm characteristics, testicular total anti-oxidant capacity and malondialdehyde as well as serum testosterone levels, pre-implantation embryo development and cyclin D1 and c-myc mRNA levels at two-cell, morula and blastocyst stages were analyzed. Spermatogenesis indices were ameliorated following RJ co-administration. Moreover, RJ co-treatment reduced germinal cells RNA damage, improved sperm characteristics, boosted pre-implantation embryo development and restored androgenesis, and oxidant/anti-oxidant status. Co-administration of RJ also decreased mRNA levels of cyclin D1 and up-regulated those of c-myc in two-cell embryos, morulas and blastocysts. The findings suggest that RJ can play a repro-protective role in PHZ-induced anemia in mice through anti-oxidant defense system reinforcement and androgenesis restoration as well as cyclin D1 and c-myc expressions regulation.

ELOVL5 Participates in Embryonic Lipid Determination of Cellular Membranes and Cytoplasmic Droplets.

Embryonic lipids are crucial for the formation of cellular membranes and dynamically participate in metabolic pathways. Cells can synthesize simple fatty acids, and the elongation of fatty acids facilitates the formation of complex lipids. The aim of this work was to investigate the involvement of the elongation of very long chain fatty acid enzyme 5 (ELOVL5) in embryonic development and lipid determination. Bovine embryos were produced in vitro using a standard protocol and randomly divided to receive one of three treatments at Day 4: morpholino (Mo) gene expression knockdown assay for ELOVL5 (ELOVL5-Mo), Mo antisense oligonucleotides for the thalassemic ß-globulin human mRNA (technical control Mo), and placebo (biological control). The phenotypes of embryonic development, cell number, ELOVL5 protein abundance, lipid droplet deposits, and lipid fingerprint were investigated. No detrimental effects (p > 0.05) were observed on embryo development in terms of cleavage (59.4 ± 3.5%, 63.6 ± 4.1%, and 65.4 ± 2.2%), blastocyst production (31.3 ± 4.2%, 28.1 ± 4.9%, and 36.1 ± 2.1%), and blastocyst cell number (99.6 ± 7.7, 100.2 ± 6.2, 86.8 ± 5.6), respectively, for biological control, technical control Mo, and ELOVL5-Mo. ELOVL5 protein abundance and cytoplasmic lipid droplet deposition were increased (p < 0.05) in ELOVL5-Mo-derived blastocysts compared with the controls. However, seven lipid species, including phosphatidylcholines, phosphatidylethanolamines, and triacylglycerol, were downregulated in the ELOVL5-Mo-derived blastocysts compared with the biological control. Therefore, ELOVL5 is involved in the determination of embryonic lipid content and composition. Transient translational blockage of ELOVL5 reduced the expression of specific lipid species and promoted increased cytoplasmic lipid droplet deposition, but with no apparent deleterious effect on embryonic development and blastocyst cell number.

Control of nucleus positioning in mouse oocytes.

The position of the nucleus in a cell can instruct morphogenesis in some cases, conveying spatial and temporal information and abnormal nuclear positioning can lead to disease. In oocytes from worm, sea urchin, frog and some fish, nucleus position regulates embryo development, it marks the animal pole and in Drosophila it defines the future dorso-ventral axis of the embryo and of the adult body plan. However, in mammals, the oocyte nucleus is centrally located and does not instruct any future embryo axis. Yet an off-center nucleus correlates with poor outcome for mouse and human oocyte development. This is surprising since oocytes further undergo two extremely asymmetric divisions in terms of the size of the daughter cells (enabling polar body extrusion), requiring an off-centering of their chromosomes. In this review we address not only the bio-physical mechanism controlling nucleus positioning via an actin-mediated pressure gradient, but we also speculate on potential biological relevance of nuclear positioning in mammalian oocytes and early embryos.

The associations of urinary phthalate metabolites with the intermediate and pregnancy outcomes of women receiving IVF/ICSI treatments: A prospective single-center study.

BACKGROUND: Phthalate exposure was reported to induce defects in ovarian function, and further influence embryo development and pregnancy outcomes. However, the data about the associations of phthalates with intermediate and pregnancy outcomes of in vitro fertilization (IVF) cycles are scarce in the Chinese population. METHODS: A total of 663 women receiving IVF/intracytoplasmic sperm injection (ICSI) treatments in our center were enrolled in this analysis. They provided one urine sample on the day of oocyte retrieval. We measured urinary concentrations of eight phthalate metabolites. Generalized linear models were used to analyze the associations of urinary phthalate metabolites with ovarian response, fertilization, early embryo development, and pregnancy outcomes. RESULTS: Among all the phthalate metabolites, mono-n-butyl phthalate (MBP) had the highest urinary concentration with a median level of 101.51 µg/g creatinine (Cr). MBP concentration was inversely associated with normal fertilization odds (overall P-trend < 0.01). There was a significant correlation of monoethyl phthalate (MEP) with decreased odds of normal fertilization in medium-concentration group compared to low-concentration group (overall P-trend = 0.02). No significant associations of metabolite concentrations with the odds of good-quality embryos on day 3 or blastocyst formation were found. Monomethyl phthalate (MMP) and MEP in medium-concentration group reduced 22.4% (95% CI: 0.64-0.94, overall P-trend = 0.04) and 21.9% (95% CI: 0.64-0.95, overall P-trend = 0.05) of the odds to gain good-quality blastocyst compared to low-concentration group. The eight phthalate metabolites were not correlated to clinical pregnancy rate, live birth rate, or early miscarriage rate. There was no significant association of di (2-ethylhexyl) phthalate (DEHP) metabolites observed with any clinical outcomes in the total population. After excluding male infertility, mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) in medium-concentration group turned to be associated with a higher number of retrieved oocytes (overall P-trend = 0.04), whereas mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) in medium-concentration group was associated with a lower odds of normal fertilization compared to low-concentration group (overall P-trend = 0.02). CONCLUSIONS: Urinary MBP concentration was much higher compared to other phthalate metabolites in this cohort of Chinese IVF/ICSI women, and also higher than it was reported by studies in other countries. MBP showed adverse impacts on fertilization. MMP and MEP could affect blastocyst quality, but not embryo quality on day 3. DEHP metabolites didn't show consistent reproductive toxicities as demonstrated in previous studies.

Adipokines Expression and Effects in Oocyte Maturation, Fertilization and Early Embryo Development: Lessons from Mammals and Birds.

Some evidence shows that body mass index in humans and extreme weights in animal models, including avian species, are associated with low in vitro fertilization, bad oocyte quality, and embryo development failures. Adipokines are hormones mainly produced and released by white adipose tissue. They play a key role in the regulation of energy metabolism. However, they are also involved in many other physiological processes including reproductive functions. Indeed, leptin and adiponectin, the most studied adipokines, but also novel adipokines including visfatin and chemerin, are expressed within the reproductive tract and modulate female fertility. Much of the literature has focused on the physiological and pathological roles of these adipokines in ovary, placenta, and uterine functions. The purpose of this review is to summarize the current knowledge regarding the involvement of leptin, adiponectin, visfatin, and chemerin in the oocyte maturation, fertilization, and embryo development in both mammals and birds.

Absence of mitochondrial DNA methylation in mouse oocyte maturation, aging and early embryo development.

Mitochondrial DNA (mtDNA) is important for oxidative phosphorylation; dysfunctions can play a role in many mitochondrial diseases and can also affect the aging of cells and individuals. DNA methylation is an important epigenetic modification that plays a critical role in regulating gene expression. While recent studies have revealed the existence of mtDNA methylation there are still controversies about mtDNA methylation due to the special structure of mtDNA. Mitochondria and DNA methylation are both essential for regulating oocyte maturation and early embryo development, but whether mtDNA methylation changes during this process is unknown. By employing bisulfite sequencing, we found that in the process of mouse oocyte maturation, postovulatory oocyte aging, and early embryo development, all analyzed mitochondrial genes, including 16S-CpGI, DCR, ND6, 12S, and ATP8, lacked 5'mC. Thus, mtDNA methylation does not occur in the oocyte and early embryo.

Insights of the tubulin code in gametes and embryos: from basic research to potential clinical applications in humans†.

Microtubules are intracellular filaments that define in space and in time a large number of essential cellular functions such as cell division, morphology and motility, intracellular transport and flagella and cilia assembly. They are therefore essential for spermatozoon and oocyte maturation and function, and for embryo development. The dynamic and functional properties of the microtubules are in large part defined by various classes of interacting proteins including MAPs (microtubule associated proteins), microtubule-dependent motors, and severing and modifying enzymes. Multiple mechanisms regulate these interactions. One of them is defined by the high diversity of the microtubules themselves generated by the combination of different tubulin isotypes and by several tubulin post-translational modifications (PTMs). This generates a so-called tubulin code that finely regulates the specific set of proteins that associates with a given microtubule thereby defining the properties and functions of the network. Here we provide an in depth review of the current knowledge on the tubulin isotypes and PTMs in spermatozoa, oocytes, and preimplantation embryos in various model systems and in the human species. We focus on functional implications of the tubulin code for cytoskeletal function, particularly in the field of human reproduction and development, with special emphasis on gamete quality and infertility. Finally, we discuss some of the knowledge gaps and propose future research directions.

Melatonin promotes human oocyte maturation and early embryo development by enhancing clathrin-mediated endocytosis.

Embryo development potential and reproductive clinical outcomes are all deeply rooted in oocyte maturation. Melatonin has been reported to promote oocyte maturation as an antioxidant in nonprimate species. Its antioxidative functions also help reduce plasma membrane rigidity, which facilitates clathrin-mediated endocytosis (CME). Whether melatonin has effects on human oocyte maturation by regulating CME is worthy of exploration. In this study, we found that the optimal melatonin concentration for human oocyte maturation was 10-11  M, and the maturation rate of this group was 71.9% (P = .03). The metaphase II (MII) stage oocytes obtained by in vitro maturation with 10-11  M melatonin had a significantly higher fertilization rate (81.4% vs 61.4%, respectively, P = .017) and blastocyst rate (32.2% vs 15.8%, respectively, P = .039) compared to controls. During maturation, antioxidative melatonin greatly enhanced CME and decreased intra-oocyte cAMP level. The former was evidenced by the increasing numbers of coated pits and vesicles, and the upregulated expression of two major CME markers-clathrin and adaptor protein-2 (AP2). CME inhibitor dynasore increased intra-oocyte cAMP level and blocked oocyte maturation, and melatonin could partly rescue oocyte maturation and significantly elevate the expression of clathrin and AP2 in the presence of dynasore. Therefore, we conclude that melatonin could promote human oocyte maturation and early embryo development through enhancing CME.

Royal jelly protects male rats from heat stress-induced reproductive failure.

Royal jelly (RJ) as an antioxidant has been shown to have attenuated oxidative stress damages in reproductive organs. The objective was carried out the effects of RJ on sperm characteristics, sperm malondialdehyde (MDA) concentration and in vitro fertilisation (IVF) outcome in heat stress (HS) exposed male rats. Forty-eight male rats were randomly divided into eight groups; group 1 received normal saline, group 2 received RJ (100 mg kg-1  day-1 ; PO), groups 3, 4 and 5 were heat-stressed (43, 39 and 37°C for 20 min per day respectively) and groups 6, 7 and 8 were heat-stressed along with RJ (43, 39 and 37°C for 20 min per day, respectively, plus RJ at a dose of 100 mg kg-1  day-1 ; PO). The HS was induced through immersion of experimental rat scrotums in a water bath. After 48 days, the HS induced remarkable diminish in sperm motility, viability and fertilising potential along with reduced blastulation rate and enhanced sperm chromatin abnormality, MDA levels and DNA damage. Nevertheless, RJ co-administration improved sperm characteristics and early embryo development as well as sperm lipid peroxidation level. Our data suggest that RJ can effectively ameliorate the experimental HS-induced infertility in rats through MDA concentration restoration and sperm characteristics and pre-implantation embryo development improvement.

MicroRNA-29b regulates DNA methylation by targeting Dnmt3a/3b and Tet1/2/3 in porcine early embryo development.

MicroRNA-29b (miR-29b) is a member of the miR-29 family, which targets DNA methyltransferases (DNMTs) and ten eleven translocation enzymes (TETs), thereby regulating DNA methylation. However, the role of miR-29b in porcine early embryo development has not been reported. In this study, we examined the effects of miR-29b in porcine in vitro fertilization (IVF) embryos to investigate the mechanism by which miR-29b regulated DNA methylation. The interference of miR-29b by its special miRNA inhibitor significantly up-regulated Dnmt3a/b and Tet1 but downregulated Tet2/3; meanwhile it increased DNA methylation levels of the global genome and Nanog promoter region but decreased global DNA demethylation levels. The inhibition of miR-29b also resulted in a decrease in the development rate and quality of blastocysts. In addition, the pluripotency genes Nanog and Sox2 were significantly downregulated, and the apoptosis genes Bax and Casp3 were upregulated, but anti-apoptosis gene Bcl-2 was downregulated in blastocysts. Our study indicated that miR-29b could regulate DNA methylation mediated by miR29b- Dnmt3a/b - Tet1/2/3 signaling during porcine early embryo development.

The Potential Roles of the Apoptosis-Related Protein PDRG1 in Diapause Embryo Restarting of Artemia sinica.

High salinity and low temperatures can induce Artemia sinica to enter the diapause stage during embryonic development. Diapause embryos stop at the gastrula stage, allowing them to resist apoptosis and regulate cell cycle activity to guarantee normal development after diapause termination. P53 and DNA damage-regulated gene 1 (pdrg1) is involved in cellular physiological activities, such as apoptosis, DNA damage repair, cell cycle regulation, and promotion of programmed cell death. However, the role of pdrg1 in diapause and diapause termination in A. sinica remains unknown. Here, the full-length A. sinica pdrg1 cDNA (As-pdrg1) was obtained and found to contain 1119 nucleotides, including a 228 bp open reading frame (ORF), a 233 bp 5'-untranslated region (UTR), and a 658-bp 3'-UTR, which encodes a 75 amino acid protein. In situ hybridization showed no tissue specific expression of As-pdrg1. Quantitative real-time PCR and western blotting analyses of As-pdrg1 gene and protein expression showed high levels at 15-20 h of embryo development and a subsequent downward trend. Low temperatures upregulated As-pdrg1 expression. RNA interference for the pdrg1 gene in Artemia embryos caused significant developmental hysteresis. Thus, PDRG1 plays an important role in diapause termination and cell cycle regulation in early embryonic development of A. sinica.