Environmentally relevant estrogens impaired spermatogenesis and sexual behaviors in male and female zebrafish.

Environmental estrogens (EEs) are found extensively in natural waters and negatively affect fish reproduction. Research on the reproductive toxicity of EEs mixtures in fish at environmentally relevant concentrations is scarce. In this study, adult male zebrafish were exposed for 60 days to EES (a mixture of EEs), EE2-low (5.55 ng/L, with an estrogenic potency equal to EES), and EE2-high (11.1 ng/L). After exposure, the expression levels of vtg1, vtg3, and esr1 in the livers in EES-treated fish remained unaltered, whereas they were significantly increased in EE2-treated fish. Both EE2-high and EES exposures notably reduced the gonad somatic index and sperm count. A disrupted spermatogenesis was also observed in the testes of EE2-high- and EES-exposed fish, along with an alteration in the expression of genes associated with spermatogonial proliferation (pcna, nanog), cell cycle transition (cyclinb1, cyclind1), and meiosis (aldh1a2, cyp26a1, sycp3). Both EE2 and EES significantly lowered plasma 11-ketotestosterone levels in males, likely by inhibiting the expression level of genes for its synthesis (scc, cyp17a1 and cyp11b2), and increased 17ß-estradiol (E2) levels, possibly through upregulating the expression of cyp19a1a. A significant increase in tnfrsf1a expression and the tnfrsf1a/tnfrsf1b ratio in EE2-high and EES-treated males also suggests increased apoptosis via the extrinsic pathway. Further investigation showed that both EE2-high and EES diminished the sexual behavior of male fish, accompanied with reduced E2 levels in the brain and the expression of genes in the kisspeptin/gonadotropin-releasing hormone system. Interestingly, the sexual behavior of unexposed females paired with treated males was also reduced, indicating a synergistic effect. This study suggests that EES have a more severe impact on reproduction than EE2-low, and EEs could interfere not only with spermatogenesis in fish, but also with the sexual behaviors of both exposed males and their female partners, thereby leading to a more significant disruption in fish reproduction.

Bisphenol A interferes with lncRNA Fhadlos2 and RUNX3 association in adolescent mouse ovary.

Bisphenol A (BPA) has a number of adverse effects on the reproductive development of females. In particular, the mechanism of disruption of ovarian development in adolescent mice is still unclear. Based on transcriptome sequencing results, a differentially expressed lncRNA, Fhad1os2, was detected in the ovaries of BPA-exposed pubertal mice. In our study, the lncRNA Fhad1os2, localized in the ovarian granulosa cell cytoplasm, could regulate the proliferation of mouse ovarian granulosa cells. Mechanistically, the results of RNA pull-down experiments as well as mass spectrometry analysis showed that ERα, an interfering signaling molecule of BPA, could directly bind lncRNA Fhad1os2 and decrease the transcription of lncRNA Fhad1os2 in response to the estrogen-like effect of BPA. BPA exposure also caused abnormal lncRNA Fhad1os2 pulldown protein-related signaling pathways in the ovaries of adolescent mice. Furthermore, lncRNA Fhad1os2 interacted with RUNX3, a transcription factor related to follicle development and hormone synthesis. As a negative regulator, lncRNA Fhad1os2 transactivated the expression of Runx3, which in turn induced RUNX3 to positively regulate aromatase (Cyp19a1) expression in mouse ovarian granulosa cells and promote estrogen synthesis. In conclusion, our study indicates that BPA exposure interferes with ERα-regulated lncRNA Fhad1os2 interactions with RUNX3 in pubertal mice, affecting estrogen synthesis in mouse granulosa cells and contributing to premature ovarian maturation in pubertal mice.

BPA exposure enhances the metastatic aggression of ovarian cancer through the ERα/AKT/mTOR/HIF-1α signaling axis.

Long-term exposure to bisphenol A (BPA) in humans may promote ovarian cancer development. In present study, the mechanisms by which BPA mediates the aggression metastatic behavior of ovarian cancer were investigated in vitro/in vivo. The results showed that BPA (10 µM) significantly promoted the proliferation, migration and invasion of human ovarian cancer cells (ES-2 and OVCAR-3 cells); moreover, it promoted ES-2 and OVCAR-3 cell glucose uptake, lactic acid release and intracellular ATP synthesis. After administration of 5 µg/kg/day BPA, tumor volume was increased compared with that in control group. KEGG and GO enrichment analyses showed that the genes from ES-2 cell in 10 µM BPA-treated group were enriched mainly in central carbon metabolism and PI3K-AKT signaling pathway. Then, qRT‒PCR and western blotting results showed that BPA (10 µM) increased the mRNA and protein expression levels of glycolysis-related genes and mTOR, p-AKT HIF-1α and ERα in vitro/vivo; whereas this effect was reduced after treatment with the ERα inhibitor methyl-piperidino-pyrazole. Furthermore, coimmunoprecipitation and mass spectrometry showed that BPA promoted the direct interaction of ERα with lactate dehydrogenase A. These results show that BPA directly promoted the proliferation, migration and invasion of ovarian cancer cells through the ERα/AKT/mTOR/HIF-1α signaling axis to enhance glycolysis.

CircRNF144B/miR-342-3p/FBXL11 axis reduced autophagy and promoted the progression of ovarian cancer by increasing the ubiquitination of Beclin-1.

Circular RNAs (circRNAs) can regulate autophagy and ovarian cancer (OC) progression. However, autophagy-associated circRNAs involved in OC progression are largely unknown. Bioinformatics, RNA sequencing, and qRT-PCR were conducted to detect circRNF144B expression in OC as well as its relationship with patient prognosis. Functional experiments were used to determine the effects of circRNF144B on the proliferation, mobility and autophagy of OC. Double luciferase reporter assays, immunoprecipitation, and ubiquitination detection were performed to determine the molecular mechanisms of circRNF144B in autophagy and OC progression. CircRNF144B was elevated in OC tissues with low autophagy levels, and associated with poor prognosis. CircRNF144B promoted the malignant biological properties of OC cells, and inhibited the autophagy. Mechanistically, circRNF144B acts as a sponge for miR-342-3p and inhibits miR-342-3p-induced degradation of lysine demethylase 2 A (FBXL11) mRNA, leading to elevated FBXL11 protein levels. Elevated FBXL11 promoted the ubiquitination and degradation of Beclin-1, thus inhibiting autophagy. In conclusion, CircRNF144B increased FBXL11 level by sponging miR-342-3p, whereas elevated FBXL11 promoted the ubiquitination and protein degradation of Beclin-1, thus suppressing autophagy flux and promoting OC progression. Thus, circRNF144B may be an effective target for OC therapy.

Maternal DDB1 regulates apoptosis and lineage differentiation in porcine preimplantation embryos.

CONTEXT: Maternal-effect genes (MEGs) play a critical role in modulating both cellular and molecular biology events in preimplantation embryonic development. Damage-specific DNA binding protein 1 (DDB1) is a gene that participates in meiotic resumption, ovulation, and embryonic stem cell maintenance. Its function in preimplantation development is not well-studied. AIMS: We aimed to explore the expression pattern, genomic heritage, and potential molecular mechanisms of DDB1 in preimplantation embryos in porcine. METHODS: In this study, RNA interference, microinjection, RT-qPCR, immunofluorescence staining and single-cell RNA sequencing were used to explore the molecular function of DDB1 in porcine preimplantation embryos. KEY RESULTS: DDB1 was found to be expressed in germinal vesicle (GV) and Meiosis II (MII) oocytes and in preimplantation embryos. We confirmed it is a MEG. DDB1 -deficient blastocysts had a significantly reduced number of trophectoderm cells, an increased apoptotic cell number and increased apoptosis index. According to a next-generation sequencing (NGS) analysis, 236 genes (131 upregulated and 105 downregulated) significantly changed in the DDB1 -deficient morula. The myeloid leukaemia factor 1 (MLF1 ) and yes-associated protein 1 (YAP1 ) expressions were significantly upregulated and downregulated respectively, in the DDB1 -deficient morula. In combination with the decreased expression of TEAD4 , CDX2 , GATA3 , OCT4 , and NANOG and the increased expression of SOX2 in the blastocyst, DDB1 may play a role in determining lineage differentiation and pluripotency maintenance. CONCLUSIONS: DDB1 is a MEG and it plays a crucial role in porcine preimplantation embryonic development. IMPLICATIONS: This study provides a theoretical basis for further understanding the molecular mechanisms of preimplantation embryo development.

AgNPs reduce reproductive capability of female mouse for their toxic effects on mouse early embryo development.

Silver nanoparticles (AgNPs) are widely applied in the field of personal protection for their powerful toxic effects on cells, and recently, a new type of vaginal gel with AgNPs is used to protect the female reproductive tract from microbes and viruses. However, a high risk of AgNPs to the fetus and the underlying mechanism of AgNPs to interfere in embryo development still remain unclear. Thus, this study investigated the impact of two drugs of vaginal gel with AgNPs on reproductive capability of the female mouse by animal experiment. Then, kinetics of AgNPs affecting embryo development was investigated by in vitro embryos culturing, and cell membrane potential (CMP) of zygotes was analyzed by DiBAC4(3) staining. Results indicated that one of the drugs of vaginal gel certainly injured embryo development in spite of no apparent histological change found in ovaries and uteruses of drug-treated mice. In vitro embryo culturing discovered that the toxic effect of AgNPs on embryo development presented particle sizes and dose dependent, and AgNP treatment could rapidly trigger depolarization of the cell membrane of zygotes. Moreover, AgNPs changed the gene expression pattern of Oct-4 and Cdx2 in blastocysts. All these findings suggest that AgNPs can interfere with normal cellular status including cell membrane potential, which has not been noticed in previous studies on the impact of AgNPs on mammalian embryos. Thus, findings of this study alarm us the risk of applying vaginal gel with AgNPs in individual caring and protection of the female reproductive system.

AgNPs reduce reproductive capability of female mouse for their toxic effects on mouse early embryo development.

Silver nanoparticles (AgNPs) are widely applied in the field of personal protection for their powerful toxic effects on cells, and recently, a new type of vaginal gel with AgNPs is used to protect the female reproductive tract from microbes and viruses. However, a high risk of AgNPs to the fetus and the underlying mechanism of AgNPs to interfere in embryo development still remain unclear. Thus, this study investigated the impact of two drugs of vaginal gel with AgNPs on reproductive capability of the female mouse by animal experiment. Then, kinetics of AgNPs affecting embryo development was investigated by in vitro embryos culturing, and cell membrane potential (CMP) of zygotes was analyzed by DiBAC4(3) staining. Results indicated that one of the drugs of vaginal gel certainly injured embryo development in spite of no apparent histological change found in ovaries and uteruses of drug-treated mice. In vitro embryo culturing discovered that the toxic effect of AgNPs on embryo development presented particle sizes and dose dependent, and AgNP treatment could rapidly trigger depolarization of the cell membrane of zygotes. Moreover, AgNPs changed the gene expression pattern of Oct-4 and Cdx2 in blastocysts. All these findings suggest that AgNPs can interfere with normal cellular status including cell membrane potential, which has not been noticed in previous studies on the impact of AgNPs on mammalian embryos. Thus, findings of this study alarm us the risk of applying vaginal gel with AgNPs in individual caring and protection of the female reproductive system.

Distal bare metal stent implantation during thoracic endovascular aortic repair is beneficial to treat complicated type B aortic dissection: A systematic review and meta-analysis.

OBJECTIVE: To explore whether thoracic endovascular aortic repair (TEVAR) plus distal bare metal stent (BMS) implantation leads to favorable clinical outcomes compared with standard TEVAR in treating acute complicated type B aortic dissection. METHODS: Relevant publications were found through a precise search of PubMed, Cochrane Library, and EMBASE. Count data were calculated as the odd ratio (OR)and 95% confidence interval (CI) using the Mantel-Haenszel statistical method, quantitative data were calculated as mean difference and 95% CI using Inverse Variance statistical method. When the data heterogeneity was large, with an I2 > 50%, a random-effects model and sensitivity analysis were performed. The analysis tool we used was the software Revman 5.3 and G*power 3.1. RESULTS: There were 7 publications involving 958 patients who were enrolled ultimately. The incidence of unplanned secondary intervention and postoperative adverse events in the TEVAR + BMS were lower than standard TEVAR (OR, 0.42, (95% CI, 0.23 to 0.75); OR, 0.57, (95% CI, 0.37 to 0.90)), and the pooled number needed to treat was 15 for unplanned secondary intervention and 15 for postoperative adverse events. There were no significant difference in the aortic-related or all-cause 30-day mortality (OR, 0.81, (95% CI, 0.25 to 2.61); OR, 0.47, (95% CI, 0.18 to 1.22)), aortic-related, all-cause mortality at least 6 months or incidence of the postoperative endoleak (OR, 0.47, (95% CI, 0.17 to 1.32); OR, 0.42, (95% CI, 0.17 to 1.06); OR, 0.81, (95% CI, 0.32 to 2.05)). CONCLUSION: There is no significant outcome difference except for reduced reintervention but this does not seem to adversely affect survival. It is unclear whether this justifies the additional cost of placing it in every complicated type B aortic dissection regardless of anatomy after standard TEVAR alone. Besides, more data are needed to verify the adjunctive distal bare metal stents' performance at different dissection stages.

Identification of transcriptome differences in goat ovaries at the follicular phase and the luteal phase using an RNA-Seq method.

The ovaries, the main female reproductive organs, directly mediate ovulation and reproductive hormone secretion. These complex physiological processes are regulated by multiple genes and pathways. However, there is a lack of research on goat ovaries, and the molecular mechanisms underlying the signaling pathways remain unclear. In this study, Illumina HiSeq 4000 sequencing was used to sequence the transcriptomes of goat ovaries. The expression patterns of differentially expressed mRNAs in goat ovaries at both the follicular and luteal phases were determined by bioinformatics analysis. A total of 1,122, 014, 112 clean reads were obtained, and 3770 differentially expressed mRNAs were identified for further analysis. There were 1727 and 2043 upregulated mRNAs in the luteal phase and follicular phase, respectively. According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, some mRNAs that were highly expressed in ovaries during the luteal phase, such as HSD17B7, 3BHSD, and SRD5A2, may be related to the synthesis of progesterone. In addition, some mRNAs that were highly expressed in ovaries during the follicular phase, such as RPL12, RPS13 and RPL10, are related to the growth and maturation of oocytes. Taken together, the findings of this study provide genome-wide mRNA expression profiles for goat ovaries at the follicular and luteal phases and identify mRNAs associated with goat hormone secretion and follicular development. In addition, this study provides a theoretical basis for further investigation of goat reproductive regulation.

Bisphenol A affects ovarian development in adolescent mice caused by genes expression change.

Many human epidemiology and animal model studies have reported that bisphenol A (BPA) exerts adverse effects on reproduction through different regulatory mechanisms and signaling pathways in adults. In recent years, the exposure risk has increased for the general population, and little is known about how BPA affects ovarian development in adolescent animals and humans. In the present study, we aimed to investigate the effects of BPA exposure on ovarian development and the transcriptome in adolescent mice. Four-week-old ICR female mice were randomly divided into two groups and orally administered BPA (200 ng/kg/day) by gavage for 4 weeks. The BPA and estrogen (E2) levels in sera from the two groups were subsequently determined by using enzyme-linked immunosorbent assays (ELISAs). An immunohistochemical study showed that several obvious ovarian structural and developmental abnormalities were observed in the treatment group with changes in the E2 receptor gene and protein expression levels. A total of 4266 differentially expressed genes (DEGs) were identified, and the possible functions of these DEGs were explored by bioinformatics analyses based on the RNA-Seq data. The two most significant expression profiles were identified by Short Time-series Expression Miner (STEM) software, and the genes in these two profiles were enriched in actin filament-based processes, behaviour and membrane potential regulation according to Gene Ontology (GO) enrichment analysis. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs are particularly involved in the endocrine system, the calcium and cAMP signaling pathways.

Filtered reproductive long non-coding RNAs by genome-wide analyses of goat ovary at different estrus periods.

BACKGROUND: The goat is an important farm animal. Reproduction is an important process of goat farming. The ovary is the most important reproductive organ for goats. In recent years, an increasing number of long non-coding RNAs (lncRNAs) have been implicated in the regulation of mammal reproduction. However, there are few studies on the function of lncRNAs in reproduction, particularly lncRNAs in the ovary. RESULTS: The sequencing of goat ovaries generated 1,122,014,112 clean reads, and 4926 lncRNAs and 1454 TUCPs (transcripts of uncertain coding potential) were identified for further analysis by using the coding potential analysis software, CNCI, CPC and Pfam-sca. There were 115 /22 differential lncRNAs /TUCPs transcripts between the ovaries of the luteal phase and the follicular phase. We predicted the related genes of lncRNA /TUCP based on co-expression and co-localization methods. In total, 2584 /904 genes were predicted by co-expression, and 326/73 genes were predicted by co-localization. The functions of these genes were further analyzed with GO and KEGG analysis. The results showed that lncRNAs /TUCPs, which are highly expressed in goat ovaries in the luteal phase, are mainly associated with the synthesis of progesterone, and we filtered the lncRNAs /TUCPs, such as XR_001918177.1 and TUCP_001362, which may regulate the synthesis of progesterone; lncRNAs /TUCPs, which are highly expressed in goat ovaries in the follicular phase, are mainly associated with oogenesis and the maturation of oocytes, and we filtered the lncRNAs /TUCPs that may regulate the oogenesis and maturation of oocyte, such as XR_001917388.1 and TUCP_000849. CONCLUSION: The present study provided the genome expression profile of lncRNAs /TUCPs in goat ovaries at different estrus periods and filtered the potential lncRNAs /TUCPs associated with goat reproduction. These results are helpful to further study the molecular mechanisms of goat reproduction.

Transcriptional defects and reprogramming barriers in somatic cell nuclear reprogramming as revealed by single-embryo RNA sequencing.

BACKGROUND: Nuclear reprogramming reinstates totipotency or pluripotency in somatic cells by changing their gene transcription profile. This technology is widely used in medicine, animal husbandry and other industries. However, certain deficiencies severely restrict the applications of this technology. RESULTS: Using single-embryo RNA-seq, our study provides complete transcriptome blueprints of embryos generated by cumulus cell (CC) donor nuclear transfer (NT), embryos generated by mouse embryonic fibroblast (MEF) donor NT and in vivo embryos at each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst). According to the results from further analyses, NT embryos exhibit RNA processing and translation initiation defects during the zygotic genome activation (ZGA) period, and protein kinase activity and protein phosphorylation are defective during blastocyst formation. Two thousand three constant genes are not able to be reprogrammed in CCs and MEFs. Among these constant genes, 136 genes are continuously mis-transcribed throughout all developmental stages. These 136 differential genes may be reprogramming barrier genes (RBGs) and more studies are needed to identify. CONCLUSIONS: These embryonic transcriptome blueprints provide new data for further mechanistic studies of somatic nuclear reprogramming. These findings may improve the efficiency of somatic cell nuclear transfer.

Long non-coding RNAs potentially function synergistically in the cellular reprogramming of SCNT embryos.

BACKGROUND: Long non-coding RNAs (lncRNAs), a type of epigenetic regulator, are thought to play important roles in embryonic development in mice, and several developmental defects are associated with epigenetic modification disorders. The most dramatic epigenetic reprogramming event occurs during somatic cell nuclear transfer (SCNT) when the expression profile of a differentiated cell is abolished, and a newly embryo-specific expression profile is established. However, the molecular mechanism underlying somatic reprogramming remains unclear, and the dynamics and functions of lncRNAs in this process have not yet been illustrated, resulting in inefficient reprogramming. RESULTS: In this study, 63 single-cell RNA-seq libraries were first generated and sequenced. A total of 7009 mouse polyadenylation lncRNAs (including 5204 novel lncRNAs) were obtained, and a comprehensive analysis of in vivo and SCNT mouse pre-implantation embryo lncRNAs was further performed based on our single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs were expressed in a developmental stage-specific manner during mouse early-stage embryonic development, whereas a more temporal and spatially specific expression pattern was identified in mouse SCNT embryos with changes in the state of chromatin during somatic cell reprogramming, leading to incomplete zygotic genome activation, oocyte to embryo transition and 2-cell to 4-cell transition. No obvious differences between other stages and mouse NTC or NTM embryos at the same stage were observed. Gene oncology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and weighted gene co-expression network analysis (WGCNA) of lncRNAs and their association with known protein-coding genes suggested that several lncRNAs and their associated with known protein-coding genes might be involved in mouse embryonic development and cell reprogramming. CONCLUSIONS: This is a novel report on the expression landscapes of lncRNAs of mouse NT embryos by scRNA-seq analysis. This study will provide insight into the molecular mechanism underlying the involvement of lncRNAs in mouse pre-implantation embryonic development and epigenetic reprogramming in mammalian species after SCNT-based cloning.

Oct4 regulates DNA methyltransferase 1 transcription by direct binding of the regulatory element.

BACKGROUND: The transcription factor Oct4 plays a pivotal role in the pre-implantation development of the mouse embryo. DNA methyltransferase 1 (Dnmt1) maintains the changes in DNA methylation during mammalian early embryonic development. Little is known of the role of Oct4 in DNA methylation in mice. In this study, Kunming white mice were used as an animal model to reveal any correlation between DNA methylation and Oct4 during mammalian embryonic development. RESULTS: The expressions of Dnmt1 and Oct4 were initially studied using real-time PCR. They exhibited different patterns during the pre-implantation stage. Moreover, by using a promoter assay and ChIP analysis, we found that the transcriptional activities of Dnmt1 in mouse NIH/3 T3 cells and CCE cells were regulated by Oct4 through direct binding to the - 554 to - 294 fragment of the upstream regulation element of Dnmt1. The downregulation of Dnmt1 expression and enzyme activity by mouse Oct4 were further confirmed by transfecting Oct4 siRNA into mouse CCE cells. CONCLUSION: Our results indicate that Oct4 is involved in DNA methylation through the regulation of Dnmt1 transcription, especially during the early stages of mouse pre-implantation embryo development.

Duplication and gene expression patterns of ß-catenin in Nile tilapia.

ß-Catenin, a key transcriptional coactivator of the Wnt pathway, plays an important role in animal embryonic development and organogenesis. In our earlier study, we have reported that two types of ß-catenin (ß-catenin-1 and ß-catenin-2) were ubiquitously expressed in almost all the tissues examined in tilapia. However, the immunolocalization of ß-catenin in those tissues, especially in extra-gonadal tissues, remains unclear. In the present study, we further confirm that these two types of ß-catenin gene exist only in teleosts and are derived from 3R (third round of genome duplication) by phylogenetic and syntenic analyses. Moreover, the transcriptome analysis conducted in this investigation reveals that two ß-catenins exhibited similar expression patterns in seven adult tissues and four key developmental stages of XX and XY gonads. Finally, immunohistochemistry analysis was performed to detect the cell localization of ß-catenin. A positive signal of ß-catenin was observed in various tissues of tilapia, such as the intestine, liver, kidney, spleen, eye, brain, and gonads. The results of our study indicate that tilapia ß-catenin might be involved in the organ development and play some specific functions in biological processes; all these data will provide basic reference for understanding the molecular mechanism of ß-catenin in regulating of teleost organogenesis.

Supplement of Betaine into Embryo Culture Medium Can Rescue Injury Effect of Ethanol on Mouse Embryo Development.

Mammal embryos can be impaired by mother's excessive ethanol uptake, which induces a higher level of reactive oxygen species (ROS) and interferes in one carbon unit metabolism. Here, our analysis by in vitro culture system reveals immediate effect of ethanol in medium on mouse embryo development presents concentration dependent. A preimplantation embryo culture using medium contained 1% ethanol could impact greatly early embryos development, and harmful effect of ethanol on preimplantation embryos would last during the whole development period including of reducing ratio of blastocyst formation and implantation, and deteriorating postimplantation development. Supplement of 50 µg/ml betaine into culture medium can effectively reduce the level of ROS caused by ethanol in embryo cells and rescue embryo development at each stage damaged by ethanol, but supplement of glycine can't rescue embryo development as does betaine. Results of 5-methylcytosine immunodetection indicate that supplement of betaine into medium can reduce the rising global level of genome DNA methylation in blastocyst cells caused by 1% ethanol, but glycine can't play the same impact. The current findings demonstrate that betaine can effectively rescue development of embryos harmed by ethanol, and possibly by restoring global level of genome DNA methylation in blastocysts.

Rex Rabbit Somatic Cell Nuclear Transfer with In Vitro-Matured Oocytes.

Somatic cell nuclear transfer (SCNT) requires large numbers of matured oocytes. In vitro-matured (IVM) oocytes have been used in SCNT in many animals. We investigated the use of IVM oocytes in Rex rabbit SCNT using Rex rabbit ovaries obtained from a local abattoir. The meiotic ability of oocytes isolated from follicles of different diameters was studied. Rex rabbit SCNT was optimized for denucleation, activation, and donor cell synchronization. Rex rabbit oocytes grew to the largest diameter (110 µm) when the follicle diameter was 1.0 mm. Oocytes isolated from <0.5-mm follicles lacked the ability to resume meiosis. More than 90% of these oocytes remained in the germinal vesicle (GV) stage after in vitro culture (IVC) for 18 h. Oocytes isolated from >0.7-mm follicles acquired maturation ability. More than 90% of these oocytes matured after IVC for 18 h. The developmental potential of oocytes isolated from >1-mm follicles was greater than that of oocytes isolated from 0.7- to 1.0-mm follicles. The highest activation rates for IVM Rex rabbit oocytes were seen after treatment with 2.5 µM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) and 5 µg/mL cycloheximide (CHX) for 1 h. Ionomycin induced the chromatin of IVM oocytes to protrude from the oocyte surface, promoting denucleation. Fetal fibroblast cells (FFCs) and cumulus cells (CCs) were more suitable for Rex rabbit SCNT than skin fibroblast cells (SFCs) (blastocyst rate was 35.6 ± 2.2% and 38.0 ± 6.0% vs. 19.7 ± 3.1%). The best fusion condition was a 2DC interval for 1 sec, 1.6 kV/cm voltages, and 40 µsec duration in 0.28 M mannitol. In conclusion, the in vitro maturation of Rex rabbit oocytes and SCNT procedures were studied systematically and optimized in this study.

Synergistic role of ß-catenin1 and 2 in ovarian differentiation and maintenance of female pathway in Nile tilapia.

Two ß-catenin (ß-cat) genes exist in teleosts but little is known about their expression and function in ovarian development. We identified ß-cat1 and ß-cat2 from the Nile tilapia. ß-cat1 and ß-cat2 displayed a similar expression pattern in the ovary during development, and were mainly expressed in the oogonia and oocytes. In luciferase assays, ß-cat1 activated the TOPFlash reporter dose-dependently, whereas ß-cat2 failed to do so. Cotransfection of ß-cat1 and ß-cat2 synergistically enhanced the expression of the reporter. A specific interaction between ß-cat1 and ß-cat2 was also observed in a mammalian two-hybrid assay. Furthermore, tilapia recombinant Dkk1, an inhibitor of the ß-cat pathway, decreased ß-cat1 and ß-cat2, while increased sox9, dmrt1, cyp11b2 and foxl2 expression in the in vitro cultured tilapia ovary, which could be abolished by simultaneous treatment with Bio, an agonist of ß-cat. Consistently, ß-cat1 or ß-cat2 knockdown in XX fish by TALENs caused the retardation of ovarian differentiation and masculinization, as reflected by the upregulation of dmrt1, cyp11b2, sox9, and serum 11-KT level. On the contrary, serum E2 level was unchanged even though foxl2 transcription was upregulated. These data suggestes that both ß-cat1 and ß-cat2 are important members and play synergistic roles in the canonical Wnt signal pathway in fish. Independent of Foxl2-leading estrogen pathway, they might be involved in ovarian differentiation and repression of the male pathway gene expression in tilapia.

Effects of alcohol on H3K9 acetylation in mouse pre-implantation embryos.

It is well known that excessive long-term alcohol consumption is harmful, especially in pregnant women. In the present study, the Kunming white mouse was used as an animal model and indirect immunofluorescence was performed to analyze the toxic effects of alcohol on early pre-implantation embryos. H3K9 acetylation immunofluorescence could not be detected in MII oocytes. H3K9 acetylation levels in the treatment group were higher than in the control group during the morula stage, and contrary to results during the blastocyst stage. Other stages showed no obvious differences for in vivo embryos. For in vitro embryos, almost no difference was found between the two experimental groups across all stages, and both groups showed increasing H3K9 acetylation levels (except at the 2-cell stage). This study shows that H3K9 acetylation levels in early pre-implantation embryos are notably impacted by excessive alcohol ingestion by females. These data are the first step in understanding the epigenetic mechanism of alcohol toxicity in early pre-implantation mouse embryos.

Comparative analysis of H3K9 acetylation level in parthenogenetic, and in vitro and in vivo developed mouse embryos.

The developmental rate of parthenogenetic embryos is slower than that of embryos generated in vitro and in vivo. To detect the effects of epigenetic modification on embryo development, we compared the H3K9 acetylation level in these three types of embryos as well as parthenogenetic embryos treated with a histone deacetylase inhibitor trichostatin (TSA) by indirect immunofluorescence. Our results showed that fluctuations in the level of acetylated H3K9 detected during embryo development are similar among different types of mouse embryos. However, the level of H3K9 acetylation in parthenogenetic embryos is significantly higher while the level in embryos generated in vitro is lower when compared with that in embryos derived from in vivo. Treatment of parthenogenetic embryos with TSA increases the developmental rate but further elevates the level of H3K9 acetylation, especially from pronuclear to 8-cell stages. These results suggest that the promoters of genes that should be silenced during pre-implantation embryo development may be hyperacetylated in parthenogenetic embryos which inhibit normal embryo development. However, the positive effect of TSA on embryo development is not through altering the H3K9 acetylation level.