Kyeong-Lim Lee

Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.

Coagulansin-A has beneficial effects on the development of bovine embryos in vitro via HSP70 induction

Treatment with the steroidal lactone, coagulansin-A, improves bovine oocyte maturation and embryo development in vitro by inducing heat shock protein 70 (HSP70), which reduces the levels of reactive oxygen species (ROS), DNA damage and inflammation.

Improvement of in vitro-produced bovine embryo treated with coagulansin-A under heat-stressed condition

Heat stress has large effects on reproduction including conception rate in cattle. In this study, we examined the effects of coagulansin-A (coa-A), a steroidal lactone, on acquired thermo tolerance during in vitro production of bovine embryos. Oocytes were incubated in in vitro maturation (IVM) media with or without coa-A at two different temperatures, 40.5˚C and 42˚C, for 20 h. The treatment of coa-A significantly improved blastocyst development only at 40.5˚C (P < 0.05). Interestingly, immunofluorescence analysis demonstrated that coa-A induced heat shock protein 70 (HSP70) and phosphatidylinositol-3-kinase (PI3K), but significantly attenuated nuclear factor kappa B (NF-κB) and cyclooxygenase-2 (COX2). To determine the expression patterns of related genes at the transcription level, qRT-PCR was performed. Expression of HSP70 and PI3K was elevated, whereas expression of NF-κB, COX2 and inducible nitric oxide synthase (iNOS) was significantly (P < 0.05) downregulated in the coa-A-treated group compared with the control group. Moreover, pro-apoptotic genes were downregulated, and antiapoptic genes were upregulated in the coa-A group. We also counted the total cell number and apoptotic nuclei at the blastocyst and found that more cell numbers (143.1 ± 1.5) and less apoptotic damages (6.4 ± 0.5) in the coa-A treatment group comparing to control group (131.4 ± 2.0 and 10.8 ± 0.5), indicating the enhanced embryo quality. In conclusion, our results demonstrate that the coa-A not only improved the blastocyst development in vitro but also increased their resistance to heat stress condition through induction of HSP70/PI3K.

Mitochondrial content and gene expression profiles in oocyte-derived embryos of cattle selected on the basis of brilliant cresyl blue staining

The aim of this study was to investigate the developmental rate, lipid and mitochondrial distribution and gene expression in oocyte-derived embryos selected on the basis of brilliant cresyl blue (BCB) staining. Lipid content and mitochondrial distribution in Day 8 blastocysts were evaluated by fluorescence intensity, while gene expression was analyzed by real-time PCR. The proportion of blastocysts (30.9%) was greater (P<0.05) in BCB+ than in BCB- oocytes (13%) but not different (P>0.05) from the control group (28.2%). Total cell number was also greater in BCB+ (155.1 ± 36.2) than in BCB- (116.6 ± 40.5) and control (127.5 ± 35.7) blastocysts. Furthermore, the apoptotic cell number was less in BCB+ (3.7 ± 4.4) than in BCB- blastocysts (8.7 ± 8.7) but not different from the control group (5.9 ± 3.9). BCB+ embryos contained more mitochondria compared to BCB- embryos (P<0.05). There was no significant difference in intercellular lipid accumulation in embryos from all groups. Interferon-τ (IFNτ), transforming growth factor β1 (TGFB1) and secreted seminal-vesicle Ly-6 protein 1 (SSLP1) gene expression was greater in BCB+ than in BCB- blastocysts. By contrast, Bcl2-associated X protein (BAX) and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) gene expression was greater in BCB- than in BCB+ and control embryos. In conclusion, oocyte-derived embryos selected on the basis of BCB staining showed differences in developmental rate, quality, mitochondrial content and target gene expression compared to control embryos.

Differential gene-expression profiles from canine cumulus cells of ovulated versus in vitro-matured oocytes

We compared the nuclear maturation status and gene-expression profiles of canine cumulus cells (CCs) derived from cumulus-oocyte complexes (COCs) that were spontaneously ovulated versus those that were matured in vitro. Cumulus-oocyte complexes were retrieved from uteri by surgical flushing (after spontaneous ovulation) or by ovariectomy follicle aspiration and in vitro maturation. The objective of Experiment 1 was to investigate the nuclear maturation status of in vivo- versus in vitro-matured oocytes. The objective of Experiment 2 was to compare gene-expression profiles of CCs derived from in vivo- versus in vitro-matured COCs. Genes analysed are related to cell maturation, development and apoptosis, including GDF9, MAPK1, PTX3, CX43, Bcl2 and BAX; mRNA expression for all of these genes, except for GDF9, differed (P<0.05) between in vivo- and in vitro-matured CCs. In conclusion, we found that gene-expression profiles are related to the quality of CCs and therefore posit that monitoring gene expression could be a useful strategy to guide attempts to improve in vitro culture systems.

Effect of peroxiredoxin II on the quality and mitochondrial activity of pre-implantation bovine embryos

Endogenous peroxiredoxin II (PRDX II) protein plays a vital role in early embryonic development. This study assessed the beneficial effects of exogenous PRDX II on bovine embryo development at the cellular and molecular levels. To this end, in vitro maturation (IVM) medium was supplemented with various concentrations of PRDX II (0, 6.25, 12.5, 25, 50, and 100μg/mL). Of these, 12.5μg/mL PRDX II was the most effective and significantly promoted embryonic development. Therefore, this concentration of PRDX II was used in subsequent experiments. The percentage of embryos that developed into Day 8 blastocysts and the total number of cells per blastocyst (38.2% and 150.6±5.1) was higher in the PRDX II-treated group than in the control (26.4% and 128.9±3.9, respectively). Moreover, the percent of TUNEL positive cells was higher (P<0.05) in the control than in the PRDX II-treated. Furthermore, PRDX II added to the IVM media increased mitochondria content in blastocysts and decreased the intracellular ROS levels in oocytes and blastocysts compared with the control (P<0.05). The expression of genes associated with blastocyst quality (CDX2 and IFNτ), antioxidant activity (SOD2), and mitochondrial activity (TFAM) was higher, whereas the expression of a gene involved in the apoptotic pathway (c-FOS) was lower, in the PRDX II-treated than in the control group. In conclusion, supplementation of IVM medium with PRDX II promotes development to the blastocyst stage and improves blastocyst quality through reducing ROS, enhancing embryonic mitochondrial activity, and modulating development- related target genes expression.

Improved developmental competence in embryos treated with lycopene during in vitro culture system

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 μM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p < 0.05) increase in cleavage and blastocyst development rates compared to the corresponding controls (84.3 ± 0.6% vs. 73.1 ± 1.9% and 41.0 ± 1.4% vs. 33.4 ± 0.7%, respectively). Lycopene also significantly reduced (p < 0.05) intracellular reactive oxygen species concentrations in oocytes and blastocysts, whereas lipid peroxidation and mitochondrial activity increased compared to control conditions. The number of apoptotic nuclei was significantly reduced in the lycopene‐treated compared to the control group (1.7 ± 0.1 vs. 4.7 ± 0.3), and the quantity of cells in the trophectoderm (207.1 ± 1.6 vs. 171.3 ± 1.0, respectively) and inner cell mass (41.9 ± 0.4 vs. 36.7 ± 0.4, respectively) was higher following treatment—although the inner cell mass‐to‐trophectoderm ratio was unchanged (1:3.3 vs. 1:3.4 for lycopene vs. control, respectively). Lycopene supplementation also significantly (p < 0.05) attenuated expression of IKBKB (Inhibitor of nuclear factor kappa B kinase, subunit beta) and reduced Caspase 9 and Caspase 3 protein abundance, while up‐regulating GDF9 (Growth and differentiation factor 9), BMP15 (Bone morphogenetic protein 15), SOD2 (Superoxide dismutase 2), NDUFA2 (NADH dehydrogenase), ACADL (Acyl‐CoA dehydrogenase, long chain), and ACSL3 (Acyl‐CoA synthetase 3, long‐chain membrane 3) transcription compared to control. Therefore, co‐culturing with lycopene during oocyte maturation improved bovine embryo developmental potential during in vitro culture by improving embryonic resilience to stress.

Effects of Co‐culture of Cumulus Oocyte Complexes with Denuded Oocytes During In Vitro Maturation on the Developmental Competence of Cloned Bovine Embryos

This study evaluated the effects of co-culture of immature cumulus oocyte complexes (COCs) with denuded immature oocytes (DO) during in vitro maturation on the developmental competence and quality of cloned bovine embryos. We demonstrated that developmental competence, judged by the blastocyst formation rate, was significantly higher in the co-cultured somatic cell nuclear transfer (SCNT+DO, 37.1 ± 1.1%) group than that in the non-co-cultured somatic cell nuclear transfer (SCNT-DO, 25.1 ± 0.9%) group and was very similar to that in the control IVF (IVF, 38.8 ± 2.8%) group. Moreover, the total cell number per blastocyst in the SCNT+DO group (101.7 ± 6.2) was higher than that in the SCNT-DO group (81.7 ± 4.3), while still less than that in the IVF group (133.3 ± 6.0). Furthermore, our data showed that mRNA levels of the methylation-related genes DNMT1 and DNMT3a in the SCNT+DO group were similar to that in the IVF group, while they were significantly higher in the SCNT-DO group. Similarly, while the mRNA levels of the deacetylation-related genes HDAC2 and HDAC3 were significantly higher in the SCNT-DO group, they were comparable between the IVF and SCNT+DO groups. However, the mRNA levels of HDAC1 and DNMT3B were significantly higher in the SCNT+DO group than in the other groups. In conclusion, the present study demonstrated that co-culture of COCs with DO improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell number.

Feline APOBEC3s, Barriers to Cross-Species Transmission of FIV?

The replication of lentiviruses highly depends on host cellular factors, which defines their species-specific tropism. Cellular restriction factors that can inhibit lentiviral replication were recently identified. Feline immunodeficiency virus (FIV) was found to be sensitive to several feline cellular restriction factors, such as apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) and tetherin, but FIV evolved to counteract them. Here, we describe the molecular mechanisms by which feline APOBEC3 restriction factors inhibit FIV replication and discuss the molecular interaction of APOBEC3 proteins with the viral antagonizing protein Vif. We speculate that feline APOBEC3 proteins could explain some of the observed FIV cross-species transmissions described in wild Felids.

210 GENERATION OF APOBEC3CH AND APOBEC3H DOUBLE-KNOCKOUT CATS BY SITE-SPECIFIC GENE TARGETING

The domestic cat (Felis catus) is a useful animal model for biomedical research because it shares many devastating diseases with humans. For example, feline immunodeficiency virus-1 is structurally and functionally similar to human immunodeficiency virus-1 (HIV-1). In vitro, the replication of HIV-1 in cat cells is restricted by the feline APOBEC3H (fA3H) and APOBEC3CH (fA3CH). Accordingly, we hypothesised that cats could be used to study HIV-1 infection in humans if fA3H and fA3CH were knocked out. However, due to limited availability of genomic editing tools in the cat, genetic modification has not been widely reported in this species. Here, we show that the fA3H/fA3CH locus could be knocked out in cat using the CRISPR/Cas9 system. Taking advantage of the fact that the fA3H and fA3CH genes share their last four exons (exons 2-5 for fA3H and exons 5-8 for fA3CH), we PCR-amplified and analysed the partial sequence of fA3H and fA3CH and designed a single guide (sg) RNA targeting exon 3 of fA3H (exon 6 of fA3CH) to achieve double knockout of these 2 genes. After transfecting cells with a sgRNA/Cas9 expression DNA vector or co-transfecting the sgRNA along with Cas9 mRNA, which were prepared by in vitro transcription into cat embryonic fibroblast cells, in vitro gene-targeting analysis revealed that the CRISPR/Cas9 system could introduce indels with high efficiency: 23 and 41% of cat fibroblast cells were targeted in the fA3H/fA3CH locus when Cas9 was introduced in the DNA and mRNA forms, respectively. We chose to perform cytoplasmic microinjection injections 6h post-IVF with 10 pL of injection solution containing 50ngµL-1 for sgRNA and 100ngµL-1 for Cas9 mRNA at a volume ratio of 1:1. We achieved a 95.5% embryo survival rate and a 22.9% blastocyst formation rate. High gene-targeting efficiency (62.5%, 15/24) from single blastocyst was achieved in targeting the fA3H/fA3CH locus by PCR-RFLP assay. We transferred 45 injected embryos to the uterine tubes of 4 pseudo-pregnant queens within 2h of injection. Two of the recipients were determined to be pregnant by ultrasound on Day 30 after embryo transfer, and gave birth to 6 live kittens on Day 65. Genotyping analysis revealed that 2 kittens were successfully targeted in the fA3H/fA3CH locus: kitten 1 was biallelically targeted, whereas kitten 5 was monoallelically targeted. Sanger sequencing of the PCR products subcloned into TA cloning vectors showed that kitten 1 carried the same 1-nucleotide deletion on both alleles, and that kitten 5 also carried the same 1-nucleotide deletion monoallelically. Therefore, we conclude that the fA3H/fA3CH locus in the 2 kittens is specifically targeted by the CRISPR/Cas9 system. To our knowledge, this is the first report of successful site-specific genetic modification in the domestic cat achieved using the CRISPR/Cas9 system and the production of live fA3H/fA3CH double-knockout kittens to study the HIV-1 infected diseases.