Jong-In Jin

Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: efficiency and in vitro developmental competence.

The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in the ssIVF group was less than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, respectively; P < 0.05). Importantly, we reported that the blastocyst formation rate in the ssICSI group was similar to that in the usICSI group, which indicated that ICSI can rescue the damage introduced to sperm by flow cytometry-mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex.

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods ☆

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142 ± 21.8 vs. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P < 0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P < 0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading.

Postthaw survival of in vitro-produced bovine blastocysts loaded onto the inner surface of a plastic vitrification straw

In this study, we investigated whether vitrification of an embryo by attachment to the inner surface of a plastic straw, which requires a small volume of vitrification solution, improves the survival of thawed embryos. In vitro-produced Korean native cattle blastocysts were randomly assigned into four groups: (1) blastocysts attached to the inner surface of a plastic straw (aV); (2) blastocysts loaded into the column of a plastic straw (cV); (3) blastocysts directly dropped into liquid nitrogen (dV); and (4) nonvitrified blastocysts (control). The postthaw recovery rate did not significantly differ among the aV, dV, and cV groups (98.3% vs. 81.5% vs. 91.4%). The postthaw survival rate was greater in the control, aV, and dV groups than in the cV group (100%, 87.7%, and 81.8% vs. 26.4%, P < 0.05), but did not significantly differ among the control, aV, and dV groups. The total number of cells per blastocyst did not significantly differ among the groups (134.4 ± 38.9 in control vs. 114 ± 48.1 in aV, 105.6 ± 33.9 in dV, and 102 ± 35.1 in cV group). However, the number of apoptotic cells per blastocyst was higher in the dV and cV groups than in the control group (10.9 ± 9.6 and 14.5 ± 9.5 vs. 0.4 ± 1.4; P < 0.05), but did not significantly differ between the control and aV groups (0.4 ± 1.4 vs. 6.6 ± 9.5). In addition, the blastocoel of each blastocyst was left intact or was mechanically punctured to reduce its volume, and the blastocysts were then vitrified using the aV method. At 12 hours after thawing, the re-expansion rate did not significantly differ among the control, punctured aV, and nonpunctured aV groups (93.3% vs. 85.2% vs. 82.8%). However, at 24 hours after thawing, the hatching rate was greater in the control and punctured aV groups than in the nonpunctured aV group (75% and 62.9% vs. 37.1%; P < 0.05). The total number of cells per blastocyst was greater in the control group than in the nonpunctured aV group (143 ± 37.2 vs. 94.5 ± 18.6; P < 0.05), but did not significantly differ between the control and punctured aV groups (143 ± 37.2 vs. 119.4 ± 19.7). The number of apoptotic cells per blastocyst was greater in the nonpunctured aV group than in the control and punctured aV groups (11.3 ± 6.1 vs. 5.9 ± 5.8 and 6.3 ± 4.4; P < 0.05). Taken together, the data show that the aV method improved the survival and quality of vitrified-thawed blastocysts. Furthermore, puncture of the blastocoel increased the hatching rate and reduced the number of apoptotic cells in vitrified-thawed blastocysts generated using the aV method.

Effect of charcoal:dextran stripped fetal bovine serum on in vitro development of bovine embryos

This study investigated the ability of charcoal:dextran stripped fetal bovine serum (CDS FBS) and heat-inactivated fetal bovine serum (HI FBS) to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentage of embryos that formed a blastocyst was significantly (P<0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84±0.78% vs. 36.85±0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly (P<0.05) increased mitochondrial activity, as identified by MitoTracker Green, as well as a reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. Quantitative reverse transcription PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly (P<0.05) increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and the anti-apoptotic gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS-supplemented group than in the HI FBS-supplemented group, whereas that of the pro-apoptotic gene BCL2-associated X protein was significantly lower. Taken together, these data suggest that supplementation of medium with CDS FBS improves the in vitro developmental competence and cryo-tolerance of bovine embryos.

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.

81 IMPROVEMENT OF DEVELOPMENTAL COMPETENCE OF BOVINE IN VITRO-PRODUCED EMBRYOS BY ADDING 2-METHOXYSTYPANDRONE IN MATURATION MEDIA

The 2-methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum. The objective of this study was to investigate the effects of 2-MS on oocyte maturation, blastocyst development, and embryo quality in terms of cell number and gene expression in vitro. A total of 2364 oocytes were cultured in TCM-199 supplemented with 10% fetal bovine serum, 1μgmL-1 oestradiol-17β, 10μgmL-1 FSH, 10ngmL-1 epidermal growth factor, 0.6mM cysteine, and 0.2mM sodium pyruvate and supplemented with different concentrations of 2-MS as following: 1.5μM (n=458), 1.0 (n=493), 0.5 (n=468), 0.1 (n=470), and 0μM (control, n=475) followed by IVF and then culture in CR1-aa medium supplemented with 44μgmL-1 sodium pyruvate, 14.6μgmL-1 glutamine, 10μLmL-1 penicillin-streptomycin, 3mgmL-1 BSA, and 310μgmL-1 glutathione for the first 3 days, and then the BSA was replaced with 10% FBS until Day 8. The differences in embryo development between experimental groups were analysed by one-way ANOVA. The Duncans multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P<0.05. The results showed that the addition of 2-MS at 1.0mM significantly improved (P<0.05) the percentage of MII oocytes, which were identified by aceto-orcein staining, compared with that in the control (76.5v. 65.4%, respectively), and remarkably (P<0.05), improved blastocyst development rates (45.29%) compared with control (32.21%). Additionally, TUNEL assay demonstrated that treatment with 1.0μM of 2-MS significantly improved the embryo quality by increasing total number of cells and reducing DNA damage. Immunofluorescent analysis showed that the protein levels of nuclear factor-kappa B (NFkB), inhibitor of kappa B kinase β (IkKβ), 8-oxoguanine, and cyclooxygenase-2 (COX2) declined significantly (P<0.05) after 2-MS treatment compared with the control. These results were confirmed by qRT-PCR, which showed a significant decrease in the mRNA levels of NFkB, IkKβ, COX2, inducible nitric oxide synthase (iNOS), BCL2-associated X protein (BAX), caspase-3, and Janus kinase2 (JAK2) after 2-MS treatment; however, the mRNA level of the anti-apoptotic gene B-cell lymphoma2 (BCL2) was significantly higher than that in the control. In conclusion, the addition of 2-MS at the indicated concentration dramatically improves the developmental competence of bovine in vitro-produced embryos.