Transcriptome profiling of in vitro-matured oocytes from a korean native cow (hanwoo) after cysteamine supplementation.

This study elucidated the molecular markers that decrease oocyte quality during in vitro culture, restricting optimal developmental potential. Here, we evaluated the transcriptomic differences between cysteamine-treated and non-treated bovine cumulus oocyte complexes (COCs) after 22 h of co-culture in the maturation media using RNA sequencing. In total, 39,014 transcripts were sequenced between cysteamine-treated and non-treated mature COCs. We evaluated the relative expression of 21,472 genes, with 59 genes showing differential expression between the two COC groups. The cysteamine-treated group had 36 up-regulated gene transcripts and 23 down-regulated gene transcripts. Moreover, gene ontology (GO) enrichment analysis revealed that multiple biological processes were significantly enriched after cysteamine supplementation. Differentially expressed genes appeared to maintain normal oocyte physiology, regulation of apoptosis, differentiation, ossification or bone formation, cardiac and muscle physiology, hormonal secretion, and membrane construction for further embryonic development. In conclusion, cysteamine affects the mRNA level of COCs during oocyte maturation by upregulating potential molecular markers and downregulating genes that affect further embryonic development.

Improves the In Vitro Developmental Competence and Reprogramming Efficiency of Cloned Bovine Embryos by Additional Complimentary Cytoplasm.

Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p < 0.05) in the CICT group than in the SCNT group (28.9 ± 0.8% vs. 20.2 ± 1.3%, respectively). Furthermore, the total cell number per day 8 blastocyst was significantly higher in the CICT group than in the SCNT group (176.2 ± 6.5 vs. 119.3 ± 7.7, p < 0.05). Moreover, CICT increased mitochondrial activity, as detected using MitoTracker® Green. The mRNA levels of DNA methyltransferase 1 and DNA methyltransferase 3a were significantly lower (p < 0.05) in the CICT group than in the SCNT group. The mRNA level of DNA methyltransferase 3b was lower in the CICT group than in the SCNT group; however, this difference was not significant (p > 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.

Production of Handmade Open Pulled Straw (OPS) Using Digital Heating Gun for the Vitrification Process.

BACKGROUND: Vitrification is the most popular technique for the cryopreservation of oocytes and embryos, replacing slow freezing methods. MATERIALS AND METHODS: This study evaluated the efficient manufacturing methods of handmade open pulled straw (OPS) with a digital heating gun that could be proposed for vitrification. RESULTS: Production efficiency of OPS using 0.5 mL straw was detected at 0, 66.1, 90.5 and 85.7% for 1~2 s and 9.5, 33.3, 47.6 and 23.8% for 2~3 s of heating time at 250, 350, 400 and 450°C respectively. The production rate of OPS using 0.25 mL straw was perceived at 33.3, 76.2, 83.3, 95.2 and 57.6 % for 1.5~2.5 s time with the optimized heat setting at 330, 340, 350, 360 and 370°C respectively. The desired inner diameter (200~300 µm) of OPS could be varied according to the gamete size, embryo developmental stages or cell lines of different species. Based on our data, the production efficiency of OPS using 0.25-mL straw were increased beyond using a 0.5-mL straw. CONCLUSION: Handmade OPSs could be efficiently produced with a digital heating gun to generate a vitrification device for freezing gametes, embryos and cell lines.

A combination of bovine serum albumin with insulin-transferrin-sodium selenite and/or epidermal growth factor as alternatives to fetal bovine serum in culture medium improves bovine embryo quality and trophoblast invasion by induction of matrix metalloproteinases.

This study investigated the use of bovine serum albumin (BSA) plus insulin-transferrin-sodium selenite (ITS) and/or epidermal growth factor (EGF) as alternatives to fetal bovine serum (FBS) in embryo culture medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, gene expression and cryotolerance, as well as the invasion ability of trophoblasts. The percentage of embryos that underwent cleavage and formed a blastocyst was higher (P<0.01) in medium containing ITS plus EGF and BSA than in medium containing FBS. Culture with ITS plus EGF and BSA also increased the hatching ability of blastocysts and the total cell number per blastocyst. Furthermore, the beneficial effects of BAS plus ITS and EGF on embryos were associated with a significantly reduced intracellular lipid content, which increased their cryotolerance. An invasion assay confirmed that culture with ITS plus EGF and BSA significantly improved the invasion ability of trophoblasts. Real-time quantitative polymerase chain reaction analysis showed that the mRNA levels of matrix metalloproteinase-2 (MMP2) and MMP9, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain and hydroxymethylglutaryl-CoA reductase significantly increased upon culture with ITS plus EGF and BSA. Moreover, protein expression levels of matrix metalloproteinase-2 and -9 increased (P<0.01) in medium supplemented with ITS plus EGF and BSA compared with medium supplemented with FBS. Taken together, these data suggest that supplementation of medium with ITS plus EGF and BSA improves invitro bovine embryo production, cryotolerance and invasion ability of trophoblasts.

In vitro production of sex preselected cattle embryos using a monoclonal antibody raised against bull sperm epitopes.

Sex preselection has always generated great interest among livestock producers. Among the prevalent sperm sorting methods, there is much evidence that sex sorting has a negative effect on sperm quality with an altered pattern of sperm motility, ultimately reducing the period of cell viability. In this study, we have established a new approach for the preselected embryo production by using WholeMom®; a monoclonal antibody developed against bull sperm epitopes for simple and easy separation of X- and Y-sperm. There were no significant differences (P > 0.05) in the percentage of presumptive zygotes between the control and the X-sperm sorted group, but there was a difference in early cleaving embryos with there being 81.2 ± 1.4%, 78.3 ± 1.0%, and 66.7 ± 1.1% for the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Similarly, the percentage of embryos that developed to the blastocyst stage (Day 7) were also greater (P < 0.05) in the control and X-sperm sorted group compared with the Y-sperm sorted group being 34.8 ± 1.0%, 32.1 ± 0.8%, and 23.7 ± 1.0% in the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Furthermore, B-SRY F2 and B-SRY R2 gene expression data indicated there was a detection accuracy of 81.0% for the female embryos and 72.5% for the male embryos produced in vitro. In conclusion, in cattle in vitro derived embryo production using pre-selected sexed semen and subsequent embryo transfer can facilitate the mass production of individuals that are genetically superior.

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.

Lupeol supplementation improves the developmental competence of bovine embryos in vitro.

Lupeol is a triterpene with various pharmacological properties. This study investigated the effect of lupeol on the in vitro development of bovine embryos. Oocytes (270 per group, 1620 in total) obtained from slaughterhouse-derived ovaries were matured and fertilized in vitro and then cultured for 8 days in a humidified atmosphere of 5% CO2 in air at 38.5 °C. The in vitro maturation medium was supplemented with 0.5, 1.0, 2.0, 3.0, and 4.0 µM lupeol. Treatment with 2.0 µM lupeol significantly (P < 0.05) improved blastocyst development. Hoechst 33342 staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling showed that treatment with 2.0 µM lupeol improved blastocyst quality by increasing the total cell number and reducing the apoptotic cell number. Confocal microscopy confirmed that treatment with 2.0 µM lupeol significantly (P < 0.05) reduced the level of 8-oxoguanine, an indicator of reactive oxygen species. Lupeol treatment also significantly attenuated protein expression of nuclear factor-kappa B subunit 1 (NFKB1), cyclooxygenase (COX) 2, and CASP3. Real-time PCR analysis of nitric oxide synthase 2, NFKB1, COX2, CASP3, and BCL2-associated X protein supported the immunofluorescence data. In conclusion, lupeol is a potent antioxidant that improves bovine embryo development in vitro.

Supplementation of lycopene in maturation media improves bovine embryo quality in vitro.

This study sought to modulate factors that reduce embryo quality in in vitro culture (IVC) systems. Over eight replicates, 3075 oocytes were cultured in in vitro maturation media containing various concentrations of lycopene, followed by in vitro fertilization and culture. The percentages of MII-stage oocytes, the presumptive zygotes that underwent cleavage and developed into blastocysts were significantly (P < 0.05) higher, the intracellular ROS concentrations reduced significantly (P < 0.05) in oocytes/blastocysts, TUNEL assay demonstrates reduced apoptosis and increased total cell number per blastocyst (P < 0.05), Immunocytochemistry confirmed that diminished protein expression of nuclear factor kappa B (NFκB), cyclooxygenase-2 (COX2), and 8-oxoguanine (indicated by ROS) and relative mRNA expression of the Caspase-3, NFκB, COX2, iNOS and BCL2-associated X (BAX) was significantly (P < 0.05) lower whereas the anti-apoptotic gene BCL2 was significantly (P < 0.05) higher in the 0.2 µM lycopene-supplemented group than the control. In conclusion, lycopene improves blastocyst quality by overcoming unfavorable conditions in in vitro culture systems.

2-Methoxystypandrone improves in vitro-produced bovine embryo quality through inhibition of IKBKB.

2-Methoxystypandrone (2-MS), a naphthoquinone from Polygonum cuspidatum, has been reported to exhibit antioxidant activities. However, its pharmacological properties have not been fully studied in vitro culture system of oocytes and embryos development. In this study, we examined the effects of 2-MS on bovine blastocyst developmental competency during preimplantation using TUNEL assay, qRT-PCR and immunocytochemistry. Oocytes were treated with 1 µM 2-MS during maturation, followed by in vitro fertilized and the zygotes were cultured. We observed that blastocyst development was significantly higher in the 2-MS treated oocytes (45.29%) compared to control groups (32.21%). We also found that 2-MS treatment significantly increased total cell number and decreased the apoptotic cells in Day-8 blastocyst. Finally, we examined the signal pathway affecting the embryo development after the 2-MS treatment. We detected a significant decrease in the mRNA levels of NFKB1, IKBKB, COX2, NOS2, BAX, CASP3, and JAK2 after 2-MS treatment, however, the mRNA level of the anti-apoptotic gene BCL2 was significantly higher than that in the control. Moreover, protein expression levels of NFKB1, IKBKB, and COX2, and 8-oxoG DNA lesion decreased significantly in the 2-MS treatment group, compared to the control. In summary, our findings suggested that treatment of oocytes with a potential anti-oxidant, 2-MS during IVM improved in vitro developmental competence of bovine embryos.

Polydatin improves the developmental competence of bovine embryos in vitro via induction of sirtuin 1 (Sirt1).

The aim of the present study was to investigate the beneficial effect of polydatin (PD), the glycoside form of resveratrol, on embryo development in vitro. Oocytes were aspirated from ovaries of Korean Hanwoo cows and cultured until Day 8 in a humidified atmosphere of 5% CO2 in air at 38.5°C. Protein and gene expression levels were determined through confocal microscopy and reverse transcription-polymerase chain reaction respectively, whereas the number of total and apoptotic cells in Day 8 blastocysts was determined using Hoechst 33342 staining and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling. Of the different concentrations of PD (0.5, 1.0 and 2.0µM) added to the IVM medium, only 1.0µM PD significantly improved blastocyst development. Immunofluorescence analysis confirmed that protein levels of sirtuin 1 (Sirt1) increased significantly (P<0.05) after PD treatment, whereas levels of reactive oxygen species (ROS) were significantly (P<0.05) decreased, as evidenced by reductions in 8-oxoguanine immunoreactivity. Similarly, protein levels of nuclear factor (NF)-κB and cyclo-oxygenase (COX)-2 were significantly (P<0.05) lower in the PD-treated group than in the control group. Treatment with 1.0µM PD reduced gene expression of BCL2-associated X protein, inducible nitric oxide synthase, COX2 and Nfkb, but increased the expression of Sirt1, supporting the immunofluorescence data. PD possesses antioxidant activity and is useful for embryo development in vitro. We conclude that supplementation of IVM medium with PD improves embryo developmental competence via Sirt1.

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.