Oocytes Selected Using BCB Staining Enhance Nuclear Reprogramming and the In Vivo Development of SCNT Embryos in Cattle

Abstract

The selection of good quality oocytes is crucial for in vitro fertilization and somatic cloning. Brilliant cresyl blue (BCB) staining has been used for selection of oocytes from several mammalian species. However, the effects of differential oocyte selection by BCB staining on nuclear reprogramming and in vivo development of SCNT embryos are not well understood. Immature compact cumulus–oocyte complexes (COCs) were divided into control (not exposed to BCB), BCB+ (blue cytoplasm) and BCB− (colorless cytoplasm) groups. We found that BCB+ oocytes yielded a significantly higher somatic cell nuclear transfer (SCNT) blastocyst rate and full term development rate of bovine SCNT embryos than the BCB− and control oocytes. BCB+ embryos (embryos developed from BCB+ oocytes) showed increased acetylation levels of histone H3 at K9 and K18 (AcH3K9, AcH3K18), and methylation levels of histone H3 at K4 (H3K4me2) than BCB− embryos (embryos developed from BCB− oocytes) at the two-cell stage. Furthermore, BCB+ embryos generated more total cells, trophectoderm (TE) cells, and inner cell mass (ICM) cells, and fewer apoptotic cells than BCB− embryos. The expression of SOX2, CDX2, and anti-apoptotic microRNA-21 were up-regulated in the BCB+ blastocysts compared with BCB− blastocysts, whereas the expression of pro-apoptotic gene Bax was down-regulated in BCB+ blastocysts. These results strongly suggest that BCB+ oocytes have a higher nuclear reprogramming capacity, and that BCB staining can be used to select developmentally competent oocytes for nuclear transfer.

Recombinant Human Bone Morphogenetic Protein 6 Enhances Oocyte Reprogramming Potential and Subsequent Development of the Cloned Yak Embryos

“…SCNT, fusion and activation of reconstructed yak oocytes, and cloned embryo cultures were performed using methods described in previous studies (Su et al, 2012(Su et al, , 2014a. Briefly, after 24 h of IVM, the COCs were denuded of the cumulus cells by treating with the COCs with 0.1% bovine testicular hyaluronidase.…”

“…Image-Pro Plus software (v. 6.0, Media Cybernetics, Silver Spring, MD, USA) was used to quantify the signal intensities of H3K9ac, H3K18ac, and H3K9me3 fluorescence as previously described (Su et al, 2012(Su et al, , 2014b. The color images were converted to grayscale images using Image-Pro Plus.…”

Metabolism throughout follicle and oocyte development in mammals

“…suggested to be linked to oocyte cytoplasmic maturation (Su et al, 2012;Ishizaki et al, 2009). Furthermore, it is well established that glucose is required to support FSH-dependent cumulus expansion and oocyte maturation (Fagbohun and Downs, 1992), while denuded mouse oocytes require pyruvate or oxaloacetate to sustain spontaneous nuclear maturation (Biggers et al, 1967;Downs and Hudson, 2000).…”

Buffalo Embryos Produced by Handmade Cloning from Oocytes Selected Using Brilliant Cresyl Blue Staining Have Better Developmental Competence and Quality and Are Closer to Embryos Produced byIn VitroFertilization in Terms of Their Epigenetic Status and Gene Expression Pattern

“…Despite availability of a plethora of information on the use of BCB staining for selection of developmentally competent oocytes for IVF, there are only two reports available regarding the use of BCB staining of cytoplasm for selection of competent oocytes for SCNT (Bhojwani et al, 2007;Su et al, 2012). In both of these studies, conventional micromanipulation-based SCNT was used in which there is minimal loss of cytoplasm because the enucleation of zona-enclosed oocytes is carried out with the help of a micromanipulator.…”

“…In both of these studies, conventional micromanipulation-based SCNT was used in which there is minimal loss of cytoplasm because the enucleation of zona-enclosed oocytes is carried out with the help of a micromanipulator. However, the conventional micromanipulation-based SCNT has now been replaced by handmade cloning (HMC) technique due to its overwhelming advantages, such as simplicity, higher efficiency, and lack of need for skilled manpower and expensive equipment (Vajta et al, 2003). We have found that almost 15-50% of cytoplasmic volume is lost in using the protrusion cone-guided enucleation process during HMC (Panda et al, 2011).…”