Brilliant Cresyl Blue Staining Research Articles

Safety of brilliant cresyl blue staining protocols on human granulosa and cumulus cells.

The selection of human immature oocytes destined for in vitro maturation (IVM) is performed according to their cumulus-oocyte complex (COC) morphology. In animal models, oocyte pre-selection with brilliant cresyl blue (BCB) staining improves fertilization and blastocyst rates and even increases the number of calves born. As the granulosa cells and cumulus cells (GCs and CCs) have a close relationship with the oocyte and are available in in vitro fertilization (IVF) programs, applying BCB staining to these cells may help to elucidate whether BCB shows toxicity to human oocytes and to determine the safest protocol for this dye. GCs and CCs were isolated from 24 patients who underwent controlled ovarian stimulation. After 48 h, cells were exposed to: Dulbecco's Modified Eagle Medium (DMEM) with or without phenol red, DPBS and mDPBS for 60 min; 13, 20 and 26 μM BCB for 60 min; and 60, 90 or 120 min to 13 μM BCB. Cellular viability was tested using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. The 20 and 26 μM BCB exposures resulted in lower cell viability, similar to when cells were exposed to BCB for 90 or 120 min. GCs and CCs viabilities were equal among control group and 13 μM BCB group after 60 min. BCB staining was not toxic to GCs and CCs when the regime of 13 μM BCB for 60 min was used. Due to the close molecular/biochemical relationship between these cells and the gamete, we propose that it is unlikely that the use of BCB could interfere with the viability/health of human oocytes.

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 by in vitro fertilization in terms of their epigenetic status and gene expression pattern.

We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB+) or colorless cytoplasm (BCB-). The blastocyst rate was higher (p<0.001) for BCB+ than for BCB- oocytes (43.41 ± 2.54 vs. 22.74 ± 1.76%). BCB+ blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher (p<0.05) than that of BCB- blastocysts. The global level of H3K9me2, which was similar in BCB+ and BCB- blastocysts, was higher (p<0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher (p<0.05) and that of GATA2 was lower (p<0.05) in BCB+ than that in BCB- blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower (p<0.05) and that of CDX2 was higher (p<0.05) in BCB+ than in IVF blastocysts. In conclusion, because BCB+ blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB- blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.

Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development.

Brilliant cresyl blue (BCB) is a super-vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocyte's developmental potential, and the transcript abundance for select TGFβ-superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus-cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low-quality oocytes, based on BCB staining, was also determined. Cumulus-oocyte complexes (COCs) from abattoir-derived ovaries were subjected to BCB staining, and germinal-vesicle-stage oocytes and cumulus cells were harvested from control, BCB+, and BCB- (low-quality oocyte) groups for real-time PCR or Western-blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB- oocytes. Western-blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB- oocytes. Embryo-culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY-1 treatment, improve the developmental competence of BCB- oocytes. These results contribute to a better understanding of molecular indices of oocyte competence.

Effect of Vitrification by Straw and Cryotop on DNA Integrity Using Comet Assay with Reference To Brilliant Cresyl Blue Exposure in Buffalo Oocytes

To improve the effectiveness of vitrification procedure of buffalo oocytes, two experiments were conducted. The first experiment evaluated the effect of cryodevices on DNA integrity of vitrified, matured buffalo oocytes. The in vitro matured oocytes were divided into two groups, the first was vitrified using conventional French straws, while the other was vitrified using Cryotops. DNA fragmentation level was evaluated by means of comet assay. There was a significant increase in the incidence of DNA damage after vitrification with both methods compared to control. The percentages of DNA damage of vitrified thawed buffalo oocytes were significantly higher in straws than Cryotops. The second experiment evaluated the effectiveness of application of the brilliant cresyl blue (BCB) stain on the selection of immature buffalo oocytes quality. Oocytes were classified according to their cytoplasmic coloration: blue coloration (BCB+) and colorless (BCB-). Both types were matured then vitrified and compared with non stained mature oocytes vitrified by straw methods. Significantly higher DNA damage was noted in both BCB+ (P < 0.05) and BCB- (P < 0.001) oocytes, compared to non-stained vitrified oocytes. The percentages of damage were significantly higher (P < 0.05) in BCB- than BCB+ oocytes. In conclusion, DNA is a target of cryoinjury in vitrified buffalo oocytes and the damage decreased when using Cryotop than straw. Immature oocytes subjected to brilliant cresyl blue staining show a tendency towards DNA damage.

293 EFFECT OF DIFFERENT CONCENTRATIONS OF LH, FSH, AND E2 ON THE MATURATIONAL RATE OF INDIGENOUS SOUTH AFRICAN CATTLE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE STAINING

In vitro maturation of indigenous African cattle oocytes is a major challenge even though different maturation protocols work successfully in other breeds. The objective of this study was to determine the maturation rate of indigenous South African cattle oocytes following in vitro maturation in media supplemented with different concentrations of hormones and selected using brilliant cresyl blue (BCB) staining. Indigenous cattle ovaries were collected from the slaughterhouse and then oocytes were retrieved by aspiration method. A total of 966 oocytes were exposed to 26 µM BCB stain and 700 oocytes were not exposed to the BCB stain. Thereafter, oocytes exposed to the BCB stain were grouped according to the colour of their cytoplasm BCB+ (oocytes with blue cytoplasm, low G6PDH) and BCB– (unstained oocytes, increased G6PDH). The BCB exposed (BCB+ and BCB–) and the oocytes not exposed to BCB were then randomly allocated into tissue culture medium (TCM199) + 10% (vol/vol) fetal bovine serum (FBS) supplemented with 3 different concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 µg mL–1 of FSH, 5 mg mL–1 of LH, and 2 µg mL–1 of E2; the T2 group was matured in the presence of 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2; and the T3 group was matured in the presence of 1.5 µg mL–1 of FSH, 7 mg mL–1 of LH, and 4.5 µg mL–1 of E2. For IVM, 20 to 25 COC were placed in 50-µL droplets of IVM medium containing the 3 different levels of hormones. Maturation rate of oocytes was determined by the extrusion of the first polar body after 24 h of incubation in maturation medium. Data was analysed by ANOVA using SAS with 4 replicates per treatment. Treatment 2 yielded higher maturation rate for both BCB+ (65.6%) and not exposed to BCB (60.3%) oocytes compared to T1 (22, 3.03, and 16% for BCB+, BCB–, and not exposed to BCB, respectively) and T3 (48, 2.2, and 48% for BCB+, BCB–, and not exposed to BCB respectively). However, BCB– oocytes had lower polar body extrusion for T1, T2, and T3 (3.03, 8.1, and 2.2%, respectively) compared to BCB+ oocytes (22, 65.6, and 48% for T1, T2, and T3, respectively). In conclusion, immature oocytes that were cultured into TCM199 supplemented with 10% FBS, 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2 showed maturation rate for BCB+ oocytes and those not exposed to BCB. Oocytes selection using BCB staining was a useful test to classify good quality cattle oocytes. Therefore, it is suggested that treatment 2 is a suitable in vitro-maturation medium to mature indigenous South African cattle oocytes.

307 MATURATION RATE AND GENE EXPRESSION ANALYSIS OF GOAT OOCYTES SELECTED BY FOLLICLE SIZE AND BRILLIANT CRESYL BLUE STAINING

Oocyte quality plays a critical role in determining the success of embryo development. Studies on cattle and goats indicate that oocytes derived from large follicles (LFO) have greater developmental competence than those derived from small follicles (SFO). Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase and is a commonly used noninvasive marker of oocyte competence. Studies in pigs, goats, cows, mice, and dogs showed that the maturation and blastocyst developmental rate of BCB+ oocytes is significantly higher than BCB– oocytes. The aim of this study was to evaluate the maturation rate of goat oocytes selected based on follicular size and BCB staining and compare their relative patterns of gene expression. Maturation rate and gene expression profile were expected to be different in these oocyte groups. Cumulus-oocyte complexes were recovered from abattoir-derived ovaries using a slicing technique. Eleven rounds of oocyte maturation and 4 rounds of BCB staining were carried out. During each replicate, oocytes from large (≥3 mm) and small (&lt;3 mm) follicles were collected separately from the same group of ovaries. Oocyte maturation rates were 54.3 ± 5.4% for LFO (n = 378) and only 33.5 ± 3.7% for SFO (n = 981; P &lt; 0.01). The BCB+ (n = 223) oocytes yielded a significantly higher maturation rate than the BCB– (n = 194) oocytes (56.1 ± 1.8 v. 20.6 ± 3.8%, respectively; P &lt; 0.001). Gene expression analysis was conducted on individual MII oocytes (21 oocytes per group). Specific target amplification was performed on a single oocyte directly by using the CellsDirect One-Step qRT–PCR Kit (Invitrogen). Quantitative real-time PCR was then performed using the 48.48 BioMark platform from Fluidigm. Forty two genes were selected from the following categories: growth factors, transcription factors, metabolism, pluripotency, cell cycle, apoptosis, and oocyte-specific genes. Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. The significance was assigned at P &lt; 0.05. The relative expression of CCNA2, CDK2, CCNB1, POU5F1, SOX2, EGF, FGF2, GDF9, ZP3, BCL2, GJA1, DDR1, PFKFB3, IGF2R, and GRB10 was significantly greater (P &lt; 0.05) in both LFO and BCB+ oocytes compared to SFO and BCB– oocytes, respectively. The proapoptotic gene BAX, the ACSL3 gene involved in fatty acid oxidation, and the growth factor IGF1 were expressed significantly higher (P &lt; 0.05) in SFO compared to LFO. By investigating these differentially expressed transcripts, we will better understand pathways involved in oocyte developmental competence and potentially use them as markers of oocyte quality. We expect that the ability to select oocytes of better quality based on BCB staining will improve outcomes of IVF and SCNT.

Relative abundance of extra-cellular miRNAs in bovine follicular fluid: Implication for cell–cell communication during oocyte growth

s from the 4th Mammalian Embryo Genomics meeting Relative abundance of extra-cellular miRNAs in bovine follicular fluid: Implication for cell–cell communication during oocyte growth M.M.H. Sohel, S. Seifi Noferesti, D. Salilew-Wondim, M. Hoelker, F. Rings, E. Tholen, J. Udin, C. Looft, K. Schellander, D. Tesfaye ∗ Animal Breeding and Husbandry Group, Institute of Animal Science, University of Bonn, Germany Here we aimed to investigate the expression pattern of the circulating extra-cellular miRNAs in exosome and non-exosomal fraction of follicular fluid consisted of fully grown or growing oocytes and to validate exosome mediated cell–cell communication between follicular cells. For this, follicles of 5–8mm diameter (n=120) were isolated and individual COCs were subjected to brilliant cresyl blue (BCB) staining and classified as BCB+ (fully grown, n=60) and BCB− (growing, n=60) groups. The corresponding follicular fluid, granulosa cells and theca cells were used for further molecular analysis. miRNAs isolated from exosomal and nonexosomal portion of follicular fluid from the two categories was used for cDNA synthesis and subsequent analysis using a humanmiRNA PCR array (with 745miRNA). Results revealed that 25miRNAs (16 up and 9 down regulated) in exosomes and 30miRNAs (21 up and 9 down regulated) in non-exosomal portion of follicular fluid were differentially expressed (fold change≥2 and p<0.05) between growing vs. fully grown oocyte group. Among these miRNAs, miR-654-5p and miR-640 were found to be enriched in exosomal portion of follicular fluid containing growing oocytes, while miR-526b* and miR-373 were highly abundant in exosomal portion of follicular fluid This paper is part of a special issue entitled: 4th Mammalian Embryo Genomicsmeeting, Guest Edited byMarc-Andre Sirard, ClaudeRobert and Julie Nieminen. containing fully grown oocytes. In-silico analysis of miRNAs enriched in follicular fluid containing growing oocytes revealed that genes are involved indifferent signalingpathways like ubiquitin mediated proteolysis, focal adhesion, oocyte meiosis, MAPK signaling pathways to be potential targets, which are crucial for follicular development. Co-culture of granulosa cells with PKH67 dye labeled exosomes showedsuccessfuluptakeof exosomesby thosecells and subsequently elevated the endogenous miRNAs level in 2.5–5.5 fold. In conclusion, the present study highlighted the oocyte growth status dependent differences of circulatingmiRNAprofiles in follicularfluidandexosomemediated uptake of thosemolecules by surrounding cells in follicular microenvironment. http://dx.doi.org/10.1016/j.anireprosci.2014.06.011 Global transcriptome analysis of bovine blastocysts developed under alternative vivo/vitro culture conditions during specifics stages of development A. Gad1,4,∗, U. Besenfelder2, V. Havlicek2, M. Holker1, F. Rings1, I. Dufort3, M.A. Sirard3, K. Schellander1, D. Tesfaye1 1 Institute of Animal Science, University of Bonn, Bonn, Germany 2 Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria 3 Centre de recherche en biologie de la reproduction, Universite Laval, Quebec, Canada 4 Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt During the period frommaturation until blastocyst formation, several critical events occur in embryos which are regulated by a harmonized expression of genes. However, the exact influence of in vitro culture conditions 0378-4320/$ – see front matter

Expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocytes extract and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized embryos.

To compare the expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocyte extracts and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized (IVF) embryos. Hand-made cloned embryos were produced using oocyte extracts treated donor cells and brilliant cresyl blue (BCB) stained recipient cytoplasts. IVF embryos were produced by culturing 15-20 COCs in BO capacitated sperms from frozen thawed buffalo semen and the mRNA expression patterns of genes implicated in metabolism (GLUT1), pluripotency (OCT4), DNA methylation (DNMT1), pro- apoptosis (BAX) and anti-apoptosis (BCL2) were evaluated at 8- to16- cell stage embryos. A significantly (P < 0.05) higher number of 8- to16- cell and blastocyst stages (73.9 %, 32.8 %, respectively) were reported in hand-made cloning (HMC) as compared to in vitro fertilization (49.2 %, 24.2 %, respectively). The amount of RNA recovered from 8- to 16- cell embryos of HMC and in vitro fertilization did not appear to be influenced by the method of embryo generation (3.76 ± 0.61 and 3.82 ± 0.62 ng/μl for HMC and in vitro fertilization embryos, respectively). There were no differences in the expression of the mRNA transcripts of genes (GLUT1, OCT4, DNMT1, BAX and BCL2) were analysed by real-time PCR between hand-made cloned and IVF embryos. Pre-treatment of donor cells with oocyte extracts and selection of developmentally competent oocytes through BCB staining for recipient cytoplast preparations may enhance expression of developmentally important genes GLUT1, OCT4, DNMT1, BAX, and BCL2 in hand-made cloned embryos at levels similar to IVF counterparts. These results also support the notion that if developmental differences observed in HMC and in vitro fertilization produced foetuses and neonates are the results of aberrant gene expression during the pre-implantation stage, those differences in expression are subtle or appear after the maternal to zygotic transition stage of development.

Association of glucose-6-phosphate dehydrogenase activity with oocyte cytoplasmic lipid content, developmental competence, and expression of candidate genes in a sheep model.

To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH) activity in sheep oocytes with cytoplasmic lipid content, maturational competence, developmental competence to the blastocyst stage, and gene expression of certain molecular markers. Before brilliant cresyl blue (BCB) staining test, oocytes were classified as high, middle, and low cytoplasmic lipid content (HCLC, MCLC, and LCLC) and after the test as having low or high G6PDH-activity (BCB(+) and BCB(-), respectively). After maturation in vitro, a group of oocytes were subjected to IVF followed by in vitro embryo culture and another group was used for evaluation of expression of candidate genes. The cleavage and blastosyst rates were lowest (P < 0.05) in LCLC group, intermediate (P < 0.05) in MCLC group and highest (P < 0.05) in HCLC group. More (P < 0.05) oocytes in HCLC group were BCB(+), and higher (P < 0.05) maturation, cleavage, and blastocyst rates were seen for BCB(+) oocytes than the BCB(-) oocytes. Our gene expression data indicated that mRNA transcript abundance of ITGB2, pZP3, BMP15, and GDF9 genes was similar between BCB oocytes groups. However, the expression of ATP1A1 was higher (P < 0.05) for BCB(+) oocytes compared to BCB(-) oocytes. In addition, BAX transcript abundance was similar (P > 0.05) among BCB(+), BCB(-), and control groups, before and after maturation in vitro. Activity of G6PDH in sheep oocytes is highly associated with lipid content, and compared with the morphological parameters might be a more precise and objective predictor for subsequent developmental competence in vitro.

Brilliant cresyl blue staining negatively affects mitochondrial functions in porcine oocytes

The aim of the present study was to examine the effects of brilliant cresyl blue (BCB) staining on mitochondrial functions in porcine oocytes. Cumulus-oocyte complexes (COCs) collected from slaughterhouse-derived porcine ovaries were cultured with (13 μM) or without (0 μM, control) BCB for 60 min. Mitochondrial functions in oocytes were examined immediately after staining or after in vitro maturation. The BCB-stained oocytes produced reactive oxygen species (ROS) at higher levels than control oocytes immediately after staining (2.2-fold, P < 0.001) and after maturation (1.7-fold, P < 0.001). The adenosine triphosphate (ATP) content and mitochondrial membrane potential (MMP) in oocytes were similar for the two groups immediately after staining. However, ATP and relative MMP levels were significantly (P < 0.05) lower in BCB-treated oocytes than in the control (2.18 versus 2.83 pM and 0.82 versus 1.0, respectively). There was no difference in mitochondrial DNA copy number between the two groups after maturation. The ATP content in early developmental stage embryos (3 days after parthenogenetic activation) was lower in the BCB-stained group than that in the control group but the difference was not significant. In conclusion, BCB staining of oocytes at the immature stage compromises mitochondrial functions throughout oocyte maturation, but function is restored during early embryo development.

25 PORCINE OOCYTES SELECTION USING BRILLIANT CRESYL BLUE AND EMBRYO DEVELOPMENT AFTER SOMATIC CELL NUCLEAR TRANSFER

The efficiency of animal cloning through somatic cell nuclear transfer (SCNT) technology is affected by numerous factors such as oocyte quality and donor cell type. Among the factors, oocyte quality can be enhanced by identification and selection of developmentally competent oocytes before in vitro maturation (IVM). Morphological criteria following homogeneous ooplasm, more than 3 layers of cumulus cells, and oocyte diameter have been used. However, the criteria vary between examiners and even within individual. In contrast, use of Brilliant cresyl blue (BCB), a marker of oocyte growing status related to activity of glucose-6-phosphate dehydrogenase (G6PD), can be a more objective selection method for the detection of fully grown oocytes in bovine, equine and porcine. To our knowledge, BCB has been used to select oocytes in parthenogenesis, intracytoplasmic sperm injection (ICSI), and IVF, excluding SCNT in porcine. The aim of this study was to investigate whether oocytes selected by BCB have better ability to develop into blastocysts than oocytes selected by morphological criteria. After aspirated cumulus–oocyte complexes (COC) from porcine ovaries were washed in HEPES-buffered TCM-199 2 times, the COC were selected through morphological criteria and divided randomly into 2 groups. Group 1 (control; 304 COC) was directly transferred into IVM medium and Group 2 was incubated in TALP supplemented with 26 μM BCB for 90 min at 38.5°C in air. Then, COC of the second group were washed twice in TALP, and COC displaying a blue-coloured ooplasm (BCB+) were selected. The 342 BCB+ COC also were transferred into IVM medium and cultured with 5% CO2 in air at 38.5°C, with hormonal supplementation for 22 h and without the hormones for another 22 h. After denudation, the rate of degenerated oocytes and maturation rate were determined. The matured oocytes were used for SCNT and the development and total cell number of blastocysts were observed. Each experiment was repeated at least 3 times. Data were analysed by unpaired Student's t-test using Graphpad Prism (GraphPad, San Diego, CA, USA). No difference was observed between Groups 1 and 2 in the rate of degenerated oocytes (9.13 ± 0.47% and 10.68 ± 2.73%, respectively), metaphase II rate (94.60 ± 2.30% and 89.26 ± 3.76%, respectively), cleavage rate (77.02 ± 1.56% and 80.05 ± 2.31%, respectively), or blastocyst formation rate (9.74 ± 1.91% and 10.74 ± 1.30%, respectively). However, total cell number of blastocyst showed significant difference between Groups 1 and 2 (57.67 ± 1.76 and 77.50 ± 1.50, respectively; P &lt; 0.01). In conclusion, selection of oocytes through BCB staining does not improve their developmental ability with respect to cleavage and blastocyst formation rate, but enhances embryo quality by means of increased total cell number per blastocyst in porcine SCNT. This study was supported by IPET (#311011-05-2-SB010), MKE (#10033839-2012-21) and TS Corporation.

Differences in Cytoplasmic Maturation Between the BCB+ and Control Porcine Oocytes Do Not Justify Application of the BCB Test for a Standard IVM Protocol

The Brilliant Cresyl Blue (BCB) test relies on G6PDH activity and a simple protocol for the selection of higher quality oocytes. Although the BCB+ oocytes of all the species that have been investigated are characterized by superior quality when compared to BCB- counterparts, application of the test for embryo production still remains an open issue. The aim of our study was to compare BCB+ and the control oocytes (not subjected to the BCB test) in terms of selected aspects of cytoplasmic maturation (mtDNA copy number, mitochondria distribution, relative transcript abundance of six marker genes). The results of our study revealed more relevant differences within the BCB+ and the control oocytes (before and after IVM) than between the two categories of oocytes. There was no difference in the transcript abundance of the BCB+ and the control oocytes in 5 out of 6 analyzed genes (BMP15, GDF9, ATP5A1, EEF1A, ZAR1) and in mtDNA content (pre-IVM 179609 vs. 176595 and post-IVM 187243 vs. 246984, respectively). With regard to mitochondria distribution in pre- and post-IVM oocytes, there was nonsignificant tendency for a more frequent occurrence of the expected patterns in the BCB+ group. The results of the present study do not support the application of BCB staining in a routine IVM protocol due to relatively high similarity in selected parameters characterizing cytoplasmic maturation of BCB+ and control oocytes. This high similarity may results from the limited amount of less competent BCB- oocytes (10%) still present among nonselected oocytes of proper morphology.

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.

On-Spot Brilliant Cresyl Blue Staining in Fine Needle Aspiration, a Novel Technique for Cytological Diagnosis

Objective: Application of on-spot staining with brilliant cresyl blue, a supravital stain, was investigated in fine needle aspirates from breast, lymph node and thyroid. Study Design: Patients with palpable breast lumps, lymph nodes and thyroid nodules were included in the study. Brilliant cresyl blue was used to stain the smears, which were evaluated for smear adequacy as well as cytological diagnosis. Smears were decolorized and subjected to Pap and/or HE staining for comparison. Results: Smears which were deemed hemorrhagic on the first attempt with brilliant cresyl blue correlated with Pap staining; then sites were reaspirated within minutes to obtain satisfactory cellular smears. Morphologically, cells which were designated malignant on supravital staining were mostly malignant on Pap/HE staining. Conclusion: On-spot brilliant cresyl blue staining increases the diagnostic potential on preliminary examination by decreasing the staining time and is as good as conventional stains in exhibiting cellular morphology.

Expression of cyclin-dependent kinase inhibitors (CDKN1, CDKN5) in developmentally competent and incompetent porcine oocytes

Although several reports have been published regarding the cyclin-dependent kinases (Cdks) in developmentally competent mammalian oocytes, still little is known about their expression pattern in developmentally incompetent female gametes. Since Cdks are the main cell cycle division regulators, also described as &amp;ldquo;checkpoints&amp;rdquo; in the MI to MII transition, the aim of this study was to investigate the differential mRNA expression of genes encoding Cdkn1 and Cdkn5 in developmentally competent and incompetent porcine oocytes. Porcine cumulus-oocyte complexes (COCs) were collected from crossbred Landrace gilts after slaughter and partly subjected to brilliant cresyl blue (BCB) staining and in vitro maturation (IVM). Three groups of COCs were analysed: (i) oocytes soon after collection without BCB staining, (ii) oocytes which remained colourless after staining (BCB&lt;sup&gt;&amp;ndash;&lt;/sup&gt;) and (iii) COCs which stained blue (BCB&lt;sup&gt;+&lt;/sup&gt;). BCB&lt;sup&gt;+&lt;/sup&gt; COCs were additionally cultured in standard porcine medium (TCM199) for 44 h. All oocytes were analysed using QT-PCR targeting CDKN1 and CDKN5 mRNA expression. The highest CDKN1 mRNA expression was found in oocytes without BCB staining and IVM (Group I) compared to BCB&lt;sup&gt;&amp;ndash;&lt;/sup&gt; and BCB&lt;sup&gt;+&lt;/sup&gt; oocytes, (P &amp;lt; 0.001, P &amp;lt; 0.01, respectively). An increased CDKN5 mRNA level was observed in BCB&lt;sup&gt;+&lt;/sup&gt; oocytes (Group III) compared to oocytes of Group I and Group II (P &amp;lt; 0.01), respectively. The mRNA expression of both genes was always higher in BCB&lt;sup&gt;+&lt;/sup&gt; compared to BCB&lt;sup&gt;&amp;ndash;&lt;/sup&gt; oocytes. Based on this study it can be supposed that CDKN1 and CDKN5 are differentially expressed in developmentally distinct porcine oocytes and in a maturation stage-dependent manner. Moreover, CDKN1 may play a role as a molecule which regulates cell cycle arrest and the specific &amp;ldquo;block of maturation&amp;rdquo; from MI to MII. &amp;nbsp;

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.

Influence of caffeine pretreatment on biphasic in vitro maturation of dog oocytes

Phosphodiesterase (PDE) inhibitors have been utilized for in vitro maturation (IVM) of oocytes to manipulate the meiotic resumption and progression. Premature chromatin condensation and DNA replication of the oocytes, immediately after the decrease in the cAMP level, are the difficulties in canine IVM. Caffeine, a nonselective competitive PDE inhibitor, due to its structural similarity to adenosine molecule maintains the cAMP level by occupying PDE enzymes such as PDE-3A inside the oocyte and PDE-4 and PDE-5 in the cumulus cells. In this study, the effects of 12-hour caffeine pretreatment in a biphasic IVM protocol were assessed on maturation rates of canine oocytes. Sixty hours of culture after a 12-hour of 10 mM caffeine pretreatment resulted in 16.9% ± 2.4 of the oocytes reaching metaphase II stage (MII) and 25.9% ± 5.2 degeneration rate compared with the control group with 2.2% ± 2.2 MII and 37.6% ± 4.3 degeneration rates (P < 0.05). Caffeine pretreatment induced higher mitogen-activated protein kinases (MAPK1 and MAPK3) phosphorylation and maturation-promoting factor activity at 12 hours and activated MAPK1 and maturation-promoting factor at 48 hours after culture in cumulus-oocyte complexes (COCs) compared with the control group (P < 0.05). Fresh canine COCs were also analyzed before IVM using brilliant cresyl blue (BCB) staining. Oocytes showed difference in meiotic resumption (MI-MII) (BCB+ = 16.11% ± 5.5, BCB− = 9.86% ± 5.0; P < 0.05) after 60 hours of culture following 12-hour caffeine pretreatment. The BCB+ canine oocytes had higher MII rate than the BCB− group under caffeine pretreatment (10.2% ± 2.9 vs. 1.1% ± 1.1, respectively; P < 0.05). Results indicated that 12-hour caffeine pretreatment of canine COCs improves the MII maturation rates at 72 hours and BCB+ oocytes have higher competency in vitro for nuclear maturation.

Differential response of IVP, parthenogenetic and nuclear transfer derived bovine embryos upon environmental heat stress-implications for expression of autosomal and X-linked genes

The present study was conducted to examine the metabolic activities and expression differences of X-linked and autosomal genes upon exposure to heat stress using different types of bovine embryos. Embryos from in vitro fertilized (IVF), parthenogenetic (PA) and nuclear transfer (NT) were given heat treatment at morula stages at 40°C for 6 hrs. Treated embryos were studied for metabolic activities by quantification of G6PD-activity through Brilliant Cresyl Blue (BCB) staining and were used for X-linked and autosomal gene expression studies. Quantification of G6PD, PGK1, XIST as sex linked and SOX, BAX, OCT4, HSP70.1 as autosomal genes and GAPDH (endogenous control) mRNAs in each cDNA sample from heat treated and untreated embryos were assessed by quantitative PCR. Results of the present study indicate sexually dimorphic expression of selected X-linked and autosomal genes upon exposure to heat stress at morula stage in IVF, PA and NT embryos. Moreover, our study showed that female IVP derived embryos show highly decreased levels of XIST after heat shock. That could be one of the reasons for reduced in vitro developmental competence of female bovine embryos since XIST is the responsible gene for X-chromosome inactivation. Any disturbance would result in dysregulation of all X-Chromosomal encoded genes. DOI: http://dx.doi.org/10.3329/bjas.v42i1.15759 Bang. J. Anim. Sci. 2013. 42 (1): 1-10

Pentose phosphate pathway activity: effect on in vitro maturation and oxidative status of bovine oocytes

The relationship between pentose phosphate pathway (PPP) activity in cumulus-oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10mM for 22h) was added to the maturation medium (P<0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P<0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5mM for 22h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5mM (the highest concentration tested; P<0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P<0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P<0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9h maturation (P<0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte.

Developmental competence of equine oocytes: impacts of zona pellucida birefringence and maternally derived transcript expression

In the present study, equine oocytes were classified into groups of presumably high and low developmental competence according to cumulus morphology, as well as oocyte ability to metabolise brilliant cresyl blue (BCB) stain. All oocytes were evaluated individually in terms of morphometry, zona pellucida birefringence (ZPB) and relative abundance of selected candidate genes. Oocytes with an expanded cumulus (Ex), representing those with presumably high developmental competence, had a significantly thicker zona (18.2 vs 17.3µm) and a significantly higher ZPB (64.6 vs 62.1) than oocytes with a compacted cumulus (Cp). Concurrently, oocytes classified as highly developmentally competent (BCB+) had a significantly thicker zona (18.8 vs 16.1µm) and significantly higher ZPB (63.1 vs 61.3) compared with oocytes classified as having low developmental competence. Expression of TFAM, STAT3 and CKS2 was significantly higher in Ex compared with Cp oocytes, whereas expression of COX1, ATPV6E and DNMT1 was lower. Together, the data reveal that developmentally competent equine oocytes are larger in size, have higher ZPB values and exhibit a typical genetic signature of maternally derived transcripts compared with oocytes with lower in vitro developmental competence.