bluuebunny/crossref_metadata_2025 huggingface dataset:
192 RESVERATROL, A NATURAL FOOD COMPOUND, SUPPRESSED THE CELL GROWTH OF BG-1 OVARIAN CANCER CELLS INDUCED BY 17β-ESTRADIOL OR BISPHENOL A THROUGH DOWNREGULATING ESTROGEN RECEPTOR α AND INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR
Authors: Kang N.-H. , Choi K.-C.
Published: December 2012

Resveratrol (trans-3,4,5-trihydroxystilbene; RES) was adopted in this study as a novel phytoestrogen displaying antioxidant, antiinflammatory, and anticancer effects. In this study, we evaluated the inhibitory effect of RES on the cell growth induced by 17β-oestradiol (E2), a typical oestrogen, and bisphenol A (BPA), an endocrine-disrupting chemical (EDC) in BG-1 ovarian cancer cells expressing oestrogen receptors (ER) through down-regulating oestrogen receptor α (ERa) and insulin-like growth factor-1 receptor (IGF-1R). The EDC and oestrogen appear to promote the development of the oestrogen-dependent cancers. Thus, we need to develop therapeutic methods for EDC-dependent cancers. In in vitro experiments, we examined the cell viability and mRNA expression of ERa ± IGF-1R genes following the treatments with E2 or BPA in the presence or absence of RES or ICI 182 780, an ER antagonist, by MTT assay and RT-PCR, respectively. We also examined the protein level of ERa, phosphorylated insulin receptor substrate-1 (IRS-1), phosphorylated Akt1/2/3, p21, and cyclin D1 by Western blot analysis. Treatment with E2 or BPA remarkably increased the growth of BG-1 ovarian cancer cells, and their enhanced cell growth appeared to be mediated by ERa. In addition, the treatment of BG-1 ovarian cancer cells with E2 or BPA resulted in an increase in ERa and IGF-1R gene expressions. However, co-treatment of RES reversed E2- or BPA-induced ovarian cancer cell growth and mRNA expressions of ERa and IGF-1R. The protein levels of phosphorylated IRS-1 and Akt were upregulated by E2 or BPA, whereas these levels were downregulated by co-treatment of RES in the presence of E2 or BPA. Taken together, these results indicate that RES may effectively inhibit ovarian cancer cell growth via downregulating cross-talk between ERa and IGF-1R.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) of Korea government (no. 2011-0015385).
201 IDENTIFICATION OF CANDIDATE X (INACTIVE)-SPECIFIC TRANSCRIPT (XIST) CODING REGION BY HOMOLOGY ANALYSIS OF SEQUENCE AND RELATIVE EXPRESSION ANALYSIS ON FERTILIZED EMBRYO AND PARTHENOTES IN PIG
Authors: Hwang J.-Y. , Kim E.-B. , Lee D.-K. , Oh J.-N. , Lee C.-K.
Published: December 2012

X (inactive)-specific transcript (XIST) has been known as long noncoding RNA, which regulates X-chromosome inactivation in female mammals to provide dosage equivalence between male and females. Its important roles in early embryo and stem cells development have been suggested in humans and mice. However, its coding region has been unclear in pigs. To determine the coding region of XIST in pigs, we examined candidate XIST coding region using BLAST, PCR, and sequencing techniques. By comparing pig whole genome sequence (Sus scrofa 10.2) with human, murine, and bovine XIST transcript sequence using BLAST, we selected candidate coding regions of XIST in pig. The result showed that XIST is coded on the minus strand of NW_003612825 contig, and its length was nearly 32 kb, which was similar to the length of human and bovine XIST genes. In order to identify the coding region of XIST in this candidate sequence, we designed 24 primer pairs and RT-PCR was performed using porcine embryonic fibroblast cells (PEF) established from porcine male and female lines.The XIST transcripts were expressed in female PEF but not in male PEF. Thus, by designing candidate intron-spanning primers, we confirmed the existence of introns between the first and last exons. The amplicon sequence was analysed and 5 short exons (<400 bp) that have similarity to the XIST coding region of other characterised species were identified. Moreover, to examine the XIST coding strand in pig genome, we conducted strand-specific reverse transcription. With BLAST homology searches, we confirmed that XIST was coded on the negative strand of the contig on the X chromosome. To analyse stage and origin of XIST transcript in the pig, we subsequently performed real-time PCR using cDNA template synthesised from sperm, oocyte, 1, 2, 4, 8, morular, and blastocyst of fertilized embryo and parthenotes, and intron-spanning primer sets were used. The results revealed that XIST was expressed after maternal to zygote transition (4- to 8-cell stage in pig) and not expressed in mature germ cells (sperm and oocyte). Moreover, parthenotes showed a higher expression level compared with fertilized embryos after the 8-cell stage. These results verify the existence of XIST in pig early embryos and may contribute to the comparative analysis of XIST mechanisms in different species. Further study using FISH and methylation analysis may reveal its mechanisms of action.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; No. 2012006276) and the Biogreen 21 program (PJ0081382011) of the Rural Development Administration, Republic of Korea.
203 IDENTIFICATION OF A POTENTIAL MULTILINEAGE-DIFFERENTIATING STRESS ENDURING CELL POPULATION IN PORCINE FETAL FIBROBLASTS
Authors: Li D. , Hall V. J. , Freude K. , Rasmussen M. , Hyttel P.
Published: December 2012

Physiologically, humans and pigs are very similar, which makes cell model systems such as porcine-induced pluripotent stem (iPS) cells an attractive model for studying human diseases. Recently, it has been shown that a finite population of cells, termed multilineage-differentiating stress enduring (MUSE) cells, which are particularly prone for reprogramming, exists in human fibroblasts. Human MUSE cells co-express the cell surface markers, SSEA-3 and CD105; however, it is unknown if a similar subpopulation of cells exists in porcine fibroblasts. Therefore, we examined the expression level of varying cell surface markers, including SSEA-1, SSEA-3, CD105, and Vimentin (positive control), in 9 porcine fetal fibroblast lines from 3 different breed backgrounds (Danish Landrace lines 2, 4, 5; Gottingen minipig lines A1, B2, B3; Yucatan minipig lines 1, 3, 4) by performing chromogen immunocytochemistry. The results revealed that all the 9 cell lines were 100% positive for Vimentin and negative for SSEA-3. However, a small positive population of SSEA-1 cells could be detected in Danish Landrace lines 2, 4, 5 (0.267%, 0.441%, 0.292%, respectively) and in Gottingen minipig lines A1, B2, B3 (0.207%, 0.214%, 0.325%, respectively). Furthermore, CD105 could only be detected in the Yucatan minipig lines (0.170, 0.177, and 0.233%, in lines 1, 3, and 4, respectively). To identify if any of these lines were more receptive to reprogramming, we performed non-integrating vector-based reprogramming. A total of 1 × 105 cells from each cell line was electroporated with 3 episomal plasmids (pCXLE-hOCT3/4-shp53; pCXLE-hSK; pCXLE-hUL), which were diluted to 1 µg µL–1, and then plated in DMEM-AQ + 10% FBS without antibiotics. The medium was replaced with the addition of penicillin/streptomycin on Day 2, and on Day 7 cells were trypsinized and passaged onto mitomycin C-treated mouse embryonic fibroblasts in iPS culture medium (DMEM-F12 + 20%KSR + NEAA + Pen/strep + 2 µL mL–1 β-Me + 10 ng mL–1 bFGF). Only a select number of Danish Landrace lines 2 and 4 formed ES-like colonies after 14 days, and few colonies with neural-like morphology emerged in Yucatan minipig line 3, but all these colonies lost their proliferative potential after a few passages. The reprogramming efficiency observed on Day 21 was 0.0787, 0.0170, and 0.0033% for Danish Landrace 2, Danish Landrace 4, and Yucatan 3 lines, respectively. The NANOG immunostaining was also performed at passage 1, and a minor proportion of the colonies in Danish Landrace line 2 was found to contain small populations of cells with nuclear staining for NANOG. Interestingly, Danish Landrace lines 2 and 4, which formed ES-like colonies, also expressed SSEA1, which may suggest that SSEA1 could potentially be a marker of a MUSE cell population. Further studies are ongoing to investigate this hypothesis. This research may further support the elite hypothesis for how reprogramming may occur.
206 EFFECT OF OVARIAN HYPERSTIMULATION ON CIRCULATORY microRNA PROFILE IN BOVINE FOLLICULAR FLUID AND BLOOD PLASMA
Authors: Seifi Noferesti S. , Hoelker M. , Sohel M. H. , Salilew-Wondim D. , Rings F. , Schellander K. , Tesfaye D.
Published: December 2012

MicroRNAs (miRNA) are noncoding small RNA that are known to play a role in posttranscriptional regulation of genes involved in various physiological processes including disease and reproduction. The circulatory forms of miRNA, which are present in body fluids, are reported to be used as biomarkers for disease and pregnancy. This study was conducted to investigate the effect of ovarian hyperstimulation on the expression pattern of circulatory miRNA in blood plasma and follicular fluid. For this, one group of Simmental heifers (n = 6) was hyper-stimulated using a superovulation (SO) protocol of 8 consecutive FSH injections over 4 days in decreasing doses, and others (n = 6) were synchronized (SY) using a standard synchronization protocol. Following this, the blood samples were collected at Day 0 (onset of oestrus) and Day 7, and follicular fluid was collected from both groups at Day 0 of the oestrous cycle. Total RNA, including small RNA, was then isolated from follicular fluid and blood plasma using a miRNeasy mini kit (Qiagen, Valencia, CA, USA) and subsequently used for circulatory miRNA expression studies using the human miRCURY LNA™ Universal RT miRNA PCR array system (Exiqon, Woburn, MA, USA). Following the miRNA PCR array run, data analysis was performed using a delta threshold cycle (ΔCT) method. Results showed that 23 miRNAs were found to be differentially expressed in blood plasma (fold change ≥2 and P ≤ 0.05) between SO and SY groups. Among these, 8 miRNAs including miR-127-3p, miR-494, miR-147, miR-134, and miR-153 were downregulated and 15 miRNAs including miR-34a, miR-103, miR-181c, miR-24-2-3p, miR-18a-3p, miR-20b-3p, and miR-708-3p were found to be upregulated in SO groups. Similarly, in follicular fluid, 71 miRNAs were found to be differentially expressed between the SO and SY groups. Of these, 33 miRNA including miR-100, miR-877, miR-659, miR-200c, miR-29b-2-3p, miR-361-5p, and miR-145 were downregulated, whereas 38 miRNAs including miR-374a, miR-720, miR-155-3p, miR-202-3p, miR-33b, miR-12-3p, and miR-224-3p were upregulated in follicular fluid aspirated from SO cows compared with the SY animals. Ingenuity pathway analysis of predicted target genes of miRNA, which are dysregulated due to ovarian hyperstimulation, showed the dominance of pathways related to neuregulin signalling, axonal guidance signalling, GNRH signalling, and Wnt β-catenin signalling pathways. In conclusion, this study revealed alternation in circulatory miRNA expression profile both in blood plasma and follicular fluid as a result of ovarian hyperstimulation.
208 COMPARISON OF THE EXPRESSION OF SOX2 AND Stat3 IN BOVINE BLASTOCYST AND HATCHED BLASTOCYST
Authors: Rascado T. S. , Guastali M. D. , Paschoal D. M. , Sudano M. J. , Vergara L. E. , Mazieiro R. R. , Landim F. C. , Araujo J. P.
Published: December 2012

The investigation of the transcription factors involved in the regulation of pluripotency in pre-implantation embryos of cattle provides important information regarding the early embryonic development and derivation of embryonic stem cells. The present experiment aimed to compare the level of expression of SOX 2 and Stat3, pluripotency markers, in bovine blastocysts (D7) and hatched blastocysts (D10), because it is known that around hatching, the inner cell mass (ICM) differentiates into hypoblast and epiblast. Cumulus–oocyte complexes were matured in TCM 199 for 24 h and fertilized with frozen–thawed sperm. Presumptive zygotes were cultured in SOFaaci for 7 days (group 1) and for 10 days (group 2). All embryos were washed 3 times in PBS, pooled, and frozen at –80°C until RNA extraction. For quantification of SOX2 and Stat3 mRNA levels, total RNA was isolated from pools of 7 embryos per replicate (n = 3) and for each examined developmental stage using RNeasy Micro Kit (Qiagen). The reverse transcriptase Superscript III (Invitrogen) was used for the synthesis of cDNA (cDNA), and the qPCR was performed with the Gotaq qPCR Master Mix (Promega, Madison, WI, USA). As negative control, cDNA was replaced by nuclease-free water in the qPCR reaction. Quantification of expression was determined by the relative standard curve method and normalized to the housekeeping gene YWHAZ. Standard curves for SOX2, Stat 3, and YWHAZ were derived from 10-fold serial dilutions of bovine DNA and gave correlation coefficients greater than 0.99 and efficiencies greater than 94%. The data from 3 replicates were analysed by ANOVA, and it was found that the level of SOX2 abundance is 9.8-fold higher in D7 blastocyst cells compared with D10 blastocyst cells, and the level of Stat 3 transcription is 1.5-fold higher in blastocyst cells than in hatched blastocysts. The expression of pluripotency markers SOX2 and Stat3 was significantly higher in embryos with 7 days of development because at this stage the MCI had not yet begun the process of differentiation.
Support of FAPESP is acknowledged.
216 EFFECTS OF DIFFERENT POLYUNSATURATED FATTY ACID SUPPLEMENTATION DURING THE POSTPARTUM PERIODS OF EARLY LACTATING DAIRY COWS ON INSULIN RESISTANCE AND SOMATOTROPIC AXIS
Authors: Dirandeh E. , Towhidi A. , Ansari Pirsaraei Z. , Ganjkhanlou M. , Zeinoaldini S. , Rezaei Roodbari A. , Najafi M.
Published: December 2012

During periods of negative energy balance, GH cannot stimulate hepatic IGF-1; the condition is termed GH resistance (Donaghy and Baxter, 1996). Studies show that insulin may be a key metabolic signal that regulates the coupling of the somatotropic axis (Butler et al., 2003). The objectives were to test the hypothesis that α-linoleic acid (n-3), or linoleic acid (n-6) could promote insulin resistance in dairy cows. For this goal, Holstein cows were assigned randomly to be fed either 1-soybean whole roast (S, n = 4), or 2-linseed extruded (L, n = 4), or 3-palm oil use a source of saturated fatty acid (C, n = 4), respectively, from calving to 60 DIM. Three diets were formulated to have equal concentrations of DM, ME, and CP, but to differ in ratio of n-3/n-6 PUFA (4.2, 1.2, and 3.2 for S, L, and C treatments, respectively). Supplementation of FA was at 1.5% of dietary dry matter. There was no difference between groups (mean ± SEM) in parity (3.0 ± 1.90) or BCS at calving day (3.2 ± 0.07). At Day 60 postpartum, liver tissues (~100 mg) were collected by needle biopsy as described by Rhoads et al. (2008) with minor modifications. Liver tissue was washed with sterile PBS, placed in a screw-cap micro centrifuge tube, snap-frozen in liquid nitrogen, and stored at –80°C until RNA extraction. We applied real-time PCR for measurement of mRNA abundance. Real-time PCR was executed in triplicate. Reaction mixtures for real-time PCR included 1.5 µL of cDNA as template, 7.5 µL of SYBRR Green PCR Master Mix (ABI, USA), 1 µL of forward primer, 1 µL of reverse primer, and 4 µL of double distilled water. The efficiencies for GHR1A, insulin receptor (IR), IGF-I, and IGFBP-2 were 1.22, 0.99, 1.02, and 1 with an R2 of 0.99 or above for the regression. We used the 2–ΔΔCt method in this model. Rotor Gene analysis software (version 6.0) was used to analyse all the results from the PCR assays. Data were analysed using the MIXED procedure of SAS (2000, SAS Institute Inc., Cary, NC, USA), in a completely randomised design. Results showed that supplementation with a high level of flaxseed on treatment L for 2 months increased levels of IR mRNA compared with S and C treatments (P < 0.05). The levels of IR mRNA were not different between S and C treatments (P = 0.202). There was also a strong tendency towards a 2.2-fold increase (P = 0.06) in GHR1A gene expression in cows on the flaxseed diet compared with the other diets, also there were no significant difference between S and C treatments. The IGF-I and IGFBP mRNA were not different between the treatments (P > 0.05). In conclusion, n-3 fatty acids promoted insulin resistance via increasing insulin receptor signalling that could improve milk production and specifically fertility in lactating dairy cows.
223 THE EFFECT OF IN VITRO FERTILIZATION MEDIA AND THE CAPACITATION AGENT ON EPIDIDYMAL WHITE-TAILED DEER SPERMATOZOA
Authors: Lambe-Steinmiller J. , Bondioli K. , Godke R. A. , Gentry G.
Published: December 2012

To determine the requirements for in vitro production of white-tailed deer embryos, media components that induce sperm capacitation and an acrosome reaction (AR) should first be identified. Typically, in vitro production protocols are species specific with regard to media composition and capacitating agents (CA). Comparison of the bovine and red deer systems revealed a distinct difference in the CA utilised [heparin v. 20% sheep serum (SS), respectively] and in the amount of bicarbonate and Ca2+ necessary for fertilization to occur. The aim of this study was to evaluate the ability of 2 CA (heparin and SS) to induce the capacitation of white-tailed deer epididymal sperm in either a standard bovine (BIVF) or red deer-formulated (DSOF) fertilization medium. Epididymal spermatozoa were collected from fair-chase bucks (n = 15), extended with Triladyl (48 to 147 × 106 sperm mL–1), and frozen in LN2 vapor. Live spermatozoa were selected from frozen–thawed semen samples from each buck and reconstituted (55 × 106 sperm mL–1) in either BIVF or DSOF and supplemented with either heparin (10 µg mL–1) or SS, or were nontreated controls. Samples were then incubated at 37°C for either zero [treatment (T) 0], 2 (T2), or 4 (T4) hours and the AR was artificially induced using lysophosphatidylcholine (100 µg mL–1). After AR induction, treatments were labelled with PI/PNA and M540/YoPro-1 so that live cells, acrosomal status, and membrane lipid disorders could be analysed by flow cytometry after each incubation period (T0, T2, and T4). The media (P = 0.42), CA (P = 0.38), or their interaction (P = 0.68) did not affect the percentage of live acrosome-intact spermatozoa across time (P = 0.73). The percentage of lipid disruption was higher (P < 0.001) in SS and changed (P = 0.005) across time. Data obtained from this study showed that unlike for red deer, increased bicarbonate and Ca2+ are not necessary to induce capacitation of white-tailed deer epididymal spermatozoa. Although SS did not appear to affect the number of live acrosome-intact spermatozoa, its presence did alter the lipid architecture of the sperm plasma membrane.
224 SILDENAFIL ACCELERATES SPERM PENETRATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM
Authors: Funahashi H. , Wu Q.-S.
Published: December 2012

Sperm capacitation, a cyclic-adenosine monophosphate-dependent phenomenon, is an important initiation step for penetration into oocytes. In porcine IVF, the use of caffeine, as a nonspecific phosphodiesterase (PDE) inhibitor, is common to accelerate sperm capacitation and penetration. The objective of this study was to examine the effects of PDE inhibitors (cilostamide, rolipram, and sildenafil as PDE type 3-, type 4-, and type 5-specific inhibitors, respectively) on the capacitation of boar sperm and the penetration into porcine oocytes in the absence of caffeine and other capacitation inducers in a chemically defined medium. After washing sperm samples collected from an ejaculated sperm-rich fraction of different individual Berkshires, the sperm were resuspended in capacitation inducer-free (theophylline- and adenosine-free) PGM-tac4 (mPGM-tac) at 5 × 105 cells mL–1. The suspension was cultured in mPGM-tac nonsupplemented or supplemented with 2.5 mM cilostamide, rolipram, or sildenafil for 90 min at 39°C in an atmosphere of 5% CO2 in air, and then the capacitation status was assessed by chlortetracycline fluorescence assay. Other sperm suspensions were used to co-culture with denuded in vitro-matured oocytes in the same medium for 8 h in an atmosphere of 5% CO2 in air and fixed, and sperm penetration was then examined. Statistical analyses of results from 4 replicated trials were performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In our result from the chlortetracycline fluorescence assay, although the incidence of intact sperm was significantly reduced in the presence of rolipram (54.3%) and sildenafil (52.7%) as compared with controls (66.7%), there were no differences in capacitated sperm among experimental groups (24.3 to 34.3%). The incidence of acrosome-reacted sperm was higher in the presence of cilostamide (17.3%) than in the others (9.0 to 13.0%). High sperm penetration was observed only in the presence of sildenafil (76.6%) as compared with the control (0%) or the presence of rolipram (4.4%) or cilostamide (1.8%). These results demonstrate that inhibition of PDE type 5, but not PDE type 3 and type 4, significantly accelerates the penetration of boar sperm into the oocytes in a capacitation inducer-free chemically defined medium, whereas inhibition of PDE type 3 may induce an acrosome reaction.
227 CALRETICULIN, A 60-kDa PROTEIN, PREVENTS POLYSPERMY IN ZONA PELLUCIDA-FREE PIG OCYTES
Authors: Romar R. , Soriano-Úbeda C. , Saavedra M. D. , Gadea J. , Avilés M. , Coy P.
Published: December 2012

After gamete membrane fusion or artificial oocyte activation, cortical granules undergo exocytosis and the released content modifies the zona pellucida (ZP), preventing polyspermy. Calreticulin (CRT), a calcium-binding highly conserved protein of 60 kDa, is contained in cortical granules from hamster eggs (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60), and we recently showed it is exocytosed from chemically activated ZP-free pig oocytes (Romar et al. 2012 Reprod. Fertil. Dev. 24). When pig ZP-enclosed oocytes were incubated with CRT, monospermy was not improved (Romar et al. 2011, Maternal communication with gametes and embryo, p. 72), suggesting that the likely role of CRT in preventing polyspermy might be carried out at the oolemma level. Our objective was to evaluate whether CRT prevents polyspermy in pig ZP-free oocytes by treating the cells with this protein before being inseminated. In vitro-matured cumulus–oocyte complexes (44 h, NCSU-37 medium) were decumulated and ZP was digested with Tyrode’s acid. The ZP-free oocytes were incubated for 30 min in TALP medium supplemented with 0, 100, 1000, and 5000 pg of CRT (ab91577, Abcam, Cambridge, MA, USA) per oocyte. After washing, ZP-free oocytes were inseminated (25 000 sperm mL–1) and gametes were co-cultured for 18 h. Putative zygotes were fixed and stained with Hoechst 33342 to analyse the fertilization results. Four replicates with 30 to 35 oocytes per group were done, and results were analysed by one-way ANOVA. A P-value ≤0.05 was taken to denote statistical significance. Incubation with CRT did not affect penetration rates that were similar among groups (77.12 ± 3.88 and 72.73 ± 4.07, respectively, for the 0- and 5000-pg CRT groups). However, the mean number of sperm per penetrated oocyte decreased from 3.01 ± 0.28 (0-pg group) to 2.07 ± 0.16 (5000-pg group), and monospermy rate increased from 30.77 ± 4.87 (0-pg group) to 52.27 ± 5.36 (5000-pg group; P ≤ 0.05). Incubation with CRT did not affect the number of sperm attached to oolemma, which was similar among all groups (11.45 ± 1.16 v. 10.75 ± 1.17, respectively, for 0 and 5000 pg of CRT). These preliminary data suggest that CRT, a protein exocytosed after oocyte activation, participates in the membrane block to polyspermy in pigs. Future studies to describe the exact mechanism of action of this chaperone protein are necessary.
Supported by MEC and FEDER (AGL2009-12512-C02-01).
233 ASSOCIATION OF THE BOVINE 2-CELL-STAGE BLASTOMERE TRANSCRIPTOME PROFILE WITH THE INDIVIDUAL DEVELOPMENTAL POTENTIAL OF THE SISTER BLASTOMERE
Authors: Held E. , Salilew-Wondim D. , Tesfaye D. , Schellander K. , Hoelker M.
Published: December 2012

Transcriptome profiling has been used to identify genes related to developmental competence in bovine embryos and oocytes for several years. However, the direct relationship between the transcriptome profile and developmental potential of the same pre-implantation embryo is missing. Therefore, in the present study, one blastomere of a 2-cell-stage embryo was taken as a biopsy and immediately snap-frozen for transcriptome analysis; the sister blastomere was cultured individually in a well-of-the-well culture system. Frozen individual blastomeres taking the form of 2-cell-stage embryos were pooled together, depending on the developmental destination of the sister blastomeres. Accordingly, three groups were defined: 1) embryos that did not cleave after separation (2CB), 2) embryos arrested before embryonic genome activation (8CB), and 3) embryos that reached the blastocyst stage (BL). On the basis of these developmental phenotypes, blastomeres were pooled and used for transcriptome analysis, using a bovine EmbryoGene microarray platform (Agilent Technologies, Santa Clara, CA, USA). Results revealed 632 genes to be differentially regulated (fold change, P ≥ 1.5, P ≤ 0.05; FDR, P ≤ 0.1) between competent (BL group) and incompetent 2-cell-stage embryos (2CB) as well as 150 genes between the BL group and 8CB. Seventy-seven genes were commonly differentially regulated. Functional annotation analyses of differentially regulated genes indicated those genes to be involved in the protein ubiquitination pathway on the one hand and in the oxidative stress response, including oxidoreductase, peroxidase, and antioxidant activity, as well in oxidative phosphorylation (e.g. NDUFS1, MAPK14, CAT, PRDX1, and PRDX6) on the other hand. Furthermore, selected candidate genes known to function as direct and indirect scavengers of reactive oxygen species (ROS) were analysed for their expression in an independent model for developmental competence; namely, early- and late-cleaved 2-cell-stage embryos. Moreover, ROS detection was performed, showing higher accumulation of ROS in late-cleaved 2-cell-stage embryos, whereas lower ROS levels were detected in early-cleaved 2-cell-stage embryos associated with higher expression of ROS scavengers. The overall findings of the present study indicate the potential of using blastomere biopsies at 2-cell-stage embryos for molecular analysis to understand the molecular mechanisms associated with the further developmental competence of early-stage embryos.
237 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED EARLY BOVINE EMBRYOS: USE OF HOMOLOGOUS FOLLICULAR FLUID SUPPLEMENTATION IN THE OOCYTE MATURATION MEDIA
Authors: Demyda-Peyrás S. , Hidalgo M. , Dorado J. , De Luca L. , Genero E. , Ortiz I. , Ramirez L. , Acha D. , Urbano M. , Alcaraz L. , Galvez M. J. , Moreno M.
Published: December 2012

Chromosomal abnormalities were described as a possible cause of embryo failures in cattle, even more so when they are in vitro produced. It has been widely demonstrated that the post-fertilization culture environment affects the frequency of blastomeric aneuploidies. However, the literature concerning the effect of the oocyte maturation techniques on in vitro-produced embryos is scarce. The aim of this study was to determine the effect of homologous bovine follicular fluid (BFF) as a possible replacement for commercial sera in the appearance of chromosomal abnormalities on early IVF-produced embryos. Cumulus–oocyte complexes obtained from ovarian puncture were maturated in modified bicarbonate-buffered TCM-199 media, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin in three different groups. Two treatments were performed: 1) base media supplemented with BFF, obtained aseptically from follicles between 4 and 10 mm in diameter (10 and 20%), and 2) a control group, with base media supplemented with 10% FCS without BFF. After 20 h of culture at 38.5°C in a 5% CO2 humid atmosphere, cumulus–oocyte complexes from both treatments were fertilized in IVF media and then cultured for 72 h in SOF media, according to our laboratory techniques. A total of 152 early embryos were cytogenetically evaluated following our standard laboratory techniques. Developed embryos were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated in each embryo by direct observation at 1250× magnification using a bright field microscope. Results were statistically compared among treatments by the expected proportion test. No significant differences (P > 0.05) were found between different culture media on the percentages of normal diploid embryos or each kind of numerical abnormality. According to our results (Table 1), the use of homologous follicular fluid as a supplement on the oocyte maturation media did not influence the appearance of abnormal complements in the embryos produced compared with the use of FCS. In conclusion, homologous follicular fluid may be considered a valid serum replacement in the maturation media on IVF-produced bovine embryos.

Table 1.Analysis of chromosomal complements of Day 3 in vitro-produced bovine embryos derived from oocytes maturated in culture media with different serum supplementation1
241 METHYLATION PROFILE OF XIST GENE EXON 1 IN BOVINE SPERMATOGENIC CELLS
Authors: Nishimura R. C. , Bessa I. R. , Dode M. A. N. , Franco M. M.
Published: December 2012

Mammalian embryos undergo two waves of genome reprogramming, the first in the zygote, when paternal and maternal genomes are demethylated and, shortly thereafter, remethylated, and the second in the primordial germ cells. It is believed that remethylation is reestablished in male germ cells before birth; however, it is not clear when and how this process occurs in the bovine. Thus, this work aimed to verify whether epigenetic reprogramming occurs during spermatogenesis by evaluating the methylation profile of XIST gene exon 1 in different spermatogenic cells. Spermatocytes, elongated spermatids, and spermatozoa from the epididymis head and tail were collected from bovine testes obtained from a slaughterhouse. The DNA was extracted from the collected cells and treated with bisulfite. The treated DNA was amplified through nested PCR, and the product was inserted on a vector and cloned in bacteria. The plasmid DNA was extracted and sequenced. The sequences of the experiment were compared with the sequence of XIST gene from GenBank, and those that presented a bisulfite conversion rate of ≥95% and a homology of ≥97% with the sequence from GenBank were used. To compare the methylation pattern among groups, the total percentage of methylated CpG was calculated in each replicate and each group. The methylation pattern was compared using Kruskal-Wallis and Mann–Whitney tests because the data did not show normality. All data were compared using the Prophet Program, version 5.0 (1996; BBN Systems and Technologies, Cambridge, MA, USA), and are shown as the mean ± SEM. The significance level used was P < 0.05. The spermatocytes, elongated spermatids, and spermatozoa from the epididymis head and tail groups presented 15.70 ± 14.54%, 1.96 ± 1.96%, 74.09 ± 6.78%, and 45.09 ± 20.19% of methylation, respectively. The epididymis head group was more methylated than the spermatocyte and elongated spermatid groups (P < 0.01). When arranged in two groups, one with spermatocytes and elongated spermatids, and the other with epididymidal spermatozoa, and compared, it was observed that the methylation level was different (P < 0.01) between them. These results suggest that epigenetic reprogramming is still occurring during spermatozoa formation.
242 PROTECTIVE EFFECT OF VITAMIN E TREATMENT IN HEAT-STRESSED BOS TAURUS BULLS
Authors: Barnabe V. H. , Barnabe R. C. , Goes P. , Perez E. G. A. , Losano J. D. A. , Rocha C. C. , Dalmazzo A. , Tsunoda R. H. , Rodrigues M. P. , Pacheco Filho P. I. , Lezcano F. A. , Nichi M.
Published: December 2012

Bos taurus bulls, when raised under tropical conditions, are highly susceptible to heat stress, which leads to impaired semen quality, leading to significant economical losses because, in these regions, the reproductive mounting season occurs mainly during the summer. Previous studies have indicated that oxidative stress (i.e. attack by reactive oxygen species) may be the main mechanism of sperm damage in such conditions. Therefore, treatment with antioxidants may be an important alternative to improve semen quality in heat-stressed B. taurus bulls. The objective of the present study was to evaluate whether the treatment with vitamin E, an important antioxidant, could improve sperm quality in insulated bulls. Towards this aim, eight adult Holstein bulls were submitted for semen collection, and the sperm was submitted for motility evaluation by computer-assisted sperm analysis (Ivos, Hamilton Thorne Inc., Beverly, MA, USA), examination of membrane and acrosomal integrity (eosin/nigrosin and fast green/bengal rose stain, respectively), mitochondrial activity (diaminobenzidine stain; full mitochondrial activity or no mitochondrial activity), and sperm susceptibility to oxidative stress (thiobarbituric acid-reactive substances). Bulls were then insulated (testicles covered in a thermal bag for 3 days) and randomly assigned to two treatment groups: no vitamin E (placebo) and vitamin E (subcutaneous injection of 3000 IU of α-tocopherol each of 10 days). Subsequent semen analysis was performed 1 and 60 days after the insulation. Statistical analysis was performed with SAS (SAS Institute Inc., Cary, NC, USA) repeated-measures ANOVA, and significance of P < 0.05 was adopted. No differences were found on any of the variables before insulation. One day after insulation, animals treated with vitamin E showed a lower percentage of static sperm and a higher percentage of motile sperm when compared with animals treated with the placebo (28 and 63% v. 56 and 34%, respectively; P < 0.05). Also at this time, sperm susceptibility to oxidative stress was lower in animals treated with vitamin E (vitamin E: 410 ng/106 sperm; no vitamin E: 1760 ng/106 sperm; P < 0.05). Sixty days after insulation, sperm susceptibility to oxidative stress was still lower in animals treated with vitamin E when compared with the placebo group (1176 and 192 ng/106 sperm, respectively; P < 0.05). However, no differences were found on the other variables. Results indicate that vitamin E, an antioxidant whose main function is protection of the plasma membrane, may be an alternative to avoid the acute deleterious effects of the heat stress in B. taurus bulls raised under tropical conditions. In addition, even with no heat stress involved, vitamin E treatment may provide constant protection, increasing the resistance of the sperm against the reactive oxygen species.
244 PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF SPERM BOUND TO THE PORCINE OVIDUCT DURING THE PERIOVULATION STAGE
Authors: Luño V. , López-Úbeda R. , Lefièvre L. , Matás C.
Published: December 2012

The interaction of spermatozoa and oviductal epithelial cells (OEC) is a controlled process that regulates sperm capacitation and the acquisition of fertilizing ability until the time of ovulation. A crucial signalling event involved in capacitation is protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm bound or unbound to the oviduct of the sow during the periovulation stage. Eight crossbred multiparous sows were inseminated with 3 × 109 sperm. The animals were anesthetized and laparotomies were performed at 36 h after insemination. Ovaries and oviducts were exposed through a midventral incision for collection. Each oviduct was divided into four parts: the ampulla, ampullary-isthmic junction, isthmus, and utero-tubal junction. All segments of the oviduct were flushed to recover spermatozoa, which were subsequently fixed. Tissue obtained from each of the oviduct segments were fixed and embedded in a paraffin block. Sections were mounted on poly-l-lysine-coated slides and deparaffinized. Flushed sperm and oviductal sections were analysed by indirect immunofluorescence using monoclonal antiphosphotyrosine antibodies. Three different sperm subpopulations were determined according to the distribution of protein tyrosine phosphorylation observed: nonphosphorylated spermatozoa (pattern 1), subequatorial segment or subequatorial segment and flagellum phosphorylation (pattern 2), and subequatorial segment and head or flagellum phosphorylation, or both (pattern 3). Data were analysed with SPSS (IBM, Armonk, NY, USA) using one-way ANOVA. After flushing, most sperm were recovered from the utero-tubal junction segment of the oviduct, and sperm exhibited a higher proportion of pattern 2 (81.62%). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment, head and flagellum in the isthmus (32.34%), ampullary-isthmic junction (37.70%), or ampulla region (35.11%; P < 0.05). Very few sperm were attached to OEC, and sperm oviduct binding was mainly found in the isthmus region. The most common tyrosine phosphorylation distribution observed in sperm attached to OEC was pattern 1 (84.21%), although labelling to the subequatorial segment was also observed. Our results showed that only sperm that did not display tyrosine phosphorylation on the sperm acrosome region (head) were found bound to OEC. In conclusion, distinct protein tyrosine phosphorylation patterns were found on sperm bound to OEC. This interaction could be used as a tool for selecting a population of sperm containing low levels of tyrosine phosphorylation.
245 EFFECT OF INCUBATION IN OVIDUCTAL EPITHELIAL CELLS AND CO-CULTURE WITH OOCYTES IN THE PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF BOAR SPERM
Authors: López-Úbeda R. , Matás C.
Published: December 2012

The oviductal epithelial cells (OEC) play an important role in the process of sperm capacitation and the acquisition of fertilizing capacity. One of the most important changes that can be observed in spermatozoa is the increase in protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm after incubation in oviductal cells in the presence of oocytes. Epithelial cells were stripped from the oviducts of cycling sows and cultivated for 7 days on a petri dish. Sperm cells treated by a discontinuous gradient of isotonic Percoll were added to the OEC and incubated for 30 min. After 30 min, the spermatozoa bound and unbound were separated and co-cultured with oocytes for 18 h. Five different samples were analysed by indirect immunofluorescence to determine the localization of proteins phosphorylated in tyrosine residues (Tardif et al., 2001 Biol. Reprod. 65, 784–792): ejaculated control, sperm unbound OEC after 30 min, sperm unbound after 30 min with oocytes, sperm bound after 30 min with oocytes, and sperm bound after 30 min and detached after culture with oocytes. Five patterns were determined according to their surface distribution: nonphosphorylated spermatozoa (pattern 1), head, tail, or both phosphorylated (pattern 2), equatorial segment phosphorylated (pattern 3), equatorial segment and head, tail, or both phosphorylated (pattern 4), and phosphorylation of the tail alone and in combination with other areas of the sperm (pattern 5). Sperm without any treatment showed lower tyrosine phosphorylation levels (87.04%; P < 0.05). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment and head, tail, or both phosphorylated (47.71%; P < 0.05), and this pattern was associated with a high level of capacitation. The same pattern was reduced when unbound sperm were co-cultured with oocytes for 18 h (25.67%; P < 0.05), and was even lower in sperm bound with oocytes. The study of phosphorylation of the tail (pattern 5) showed no significant differences between different samples. We concluded that sperm-bound OEC have lower levels of phosphorylation, indicating a selective function in the sperm-OEC interaction. In addition, phosphorylation of the tails appears to be related more to the hyperactivation of sperm than to the different culture conditions.
26 REPROGRAMMING OF TWO SOMATIC NUCLEI IN THE SAME MOUSE OOPLASM LEADS TO PLURIPOTENT NOT TOTIPOTENT EMBRYOS
Authors: Pfeiffer M. J. , Esteves T. C. , Balbach S. T. , Arauzo-Bravo M. J. , Stehling M. , Jauch A. , Houghton F. D. , Boiani M.
Published: December 2012

The use of cloned embryos to derive pluripotent embryonic stem cells (ESC) has raised expectations among many for its potential to benefit regenerative medicine. However, diploid cloned embryos also have the ability for totipotency and they occasionally develop to term. Thus, a therapeutic application cannot be kept totally separate from reproductive cloning. In contrast to diploidy, tetraploidy is incompatible with full development in mammals; yet, tetraploid cells arise after normal diploid development and contribute to certain organs of the adult body. In this context, we asked if single oocytes are able to reprogram 2 simultaneously transplanted somatic nuclei, and whether resultant embryos may serve as a source for functional pluripotent cell lines. We modified the conventional nuclear cloning method by simultaneously injecting 2 nuclei of cumulus cells into a single ooplasm of the B6C3F1 mouse strain. Following activation of these reconstructed oocytes, the blastocyst rates were lower compared with conventional cloned embryos (1 nucleus injection), but similar to tetraploid fertilized embryos generated by intracytoplasmic sperm injection (ICSI) of 2 sperm heads and prevention of second polar body extrusion using cytochalasin B. Upon transfer of tetraploid cloned blastocysts to pseudopregnant recipients, no fetuses were found at midgestation (Table 1). Tetraploid cloned blastocysts supported the derivation of ESC (Table 1), which maintain a stable tetraploid karyotype over more than 20 passages. These tetraploid ESC possess the major hallmarks of pluripotency, including matching gene expression profiles (MouseRef-8 v2 expression BeadChip, Illumina) compared with those of diploid counterparts (>99% similarity) as well as the ability to differentiate into derivatives of the 3 germ layers when forming teratomas in severe-combined immunodeficient mice. Upon injection into wild-type blastocysts, tetraploid ESC populated the inner cell mass; however, after transfer of these chimeras to pseudopregnant recipients, derivatives of tetraploid ESC were found only in extraembryonic tissues of midgestation fetuses. In sum, we have shown that the reprogramming capacity of mouse oocytes is sufficient for 2 somatic nuclei, and that resultant cloned embryos support pluripotency but not totipotency. Our approach to therapeutic cloning is free of reproductive implications.

Table 1.Developmental competence of single (2N) and double nucleus transplanted (4N) cloned mouse embryos compared with fertilized controls
267 EFFECT OF FOLLICULAR FLUID CONCENTRATION ON IN VITRO DEVELOPMENT OF PORCINE OOCYTES AND THE EXPRESSION OF GENES RELATED TO CUMULUS EXPANSION AND EMBRYO QUALITY
Authors: Park J. E. , Oh H. J. , Kim M. J. , Kim G. A. , Park E. J. , Choi J. , Moon J. H. , Lee B. C.
Published: December 2012

It is well known that the presence of porcine follicular fluid (PFF) in in vitro maturation media enhances the developmental competence of porcine oocytes. However, it is also suggested that the action of PFF can be modulated positively or negatively by its components. In this study, we investigated the effects of PFF concentration (10 v. 1%) and protein-free media (PFF 0%) on the maturation of porcine oocytes in vitro, and analysed the difference in gene expression in the resulting cumulus cells and blastocysts after parthenogenetic activation. Three groups were tested: 1) 10% PFF: TCM-199 + 10% PFF (n = 638); 2) 1% PFF: TCM-199 + 3.05 mM d-glucose + 1% PFF (n = 418); and 3) 0.1% polyvinyl alcohol: TCM-199 + 3.05 mM d-glucose + 0.1% polyvinyl alcohol (n = 693). Cumulus–oocyte complexes were cultured for 20 to 22 h in the respective media that contained gonadotrophin (1 µg mL–1), epidermal growth factor (10 ng mL–1), cysteine (0.57 mM), sodium pyruvate (0.91 mM), insulin (5 µg mL–1), and 9-cis retinoic acid (5 nM). They were then cultured for an additional 20 to 22 h without hormonal supplements. Data was analysed by one-way ANOVA using the SAS program (SAS Institute Inc., Cary, NC, USA). No significant difference in oocyte maturation rate was observed. However, significantly higher (P < 0.05) proportions of embryos developed in the blastocyst stage when the oocytes were matured in 10% PFF group (45%) than in the 1% PFF group (31.1%). The total cell numbers were not significantly different among groups (52 ± 1.3 v. 54.6 ± 3.1 v. 54.4 ± 2.5, respectively). In addition, the expression of matrix molecule (HAS2, GREM1), steroidogenesis (HSD3B), epidermal growth factor signalling (AREG, BTC), and cell cycle regulator (CCND2) genes were upregulated in the cumulus that was obtained from oocytes that matured in 10% PFF. The expression of the anti-apoptotic gene (BclxL) was upregulated, and the expression of the pro-apoptotic gene (Bax) and metabolism-related genes (GLUT1, LDHA) were downregulated in blastocysts that developed from the 10% PFF group. Therefore, it can be concluded that supplementation of 10% PFF during in vitro maturation improves embryo development by increasing matrix molecules and maturation-enabling factors in the cumulus and by reducing apoptosis.
This study was supported by IPET (No. 311011-05-1-SB010), MKE (No. 10033839-2012-21), the Research Institute for Veterinary Science, the BK21 Program, and the TS Corporation.