bluuebunny/crossref_metadata_2025 huggingface dataset:
100 EMBRYO SURVIVAL AND CONCEPTUS ELONGATION FOLLOWING ASYNCHRONOUS EMBRYO TRANSFER IN CATTLE
Authors: Randi F. , Fernandez B. , McDonald M. , Johnson C. , Forde N. , Lonergan P.
Published: December 2014

Maternal progesterone (P4) regulates early conceptus growth and development in ruminants. Early embryo transfer studies in sheep and cattle demonstrated a need for close synchrony between the embryo and the uterine environment of the recipient. However, manipulating P4 may be one way of strategically regulating the temporal changes that normally occur in the uterine environment in order to allow flexibility in the timing of embryo transfer. For example, previous studies have demonstrated that P4 administration during the first few days of the oestrous cycle facilitates pregnancy establishment with older embryos. The aim of this study was to examine the effect of embryo-uterine synchrony on conceptus elongation in cattle. Oestrous cycles of crossbred beef heifers were synchronised using an 8-day P4-Releasing Intravaginal Device (PRID Delta®, CEVA, Mountain View, CA, USA) with administration of a prostaglandin F2α analogue (Enzaprost®, CEVA; 5 mL equivalent to 25 mg of dinoprost) given on the day before PRID removal. Heifers were checked for signs of oestrus 4 times per day commencing 30 h after PRID withdrawal. Only those seen in standing oestrus (n = 50) were randomly assigned to 1 of 5 treatment groups to receive Day 7 in vitro-produced blastocysts (n = 10 per recipient) (1) on Day 5 post-oestrus; (2) on Day 5, with P4 supplementation via PRID from Day 3 to 5 + 750 IU of eCG at PRID insertion; (3) on Day 5, PRID Delta from Day 3 to 5 plus 3000 IU of hCG at PRID insertion; (4) on Day 7, or (5) on Day 9. At embryo age Day 14, all heifers were slaughtered and the uterus was flushed to recover and measure conceptuses. Data are summarised in Table 1. Fewer recipients yielded conceptuses (P < 0.05) and fewer conceptuses overall were recovered (P < 0.05) following transfer on Day 5 compared with Day 7 or Day 9. Supplementation with P4 resulted in short cycles (evidenced by corpus luteum regression and/or a recent ovulation at slaughter) in 33.3 to 54.5% of recipients receiving embryos on Day 5. Mean conceptus length was greater (P < 0.05) following transfer to an advanced uterus. In conclusion, transfer of embryos to a retarded (Day 5) uterine environment results in poor embryo survival. Supplementation with P4 shortened the interoestrous period in a significant number of heifers. Transfer to an advanced uterine environment promotes conceptus elongation, presumably driven by P4.

Table 1.Embryo survival and conceptus length data
104 EXPRESSION OF ACTIVIN A AS A LOCAL REGULATOR IN THE BOVINE OVIDUCT
Authors: Yamamoto Y. , Kobayashi Y. , Yoshimoto Y. , Okuda K.
Published: December 2014

Activin (ACV) is known as a local regulator of several reproductive functions including follicular development and implantation in mammals. ACVA is a glycopeptide belonging to the transforming growth factor β superfamily, and is a homodimer of inhibin ßA (INHBA) subunits. Follistatin (FST), an ACV-specific binding protein, inhibits ligand-receptor binding. ACV receptor (ACVR) is a hetero-tetramer consisting of 2 kinds of protein, ACVR1 or ACVR1B and ACVR2A or ACVR2B. The oviduct provides an optimal environment for sperm capacitation, fertilization, and early embryonic development. Previous reports have demonstrated that ACVRs were expressed in bovine oocytes and embryos, and that early embryonic development is facilitated by ACVA in vitro. ACVA produced by the bovine oviduct may affect gametes and embryos as well as oviductal cells as a local regulator in cow. Bovine oviductal samples were classified into 6 stages of the oestrous cycle (day of ovulation; Days 2–3 after ovulation; Days 5–6; Days 8–12; Days 15–17; Days 19–21). We examined (1) protein expression of ACVA and FST in oviductal fluid collected from the ampulla and isthmus, (2) mRNA expression of INHBA and FST in the ampullary and isthmic oviductal tissues during the oestrous cycle, (3) the effects of oestradiol-17β (E2: 0.1, 1, 10 nM) and progesterone (P4: 1, 10, 100 nM) on mRNA expressions of INHBA and FST in cultured oviductal epithelial cells isolated from the ampulla and isthmus, and (4) mRNA expression of ACVRs in tissues and in cultured epithelial and stromal cells. The main findings were as follows: (1) Both ACVA and FST were detected throughout the oestrous cycle in the oviductal fluid of the ampulla and isthmus. (2) INHBA expression was higher in the isthmus than in the ampulla. FST expression in the ampulla was lowest at peri-ovulation, INHBA expression in the isthmus was highest on the day of ovulation and FST in the isthmus during Days 2–6 was highest. Because an increase of ACVA production and a decrease of FST production raise ACVA bioactivity, ACVA seems to be most active at peri-ovulation in both the ampulla and isthmus. (3) In the cultured isthmic oviductal epithelial cells, 10 nM E2 significantly stimulated INHBA expression, but tended to suppress FST expression. Therefore, the bioactivity of ACVA seems to be controlled by E2 during the oestrous cycle in the isthmus. (4) The expression of ACVR1B and ACVR2A was clearly detected in the tissues as well as in cultured epithelial and stromal cells. The overall findings suggest that ACVA secreted into oviductal fluid plays an important role in oviductal functions, including fertilization in the ampulla and sperm motility and viability in the isthmus. It is also suggested that ACVA acts on both epithelial and stromal cells as a local regulator of cellular functions, such as cellular proliferation and secretion in the cow.
112 EFFECT OF ASYNCHRONOUS EMBRYO TRANSFER ON GLUCOSE TRANSPORTER EXPRESSION IN EQUINE TROPHECTODERM
Authors: Stout T. A. , Gibson C. , de Ruijter Villani M.
Published: December 2014

Equine pregnancy is characterised by an unusually long pre-implantation period (40 days) during which the conceptus is entirely dependent on uterine secretions for nutrient provision; although glucose is an important nutrient during development post-blastocyst formation, little is known about its transport into the early horse conceptus. Equine embryos are also known to tolerate an unusually large degree of uterine asynchrony following embryo transfer (ET). However, negative asynchrony (recipient behind the donor) of more than 5 days markedly retards conceptus growth and development, and thereby offers a unique tool for studying the effect of the uterine environment on early development. In a preliminary study, we detected abundant mRNA expression for the facilitative glucose transporters (SLC2As) 1–3, 5, 8 and 10 and sodium-glucose co-transporter (SLC5A)11 in Day 14 to 28 equine conceptus membranes. In the current study, we evaluated the effect of uterine asynchrony on trophectodermal glucose transporter expression. Day 8 horse embryos were transferred to recipient mares that ovulated on the same day (synchronous; n = 10) or 5 days after (asynchronous; n = 10) the donor mare. The conceptuses were collected 6 or 11 days after ET (Day 14 or 19 of embryo development: n = 5 per group). Trophectodermal mRNA expression for glucose transporters was evaluated by RT-qPCR, and the effects of asynchronous ET and stage of pregnancy were analysed by two-way ANOVA followed by independent-samples t-tests. Gene expression for SLC2A3 and 8 was stable over time and treatment. Expression of SLC2A1 and SLC5A11 decreased between Days 14 and 19 in synchronous pregnancies only (P < 0.05). SLC2A2 expression increased markedly on Day 19 in synchronous (P < 0.01) but not asynchronous pregnancies (P < 0.05). SLC2A5 expression was lower in the asynchronous group on Day 14, but increased beyond expression levels in synchronous pregnancies by Day 19 (P < 0.05). In summary, expression of SLC2A1 and 3, the major placental glucose transporters, was not affected by asynchronous ET. The marked up-regulation of SLC2A2 expression between Days 14 and 19 of synchronous but not asynchronous pregnancy suggests a stage-specific function, whereas the increase in SLC2A5 at Day 19 after asynchronous ET could be a compensatory response to growth retardation.
This study was funded by EpiHealthNet (Project number 317146).
118 COMPARISON OF TRANSCRIPTOME PROFILES FROM ENDOMETRIAL CARUNCLES AND PERIPHERAL BLOOD MONONUCLEAR CELLS REVEAL COMMON AND TISSUE-SPECIFIC BIOLOGICAL PROCESSES REGULATED AT IMPLANTATION IN SHEEP
Authors: Mauffré V. , Sandra O. , Giraud-Delville C. , Urien C. , Jouneau L. , Loup B. , Valour D. , Cotinot C. , Schwartz-Cornil I. , Grimard B. , Constant F.
Published: December 2014

In mammals, implantation is associated with major changes in gene profiles in the female reproductive tract. Molecular signatures of the endometrium have also been shown to vary according to the ability of the embryo to develop to term. Nevertheless, analysing endometrial gene patterns during implantation is incompatible with the maintenance of pregnancy. Therefore early determination of pregnancy issue requires a noninvasive method. Peripheral blood mononuclear cells (PBMC) could represent such an alternative but their reaction to the presence of an implanting embryo has to be investigated. The aim of this study was to investigate gene expression profiles of endometrial caruncular tissue (CAR) and PBMC collected from pregnant ewes (n = 4) and nonpregnant ewes inseminated with inactivated sperm (n = 4) at Day 15 after oestrus. Differentially expressed genes (DEG) were identified using an ovine custom-designed array derived from the ovine 15K Agilent array (Ruscanu et al. 2013 J. Virol. 87, 9333–9343). Data were normalized by Loess and analysed by a linear model in the Limma R package. P-values were corrected using the Benjamini and Hochberg procedure. Comparing pregnancy versus nonpregnancy led to the identification of 2826 DEG in CAR (P < 0.05) and 396 DEG in PBMC (P < 0.10; 265 DEG common with CAR). Ingenuity Pathway Analysis (IPA; Ingenuity Inc., Mountain View, CA, USA) analysis of the 396 PBMC-related DEG revealed 72 overrepresented biological functions (P < 0.001). Among the 15 most overrepresented functions, 13 were common between CAR and PBMC and were mostly related to the immune system, as “infectious disease”, “cell-to-cell signalling and interaction”, “immunological disease”, “immune cell trafficking and inflammatory response”. Using the downstream effect analysis (DEA) of IPA, we identified 163 functions predicted to be increased and 8 functions predicted to be decreased for the CAR DEG dataset, whereas 12 functions were predicted to be increased and 40 functions predicted to be decreased for the PBMC DEG dataset. Interferon (IFN) signalling was strongly present in both datasets, with 44% of PBMC DEG and 29% of CAR DEG found to be related to IFN type I response according to the Interferome database (www.interferome.org). A selection of 12 DEG was validated by qRT-PCR in CAR, intercaruncular areas, and PBMC using 8 pregnant and various groups of nonpregnant ewes (n = 7–9/group). Our data show that PBMC transcriptome is influenced by early pregnancy in sheep, including a major impact of IFN type I such as IFN tau, the signal of maternal recognition of pregnancy. Identifying relevant circulating biomarkers reflecting the quality of the embryo will require further investigation.
The authors thank UCEA team (INRA), B. Jost (IGBMC) and F. Moreews (Sigenae).
135 EFFECT OF L-ERGOTHIONEINE SUPPLEMENTATION DURING CULTURE ON IN VITRO EMBRYO DEVELOPMENT IN BUFFALO (BUBALUS BUBALIS)
Authors: Zullo G. , Salzano A. , Bifulco G. , Longobardi V. , Albero G. , Neglia G. , Gasparrini B.
Published: December 2014

It is known that in vitro mammalian embryo development is negatively affected by the increased oxidative stress occurring under culture conditions. The oxidative damage of cell components via reactive oxygen species interferes with proper cell function. Buffalo embryos are particularly sensitive to oxidative stress because of their high lipid content (Boni et al. 1992 Acta Med. Vet. 38, 153–161). l-Ergothioneine (LE) is a powerful scavenger of hydroxyl radicals (OH) and an inhibitor of iron or copper ion-dependent generation of OH from hydrogen peroxide (H2O2). The aim of this study was to evaluate whether enriching the in vitro-culture medium with LE improves in vitro embryo production efficiency in buffalo. Abattoir-derived buffalo oocytes (n = 854, over 6 replicates) were in vitro matured and fertilized according to standard procedures (Gasparrini et al. 2006 Theriogenology 65, 275–287). Twenty hours after IVF presumptive zygotes were cultured in SOFaa supplemented by 8 mg mL–1 BSA in a controlled gas atmosphere consisting of 5% CO2, 7% O2, 88% N2, in humidified air, at 38.5°C with 0 (control; n = 214), 0.05 mM LE (n = 217), 0.1 mM LE (n = 204), and 1 mM LE (n = 219). Cleavage rate was assessed at the time of change of culture (Day 5) and the cleaved elements were cultured for a further 2 days. The embryos obtained by the end of culture, i.e. on Day 7 post-IVF, were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson, Nelson 2010 Manual of the International Embryo Transfer Society 86–105). The percentages of total transferable embryos and Grade 1 and 2 blastocysts in relation to cleaved oocytes were recorded. Because the chronology of development is known to be one of the most reliable parameters for assessing quality, the percentage of fast-developing embryos, i.e. hatched and expanded blastocysts, was also recorded. Data were analysed by Chi-squared test. Cleavage rate was not affected by the treatment (71.4, 66.8, 68.7, and 63.0%, respectively, with 0, 0.05, 0.1, and 1 mM LE). The total embryo output increased in groups supplemented with 0.05 and 0.1 mM LE (31.3, 42.2, 43.8, and 21.7%, respectively, with 0, 0.05, 0.1, and 1 mM LE; P < 0.05). However, the enrichment of in vitro culture with 0.1 mM LE also increased the percentage of Grade 1 and 2 blastocysts compared with the control and to 1 mM LE (21.6, 30.9, 33.9, and 21.7%, respectively, with 0, 0.05, 0.1, and 1 mM LE; P < 0.05). Likewise, 0.1 mM LE gave higher percentages of fast developing embryos than the control and 1 mM LE groups. In conclusion, these results demonstrated a beneficial effect of LE during culture on buffalo in vitro embryo development. The dose response trial indicated that the optimal concentration is 0.1 mM that also influenced the chronology of development and hence embryo viability.
142 MORPHOLOGICAL EVALUATION OF CANINE EMBRYOS OBTAINED IN VIVO
Authors: Guaitolini C. R. de F. , Derussi A. A. P. , Volpato R. , Ackermann C. L. , Matsubara L. M. , Maziero R. R. D. , Sudano M. J. , Agostinho L. M. , Landim-Alvarenga F. da C. , Lopes M. D.
Published: December 2014

The aim of this study was to evaluate re-expansion and ultrastructure of in vivo-produced canine embryos immediately after collection (Co; n = 6) and 24 h after in vitro culture (Co24; n = 6). For embryo collection, two bitches in pro-oestrous were monitored every 48 h by vaginal cytology up to the detection of 80 to 90% of superficial cells. Then, they were inseminated 3 times on alternate days with fresh semen from 1 fertile male. Embryo collections were performed after ovariohysterectomies and uterine flushing 12 days after the first artificial insemination. Each uterine horn was flushed 3 times with 60 mL of DPBS at 36 to 37°C and collecting embyos in Petri dishes. Embryos culture were performed at 38.5°C under an atmosphere with 5% CO2 and maximum humidity, using SOFaa media added with 20% of fetal bovine serum (FBS). Embryo reexpansion was evaluated after 24 h of culture and the embryos were classified as re-expanded or not re-expanded, according the appearance of the blastocoele by stereomicroscopy (Leica MZ 12.5, Leica Microsystems, Wetzlar, Germany). Twelve embryos were collected, 5 from bitch A and 7 from bitch B; 100% of embryos (6/6) showed re-expansion after 24 h of culture. Blastocoele reexpansion was used as an embryo viability marker. The group Co showed perivitelline space and apical surface of blastomeres covered with microvilli, elongated mitochondria, rough endoplasmic reticulum (RER), tight junctions, and a large amount of lipid droplets that was similar to the results previously described in others mammals species. The Co24 group showed the same characteristcs of the group Co at the time of collection, however with a reduction of lipid droplets and the presence of myelinic structures. In conclusion, the lipid droplet reduction and presence of myelinic structures after 24 h of in vitro culture may indicate lipid consumption associated with embryo expansion.
149 IN VITRO BOVINE EMBRYOS FROZEN BY DIRECT TRANSFER METHOD WITH ETHYLENE GLYCOL
Authors: Kizil S. H. , Satilmis M. , Akyol N. , Karasahin T.
Published: December 2014

The objective of this study was to search for capability of freezing by ethylene glycol direct transfer method of in vitro-produced cattle embryos. Fifty-six in vitro-produced good-quality cow embryos were frozen by direct transfer method with ethylene glycol in this study. Cattle ovaries were collected from a slaughterhouse and oocytes were aspirated from follicles with 2 to 8 mm diameters. Then oocytes were let for maturation of 20 to 22 h in 100-μL microdroplets of TCM-199 with 0.1 mM β-mercaptoethanol and 20% FCS. After 5 to 6 h of fertilization in Bracket Oliphant (BO), they were cultured for 7 days in 100 µL of CR1aa medium with 5% FCS under 5% CO2, 98% relative humidity, and 38.5°C in a CO2 incubator. Embryos were equilibrated for 15 min in room temperature in 1.8 M ethylene glycol + 0.1 M sucrose in Dulbecco's phosphate buffered saline (D-PBS) supplemented with 20% FCS. Embryos were then loaded individually into a 0.25-mL straw and placed directly into a cooling chamber of a programmable freezer with methyl alcohol precooled to –7°C. After 2 min, the straw was seeded and maintained at –7°C for 8 min more. Then it was cooled to –30°C at 0.3°C min–1 before plunging into liquid nitrogen. The frozen embryos were thawed by allowing the straw to stand in air for 5 to 6 s and then immersing them in a 30°C water bath for 10 s. After thawing, embryos were transferred into TCM-199 + 0.1 mM β-mercaptoethanol + 20% FCS medium to check in vitro survival rates at 48 h post-thawing. The re-expansion and hatching rate of blastocysts was 64.28% (36 blastocysts). This result indicated that ethylene glycol can be used effectively for cow embryo freezing as a suitable cryoprotectant for direct transfer method.
180 FATTY ACID COMPOSITION IN FOLLICULAR FLUID OF PREPUBERTAL GOAT OVARIES IN WINTER AND AUTUMN
Authors: Paramio M. T. , Roura M. , Hammami S. , Izquierdo D. , Catalá M. G.
Published: December 2014

Fatty acids (FA) in follicular fluid (FF) play an important role on oocyte quality and embryo development (Fouladi-Nashta et al. 2007 Biol. Reprod. 77, 9–17). In our laboratory, we have shown in prepubertal goat differences in the percentage of blastocysts produced in vitro according to season. Thus, we have found in winter 15.8% and in autumn a decrease up to 4.7% of blastocysts that were produced from oocytes of 1 month old suckling Murciano-Granadina goat females and IVF with fresh semen. The aim of this study was to analyse composition of FF in order to find an explanation to seasonal changes in in vitro embryo production. Ovaries were recovered in winter and autumn from 1 month suckling goats (Murciano-Granadina) from a local slaughterhouse and the FF of all visible follicles was recovered using a sterile syringe. Each sample containing a pool of FF of different ovaries was frozen at –80°C until chromatography analysis. For the FA analysis, the Sukhija and Palmquist (1988 J. Agric. Food Chem. 36, 1202–1206) protocol with some adaptations was used. Briefly, 200 μL of FF sample was vortexed for 60 s with 250 μL of toluene and 1 mL of HCL (5%) and then warmed in a water bath for 1 h at 70°C. Subsequently 1.25 mL of K2CO3 (12%) and 500 μL of toluene was added, vortexed for 30 s and centrifuged for 5 min (3000 rpm). Finally the supernatant was recovered and dried with Na2SO4. The extracted samples were maintained in –20°C until gas chromatographic analysis (123–2362, Agilent Technologies Inc., Santa Clara, CA). The results in Table 1 express the mean of 3 replicates of follicular fluid pool as micromolar concentration of FA in FF. The FA profile in FF showed significant higher concentrations of α-linolenic (C18:3n3), eicosapentaenoic (EPA), docosahexaenoic (DHA), and omega-3 (n-3 polyunsaturated fatty acid; PUFA) in winter compared to autumn. This could be indicating that these PUFA have a positive effect on oocyte quality because of the higher embryo development of these oocytes during winter. Studies in our laboratory have shown that sperm penetration and normal zygotes were similar in both seasons even though the blastocyst yield was statistically higher in winter. We can speculate that fatty acids in the follicular environment are affecting the oocyte quality, increasing the possibility of reaching the blastocyst stage in prepubertal goat according to season. Further studies should be done to reach a more accurate conclusion.

Table 1.Concentration (µM) of fatty acids in FF of prepubertal goat during winter and autumn (3 replicates)
184 FOLLICULOGENESIS AND DONOR AGE INFLUENCE MATRIX METALLOPROTEINASE-2 EXPRESSION IN THE DOMESTIC CAT
Authors: Songsasen N. , Fujihara M. , Yamamizu K. , Wildt D. E.
Published: December 2014

Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are known to play key roles in the remodelling of extracellular matrix during ovarian folliculogenesis, especially during the final stage of follicle development. To date, little is known about the significance of MMPs and TIMPs during preantral follicle development. This study determined the expression of MMPs and TIMP-1 during various stages of cat folliculogenesis, largely for the purpose of securing information useful to improving in vitro follicle culture. Primordial (~10 follicles/cat), primary (~5 follicles/cat), secondary (~9 follicles/cat), early antral (~9 follicles/cat), and antral (~4 follicles/cat) follicles were physically isolated from ovaries recovered from 15 cats (5 months to 3 years old during follicular stage) undergoing ovariohysterectomy and assessed for expression of MMP-1, -2, -3, -7, -9, and -13 as well as TIMP-1 using real-time quantitative polymerase chain reaction (q-PCR; 2–4 replicates/follicle stage). Additional ovaries were obtained from three prepubertal (6 months old) and three adult (1 year old) cats and ovarian pieces were fixed in Bouin's solution and assessed for MMP-2 and -13 localization using immunohistochemistry. MMP expression data were analysed using the Kruskull-Wallis one-way ANOVA. Follicles from all stages of development expressed MMPs and TIMP-1. Specifically, expression of MMP-2 increased (P < 0.05) as folliculogenesis progressed (10-fold increases from primordial to early-antral and antral stage). There were no differences (P > 0.05) in the expression of other MMPs among follicular classes. For TIMP-1, there was a tendency (P = 0.07) for increased expression after antrum formation (early antral and antral stages). Immunohistochemistry analysis revealed that MMP-2 was expressed in both the oocyte and somatic cells of all follicular stages in prepubertal cats. However, MMP-2 expression was limited to granulosa and theca cells of antral follicles in adult females. MMP-13 was expressed in the granulosa and theca cells of primary, secondary, and antral stage follicles, and there were no differences (P > 0.05) in localization patterns for this protein between prepubertal and adult females. In summary, the study is the first to report the expression of MMPs as well as TIMP-1 in isolated cat follicles. The difference in MMP-2 expression between prepubertal and adult cats suggests that there may be age-specific requirements for in vitro follicle growth. We are keenly interested in this information for underpinning the development of new in vitro microenvironments for growing immature cat follicles. We suspect that such information will be crucial for understanding how to promote the remodelling of the extracellular matrix by creating degradable biomaterials containing MMP-sensitive peptides to allow optimal follicle expansion.
197 INFLUENCE OF PLASMID CONSTRUCTION AND CELL TYPE ON THE GENERATION OF GENETICALLY MODIFIED CELL LINES IN CATTLE
Authors: Curcio A. G. , Bressan F. F. , Viana K. S. , Rios A. F. L. , Meirelles F. V. , Dias A. J. B.
Published: December 2014

Several factors may influence transgenic animal production efficiency, and among them gene construction and the cell type used are of great importance. For a long time, fetal fibroblasts were largely used in generation of transgenic cattle production by nuclear transfer, however adult cells are very useful for cloning once the genotype of the donor nuclei is known, and derivation of such cells is technically simple, efficient, and reproducible. Thus, this study aimed to evaluate the effect of cell type on the percentage of GFP+ cells and fluorescence intensity, using two plasmids constructs encoding for green fluorescent protein (GFP). Transfections were performed in bovine fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) transfected by use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for the internalization of FUGW or pEGFPN2 plasmid. Forty-eight hours after transfection, the number and the fluorescence intensity (arbitrary units) of GFP+ cells was measured by flow cytometry (FACSAria, FACSDiva Software, BD Biosciences, Franklin Lakes, NJ). Non-transfected cells were used as controls. Means were compared by the Student–Newman–Keuls test (SNK; P < 0.05). The FUGW plasmid promoted a higher rate of transfection and fluorescence intensity than pEGFPN2 in all cell types evaluated. When the FUGW plasmid was used, higher transfection rates were obtained with fetal fibroblasts (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65, CC: 3.9 ± 1.97), while higher fluorescence intensity was observed in adult fibroblasts (FF: 4542 ± 497.09; AF: 9367.5 ± 3490.9, CC: 3496 ± 2638.92). The pEGFPN2 plasmid showed percentage of transfected cells and fluorescence intensity significantly higher than the control only in cumulus cells (pEGFPN2 – FF: 4.9 ± 0.14 and 206.47 ± 755; AF: 760 and 2.4 ± 0.70 ± 330.92; CC: 3.9 ± 1.97, and 1418 ± 36.06, respectively; control – FF: 0.15 ± 0.07 and 249 ± 6 : 36; AF: 0.15 ± 0.07 588 ± 213.54, and CC 0.05 ± 0 214 ± 0.07, respectively). We conclude that the plasmid construction may influence the overall efficiency in transfected cells; however, the transfection percentage and fluorescence intensity is greatly influenced by the cell type. We suggest that transgenesis of a specific cell type may be enhanced by the proper choice of the expression vector.
204 A COMPARATIVE ANALYSIS OF CALCIUM TRANSPORT GENES EXPRESSION IN HOLSTEIN AND HANWOO (KOREAN CATTLE) IN THE DUODENUM AND KIDNEY
Authors: Cho H. , Jeung E.-B.
Published: December 2014

Transmembranous calcium (Ca2+) channels such as Trpv5/6,Ncx1,Pmca1b are known to play an important role in maintaining homeostasis and metabolizing Ca2+ ions. Transient receptor potential cation channel subfamily V members 5 and 6 (Trpv5/6) play an important role in Ca2+ absorption. Na+/Ca2+ exchanger 1 (Ncx1) and plasma membrane Ca2+-transporting ATPase1 (Pmca1b) play a role in Ca2+ excretion. Holstein cattle are known to provide higher milk production than other cattle breeds. In this respect, a higher susceptibility to hypocalcaemia that is a risk factor for many of calcium-related diseases such as milk fever has been observed. In contrary, Hanwoo cattle are relatively resistant to the calcium-related diseases. The aim of this study was to evaluate the expression of calcium transport genes in the duodenum and kidney of Hanwoo (Korean cattle) and Holstein cattle. All samples were obtained from Holstein and Hanwoo cattle from 36 to 60 months of age at a local slaughterhouse. The collected duodenum and kidney were rapidly excised and washed in cold sterile saline (0.9% NaCl). Total RNA was prepared from duodenum and kidney tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. Protein was extracted using Pro-prep (iNtRON Bio), according to the manufacturer's protocol. Expression of Trpv5/6, Ncx1 and Pmca1b was analysed by real-time RT–PCR, Western blot analysis, and immunohistochemistry. In dairy cattle's kidney (Holstein), the level of Trpv5 mRNA was ~3-fold more than Hanwoo's kidney while Pmca1b mRNA was one-fifth less than Hanwoo's kidney. However, no difference in Trpv6 and Ncx1 mRNA expression was observed in the kidney of Holstein compared with Hanwoo. Duodenal Trpv5 and Trpv6 mRNA were scarcely expressed in both Hanwoo and Holstein cattle. In addition, the mRNA expression of Pmca1b and Ncx1 were also not different between Hanwoo and Holstein. The protein expression of calcium transport genes were similar to the mRNA levels of calcium transport genes in both Hanwoo and Holstein. Localization of calcium transporter genes were identified, the glomerulus, and proximal and distal convoluted tubules expressed TRPV5, 6, Pmca1b, and Ncx1 in the kidney. These four calcium transport genes may play an important role in the bovine duodenum and kidney. The difference between Holstein and Hanwoo animals which express different gene expression patterns may be helpful in studying diseases associated with calcium metabolism, e.g. milk fever.
213 DIOXIN SIGNIFICANTLY ENHANCED THE TUMOR MASS FORMATION IN A XENOGRAFTED MOUSE MODEL TRANSPLANTED WITH BG-1 OVARIAN CANCER CELLS
Authors: Go R.-E. , Choi K.-C.
Published: December 2014

Previous studies suggest that environmental factors, such as high levels of meat consumption, caffeine, cigarette smoking, and endocrine disrupting chemicals (EDC), may enhance a high risk of ovarian cancer. Cytochrome P450 (CYP) 1A1 may play a major role in metabolic activation of procarcinogens to carcinogens. For example, polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) occur by pyrolysis of fossil fuels and flow into the body by organic matter, such as tobacco leaves and contaminated water by pesticides. 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) is a commonplace pollutant and promoter of carcinogenesis as the most potent substance. In this study, we examined the effects of 17β-oestradiol (E2), TCDD, and TCDD in the presence of E2 on the expressions of CYP1A1, CYP1B1, and aryl hydrocarbon receptor (AhR) by RT–PCR analysis and Western blot analysis. In addition, the cell viability by TCDD and E2 were examined in BG-1 human ovarian cancer cells by MTT assay. To evaluate the cell viability, BG-1 cells were cultured with a negative control (0.1% DMSO), E2 (1 × 10–9 M) or TCDD (10–6–10–8 M). E2 markedly increased BG-1 cell viability ~5 times, and TCDD also induced BG-1 cell viability highest at the concentration of 1 × 10–8 M compared to a control (0.1% DMSO) (P < 0.05). When respective treatment was co-treated with ICI 182 780, an ER antagonist, BG-1 cell viability was reversed to the level of a negative control. Although the mRNA expression of CYP1B1 was not altered by E2, TCDD, or TCDD plus E2, the transcriptional level of CYP1A1 appeared to be increased by E2 and TCDD in a time-dependent manner. When the cells were treated with TCDD plus E2, the mRNA level of CYP1A1 was more greatly increased than by only TCDD or E2 treatment. In xenograft mouse models transplanted with BG-1 cells, E2 treatment significantly increased the tumour mass formation ~6 times, and TCDD also induced tumour formation ~4 times compared to a vehicle (0.1% DMSO in PBS) during 8 weeks in this xenograted mouse model. In addition, TCDD plus E2 treatment also greatly induced ovarian tumour formation compared to only E2 treatment in this mouse model. Taken together, these results indicate that TCDD may induce ovarian cancer cell growth via CYP1A1 gene expression and have disruptive effect in oestrogen receptor (ER) expressing cells or tissues.
214 TRICLOSAN-INDUCED CELL CYCLE RELATED AND APOPTOTIC RELATED GENES WERE REVERSED BY KAEMPFEROL IN IN VITRO BREAST CANCER AND XENOGRATED MOUSE MODELS
Authors: Kim S.-H. , Choi K.-C.
Published: December 2014

Triclosan (Tri) is one of many endocrine-disrupting chemicals (EDCs) that are scattered with environment agents, such as toothpastes, deodorants, and cleaning supplies. As a phytoestrogen, kaempferol (Kae) is one of bioflavonoids, which has been found in a variety of vegetables including broccoli, tea, and tomatoes. Although Kae may have anti-cancer activity, its exact mechanism is under investigation, and might be the induction of apoptosis and inhibition of cell proliferation or angiogenesis. In this study, we examined the anti-proliferative effects of Kae in Tri-induced cell growth in MCF-7 breast cancer cells. A proper concentration and co-treatment effect of Tri and Kae were determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay to measure cell viability in vitro. MCF-7 cells were cultured with a negative control (0.1% DMSO), E2 (1 × 10–9 M), Tri (10–5–10–8 M) and Kae (50, 70, and 90 mM). In this study, treatment with Tri (10–6 M) increased the cell viability of MCF-7 cells, while Kae (50 mM) significantly reduced the cell viability compared to the negative control (P < 0.05). In addition, Kae significantly reversed Tri-induced MCF-7 cell growth at 50 mM compared with a higher concentration (100 mM; P < 0.05). To confirm that Kae inhibited Tri-induced cell growth, we examined the transcriptional levels of cell growth and apoptosis-related markers, i.e. cyclin D, p21, cyclin E, p27 and bcl-2, and bax genes, using reverse transcription (RT)-PCR. The expression levels of cyclin D, cyclin E, and bax/bcl-2 ratio were increased, while those of p21 and p27 mRNAs were decreased by Tri in MCF-7 cells. In addition, Kae treatment significantly reversed Tri-induced gene expressions in an opposite manner. In parallel with its mRNA level, the protein level of cyclin E, p-ERK and p-MEK1/2 were induced by Tri while it was reversed by Kae as shown by Western blot analysis. The expression levels of p21 and bax genes were altered by Tri and reversed by Kae treatment in this study. As an in vivo model, a xenografted mouse model was generated following injection with MCF-7 breast cancer cells in 6 weeks. In parallel with in vitro results, tumour volumes following treatment with E2 and Tri were continually increased compared to a vehicle (corn oil). It was of interest that treatment of the mice with combination of E2 plus Kae or Tri plus Kae showed less tumour formation rather than that of singly treated mice with E2 or Tri. Taken together, these results indicate that Kae may inhibit the growth of MCF-7 cells via regulating of cell cycle and apoptosis-related genes. In addition, EDC-induced progression of breast cancer may be suppressed by a phytoestrogen, i.e. Kae, in a specific manner.
216 HORMONAL REGULATION OF CLAUDIN-4 TIGHT JUNCTION MOLECULE IN MOUSE PLACENTA
Authors: Ahn C. , Lee D. , Jeung E.-B.
Published: December 2014

Tight junctions (TJ) are composed of a branching network of sealing strands. TJ regulate paracellular conductance and ionic selectivity. TJ components include the peripheral protein ZO-1, junctional adhesion molecules (JAM) and the integral proteins such as occludin and claudin. Claudins are a family of proteins that are the most important components in the tight junctions. The established paracellular transport barriers that control transportation of molecules within intercellular space. The present study focused on the expression of claudin, suggesting as major working molecules in the paracellular transport system. To study the regulation and roles of claudin family, we examined expression of mouse placental claudin family. Fifteen pregnant C57/BL6 mice were used in this study and TJ proteins including Claudin-1 to Claudin-24 expressions by real-time RT–PCR and Western blotting. The mice were divided into 3 groups depending on the gestational day (on Days 12, 16, and 20 of gestation).The localization of TJ proteins were examined by immunohistochemistry. After we identified the fluctuation of claudin expression during pregnancy, we assumed that the hormones are one regulator for claudin family. Therefore, we performed an in vivo study with hormone receptor antagonists (ICI 182, 780, and RU-486) for examining hormonal effect on claudin expression in the placenta. Forty-nine mice were divided into 7 groups. The changes of claudin expression were examined with real-time RT-PCR and Western blotting. In the transcription levels, Claudin-1, claudin-2, claudin-4, and Claudin-5 expression levels were relatively high compared to others in the claudin family in all periods of the pregnancy. The claudin-4 expression, which reduces permeability of ions, increased over a period of time. However, caludin-5 expression that is the responsive protein for a decrease in paracellular conductance, were decreased. Claudin-1 and -4 have been known as responsive genes for a decrease in paracellular conductance. On the other hand, claudin 2 and 5 have been known as increasing paracellular conductance. In addition, immunohistochemistry was performed to identify their localization for inferring permeability in placenta. In summary, we analysed the claudin expressions and presented possible important claudins among its family. Furthermore, their localization was also examined in the mouse placenta. In addition, the regulation of critically expressed claudins by pregnancy-associated hormones, E2 and P4, was examined. These results may provide functional and structural roles of claudins and their involvement in the maternal-fetal interaction and in the transportation of placental materials.
222 ANTIFUNGAL AGENTS AS AGRICULTURAL PRODUCTS, FENHEXAMID, FLUDIOXONIL, AND CYPRODINIL, INDUCED THE EXPRESSION OF CYTOCHROME P450 FAMILY AND CELL CYCLE-RELATED GENES IN ESTROGEN RECEPTOR EXPRESSING BG-1 OVARIAN CANCER CELLS
Authors: Go R.-E. , Choi K.-C.
Published: December 2014

Fenhexamid, fludioxonil, and cyprodinil are antifungal agents used in agricultural applications, which are present at measurable amounts in fruits and vegetables. The induction of CYP gene expression including CYP1A1 and CYP1B1 is mediated by the transformation of polycyclic aromatic hydrocarbons (PAHs), and modulated by aryl hydrocarbon receptor (AhR). CYP1A1 is expressed in the liver, pancreas, thymus, uterus, and small intestine. CYP1B1 is abundant in the prostate, breast, and uterus. Expression levels of CYP1A1 and CYP1B1 indicate PAH-induced immunotoxicity, oxidative stress, and activation of environmental carcinogens. In this study, the ability of cell viability was examined as MTT assay by pesticides; fenhexamid, fludioxonil and cyprodinil. In addition, expression levels of mRNA and protein of AhR, CYP1A1, and Cyclin D1 were analysed by RT-PCR and Western blot analysis in BG-1 ovarian cancer cells with oestrogen receptors (ER). To evaluate the ability of cell viability, BG-1 cells were cultured with a negative control (0.1% DMSO), 17β-oestradiol (E2; 1 × 10–9 M), fenhexamid, fludioxonil or cyprodinil (10–5–10–8 M). To evaluate the expression levels of mRNA and protein, BG-1 cells were cultured with a negative control (0.1% DMSO), 17β-oestradiol (E2; 10–9 M) and these pesticides (10–5 M). As results, E2 as a positive control markedly increased BG-1 cell viability ~5 times compared to a negative control (P < 0.05). In addition, treatments with these pesticides increased BG-1 cell viability at the concentrations of 10–8 and 10–5 M about from 1.5 to 2 times, respectively (P < 0.05). When respective treatment co-treated with ICI 182 780, an ER antagonist, BG-1 cell viability was reversed to the level of a negative control. The mRNA expression of CYP1A1 was increased by E2, fenhexamid, and cyprodinil in a time-dependent manner but not by fludioxonil, while its level was reversed in the presence of ICI 182 780. In parallel with their transcriptional levels, protein levels of CYP1A1 and cyclin D1 were induced by E2 and these pesticides, while the level of AhR was not altered by E2 and these pesticides. Taken together, these results imply that the pesticides, fenhexamid, fludioxonil, and cyprodinil, may have disruptive effects on ER expressing cells or tissues by alteration of CYP1A1 and cyclin D1 via an ER-dependent pathway.
225 VALIDATION OF REFERENCE microRNAs FOR NORMALIZING EXPRESSION DATA GENERATED BY QUANTITATIVE PCR
Authors: Mahdipour M. , Van Tol H. T. A. , Stout T. A. E. , Roelen B. A. J.
Published: December 2014

Small noncoding RNAs such as microRNAs (miRNA) act as posttranscriptional regulators of numerous gene targets. The level of expression of these epigenetic factors can regulate the stability and translation of target mRNA molecules. Quantification of miRNA expression levels can therefore help us understand biological processes such as those regulating oocyte and pre-implantation embryo development. To date, most studies that have examined miRNA expression levels have used expression of either reference mRNAs or U6 spliceosomal RNA or miRNA Let-7a for normalization. We analysed the suitability of several miRNAs as potential expression normalizers in bovine oocytes and early embryos. Bovine oocytes at the germinal vesicle and metaphase II stages, and zygotes, 2-cell, 4-cell and 8-cell embryos, morulae, and blastocysts were collected during three in vitro fertilization replicates. qPCR was performed to quantify expression of miR-93, miR-103a, miR-26a, miR-191, miR-23b, Let-7a, and U6. The average starting material for each sample was determined with the help of specific standard curves made for each primer set. Subsequently, geNorm software was used to identify a set of stably transcribed miRNAs. Surprisingly, U6 spliceosomal RNA and Let-7a, which are widely used to normalise miRNA expression levels, were not stably expressed and were therefore considered unsuitable for normalization. Stepwise removal to determine the optimal number of reference miRNAs identified miR-93 and miR-103a as the most stably expressed. It is concluded that the combination of miR-93 and miR-103a can be used for normalizing miRNA expression for qPCR experiments on bovine oocytes and pre-implantation embryos.
235 DEVELOPMENT OF IN VIVO-MATURED OOCYTES COLLECTED FROM JAPANESE BLACK CATTLE STIMULATED WITH DIFFERENT DURATIONS OF FOLLICULAR GROWTH
Authors: Yamanouchi T. , Matsuda H. , Ohtake M. , Aikawa Y. , Kobayashi S. , Imai K. , Hashiyada Y.
Published: December 2014

We demonstrated that in vivo-matured oocytes (mOC) collected by ovum-pick up (OPU) from cows after stimulation of follicular growth (FG) are suitable for producing good quality blastocysts (BL). However, it is not known whether duration of FG affects developmental competence of mOC. The purpose of this study was to examine development of mOC after stimulation with different duration of FG. Japanese black donor cows (n = 4 per each group), were treated with a CIDR at Day 0. Follicle of diameter >8 mm were removed on Day 5. A total 20 AU of FSH was administrated to cows twice daily with decreasing doses from the evening of Day 6 to the morning of Day 10. In the conventional group (48PG), a administration of PGF2α (0.75 mg of cloprostenol), CIDR withdrawal, and administration of GnRH (0.2 mg of fertirelin acetate) were performed on the evening of Day 8, morning of Day 9, and morning of Day 10, respectively. In the experimental group (72PG), administration of PGF2a, CIDR withdrawal, and administration of GnRH were performed on the evening of Day 9, the morning of Day 10, and the morning of Day 11, respectively. The mOC were collected from follicles >5 mm by OPU at 25 to 26 h following GnRH administration. Collected mOC were inseminated with 3 × 106 sperm mL–1 in BO solution on 30 h after GnRH. After 6 h of IVF, presumptive zygotes were cultured for 168 h in 5% CS + CR1aa, using a micro-well culture dish (Dai-Nippon-Print) and time-lapse cinematography (CCM-1.4MZS; Astec) for individual embryo observation. The kinetics of early embryo was analysis by CCM-1.4 software. To assess the quality of BL, prognostic factors were used as follows: (1) less than 27 hpi (hours post-insemination) at the first cleavage (1st CD), (2) 2 blastomeres at the end of 1st CD, and (3) absence of multiple fragments at the end of the 1st CD (Sugimura et al. 2012 PLoS ONE 7, e36627; Imai et al. 2014 Reprod. Fertil. Dev. 26, 182). Data were analysed by Student's t-test or chi-square test. The number of mOC were 12.5 ± 4.7 and 10.3 ± 2.7 (means ± s.e.) oocytes per session in 48PG and 72PG. There was no significant difference in cleavage rate or BL formation rate (97.5 ± 1.5 v. 98.2 ± 1.8%, 66.3 ± 8.2 v. 66.8 ± 3.5%, respectively). The time for 1st CD was shorter in 48PG (26.1 ± 0.3 v. 27.8 ± 0.4; P < 0.01), and the rate of 1st CD less than 27 hpi was superior in 48PG compared with 72PG (74.3 v. 42.9%; P < 0.05). However, the rate of 2 blastomeres and absence of multiple fragments were not different between 48PG and 72PG. The number of BL tended to decrease in 72PG compared with 48PG (28.6 v. 48.6%; P = 0.087). These results indicate that duration of FG did not affect the rate of cleavage and BL formation. However, extension of duration of FG might reduce the quality of BL.
268 ROLE OF ATPase MOTOR CYTOPLASMIC DYNEIN AND PRIMARY CILIA IN TESTICULAR MORPHOGENESIS
Authors: Dores C. , Dobrinski I.
Published: December 2014

In vertebrates, the primary cilium is a nearly ubiquitous organelle present in somatic cells, but little is known about its function in the male gonad. We investigated the role of primary cilia in testis cells using in vitro formation of seminiferous tubules and in vitro culture of testicular somatic cells by inhibiting the primary cilium with CiliobrevinD, a cell-permeable, reversible chemical modulator that inhibits the major component of the organelle: ATPase motor cytoplasmic dynein. We analysed in vitro cultures for the presence of primary cilia and the activation of hedgehog signalling through translocation of Gli2 to the nuclei; in vitro tubule formation was evaluated by length and width of tubules formed. Methods: testicular cells were harvested from neonatal pigs by 2-step enzymatic digestion. Cells (50 × 106 mL–1) were plated on 100 mm Petri dishes in 15 mL of DMEM + 5% FBS + 50 U of penicillin and incubated at 37°C in 5% CO2 in air overnight, cells remaining in suspension and those slightly attached were removed and the somatic cells attached were trypsinized to obtain a single cell suspension, and then submitted to two different protocols: in vitro culture (A) or in vitro tubule formation (B), n = 5 replicates each. For A, somatic cells were replated on coverslips in 24-well plates and cultured in serum free media for 48 h, then for the treated group, 10 mM of CiliobrevinD was added for 24 h, attached cells from control and treated groups were fixed in 4% PFA and characterised by immunocytochemistry for ARL13B, Vimentin, and Gli2. For B: 1 × 106 cells were added to 24-well plates coated with 1 : 1 diluted Matrigel, the control group was kept in serum free media and to the treated group was added 20 mM CiliobrevinD at Day 0. Results: A) primary cilia were present in 89.3 ± 2.3% of cells cultured in serum-free media for the control group and Gli2 was located in the nuclei of 90.2 ± 1.2% of cells; in the CiliobrevinD-treated group the percentage of primary cilia decreased (P < 0.05) to 3.1 ± 2.5% and nuclear Gli2 to 3.9 ± 0.7; B) tubules formed in the control group were significantly longer and wider than the ones formed when CiliobrevinD was added (9.91 ± 0.35 v. 5.540 ± 1.08 mm and 339.8 ± 55.78 v. 127.2 ± 11.9 µm, respectively, P < 0.05 by Student's t-test). In conclusion, the inhibition of ATPase motor cytoplasmic dynein perturbs formation of primary cilia in testicular somatic cells, blocks Hedgehog signalling, and impairs in vitro tubule formation. Therefore, primary cilia on testicular somatic cells appear to be essential for testicular morphogenesis.Research was supported by 5 R01 OD016575-13.