bluuebunny/crossref_metadata_2025 huggingface dataset:
215 SNAIL AND SLUG, MARKERS OF EPITHELIAL MESENCHYMAL TRANSITION, APPEARED TO BE ALTERED BY ALKYL-PHENOLS, BISPHENOL A AND NONYL-PHENOL, IN OVARIAN CANCER CELLS EXPRESSING ESTROGEN RECEPTORS
Authors: Kim Y.-S. , Choi K.-C.
Published: December 2014

The ovary is the important organ to produce oocytes. Any disorder will affect embryo production. Ovarian cancer is one of gynecologic cancers in women which can affect ovarian functions. Oestradiol (E2) may be involved in ovarian cell growth and epithelial-mesenchymal transition (EMT) for diverse functions. EMT is an important process in embryo development and tumour migration or progression. Bis-phenol A (BPA) and nonyl-phenol (NP) have an estrogenic property, which can be suspected as endocrine disrupting chemicals (EDC). In this study, it has been examined whether BPA and NP can cause EMT process and migration in BG-1 ovarian cancer cells. To confirm the effect of these EDCs, BG-1 ovarian cancer cells were cultured and treated with DMSO (0.1%), E2 (10–7 M), BPA (10–6 M) and NP (10–6 M) for 0, 6, and 24 h. The mRNAs were extracted to perform reverse-transcription (RT)-PCR and the changes in the mRNA expressions were analysed by ANOVA test. Following treatments with BPA and NP, alterations of EMT markers; that is, vimentin and E-cadherin, were examined at mRNA levels by RT-PCR. The levels of vimentin were up-regulated by E2, BPA, or NP in a time-dependent manner. In addition, transcriptional factors of EMT response, i.e. snail and slug, were enhanced by these treatments more than 2 times. BG-1 cells were exposed to these EDCs for 0, 24, and 48 h. Vimentin and snail proteins were induced by E2, BPA, or NP, while the expression of E-cadherin was decreased by them. To reveal that this EMT response is affected by oestrogen receptor (ER), the cells were treated with these EDCs in the presence of an ER antagonist, ICI 182 780 (10–6 M). Treatment with ICI 182 780 reversed EDC-induced alteration of these EMT markers, E-cadherin, vimentin, and snail. Since EMT response can cause metastasis, a scratch assay was performed to show migration caused by BPA or NP. BPA or E2 enhanced migratory capability of these BG-1 cells. Taken together, these results indicate that BPA and NP, potential EDC, may have an ability to influence ovarian cancer metastasis via regulating snail and slug genes in ER-positive ovarian cancers. In a future study, their effects in inducing EMT and migration will be tested in a xenograft mouse model.This work was supported by a grant from the Next-Generation BioGreen 21 Program (no. PJ009599), Rural Development Administration, Republic of Korea.
217 THE EXPRESSION OF β-OXIDATION RELATING GENES UNDER HYPOXIC STRESS IN HUMAN PLACENTAL CHORIOCARCINOMA CELL (BeWo)
Authors: Shin E.-K. , Jeung E.-B.
Published: December 2014

Preeclampsia (PE) is thought in many cases to be caused by a shallowly implanted placenta that becomes hypoxic. Hypoxia can result from a failure at any stage in the delivery of oxygen to the cells. In peripheral tissues, oxygen diffuses down a pressure gradient into cells and moves into their mitochondria, where it is used to produce energy in conjunction with the breakdown of glucose, fats, and some amino acids. The aim of the study was to investigate the question of whether hypoxic stress is involved in β-oxidation of human placental BeWo cells. One of the β-oxidation related genes, ACADVL was detected by gene-fishing technology using the preeclamptic placenta of human. We conducted in vitro experiments to confirm a preliminary study by inducing hypoxic stress in the human placental BeWo cell. BeWo cells were cultured at 37°C in a 20% O2, 5% CO2 humidified tissue culture incubator. And then we induced hypoxic stress in BeWo cell cultured under 1% O2, 5% CO2, and balanced with N2. The BeWo cells were divided into three groups: normoxia, hypoxia 24 h, and hypoxia 48 h. The expression of β-oxidation related genes (ACADVL, EHHADH, HADH, ACAA) were observed under hypoxic condition in BeWo cells by using real-time RT-PCR. Relative quantification with HPRT1 was based on the comparison of CT at a constant fluorescent intensity. The amount of transcript is inversely related to the observed CT, and for every 2-fold dilution in the transcript, CT is expected to increase by 1. Relative expression was calculated using the equation R = 2–(CTsample – CTcontrol). Data were analysed with a nonparametric one-way ANOVA, followed by Tukey's test for multiple comparisons. All statistical analyses were performed using GraphPad Prism software (v 4.0, GraphPad Software, La Jolla, CA, USA). P-values <0.05 were considered statistically significant. The expression of a gene known as a biomarker for hypoxia, HIF-1a, was significantly increased in BeWo cells which induced preeclampsia. The elevated level of HIF-1a indicates that our experimental conditions closely mimicked preeclampsia. The β-oxidation related genes, ACADVL, EHHADH, and HADH expressions were significantly increased under hypoxic condition in BeWo compared with normoxic control. These results indicate that changes of β-oxidation related genes observed under hypoxic BeWo cells are similar to ones associated with preeclampsia, and the expression of β-oxidation related genes were up-regulated by hypoxic stress. They might be involved in pathogenesis of preeclampsia during gestation. Taken together, increase of β-oxidation-related genes under hypoxic stress may cause a compensation of energy metabolism deficiency through lipid metabolism.
223 PROGRESSION OF BREAST CANCER WAS CAUSED BY TREATMENT WITH BENZOPHENONE-1 AND NONYL-PHENOL VIA ALTERATION OF CELL CYCLE AND METASTASIS-RELATED GENES IN A CELLULAR MODEL
Authors: In S.-J. , Hwang K.-A. , Kim S.-H. , Choi K.-C.
Published: December 2014

Endocrine disrupting chemicals (EDC) are defined as environmental compounds that may result in adverse health problems such as cancer proliferaition and metastasis in humans. Benzophenone-1 (2,4-dihydroxybenzophenone, BP-1) and nonyl-phenol (NP) are known as typical EDCs. They are discharged from numerous industrial products including plastics, pesticides, drugs, detergents, and cosmetics. In this study, we examined the effect of BP-1 and NP on the growth of MCF-7 human breast cancer cells expressing oestrogen receptors (ER) in comparison with E2 to assess their risk in cancer progression. In cell viability assay, BP-1 (10–5, 10–6, and 10–7 M) and NP (10–6 and 10–7 M) were determined to induce the proliferation of MCF-7 cells as well as E2 (10–9 M) was compared to a negative control treated with DMSO (P < 0.05). Next, to confirm that BP-1 and NP increase growth and metastasis of MCF-7 cells, the alterations in transcriptional and translational levels of related markers, i.e. cyclin D1, p21, and cathepsin D, were examined by reverse-transcription (RT)-PCR and Western blot assay. Cyclin D1 is a factor responsible for G1/S cell cycle transition and p21 is a potent cyclin-dependent kinase (CDK) inhibitor that arrests cell cycle in G1 phase. Cathepsin D is one of the proteases that are responsible for cancer progression and metastasis. Treatment of MCF-7 breast cancer cells with BP-1 (10–5 M) or NP (10–6 M) resulted in up-regulation of cyclin D1 and cathepsin D and down-regulation of p21 at transcriptional and translational levels as well as E2 (10–9 M) compared to a negative control treated with DMSO (P < 0.05). In addition, E2, BP-1, or NP-induced alterations of these genes were reversed by the presence of ICI 182 780 (10–8 M), an ER antagonist, suggesting that the changes in these gene expressions may be regulated by ER-dependent signalling pathway. In conclusion, these results suggest that BP-1 and NP, like E2, may accelerate the growth of MCF-7 breast cancer cells by regulating cell-cycle-related genes through ER-mediated signalling pathway. Furthermore, these EDCs can adversely affect human health by promoting cancer metastasis through the amplification of cathepsin D via ER-dependent signalling pathway.
234 NEW CULTURE MEDIA AFFECTS BLASTOCYST DEVELOPMENT AND GENE EXPRESSION LEVELS IN IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Nielsen J. M. K. , Wrenzycki C. , Hyttel P. , Poppicht F. , Strøbech L.
Published: December 2014

The purpose was to examine effects of different media for bovine in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on blastocyst rates, morpho-kinetics, and relative abundance of mRNA of 8 genes associated with critical processes and developmental competence in the embryo. Abattoir-derived cumulus-oocyte complexes (COC) were in vitro matured (IVM) in either TCM199 [+0.5% BSA and gonadotropins (Suigonan Vet 150 I.E. mL–1)] or in a novel commercially available media (Bo-IVM), and a total of 1196 presumptive zygotes, from 4 replicates, were submitted to in vitro culture (IVC) and cultured in either SOF (+0.5% BSA) or in a novel commercially available media (Bo-IVC). Blastocyst rates and morpho-kinetics were assessed on Day 8 after fertilization. The high-quality blastocysts from each group were analysed by RT-qPCR, on single blastocysts using earlier verified primers, for BAX, BCL2L1, DNMT3A, FASN, G6PD, HSPA1A, SLC2A1, and SLC2A3. Data on blastocyst rates were analysed for statistical differences using a linear regression model, using a binary reproach and general estimating equations. One-way ANOVA was used to detect differences in the relative abundance of mRNA between groups, whereas differences between maturation and culture media were analysed by a 2-way ANOVA. Blastocyst rates in the Bo-IVM/Bo-IVC (37%), TCM199/Bo-IVC (33%), Bo-IVM/SOF (26%), and TCM199/SOF (28%) groups were significantly different from each other (P < 0.0001). Specifically, the Bo-IVM/Bo-IVC group differed significantly from both SOF-cultured groups (P < 0.01). Subjectively, this group also had embryos of the highest quality and most advanced development. Significantly increased levels of mRNA transcripts were found for embryos cultured in Bo-IVC for all genes (P < 0.05) except BCL2L1. In conclusion, the developmental rates and gene expression of in vitro-produced bovine blastocysts were affected by the use of different culture media. Increased blastocyst rates, apparently superior embryo quality, and more abundant gene expression were achieved when blastocysts were cultured in Bo-IVC culture media compared with SOF.The project was supported by The Danish Council for Strategic Research.
238 THE ROLE OF PHOSPHODIESTERASE (PDE) 5A AS MEDIATOR OF LIPOLYSIS IN BOVINE OOCYTES AND ITS EFFECT ON THE DEVELOPMENT AND LIPID CONTENT OF EMBRYOS PRODUCED IN VITRO
Authors: Schwarz K. R. L. , Adona P. R. , Botigelli R. C. , Del Collado M. , Elias C. , Chiaratti M. R. , Castro F. C. , Cunha M. C. R. V. , Leal C. L. V.
Published: December 2014

Intracellular levels of cyclic adenosine monophosphate modulators (cAMP) and cGMP, in adipocytes, are important for the regulation of the lipolysis rate. The phosphodiesterases (PDE) control cGMP and cAMP levels by degradation. Different PDE isoforms are expressed in bovine oocytes and cumulus cells. Previously, we found that using an inhibitor of PDE5A (sildenafil, SILD) increased cGMP levels in bovine oocytes during in vitro maturation (IVM). In the current study we investigated if inhibition of PDE5A during maturation reduces the lipid content in IVF embryos. For this, oocytes were cultured for 24 h in maturation medium with 10% FCS and 10–7 M SILD (treatment I), 10% FCS (treatment II) and 0.4% BSA (control; N ± 160 COC/groups submit to IVF). After COC were in vitro fertilized, cleavage (Day 4) and blastocyst rates (Day 7) were measured. Blastocysts were stained with Nile Red (1 μg mL–1) for lipid content quantification, by mean fluorescence intensity per μm2, measured in the ImageJ program (fluorescence intensity, f.i.). Four replicates were transformed to log10 and subjected to statistical analysis using the SAS system (SAS Institute Inc., Cary, NC, USA) by ANOVA followed by Tukey test with a significance level of 5%. No difference in cleavage (Day 4) and blastocyst (Day 7) rates were observed in all groups (82 and 41.9%, respectively), showing that presence of FCS, SILD, or both in IVM medium did not affect embryo development. Treatment I had higher lipid content (40.35 f.i.) than treatment II (31.12 f.i.), which in turn was also superior to control (22.31 f.i.). According to the results, the presence of FCS in IVM media generates embryos with higher lipid content, and association of FCS and SIL further increased lipid content. Although inhibition of PDE5 increases cGMP levels and leads to higher lipolysis, such an effect was not observed when SIL was used as the PDE5 inhibitor. Reasons for such findings are still unclear, but a possibility would be the activation of a negative feedback mechanism by the increased cGMP generated by SIL, because this nucleotide activates PKG, which in turn inhibits cGMP synthesis by guanylate cyclase. During development the lower cGMP levels could reduce lipolysis, resulting in increased lipid accumulation in embryos. Further studies are needed to address this possibility.
239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS
Authors: Fanti T. , Ortega N. M. , Garaguso R. , Franco M. J. , Herrera C. , Mutto A. A.
Published: December 2014

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos.
Table 1.Effect of treatment on maturation, cleavage, and cryotolerance
242 PROMISING TWO-STEP EVALUATION SYSTEM FOR SELECTING HIGH DEVELOPMENTAL COMPETENCE IN VITRO FERTILIZED EMBRYOS IN CATTLE USING WELL-OF-THE-WELL DISH
Authors: Taniai M. , Takayama M. , Dochi O. , Imai K.
Published: December 2014

Bovine IVF embryos are evaluated morphologically using light microscopy just before transfer. However, this evaluation method is subjective, and an objective method with more certainty is needed. Sugimura et al. (PLoS ONE 2012 7, e36627) reported a promising system for selecting healthy IVF bovine embryo by using time-lapse cinematography and 5 prognostic factors. This study was to investigate the efficacy of a 2-step evaluation system of IVF embryos using microscopy for selecting high developmental competence IVF embryos. Cumulus-oocyte complexes (COC) were collected by ovarian follicular aspiration (2 to 5 mm diameter) obtained from a local abattoir. The COC (n = 488) were matured in TCM-199 medium supplemented with 5% calf serum (CS) and 0.02 IU mL–1 of FSH at 38.5°C for 20 h in an atmosphere of 5% CO2 (20 COC 100 µL–1 droplets). After 10 h of gametes co-culture (5.0 × 106 sperm cells mL–1), the presumptive zygotes were cultured in 125 µL of CR1 aa medium supplemented with 5% CS in well of-the-well culture dishes (AS ONE, Japan; 25 zygotes well–1) at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days. Two-step evaluations of embryos were done at 27 and 55 h post-IVF (hpi). In the first step of evaluation, cleavage patterns at 27 hpi were categorized as mono-cell, 2-cell with even blastomeres and without fragments (normal cleavage), 2-cell with uneven blastomeres, and ≥3 blastomeres. During the second step of evaluation, embryos were classified by their number of blastomeres (2 to 5 cells, 6 to 8 cells, and >8 cells) and the absence or presence of multiple fragments (<20 or >20%) at 55 hpi. The data were analysed by chi-square test. The blastocyst rate (BL%) of embryos cleaved before 27 hpi (56.6%, n = 106) was higher (P < 0.01) than those of embryos cleaved after 27 hpi (37.0%, n = 235). A greater percentage (P < 0.05) of 2-cell embryos with normal cleavage (68.0%, n = 50) developed to blastocysts than from with =3 blastomeres at 27 hpi (40.6%, n = 32). Superior BL% (P < 0.01) was obtained from embryos categorized as 6- to 8-cell stage (58.6%, n = 140) and >8 cell stage (70.6%, n = 25) compared with those embryos at the 2- to 5-cell stage at 55 hpi (26.1%, n = 176). Embryos with no fragments (58.0%, n = 467) had higher BL% (P < 0.01) compared with those with <20% fragments (30.7%, n = 127) and having with >20% fragments (17.5%, n = 25) at 55 hpi. The highest of BL% was observed in embryos showing a normal cleavage to 2-cells with at 27 hpi and having >6 cells with no fragments at 55 hpi (95.2%, n = 21, P < 0.01). These results demonstrate that the 2-step evaluation system at 27 and 55 hpi using microscopy is an effective method for selecting IVF embryos with high developmental competence.
244 IN VITRO EMBRYO PRODUCTION WITH CONVENTIONAL OR Y-SEXED SEMEN IN BEEF CATTLE
Authors: Tribulo H. E. , Carcedo J. , Tribulo R. J. , Bernal B. , Garzon J. , Tribulo A. , Bó G. A.
Published: December 2014

An experiment was designed to evaluate in vitro embryo production following the use of frozen-thawed conventional or Y-sexed semen from a single Brangus and a single Braford bull of proven fertility. Semen was obtained by splitting the same ejaculate to be frozen directly or sex-sorted and then frozen. Oocytes were obtained from 69 ultrasound-guided follicle aspiration (ovum pickup) sessions performed at random stages of the oestrous cycle without superstimulation in 24 Brangus and 10 Braford cows and heifers. Viable oocytes (n = 1120) were matured in TCM-199 medium with NaHCO3 and supplemented with 1% fetal bovine serum. Frozen-thawed sperm from the Brangus and Braford bulls were selected with Percoll for IVF, capacitated in Fert Medium, and used at a final concentration of sperm per milliliter for conventional (non-sexed) semen and 2 × 106 sperm mL–1 for Y-sexed semen. After 16 h (sexed) or 18 h (conventional) of co-incubation with oocytes in Fert Medium, presumptive zygotes were denuded and cultured in SOF supplemented with 0.4% BSA under oil at 37°C, 5% CO2, and saturated humidity for 7 days. The total number of oocytes matured and fertilized from the Brangus donors was 538 and 318 for conventional and sexed semen, respectively. The total numbers of oocytes matured and fertilized from the Braford donors were 139 and 125 for conventional and sexed semen, respectively. Data were compared by ANOVA for mixed models, using breed and type of semen as fixed variables and cow (i.d.) as a random variable. Cleavage and blastocyst rates were first transformed by square root and then analysed by ANOVA for mixed models. Mean (± s.e.m.) number of total viable oocytes collected, cleaved zygotes, and blastocysts produced per ovum pickup session did not differ (P = 0.18) between breeds (Brangus: 17.1 ± 1.6, 10.0 ± 0.9, and 6.2 ± 0.7 v. Braford: 13.9 ± 2.8, 7.6 ± 1.5, and 4.0 ± 0.8), and there was no breed × semen interaction on the mean number of cleaved zygotes and blastocysts produced. However, the mean (± s.e.m.) number of cleaved zygotes and blastocysts produced was significantly higher (P < 0.05) when the oocytes were fertilized with conventional semen (10.7 ± 1.2 and 6.5 ± 0.8) than with sexed semen (7.7 ± 0.7 and 4.3 ± 0.6). The mean cleavage rate was also significantly higher (P < 0.05) when the oocytes were fertilized with conventional semen (76.8 ± 3.9) than with sexed semen (54.1 ± 4.2). Blastocyst rate tended to be higher (P = 0.1) with conventional semen (40.5 ± 3.3) than with sexed semen (33.6 ± 4.2). Although in vitro production may be the preferred alternative for the production of embryos of a known sex, the number of blastocysts produced might be reduced as compared with the use of non-sexed semen from the same bull.
260 EVALUATION OF PORCINE IN VITRO PRODUCTION WITH THE ADDITION OF CHITOSAN TO CULTURE MEDIA
Authors: García F. , Ducolomb Y. , Miranda-Castro S. P. , De la Torre-Sánchez J. F. , Romo S.
Published: December 2014

Chitosan is a partially deacetylated polymer obtained from the alkaline deacetylation of chitin, which is a glucose-based unbranched polysaccharide widely distributed in nature as the main component of exoskeletons of crustaceans and insects. Chitosan has a variety of physicochemical and biological properties resulting in numerous applications. In addition to its lack of toxicity and allergenicity, its biocompatibility, biodegradability, and bioactivity make it a very attractive substance for diverse applications as a biomaterial in pharmaceutical and medical fields. Chitosan stimulates cell growth and it has been used in fibroblast culture, increasing cell proliferation. For these reasons, it is important to evaluate if this polymer has a positive effect on embryo production. The aim of this study was to evaluate porcine oocyte maturation and embryo development, comparing the effect of supplementing different concentrations of chitosan to the maturation (MM) and development media (DM). Cumulus-oocyte complexes (COC) were aspirated from ovarian follicles of slaughtered sows. The COC were matured in supplemented TCM-199 (MM) and incubated for 44 h. All incubations were performed at 38.5°C, with 5% CO2 in air and humidity at saturation. After maturation IVF was performed, frozen-thawed semen from the same boar was used and gametes were co-incubated in MTBM for 7 h. Then, putative zygotes were cultured in NCSU-23 (DM) for 144 h. The following experiments were performed: 1) addition of 0 (control), 35, 50, 100, and 150 ppm chitosan to the MM (n = 1353), 2) addition of 0, 50, 100, and 150 ppm chitosan to the DM (n = 739), 3) addition of 0, 50, 100, and 150 ppm of chitosan to the MM first and then the same concentrations to the DM (n = 702). When chitosan was added to the MM, the highest percentage of matured oocytes (metaphase II) was obtained in the 50 ppm treatment (87%, P < 0.05) when compared with the control, 100, and 150 ppm groups (78, 78, and 82%, respectively). Regarding the percentage of blastocysts, there were no differences when comparing the treatment and the control groups (ranging from 12 to 13%). After addition of chitosan to the putative zygotes in the DM, the percentage of morulae in the 150 ppm treatment was significantly increased with regard to the other groups (54 v. 46%, respectively, P < 0.05). When adding chitosan to both MM and DM, there was no effect on embryo development. It is concluded that the addition of chitosan to the MM at a concentration of 50 ppm significantly improved oocyte maturation and a concentration of 150 ppm in the DM increased the percentage of morulae. Chitosan had a positive effect on oocyte maturation and embryo development. These results justify further investigations to find out if chitosan can be useful as a supplement for chemically defined media.
278 INFLUENCE OF NITRIC OXIDE AND PHOSPHODIESTERASE INHIBITORS ON CYCLIC NUCLEOTIDES AND MEIOSIS RESUMPTION IN BOVINE OOCYTES IN VITRO MATURED
Authors: Botigelli R. C. , Schwarz K. R. L. , Zaffalon F. G. , Leal C. L. V.
Published: December 2014

Nitric oxide (NO) is a local action mediator of essential hormones and neurotransmitters that regulate several physiological processes in reproduction. Nitric oxide activates soluble guanylate cyclase (sGC), and results in the production of cyclic guanosine monophosphate (cGMP). This nucleotide is involved in oocyte maturation and, consequently, in the success of fertilization. In addition to cGMP, another cyclic nucleotide, cAMP, is also related to maturation. The levels of these nucleotides are balanced by synthesis and degradation is made by phosphodiesterases (PDE). The aim of this study was to analyse the effect of NO and PDE inhibitors on cyclic nucleotides concentrations and meiosis resumption in bovine oocytes matured in vitro. In experiment 1, cumulus-oocyte complexes (COC) were cultured in maturation medium with a NO donor (0.1 µM S-nitroso-N-acetylpenicillamine; SNAP) associated or not with a PDE5 inhibitor (10 µM sildefanil; SILD) for 1 h; after this period, cGMP levels (40 COC/group) were measured in COC using commercial enzyme immunoassay kits (EIA). Immature (0 h) and control COC (untreated) were measured as well. In experiment 2, COC were cultured for 9 h in maturation medium with an NO donor (0.1 µM SNAP) associated or not with inhibitors the PDE5 (10 µM SILD), PDE3 (20 µM cilostamide; CIL), or PDE8 (50 µM dipyridamole; DIP) and assessed for meiosis resumption (%GVBD; ± 100 COC/treatment). In experiment 3, COC were cultured as in experiment II and cAMP levels (10 COC/group) were measured after 1 h in culture using commercial EIA kits. Immature COC were also measured. Statistical analyses were performed using the SAS system. Data were tested for normal distribution and were transformed (arcsine; GVBD). The percentages of GVBD were analysed by one-way ANOVA followed by Bonferroni post-hoc test (4 replicates). The cGMP and cAMP levels were analysed by two-way ANOVA followed by Bonferroni post-hoc testing (5 replicates). Differences with probabilities of P < 0.05 were considered significant. In experiment 1, cGMP levels decreased from 0 to 1 h in control and SNAP groups (P < 0.05). When SNAP was associated with SILD, cGMP levels were similar to immature oocytes (P > 0.05%). In experiment 2, all treatments delayed meiosis by decreasing GVBD rates (24.7 ± 2.8 to 56.9 ± 8.6%) when compared to controls (77.1 ± 1.8%, P < 0.05). However, SNAP + CIL had the lowest GVBD rates (24.7 ± 2.8%, P < 0.05). In experiment 3, cAMP levels declined from imature COC (93 ± 7 fmol/COC) to 1 h in all treatments (21 ± 6 to 10 ± 5 fmol/COC; P < 0.05). The results indicate that NO and PDE inhibitors during maturation were ineffective in preventing the decrease in cyclic nucleotides levels during the first hour of maturation, but were still effective in delaying meiosis resumption, and the effectiveness depends on the PDE isoform which is inhibited (SNAP + CIL was most effective).
29 SCRIPTAID IMPROVES SOMATIC NUCLEAR TRANSFER EFFICIENCY DURING IN VITRO CULTURE OF PORCINE EMBRYOS DERIVED FROM INBRED MINIATURE PIG FETAL FIBROBLASTS
Authors: Koppang R. , Mtango N. R. , Barcelo-Fimbres M. , Verstegen J. P.
Published: December 2014

Porcine somatic cell nuclear transfer (SCNT) is limited to the same or next day surgical embryo transfer due to poor culture conditions in vitro. In this study, we aimed to overcome this problem by treating SCNT embryos with scriptaid, an inhibitor of histone deacetylase (HDACi) that helps with epigenetic reprogramming of the somatic nuclei. Scriptaid was chosen over other HDACi because it has been shown to improve histone acetylation in the same pattern as that of IVF embryos as well as its low toxicity characteristic (Zhao et al. 2009 Biol. Reprod. 81, 525–530; Zhao et al. 2010 Cell Reprogram. 12, 75–78). An inbred miniature pig fetal fibroblast cell line that is known to give low blastocyst rate in culture was used as a source of donor cells transferred into enucleated oocytes. Traditional SCNT was performed; embryos were fused and chemically activated in 10 µM ionomycin for 5 min and 2 mM DMAP for 3 to 4 h before being transferred into scriptaid. Embryos were treated with 500 nM scriptaid (Zhao et al. 2010) for 18 h and the untreated group was used as control. A total of 806 oocytes were used in 8 replicates. The constructed embryos were cultured in modified porcine zygote medium 5 (mPZM-5) for 7 days at 39°C in 5% O2, 5% CO2, 90% N2 atmosphere. Cleavage rates were assessed at 2.5 days and blastocyst rates at Day 7 after activation. Data were analysed by ANOVA using GLM, and percentages were transformed using arcsin square root using Statistix 10 software (Tallahassee, FL, USA). There were no differences in cleavage rates for control group v. scriptaid (55.3 v. 49.9%; P > 0.1; Table 1). The blastocyst rate per construct showed remarkable increase in the scriptaid treated group compared with the control group (12.8 v. 2.2%; P < 0.01; Table 1). Similarly, a significant effect was observed for blastocyst per embryos cleaved where scriptaid had higher rates compared with control (25.8 v. 5.8%; P < 0.01). These results indicated that improving nuclear reprogramming of miniature porcine SCNT clones by scriptaid treatment enhanced blastocyst production during the in vitro culture of porcine embryos.

Table 1.Mean (± s.e.m.) measures of embryonic development of SCNT embryos
303 HUMAN RECOMBINATION GRANULOCYTE-COLONY STIMULATING FACTOR (HRG-CSF) HAVE BENEFICIAL EFFECTS ON PORCINE OOCYTES QUALITY DURING IN VITRO MATURATION AND SUBSEQUENT VIABILITY OF EMBRYONIC DEVELOPMENT
Authors: Cai L. , Lee E. , Hyun S.-H.
Published: December 2014

Granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3 (CSF3), is required for the proliferation, differentiation, and survival of cells. In humans, G-CSF is a biomarker of human oocyte developmental competence for embryo implantation. Furthermore, G-CSF concentration increases during the menstrual cycle and levels were significantly higher during ovulatory phase than the other phases. In this study, we examined G-CSF and its receptor gene expression in the porcine granulosa cells, corpus luteum, cumulus cells, and oocytes. The cumulus-oocyte complexes (COC) were aspirated from antral follicles 1 to 3 mm (small follicle) and 3 to 6 mm (medium follicle). The COC from 2 kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of hrG-CSF (0, 10, and 100 ng mL–1, respectively). Statistical analyses were done by one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. After real time-PCR was performed, the CSF3 and its receptor (CSF3R) were observed all of granulosa cells, corpus luteum, cumulus cells, and oocytes. Interestingly, the CSF3 transcript levels were significantly lower in oocytes compared with other cell types, but the CSF3R transcript levels in oocytes were almost similar with granulosa cells. After 44 h of IVM, the rates of nuclear maturation had no difference, and the intracellular ROS levels of oocytes from both kind of follicle groups matured with 10 ng mL–1 were significantly decreased compared to other groups (P < 0.05). After PA, the cleavage and blastocyst formation rates were significantly (P < 0.05) increased for the 100 ng mL–1 of small follicle (SF; 63.29 and 31.18%) group compared to control and 10 ng mL–1 of SF (38.64, 10.4, and 49.0, 15.6%, respectively) group, and significantly (P < 0.05) increased in the 10 ng mL–1 of medium follicle (MF; 76.32 and 45.61%) group compared with control and 100 ng mL–1 of MF (52.1, 32.8 and 61.3, 33.9%, respectively). The total cell numbers of blastocyst from SF and MF groups were significantly increased in the 10 ng mL–1 (73.67 and 106.52) groups. At IVF, the blastocysts formation rates were significantly increased in the 10 ng mL–1 of MF group compared to control, 100 ng mL–1 of SF, and control of MF (21.1, 22.8, and 27.8%, respectively). We also examined the Bcl2 and ERK2 transcript levels, which were significantly increased at 100 ng mL–1 of SF and 10 ng mL–1 of MF. These results suggest that hrG-CSF improved the quality of porcine oocyte and embryonic viability.This was supported, in part, by a grant from the National Research Foundation of Korea Grant Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.
316 GENOMIC EVALUATION OF BOVINE EMBRYOS WITHIN 24 HOURS
Authors: Jung S. , Reichenbach M. , Fries R. , Wolf E. , Gschoederer C. , Scherzer J. , Grupp T. , Reichenbach H.-D.
Published: December 2014

The aim of this study was to develop a reliable procedure for genomic evaluation of bovine embryos to determine gender, polled status, and hereditary defects within 24 h after collection. German Simmental animals (n = 15) were superovulated (n = 25) using a standard protocol. Embryos were recovered on Day 7 (Day 0 = oestrus). A total of 217 embryos (morula, n = 130; early blastocyst, n = 43; blastocyst, n = 44) were biopsied with a steel blade attached to a micromanipulator. Biopsied cells were immediately transferred into 1 µL TE buffer to a 500 µL reaction tube and embryos were in vitro cultured until genomic results were available. For commonly used molecular genetic methods (e.g. 5′-exonuclease genotyping, PCR or high density genotyping) DNA amounts of 2–200 ng are required. However, the DNA quantity of a single diploid cell amounts to 6 pg only. The embryo biopsies used, usually consists of 10–30 cells, necessitating an artificial amplification of the embryonic genome. Taking all vital measures to avoid external DNA contamination, isothermal whole genome amplification was performed with the REPLI-g Mini Kit (Qiagen, Valencia, CA, USA) using random hexamers and Phi29-Polymerase. Depending on the number of cells, a total DNA amount of 4–7 µg was achieved. Polled status and gender was determined using PCR with subsequent gel-electrophoresis. 5′-exonuclease assays were used to obtain genotypes for the detection of genetic defects. At present, eight, mostly Simmental-specific genetic disorders can be examined: three traits associated with severe growth retardation, dwarfism (DW), Braunvieh-haplotype 2 (BH2) and stunted growth (FH2), the lethal skin disorder zinc deficiency-like syndrome (ZDL), a fertility trait bovine male subfertility (BMS), embryonic death Fleckvieh-haplotype 4 (FH4), a bleeding disorder thrombopathia (TP) and arachnomelia (A), within 24 h. On average, 8.7 embryos were biopsied per embryo recovery, i.e. 93.9% of the total number of transferable embryos. Fourteen embryo samples (6.5%) totally failed during analysis, possibly due to the loss of samples. In successful analyses, gender was undetermined in two embryos; remaining embryos were 52.2% female and 47.8% male. Polled status could be analysed in 92.6% of the embryos. The analyses of embryos for possible inherited genetic disorders (healthy, heterozygote, or homozygote; n = 578) were successful in 90.1%. The transfer of biopsied embryos (n = 30) led to 63.3% pregnancies (Day 42). A validation of the present results has to be done as soon as the produced calves are born, demonstrating the reliability of the procedure.Research was funded by the Bayerische Forschungsstiftung (AZ-1031-12).
32 ALLOCATION OF INNER CELL MASS AND TROPHECTODERM CELLS OF NUCLEAR TRANSFER EMBRYOS CULTURED IN MEDIUM SUPPLEMENTED WITH EPIDERMAL GROWTH FACTOR AND INSULIN-LIKE GROWTH FACTOR-I
Authors: Caputcu A. T. , Arat S. , Cevik M. , Akkoc T. , Cetinkaya G. , Bagis H.
Published: December 2014

This study was conducted to determine the additive effects of exogenous growth factors and different macromolecules during in vitro oocyte maturation (IVM) and sequential embryo culture of nuclear transfer (NT) embryos. Oocytes were matured in TCM-199 supplemented with 10% fetal calf serum (FCS), 50 mg mL–1 sodium pyruvate, 1% penicillin/streptomycin (10 000 U mL–1 penicillin G, 10 000 mg mL–1 streptomycin), 5 mg mL–1 LH, and 0.5 mg mL–1 FSH without growth factors (Treatment 1) or with 50 ng mL–1 epidermal growth factor (EGF; Treatment 2) or with 50 ng mL–1 EGF and 100 ng mL–1 insulin-like growth factor-I (IGF-1; Treatment 3). Cloned bovine embryos were produced by transferring granulosa cells into enucleated meiosis II oocytes. Following activation, reconstructed embryos were cultured in Quinn's Advantage Cleavage Medium (QACM) supplemented with 8 mg mL–1 essentially fatty-acid free (FAF) BSA for 72 h. Then, developing embryos from granulosa cells were cultured in Sequential Quinn's Advantage Blastocyst Medium (QABM) supplemented with 4 mg mL–1 essentially FAF-BSA (Sigma-Aldrich, St. Louis, MO, USA) + 5% FCS (Group 1), 4 mg mL–1 BSA + 5% FCS+100 ng mL–1 IGF 1 (Group 2), and 4 mg mL–1 BSA + 5% FCS+100 ng mL–1 IGF-1+50 ng mL–1 EGF (Group 3) for an additional 5 days under low oxygen tension (5% CO2, 5% O2, 90% N2) at 38.5°C in high humidity conditions. Maturation rates of oocytes matured in the presence of EGF (75.5%) and EGF+IGF-I combination (75.0%) were significantly higher than those of oocytes matured (63.8%) in the presence of only FCS (P < 0.05). The developing NT embryos derived from granulosa cells of the Anatolian Grey Cattle showed no significant differences in fusion (53.62%, 53.25%, 57.36%), cleavage (67.98%, 74.20%, 66.80%), or blastocyst rates (32.65%, 29.47%, 41.77%) among culture groups (P > 0.05). When 13 to 23 embryos per group were examined by using differential staining, the results showed that the IGF-I alone and combination with EGF in the sequential embryo culture medium (Group 2: 46.61%. and Group 3: 41.37%) significantly increased the number of inner cell mass (ICM)/total blastocyst cell ratio in comparison with Group 1 (29.32%, no IGF-I and EGF; P < 0.05). Our results showed that the addition of growth factors to IVM medium and sequential culture medium changed the cell ration of cloned bovine embryos to the advance of ICM without changing total cell number. Supplementation of media with growth factors can alter the allocation of ICM and trophectoderm cells in NT embryos.
337 PROMOTER-DEPENDENT SILENCING OF REPROGRAMMING TRANSCRIPTION FACTORS IN MOUSE INDUCED PLURIPOTENT STEM CELLS PRODUCED WITH SLEEPING BEAUTY TRANSPOSON VECTORS
Authors: Petkov S. G. , Kues W. A. , Niemann H.
Published: December 2014

Epigenetic silencing of the transgenes has been considered a prerequisite for complete reprogramming of mouse somatic cells to induced pluripotent stem cells (miPSC). Here, we examined the activity status of the reprogramming transcription factors in miPSC produced with Sleeping Beauty (SB) transposon vectors carrying expression cassettes with the porcine OCT4, SOX2, c-MYC, and KLF4 (pOSMK) under the control of doxycycline (DOX)-inducible (TetO) or constitutive (CAG) promoters. Mouse embryo fibroblasts (MEF) were electroporated with SB-TetO-rTA-SV40pA-TetO-pOSMK-IRES-tdTomato-bGHpA (TetO group) or with SB-loxP-CAG-pOSMK-IRES-tdTomato-SV40pA-loxP (CAG group) together with SB100x (SB transposase). The cells were cultured on mitotically inactivated MEF feeders with DMEM supplemented with 20% knockout serum replacement, 2 mM l-glutamine, penicillin-streptomycin, nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1000 U mL–1 of ESGRO, and 5 µg mL–1 of DOX. The miPSC colonies were individually picked, disaggregated to single cells, and propagated further under the same culture conditions. Three cell lines from each experimental group were examined for pluripotency characteristics, and the activity of the transgenes was monitored by the presence of tdTomato fluorescence and by RT-PCR. The miPSC produced with TetO vector silenced the transgene expression within 11 days post-transfection (in the presence of DOX) and upregulated the endogenous pluripotency genes Oct4, Sox2, Nanog, Rex1, and Utf1. These cells showed typical miPSC morphology and ability to differentiate into cells from the 3 primary germ layers in vitro and in vivo (teratomas). At the same time, the miPSC from the CAG group did not silence the transgenes even after 20 passages of continuous propagation, although they upregulated the endogenous pluripotency genes similarly to the TetO group. Moreover, these cells also showed ability to differentiate in vitro into cells from the 3 germ layers (contracting cardiac myocytes, neurons, epithelia) expressing differentiation markers Afp, Sox17, Gata4, Gata6, cardiac troponin, nestin, and PGP 9.5. Following Cre-mediated excision of the reprogramming cassette, the miPSC from the CAG group continued to self-renew and the expression of pluripotency markers Oct4, Sox2, Nanog, and Rex1 did not change significantly, as evidenced by real-time RT PCR (all P > 0.1), showing that these cells were not dependent on the transgenes for maintaining their pluripotency characteristics. Currently, we are investigating the ability of the miPSC from the CAG group to differentiate in vivo by producing teratomas and chimeras. The results from our preliminary investigations suggest that porcine transcription factors can be used for production of miPSC and that the silencing of the reprogramming transcription factors in miPSC is promoter-dependent, but may not be absolutely necessary for complete reprogramming to pluripotency.
338 IN VITRO GENERATION OF LENTOID BODIES FROM INDUCED PLURIPOTENT STEM CELLS OF TRANSGENIC MICE
Authors: Anand T. , Kumar D. , Talluri T. R. , Niemann H. , Kues W. A.
Published: December 2014

Pluripotent cells have the developmental potential to generate all adult cell types, so ocular diseases resulting from the failure of specific cell types could be potentially treatable through the transplantation of differentiated cells derived from stem cells. The present study was conducted with the aim of generating a cataract model. We attempted to derive the induced pluripotent stem (iPS) cells from fibroblast cells of transgenic (crytom) mice carrying a transgenic construct-alphaA crystallin promoter driving the tandem dimer (td) Tomato marker transgene, integrated in the genome. The 4- to 6-week-old female crytom mice were selected, superovulated, and mated. The fetuses were recovered and examined on various different days (10.5 to 15.5 days postfertilization), and the reporter expression was found to be initiated 12.5 days postfertilization and the intensity was increased thereafter. The expression of tdTomato was confirmed in the fetuses by Western blotting. Murine embryonic fibroblast (MEF) cultures were generated and electroporated with a reprogramming transposon cassette carrying Yamanaka factors (OCT4, SOX2, KLF4, and MYC) and Sleeping Beauty transposase to generate iPS cells which were picked up and clonally expanded. The cells were confirmed by PCR for tdTomato in the genome and characterised for the expression of Oct4 and cryAB by immunofluorescence. The iPS cells were also injected into the nude CD1 mice to test for teratoma formation. The generated cells were allowed to differentiate spontaneously on 3 different types (viz. P19, NTERA, and STO) of cell lines as feeders, in the absence of LIF, and cells were expected to fluoresce if differentiated to eye lens lineage. After long-term cultures, the iPS cells were found to differentiate and form lentoid bodies which expressed tdTomato. Thus, alphaA crystallin-tdTomato construct was allowed following lens cell formation by specific fluorescence excitation in a spatial and temporal manner. The employment of cell type-specific reporters for establishing and optimizing targeted differentiation in vitro seems to be an efficient and generally applicable approach for developing differentiation protocols for desired cell populations. Hence a transgenic murine iPS cell line was generated which exhibited potential to be used as a model for eye cataracts and other eye abnormalities.
350 EFFECT OF DIFFERENT SUB-DOSES OF EQUINE CHORIONIC GONADOTROPIN ON PREGNANCY AND TWINNING RATES OF CYCLIC ZEBU COWS SUBMITTED TO FIXED-TIME ARTIFICIAL INSEMINATION
Authors: Alvarez R. H. , Natal F. L. N. , Pires R. M. L. , Duarte K. M. R. , Oliveira C. A.
Published: December 2014

The injection of a low dose of eCG has the potential to induce multiple ovulation and pregnancies in cattle. The present study aimed to evaluate the ovarian response, conception rate and incidence of twin pregnancies of cyclic cows receiving 1 of 2 low doses of eCG. Multiparous Nellore (Bos t. indicus) cows with plasma progesterone levels >1 ng∙mL–1 on at least one of 2 blood samples collected at 10-day intervals (Day –10 and Day 0) received an intramuscular (IM) injection of 2 mg of oestradiol benzoate (EB; Estrogin®, AUSA, São Paulo, Brazil) and a vaginal device (DIP) containing 1 g of progesterone (Primer®, Tecnopec, São Paulo, SP, Brazil) on Day 0. On Day 8, the DIP was removed and cows received an IM injection of 150 μg of cloprostenol (Veteglan®, Hertape Calier, Juatuba, MG, Brazil). At this time, the animals were randomly distributed into 3 groups. Group 1 (n = 30) received an IM injection of 2 mL of saline, whereas groups 2 (n = 41) and 3 (n = 23) received 600 IU and 900 IU of eCG (Novormon® MSD Saude Animal, São Paulo, Brazil), respectively. Twenty-four hours later (Day 9), all groups received 1 mg of EB and were submitted to fixed-time artificial insemination (FTAI) 30 h later (i.e. 54 h after DIP removal). Oestrus observation was performed daily from the time of the withdrawal of the DIP until the day of FTAI. Ovaries were examined ultrasonically at the time of FTAI, the following day and 7 days after FTAI. Pregnancy diagnosis was done by ultrasonography 30 days after FTAI and the incidence of twin or single calves was recorded at birth. Data were analysed by chi-square test. The rate of expression of oestrus was 70.0% (group 1), 82.9% (group 2), and 78.2% (group 3; P = 0.25). Cows that had 2 or more large follicles at the time of FTAI was 0% (group 1), 14.6% (group 2), and 34.8% (group 3; P < 0.05). The ovulation rate of cows in group 1 (80.0%) was higher than cows in groups 2 (48.8%) and 3 (52.2%; P < 0.05). The conception rates for groups 1, 2, and 3 were 50.0, 26.8, and 39.1%, respectively (P < 0.05). Two animals in group 3, one in group 2, and none of group 1 had twin pregnancies on Day 30 after FTAI. Only one of these cows (group 3) had a twin calving. It was concluded that the injection of 600 or 900 IU eCG, in an oestradiol/progestogen FTAI protocol does not result in an increase in the rate of twin calvings, but may negatively affect pregnancy rates of cyclic Nellore cows.Financial support was provided by FAPESP (proc. 2011/13096–0).