bluuebunny/crossref_metadata_2025 huggingface dataset:
137 EFFECTS OF X-SORTED SPERM IN QUALITY OF BOVINE BLASTOCYST DERIVED FROM IN VIVO-MATURED OOCYTES
Authors: Imai K. , Ohtaku M. , Aikawa Y. , Matsuda H. , Kobayashi S. , Horiguchi E. , Matoba S. , Hashiyada Y.
Published: December 2013

Recently, we reported on a promising system for selecting healthy IVF embryos in cattle using kinetics of early embryo development and oxygen consumption of blastocyst [Sugimura et al. 2012 PLoS ONE 7, e36627]. The present study was conducted to examine the differences in embryo quality of bovine blastocysts obtained after IVF of in vivo-matured oocytes with X-sorted and unsorted sperm. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two ovum pickup (OPU) sessions were conducted in each cow to fertilize with or without X-sorted sperm. In vivo-matured oocytes were collected by OPU just before ovulation after superstimulation treatment. The oocytes were inseminated with 5 × 106 sperm mL–1 of each sperm, and presumptive zygotes were cultured in CR1aa supplemented with 5% newborn calf serum and 0.25 mg mL–1 of linolenic acid albumin at 38.5 C in 5% CO2, 5% O2, and 90% N2 for 168 h. Embryo kinetics were observed individually using a microwell culture dish (Dai-Nippon Print) and time-lapse cinematography (CCM-1.4MZS; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken every 15 min during the in vitro culture period and images were analysed by CCM-1.4 software (Astec). By assessing the quality of blastocysts, a combination of identified prognostic factors were used: (1) timing of the first cleavage (less than 27 h post-insemination); (2) two blastomeres at the end of the first cleavage; (3) absence of fragments at the end of the first cleavage; and (4) six or more blastomeres at the onset of the lag-phase. Data were analysed by ANOVA. In total, 34.1 ± 18.4 oocytes per session per donor were collected by OPU, and 23.7 ± 13.4 oocytes had an expanded cumulus cell. Oocyte recovery rates were recorded at 77.1 ± 15.1%. After IVF and in vitro culture, 10.6 ± 7.7 blastocysts per session per donor were produced in this study. There was no significantly difference in cleavage rates and blastocyst formation rates between X-sorted sperm and unsorted sperm (87.1 ± 10.8 and 82.6 ± 12.1% and 38.4 ± 23.6 and 57.1 ± 23.4%, respectively). However, blastocysts derived from X-sorted sperm showed significantly (P < 0.05) lower quality in the prognostic factor (1) and combined (1) to (4) than that in unsorted sperm (35.3 v. 54.0 and 14.7 v. 42.9%, respectively). Pregnancy rates were higher for the blastocysts that had a high score in the prognostic factors (1) to (4) compared to those that had a low score (75.0%, n = 8 v. 36.4%, n = 22). These results suggest that quality of blastocysts, based on the prognostic factors studied, derived from X-sorted sperm is lower than that from unsorted sperm.
Supported by the Research and Development projects for application in promoting new policy of agriculture, forestry and fisheries (22016).
139 PERTURBATION OF THE DYNAMICS OF DNA METHYLATION IN THE PATERNAL GENOME FOLLOWING IN VITRO FERTILIZATION IN CATTLE: THE SECRETS OF HEAT STRESS EFFECTS
Authors: Rahman M. B. , Rijsselaere T. , Van Soom A.
Published: December 2013

Ejaculates collected from breeding bulls during the summer season under tropical or subtropical conditions are often reported to have a lower fertilization potential. We recently reported that sperm cells at post-meiotic stages of development were more susceptible to heat stress (scrotal insulation technique). In post-meiotic stages of sperm cell development, extensive incorporation of histone variants and hyperacetylation confirms unstable chromatin. The unstable forms of chromatin are more vulnerable to heat stress and affect sperm DNA-protamine condensation. By using a special stain and nuclear morphometric assessment, Chromomycin A3 and Fourier harmonic analysis, respectively, we observed protamine-deficient spermatozoa with altered chromatin condensation in the heat-stressed ejaculates. In a recent study, we investigated the patterns of global DNA methylation in pronuclear development and fertilization potential of such altered chromatin condensed spermatozoa. To this end, 1239 in vitro-matured oocytes were fertilized with spermatozoa of 3 groups (i.e. standard IVF control, non-heat-stressed control, and heat-stressed spermatozoa). Data were analysed by means of ANOVA. The results showed that heat-stressed spermatozoa with altered chromatin condensation perturb the dynamics of DNA methylation reprogramming in the paternal pronucleus (i.e. disordered the active demethylation followed by a de novo methylation pattern during one cell stage). In addition, there was a tendency for a decrease in the size of both paternal and maternal pronuclei developed after fertilization with heat-stressed spermatozoa in comparison with standard IVF control spermatozoa, leading to lower fertilization rates (54 v. 82%). Future studies should focus on the mechanisms of perturbed DNA demethylation in the paternal genome after in vitro fertilization of oocytes with spermatozoa having altered chromatin condensation.
146 EFFECT OF PROTEIN SYNTHESIS INHIBITOR ON BOAR SPERM CAPACITATION AND FERTILIZATION IN VITRO
Authors: Okudaira Y. , Funahashi H.
Published: December 2013

In human, bovine, mouse, and rat sperm, translation of RNA to proteins in the mitochondrial ribosome during capacitation has been reported to be important for fertilization. The objective of this study was to examine effect of protein synthesis inhibitor (ribosome inhibitor) on boar sperm capacitation and IVF. Sperm from an ejaculated sperm-rich fraction of Berkshire boars were washed by centrifugation (1500 rpm for 35 min) in a Percoll gradient (45/90%) and then incubated in modified Medium-199 containing 0.4% BSA and 5 mM caffeine sodium benzoate, supplemented with or without a mitochondrial ribosome-specific (55S ribosome) inhibitor, chloramphenicol (CP; 0.3 mM), or a cytoplasmic ribosome-specific (80S ribosome) inhibitor, cyclohexide (CH; 3.6 mM), in an atmosphere of 5% CO2 in air at 39°C for 45 or 90 min. At 45 and 90 min after culture, sperm viability, motility, and chlortetracyclin-stained patterns (to assess the sperm functional status, capacitation, and acrosome reaction) were examined. Porcine oocytes were matured in vitro for 44 h in porcine oocyte medium supplemented with eCG, hCG, and dibutyryl cyclic adenosine monophosphate for the first 20 h. Matured oocytes after the removal of cumulus cells were co-cultured with sperm (final conc.: 2.5 × 105 cells mL–1) in the absence or presence of CP or CH for 8 h. Sperm penetrability was also determined. Statistical analyses of data from 4 replicated trials were performed by ANOVA. After 45 and 90 min of culture, neither CP nor CH affected sperm viability and motility (P > 0.05). The addition of CP after 45 and 90 min of culture significantly (P < 0.05) decreased capacitated and acrosome-reacted sperm rates, as detected by chlortetracyclin fluorescence assay (capacitated: control 9.6 v. CP 5.6%, control 17.8 v. CP 10.2%; acrosome reacted: control 4.6 v. CP 2.2%, control 9.2 v. CP 4.8%, respectively; P < 0.05). In the presence of CH, IVF rate and number of sperm per penetrated egg were decreased (control 80.8 v. CH 46.8%, 2.2 v. 1.4, respectively; P < 0.05). In the presence of CH, however, the percentage of metaphase II oocytes after co-culture with sperm for 8 h was lower than other 2 groups (control 87.6 v. CP 85.5 v. CH 74.0%; P < 0.05), and the percentage of A/T-II oocytes was higher than in the other 2 groups (control 1.1 v. CP 0 v. CH 9.4%; P < 0.05). From these results, we conclude that mitochondrial ribosome-specific inhibitor, chloramphenicol, affects capacitation and acrosome reaction but not penetration, whereas cytoplasmic ribosome-specific inhibitor, cyclohexide, decreases the number of oocytes that reach metaphase II stage and are penetrated.
148 EXPRESSION OF OVIDUCT-SPECIFIC GLYCOPROTEIN IN THE CANINE OVIDUCT DURING THE PERIOVULATORY PERIOD
Authors: Marnier C. , Saint-Dizier M. , Tahir M. Z. , Chastant-Maillard S. , Reynaud K.
Published: December 2013

In the canine species, the oocyte is ovulated at the immature germinal vesicle (GV) stage and will reach metaphase II stage after 3 to 4 days spent in the oviduct. Fertilization and embryonic development to the blastocyst stage also take place in the oviduct. In a previous study (Tahir et al. 2012 Reprod. Domest. Anim. 47, 487), we reported the expression of oviductin (oviduct-specific glycoprotein) mRNA in the oviduct. The present study aimed to describe the oviductin protein expression (immunolocalization and Western blot quantification) and the effect of the oviducal region and the ovarian cycle. Beagle bitches were ovariectomized at 6 stages (6 bitches/stage): anestrus, after the onset of proestrus and before the LH peak (Pre-LH), after the LH peak and before ovulation (Pre-ov), 1 day (Day 1), 4 days (Day 4), and 7 days (Day 7) after ovulation. Three oviducal regions were collected [i.e. ampulla, isthmus, and ampulla-isthmus junction (AIJ)]. Ampulla and isthmus were fixed in paraformaldehyde, embedded in paraffin, and 7-μm sections were used for immuno-histochemistry using a goat polyclonal anti-human oviductin (N20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the ImmPress kit (Vector Laboratories, Burlingame, CA, USA). Total protein from the AIJ was extracted and used for Western Blot using a mouse monoclonal anti-mouse antibody (H8; Santa Cruz Biotechnology). The expression of oviductin in AIJ was quantified in duplicate on blots using ImageJ software and normalized with actin levels. Relative amounts of oviductin were compared between stages by ANOVA followed by a Tukey test. Immuno-histochemistry revealed that oviductin was specifically expressed in the nonciliated cells of the oviducal epithelium from Pre-LH to Day 7, with a stronger staining in the isthmus than in the ampulla at all stages. Furthermore, the expression of oviduct-specific glycoprotein, detected by Western Blot, varied significantly with the stage (P < 0.0001). The oviductin protein expression was at its lowest level at anestrus, then increased significantly at Pre-LH and Pre-ov (35- and 41-fold higher levels than anestrus, respectively), reached a maximal level at Day 1 (66-fold higher than anestrus), then decreased at Days 4 and 7 (47- and 20-fold higher than anestrus, respectively). In conclusion, this is the first report of oviductin protein expression in the canine oviduct. The region-specific higher expression of oviductin at Day 1 post-ovulation suggests a potential role of this glycoprotein in gamete maturation and fertilization in the bitch.
162 EFFECT OF MEIOTIC ARREST USING BUTYROLACTONE I AND ROSCOVITINE IN RESISTANCE TO EMBRYO CRYOPRESERVATION
Authors: Maziero R. R. D. , Paschoal D. M. , Guastali M. D. , Sudano M. J. , Guasti P. N. , Papa P. M. , Rascado T. S. , Papa F. O. , Basso A. C. , Landim-Alvarenga F. C.
Published: December 2013

In vitro embryo production by maturation, fertilization, and culture has become a valuable assisted reproductive technology in cattle breeding systems. However, even with the notable innovations in this system, the greatest obstacles for the complete success of this biotechnology are the low pregnancy rates after transfer and the greater sensitivity to cryopreservation. Thus, this study evaluated the effect of the addition of a meiotic arrest using butyrolactone I and roscovitine on in vitro production of bovine embryos. Nelore oocytes were matured in TCM-199 with Earle's salt + 10% FCS, FSH, and LH, in 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium in presence of butyrolactone I (BL-I) 25 μM + roscovitine (ROS) 6.25 μM. Then, the oocytes (Control n = 405; BL-I + ROS n = 386) were cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the conditions above. Semen was selected through Percoll gradient and the concentration adjusted to 2 × 106 sperm mL–1. The presumed zygotes were culture in 90-μL droplets of SOFaa + 0.6% BSA + 2.5% FCS in 5% CO2 until Day 7, when the blastocyst rate was evaluated. We performed 5 replicates. Data were analysed with ANOVA, followed by Tukey test using the general linear model (PROC GLM) of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. Statistical differences were observed in blastocyst production rate: Control: 42.64% and BL-I + ROS: 55.96% (P < 0.05). The remaining embryos (Control n = 163; BL-I + ROS n = 182) were vitrified, warmed, and recultured in SOFaa with 10% FCS for 12 h. After this period, re-expansion rate was determined. We found difference in re-expansion rates of groups (Control: 84.66% and BL-I + ROS: 93.40%; P < 0.05). Additionally, differences were found among total number cell groups: Control: 54.33 and BL-I + ROS: 97.5; P < 0.05). The use of BL-I + ROS is able to better a producing rate of in vitro embryos and improve the cryotolerance.
The authors acknowledge FAPESP 2010/18994-4 and 2011/08926-4 for financial support.
168 INHIBITION OF BOVINE OOCYTE CUMULUS CELL PROGESTERONE SYNTHESIS DURING IN VITRO MATURATION AFFECTS CHROMOSOME ALIGNMENT AND SPINDLE INTEGRITY IN FERTILIZED EGGS AND EARLY EMBRYOS
Authors: Daly E. , Fahey A. G. , Herlihy M. M. , Fair T.
Published: December 2013

We have previously demonstrated the importance of progesterone (P4) synthesis by cumulus cells during oocyte maturation in vitro (IVM) for bovine oocyte acquisition of developmental competence and subsequent embryo development (Aparicio et al. 2011 Biol. Reprod. 84). The aim of this study was to identify key processes that may be deregulated by the inhibition of P4 signalling in the cumulus–oocyte complex (COC) during IVM. To this end, good quality immature COC were placed in IVM medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of 10 μM trilostane (which blocks P4 synthesis by inhibiting 3 β-hydroxysteroid dehydrogenase; Stegram Pharmaceuticals Ltd., Surrey, UK). Matured COC were washed and placed in 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization (IVF) was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Subsets of presumptive fertilized eggs and developing embryos were recovered at 6, 72, 120, and 192 h postinsemination (hpi) and processed for confocal whole-mount immunocytochemistry. The meiotic and mitotic spindles and chromosomes were visualised by immunofluorescent labelling of α-tubulin and 4′,6-diamindino-2-phenylindole (DAPI), respectively, and classified as normal if the chromosomes were correctly aligned or appropriately segregated, or abnormal if lagging chromosomes or abnormal chromosome segregation were observed. Samples were collected from 5 replicates (n = 50 zygotes/embryos per treatment, per timepoint) and a total of 157 spindles were observed. Logistic regression analysis was conducted to determine the probability of abnormal spindle formation. The incidence of spindle abnormality was regressed on time, treatment, and treatment by time. For all time points, there was significant reduction in the odds of abnormal spindle formation in control samples versus trilostane-treated samples (P < 0.001). In conclusion, our data imply a role for P4 signalling in maintaining spindle integrity during oocyte meiotic maturation and progression through the initial mitotic divisions of early embryo development in cattle.
171 IN VIVO AND IN VITRO MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS–OOCYTE COMPLEXES DURING THE OVULATORY SEASON
Authors: Cervantes M. P. , Anzar M. , Mapletoft R. J. , Palomino J. M. , Adams G. P.
Published: December 2013

Technologies are being developed to conserve the genetic diversity of wood bison. Knowledge of the characteristics of in vivo and in vitro maturation of the cumulus–oocyte complex (COC) are needed in wood bison to design efficient in vitro embryo production protocols. The objectives were to (1) determine the optimal interval after hCG treatment for in vivo maturation of COC in superstimulated wood bison, and (2) compare the characteristics of COC after in vitro and in vivo maturation. Ovarian synchronization was induced in 25 bison during October and November by giving a luteolytic dose of prostaglandin followed 8 days later by follicular ablation (Day –1). Ovarian superstimulation was induced with FSH (Folltropin-V) given i.m. on Day 0 (300 mg) and Day 2 (100 mg). A second luteolytic dose of prostaglandin was given on Day 3. Bison were assigned randomly to 5 groups (n = 5/group). The COC were collected by transvaginal follicle aspiration on Day 4 and were either assessed immediately (0 h, control), or matured in vitro for 24 or 30 h (in vitro maturation), or collected on Day 5 (in vivo maturation), 24 or 30 h after bison were given 2000 IU of hCG i.m. on Day 4. In vitro maturation was done in TCM-199 with 5% calf serum, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin, at 38.5°C and in a 5% CO2 humidified atmosphere. Nuclear maturation was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII) with anti-lamin AC/DAPI staining. Groups were compared by analysis of variance and Fisher's exact test (Table 1). A mean (±s.e.m.) of 7.3 ± 1.7 COC were collected per bison, with no difference among groups. The COC in the control (0 h) group were at the nonexpanded GV stage. Cumulus cells were more expanded after in vivo than in vitro maturation, and the percentage of fully expanded COC was the highest in the 30-h in vivo maturation group (87%; P < 0.05). The greatest number of oocytes reached MII stage after 24 h of in vitro maturation, and 30 h of in vivo maturation. In conclusion, nuclear maturation occurred more quickly in vitro compared with in vivo, but the degree and incidence of cumulus expansion was greater after in vivo maturation. The competence of oocytes to undergo fertilization and develop into embryos remains to be investigated.

Table 1.Cumulus expansion and nuclear maturation of wood bison oocytes
176 IN VITRO MATURATION OF DOG OOCYTES IN CANINE FOLLICULAR FLUID
Authors: Reynaud K. , Canguilhem S. , Thoumire S. , Chastant-Maillard S.
Published: December 2013

In the canine species, assisted reproductive technologies, especially in vitro maturation (IVM) and IVF, are still ineffective. The main limiting factor remains the immaturity of the oocytes collected from anestrus ovaries. The ability of an oocyte to reach the MII stage in vitro is linked to the diameter of its follicle and anestrus oocytes, collected from small (<1 mm) follicles, are profoundly immature (De Lesegno et al. 2008). The objective of this study was to improve cytoplasmic quality by mimicking in vivo conditions; that is, to test the effect of pure preovulatory follicular fluid (FF) on survival and IVM rates of anestrus dog oocytes, in order to improve the nuclear and cytoplasmic maturation of these immature oocytes. Follicular fluids samples were collected from 54 Beagle bitches at 2 stages: before the LH peak (n = 23 bitches) and after the LH peak (n = 31 bitches). Only follicular fluid samples from large (>4 mm) follicles were collected and pooled by stage. Control oocytes were matured in 20% FCS/M199 medium. Groups of 5 oocytes were in vitro matured in 30 μL of follicular fluid, in half-area 96-well plates (5% CO2, 38°C). After 72 h of IVM, oocytes were denuded, fixed, and stained for DNA and tubulin before observation by confocal microscopy, and nuclear stages were classified as GV-A to GV-E, MI, and MII (Reynaud et al. 2012). A total of 460 oocytes were collected from 13 anestrus bitches and allocated to either the control medium (n = 155), the Pre-LH FF (n = 145) or the Post-LH FF (n = 160) groups. After 72 h of IVM, the morphology of the cumulus–oocyte complexes (COC) in the post-LH group was different from that of the others: cumulus cells appeared more compact and darker. Analysis of the nuclear stages showed that the degeneration rate was significantly higher (P < 0.05) in the post-LH group (58.7%) than in the pre-LH (40.9%) or in the control group (34.4%). No significant differences (P > 0.05) were observed between the 3 groups in the rate of immature GVA-B oocytes (36.4, 28.5, and 25.3% in the control, Pre-LH, and Post-LH groups, respectively), in the rate of meiotic resumption (GV-C/D/E, MI, MII stages, 44.4, 51.9, and 38.7% in the control, Pre-LH, and Post-LH groups, respectively). Metaphase II rates were not significantly different (12.1, 8.6, and 4.8% in the control, Pre-LH, and Post-LH groups, respectively). In conclusion, canine COC may survive when exposed to IVM in pure follicular fluid, but the degeneration rate was higher in the post-LH group. The presence of follicular fluid did not inhibit meiosis resumption, but did not significantly improve IVM rates. To better mimic in vivo conditions, IVM in a sequence of media, such as IVM in follicular fluid followed by IVM in oviducal fluid remains to be tested.
182 EFFECTS OF MITOCHONDRIAL ACTIVITY OF INJECTED SPERM ON EARLY DEVELOPMENT IN BOVINE INTRACYTOPLASMIC SPERM INJECTION-DERIVED EMBRYOS
Authors: Nagao Y. , Yamamoto H. , Sarentonglaga B. , Ogata K. , Yamaguchi M. , Kato Y.
Published: December 2013

Intracytoplasmic sperm injection (ICSI) has become the method of choice for bovine ovum pick-up and IVF. However, there are many difficulties with the ICSI technique to obtain viable fetuses. One of the major problems associated with this technique is our lack of knowledge concerning the status of the sperm mitochondria when injected into the oocyte and its effect on embryo development. First, we examined the mitochondrial activity of sperm that had been activated by culturing with methyl-β cyclodextrin (MBCD), in ICSI and in IVF. In vitro-matured oocytes and JC1-labelled sperm were used for the ICSI and IVF. The fluorescence intensity of injected/penetrated sperm mitochondria was measured using confocal laser scanning microscopy. Then, the relative membrane potential of the mitochondria was analysed by a ratiometric method. Second, the reactive oxygen species (ROS) production and capacitation status of the sperm exhibiting normal motility and of the sperm that had been activated by culturing with MBCD were analysed. The ROS levels produced by the sperm were estimated using the luminol assay. The chlortetracycline stain was used to evaluate capacitation status of the sperm. Third, the effect of ROS produced by these sperm types upon embryogenesis following ICSI and IVF was studied. Early developing embryos were examined with a stereomicroscope for cleavage and development to the blastocyst stage after 7 days of culture. Chromosome samples stained with Giemsa solution from the blastocysts were used to analyse the chromosomal integrity. Data were analysed by t-test for Experiments 1 and 2, and ANOVA with Fisher's PLSD test for Experiment 3. The mitochondrial activity immediately after ICSI was higher than at 3 h after insemination (immediately after sperm penetration) in IVF (P < 0.05). The sperm exhibiting activation were capacitated and produced more ROS than the sperm exhibiting normal motility (P < 0.05). The rates of cleaved embryo and blastocyst after ICSI with activated sperm were the same as that in ICSI with normal motility sperm and in IVF (cleaved rate: 66.7, 71.8, and 85.0%, respectively; blastocyst rate: 24.4, 23.3, and 32.0%, respectively). However, chromosomal integrity of blastocysts derived from ICSI with activated sperm was lower than that for ICSI with normal motility sperm or for IVF (23.1, 75.0, and 63.6%, respectively; P < 0.01). In conclusion, capacitated, activated sperm induced chromosomal aberrations during early embryo development following ICSI. Conceivably, the selection of sperm exhibiting progressive motility, which is expected to be activated and to fertilize, would not always be better for early embryo development and fetal growth following ICSI due to the ROS derived from the sperm mitochondria. Injection of sperm exhibiting normal motility, or of mitochondria reduced activated sperm, could improve the quality of ICSI-derived embryos.
189 SMALL MOLECULE INHIBITORS PD0325901 AND CHIR99021 CAUSE REDUCED EXPRESSION OF PLURIPOTENCY GENES IN PUTATIVE PORCINE INDUCED PLURIPOTENT STEM CELLS
Authors: Petkov S. , Niemann H.
Published: December 2013

Small molecule inhibitors acting on the MEK and Wnt signalling pathways (PD0325901 and CHIR99021, respectively) have been used successfully for the maintenance of murine and rat pluripotent stem cells in the in vitro culture. The effects of these compounds in other species have not been conclusively determined, and some reports suggest that they may actually cause loss of pluripotency in human embryonic stem cells and decrease of OCT4 expression levels in porcine induced pluripotent stem cells (piPSC). In our experiments, putative piPSC lines derived from transgenic porcine fetal fibroblasts (pFF) and adipose mesenchymal stem cells (pAMSC) that harbor the OCT4-EGFP reporter construct were maintained in piPSC culture medium [DMEM supplemented with 10% Knockout Serum Replacement (Life Technologies, Carlsbad, CA, USA), 10% fetal bovine serum, nonessential amino acids, L-glutamine, penicillin-streptomycin, and 1000 U mL–1 murine leukemia inhibitory factor (LIF)], with or without addition of 1 μM PD0325901 and 3 μM CHIR99021. After five passages in the two experimental culture conditions, three pFF-derived putative piPSC lines were evaluated morphologically and the expression of various pluripotency genes was analysed by real-time quantitative PCR. The results showed decrease of OCT4-EGFP expression and loss of compact colony morphology of the cells cultured in the presence of PD0325901 and CHIR99021. Moreover, the transcriptional expression levels of OCT4, SOX2, NANOG, REX1, UTF1, and TDH were reduced by 12-, 9-, 10-, 3-, 5-, and 20-fold, respectively, compared with the cells cultured without these inhibitors. When putative piPSC derived from pAMSC were cultured in medium supplemented with small molecule inhibitors, the OCT4-EGFP expression was completely lost within a few days. The cells also lost their iPSC-like colony morphology and were further propagated as single, mesenchymal-like cells. The same effects were observed when the cells were cultured with CHIR99021 alone, whereas there were no such changes when we used only PD0325901. This suggests that, similarly to human embryonic stem cells (ESC), the activation of the Wnt pathway in pAMSC-derived iPSC may lead to differentiation in these cells. The effects of the CHIR99021 alone on the pFF-derived piPSC-like cells are still to be determined. In conclusion, the results of our preliminary investigations call into question the effectiveness of PD0325901 and CHIR99021 in the maintenance of piPSC in culture.
194 EPIGENETIC REMODELING OF ADULT SOMATIC CELLS
Authors: Pennarossa G. , Maffei S. , Gandolfi F. , Brevini T. A. L.
Published: December 2013

Mammalian differentiation is obtained through epigenetic regulations that shape the genome, which is identical in all cells, to distinct phenotypes and tissue specific identities. The differentiated state of mature cells in an adult organism is therefore acquired through epigenetic restrictions that lead to a gradual loss of differentiative potency. In agreement with this, recent experiments demonstrate that terminally differentiated cells can be induced to de-differentiate in vitro and increase their plasticity in response to epigenetic modifiers that are capable of reverting cells from their lineage commitment to a more plastic state. Here we describe experiments where we prepared porcine skin fibroblasts and granulosa primary cultures and exposed them to an inhibitor of DNA methylation, the 5-aza-cytidine (5-aza-CR), to increase cell plasticity. Taking advantage of the obtained increased permissivity window, we investigated the ability of 5-aza-CR treated cells to respond to specific differentiation conditions and be re-addressed to a different cell lineage either within the same germ layer or to a different germ layer. Cells were evaluated for their morphological changes and assessed using RT-PCR and immunocytochemical studies during the treatment. Following the exposure to 5-aza-CR the phenotype of both cell types changed. Treated cells displayed an oval or round shape, and appeared smaller with larger nuclei and granular and vacuolated cytoplasm. This was accompanied by an active expression of the main pluripotency-related genes OCT4, NANOG, SOX2, and REX1, originally undetectable in untreated fibroblasts and granulosa cells. 5-aza-CR treated granulosa cells cultured with recombinant human vascular endothelial growth factor to induce myogenic specification (different lineage within the same germ layer) suppressed the expression of granulosa specific marker (Cytokeratin) as well as of the pluripotency genes, and expressed MYOD, MYF5, and MYOG (earliest myogenic markers that are involved in the coordination of skeletal muscle development or myogenesis). In order to trans-differente 5-aza-CR treated fibroblasts to cells of a different germ layer, they were exposed to activin A to promote endoderm commitment. Cells down-regulated Vimentin (fibroblast marker) as well as pluripotent gene expression and transcribed Nestin (transiently involved in multi-lineage progenitor cell differentiation), SOX17, FOXA2 (induction of definitive endoderm), and HNF4A, HNF1 (primitive gut tube specific genes). Altogether these results suggest that it is possible to obtain a direct inter-lineage conversion by removing epigenetic restriction, using demethylating agents such as 5-aza-CR, and avoiding a stable pluripotent state. This novel approach may represent a promising tool for regenerative medicine because it does not involve the use of any transgenic modifications, retroviral transfection, or both.
Supported by Network Lombardo iPS (NetLiPS) Project ID 30190629.
206 ESTIMATION OF EMBRYO DEVELOPMENTAL TOXICANTS IN HUMAN EMBRYONIC STEM CELL
Authors: Jung E. M. , Choi Y. , Kang H. S. , Shin E. K. , Jeung E. B.
Published: December 2013

An embryonic stem cell test (EST) has been developed to evaluate the embryotoxic potential of chemicals with an in vitro system. In the present study, novel methods to screen toxic chemicals during the developmental process were evaluated using undifferentiated human embryonic stem (hES: H9) cells. hES H9 cells were obtained from WiCell Research Institute, Inc., (Madison, WI, USA) and cultured according to the manufacturer's protocol. The cells were cultured on mouse embryonic fibroblast (mEF) cells in hES cell medium composed of DMEM/F12 supplemented with 20% defined fetal bovine serum (FBS), 1% minimal essential medium (MEM) nonessential amino acids, 0.1 mM b-mercaptoethanol, 2 mM L-glutamine, 50 U mL–1 penicillin/streptomycin, and 4 ng mL–1 human recombinant basic fibroblast growth factor. By using surface marker antigens (SSEA-4, TRA-1-60, and TRA-1-81), we confirmed undifferentiated conditions of the used hES cells by immunocytochemistry. We assessed the developmental toxicity of well-known embryotoxic chemicals (cytosine arabinoside, 5-fluorouracil, hydroxyurea, and indomethacin) by expression of pluripotent ES cell markers (OCT-4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2(SFRP2), teratocarcinoma-derived growth factor 1 (TDGF1), and phosphatase, and tensin homologue (PTEN)) in different concentrations for up to 7 days. Expression of pluripotent ES cell markers were determined by quantitative real-time PCR. Data are presented as the mean ± standard error of the mean (s.e.m.), and were analysed with a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. P-values <0.05 were considered to be statistically significant. Although expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers. Most of the pluripotent ES cell markers were down-regulated in dose-dependent manner following treatment with embryotoxic chemicals. After treatment with 5-fluorouracil, indomethacin, and penicillin G, we observed a remarkable convergence in the degree of up-regulation of development, cell cycle, and apoptosis-related genes by gene expression profiles using Affymetrix GeneChips (Santa Clara, CA, USA). Taken together, these results suggest that embryotoxic chemicals have cytotoxic effects, and modulate the expression of ES cell markers as well as development-, cell cycle-, and apoptosis-related genes that have pivotal roles in undifferentiated hES cells. Therefore, we suggest that hES cells may be useful for testing the toxic effects of chemicals that could affect the embryonic developmental stage.
212 EFFECT OF A LOW DOSE OF eCG ON SUPEROVULATION AND EMBRYO COLLECTION IN WOOD BISON DURING THE BREEDING SEASON
Authors: Palomino J. M. , Mapletoft R. J. , Woodbury M. R. , Cervantes M. P. , Adams G. P.
Published: December 2013

In an effort to conserve a threatened Canadian species, Bison bison athabascae, we developed an embryo collection protocol subsequent to superovulatory treatment involving two doses of FSH diluted in hyaluronan given 48 h apart. The follicular response to superstimulatory treatment was satisfactory, but many follicles did not ovulate, thus limiting the number of embryos collected. Based on recent results in cattle, where replacement of the final doses of FSH with a low dose of eCG resulted in the recovery of a greater number of ova/embryos, the objective of the present study was to evaluate the effect of adding eCG to the superovulatory protocol to increase ovulation rate, embryo collection, and embryo quality in wood bison during the breeding season (September). Ovarian synchronization was induced in wood bison (n = 24) by treatment with a luteolytic dose of prostaglandin (500 mcg, Cloprostenol) followed 8 days later by transvaginal ultrasound-guided follicular ablation. Follicular wave emergence (Day 0) was defined as the day after follicle ablation. Bison were assigned randomly to two groups: FSH (n = 12) and FSH+eCG (n = 12). FSH was diluted in hyaluronan (5 mg mL–1, MAP-5, Bioniche Animal Health, Belleville, ON, Canada) and given intramuscularly on Day 0 (300 mg) and Day 2 (100 mg) in both groups. Bison in the FSH+eCG group received 450 IU eCG (Pregnecol, Bioniche Animal Health) intramuscularly on Day 3, and bison in both groups were administered a luteolytic dose of prostaglandin on Day 3. On Day 5, bison were given 2500 IU hCG (Chorulon, Merck Animal Health, Summit, NJ, USA) intramuscularly to induce ovulation. The bison were artificially inseminated with chilled semen 12 and 24 h after hCG treatment. Nonsurgical embryo collection was performed on Day 13. The ovaries were examined by transrectal ultrasonography on Days 5, 7, and 13 to record the follicular response, ovulation rate, and number of corpora lutea (CL), respectively. Results were compared between groups by t-test or chi-square test (Table 1). The number of ovulatory-sized follicles (≥9 mm), ovulation rate (number ovulations/ovulatory-sized follicles), number of CL, number of ova/embryos, and number of transferable embryos were not different between groups (P = 0.23, P = 0.19, P = 0.25, P = 0.18, P = 0.09, respectively). In conclusion, the superovulatory response and embryo collection rate in wood bison approached that observed in cattle, but were not improved by the addition of a low dose of eCG.

Table 1.Response of wood bison (mean ± s.e.m.) to superovulatory treatment with or without eCG
224 CONSTRUCTION OF GENE TARGETING VECTORS FOR RAG-1 AND RAG-2
Authors: Byrne A. , Beaton B. , Wells K.
Published: December 2013

Gene targeting in mammalian cells has become a routine technique and is currently used to study gene function, create biomedical models, and generate potential tissue sources for xenotransplantation. Severe combined immunodeficiency (SCID) is a condition characterised by the absence of T cells and a lack of B cell function. Severe combined immunodeficiency affects ~1 out of every 100 000 infants. Autosomal recessive SCID can occur due to a mutation within the recombination activating genes (RAG-1/RAG-2) that play a role in recombination of immunoglobulins and T-cell receptors. Gene targeting has been used to create mouse models to study the effects of a RAG-1 or RAG-2 deficiency on the immune system. In 1992, Mombaerts et al. generated a homozygous mouse model of RAG-1 deficiency, whereas Shinkai et al. generated a homozygous mouse model of RAG-2. Both models resulted in the absence of mature T or B lymphocytes; which was concluded to be due to the lack of the ability to initiate the V(D)J recombination process. Because of the anatomical and physiological similarities between humans and pigs, a swine model of both RAG-1 and RAG-2 deficiency would have utility. We hypothesise that disruption of RAG-1, RAG-2, or both in swine will result in a SCID phenotype. A first step in the creation of a swine SCID model is to assemble targeting vectors. The objective of this work was to construct targeting vectors. To accomplish this goal, genomic DNA from porcine fetal fibroblasts was used to amplify a 6840-bp PCR product including the porcine RAG-1 gene. This fragment was cloned into TOPO pCR-XL (Invitrogen, Carlsbad, CA, USA). So that a mammalian G418 resistance cassette could be used for selection of targeting events, this plasmid was modified to remove the endogenous AphII gene (provides G418 resistance). The pKW4 contains LoxP (locus of X-ing over) sites that flank a G418 resistance cassette (based on mammalian codon usage), which is driven by a phosphoglycerate kinase (PGK) promoter (Lorson et al. 2011). This cassette was inserted into the RAG-1 gene to create the targeting construct pAB6. For RAG2, a 9466-bp PCR product i ncluding the RAG-2 gene was amplified and cloned into TOPO pCR-Blunt II (Invitrogen). The LoxP flanked G418 resistance cassette from pKW4 was inserted into the second exon of the RAG-2 gene sequence, creating the targeting construct pAB13. Further, diagnostic screening strategies were developed and validated to discriminate gene-targeting events from random integration. We report here 2 targeting vectors and validated screening methods for gene targeting in porcine fetal fibroblasts that have been validated for cloning. These vectors will be applied toward an effort to create a porcine SCID model. The implications of such a model include evaluation of basic immune function, evaluation of the innate immune system in vaccine efficacy, and organ transplantation.
225 OXIDATIVE STRESS AND MEMBRANE INTEGRITY OF CRYOPRESERVED SPERMATOZOA WITH VITAMIN C
Authors: Almeida R. D. , Zervoudakis L. K. H. , Duarte Junior M. F. , Zervoudakis J. T. , Nichi M. , Senra e Silva L. E. , Tsuneda P. P. , Wingert F. M. , Fraga A. L. C. R. , Lemos A. P. G. , Arguelo F. A. P. B. , Losano J. D. A.
Published: December 2013

Disturbances in oxidant/production in favour of oxidizing cause oxidative stress during the cryopreservation process. Vitamin C is an antioxidant non-enzymatic present in seminal plasma that protects sperm from oxidative stress. The aim of this study was to evaluate supplementation with ascorbate in a cryopreservation medium on oxidative stress and quality of cryopreserved sperm membranes. It was used in 24 bulls averaging 31 months and 732 kg, kept on pasture for this experiment. One ejaculate was collected from each breeder by electrostimulation. The treatments were CO-Control (no additives) and AS-ascorbate (0.45 mg mL–1). Ascorbate was added to diluted semen at the time of filling in 0.5-mL straws before the cryopreservation process. Thawing was performed in a water bath at 37°C for 30 s. After thawing the samples, aliquots for assessment of plasma and acrosome membrane integrity and evaluation of oxidative stress [TBARS concentration spontaneous (TE) and induced (TI)] were taken. The experiment was conducted in a completely randomised design. Data were analysed by ANOVA and compared by the Tukey average test with a significance level of 5%. No differences were observed (CO- 31.67 ± 2.81 v. AS- 31.08 ± 2.42, P > 0.05); the quality of the sperm membrane and oxidative stress (TECO: 5.98 ± 2.13 v. TEAS: 6.62 ± 2.33, P > 0.05 and TICO: 62.34 ± 8.22 v. TIAS: 58.52 ± 8.27, P > 0.05) in sperm cryopreserved with ascorbate were similar to the control group. Typically, animals under adequate nutritional conditions produce adequate amounts of ascorbate; perhaps because of that the treatment using exogenous ascorbate did not present a significant result. The supplementation of extender with ascorbate did not affect oxidative stress and the quality of the plasma membrane and acrosome sperm cryopreserved. Preliminary studies had shown that the addition of higher concentration (4,5 mg mL–1) of ascorbate has a beneficial effect on oxidative stress and membrane integrity (P < 0.05). Thus, further research should be done to better understand the effects of ascorbate in bovine semen.
226 HUMAN LACTOFERRIN EXPRESSED IN COW MILK CAN EXTEND THE SHELF LIFE OF MILK
Authors: Lotti S. N. , Cooper A. , D. Murray J.
Published: December 2013

Lactoferrin (LF) is an antimicrobial protein that is produced at high levels in human secretions, including milk, but is present at very low levels in the milk of ruminants. Lactoferrin is an iron-binding protein, and because iron is required by some bacteria to grow, lactoferrin can reduce the growth of certain bacteria. Lactoferrin can inhibit the growth of gram-negative bacteria, gram-positive bacteria, yeast, and some antibiotic-resistant pathogens. Lactoferrin also has bacteriocidal activity, killing the organism by inducing membrane perturbations often in conjunction with other antimicrobial agents such as lysozyme. Due to these properties, lactoferrin has the potential to increase the health of lactating animals as well as extend the shelf life of the milk. A herd of cows were genetically engineered to produce milk containing recombinant human lactoferrin (rhLF-milk) at approximately the same concentration found in human milk. In this study, growth of bacteria in raw milk samples from 2 control cows was compared with that from 2 cows that produce rhLF-milk. The experiment examined the shelf life of milk at room temperature over a 72-h period and the shelf life of milk left at 4°C over 3 weeks by plating aliquots on agar plates and counting the number of bacterial colonies that grew on plates after 24 and 48 h. The milk left at room temperature was plated at 0, 24, 48, and 72 h. The milk left in at 4°C was measured at 0, 4, 7, 11, 14, 18, and 21 days. Statistical evaluations were conducted using SAS statistical software, version 9.3 (SAS Institute Inc., Cary, NC, USA). Bacterial data were compared using the repeated-measures function in SAS and the Greenhouse-Geisser epsilon test to determine differences. For all tests, P-values <0.05 were considered statistically significant. The rhLF-milk showed less bacteria growth than control milk over the 4 time periods when left at room temperature (P = 0.0029), but did not inhibit the growth of bacteria when held for longer periods at 4°C (P = 0.6031). This shows that rhLF-milk may have an extended shelf life at room temperature and thus may be a means to increase food availability in developing countries. The next step in this research will be examining the populations of bacteria in the milk to determine how rhLF alters the populations of bacteria growing in milk over time, the different types of bacteria populations between the rhLF and control milk and what their effects are.
34 EFFECT OF DONOR CELLS SERUM STARVATION ON THE DEVELOPMENT OF AGGREGATED ZONA FREE CLONED EQUINE EMBRYOS
Authors: Gambini A. , De Stefano A. , Bevaqua R. J. , Salamone D. F.
Published: December 2013

Donor cell synchronization for nuclear transfer (NT) is one of the crucial steps in the cloning procedure, and it has been shown that different methods affect embryo development. The goal of the present study was to determine the effect of serum starvation in combination with growth to confluence of the somatic donor cells, on in vitro embryo development and quality of aggregated cloned equine embryos. Oocyte collection, maturation, cloning, and activation procedures were performed as described previously by (Gambini et al. 2012 Biol. Reprod. 87, 15). Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of 2 horses. They were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics. After cell multiplication, they were cryopreserved and stored in liquid nitrogen. After thawing, 2 groups of cell synchronization were established. Group I: growth to confluence for 3 to 5 days before NT followed by serum starvation in DMEM supplemented with 0.5% FBS for 24 h prior to NT; group II: growth to confluence for 3 to 5 days before NT. After activation, reconstructed embryos (RE) were cultured in SOF in the well of well system, placing 3 RE per well. Cleavage and blastocyst formation at Day 7 to 8 were assessed. At Day 7 to 8, embryos were measured and some of each group were fixed with paraformaldehyde to measure DNA fragmentation through the DeadEnd fluorometric TUNEL system (Promega, Madison, WI, USA). In vitro embryo development, on a per embryo and cleaved RE basis, and blastocyst size was compared using the chi-squared test; the proportion test was used for statistical analysis of DNA fragmentation levels (fragmented DNA cells/total cells). There were no statistic differences on cleavage per RE (I: 136/177, 80.2%; II: 142/171, 79.6%), blastocyst rates at Day 7 (I: 29/58, 49.1%, II: 22/56, 39.2%), or on blastocyst size at Day 8. Statistical differences were observed in blastocyst rate at Day 8 (I: 42/58, 72.4%, II: 30/56, 53.5%) and in blastocyst DNA fragmentation levels (I: 202/2464, 8%; II: 173/1440 12%). In conclusion, the restriction of FBS to the cell culture medium 24 h before cloning seams to improve embryo development at Day 8 and also reduces the level of apoptosis in cloned blastocyst, suggesting a better embryo quality. For these reasons, we consider that the addition of the restriction of FBS to the growth to confluence is beneficial for cloned equine embryo development.