bluuebunny/crossref_metadata_2025 huggingface dataset:
GENERATION AND CHARACTERISATION OF INDUCED PLURIPOTENT STEM CELLS (iPSCs) FROM ADULT CANINE FIBROBLASTS
Authors: Piedrahita Jorge A. , Koh Sehwon , Olby Natasha
Published: December 2011

Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can give rise to derivatives of all three germ layers and thus have great potential in regenerative medicine. In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing four transcription factors, Oct3/4, Klf4, Sox2 and c-Myc (OKSM). In his presentation we will describe the derivation of iPS cells from adult canine fibroblast by retroviral OSKM transduction. The isolated canine iPS cells were expanded in three different iPS culture media (FGF2, LIF and FGF2 plus LIF) and only the cells cultured in FGF2 plus LIF showed strong AP activity expressed pluripotency markers, POU5F1 (OCT4), SOX2, NANOG and LIN28 as well as ES cells-specific genes (PODXL, DPPA5, FGF5, REX1 and LAMP1). In vitro differentiation by formation of embryoid bodies (EBs) and directed differentiation showed cell derivatives of all three germ layers as confirmed by expression for AFP, CXCR4 and SOX17 (endoderm), desmin (DES), vimentin (VIM), MSX1 and BMP2 (mesoderm) and glial fibrillary acidic protein (GFAP), TUJ1, NCAM and bIII-tubulin (TUBB, ectoderm). In vivo, the putative canine iPS cells formed simple teratomas that expressed markers for all three germ layers. In summary, we were able to derive induced pluripotent cells from adult somatic cells by using four transcription factors. The isolated canine iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF and LIF is still unknown. This work was funded by a grant from the America Kennel Club to JAP.
45 THE EFFECT OF MEIOTIC STAGE OF BOVINE OOCYTES ON THE SURVIVAL OF VITRIFIED CUMULUS–OOCYTE COMPLEXES
Authors: Prentice J. R. , Singh J. , Anzar M.
Published: December 2011

Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0 h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6 h, metaphase I (MI; 90%) at 12 h and MII (84%) at 22 h (n > 62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22 h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose + 20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1 min in 0.5 M sucrose + 20% CS in TCM-199), COC completed 22 h of IVM and the nuclear stage was evaluated with lamin A/C-4′6-diamidino-2-phenylindole staining. Upon completion of 22 h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P < 0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22 h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0 h, 17% at 12 h and 14% at 22 h; P = 0.825). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability.
This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.
48 EFFECT OF TREHALOSE ADDITION ON IN VITRO VIABILITY OF COOLED RAM SPERMATOZOA
Authors: Dinatolo E. , Gimenez Zapiola C. , Marquez S. , Perez A. , Martinez E. , Sansinena M.
Published: December 2011

Refrigeration of ovine spermatozoa is a valuable tool for genetic progress. Sugars, in particular disaccharides, have been shown to have positive effects in semen extender, providing energy substrate during cell incubation, decreasing osmotic pressure, causing cell dehydration and ultimately reducing cold shock and cryodamage. Several authors have reported changes in plasma membrane protein organisation and membrane fluidity during cooling. Trehalose, a non-reducing disaccharide has been associated with stabilisation of cellular proteins and interaction with membrane phospholipids due to its ability to replace water at the membrane-solution interface. Many studies have confirmed the beneficial effect of the addition of trehalose during cryopreservation at –196°C; however, its effects during refrigeration (5–7°C) have not been reported. Ejaculates form four fertile Corriedale rams were collected by electroejaculation during the ovine reproductive season in the Southern Hemisphere (May–August); the study was conducted in 12 replicates. After initial quality assessment, ejaculates were pooled into heterospermic samples, diluted in Tris-buffered extender and separated into treatments consisting of (a) no refrigeration (kept at room temperature 22–25°C), (b) refrigeration (cooling rate of 2°C/3 min to reach a final temperature of 5–7°C), or (c) refrigeration with the addition of 150 mOsm trehalose. Samples were evaluated for total motility, progressive motility, hyposmotic swelling test (HOST) and eosin Y (1 step, WHO protocol) live/dead stain at 0, 6, 12, 24 and 48 h post-refrigeration. Results were analysed using 1-way ANOVA with Tukey post-hoc multiple comparison test. Compared with refrigeration without the addition of disaccharides, the refrigeration in presence of trehalose significantly increased (P < 0.05) total motility at 48 h (32 ± 0.9 vs 50 ± 1.1) and progressive motility at 24 (40 ± 0.9 vs 65 ± 0.6) and 48 h (0 vs 38 ± 1.0) post-refrigeration. Trehalose also decreased (P < 0.05) the percentage of damaged spermatozoa as evidenced by HOST and live/dead stain after 12 (42 ± 1.1 vs 29 ± 0.7), 24 (52 ± 0.9 vs 37 ± 0.6) and 48 h (74 ± 0.8 vs 60 ± 1.0) of refrigeration. Although no statistical difference was detected, there was a trend of trehalose to improve total and progressive motility parameters at the initiation of treatments (0 h). In addition, spermatozoa refrigerated in the presence of trehalose was the only treatment group to retain progressive motility after 48 h of storage. Our results indicate that trehalose could be used to extend the in vitro viability of refrigerated ovine spermatozoa for up to 48 h. Further studies are needed to assess the in vivo fertility (pregnancy rate) of refrigerated spermatozoa in the presence of this non-permeating cryoprotectant sugar.
50 EFFECTS OF VITAMINS ON THE QUALITY AND FERTILITY OF BOAR SEMEN AFTER LIQUID PRESERVATION AT 5°C
Authors: Namula Z. , Luu V. V. , Kaedei Y. , Kodama R. , Otoi T.
Published: December 2011

Liquid preservation can be used as an alternative to freeze-thawing for preserving semen for AI. The efficiency of some boar semen extenders has been studied over storage periods of 5 to 7 days. The objective of this study was to evaluate the viability and penetrability of boar spermatozoa preserved at 5°C in a modified Modena-based extender supplemented with either 100 μM vitamin C (Vc), 100 μM vitamin E (Ve), or 100 μM Vc + 100 μM Ve (Vc + e). The final sperm concentration was adjusted to cells mL–1 and the semen was then stored at 5°C for 4 weeks. In Experiment 1, the semen samples were assessed every week during the 4-week storage in each extender for the following factors: motility, by using computer-assisted semen analysis (CASA); viability, by using the Live/Dead fluorescence viability assay; plasma membrane integrity, by using the hypoosmotic swelling test (HOST); and acrosome integrity, by using fluorescein isothiocyanate (FITC)-labelled peanut agglutinin staining. In Experiment 2, we examined the penetrability of spermatozoa that had been stored in each extender for 4 weeks and the development of fertilized oocytes. Data were analysed using ANOVA. In Experiment 1, when the semen was stored for 2 weeks, the mean percentage values of total sperm motility and viability for semen stored with Ve were significantly higher than those for semen stored without Vc and Ve (control group) (84.3 vs 67.9% and 59.8 vs 51.2%, respectively; P < 0.05). Moreover, the percentage sperm motility for semen stored for 4 weeks tended to be higher in the Ve group than in the control group (44.2 vs 32.7%; P < 0.1). Storage with Vc or Vc + e did not improve sperm motility and viability of semen. The plasma membrane integrity and acrosome integrity of semen did not significantly differ among the groups during the 4-week storage. In Experiment 2, the rates of sperm penetration and of development to blastocysts of fertilized oocytes did not differ between the Ve and control groups (33.0 vs 28.5% and 14.9 vs 10.1%, respectively; P > 0.05). However, storage with Vc reduced the rate of oocyte development compared with the Ve and control groups (1.1%; P < 0.05). In conclusion, adding Ve to the semen extender may improve the motility and fertility of boar semen stored at 5°C. However, adding Vc has a harmful effect on the quality and fertility of stored boar semen.
53 VITRIFICATION OF HANDMADE CLONED BUFFALO EMBRYOS USING ETHYLENE GLYCOL AND DIMETHYL SULPHOXIDE AND SUBSEQUENT EFFECT ON CRYOSURVIVAL AND APOPTOSIS
Authors: Krishna Kanth G. , Selokar N. L. , Saini M. , Singh K. P. , Muzaffer M. , Elamaran G. , Saha A. P. , Chauhan M. S. , Manik R. S. , Palta P. , Singla S. K.
Published: December 2011

The objective of the present work was to optimize concentration of cryoprotectants ethylene glycol (EG) and dimethyl sulphoxide (DMSO) to facilitate the vitrification of buffalo embryos produced by somatic cell nuclear transfer (SCNT). Cloned embryos were produced according to standardized protocols of our laboratory using handmade cloning (Shah et al. 2008). Three different concentrations of EG and DMSO (7.5, 10 and 15%, having 0.5 M sucrose in TCM-199 containing 20% serum) were selected in combination for vitrification of cloned blastocysts in French ministraws (0.25 mL). The numbers of cloned blastocysts vitrified for each concentration were 51, 51 and 52, respectively. The post-thaw viability was accessed by re-expansion rate of blastocysts after culturing in RVCL media (K-RVCL-50, Cook® Australia, Queensland, Australia) for 18 to 24 h. On the basis of re-expansion rate, there was no significant effect of any selected concentrations (7.5, 10 and 15%) on post-thaw viability (25.33 ± 2.43%, 29.00 ± 2.52% and 30.83 ± 3.01%, respectively; P > 0.05). The effect of vitrification on apoptosis level was checked after 18 to 24 h post-thaw by TUNEL assay and the apoptosis index was calculated by dividing the total number of nuclei with DNA-fragmented positive nuclei of the respective blastocyst. We found that, 7.5%-group embryos resulted with a significantly higher apoptotic index (8.28 ± 0.57) than that of the 10 and 15% groups (5.09 ± 0.46 and 4.28 ± 0.24, respectively; P < 0.05). These results clearly indicate that a lower concentration of cryoprotectants (7.5%) increased the chance of apoptosis in blastocysts that were frozen-thawed. The quantitative expression of apoptosis-related genes (Bax, Bid, Mcl-1 and Bcl-xl) in all 3 treatment groups and fresh control embryos were determined by RT-qPCR. Three replications were performed and the mRNA level of each sample was normalized to that of glyceride-3-phosphate dehydrogenase mRNA level. Results of RT-qPCR were analysed using the 2–ΔΔCT method to compare the relative transcriptional levels of the target genes in each group. The RT-qPCR data revealed that the 7.5% vitrified group embryos possessed high expression of pro-apoptotic genes (Bax and Bid) and lower expression of anti-apoptotic genes (Mcl-1 and Bcl-xl) in comparison to the 10 and 15% groups. However, there was no significant change in gene expression between the 10 or 15% groups in comparison with fresh non-vitrified embryos. Our results conclude that the best choice is to use 10 or 15% EG and DMSO cryoprotectants for in-straw zona-free cloned buffalo embryo vitrification. However, further experiments are needed to enhance survival after vitrification.
This work was supported by research grants from the National Agriculture Innovative Project (1(5)/2007-NAIP-2) to S. K. Singla.
64 USING PORCINE GRANULOSA CELLS AS FEEDERS FOR PORCINE AND BOVINE TROPHECTODERM CELL CULTURE
Authors: Saadeldin I. M. , Elsayed A. , Kang J. T. , Park S. J. , Kim S. J. , Moon J. H. , Jang G. , Lee B. C.
Published: December 2011

The trophectoderm cells, arising from the outer side of the blastomere in the blastocyst stage, are the first differentiated embryonic cells with specific potential as stem cells. The physiology of trophectoderm cells has been studied; however, their functions still remain unclear, because the lack of definitive information of cell lineages. Here, we aimed to establish in culture different feeder-dependent trophectoderm cell lines from 9-day, preimplantation, in vitro-produced porcine and bovine embryos. We used 2 different feeders: porcine granulosa cells (PGC) and mouse embryonic fibroblasts (MEF). Both cells were mitotically inactivated by mitomycin-C and then cultured with a density of 5 × 104 mL–1 on 0.1% (wt/vol) gelatin coated 4-well dishes in DMEM-199 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), nonessential amino acids (NEAA), β-mercaptoethanol and nucleosides (Talbot et al. 2000 Biol. Reprod. 62, 235–247). Trophectoderm cells were observed by light microscopy and characterised by reverse transcription-PCR using specific primers for both species. Different feeders and trophectoderm cells growth rates were compared after trypsinization using a hemocytometer. Data were analysed using 1-way ANOVA. In results, trophectoderm cells display epithelial characteristics, cuboidal morphology and express mRNA of homebox protein CDX2, cytokeratin 8 (KRT8) and interferon (IFN) gamma or tau for porcine or bovine cells, respectively. Moreover, oestrogen receptor (ESR1) and progesterone receptor (PGR) were expressed in trophectoderm cells of both species. Porcine granulosa cells were highly proliferative with doubling time of 24 h when compared to MEF (P ≤ 0.5), easy to recover and provided a reasonable source of steroids, 17β-oestradiol (E2; 31.21 ± 3.1 ng mL–1) and progesterone (P4; 6.36 ± 0.4 ng mL–1). Moreover, trophectoderm cell colonies of both species that cultured on PGC grew faster, with a doubling time of 48 h when compared to those cultured on MEF (P ≤ 0.5). We speculate that the continuous supplement of steroids and other cytokines during the co-culture of trophoblasts with granulosa cells might help the trophectoderm cells growth more than that of MEF. Further investigations are required in this regard. In conclusion, porcine granulosa cells can be good alternative feeders to culture porcine and bovine trophectoderm.
This research was supported by MKE (Grant # 10033839-2011-13) and IPET.
70 EXPRESSION OF NUCLEAR AND MEMBRANE PROGESTERONE RECEPTORS IN THE CANINE OVIDUCT DURING THE PERIOVULATORY PERIOD
Authors: Tahir M. Z. , Reynaud K. , Thoumire S. , Chastant-Maillard S. , Saint-Dizier M.
Published: December 2011

In the bitch, the oviduct is the site of oocyte maturation (Day 1 to 3 after ovulation), sperm transport/capacitation, fertilization (Day 3 to 4) and embryo development to the morula/blastocyst stage (Day 4 to 8). Unlike other mammals, these events occur in the presence of high (>6 ng mL–1) and increasing plasma levels of progesterone (P4), but little is known about the regulation of oviductal functions by P4 in the bitch. The objective of this work was to study the mRNA expression of nuclear (PR) and membrane (PGRMC1, PGRMC2, mPRβ and mPRγ) P4 receptors in the canine oviduct during the periovulatory period. Thirty-six Beagle bitches were ovariectomized at 6 stages: anestrus, before the LH peak (pre-LH), after the LH peak (pre-ov) and after ovulation (Day 1, 4 and 7). Three oviductal regions were collected [i.e. ampulla, isthmus and utero-tubal junction (UTJ)]. Total RNA was extracted and then reverse transcribed. The expression of target genes was assessed in duplicate by quantitative PCR (LightCycler® 480; Roche Diagnostics, Meylan, France) using the relative standard curve method and normalized by the geometric mean of 2 reference genes (RPS19 and GAPDH). Relative amounts of mRNA were compared between groups by ANOVA followed, when necessary, by Duncan's test. The expression of nuclear and membrane P4 receptor mRNA varied according to the stage. Expression of PR mRNA was significantly higher at pre-LH, pre-ov and Day 1 stages [means of 1.8, 1.6 and 1.5 arbitrary units (AU), respectively] than at anoestrus, Day 4 and Day 7 (1, 0.4 and 0.5 AU, respectively) in the ampulla. Same patterns of expression were observed for PR in the isthmus and UTJ. Expression of PGRMC1 and PGRMC2 mRNA were at the lowest level during anoestrus (1 AU) and increased significantly from pre-LH to Day 7 in the ampulla (from 2.2 to 8.3 AU and from 1.3 to 5.4 AU for PGRMC1 and PGRMC2, respectively) and in the isthmus (from 0.4 to 2.6 AU and from 0.5 to 1.8 AU for PGRMC1 and PGRMC2, respectively). In the UTJ, mRNA levels for PGRMC1 and PGRMC2 were the highest at Day 4 (3.9 AU) and pre-LH (2.1 AU), respectively, compared to other stages. Expression of mPRβ mRNA did not vary according to the stage in the ampulla and the isthmus, whereas it was significantly lower at Days 4 and 7 (0.6–0.7 AU) compared to other stages (1–1.2 AU) in the UTJ. Expression of mPRγ was significantly higher at Day 7 (5.0 AU) compared to other stages (0.2–1 AU) in the ampulla and was significantly higher at both anoestrus (1 AU) and Day 7 (0.9 AU) compared to other stages (0.02–0.09 AU) in the isthmus, whereas it did not vary significantly in the UTJ. In conclusion, our data suggests that P4 may be an important regulating factor of oviductal functions and could mediate its actions through genomic as well as non-genomic pathways.
86 EFFECT OF LACTATION ON EMBRYO DEVELOPMENT DURING THE POSTPARTUM PERIOD IN DAIRY COWS
Authors: Maillo V. , Besenfelder U. , Havlicek V. , Garrett M. , Kelly A. K. , Rizos D. , Lonergan P.
Published: December 2011

The aim of this study was to examine the effect of lactation and associated metabolic profiles on the ability of the reproductive tract of postpartum dairy cows to support early embryo development. Twenty-one age-matched primiparous Holstein cows were used. Immediately after calving, half of the cows were dried off while the remainder were milked twice daily. To characterise the metabolic profile of the cows, jugular blood samples were taken twice weekly starting 15 days before calving until Day 100 postpartum. At the same time, bodyweight (BW) and body condition score (BCS) were recorded. In Experiment 1, around Day 60 postpartum, the oestrous cycles of all cows were synchronized and sixty-five 2- to 4-cell in vitro-produced embryos were endoscopically transferred on Day 2 (Day 0 = oestrus) to the oviduct ipsilateral to the corpus luteum. On Day 7, the oviduct and uterus were flushed endoscopically and the number of embryos developing to the blastocyst stage was recorded. In Experiment 2, around Day 95 postpartum, cows were re-synchronized and 15 to 20 in vitro-produced blastocysts were transferred to the uterine horn ipsilateral to the corpus luteum. On Day 14, conceptuses were recovered by flushing the reproductive tract at slaughter and were measured. Jugular blood samples were taken daily from Day 0 to 7 (Exp. 1) or 14 (Exp. 2) to measure serum concentrations of progesterone. Data were analysed by ANOVA. Concentrations of NEFA and β-HB were higher (P ≤ 0.05) and glucose, insulin and IGF-1 were lower (P ≤ 0.05) in lactating compared with dry cows. BW and BCS were significantly higher in the non-lactating cows throughout the postpartum period. Recovery rates in both experiments were similar between groups (Exp. 1: 63.9 ± 7.2 vs 65.6 ± 8.6 and Exp 2: 33.3 ± 9.6 vs 39.8 ± 9.6 for dry and milking cows, respectively). In Exp. 1, of the structures recovered, significantly more developed to the blastocyst stage in the dry than in lactating cows (49.3 ± 3.8 vs 32.6.3 ± 4.4, respectively; P ≤ 0.05). Progesterone concentrations did not differ between groups. In Exp. 2, no differences were observed in terms of conceptus dimensions on Day 14 (n = 152). Progesterone concentrations were higher in lactating cows from Day 9 to 14 (P ≤ 0.05). In conclusion, this study provides evidence that at 60 days postpartum, the reproductive tract of lactating cows is compromised in its ability to support early embryo development compared with age-matched parous non-lactating cows; however, by 95 days postpartum there was no apparent difference in conceptus development, consistent with less metabolic stress as indicated by the metabolic profile.
Funded by Science Foundation Ireland (SFI/07/SRC/B1156) and the Spanish Ministry of Science and Innovation (AGL2009-11810). VM was supported by an STSM award from the COST Action FAO7O2.
95 SHEEP ZYGOTES CULTURED WITH HYALURONAN, BOVINE SERUM ALBUMIN, OR SERUM AND THEIR EFFECTS ON PRENATAL AND POSTNATAL EMBRYO PRODUCTION AND DEVELOPMENT
Authors: Ali A. B. T.
Published: December 2011

Mammalian pre-implantation embryos are sensitive to their environment and numerous publications suggest that post-fertilization culture is the most critical period affecting embryo quality and subsequent embryo development. This study was conducted to examine the viability, twining, sex ratio and birthweight (BW) after transfer of fresh and vitrified in vitro-produced sheep embryos derived from oocytes cultured in a semi- or defined medium supplemented with fatty-acid–free bovine serum albumin (BSA) with or without hyaluronan (HA) versus fetal bovine serum (FBS) as a control. A total of 649 oocytes were matured in TCM-199 containing 2 mM glutamine, 4 mg mL–1 BSA, 100 μM cysteamine, 0.3 mM sodium pyruvate, 1 μg mL–1 oestradiol-17β and fertilized with fresh semen prepared with SOF, enriched with 20% oestrus sheep serum. Presumptive zygotes were cultured for 48 h in SOF supplemented with 1% BME-essential and 1% MEM-nonessential amino acids, 1 mM glutamine and 8 mg mL–1 BSA. On the 3rd day of culture, presumptive zygotes were divided randomly into 3 groups: (1) BSA+HA, medium supplemented with 8 mg mL–1 BSA and 6 mg mL–1 HA, (2) BSA, medium supplemented with 8 mg mL–1 BSA only, or (3) control, medium supplemented with 5% charcoal-stripped fetal bovine serum (FBS). On the 5th day of culture, media were replaced accordingly in all groups. Embryos that reached the expanded blastocyst stage by 6 or 7 days/group were recorded and transferred as fresh or vitrified. The cleavage rate, blastocyst formation, pregnancies (PR) at 40, 60 and 80 days, lambing, twinning, sex ratio and BW were all recorded. Among all groups, no significant differences were observed in cleavage and blastocyst rates. The proportion of zygotes that developed to blastocysts at 6 days was significantly higher after culture with BSA+HA vs BSA and control (67/92, 72.8%), (46/118, 39.0%) and (29/72, 40.3%), respectively. Compared with the control group, vitrified BSA blastocysts were associated with increased (P < 0.05) PRs after 60 and 80 days and lambing rates. In comparison, there were no significant differences in pregnancy and lambing rate after transfer of fresh blastocysts. There was a positive correlation between age of embryo at transfer and higher BW. The mean BWs were higher after transfer of 7-day vs 6-day embryos (3.95 and 3.37 kg), respectively. However, there were no significant differences in twining and sex ratio after transfer fresh/or vitrified treated embryos versus control group. In conclusion, the results indicate that sheep zygotes cultured with BSA+HA had an increased development to blastocysts at 6 days, but the rate of blastocyst formation was not increased, PR and lambing rate were comparable with the BSA and control groups. In addition, transfer of vitrified embryos produced in the presence of BSA significantly increased the PR at 60, 80 days and lambing rate versus control group.
An enumerative stepwise ansatz enables atomic-accuracy RNA loop modeling
Authors: Sripakdeevong Parin , Kladwang Wipapat , Das Rhiju
Published: December 2011

Atomic-accuracy structure prediction of macromolecules should be achievable by optimizing a physically realistic energy function but is presently precluded by incomplete sampling of a biopolymer’s many degrees of freedom. We present herein a working hypothesis, called the “stepwise ansatz,” for recursively constructing well-packed atomic-detail models in small steps, enumerating several million conformations for each monomer, and covering all build-up paths. By making use of high-performance computing and the Rosetta framework, we provide first tests of this hypothesis on a benchmark of 15 RNA loop-modeling problems drawn from riboswitches, ribozymes, and the ribosome, including 10 cases that are not solvable by current knowledge-based modeling approaches. For each loop problem, this deterministic stepwise assembly method either reaches atomic accuracy or exposes flaws in Rosetta’s all-atom energy function, indicating the resolution of the conformational sampling bottleneck. As a further rigorous test, we have carried out a blind all-atom prediction for a noncanonical RNA motif, the C7.2 tetraloop/receptor, and validated this model through nucleotide-resolution chemical mapping experiments. Stepwise assembly is an enumerative, ab initio build-up method that systematically outperforms existing Monte Carlo and knowledge-based methods for 3D structure prediction.
Dopamine stabilizes tumor blood vessels by up-regulating angiopoietin 1 expression in pericytes and Krüppel-like factor-2 expression in tumor endothelial cells
Authors: Chakroborty Debanjan , Sarkar Chandrani , Yu Hongmei , Wang Jiang , Liu Zhongfa , Dasgupta Partha Sarathi , Basu Sujit
Published: December 2011

Impaired blood flow in the tumor vascular bed caused by structurally and functionally abnormal blood vessels not only hinders the delivery of chemotherapeutic agents but also aggravates tumor hypoxia, making the tumor cells further resistant to antineoplastic drugs. Therefore, normalization of tumor blood vessels may be an important approach to increase therapeutic efficacy in the treatment of cancer patients. As blood vessels are supplied by sympathetic nerves containing dopamine (DA), and DA regulates functions of normal blood vessels through its receptors present in these vessels, we investigated the effect of DA on tumor vasculature. Here we report loss of sympathetic innervation and endogenous DA in abnormal and immature tumor blood vessels in malignant colon and prostate tumor tissues. In contrast, exogenous administration of DA normalizes the morphology and improves the functions of these vessels by acting on pericytes and endothelial cells, the two major cellular components of blood vessels. DA acts through its D
2
receptors present in these cells to up-regulate directly the expression of angiopoietin 1 (Ang1) in pericytes and the expression of the zinc finger transcriptional factor, Krüppel-like factor-2 (KLF2) in tumor endothelial cells. Importantly, this vessel stabilization by DA also significantly increases the concentration of anticancer drug in tumor tissues. These results show a relationship between vascular stabilization and a neurotransmitter and indicate that DA or its D
2
receptor-specific agonists can be an option for the treatment of cancer and disorders in which normalization of blood vessels may have therapeutic benefits.
Multimodal image coregistration and inducible selective cell ablation to evaluate imaging ligands
Authors: Virostko John , Henske Joseph , Vinet Laurent , Lamprianou Smaragda , Dai Chunhua , Radhika Aramandla , Baldwin Ronald M. , Ansari Mohammad S. , Hefti Franz , Skovronsky Daniel , Kung Hank F. , Herrera Pedro L. , Peterson Todd E. , Meda Paolo , Powers Alvin C.
Published: December 2011

We combined multimodal imaging (bioluminescence, X-ray computed tomography, and PET), tomographic reconstruction of bioluminescent sources, and two unique, complementary models to evaluate three previously synthesized PET radiotracers thought to target pancreatic beta cells. The three radiotracers {[
18
F]fluoropropyl-(+)-dihydrotetrabenazine ([
18
F]FP-DTBZ), [
18
F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline (
18
F-AV-266), and (2
S
,3
R
,11b
R
)-9-(3-fluoropropoxy)-2-(hydroxymethyl)-3-isobutyl-10-methoxy-2,3,4,6,7,11b-hexahydro-1
H
-pyrido[2,1-
a
]isoquinolin-2-ol (
18
F-AV-300)} bind vesicular monoamine transporter 2. Tomographic reconstruction of the bioluminescent signal in mice expressing luciferase only in pancreatic beta cells was used to delineate the pancreas and was coregistered with PET and X-ray computed tomography images. This strategy enabled unambiguous identification of the pancreas on PET images, permitting accurate quantification of the pancreatic PET signal. We show here that, after conditional, specific, and rapid mouse beta-cell ablation, beta-cell loss was detected by bioluminescence imaging but not by PET imaging, given that the pancreatic signal provided by three PET radiotracers was not altered. To determine whether these ligands bound human beta cells in vivo, we imaged mice transplanted with luciferase-expressing human islets. The human islets were imaged by bioluminescence but not with the PET ligands, indicating that these vesicular monoamine transporter 2-directed ligands did not specifically bind beta cells. These data demonstrate the utility of coregistered multimodal imaging as a platform for evaluation and validation of candidate ligands for imaging islets.
Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8
Authors: Jo Youngah , Lee Peter C. W. , Sguigna Peter V. , DeBose-Boyd Russell A.
Published: December 2011

Accumulation of sterols in membranes of the endoplasmic reticulum (ER) leads to the accelerated ubiquitination and proteasomal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids. This degradation results from sterol-induced binding of reductase to the Insig-1 or Insig-2 proteins of ER membranes. We previously reported that in immortalized human fibroblasts (SV-589 cells) Insig-1, but not Insig-2, recruits gp78, a membrane-bound RING-finger ubiquitin ligase. We now report that both Insig-1 and Insig-2 bind another membrane-bound RING-finger ubiquitin ligase called Trc8. Knockdown of either gp78 or Trc8 in SV-589 cells through RNA interference (RNAi) inhibited sterol-induced ubiquitination of reductase and inhibited sterol-induced degradation by 50–60%. The combined knockdown of gp78 and Trc8 produced a more complete inhibition of degradation (> 90%). Knockdown of gp78 led to a three to fourfold increase in levels of Trc8 and Insig-1 proteins, which opposed the inhibitory action of gp78. In contrast, knockdown of Trc8 had no effect on gp78 or Insig-1. The current results suggest that sterol-induced ubiquitination and proteasomal degradation of reductase is dictated by the complex interplay of at least four proteins: Insig-1, Insig-2, gp78, and Trc8. Variations in the concentrations of any one of these proteins may account for differences in cell- and/or tissue-specific regulation of reductase degradation.
Heterozygote advantage as a natural consequence of adaptation in diploids
Authors: Sellis Diamantis , Callahan Benjamin J. , Petrov Dmitri A. , Messer Philipp W.
Published: December 2011

Molecular adaptation is typically assumed to proceed by sequential fixation of beneficial mutations. In diploids, this picture presupposes that for most adaptive mutations, the homozygotes have a higher fitness than the heterozygotes. Here, we show that contrary to this expectation, a substantial proportion of adaptive mutations should display heterozygote advantage. This feature of adaptation in diploids emerges naturally from the primary importance of the fitness of heterozygotes for the invasion of new adaptive mutations. We formalize this result in the framework of Fisher's influential geometric model of adaptation. We find that in diploids, adaptation should often proceed through a succession of short-lived balanced states that maintain substantially higher levels of phenotypic and fitness variation in the population compared with classic adaptive walks. In fast-changing environments, this variation produces a diversity advantage that allows diploids to remain better adapted compared with haploids despite the disadvantage associated with the presence of unfit homozygotes. The short-lived balanced states arising during adaptive walks should be mostly invisible to current scans for long-term balancing selection. Instead, they should leave signatures of incomplete selective sweeps, which do appear to be common in many species. Our results also raise the possibility that balancing selection, as a natural consequence of frequent adaptation, might play a more prominent role among the forces maintaining genetic variation than is commonly recognized.
PRDM1
is a tumor suppressor gene in natural killer cell malignancies
Authors: Küçük Can , Iqbal Javeed , Hu Xiaozhou , Gaulard Phillip , De Leval Laurence , Srivastava Gopesh , Au Wing Yan , McKeithan Timothy W. , Chan Wing C.
Published: December 2011

Natural killer cell lymphoma (NKCL) constitutes a rare and aggressive form of non-Hodgkin lymphoma, and there is little insight into its pathogenesis. Here we show that
PRDM1
is a tumor suppressor gene in NKCLs that is inactivated by a combination of monoallelic deletion and promoter CpG island hypermethylation. We observed monoallelic deletion of
PRDM1
loci in 8 of 18 (44%) NKCL cases. The other allele showed significant promoter methylation in 12 of 17 (71%) cases. In support of its role as a tumor suppressor gene, the reconstitution of PRDM1 in PRDM1-null NK cell lines led to G2/M cell cycle arrest, increased apoptosis, and a strong negative selection pressure with progressive elimination of PRDM1-expressing cells, which was enhanced when IL-2 concentration is limiting. We observed a progressive increase in
PRDM1
expression—in particular,
PRDM1
α—in normal NK cells in response to IL-2 and in normal NK cells activated with an engineered NK cell target, K562-Cl9-mb21, suggesting its role in NK cell homeostasis. In support of this role, knockdown of PRDM1 by shRNA in normal NK cells resulted in the positive selection of these cells. We identified
MYC
and
4-1BBL
as targets of PRDM1 in NK cells. Disruption of homeostatic control by PRDM1 may be an important pathogenetic mechanism for NKCL.
Intercontinental differences in resource use reveal the importance of mutualisms in fire ant invasions
Authors: Wilder Shawn M. , Holway David A. , Suarez Andrew V. , LeBrun Edward G. , Eubanks Micky D.
Published: December 2011

Mutualisms play key roles in the functioning of ecosystems. However, reciprocally beneficial interactions that involve introduced species also can enhance invasion success and in doing so compromise ecosystem integrity. For example, the growth and competitive ability of introduced plant species can increase when fungal or microbial associates provide limiting nutrients. Mutualisms also may aid animal invasions, but how such systems may promote invasion success has received relatively little attention. Here we examine how access to food-for-protection mutualisms involving the red imported fire ant (
Solenopsis invicta
) aids the success of this prominent invader. Intense interspecific competition in its native Argentina constrained the ability of
S. invicta
to benefit from honeydew-producing Hemiptera (and other accessible sources of carbohydrates), whereas
S. invicta
dominated these resources in its introduced range in the United States. Consistent with this strong pattern, nitrogen isotopic data revealed that fire ants from populations in the United States occupy a lower trophic position than fire ants from Argentina. Laboratory and field experiments demonstrated that honeydew elevated colony growth, a crucial determinant of competitive performance, even when insect prey were not limiting. Carbohydrates, obtained largely through mutualistic partnerships with other organisms, thus represent critical resources that may aid the success of this widespread invasive species. These results illustrate the potential for mutualistic interactions to play a fundamental role in the establishment and spread of animal invasions.
Climate-associated phenological advances in bee pollinators and bee-pollinated plants
Authors: Bartomeus Ignasi , Ascher John S. , Wagner David , Danforth Bryan N. , Colla Sheila , Kornbluth Sarah , Winfree Rachael
Published: December 2011

The phenology of many ecological processes is modulated by temperature, making them potentially sensitive to climate change. Mutualistic interactions may be especially vulnerable because of the potential for phenological mismatching if the species involved do not respond similarly to changes in temperature. Here we present an analysis of climate-associated shifts in the phenology of wild bees, the most important pollinators worldwide, and compare these shifts to published studies of bee-pollinated plants over the same time period. We report that over the past 130 y, the phenology of 10 bee species from northeastern North America has advanced by a mean of 10.4 ± 1.3 d. Most of this advance has taken place since 1970, paralleling global temperature increases. When the best available data are used to estimate analogous rates of advance for plants, these rates are not distinguishable from those of bees, suggesting that bee emergence is keeping pace with shifts in host-plant flowering, at least among the generalist species that we investigated.
Next-Generation Systemic Acquired Resistance
Authors: Luna Estrella , Bruce Toby J.A. , Roberts Michael R. , Flors Victor , Ton Jurriaan
Published: December 2011

Systemic acquired resistance (SAR) is a plant immune response to pathogen attack. Recent evidence suggests that plant immunity involves regulation by chromatin remodeling and DNA methylation. We investigated whether SAR can be inherited epigenetically following disease pressure by Pseudomonas syringae pv tomato DC3000 (PstDC3000). Compared to progeny from control-treated Arabidopsis (Arabidopsis thaliana; C1), progeny from PstDC3000-inoculated Arabidopsis (P1) were primed to activate salicylic acid (SA)-inducible defense genes and were more resistant to the (hemi)biotrophic pathogens Hyaloperonospora arabidopsidis and PstDC3000. This transgenerational SAR was sustained over one stress-free generation, indicating an epigenetic basis of the phenomenon. Furthermore, P1 progeny displayed reduced responsiveness of jasmonic acid (JA)-inducible genes and enhanced susceptibility to the necrotrophic fungus Alternaria brassicicola. This shift in SA- and JA-dependent gene responsiveness was not associated with changes in corresponding hormone levels. Instead, chromatin immunoprecipitation analyses revealed that SA-inducible promoters of PATHOGENESIS-RELATED GENE1, WRKY6, and WRKY53 in P1 plants are enriched with acetylated histone H3 at lysine 9, a chromatin mark associated with a permissive state of transcription. Conversely, the JA-inducible promoter of PLANT DEFENSIN1.2 showed increased H3 triple methylation at lysine 27, a mark related to repressed gene transcription. P1 progeny from the defense regulatory mutant non expressor of PR1 (npr1)-1 failed to develop transgenerational defense phenotypes, demonstrating a critical role for NPR1 in expression of transgenerational SAR. Furthermore, the drm1drm2cmt3 mutant that is affected in non-CpG DNA methylation mimicked the transgenerational SAR phenotype. Since PstDC3000 induces DNA hypomethylation in Arabidopsis, our results suggest that transgenerational SAR is transmitted by hypomethylated genes that direct priming of SA-dependent defenses in the following generations.
Birth weight is inversely associated with blood pressure and serum aldosterone and cortisol levels in children
Authors: Martinez‐Aguayo Alejandro , Aglony Marlene , Bancalari Rodrigo , Avalos Carolina , Bolte Lillian , Garcia Hernan , Loureiro Carolina , Carvajal Cristian , Campino Carmen , Inostroza Andrea , Fardella Carlos
Published: December 2011

Context  Low birth weight has been independently associated with adult hypertension, and renin‐angiotensin system (RAS) plays a role in this connection.

Objective  To characterize the associations between birth weight (BW) and serum aldosterone (SA), serum cortisol, plasma renin activity (PRA) and blood pressure (BP).

Design  Cross‐sectional study.

Subjects  Children from the community born at a gestational age >32 weeks.

Methods  Systolic and diastolic BP indices (SBPi and DBPi) were calculated using the observed BP/50th percentile BP for gender, age and stature. BW was transformed to a standard deviation score (SDS) for gestational age, whereas SA, serum cortisol and PRA were transformed using the natural log.

Results  We selected 288 subjects between the ages of 4·9 and 15·5 years (Females, 50%). After adjusting for body mass index (BMI) SDS and Tanner, multiple regression analysis revealed that BW (SDS) was both independently and inversely associated with the natural log of SA (β = −0·065; P = 0·039), the natural log of serum cortisol (β = −0·064; P = 0·009), SBPi (β = −0·012; P = 0·020) and DBPi (β = −0·023; P = 0·002). An association was not observed with PRA (P = 0·178) and aldosterone renin ratio (ARR) (P = 0·452). Serum cortisol levels were positively associated with SA (r = 0·125; P = 0·034), while an association with PRA (P = 0·251) and ARR (P = 0·052) was not observed.

Conclusions  The results of this study demonstrate an inverse association between birth weight and blood pressure and serum aldosterone and cortisol levels. This association is independent of BMI and Tanner, suggesting foetal programming of the hypothalamic–pituitary–adrenal axis.
CETP‐mediated cholesteryl ester enrichment of apoB subclasses in type 1 diabetes
Authors: Bagdade John D. , Knight‐Gibson Carolyn , Simpson Nancy , Gerkin Richard , Alaupovic Petar , Reardon Christopher
Published: December 2011

Eur J Clin Invest 2012; 42 (7): 709–716

Objective  Accelerated cholesteryl ester transfer (CET) in patients with types 1 (T1D) and 2 diabetes enhances the atherogenicity of the apoB‐containing CE acceptor lipoproteins. The study of lipoprotein density fractions cannot identify which of the five immunologically distinct apoB subclasses function as CE acceptors because they are heterogeneous and present in very low‐, intermediate‐ and low density lipoproteins (VLDL, IDL and LDL, respectively). In order to design lipid‐modifying therapies that specifically target these CE‐enriched lipoprotein particles, it is necessary to first characterize their CE acceptor function.

Methods and Results  To identify the CE acceptors, we estimated CE net mass transfer to the apoB subclasses LpB:C, LpB:E + LpB:C:E, LpB and LpAII:B:C:D:E from changes in neutral lipids measured by gas chromatography following their separation by sequential immunoaffinity chromatography in the plasma of 12 patients with T1D and six control subjects. In both groups, CE was distributed equally to LpB:E + LpB:C:E and LpB:C. In the T1D CE acceptors, however, both the magnitude of the increase (18% vs. 10%; P < 0·01) and the per particle mass of CE transferred were significantly greater than in controls (T1D: 2·29 μmol ± 2·1 vs. control 0·43 ± 0·43/mg apoB; P < 0·047).

Conclusion  While LpB:E + LpB:C:E and LpB:C functioned as CE acceptors in both groups, these subclasses increased their CE content to a greater degree and accrued more CE per particle in the patients with T1D. As this disturbance in lipoprotein remodelling may increase the cholesterol burden and potential atherogenicity of these apoB subclasses, it may be a previously unrecognized factor that increases cardiovascular risk in patients with T1D.
On the brink of extinction? How climate change may affect global chelonian species richness and distribution
Authors: Ihlow Flora , Dambach Johannes , Engler Jan O. , Flecks Morris , Hartmann Timo , Nekum Sven , Rajaei Hossein , Rödder Dennis
Published: December 2011

Anthropogenic global climate change has already led to alterations in biodiversity patterns by directly and indirectly affecting species distributions. It has been suggested that poikilothermic animals, including reptiles, will be particularly affected by global change and large‐scale reptile declines have already been observed. Currently, half of the world's freshwater turtles and tortoises are considered threatened with extinction, and climate change may exacerbate these declines. In this study, we assess how global chelonian species richness will change in the near future. We use species distribution models developed under current climate conditions for 78% of all extant species and project them onto different Intergovernmental Panel on Climate Change (IPCC) scenarios for 2080. We detect a strong dependence of temperature shaping most species ranges, which coincide with their general temperature‐related physiological traits (i.e., temperature‐dependent sex determination). Furthermore, the extent and distribution of the current bioclimatic niches of most chelonians may change remarkably in the near future, likely leading to a substantial decrease of local species abundance and ultimately a reduction in species richness. Future climatic changes may cause the ranges of 86% of the species to contract, and of these ranges, nearly 12% are predicted to be situated completely outside their currently realized niches. Hence, the interplay of increasing habitat fragmentation and loss due to climatic stress may result in a serious threat for several chelonian species.