bluuebunny/crossref_metadata_2025 huggingface dataset:
119 ULTRASONOGRAPHIC MONITORING OF CANINE OVARIES CLAMPED AT SUBCUTANEOUS SITE AFTER HORMONE TREATMENT
Authors: Terazono T. , Luu V. V. , Do L. T. K. , Sato Y. , Taniguchi M. , Takagi M. , Otoi T.
Published: December 2013

Follicular growth in bitches is usually detected indirectly through behaviour observation, vaginal smears, and hormonal assay in blood. Although real-time ultrasonography can reveal the development of canine ovarian follicles, no method has been established to determine or predict ovulation accurately. Moreover, the location and small size of the ovaries make imaging technically difficult. This study was conducted to investigate follicular waves of canine ovaries stimulated by hormone treatment, in which ovaries had been clamped at a subcutaneous site. Bilateral malacotomy of 3 bitches (4 years of age) at the anestrous (2 bitches) and proestrous (1 bitch) stages of the oestrous cycle was performed using a ventral flank abdominal approach with routine techniques and materials. Each ovary that maintained blood circulation from the suspensory ligament was clamped at a subcutaneous site through muscles of the abdomen. Oestrus was induced using subcutaneous administration of 500 IU of eCG and 1000 IU of hCG (eCG/hCG). Each bitch was given 1000 IU of hCG at 11 days after eCG/hCG administration. Examinations with ovarian ultrasonography using a 7.5-MHz sector transducer, vaginal cytology, and serum progesterone assay were performed daily until 20 days after eCG/hCG administration, and every 10 days thereafter from 20 days to 60 days. Serosanguineous vaginal discharges and vaginal cytology of 2 of the bitches were observed. Follicular growth (>1.1 mm in diameter) was observed in all bitches after eCG/hCG administration. The appearance of new follicular growth was observed on 2 days, 6 days, and 8 days after eCG/hCG administration. The mean diameter of follicles reached 4.3 to 5.5 mm, and the maximum numbers of follicles in bitches were 11 to 16. However, all follicles regressed, irrespective of hCG administration. Elevation in progesterone levels (>2 ng mL–1) after eCG/hCG administration was observed from 2 days to 12 days after eCG/hCG administration. No correlation was found between follicular development, progesterone profiles, and vaginal smear characteristics. Follicular growth clamped at the subcutaneous site can be monitored easily using ultrasound without an experienced operator. Moreover, ultrasonography proved that hormonal stimulation can induce follicular growth, but the day of appearance of new follicles varied.
126 POTENTIAL EFFECTS OF ENGINEERED STEM CELLS TRANSDUCED WITH THERAPEUTIC GENES AGAINST HeLa HUMAN CERVICAL CANCER CELLS VIA A SELECTIVE TUMOR TROPISM CAUSED BY VASCULAR ENDOTHELIAL GROWTH FACTOR
Authors: Yi B.-R. , Kim S. U. , Choi K.-C.
Published: December 2013

According to the World Health Organization, cancer of cervix uteri is the second most common cancer among women worldwide. Recently, cervical cancer still remains a significant public health problem for women despite the development of the human papilloma virus vaccine. The aim of the present study was to investigate the therapeutic efficacy of genetically engineered stem cells (GESTEC) expressing bacterial cytosine deaminase (CD), human interferon-β (IFN-b) gene, or both against HeLa human cervical cancer and the migration factors of the GESTEC toward the cancer cells. A continuously dividing immortalized cell line of neural stem cells (NSC) from a single clone of human fetal brain, HB1.F3, was developed by introducing v-myc. The further introduction of these NSC with bacterial CD and human IFN-b resulted in the generation of HB1.F3.CD and HB1.F3.CD.IFN-b cells. The anticancer effect of these GESTEC was examined in a co-culture with HeLa cells using MTT assay to measure cell viability. A transwell migration assay was performed to assess the migration capability of the stem cells to cervical cancer cells. Next, several chemoattractant ligands and their receptors related to a selective migration of the stem cells towards HeLa cells were determined by real-time PCR. The cell viability of HeLa cells was decreased in response to 5-fluorocytosine (5-FC), a prodrug, indicating that 5-fluorouracil (5-FU), a toxic metabolite, was converted from 5-FC by the CD gene and it caused the cell death in a co-culture system. When IFN-b was additionally expressed with the CD gene by these GESTEC, the anticancer activity was significantly increased. In the migration assay, the GESTEC selectively migrated to HeLa cells. As results of real-time PCR, chemoattractant ligands such as MCP-1, SCF, and VEGF were expressed in HeLa cells, and several receptors such as uPAR, VEGFR2, and c-kit were produced by the GESTEC. These GESTEC transduced with the CD gene and IFN-b may provide the potential of a novel gene therapy for anti-cervical cancer treatments via their selective tumour tropism derived from VEGF and VEGFR2 expressions between HeLa cells and the GESTEC.
This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009599), Rural Development Administration, Republic of Korea.
169 INFLUENCE OF TIME BEFORE BOS INDICUS OOCYTE ASPIRATION ON EMBRYO DEVELOPMENTAL COMPETENCE, EXPRESSION OF MATER AND OCT-4, AND FOLLICULAR STEROID CONCENTRATION
Authors: Urrego R. , Herrera E. , Chavarría N. , Camargo O. , Rodriguez-Osorio N.
Published: December 2013

The ability of bovine embryos to develop to the blastocyst stage, to implant, and to generate healthy offspring, depends greatly on the oocyte contribution. Oocyte competence is attributed to its close communication with the follicular environment and to its capacity to synthesise and store great amounts of mRNA. Higher developmental competence of bovine oocytes has been associated with the expression of certain genes and with the steroid concentration in the follicular fluid. Hence, the aim of this study was to establish the influence of OCT-4 and MATER mRNA abundance in the oocyte and the influence of progesterone and oestradiol follicular fluid concentration on the competence of bovine oocytes retrieved 30 min or 4 h after slaughter. Cumulus–oocyte complexes (COC) were left in postmortem ovaries for 30 min (Group I) or 4 h (Group II) at 30°C before aspiration. Progesterone and oestradiol concentrations were measured in the follicular fluid in both groups by immunoassay using an Immulite 2000 analyzer. Immature oocytes were evaluated for MATER and OCT-4 mRNA abundance by real-time PCR (total RNA isolated from pools of 100 oocytes per repeat) or were subjected to in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC). For in vitro embryo production, 455 (Group I) and 470 (Group II) COC were used in three repeats. Progesterone concentration was lower (P ≤ 0.05) in Group II than in Group I. Conversely, oestradiol concentration did not vary between groups. Similarly, Group II oocytes exhibited the highest (P < 0.05) MATER and OCT-4 abundance. For embryo development, there were no significant differences between cleavage rates (72 h post-insemination) between both groups. However, blastocyst (168 h post-insemination) and hatching (216 h post-insemination) rates in Group II were greater (P < 0.05) with 21.3 compared with 30.7% and 54.2 compared with 75.3%, respectively. These results indicate that progesterone concentration in the follicle and the abundance of MATER and OCT-4 transcripts could be good predictors of embryo developmental competence and that retrieving COC 4 h after slaughter could increase blastocyst and hatching rates.
This work was supported by COLCIENCIAS COD 122852128473 Colombia.
183 CANINE EMBRYOS OBTAINED BY INTRACYTOPLASMIC SPERM INJECTION
Authors: Chastant-Maillard S. , Reynaud K. , Thoumire S. , Chebrout M.
Published: December 2013

In vitro fertilization encounters 2 specific difficulties in the canine species, with no puppies born to date: low penetration rates (10–50%) and high polyspermia (around 50% of fertilized oocytes; Saint-Dizier et al. 2001 J. Reprod. Fert. Suppl. 57, 147–150). The objectives of the study were to test whether intracytoplasmic sperm injection (ICSI), which overcomes these 2 obstacles, could allow production of canine embryos, using in vivo- or in vitro-matured oocytes. The time of ovulation was determined on 8 Beagle bitches from our experimental kennel by blood progesterone assay and transabdominal ultrasound examination. After ovariohysterectomy 82 to 100 h after ovulation, 58 metaphase II (MII) oocytes were collected by tubal flushing. In parallel, 88 oocytes from 6 anoestrus bitches were matured in vitro (M199 + 20% fetal calf serum for 72 h in 5% CO2 at 38°C). Sperm was collected from 1 Beagle dog with excellent fertility record at natural mating. The sperm was diluted 1 : 100 in PBS/BSA without any selection process. Intracytoplasmic sperm injection was performed at 38°C in M199 HEPES + 20% BSA (4-μm injection pipette; 120-μm holding pipette). One motile spermatozoon of normal morphology was injected per oocyte. Injected oocytes were cultured in vitro for 48 h after injection (M199 + 20% fetal calf serum in 5% CO2 at 38°C) in 4-well open dishes. Oocytes were then fixed and DNA and tubulin were stained for observation by confocal microscopy (Chebrout et al. 2012 Microsc. Microanal. 18, 483–492). Among the 58 MII oocytes recovered in vivo, 7.4% lysed at injection and 20% degenerated during the 48 h after injection. Among the 40 injected oocytes still alive, 6 fragmented (15%) and 4 developed as embryos [10%; 2-pronuclei (n = 2), 2-cell and 6-cell). None of the other oocytes showed decondensed female chromatin. Among the 88 oocytes incubated for in vitro maturation, 13 (14.8%) reached MII. These were successfully injected; 48 h after injection, 3 were embryos at the 2-cell stage and 10 were at the MII stage with a condensed sperm head. Fifty-one non-mature oocytes were injected; 31 were at the germinal vesicle (GV) stage and the stage of others was not determined. Of the GV oocytes, 71% degenerated during culture after injection. The 9 surviving oocytes were still at the GV stage with condensed sperm head 48 h after injection. In conclusion, canine embryos can be obtained through ICSI. Nevertheless, this procedure induced low activation rates. Development at later stages, especially after transfer into a recipient female, is to be evaluated, in particular for in vitro-produced MII oocytes, of lower cytoplasmic competence (Viaris et al. 2008 Reprod. Fert. Dev. 20, 626–639).
192 IDENTIFICATION AND CHARACTERIZATION OF Oct4-EGFP EXPRESSING CELLS IN TRANSGENIC PIG TESTIS
Authors: Nowak-Imialek M. , Lachmann N. , Herrmann D. , Jacob F. , Niemann H.
Published: December 2013

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.
202 EFFECT OF LEUKEMIA INHIBITORY FACTOR AND FORSKOLIN ON ESTABLISHMENT OF RAT EMBRYONIC STEM CELL LINES
Authors: Hirabayashi M. , Goto T. , Tamura C. , Sanbo M. , Hochi S.
Published: December 2013

Rat embryonic stem (ES) cell lines can be established in culture medium containing inhibitors for glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK). We confirmed reproducibility of the 2i culture system in establishing rat ES cell lines (Hirabayashi et al. 2010 Mol. Reprod. Dev. 77, 94) and the likelihood of successful germline transmission (genuine) of rat ES cell lines established in leukemia inhibitory factor (LIF)- and forskolin (FK)-supplemented 2i medium (Hirabayashi et al. 2013 Transgenic Res. 22, 411–416). This study was designed to investigate whether LIF and/or FK supplemented to the 2i medium support establishment of germline-competent rat ES cell lines. E4.5 blastocysts were recovered from BLK rat females, and zona-free embryos were plated on mitomycin-treated mouse embryonic fibroblasts in N2B27 medium containing 1 mM MEK inhibitor PD0325901 and 3 mM GSK3 inhibitor CHIR99021, with rat 1000 U mL–1 LIF and/or 10 μM FK. Outgrowth rate of the blastocysts after 1 wk culture and establishment efficiency of ES cell lines after third passage were analyzed by Fisher's exact probability test. Arcsin-transformed percentage data on full-term development of ES cell-injected blastocysts, chimeric rat production, and germline-competent chimeras were analyzed by Fisher's least significant difference test after one-way ANOVA. Because of the higher outgrowth rates of blastocysts, supplementation of rat LIF, FK, or both contributed to the higher (P < 0.05) establishment efficiency of ES cell lines in BLK rat strain (76% to 92% v. 50% in LIF/FK-free 2i medium). Neither efficiency of producing chimeric rats (14% to 39% of blastocysts injected) nor germline transmission competency of the chimeric rats (67% to 100% of cell lines analyzed) was influenced by the pre-treatment of ES cell lines. When the LIF/FK-supplemented 2i medium was used, rat strain for blastocyst donor such as F344 or WI was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, however, all of them (9/9 in overall) were found to be germline-competent by progeny test of chimeric rats. In conclusion, both LIF and FK are not essential, but can play a beneficial role, for the establishment of genuine rat ES cell lines.
This work was supported by a grant-in-aid for basic research from Japan Society for the Promotion of Science (No. 25290037; to M.H.).
216 COMPLETE REDUCTION OF p53 EXPRESSION BY RNA INTERFERENCE FOLLOWING HETEROZYGOUS KNOCKOUT IN PORCINE FIBROBLASTS
Authors: Kim Y. J. , Kim M. J. , Kim T. H. , Kim H. W. , Shim H.
Published: December 2013

Because p53 has a critical role in regulation of cell cycle and apoptosis in mammals, mutations of p53 often cause various cancers in mammals. Murine models have contributed to our understanding in cancer related to p53 mutations. Mice, however, have different characteristics from humans in many ways. For instance, the short lifespan of the mouse gives rise to limitations in clinical application of the data obtained from this species. Hence, it would be beneficial to establish a more suitable model in species other than the mouse. The porcine model could be an appropriate alternative because pigs share many anatomical and physiological similarities with humans. However, the production of pigs with homozygous knockout (KO) requires years of breeding heterozygous KO animals. Here, we completely reduced the expression level of p53 mRNA and protein in miniature pig fetal fibroblasts using a combination of gene targeting and RNA interference technique. These cells may be used for nuclear transfer to directly produce pigs without expression of the gene of interest. First, we disrupted the exon 2 region of p53 gene to produce p53 heterozygous KO cells. Miniature pig fetal fibroblasts were transfected with the p53 gene targeting vector. After Geneticin treatment for 2 weeks, a total of 48 surviving colonies were screened by PCR, and one was identified as a homologous recombinant (1/48, 2.08%). Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. Transfected fibroblasts were treated with Zeocin for 3 weeks, and the shRNA integrations were confirmed by PCR. We obtained p53 KO, KD, and KOKD fibroblasts which involve p53 KO and KD either separately or simultaneously. In mRNA expression based on RT-PCR, p53 KO fibroblasts showed no difference with wild-type control (91.8 v. 100%). However, the expression levels of KD and KOKD cells significantly decreased (35.5 and 34.7%) compared with the control. In p53 protein levels analysed by Western blot, reduction of the protein was observed in p53 KD, whereas no reduction in p53 KO that might be due to heterozygous mutation. Interestingly, no p53 protein was detected in KOKD, suggesting complete reduction of the protein by synergistic effect of KO and KD. In this study, we demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference technique. Moreover, complete abolishment of protein expression is feasible using combination of the 2 techniques. In further research, cloned miniature pigs with various levels of p53 expression or even lacking p53 expression may be produced using the fibroblasts isolated in the present study as nuclear donors. These pigs may provide new animal models and insights in cancer with respect to the effect of p53.
23 IMPROVING EFFICIENCY IN WORK WITH TRANSFER OF CLONED PIG EMBRYOS
Authors: Callesen H. , Liu Y. , Li R. , Schmidt M.
Published: December 2013

Cloning is a quite inefficient procedure with only around 10% of offspring born based on number of cloned embryos transferred. Every step to increase this level is therefore welcomed. Our group has worked with cloning of pig embryos since 2006, with the main purpose to establish a well-functioning cloning system to have transgenic piglets born as animal models for important human diseases. Here we report results from our attempts to improve efficiencies in several steps in the whole cloning procedure. Over 7.5 years, donor cells from 3 breeds were nontransgenic (50%, 4 types) or transgenic with 1 of 6 different types of gene. Oocytes from Large White (LW) sows or gilts were handmade cloned, so the zona-free cloned embryos were in vitro cultured until Day 5 to 6 to select 13 311 embryos (morulae or blastocysts) for transfer to 171 LW recipient sows or gilts. Of these, 126 were pregnant (74%; Day 35), but 20 aborted before term. A total of 704 offspring were delivered; half of the piglets were alive after 4 weeks and developed normally after that. Frequencies were compared using Chi-squared test; mean values by one-way ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Specific improvements were tested in 4 areas: (1) donor cells: stimulating reprogramming using Xenopus egg extract (Liu et al. 2013 Reprod. Fertil. Dev. http://dx.doi.org/10.1071/RD13147); (2) oocytes: preferably from sows, but also using larger gilt oocytes (Li et al. 2013 Zygote http://dx.doi.org/10.1017/S0967199412000676); (3) transfer: using both uterine sides (Theriogenology 74 : 1233); (4) number of embryos transferred: after cloning with same nontransgenic cells, embryo numbers per recipient were reduced from 90 to 30 (see Table 1). As a consequence of these different activities, overall results improved over the 7.5-year period [first 3.5 years v. last 4 years: 48% (32/67) v. 90% (94/104) recipients pregnant after transfer (P < 0.05); 5.6 ± 0.6 (n = 22) v. 6.9 ± 0.5 (n = 84) piglets/litter]. In our system, one good cloning person can now produce all embryos needed for one recipient in one good working day. Transfer of fewer cloned embryos results in fewer piglets, but it reduces the workload to produce cloned embryos and does not reduce efficiency. Further work is still needed to better understand the biological and technical challenges in work with cloning; 2 important areas are quality evaluation of the donor cells used for cloning and the recipient's reaction to transfer of many embryos. In conclusion, a reasonable increase in the overall efficiency in pig cloning work was achieved, which reduces the need for personnel, time, and material when working with this technology.

Table 1.Results of improving efficiency of cloning
52 VITRIFICATION OF DAY-8 EQUINE EXPANDED BLASTOCYST: AN IN VITRO COMPARISON OF DIRECT VERSUS INDIRECT MECHANICAL INTRODUCTION OF CRYOPROTECTANT
Authors: Diaz F. A. , Paccamonti D. L. , Bondioli K. R. , Gentry G. T.
Published: December 2013

The cryopreservation of equine expanded blastocysts (>300 μm) has been largely unsuccessful primarily due to the low permeability of the embryo to cryoprotectants. This low permeability has been attributed to the acellular glycoprotein capsule that develops when an embryo approaches approximately 300 μm in diameter. Mechanical alternatives may provide a means to overcome the capsule barrier and the relative large embryo size to successfully cryopreserve equine embryos. The objective of this experiment was to compare re-expansion rates of vitrified equine expanded blastocysts following either direct or indirect mechanical introduction of cryoprotectants using a coaxial microinjection system (Dracula pipette). Twenty-six Day-8 expanded blastocysts were subjected to capsule puncture, cryoprotectant injection, and blastocoele fluid extraction (direct treatment) or capsule puncture and blastocoele fluid extraction (indirect treatment) before cryopreservation. The Dracula pipette incorporates the injection pipette within the holding pipette, facilitating aspiration of blastocoele fluid or injection of cryoprotectant in a single unit. A standard vitrification protocol using a final concentration of 3.4 M glycerol and 4.6 M ethylene glycol at cryopreservation was used. Vitrified embryo re-expansion was assessed following in vitro culture at 24, 48, and 72 h post-warming. Differences across treatments were analysed using the Student's t-test for re-expansion and the chi-squared test of independence for capsule loss. Pre-vitrification embryo mean diameter (mean ± standard error) for direct and indirect treatment groups were not different, 979 ± 85.6 μm and 912 ± 101.4 μm, respectively (P = 0.62). Post-vitrification embryo mean diameters were not different for the direct and indirect treatments (688 ± 63 and 662 ± 75 μm, respectively; P = 0.79). Following 72 h of in vitro culture, there was no difference in mean embryo diameter (1813.16 ± 209 μm v. 1383.88 ± 198 μm; P = 0.21), or re-expansion rates (69 v. 69%) for direct and indirect treatment groups, respectively. However, partial or total capsule loss was 69% (9/13) for direct treatment embryos compared with 30% (4/13) for indirect treatment embryos (P = 0.049). Results from this experiment demonstrate that capsule puncture and blastocoele fluid extraction before vitrification resulted in high re-expansion rates of Day-8 equine expanded blastocysts after warming. More importantly, the relatively large percentage of capsule failure when directly introducing cryoprotectant into the embryo may interfere with maternal recognition of pregnancy following embryo transfer. Nonetheless, based on the embryonic re-expansion rate of vitrified equine embryos following the indirect technique, we anticipate that a relatively high pregnancy rate can be obtained if this technique is used.
57 EFFECT OF CHOLESTANOL OR CHOLESTEROL ON THE MOTILITY OF FROZEN GOAT SPERMATOZOA AFTER A THERMAL RESISTANCE TEST
Authors: Silva B. G. , Moraes E. A. , Matos W. C. G. , Oliveira C. S. , Ferrari Junior W. D. , Graham J. K.
Published: December 2013

The objective of the present study was to determine the concentration of cholesterol or cholestanol-loaded-cyclodextrin that needs to be added to goat sperm before cryopreservation to optimize its survival. The cholesterol or cholestanol loaded methyl-β-cyclodextrin was prepared as described by Moraes et al. (2010 Anim. Reprod. Sci. 118, 148–154). A working solution of the cholesterol or cholestanol-loaded cyclodextrin was prepared by adding 50 mg of each one to 1 mL of TALP at 37°C and mixing the solution briefly using a vortex mixer. Ejaculates (n = 24) from 5 bucks were used for this experiment. Sperm from each ejaculate were diluted 1 : 1 (vol : vol) in Tris diluent (200 mM Tris, 65 mM citric acid, and 55 mM glucose) and centrifuged at 800 × g for 10 min. The pellets were resuspended to a concentration of 120 × 106 sperm mL–1 in Tris and subdivided into 7 aliquots of 5 mL each (600 × 106 total sperm). Sperm were treated in 7 treatment groups that received no additive (0 mg; control) or different levels of cholesterol or cholestanol (0.75, 1.5, or 3.0 mg/120 × 106 sperm). All treatments were incubated for 15 min at room temperature and then cooled to 4°C over 2 h. The samples were diluted with Tris-egg yolk diluent containing 2% glycerol. The sperm were packaged into 0.5-cc straws and frozen in static liquid nitrogen vapor for 20 min and then straws were plunged into liquid nitrogen and stored until analysed for motility and thermal resistance test using a computer-assisted semen analysis system (CASA). Two straws from each treatment were thawed in a 37°C water bath for 30 s and extended in Tris. For the thermal resistance test, after thawing, 0.5 mL of semen from each treatment was placed in 1.5-mL tubes in a water bath at 37°C for 3 h. At 0, 60, 120, and 180 min, subsamples were evaluated for sperm total and progressive motility using a computer-assisted sperm motion analyzer. A total of 200 spermatozoa were counted in at least 5 different fields. Data were analysed using ANOVA and treatment means were separated, using the SNK test at 5% probability. Cholesterol (0.75 mg; 46.7%) and cholestanol (1.5 mg; 40.5%) produced an increase in progressive motility compared with other treatments after 1 h of incubation (P < 0.05). However, cholestanol (0.75 mg; 39.5 and 31%) was higher for total and progressive motility after 3 h of sperm incubation compared with the control (27 and 17.8%; P < 0.05), respectively. The addition of 0.75 mg of cholestanol in fresh sperm before cryopreservation improved the motility of freeze-thawed goat sperm compared with cholesterol. Therefore, adding cholestanol to goat sperm membranes improved cell cryosurvival.
Supported by Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
84 THE EFFECT OF β-MERCAPTOETHANOL ON CLEAVAGE RATES, DEVELOPMENTAL COMPETENCE AND QUALITY OF IN VITRO PRODUCED BOVINE EMBRYOS
Authors: Lliteras E. R. , Chong M. , Andries S. , Merckx E. , Jorssen E. P. A. , Leroy J. L. M. R. , Bols P. E. J.
Published: December 2013

The production of excessive levels of reactive oxygen species can be a major problem during in vitro embryo culture. Although studies have shown that supplementation with exogenous antioxidants can improve embryo quality, the results are controversial among researchers. In this study, we examined the effects of different concentrations of β-mercaptoethanol (β-ME) added to the culture media, on cleavage rates, the quality and developmental competence of in vitro-produced bovine embryos. The embryos were produced in vitro as described previously (Van Hoeck et al., 2013). Briefly, in total, 753 grade I cumulus–oocyte complexes (COC) from 2- to 6-mm-diameter follicles were matured in groups of 50 in 500 μL of TCM with 20 ng mL–1 EGF for 24 h, fertilized in groups of 100 in 500 μL of fertilization medium for 20 h (5% CO2, 38.5°C). Presumptive zygotes were denuded and randomly assigned to 4 treatments with different concentrations of β-ME: 0 μM (control), 50 μM, 100 μM, and 150 μM. They were cultured in groups of  ±25 in 50 μL of SOF supplemented with ITS (10 μg mL–1 insulin; 5.5 μg mL–1 transferrin; 6.7 ng mL–1 selenium) and 2% BSA and covered with mineral oil (5% O2, 5% CO2, 38.5°C). At 48 h post-insemination (p.i.), cleavage rate was evaluated and expressed as the number of cleaved embryos on total number of oocytes. At Day 7 p.i., blastocyst rate was determined (number of blastocysts on total number of oocytes), blastocysts were fixed in 4% paraformaldehyde, and total cell number was determined by DAPI staining. Data were analysed by ANOVA and post hoc test. Comparable cleavage rates were obtained in treated groups: control (80.8%), 50 μM (77.7%), 100 μM (77.9%), and 150 μM (73.6%; P > 0.05). Also, no significant effect of treatment could be found on blastocyst rates: control (36%), 50 μM (36.5%), 100 μM (38.4%), and 150 μM (30.4%). The total cell number per blastocyst increased significantly (P < 0.05) using 100 μM of β-ME compared with the controls (158.0 ± 24.3 v. 123.2 ± 9.72, respectively). These results suggest that the inclusion of 100 μM β-ME during in vitro embryo culture could be used for production of high quality bovine blastocysts.
98 COMMERCIAL APPLICATION OF A NEW COW-SIDE LH ASSAY FOR DETERMINING OPTIMUM AI INTERVALS IN SUPEROVULATED BEEF DONORS IN THE USA: A PRELIMINARY STUDY
Authors: Devine T. L. , Looper M. L. , Pryor J. H. , Kara E. , Maurel M.-C. , Decourtye J. , Baldauf P. P. , Jurney K. H. , Looney C. R.
Published: December 2013

Successful superovulation and embryo collection require donors to be managed for precise insemination intervals. At times, determinations of these intervals can become difficult because of oestrus detection subjectivity. The objective of this study was to determine the feasibility of performing a rapid cow-side LH assay (Predi′Bov®, ReproPharm, Nouzilly, France) on superovulated donors with emphasis on determining how to use the results in a commercial program to time inseminations. This study was conducted at Ovagenix (Bryan, TX, USA) on 24 superovulated donors. On Day 0 cows received an intravaginal progesterone-releasing device (CIDR®, Pfizer Animal Health, New York, NY, USA) and were administered 25 mg of progesterone and 2.5 mg of oestradiol 17β intramuscularly (IM, 2 mL). Superstimulation was induced using 150–300 mg of Folltropin-V® (FSH, Bioniche Animal Health Inc., ON, Canada) administered with 7 injections at 12-h intervals in decreasing dosages (Day 4–7). On Day 6, two doses of Estrumate® (equaling 1 mg, Intervet Inc., Merck Animal Health, Summit, NJ, USA) were given IM, concurrently with FSH injections 5 and 6 with CIDR removal on Day 7 (a.m.). HeatWatch® transmitters (Cow Chips LLC, Denver, CO, USA) were applied to donors on Day 7 to determine onset of standing oestrus. Blood samples were collected in heparinized tubes via tail vein puncture starting at CIDR removal continuing every 6 h until a positive Predi′Bov® test was acquired or 36 h after CIDR removal. Whole blood (0.5 mL–1) was submitted to a proprietary 3-step procedure using a tube-stick applicator (40 min) to determine whether increased concentrations of LH were present. Stick applicators that turned blue were positive for increased concentrations of LH. Donors were artificially inseminated 12 and 24 h after a positive test, with specified sires, and embryos were nonsurgically collected 7 days after insemination. The Predi′Bov® test determined 91.6% (22/24) of the LH peaks occurred within 12 to 24 h after CIDR removal; 45.8% (11/24) occurred at 18 h; and 4% (1/24) at 30 h. A total of 203 viable embryos (average 8.5/donor) was collected with 174 embryos (average 9.2 ± 4.7/donor) from donors that exhibited oestrus and produced a positive test (19/24; 79.2%) and 29 embryos (average 5.8 ± 6.3/donor) from donors exhibiting silent oestrus that produced a positive test (5/24; 20.8%). One donor exhibited oestrus and never produced a positive test, but contributed 8 viable embryos to the total. In conclusion, Predi′Bov® cow-side usage could offer commercial utility when identifying oestrus is difficult or nonexistent. Increased detection of ovulation could help to identify optimal AI intervals, thereby increasing embryo production and limiting the use of expensive semen for desired donors.
Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1
Authors: Li Shih-Hon , Reinke Ashley A. , Sanders Karen L. , Emal Cory D. , Whisstock James C. , Stuckey Jeanne A. , Lawrence Daniel A.
Published: December 2013

Significance

Serine protease inhibitors (serpins) are a protein superfamily whose members are involved in many diseases and are thus attractive drug targets. In addition to protease inhibition, serpins also bind a variety a of other biological molecules, including extracellular matrix components and cell surface receptors. The inhibitory mechanism of serpins requires a conformational change that can also alter their affinity to non-protease ligands. Here a surprising allosteric mechanism of action is revealed for a small molecule inhibitor of the serpin, plasminogen activator inhibitor 1 (PAI-1). Compound binding prevents PAI-1 interaction with both proteases and with its cofactor even though the binding sites are located 40 Å apart. These results suggest the potential for the identification other therapeutically useful serpin inhibitors.
Cellular distribution of copper to superoxide dismutase involves scaffolding by membranes
Authors: Pope Christopher R. , De Feo Christopher J. , Unger Vinzenz M.
Published: December 2013

Significance

What are the odds that a 1,000 Å
3
-sized metal-binding site will find its cargo parsing a volume of 30 trillion Å
3
by a random 3D diffusional process? Establishing a unique paradigm in the field of copper biology, we show that cells use a stunningly simple method to beat these odds by reducing the dimensionality of the problem through exploitation of cellular membranes as scaffolding components. We demonstrate that the copper delivery chaperone for the essential enzyme Cu/Zn superoxide dismutase 1 (SOD1) has the ability to bind to bilayers and that this previously unknown membrane binding interface is important for copper distribution to SOD1.
Founder niche constrains evolutionary adaptive radiation
Authors: Flohr Régis C. E. , Blom Carsten J. , Rainey Paul. B. , Beaumont Hubertus J. E.
Published: December 2013

Significance

Much of the biodiversity on Earth evolved by adaptive radiation: rapid evolution of ecologically different species from a single ancestor. It is well established that the environment shapes the dynamics of adaptive radiation; however, theory predicts that the properties of the founding ancestor also play a role. Using experimental bacterial populations, we demonstrate that adaptive radiation is constrained by the ecology of the founder. The findings advance our understanding of the evolution of biodiversity and reveal a mechanism by which chance may have influenced the tempo of adaptive radiations in natural history.
Bordetella pertussis
adenylate cyclase toxin translocation across a tethered lipid bilayer
Authors: Veneziano Rémi , Rossi Claire , Chenal Alexandre , Devoisselle Jean-Marie , Ladant Daniel , Chopineau Joel
Published: December 2013

Significance

Many bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, although how they pass through a lipid bilayer remains largely unknown.
Bordetella pertussis
adenylate cyclase (CyaA) toxin delivers its catalytic domain directly across the cell membrane. To characterize this unique translocation process, we designed an in vitro assay based on a tethered lipid bilayer assembled over a biosensor surface derivatized with calmodulin, a natural activator of the toxin. CyaA activation by calmodulin provided a highly sensitive readout for toxin translocation across the bilayer. CyaA translocation was calcium- and membrane potential-dependent but independent of any additional eukaryotic protein. This biomimetic membrane will permit in vitro studies of protein translocation in precisely defined conditions.
Retrograde transfer RNA nuclear import provides a new level of tRNA quality control in
Saccharomyces cerevisiae
Authors: Kramer Emily B. , Hopper Anita K.
Published: December 2013

Significance

Transfer RNAs (tRNAs) traffic in a retrograde direction from the cytoplasm to the nucleus, a process that is conserved from yeast to vertebrates. The biological significance of retrograde tRNA nuclear import is poorly understood. We report that the tRNA retrograde pathway serves as a quality control pathway that monitors 5′ and 3′ end processing and the modification status of cytoplasmic tRNAs. Aberrant tRNAs are imported into the nucleus where they are repaired or destroyed. This newly discovered quality control function of the tRNA retrograde nuclear pathway likely functions in parallel with the cytoplasmic 5′–3′ exonuclease tRNA quality control pathway.
Molecular basis of usher pore gating in
Escherichia coli
pilus biogenesis
Authors: Volkan Ender , Kalas Vasilios , Pinkner Jerome S. , Dodson Karen W. , Henderson Nadine S. , Pham Thieng , Waksman Gabriel , Delcour Anne H. , Thanassi David G. , Hultgren Scott J.
Published: December 2013

Significance

Gram-negative bacteria use chaperone-usher pathway (CUP) pili to colonize host tissues and mediate biofilm formation. CUP ushers are outer membrane (OM) pore proteins that catalyze pilus assembly and mediate pilus extrusion in a gated fashion to maintain OM homeostasis. Using antibiotic sensitivity and electrophysiology experiments, specific residues of two ushers, FimD of type 1 pili and PapC of P pili, were identified as crucial for proper usher gating. Further, deletion of the P pilus anchoring/terminator subunit resulted in an open usher conformation, indicating this subunit may be a target for antivirulence compounds to potentiate antibiotic treatment. Identification of mechanisms that can be used to increase bacterial sensitivity to antibiotics is crucial for development of novel compounds to fight infections.
Should psychotherapists disclose their own psychological problems?
Authors: Somers Andrea D. , Pomerantz Andrew M. , Meeks J. Thadeus , Pawlow Laura A.
Published: December 2013

Background: Self‐disclosures by psychotherapists can encompass a wide array of content, including information about the personal life of the psychotherapist. Aim: The goal of this study was to empirically assess reactions to psychotherapists disclosing their own personal problems to clients with similar problems. Methodology: This study explored participants' (n = 155) reactions to vignettes describing psychotherapists as either disclosing or not disclosing their own history of psychological problems to clients presenting with similar problems. Findings: Compared to otherwise identical vignettes featuring psychotherapists who did not self‐disclose, vignettes featuring psychotherapists who self‐disclosed were rated more positively in a variety of ways. Specifically, psychotherapists in the vignettes featuring self‐disclosure were perceived as possessing a higher level of favourable personal qualities and as more likely to establish strong working relationships with clients and achieve success in therapy. Limitations of the method and alternate explanations for the results are considered. Implications: Implications regarding the judicious use by psychotherapists of self‐disclosure regarding their own past psychological problems are discussed.
The Locus of Word Frequency Effects in Skilled Spelling-to-Dictation
Authors: Chua Shi Min , Rickard Liow Susan J.
Published: December 2013

In spelling-to-dictation tasks, skilled spellers consistently initiate spelling of high-frequency words faster than that of low-frequency words. Tainturier and Rapp's model of spelling shows three possible loci for this frequency effect: spoken word recognition, orthographic retrieval, and response execution of the first letter. Thus far, researchers have attributed the effect solely to orthographic retrieval without considering spoken word recognition or response execution. To investigate word frequency effects at each of these three loci, Experiment 1 involved a delayed spelling-to-dictation task and Experiment 2 involved a delayed/uncertain task. In Experiment 1, no frequency effect was found in the 1200-ms delayed condition, suggesting that response execution is not affected by word frequency. In Experiment 2, no frequency effect was found in the delayed/uncertain task that reflects the orthographic retrieval, whereas a frequency effect was found in the comparison immediate/uncertain task that reflects both spoken word recognition and orthographic retrieval. The results of this two-part study suggest that frequency effects in spoken word recognition play a substantial role in skilled spelling-to-dictation. Discrepancies between these findings and previous research, and the limitations of the present study, are discussed.
Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts
Authors: Wang Songyu , Romano Fabian B. , Field Christine M. , Mitchison Tim J. , Rapoport Tom A.
Published: December 2013

In metazoans the endoplasmic reticulum (ER) changes during the cell cycle, with the nuclear envelope (NE) disassembling and reassembling during mitosis and the peripheral ER undergoing extensive remodeling. Here we address how ER morphology is generated during the cell cycle using crude and fractionated Xenopus laevis egg extracts. We show that in interphase the ER is concentrated at the microtubule (MT)-organizing center by dynein and is spread by outward extension of ER tubules through their association with plus ends of growing MTs. Fusion of membranes into an ER network is dependent on the guanosine triphosphatase atlastin (ATL). NE assembly requires fusion by both ATL and ER-soluble N-ethyl-maleimide–sensitive factor adaptor protein receptors. In mitotic extracts, the ER converts into a network of sheets connected by ER tubules and loses most of its interactions with MTs. Together, these results indicate that fusion of ER membranes by ATL and interaction of ER with growing MT ends and dynein cooperate to generate distinct ER morphologies during the cell cycle.
Silent RNAs express themselves
Authors: Leslie Mitch
Published: December 2013

Study finds that RNA molecules housed in cytoplasmic granules are active.
The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast
Authors: Smardon Anne M. , Diab Heba I. , Tarsio Maureen , Diakov Theodore T. , Nasab Negin Dehdar , West Robert W. , Kane Patricia M.
Published: December 2013

The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H+-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1∆ mutants exhibit a Vma−growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma−growth defect when combined with the rav1∆ mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma−growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1∆ mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1∆ and rav1∆vph1∆, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1∆ perturbed the normal localization of Stv1–green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma−phenotypes arise from defects in Vph1p-containing complexes caused by rav1∆, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.
Identification of New Races and Deviating Strains of the Spinach Downy Mildew Pathogen Peronospora farinosa f. sp. spinaciae
Authors: Feng Chunda , Correll James C. , Kammeijer Katherine E. , Koike Steven T.
Published: December 2013

Spinach downy mildew disease, caused by the obligate pathogen Peronospora farinosa f. sp. spinaciae, is the most economically important spinach (Spinacia oleracea) disease. New races of this pathogen have been emerging at a rapid rate over the last 15 years. This is likely due to production changes, particularly in California, such as high-density plantings and year-round spinach production. As of 2004, 10 races of P. farinosa f. sp. spinaciae had been identified, and the spinach resistance locus RPF2 provided resistance to races 1 to 10. Based on disease reactions on a set of spinach differentials containing six hypothesized resistance loci (RPF1-RPF6), races 11, 12, 13, and 14 of P. farinosa f. sp. spinaciae were characterized based on samples collected in the past 5 years as part of this study. Race 11, identified in 2008, could overcome the resistance of spinach cultivars resistant to races 1 to 10. Spinach resistance loci RPF1, RPF3, and RPF6 provided resistance to race 11. Race 12 was identified in 2009 and could overcome the resistances of the RPF1 and RPF2 loci. The RPF3 locus was effective against race 12. Race 13 was identified in 2010 and could overcome the resistance imparted by the RPF2 and RPF3 loci, whereas the RPF1 locus was effective against race 13. Race 14 was similar to race 12 and caused identical disease responses on the standard differentials but could be distinguished from race 12 by its ability to cause disease on a number of newly released cultivars, including ‘Pigeon’, ‘Cello’, and ‘Celesta’. Five novel strains of P. farinosa f. sp. spinaciae were also identified. For example, isolate UA4711 of the pathogen, collected from Spain in 2011, was able to overcome the resistance imparted by the RPF1 and RPF3 loci, while RPF2 and RPF4 were effective against this strain. A total of 116 spinach cultivars, including 103 commercial lines and 13 differential cultivars, were evaluated for resistance to race 10 and the newly designated races 11, 12, 13, and 14.