bluuebunny/crossref_metadata_2025 huggingface dataset:
108 CALYCULIN-A INDUCES CHROMOSOME CONDENSATION FOR CYTOGENETIC ANALYSIS IN BOVINE AND MURINE BLASTOMERES
Authors: Kramer J. M. , Evans A. , Drury K. , Moore K.
Published: January 2008

Cytogenetic studies of preimplantation embryos have traditionally used interphase fluorescence in situ hybridization (FISH) to examine chromosome copy number, chromosomal rearrangements, or sex determination. Comprehensive analysis of chromosomes is hindered by the low mitotic index of blastomeres, combined with the technical limitations of interphase FISH. Efforts to overcome these limitations include inducing premature chromosome condensation (PCC) for metaphase FISH, by fusing blastomeres with metaphase II-stage oocytes, or with time-consuming incubations with okadaic acid or vinblastine. Our objective was to evaluate Calyculin-A (CA) as an alternative to induce PCC in blastomeres. In vitro-produced bovine 8-cell embryos and frozen–thawed mouse 8-cell embryos were cultured with CA and examined for changes in morphology before fixation. Colcemid (0.1 mg mL–1), the standard for cytogenetic analysis, and vehicle served as positive and negative controls. Blastomeres were washed in hypotonic solution, loaded onto slides, and fixed in methanol:acetic acid (3:1). The degree of chromatin condensation and quality of chromosome spreads were determined by 42,6-diamidino-2-phenylindole staining and visualization on an epifluorescence microscope (100�). Experiment 1 (1 rep, 136 cells) tested dose of CA on bovine blastomeres at 50, 100, 150, 250, 500, and 750 nm. Experiments 2 and 3 tested culture duration of CA (50 nm) at 60, 120, and 180 min in bovine blastomeres and 60, 90, and 120 min in mouse blastomeres (2 reps each, 132 and 207 cells, respectively). Data were analyzed by chi-square with significance deemed P < 0.05. Cell lysis and blebbing was observed in bovine blastomeres treated with greater than 150 nm CA. Duration of CA treatment affected the frequency of bovine and mouse blastomeres undergoing PCC. Fewer bovine blastomeres treated for 60 min underwent chromatin condensation compared to blastomeres treated for at least 120 min with 50 nm CA (25% v. 100%; P < 0.05). In mice, the frequency of blastomeres undergoing PCC was lower (43%) for 50 nm CA at 60 min than at 90 and 120 min (90 and 100%, respectively; P < 0.05). Chromatin condensation suitable for cytogenetic analysis was 20, 34, and 20%, respectively, but did not differ (P > 0.10). Further examination (Exp. 4, 5 reps, 293 cells) comparing the degree of condensation revealed 55% of the total bovine blastomeres treated with 50 nm CA for 120 min were suitable for cytogenetic analysis, as compared to 30% treated with colcemid for 16 h (P < 0.05). Bovine and mouse blastomeres treated with vehicle had lower PCC than all other treatments averaging 2 and 7%, respectively (P < 0.05). These results suggest that CA can rapidly induce PCC in blastomeres from pre-implantation bovine and murine embryos, but the degree of chromatin condensation may not always be suitable for detailed cytogenetic analysis from a single blastomere.
127 DEVELOPMENT AND QUALITY OF BOVINE MORULAE CULTURED IN SERUM-FREE MEDIUM WITH RETINOIC RECEPTOR SPECIFIC AGONISTS
Authors: Gómez E. , Caamaño J. N. , Muñoz M. , Rodríguez A. , Facal N. , Díez C.
Published: January 2008

In the cell, all-trans retinoic acid (ATRA), a vitamin A metabolite, binds to retinoic acid-receptor (RAR), whereas the ATRA isomere 9-cis-retinoic acid (9-cis-RA) binds to both RAR and the retinoid X receptor (RXR). Synthetic compounds such as LG100268 (LG; Ligand Laboratories) are highly specific to bind RXR, which allows to differentially study the RAR and RXR pathways. In previous work morulae treated with LG for 48 h showed to improve blastocyst development and to activate pro-apoptotic genes (in press), whereas ATRA for 24 h increased cell numbers in the inner cell mass (ICM) and the trophectoderm (TE) (Rodr�guez et al. 2006 Hum. Reprod. 21, 2149–2157). However, LG and ATRA were never both compared for 24 in medium with BSA, which is thought to be more appropriate to produce embryos for cryopreservation than serum-containing medium. In this work we analyze development, quality, and viability of morulae cultured with RAR and RXR agonists. Cumulus–oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro. Presumptive zygotes were cultured in synthetic oviduct fluid (SOF) +3 gL–1 BSA. On day 6, morulae were treated for 24 h with ATRA 0.7 µm, LG 0.1 µm, or no additives. Blastocyst development was monitored up to day 8. Differential cell counts were made on hatched blastocysts on days 7 and 8. Apoptosis and necrosis (TUNEL + nuclear histology) were made on day 8 expanded and hatched blastocysts. Data were analyzed by GLM and Duncan's test, expressed as LSM � SE, and development rates were expressed as percentages of cultured morulae (replicates [R] = 14 for development; R = 9 for cell counts; R = 4 for apoptosis; n = 1647 morulae). ATRA yielded more blastocysts on day 8 than LG and controls (72.2 � 2.2 v. 60.0 � 2.3 and 65.6 � 2.4, respectively; P < 0.02), and more expanded blastocysts than LG (48.6 � 2.3 v. 36.6 � 2.4; P < 0.02), but no more than controls (43.5 � 2.5). Day-7 and day-8 hatched blastocysts cultured with ATRA showed more total cells than day-7 controls (163.5 � 8.0 and 161.5 � 5.4 v. 137.7 � 8.9, respectively; P < 0.05). However, in the presence of ATRA, day-8 blastocysts showed a strong cell reduction in the inner cell mass (ICM), whereas their day-7 counterparts conserved ICM/total cells proportions comparable to day-7 controls (11.0 � 1.2 v. 19.7 � 1.7 and 20.6 � 1.9, respectively; P < 0.03). The LG increased apoptotic index (AI) and necrotic index (NI) in the ICM (AI: 14.5 � 2.4 v. 6.4 � 1.5 and 6.4 � 1.4; NI: 5.0 � 1.2 v. 0.9 � 0.8 and 1.6 � 0.7; for LG, ATRA, and controls, respectively; P < 0.02). Embryos produced with ATRA showed improved development and cell distribution without increasing apoptosis and necrosis. Vitrification of excellent day-7 and day-8 blastocysts is in course to evaluate cryosurvival and further embryo transfer to determine full developmental competence.
Grant Support: MEC, project AGL2005-04479. M. Muñoz is sponsored by FICYT.
172 IN VITRO EXPOSURE TO XENOESTROGENS INDUCES GROWTH HORMONE TRANSCRIPTION AND RELEASE VIA AN ESTROGEN RECEPTOR-DEPENDENT PATHWAY IN RAT PITUITARY GH3 CELLS
Authors: Dang V.-H. , Jeung E.-B.
Published: January 2008

The term endocrine disruptor (ED) has been used widely to characterize natural and synthetic environmental compounds that may interfere with the endocrine system(s) of humans and wildlife. In previous studies, we demonstrated that in vitro single exposure to EDs induces CaBP-9k expression, a useful biomarker for detecting the estrogenic activities of EDs in rat pituitary GH3 cells. Here we employ the identical model to examine the effects of EDs in the regulation of growth hormone (GH) gene expression, an important hormone in growth, development, and body composition. We measured levels of GH mRNA transcription and GH release using semi-quantitative RT-PCR and EIA kit, respectively. GH3 cells were treated with alkyphenols (APs), i.e., octyl-phenol (OP), nonyl-phenol (NP), and bisphenol A (BPA), in a dose-dependent manner (10–5, 10–6, and 10–7 M) and harvested following 24 h of treatment. Cells were also exposed to a high concentration (10–5 M) of OP, NP, or BPA and harvested at various time points (1, 3, 6, 12, and 24 h). An anti-estrogen, ICI 182780 (10–7 M) was used to examine the potential involvement of estrogen receptor (ER) in the induction of GH by EDs through an ER-mediated pathway. The data were analyzed by one-way ANOVA, followed by Tukey's multiple comparison. OP, NP, and BPA induced a significant increase in GH gene expression at high (10–5 M) and medium (10–6 M) doses at 24 h. ED-exposure induced a marked increase in GH gene transcription as early as 6 h and peaked at 12 h. Co-treatment with ICI 182780 significantly attenuated ED-induced GH expression in GH3 cells. Interestingly, the level of in vitro GH release was increased significantly at 24 h in response to OP, NP, or BPA, whereas co-treatment with ICI 182780 significantly diminished ED-induced GH secretion in GH3 cells, indicating that ER may play a part in both GH gene transcription and GH release in these cells. Here we demonstrate for the first time that single in vitro exposure to OP, NP, or BPA results in an increase in GH expression at 24 h in GH3 rat pituitary cells. These results may provide new insight into the mode of ED action in GH gene regulation as well as the biological pathway underlying these molecular events. Furthermore, data showing GH responsiveness evoked by EDs supports the aim to develop an assay for use in predicting adverse health effects of EDs in humans and wildlife.
177 EXPRESSION PROFILING OF HISTONE-MODIFYING GENES IN BOVINE PREIMPLANTATION EMBRYOS
Authors: Linger G. D. , Bormann C. L. , Peoples M. , Golding M. C. , Long C. R.
Published: January 2008

Global activation of the embryonic genome marks one of the most important aspects of early embryonic development and coincides with both global DNA and histone modifications necessary to control gene expression for further development. In bovine preimplantation embryos, epigenetic reprogramming occurs between fertilization and the blastocyst stage. De novo DNA methylation from the 8-cell stage to the morula represents activation of DNA methyltransferases and histone-modifying genes; however, it is still unclear how these critical events of early development and differentiation are regulated. In this preliminary study, we used quantitative real-time PCR (qRT-PCR) to examine stage-specific expression in bovine gametes (testis and ova) and preimplantation embryos of eight histone-modifying genes: LSH1 (which associates with both DNA and histone methyltransferases), LSD1 (a histone demethylase), SUV39H1, SUV420H1, SETB1, SUZ12, SMYD3, and G9a (all representative histone methyltransferases). Bovine ova and embryos were produced via in vitro maturation, fertilization, and culture from a single pool of ova as per standard laboratory protocol. Testes were harvested from a mature bull and immediately snap frozen in liquid nitrogen. RNA was isolated from testis, and groups of 10–15 in vitro-matured bovine ova or in vitro-produced embryos at the 2-, 8-cell, morula, and blastocyst stages and stored at –80�C until further use. cDNA was generated from each RNA sample using Superscript II" reverse transcriptase (Invitrogen, Carlsbad, CA, USA) under identical PCR conditions. Relative gene expression from each RNA sample was calculated in triplicate using the ΔΔCt method with both SYBR� Green and Taqman� qRT-PCR methods (Applied Biosystems, Foster City, CA, USA) and normalized to endogenous bovine GAPDH expression as per standard protocol. Due to excessive variation in a few of the qRT-PCR reactions, consistent data were collected for only SETB1, SUV39H1, SUV420H1, and G9a. SETB1 and G9a shared a similar expression pattern, with both exhibiting a dramatic relative increase in transcript level near the 8-cell stage and reduction to basal levels thereafter. Also, SUV39H1 and SUV420H1 were similar, showing a gradual increase in relative expression from the oocyte to the 8-cell stage, followed by a drop to low levels by the morula stage. These data indicate that several histone-modifying genes are expressed in distinct patterns during the critical period of zygotic genome activation and establishment of the epigenome in early preimplantation bovine embryos. Continuing studies will determine specific gene function through shRNA-induced silencing of histone-modifying genes and their role in control of gene expression.
178 RELATIVE ABUNDANCE OF Oct-4, Nanog, AND Sox-2 TRANSCRIPTS IN PORCINE OOCYTES AND CLEAVAGE-STAGE EMBRYOS PRODUCED VIA FERTILIZATION IN VITRO OR PARTHENOGENESIS
Authors: Magnani L. , Cabot R.
Published: January 2008

Parthenogenetic embryos obtained by electroactivation of mature oocytes have been used as models in developmental studies. The correct gene expression in early cleavage embryos is essential to sustain embryo development. The precise regulation of genes involved in pluripotency (Oct-4, Sox-2, and Nanog) is crucial to the formation of inner cell mass and trophoblast cells. Failure to do so can contribute to impaired development. We hypothesized that porcine embryos produced by fertilization in vitro and parthenogensis would possess a similar pattern of expression of Oct-4, Nanog, and Sox-2 during cleavage development. The objective of this study was to determine the developmental expression pattern of these three transcription factors in porcine oocytes and cleavage-stage embryos produced by either fertilization or parthenogenesis. Messenger RNAwas isolated from pools of 40-150 germinal vesicle (GV)- and MII-arrested oocytes and pools of 2-cell (2c), 4-cell (4c), 8-cell (8c), and blastocyst-stage embryos produced by in vitro fertilization (IVF) or electroactivation. Quantitative real-time PCR was performed following cDNA synthesis. Transcripts for Oct-4, Nanog, Sox-2, andYWHAG (housekeeping gene control) were amplified in duplicate across three to five experimental replicates. Transcripts were quantified using the comparative CT method using YWHAG as internal control and GV stage as normalizing stage. Fold activation and repression were analyzed with ANOVA and Tukey's post-hoc test. Our results show that porcine embryos produced by either IVF or electroactivation possess a similar pattern of pluripotent gene expression during cleavage-stage development. Oct-4 was found to be present in high abundance in the 2-cell parthenogenetic embryos and then repressed at the 8-cell stage (10-fold; P < 0.05, 2c v. 8c). In IVF embryos, Oct-4 was found in significantly higher amount at the 2-cell stage (35-fold; P < 0.05, 2c v. GV). Nanog transcripts were present at low levels from the GV oocyte until the 4-cell stage in both IVF and parthenogenetic embryos and then upregulated 10 000-fold at the 4-cell stage (P < 0.0001, GV v. 4c); at the blastocyst stage, Nanog transcript levels were similar to the levels found in the GV stage oocytes. Sox-2 transcripts were lower in MII oocytes and were significantly upregulated in 8-cell-stage embryos produced by either IVF or electroactivation (9- and 20-fold; P < 0.01, P < 0.0001, MII v. 8c, respectively). In addition, Sox-2 transcripts were significantly higher in parthenogenetic blastocysts compared to IVF-derived blastocysts (P < 0.05). This work demonstrates that cleavage-stage porcine embryos, produced by either electroactivation or IVF, undergo a similar pattern of activation of key regulatory genes; however, the activation method can have an influence on the transcript abundance of specific genes at defined stages.
188 EFFECTS OF CULTURE MEDIUM AND PROTEIN SUPPLEMENTATION ON mRNA EXPRESSION OF IN VITRO-PRODUCED PREIMPLANTATION BOVINE EMBRYOS
Authors: Purpera M. N. , Ballard C. B. , Giraldo A. M. , Hylan D. , Godke R. A. , Bondioli K. R.
Published: January 2008

Numerous studies have reported aberrant gene expression levels in embryos attributed to suboptimal culture conditions. This study investigated the effects of different culture systems and protein sources on the development of IVP embryos as measured by cleavage and blastocyst rates, cell number, and relative abundance levels of Oct-4, Connexin 43, Nanog, and glucose transporter-1 (Glut-1) when compared with in vivo embryos. Experiment (Exp) 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium (KSOMaa) supplemented with amino acids. Exp 2 compared the same two culture systems with and without the addition of calf serum (CS). Oocytes were matured for 22 h, fertilized in vitro, and then cultured in the appropriate treatment medium. RNA was extracted from pools of blastocysts, reverse transcribed to cDNA, and primer-specific amplified via Q-PCR. Exp 1 analyzed 10 pools per treatment, and either 10 (in vivo) or 11 pools were analyzed per treatment in Exp 2. One-way ANOVA followed by multiple pair-wise comparisons using Tukey's test was used to detect differences between treatments and in vivo embryos (P < 0.05). Results from both experiments indicated that, despite similar cleavage and blastocyst rates among treatments, significant differences were detected at the mRNA level and in cell numbers between treatments. In Exp 1, Oct-4 was found to have a mean abundance mRNA level significantly greater in KSOMaa-cultured blastocysts than in either SOFaa-cultured blastocysts or in vivo embryos. The same pattern of upregulation of Oct-4 in KSOMaa or KSOMaa with CS-cultured blastocysts was detected in Exp 2. In contrast to that reported by others, Connexin 43 was not expressed at detectable levels in the in vivo embryos analyzed in our studies. Connexin 43 was not detected in IVP blastocysts used in Exp 1. Conversely, Connexin 43 was detected in KSOMaa, SOFaa, and SOFaa with CS-cultured blastocysts in Exp 2. There was no significant difference in expression of the ICM-specific transcript Nanog in either experiment. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of Glut-1 when compared with other treatments and in vivo embryos. Overall, the transcript levels of the majority of the genes analyzed were altered by the in vitro culture condition. Differences continue to be observed between in vitro-cultured and in vivo embryos, and until these differences are minimized, aberrations in in vitro development will continue to arise. Further research will possibly modify the current culture conditions, allowing for the production of in vitro embryos of higher developmental potential similar to that observed in in vivo-derived embryos.
214 INFLUENCE OF THE BREED OF BULL (GIR v. HOLSTEIN) ON THE RESISTANCE OF BOVINE EMBRYOS TO HEAT SHOCK AT EARLY STAGES OF IN VITRO DEVELOPMENT
Authors: Saito D. Y. , Satrapa R. A. , Romão R. M. , Nabhan T. , Simões R. A. L. , Barros C. M.
Published: January 2008

Heat shock (HS) has negative effects on pregnancy rates in lactating dairy cows. There are genetic differences in tolerance to HS, with Bos indicus cattle and embryos being more resistant to elevated temperatures than Bos taurus. Recently, Pegorer et al. (2007 Theriogenology 67, 692–697) observed that Gir semen (Bos indicus), when compared with Holstein semen (Bos taurus), increased pregnancy rates of lactating Holstein cows during the Brazilian summer. The objective of the present study was to evaluate the influence of breed of bull (Gir or Holstein) on the resistance of embryos to HS during the early stages of in vitro development. Oocytes from Holstein ovaries obtained from a local abattoir were matured in TCM 199 medium, fertilized with semen from 1 of 12 bulls (6 Gir and 6 Holstein), and cultured in synthetic oviduct fluid to the blastocyst stage. Ninety-six hours after insemination (96 hpi), embryos with less than 16 cells were discarded; half of the embryos (e16 cells) that were fertilized by each bull were submitted to HS (41�C for 12 h) and the other half (e16 cells) were maintained at 39�C (control group). After the HS period, embryos were maintained at 39�C until the end of the experiment. Six semen straws from different Gir bulls (Bos indicus) and 6 straws from Holstein bulls (Bos taurus) were used, in a total of 6 assays, to minimize the bull effect. Each breed of bull (Gir or Holstein) was used to fertilize a total of at least 300 Holstein oocytes per treatment (control v. HS). Cleavage (48 hpi), blastocyst (144 hpi), and hatched blastocyst (216 hpi) rates of embryos submitted or not submitted to HS were analyzed by PROC GLM of SAS (SAS Institute Inc., Cary, NC, USA) to adjust the model of ANOVA, with each combination of assays used as a block. The proportion data were arcsine transformed to study the effects of breed, treatment, and their interaction. There was no difference in cleavage rate when Gir semen was used to fertilize oocytes from Holstein cows (Gir � Holstein; 76.6%) as compared with Holstein � Holstein (75.5%). However, HS significantly decreased (P < 0.01) blastocyst rates (in relation to embryos e16 cells) from both Gir (74 and 62.6%, control and HS, respectively) and Holstein bulls (69 and 55%, respectively). The same was observed for hatched blastocysts from Gir (42 and 28.3%) and Holstein bulls (37 and 25%). Nevertheless, there was no interaction of breed of bull � treatment (HS) for blastocysts (P > 0.09) and hatched blastocysts (P > 0.1). It was concluded that the breed of bull used in the present experiment (Gir v. Holstein) did not increase the resistance of bovine embryos to HS at early stages of in vitro development.
221 PREPUBERTAL MOUSE BIOASSAY FOR OVULATION-INDUCING FACTOR IN SEMINAL PLASMA
Authors: Bogle O. A. , Ratto M. H. , Adams G. P.
Published: January 2008

A substance in the seminal plasma of llamas and alpacas has been discovered that induces ovulation and growth of the corpus luteum (CL) in the female of the same species. The ovarian effects of the ovulation-inducing factor (OIF) are associated with a surge release of LH into circulation. Ultrasonographic detection of ovulation and CL development is currently the only method available for testing the bioactive effects of OIF. The purpose of this study was to determine if a superstimulatory prepubertal mouse model could be developed as an in vivo bioassay for OIF. Prepubertal female CD1 mice (n = 144), 20 days of age and weighing 20–25 g, were housed at 24�C with lights on from 0500 to 1900 h and free access to food and water. An intramuscular dose of 5 IU of eCG (Novormon, Bioniche Animal Health, Belleville, ON, Canada) was given (Day 0) for ovarian superstimulation. On Day 2, mice were assigned randomly to 4 groups (n = 36 per group) and given a single 0.1 mL intraperitoneal dose of (1) 5 IU of hCG (Chorulon, Intervet Canada, Ltd., Whitby, ON, Canada), (2) 5 µg GnRH (gonadotropin-releasing hormone: Cystorelin, Merial, Ltd., Iselin, NJ, USA), (3) llama seminal plasma, or (4) phosphate-buffered saline (negative control). On Day 3, females were euthanized by an overdose of inhaled halothane. Oviducts were collected and oocytes were counted using trans-illumination stereomicroscopy. The proportion of mice that ovulated did not differ among groups treated with hCG, GnRH, and seminal plasma (31/36, 31/36, 28/36, respectively); however, the proportion of mice that ovulated in each treatment group was greater than that in the saline-treated group (9/36) (P < 0.001). The number of oocytes counted (mean � SEM) was also similar among groups treated with hCG (25.8 � 2.9), GnRH (27.4 � 2.7), and seminal plasma (19.2 � 2.8), all of which were greater (P < 0.01) than in the saline-treated group (6.2 � 2.1). We conclude that the superstimulated prepubertal CD1 mouse model is effective as an in vivo bioassay for OIF in seminal plasma. Whether the bioassay may be used for quantitative estimates of OIF activity will require dose-response trials using serial dilutions of seminal plasma.
245 EFFECTS OF MELATONIN ON THE IN VITRO OOCYTE MATURATION OF PORCINE CUMULUS - OOCYTE COMPLEXES
Authors: Kang J. K. , Koo O. J. , Kwon D. K. , Jang G. , Kang S. K. , Kim D. Y. , Lee B. C.
Published: January 2008

In mammalian species, melatonin is a multifunctional hormone that mediates several circadian and seasonal processes, including reproduction. In addition to its hormonal actions, melatonin has a strong antioxidant effect and acts as a powerful free radical scavenger. Accordingly, the objective of this study was to investigate the local expression of the melatonin-receptor I (Mel-RI) gene and the effect of melatonin as an antioxidant on the in vitro maturation of porcine cumulus–oocyte complexes. Oocytes were obtained from aspiration of slaughterhouse ovaries and then matured in tissue culture medium 199 (TCM199) supplemented with 0.1% polyvinyl alcohol (PVA), 20 ng mL–1 epidermal growth factor (EGF), and 4 IU mL–1 pregnant mare serum gonadotropin (PMSG)/hCG. Expression of Mel-RI gene in cumulus cells, granulosa cells, or oocytes was evaluated by RT-PCR (Exp 1). The effect of various concentrations (0, 10, 50, and 100 ng mL–1) of melatonin on the in vitro maturation in terms of nuclear maturation (polar body extrusion) (Exp 2) and measurement of reactive oxygen species (ROS) by dichlorohydrofluorescein diacetate (Yang et al. 1998 Hum. Reprod. 13, 998–1002) (Exp 3) were investigated. Each experiment was replicated at least four times, and each replication included more than thirty oocytes. For statistical analysis, one-way anova in the Prizm software program (GraphPad, San Diego, CA, USA) was used. The Mel-RI gene was locally expressed in cumulus and granulosa cells but not in oocytes. Melatonin at 10 ng mL–1 had a beneficial effect on in vitro maturation (84.6%) compared to 0 ng mL–1 (75.6%), 50 ng mL–1 (80.9%), and 100 ng mL–1 (76.5%). Levels of ROS were also significantly decreased by melatonin treatment at 10 (14.07), 50 (14.42), and 100 ng mL–1 (13.03) of melatonin (P < 0.001) compared to that of the control group (28.21). The presence of melatonin receptor in cumulus cells and granulosa cells indicates a potentially important role for this hormone in regulating porcine ovarian and reproduction function. In conclusion, the present study demonstrated that melatonin had a beneficial effect on in vitro maturation of porcine cumulus–oocyte complexes, probably through binding a receptor and decreased ROS production. Further studies are required to elucidate the relationship between the appearance of melatonin receptor and mechanisms of melatonin function in the embryo.
274 LYSOLECITHIN TREATMENT OF ELAND, BONGO, AND BOVINE SPERMATOZOA AND CLEAVAGE OF BOVINE OOCYTES AFTER INTERSPECIES INTRACYTOPLASMIC SPERM INJECTION
Authors: Wirtu G. , Pope C. E. , Gomez M. C. , MacLean R. A. , Paccamonti D. L. , Dresser B. L.
Published: January 2008

Compared to success rates in human, intracytoplasmic sperm injection (ICSI) is inefficient in ungulate species. Although factors such as injection of membrane-intact sperm and toxic effects of acrosome contents are suspected causes, the reasons for the inefficiency are unclear. A recent report in mice demonstrated that ICSI using spermatozoa treated with a physiological detergent, lysolecithin, improved oocyte activation, cleavage, and offspring production after embryo transfer (Morozumi K et al. 2006 PNAS 109, 17 661–17 666). The objectives of the present study were to evaluate the effects of detergent treatment on motility and membrane integrity of frozen thawed eland, bongo and bovine spermatozoa and to examine sperm decondensation/embryonic cleavage following ICSI of in vitro-matured bovine oocytes. In experiment 1, sperm motility was observed on a warm microscope stage during exposure to 3 lecithin concentrations, 0.04, 0.02, and 0.01%, and the time at which 100% of the spermatozoa lost motility was recorded. In experiment 2, spermatozoa were exposed to 0.02% lecithin for 22 s, and the membrane integrity and acrosome status of spermatozoa were determined using a combined trypan blue-Giemsa staining (Nagy et al. 1999 Theriogenology 52, 1153–1159). In experiment 3, bovine oocytes were injected, using the piezo drill, with lecithin-treated (0.02%, immobilized) or untreated (piezo pulse immobilized) eland, bongo, or bovine spermatozoa and subsequently cultured for 2 days in CR1aa containing 3 mg mL–1 BSA. Each experiment was replicated at least 3 times. Lecithin induced time- and concentration-dependent loss of sperm motility. The average time to loss of motility in 100% of the spermatozoa at 0.04, 0.02, and 0.01% lecithin was 107, 222, and 344 s in bovine; 82, 135, and 179 s in eland; and 65, 115, and 158 in bongo, respectively. Data on membrane integrity (intact or nonintact) and acrosome status (reacted or nonreacted) of detergent-treated or control spermatozoa are shown in Table 1. Sperm head decondensation and embryonic cleavage were observed following homologous and interspecies (antelope into bovine) ICSI of lecithin-treated or control spermatozoa. In conclusion, lecithin treatment induced concentration and time-dependent loss of motility and was effective in damaging the sperm membrane and acrosome in eland, bongo, and domestic bulls. Eland and bongo spermatozoa underwent decondensation and activated bovine oocytes after interspecies ICSI.

Table 1.
4 IMPAIRED INCRETIN EFFECT IN TRANSGENIC PIGS EXPRESSING A DOMINANT NEGATIVE RECEPTOR FOR GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE IN THE PANCREATIC ISLETS
Authors: Renner S. , Keßler B. , Herbach N. , von Waldthausen D. C. , Wanke R. , Hofmann A. , Pfeifer A. , Wolf E.
Published: January 2008

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by specific cell types in the intestine and are responsible for the so-called incretin effect, the phenomenon that an oral glucose load elicits a higher insulin response than does an intravenous glucose load. In patients with type 2 diabetes mellitus the overall incretin effect is reduced. This fact is mostly attributed to a lowered insulinotropic effect of GIP, while the effect of GLP-1 is preserved. In order to better understand the consequences of impaired function of GIP, knockout mice lacking a functional GIP receptor (GIPR–/–) as well as transgenic mice expressing a dominant negative GIPR (GIPRdn) were established. While GIPR–/– mice show only relatively mild changes in glucose homeostasis, GIPRdn mice display a distinct diabetic phenotype due to disturbed development of the endocrine pancreas (Herbach et al. 2005 Regul. Pept. 125, 103–117). To further clarify the underlying mechanisms, we used a novel, highly efficient gene transfer technology based on lentiviral vectors (Hofmann et al. 2003 EMBO Rep. 4, 1054–1060; Hofmann et al. 2006 Mol. Ther. 13, 59–66) to generate transgenic pigs expressing a GIPRdn under the control of the rat Ins2 promoter (RIP). RIP-GIPRdn transgenic pigs develop normally and do not display diabetes mellitus up to at least one year of age. Weekly measured fasting blood glucose levels in transgenic animals did not show a significant difference compared to control pigs. The same was true for monthly determined fructosamine levels. However, RIP-GIPRdn transgenic pigs exhibited reduced insulin release and higher glucose levels than non-transgenic littermate controls in an oral glucose tolerance test. The area under the curve (AUC) for insulin was 49% smaller (P < 0.01) and the AUC for glucose 26% larger (P < 0.05) in RIP-GIPRdn transgenic pigs (n = 5) than in their non-transgenic littermate controls (n = 5). These findings demonstrate that expression of a GIPRdn, which was shown by RT-PCR in isolated pancreatic islets, disturbs the function of GIP in transgenic pigs. Thus we have created a novel, clinically relevant animal model for studying the roles of the GIP/GIPR system. Quantitative morphological studies of the pancreas are being performed to clarify whether GIPR function is essential for pancreatic islet development and maintenance.
81 INDUCED BLASTOCOEL COLLAPSE IMPROVES SURVIVAL RATES OF PORCINE BLASTOCYSTS AFTER VITRIFICATION
Authors: Lin L. , Du Y. , Kragh P. M. , Li J. , Bolund L. , Yang H. , Zhang X. , Kuwayama M. , Vajta G.
Published: January 2008

Cryopreservation of porcine blastocysts is regarded as a demanding task because of the high chilling sensitivity and low survival rates. Ultrarapid vitrification techniques combined with the previous artificial induction of collapse of the blastocoel have been successfully applied in mouse and human. The purpose of our work was to use the Cryotop (Kitasato Supply Co., Tokyo, Japan) vitrification method combined with induced blastocyst collapse for cryopreservation of porcine blastocysts produced with parthenogenetic activation of in vitro-matured oocytes. A total of 1200 slaughterhouse-derived porcine oocytes were matured for 41–42 h. Zona-intact or zona-free oocytes (after pronase digestion) were activated with a single electric impulse of 63 or 43 V cm–1, respectively, and incubated in 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide for 4 h (Du et al. 2007 Cryobiology 54, 181–187). Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O2, and 90% N2 and maximum humidity. On Day 6, collapse was induced manually in approximately half of the blastocysts with the use of a fire-polished holding pipette and injection pipette (Cook Australia, Brisbane, Queensland, Australia). Ten min after the manipulation, collapsed and control (not manipulated) blastocysts were vitrified with the Cryotop, and then warmed and diluted in 1, 0.5, and 0 m sucrose, respectively (Kuwayama et al. 2005, Reprod. BioMed. Online 11, 300–308). Reexpansion of blastocysts was evaluated under a stereomicroscope after 16–18 h of culture under conditions described above. SPSS 11.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical calculations; values with P < 0.05 were regarded as significant. Reexpansion rates of blastocysts are shown in Table 1. Our results indicate that manually induced collapse of the blastocoel increases reexpansion rates of both zona-intact and zona-free porcine blastocysts after Cryotop vitrification. The combination of these simple techniques may help to improve the efficiency of cryopreservation of pig embryos for both experimental and practical applications.

Table 1. Reexpansion rates after vitrification of blastocysts with or without induced blastocoel collapse

The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen, and ClausWillemoes for their help and excellent technical assistance.
98 VITRIFICATION OF ZYGOTES SUPPORTS FULL-TERM DEVELOPMENT IN RABBITS
Authors: Xu J. , Zhang J. , Chen Y. , Cater M. , Yang X. , Du F.
Published: January 2008

Rabbit can serve as an excellent model for human reproduction and relevant disease study. The objective of the study was to develop an effective procedure to preserve the rabbit genome by vitrifying zygotes. Sexually matured New Zealand White rabbits maintained under a 16 h light:8 h dark cycle were superovulated with a regime of two 0.3-mg, two 0.4-mg, and two 0.7-mg injections of FSH with an interval of 12 h between, followed by an injection of 200 IU hCG and mating. Presumptive zygotes were flushed from the oviducts and collected by midventral lapartomy 18 h after hCG injection. One-cell-stage embryos were assigned to pre-equilibration at 38.5�C in HEPES-buffered TCM199 supplemented with 20% FBS + 7.5% ethylene glycol (EG) (v/v) + 7.5% dimethylsulphoxide (DMSO) (v/v) (dehydration medium) for 3 min, and subsequently to 1.0 ml of vitrification medium (TCM199 supplemented with 20% FBS + 20% EG and 20% DMSO), as described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58), at room temperature for 3 min. Five to six embryos per group were then vitrified in a micro-droplet (approximately 1–2 µL) by directly dropping them into a thin layer of liquid nitrogen on the solid surface that generated a super cold surface for vitrification. Vitrified embryos were sequentially warmed, rehydrated in 20% FBS M199 with different concentrations of sucrose, and washed in 20% FBS M199 for 5 min. Warmed embryos were assigned either for further in vitro culture or for embryo transfer to test corresponding developmental potentials. Dutch rabbits were used as recipients in the procedure of asynchronous ovulation induction (22-h delay to zygote donors) by an intramuscular injection of 15 µg of GnRH per doe. Three or four warmed zygotes were surgically transferred into the recipients on the same day of thawing. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post-embryo transfer (ET); C-sections were performed on Day 31 to retrieve full-term developed newborns. Preliminary results showed that a 96% (n = 35) post-warming survival rate was achieved with vitrified rabbit zygotes; subsequent cleavage and blastocyst development were 41.6 and 33.3%, respectively. Transfer of a total of 23 warmed embryos into 6 recipients resulted in 5 pregnancies (83.3%, 5/6). Five live kits (21.7%, 5/23) were delivered. Our study suggests that vitrification of rabbit zygotes is a feasible approach for preserving rabbit genetic material. The establishment of suitable conditions for the vitrification of rabbit zygotes could be useful as a model for vitrification of human 1-celled embryos.
The authors thank the staff of the animal facility at the University of Connecticut for animal care and maintenance. This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261-01.
Counting membrane-embedded KCNE β-subunits in functioning K
+
channel complexes
Authors: Morin Trevor J. , Kobertz William R.
Published: January 2008

Ion channels are multisubunit proteins responsible for the generation and propagation of action potentials in nerve, skeletal muscle, and heart as well as maintaining salt and water homeostasis in epithelium. The subunit composition and stoichiometry of these membrane protein complexes underlies their physiological function, as different cells pair ion-conducting α-subunits with specific regulatory β-subunits to produce complexes with diverse ion-conducting and gating properties. However, determining the number of α- and β-subunits in functioning ion channel complexes is challenging and often fraught with contradictory results. Here we describe the synthesis of a chemically releasable, irreversible K
+
channel inhibitor and its iterative application to tally the number of β-subunits in a KCNQ1/KCNE1 K
+
channel complex. Using this inhibitor in electrical recordings, we definitively show that there are two KCNE subunits in a functioning tetrameric K
+
channel, breaking the apparent fourfold arrangement of the ion-conducting subunits. This digital determination rules out any measurable contribution from supra, sub, and multiple stoichiometries, providing a uniform structural picture to interpret KCNE β-subunit modulation of voltage-gated K
+
channels and the inherited mutations that cause dysfunction. Moreover, the architectural asymmetry of the K
+
channel complex affords a unique opportunity to therapeutically target ion channels that coassemble with KCNE β-subunits.
A phosphopantetheinylating polyketide synthase producing a linear polyene to initiate enediyne antitumor antibiotic biosynthesis
Authors: Zhang Jian , Van Lanen Steven G. , Ju Jianhua , Liu Wen , Dorrestein Pieter C. , Li Wenli , Kelleher Neil L. , Shen Ben
Published: January 2008

The enediynes, unified by their unique molecular architecture and mode of action, represent some of the most potent anticancer drugs ever discovered. The biosynthesis of the enediyne core has been predicted to be initiated by a polyketide synthase (PKS) that is distinct from all known PKSs. Characterization of the enediyne PKS involved in C-1027 (SgcE) and neocarzinostatin (NcsE) biosynthesis has now revealed that (
i
) the PKSs contain a central acyl carrier protein domain and C-terminal phosphopantetheinyl transferase domain; (
ii
) the PKSs are functional in heterologous hosts, and coexpression with an enediyne thioesterase gene produces the first isolable compound, 1,3,5,7,9,11,13-pentadecaheptaene, in enediyne core biosynthesis; and (
iii
) the findings for SgcE and NcsE are likely shared among all nine-membered enediynes, thereby supporting a common mechanism to initiate enediyne biosynthesis.
Uses of Coercion in Addiction Treatment: Clinical Aspects
Authors: Sullivan Maria A. , Birkmayer Florian , Boyarsky Beth K. , Frances Richard J. , Fromson John A. , Galanter Marc , Levin Frances R. , Lewis Collins , Nace Edgar P. , Suchinsky Richard T. , Tamerin John S. , Tolliver Bryan , Westermeyer Joseph
Published: January 2008

Coerced or involuntary treatment comprises an integral, often positive component of treatment for addictive disorders. By the same token, coercion in health care raises numerous ethical, clinical, legal, political, cultural, and philosophical issues. In order to apply coerced care effectively, health care professionals should appreciate the indications, methods, advantages, and liabilities associated with this important clinical modality. An expert panel, consisting of the Addiction Committee of the Group for the Advancement of Psychiatry, listed the issues to be considered by clinicians in considering coerced treatment. In undertaking this task, they searched the literature using Pubmed from 1985 to 2005 using the following search terms: addiction, alcohol, coercion, compulsory, involuntary, substance, and treatment. In addition, they utilized relevant literature from published reports. In the treatment of addictions, coercive techniques can be effective and may be warranted in some circumstances. Various dimensions of coercive treatment are reviewed, including interventions to initiate treatment; contingency contracting and urine testing in the context of psychotherapy; and pharmacological methods of coercion such as disulfiram, naltrexone, and the use of a cocaine vaccine. The philosophical, historical, and societal aspects of coerced treatment are considered.
Jenti: an efficient tool for mining complex inbred genealogies
Authors: Falchi Mario , Fuchsberger Christian
Published: January 2008

Summary: An efficient tool for mining complex inbred genealogies that identify clusters of individuals sharing the same expected amount of relatedness is described. Additionally it allows for the reconstruction of sub-pedigrees suitable for genetic mapping in a systematic way.

Availability:  http://www.jenti.org

Contact:  m.falchi@imperial.ac.uk
Testing for genetic trade-offs between early- and late-life reproduction in a wild red deer population
Authors: Nussey Daniel H , Wilson Alastair J , Morris Alison , Pemberton Josephine , Clutton-Brock Tim , Kruuk Loeske E.B
Published: January 2008

The antagonistic pleiotropy (AP) theory of ageing predicts genetically based trade-offs between investment in reproduction in early life and survival and performance in later life. Laboratory-based research has shown that such genetic trade-offs exist, but little is currently known about their prevalence in natural populations. We used random regression ‘animal model’ techniques to test the genetic basis of trade-offs between early-life fecundity (ELF) and maternal performance in late life in a wild population of red deer (
Cervus elaphus
) on the Isle of Rum, Scotland. Significant genetic variation for both ageing rates in a key maternal performance measure (offspring birth weight) and ELF was present in this population. We found some evidence for a negative genetic covariance between the rate of ageing in offspring birth weight and ELF, and also for a negative environmental covariance. Our results suggest rare support for the AP theory of ageing from a wild population.
Particle acceleration by electromagnetic waves
Authors: Bingham R
Published: January 2008

We consider the symmetry in the interaction of photons and electrons, which has led to a common description of electron and photon accelerations; effects such as photon Landau damping arise naturally from such a treatment. Intense electromagnetic waves can act as a photon mirror to charged particles. The subsequent acceleration is equivalent to the photon pulse accelerating electrons. During the interaction or reflection process, the charged particle can emit bursts of radiation similar to the radiation emitted from the particles during wave breaking of plasma waves.
Underwater digital holography for studies of marine plankton
Authors: Sun H , Benzie P.W , Burns N , Hendry D.C , Player M.A , Watson J
Published: January 2008

Conventional and digital holographies are proving to be increasingly important for studies of marine zooplankton and other underwater biological applications. This paper reports on the use of a subsea digital holographic camera (eHoloCam) for the analysis and identification of marine organisms and other subsea particles. Unlike recording on a photographic film, a digital hologram (e-hologram) is recorded on an electronic sensor and reconstructed numerically in a computer by simulating the propagation of the optical field in space. By comparison with other imaging techniques, an e-hologram has several advantages such as three-dimensional spatial reconstruction, non-intrusive and non-destructive interrogation of the recording sampling volume and the ability to record holographic videos. The basis of much work in optics lies in Maxwell's electromagnetic theory and holography is no exception: we report here on two of the numerical reconstruction algorithms we have used to reconstruct holograms obtained using eHoloCam and how their starting point lies in Maxwell's equations. Derivation of the angular spectrum algorithm for plane waves is provided as an exact method for the in-line numerical reconstruction of digital holograms. The Fresnel numerical reconstruction algorithm is derived from the angular spectrum method. In-line holograms are numerically processed before and after reconstruction to remove periodic noise from captured images and to increase image contrast. The ability of the Fresnel integration reconstruction algorithm to extend the reconstructed volume beyond the recording sensor dimensions is also shown with a 50% extension of the reconstruction area. Finally, we present some images obtained from recent deployments of eHoloCam in the North Sea and Faeroes Channel.
Predicting the properties of micro- and nanocomposites: from the microwhiskers to the bristled nano-centipedes
Authors: Guz I.A , Rodger A.A , Guz A.N , Rushchitsky J.J
Published: January 2008

The paper draws on the similarities between the well-known process of whiskerization of microfibres and the recent idea of bristled nanowires. The new method for evaluation of the effective elastic properties of such materials is suggested based on the model of four-component composition. This model assumes the transverse isotropy of continuum and predicts five elastic moduli and density as independent effective constants. An example of calculation of the constants for the particular materials is given. It shows the significant increase in the shear strength of composites with whiskerized or bristled fibres.
Analysis of theArabidopsis O-Acetylserine(thiol)lyase Gene Family Demonstrates Compartment-Specific Differences in the Regulation of Cysteine Synthesis
Authors: Heeg Corinna , Kruse Cordula , Jost Ricarda , Gutensohn Michael , Ruppert Thomas , Wirtz Markus , Hell Rüdiger
Published: January 2008

Cys synthesis in plants takes place in plastids, cytosol, and mitochondria. Why Cys synthesis is required in all compartments with autonomous protein biosynthesis and whether Cys is exchanged between them has remained enigmatic. This question was addressed using Arabidopsis thaliana T-DNA insertion lines deficient in the final step of Cys biosynthesis catalyzed by the enzyme O-acetylserine(thiol)lyase (OAS-TL). Null alleles of oastlA or oastlB alone showed that cytosolic OAS-TL A and plastid OAS-TL B were completely dispensable, although together they contributed 95% of total OAS-TL activity. An oastlAB double mutant, relying solely on mitochondrial OAS-TL C for Cys synthesis, showed 25% growth retardation. Although OAS-TL C alone was sufficient for full development, oastlC plants also showed retarded growth. Targeted affinity purification identified the major OAS-TL–like proteins. Two-dimensional gel electrophoresis and mass spectrometry showed no compensatory changes of OAS-TL isoforms in the four mutants. Steady state concentrations of Cys and glutathione and pulse-chase labeling with [35S]sulfate indicated strong perturbation of primary sulfur metabolism. These data demonstrate that Cys and also sulfide must be sufficiently exchangeable between cytosol and organelles. Despite partial redundancy, the mitochondria and not the plastids play the most important role for Cys synthesis in Arabidopsis.