bluuebunny/crossref_metadata_2025 huggingface dataset:
297A METHOD TO DRIVE CALCIUM SIGNALLING DYNAMICS IN FERTILIZED MOUSE EGGS
Authors: Banrezes B. , Toth S. , Huneau D. , Schultz R. , Ozil J.-P.
Published: February 2004

Following fertilization, eggs exhibit a series of repetitive increases in intracellular calcium that activate development. The developmental impact of the long-lasting series of Ca2+ signals is still a subject of controversy. Although several studies using parthenogenetically activated eggs suggest that Ca2+ dynamics affect post-implantation development, artificial stimulation of Ca2+ signaling after ICSI in bovine eggs shows that development still remains poor in comparison to fertilized eggs. Such divergence between parthenogenetic studies and those aimed at stimulating ICSI eggs makes it impossible to draw any conclusions regarding the function of Ca2+ signaling for two reasons. First, non-fertilized eggs do not release Ca2+ from intracellular stores and their development is compromised due to the absence of paternally-derived chromosomes. Second, because ICSI eggs are excitable, Ca2+ stimulation generates additional Ca2+ oscillations that might compromise their development. Moreover, in both cases, Ca2+ signaling is not physiological. To understand better the function of Ca2+ signaling at fertilization, we developed a new approach based on micro fluidic technology that makes it possible to drive Ca2+ signal dynamics of fertilized eggs with no apparent deleterious effects. This method relies on the fact that the properties of the IP3 receptor (IP3R) calcium channel are changed after fertilization, and IP3 and Ca2+ act as co-agonists to cause Ca2+-induced Ca2+ release (CICR) from intracellular stores. Because Ca2+ has both an inhibiting and a stimulating function, we exploited these opposing properties. First, we inhibited Ca2+ release by external washing with Ca2+-free medium;; this extra cellular washing decreases cytosolic [Ca2+]I, and facilitates dissociation of Ca2+ ions from the IP3R that in turn decreases the probability of IP3R channel opening. Second, once the IP3R is inhibited, a simple injection of Ca2+ ions by electropermeabilization triggers channel opening and induces Ca2+ release. Then, by just varying the time interval and the number of the electrical pulses, it is possible to drive the dynamics of the CICR process that initiates development. Intracellular Ca2+ imaging demonstrated that fertilized eggs subjected to 24 electrical pulses (1.45kVcm−1) every 8min for 3h in the microfluidic processor responded by exhibiting 24 induced-Ca2+ transients that are caused by calcium release from intracellular stores. All auto-regenerative responses between pulses were inhibited. Among 60 treated embryos transferred to pseudo-pregnant recipients, 40 (67%) developed to term, with birth of live offspring, thus demonstrating that this new methodology does not compromise development. Because the eggs are fertilized, it now becomes possible to study the function of Ca2+ signaling during egg activation and to evaluate its developmental impact, if any, in association with genomic approaches.
298RED DEER (CERVUS ELAPHUS) PARTHENOGENETIC BLASTOCYSTS PRODUCED
USING IONOMYCIN/6DMAP ACTIVATION
Authors: Beaumont S.E. , Berg D.K. , Asher G.W.
Published: February 2004

Successful activation of red deer oocytes is a necessary prerequisite for the cloning of red deer individuals with desirable genetic characteristics. To investigate this, an established biphasic protocol used for oocyte activation in sheep was investigated for suitability. The method chosen was 5μM Ionomycin for 5min followed by 2mM 6DMAP for 3h ( Loi P et al., 1998 Biol. Reprod. 58, 1177–1187). The medium used during activation and subsequent culture was Deer Synthetic Oviduct Fluid, which has been shown to support routine in vitro fertilization and blastocyst development (15%) of in vitro-matured red deer oocytes (DSOF, Berg D et al., 2003 Theriogenology 59, 189–205). Red deer abattoir-derived COCs were matured in vitro for 22h before random allocation across 3 treatment groups comprising a standard IVF group, the activation group and a negative control group exposed to medium only. Activation treatment oocytes were stripped of cumulus by vortexing in 0.1% hyaluronidase before selecting for first polar body extrusion. First-step activation was performed in medium comprising HEPES-buffered IVF-DSOF containing 4mM Ca2+. Second-step activation used 3mM Ca2+ early DSOF under 7% O2, 5% CO2, and 88% N2 at 38.5°C. Standard IVF was conducted at 23h post-IVM using 4mM Ca2+ IVF-DSOF and 0.5×106mL−1 final sperm concentration. Following activation and IVF, oocytes were washed 3 times in HEPES DSOF before culture for 7 days in sequential DSOF with late DSOF on Day 4 containing 1.5mM Ca2+. Cleavage was assessed 24h after activation, and all blastocysts were fixed for cell counts. Four replicates of each treatment were performed. Cleavage and blastocyst rates were examined by chi-square analysis and cell numbers by ANOVA. First polar body extrusion rate was 84%. Cleavage was similar between the activation treatment and IVF (P>0.05 ); but a significant difference was found in blastocyst development rates (P<0.05) with the Ionomycin and 6DMAP protocol being superior to the IVF treatment. Exposure to high Ca2+ media alone resulted in only 5% of the negative control oocytes cleaving to 2 cells. Results show that Ionomycin and 6DMAP are effective in activating red deer oocytes and DSOF is a suitable medium to produce parthenogenetic blastocysts.
299OOCYTE ACTIVATION IS THE RESULT OF CALCIUM SIGNAL SUMMATION
Authors: Huneau D. , Toth S. , Ozil J.-P.
Published: February 2004

Egg activation in mammals is caused by a series of cytosolic Ca2+ oscillations that are essential for development. However, the specific functional significance of the number or the frequency of the Ca2+ signals is not clear. The objective of this study was to determine whether the efficiency of egg activation relies on the frequency or on the number of signals. Given the repetitive nature of the signal, it is nearly impossible to discriminate the specific effect of the frequency from the number because increasing the time interval between signals directly reduces their number in a given time unit and vice versa. To bypass such difficulty, we subjected non-fertilized mouse oocytes, which are not capable of releasing Ca2+ from intracellular stores, to a coherent series of four treatments. Repetitive intracellular Ca2+ signals were introduced into freshly ovulated mouse oocytes by electropermeabilisation of Ca2+ ions by means of a microfluidic processor . The first regime (T1) was a monotonic [Ca2+]i increase lasting about 10min and provoked by 4 consecutive electrical pulses (1.45kVcm−1) given at 2-min intervals. This large calcium signal gave a poor activation rate: 6% (4/67). The second regime (T2) was made up of six lower pulses (1.12kVcm−1) given at 30-min intervals. The rate of egg activation reached 38% (75/195). The third treatment (T3) consisted in subjecting oocytes consecutively to the T1 and the T2 treatments. In that case, the egg activation reached 88% (121/138). The remarkable increase in egg activation efficiency can be attributed to the increase in the total [Ca2+]i load, which is the only change in T3. Thus, it appears that the large monotonic increase (T1) potentiates the response as revealed by the periodic stimulation delivered by T2. It therefore seems that the process downstream of the signal is incremental in nature, and entrance in interphase of the cell cycle is reached when the summation of individual signals reaches a sufficient level. We next evaluated whether the putative summation effect makes it possible to regularly get 100% of egg activation by increasing the cytosolic Ca2+load. Oocytes were subjected to a last treatment (T4) that consisted of T1 and then 24 pulses (1.45kVcm−1) given every 8min. In that case 100% of the oocytes were activated (80/80). Thus, it appears that all oocytes are subject to egg activation if they receive a sufficient amount of calcium signal. Moreover, the frequency does not seem to play a crucial role for entrance into interphase because reducing the time interval from 30min (T3) to 8min (T4) resulted in high activation rates in both cases (88% and 100%, respectively). The question remains as to whether the distribution of the Ca2+ signal throughout the process of egg activation might affect some epigenetic modifications that impact developmental processes.
30USE OF ADULT STEM/PROGENITOR CELLS AS NUCLEAR DONORS TO PRODUCE CLONED PORCINE EMBRYOS
Authors: Bosch P. , Pratt S.L. , Sherrer E. , Hodges C.A. , Ivy Hill E. , Kachline E. , Stice S.L.
Published: February 2004

Incomplete or defective nuclear reprogramming may be responsible for low cloning efficiencies. Less differentiated stem cells are thought to be more easily reprogrammed, resulting in improved survival of cloned mice (Rideout WM III et al., 2000 Nat. Genet. 24, 109–110). Our objective was to establish porcine mesenchymal stem cell (MSC) cultures and use these as donor cells in nuclear transfer (NT). A bone marrow (BM) aspirate was collected from an anesthetized gilt. BM mononuclear cells were isolated by centrifugation over a density gradient (Histopaque-1077; Sigma, St. Louis, MO, USA), resuspended in low glucose DMEM (Gibco) plus 10% FBS and plated on flasks; fibroblast-like MSCs were later passaged. Ear skin fibroblast (SF) cultures from the same BM donor gilt were established. Cultures of MSC and SF were exposed to lipogenic, osteogenic or chondrogenic differentiation media (Pittenger MF et al., 1999 Science 284, 143–147) for 14 days. Cells cultured in DMEM with 10% FBS served as controls. Differentiation was assessed by histochemical methods. Calcium deposits and alkaline phosphatase (AP) activity (Vector Red AP Substrate Kit, Vector Labs) were indicative of osteogenic differentiation. MSCs cultured under osteogenic conditions were positive for AP activity and developed a black color after von Kossa staining, indicative of calcium deposition. Oil red O stain identified cellular lipid accumulation. When exposed to adipogenic differentiation media, 10–15% of MSCs developed an adipocyte phenotype with lipid droplet accumulation and oil red O staining. Lipogenic differentiation was not observed in SF and control cultures. Presence of acidic mucopolysaccharides associated with cartilage formation was determined by alcian blue stain. MSCs exposed to chondrogenic conditions were alcian blue-positive, and SF and control cultures were alcian blue negative. For NT, confluent (passage 2) MSC and SF cultures were exposed to roscovitine (15μM; Sigma) for 24h. In vitro-matured oocytes were enucleated and a single cell (MSC or SF) was transferred into the periviteline space. Cell-oocyte couplets were fused in Zimmerman’s medium with a single electric pulse (250V/mm for 20μs) delivered through a needle-type electrode. NT units were electrically activated (2 pulses of 100V/mm for 60μs separated by 5s) in a chamber 1h after fusion and transferred to NCSU-23 medium. Embryos were examined for cleavage and blastocyst formation at 2 and 7 days after NT, respectively. Cleavage rates were 53.3% (40/75) for MSC and 59.7% (46/77) for SF NT embryos. Development to blastocyst stage was 6.6% (5/75) in the MSC group and 1.2% (1/77) in SF group. In conclusion, we established an adult MSC line from a live animal using a minimally invasive BM aspiration technique. Additionally, MSC donor-derived NTs developed to the blastocyst stage. Further experiments will determine nuclear reprogramming in MSC-derived NT embryos.
300MEIOTIC CELL CYCLE PROGRESSION AFTER PARTHENOGENETIC OOCYTE
ACTIVATION: EFFECTS OF STRAIN AND ACTIVATION STIMULUS
Authors: Ibanez E. , Albertini D.F. , Overstrom E.W.
Published: February 2004

Parthenogenetic activation of mammalian oocytes can be induced by several stimuli in vitro. In the mouse, ethanol and Sr2+ are commonly used artificial activating agents. The aim of this study was to investigate both the effects of these two activating agents and the genetic background of the oocyte on meiotic cell cycle progression following activation. Metaphase-II (MII) oocytes were collected from superovulated (5 IU eCG/5 IU hCG) 8–10-week-old female mice of hybrid B6D2F1, inbred C57BL/6, outbred CF-1 and nude (NU/+) strains at 16h post-hCG. Oocytes were activated either by a 5-min exposure to 7% ethanol (E), followed by culture in KSOM, or by continuous culture in Ca2+ -free KSOM containing 10mM SrCl2 (S). Starting 5min after the activation stimulus, oocytes were fixed at 5-min intervals, until 20min post-activation (pa), and then at 30-min intervals, until 6hpa. Fixed oocytes were processed for immunofluorescence analysis of chromatin (Hoechst), spindle (α/β-tubulin) and microfilaments (phalloidin) organization. The initial time course of activation (5–20min pa), assessed by exit from MII arrest, was faster in E- than in S-activated oocytes, except in the C57BL/6 strain in which anaphase (AII) onset after ethanol exposure was significantly delayed in comparison to the other three strains. Progression from AII into telophase (TII), at 50 min pa, was faster in CF-1 and C57BL/6 E-activated oocytes (78.3–87.1%) and in B6D2F1 and CF-1 S-activated oocytes (93.1–94.2%) when compared to the other strains (60–68.8% and 41–62.5%, respectively). NU/+ S-activated oocytes showed the lowest activation rates (88.5% v. 100% at 6h pa), and TII entry was also delayed both in comparison to the other strains (41% vs. 62.5–94.2% at 50min pa) and in comparison to E-activated NU/+ oocytes (68.8%). Rotation of the meiotic spindle began at 15min pa in both E- and S-activated C57BL/6 oocytes, at 20min pa in B6D2F1 and CF-1, and at 50min pa in NU/+ oocytes and, in general, spindle rotation was completed more rapidly following E activation. Second polar body (PB2) extrusion was also delayed in NU/+ oocytes (starting at 1h 20min pa) when compared to all other strains (starting at 50min pa) irrespective of the activating agent used. In E-activated oocytes, TII exit commenced earlier in C57BL/6 oocytes (1h 20min pa) than in the other three strains (1h 50min–2h 20min pa), but a significant proportion of CF-1 and NU/+ oocytes (37–49% at 5h 50min pa) remained arrested at metaphase III. S-activation not only accelerated TII exit but also increased the rate of pronuclear formation in both CF-1 and NU/+ oocytes to 100%. In conclusion, the results demonstrate that both the activating stimulus and the genetic background of the oocyte have significant effects on key events of meiotic cell cycle progression following activation. Supported by USDA NRI 2001-35205-09966 and Charles River Labs.
302A MICROFLUIDIC DEVICE TO CONTROL Ca2+ SIGNALLING IN FERTILIZED
AND NON FERTILIZED EGGS
Authors: Ozil J.-P. , Glemarec C. , Sainte-Beuve T.
Published: February 2004

The process of egg activation has been the subject of much attention for many years. It has recently gained even more interest since nuclear transfer experiments have revealed the unique and extraordinary properties of the oocyte cytoplasm to cause massive reorganization of a somatic nucleus. The transduction mechanisms by which the incoming sperm triggers a series of intracellular Ca2+ oscillations is becoming clearer, but how activation impacts the process of remodelling is still unknown. One possibility to get a better view is to experimentally drive the dynamics of Ca2+ oscillations to monitor the changes in a sperm nucleus in maternal chromosomes or in a somatic nucleus during activation. For this purpose, we have used the modern technology of microfluidics in association with electropermeabilization of the cell membrane to drive the process of egg activation. We designed a microdevice that assures precise control of [Ca2+]I and makes it possible to slow down or accelerate the dynamics of Ca2+ signalling in fertilized and non-fertilized eggs during the process of activation. This device consists of a micro-chamber 400μm wide, 0.5mm high, and 25mm long, making a volume of 5μL. The lateral walls of the chamber are made of gold to subject the cells to an electric field pulse. The bottom surface has a slot 20μm wide used to hold the oocytes by suction. The space above is left open to allow access with a mouth-pipette to lodge a maximum of a hundred eggs. At any moment eggs can be put in place or removed. This chamber is embedded in a disposable cartridge made for us by Micronics (www.micronics.net). A series of computer-driven pumps and micro-valves embedded in the cartridge makes it possible to multiplex very rapid washing with four reagents. The total reagent content of the chamber can be replaced in less than 1s while eggs are maintained aligned. This washing process makes it possible to control the electropermeabilization technique while embryos are cultured in vitro. To evaluate the survival to term of fertilized eggs cultured and stimulated in this microsystem, 60 fertilized embryos were subjected to 24 electrical pulses (1.45kVcm−1) every 8min for 3 hours in the absence of Ca2+ ions during the electrical pulse. Imaging recording showed that the ongoing Ca2+ signal dynamic triggered by fertilization is not altered by the treatment. Among the 60 treated embryos transferred to pseudopregnant recipients, 45 (75%) were capable of giving birth to viable young. Since this new microtechnology appears to be biologically safe, it becomes possible to automatically drive a great variety of signals onto and into the cell during in-vitro culture, to better evaluate how oocytes integrate signals during the process of activation, and to give a biological response at later stages of development. Moreover, this new microtechnology will provide standardization of the biological response after fertilization, ICSI or nuclear transfer experiments.
303PARTHENOGENETIC DEVELOPMENT OF PIG OOCYTES BY CHEMICAL
ACTIVATION
Authors: Park C.S. , Jin D.I. , Kim M.Y. , Chang Y.J. , Yi Y.J.
Published: February 2004

Efficient activation is essential for the success of animal cloning by nuclear transfer. The aim of this study was to investigate the effects of chemical activation agents on parthenogenetic development of pig oocytes matured in vitro. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were selected for activation. Oocytes were activated as follows. First, all oocytes were activated with 25mM HEPES buffered NCSU-23 medium containing 8% ethanol for 10min. After that, in treatment 1, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 3h. In treatment 2, oocytes were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 3h. In treatment 3, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 1.5h, and then were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 1.5h. In treatment 4, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B plus 10μgmL−1 cycloheximide for 3h. Following activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture for 20 and 144h. Activated oocytes were fixed and stained for evaluation of activation rate, cleaved oocytes, blastocyst formation rate and cell numbers per blastocyst. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of oocyte activation was higher in treatment 4 (62.1%) than in treatment 1, 2 and 3 (52.0, 49.6 and 58.0%, respectively). The percentage of cleaved oocytes was lower in treatment 1 and 2 (56.9 and 55.2%) than in treatment 3 and 4 (68.8 and 68.5%). The rate of blastocyst formation from the cleaved oocytes was higher in treatment 3 and 4 (19.8 and 22.0%) than in treatment 1 and 2 (12.1 and 11.7%). Mean cells per blastocyst were lowest in treatment 2 (21.2±0.9) compared to treatment 1, 3 and 4 (27.3±2.2, 30.4±3.8 and 30.9±3.4, respectively). In conclusion, cytochalasin B combined with cycloheximide was more efficient for parthenogenetic development of pig oocytes matured in vitro.
305DEVELOPING AN ACTIVATION PROTOCOL FOR SOMATIC CELL NUCLEAR
TRANSFER (SCNT) IN THE DOMESTIC CAT
Authors: Shin T. , Otoi T. , Kraemer D.C. , Westhusin M.E.
Published: February 2004

In order to establish an activation protocol for somatic cloning in the domestic cat, we evaluated the developmental competence of cat embryos derived from in-vitro matured ova after parthenogenetic activation treatment. The quality of parthenogenetic embryos was assessed by D3 cleavage rates, D8 rates of blastocyst formation and total nucleus numbers in expanded/hatching blastocysts. Parthenogenetic activation treatments were as follows;; Treatment I: 3.0kVcm−1 (25μs, twice) in 0.3M mannitol containing 0.1mM CaCl2· 2H2O and 0.1mM MgSO4, administered to matured cat oocytes and followed by 10μgmL−1 cycloheximide +5μgmL−1 cytochalasin B in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7h. Treatment II: The first electric stimulation was performed as described for treatment I except that the activation medium consisted of 0.3M mannitol containing Mg, but without Ca. Two hours later, pre-pulsed MII oocytes were electropulsed by applying 1.0kVcm−1 (50μs, twice, 5s apart) in 0.3M mannitol containing Ca and Mg for additional activation, followed by culture in 10μgmL−1 cycloheximide +5μgmL−1 cytochalasin B treatment in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7h. Immature cat oocytes were obtained from ovaries by mincing/dissection and matured in vitro for 26–30h as previously described (Gomez et al., 2001, Therigenology, 55, 472). Only MII oocytes with a 1st polar body were utilized for the activation procedure after removal of cumulus cells with 0.1% hyaluronidase by gentle pipetting. A total of 1120 oocytes were collected and the overall maturation rate was 49.8% (551/1120). After parthenogenetic activation of the MII oocytes, the embryos were cultured in vitro as described previously (Pope et al., 2000, Theriogenology, 53, 163–174). The results are shown in Table 1. Treatment II resulted in significantly higher (P<0.01) D3 cleavage rates;; however, there were no significant differences in D8 blastocyst formation and total nucleus numbers. These data suggest that an additional electric activation (Treatment II) may increase the in vitro cleavage rates compared to using a fusion and electrical stimulation simultaneously (Treatment I). In addition, we demonstrated the developmental competence of domestic cat embryos derived from in vitro maturation, activation, and culture for development to the pre-implantation stage. By using these procedures for SCNT, several pregnancies were established and a healthy cloned kitten resulted in our laboratory (Shin et al., 2002, Nature, 415, 859). Therefore, this protocol can be useful, not only for prediction of the developmental competence of domestic cat oocytes matured in vitro, but also when used with SCNT to produce cloned cats.

Comparison of cleavage rates and developmental competence to blastocyst stage following parthenogenetic activation treatments in domestic cat oocytes matured in vitro
307CHARACTERIZATION OF PROTEIN PHOSPHORYLATIONS IN THE COURSE OF MEIOTIC
MATURATION OF BOVINE OOCYTES
Authors: Bhojwani M. , Marx M. , Melo-Sterza F. , Kanitz W. , Leiding C. , Tomek W.
Published: February 2004

The importance of protein phosphorylations during meiotic maturation (transition from prophase I to metaphase II) of oocytes is documented by the fact that the inhibition of the M-phase kinases, cdc2k or MAPK, arrests the oocytes in the GV stage. A detailed knowledge of the targets of these kinases during this stage of development is still missing. Therefore, we have analyzed the proteome of bovine oocytes by high resolution 2D-gel electrophoresis to detect differences in the expression and phosphorylation state of proteins in the course of in vitro maturation (IVM). Bovine oocytes were matured for different times in TCM 199 containing 3% BSA and 300 oocytes each in GV stage (0-h maturation), in GVBD/M I (10-h maturation) or in M II stage (240h maturation) were separated on the gels. The proteins were visualized by staining them with silver or with the fluorescence dye Sypro Ruby, and phosphorylated proteins were detected by Western Blotting with Ser-, Thr-, or Tyr-phosphorylation specific antibodies or by staining with the phosphoprotein specific fluorescence dye Pro-Q Diamond. Gels made from oocytes at the above mentioned maturation stages were compared by a computerized gel-overlay software program (2D Decodon, Greitswald, Germany). The overall protein synthesis was statistically analysed by ANOVA (SigmaStat, Ekrath, Germany), pairwise multiple comparison procedure. Only distinct spots with a difference greater than 30% in their optical densities were considered to be differently expressed or phosphorylated. The results showed a three-fold increase in the rate of overall protein synthesis (p 0.05) during GVBD. Newly synthesized proteins were detected mainly in the higher molecular weight (MW) range (60–80kDa), and protein degradations were found mainly in the lower MW range (20–40kDa) after GVBD. Preliminary data obtained by analyzing the phosphorylation pattern showed that obviously no phosphorylated proteins could be detected in the GV-stage oocytes. Phosphorylation of different proteins was observed at the time of GVBD after 6 to 10h IVM, concomitantly with the activation of cdc2k and MAPK. A maximum of phosphorylated proteins was observed in metaphase II. The first results obtained by performing peptide mass fingerprinting using MALDI-Tof showed that members of the family of heat-shock proteins, ribosomal proteins and putative zinc finger proteins (transcription regulators) were differently expressed or phosphorylated during IVM. This work was supported by the DFG, To 178/1-1, 2 and by the Eibl-Stiftung.
31EFFECTS OF CYCLOHEXIMIDE ON CAPRINE SOMATIC CELL NUCLEAR TRANSFER
EMBRYO AND FETAL DEVELOPMENT
Authors: Butler R.E , Melican D. , Hawkins N. , Jellerette T. , Nims S. , Graslie K. , Gavin W.
Published: February 2004

The use of protein synthesis inhibitors to down regulate the levels of Maturation Promoting Factor (MPF) following fusion and activation are widely used in the field of Nuclear Transfer (NT). Cycloheximide is a protein synthesis inhibitor that blocks the levels of cyclin B, a component of MPF which is required to maintain MII stage arrest in oocytes. However, it is unclear what, if any, effects these broadbased inhibitors may have on nonspecific protein expression in the oocytes or karyoplasts. The purpose of this study was to examine the effects of treatment with and without cycloheximide on embryo and fetal development to term. Ovulated in vivo MII oocytes from superovulated does during the traditional breeding season were surgically collected and then enucleated and reconstructed with either transfected fetal or adult skin cell karyoplasts or transgenic primary somatic skin cells. Couplets were simultaneously fused and activated with a single electrical pulse between 2.6 and 3.0kVcm−1 for 20μs. An additional electrical pulse was given to fused couplets and non-fused couplets were re-fused. Reconstructed embryos were either treated with 5μgmL−1 cyclohexamide (Sigma, St. Louis, MO, USA) for a minimum of 3h or cultured directly post-fusion and activation. The embryos were cultured in SOF +BSA at 38°C in a humidified modular incubation chamber (Billrups-Rothenberg, USA) containing 6% O2, 5% CO2, and 89% N2 for 24–48h. Cleavage was assessed, and nuclear transfer embryos, with age appropriate development (up to 2 cells at 24h or 2 to 8 cells by 48h), were surgically transferred to the oviduct of surrogate recipient does. There were 18 and 11 confirmed pregnancies by Day 50 post-fusion and activation from the cycloheximide and non cycloheximide groups, respectively. A total of 12 recipients that received cycloheximide treated embryos produced term pregnancies and yielded 15 offspring. Alternatively, 9 recipients that received untreated embryos produced term pregnancies which yielded 12 offspring. A total of 27 NT offspring were produced and one offspring from a set of quadruplets died several days post-natally. While significantly more cycloheximide treated NT embryos cleaved, there was no significant difference in pregnancy rate or offspring born between treatment groups. These results suggest that the use of cycloheximide for embryo development in somatic cell nuclear transfer may not be necessary for establishing and maintaining caprine NT pregnancies.

Table 1
314EFFECT OF MACROMOLECULE SUPPLEMENTATION DURING IN VITRO MATURATION ON
THE DEVELOPMENTAL COMPETENCE OF GOAT OOCYTES
Authors: Herrick J.R. , Behboodi E. , Memili E. , Blash S. , Echelard Y , Krisher R.L.
Published: February 2004

In vitro maturation of goat oocytes has traditionally involved the use of serum or BSA. However, these products introduce variability and complicate evaluation of the effects of other medium components. The objective of this study was to examine the effects of citrate and hyaluronate in the absence or presence of BSA during IVM on the developmental competence of goat oocytes. Abattoir-derived, cumulus-oocyte complexes (COC) were matured for 20–22h (6.0% CO2 in air, 38.7°C) in modified SOF medium (1.5mM glucose, 3.0mM L-lactate, 0.1mM pyruvate, 1.0mM glutamine, 0.1mM taurine) supplemented with 1×MEM nonessential amino acids, 0.5×MEM essential amino acids, 1×MEM vitamins, 0.1mM cysteamine, 5μg mL−1 insulin, 5μgmL−1 transferrin, 5ng mL−1 selenium, 50ngmL−1 EGF, 0.01U mL−1 LH and FSH, and 50μgmL−1 gentamicin. Treatments were: (1) 1mgmL−1 PVA (protein-free, defined); (2) 4mgmL−1 BSA (semi-defined); (3) 0.5mM citrate and 0.5mgmL−1 hylauronate (C+H, defined); and (4) 0.5mM citrate and 0.5mgmL−1 hylauronate with 4mgmL−1 BSA (C+H+BSA, semi-defined). At the end of IVM, COC were transferred to modified Brackett and Oliphant’s medium with 7.7mM Ca-(l)-lactate and 20% FCS for IVF. Frozen-thawed sperm were processed through a 45%:90% Percoll gradient and added to IVF drops (50μL) containing COC at a final concentration of 14–15×106 spermmL−1. Gametes were coincubated in the presence of heparin (25μgmL−1) for 22–24h in 7% CO2 in air at 38.7°C. After coincubation, cumulus cells were removed and zygotes were cultured (6% CO2, 5% O2, 89% N2, 38.7°C) in G1 v.3 for 3 days followed by 4 days in G2 v.3. Cleavage was evaluated when embryos were moved to G2, and development to the blastocyst stage was assessed at the end of culture. All blastocysts were fixed and stained with Hoechst 33342 for total cell counts. Analysis of variance was performed using the general linear mixed model macro of SAS. Means are presented ±SEM and probability values P<0.05 were considered significant. The use of BSA did not improve (P>0.05) the developmental potential of goat oocytes (Table 1). Furthermore, a similar proportion (P>0.05) of oocytes developed to the blastocyst and hatching blastocyst stage after maturation under defined conditions compared to oocytes matured with BSA. In conclusion, developmentally competent goat oocytes can be produced by IVM under defined conditions.

Table 1
Development of goat oocytes following IVM with different macromolecules.
315THE REDUCTION OF MATURATIONAL COMPETENCE BY STREPTOMYCIN DURING IN
VITRO MATURATION OF GOAT FOLLICULAR OOCYTES
Authors: Kang J.K. , Yang J.H. , Naruse K. , Park C.S. , Min K.S. , Jin D.I.
Published: February 2004

Antibiotics are commonly added to mammalian oocyte maturation media, but their effects on oocytes maturation have not been examined thoroughly. Goat follicular oocytes were used to investigate whether penicillin, streptomycin or gentamycin affect maturational competence of oocytes and subsequent parthenogenetic activation potential in vitro. Cumulus-oocyte complexes collected from a local abattoir were matured for 24h in five treatments, and matured oocytes were cultured for 48h in five treatments after parthenogenetic activation by treatment with ionomycin, followed by immediate exposure to 6-diethlaminopurine; (1) Control: TCM-199 medium with no antibiotics, (2) TCM-199 with 100IU/mL−1 penicillin (P-4687, Sigma, St. Louis, MO, USA), (3) TCM-199 with 50μgmL−1 streptomycin (S-1277, Sigma), (4) TCM-199 with 50μgmL−1 gentamycin (G-1264, Sigma) and (5) TCM-199 with both 100IUmL−1 penicillin and 50μgmL−1 streptomycin. Maturation rates at 24h post-in vitro maturation and parthenogenetic cleavage development at 48h post-activation were evaluated. Data were analyzed by ANOVA and Student’s t-test. Penicillin and gentamicin treatment groups did not affect maturation rates and percentages of cleavage to 2–4 cell stage at 48h post-chemical oocyte activation. However, when streptomycin was present in the maturation medium, the percentages of matured oocytes at 24h post-in vitro maturation of immature goat oocytes were significantly lower than those from the other groups. However, among the five treatments, there was no significant difference in cleavage rates of matured oocytes at 48h post-activation (Table 1). Therefore, streptomycin did interfere with the maturation of immature goat oocytes, but did not affect the subsequent development of matured goat oocytes. The mechanism by which streptomycin affects the maturation of goat follicular oocytes needs to be investigated further. We conclude that streptomycin in oocyte maturation medium can be detrimental during in vitro maturation of goat follicular oocytes.

Table 1
Effect of antibiotics on maturational competence of goat follicular oocytes and subsequent parthenogenetic activation potential in vitro
32ULTRASOUND MEASUREMENTS OF BOVINE SOMATIC CLONES AT DAY 50 AND DAY 64 OF
PREGNANCY
Authors: Chavatte-Palmer P. , Laigre P. , Juillien C. , Heyman Y. , Constant F. , Guillomot M. , LeBourhis D. , Renard J.P.
Published: February 2004

The overall efficiency of cloning has stayed low since this technique appeared. Most of the losses in the bovine species take place in early gestation between Day 35 and Day 70, when deficient placental development has been described. Late fetal losses are associated with excessive fetal growth and placental hydrops (Large Offspring Syndrome or LOS). One study measuring crown-rump length (CRL) by ultrasound (US) did not show any difference between clones and controls (Pace et al., 2002). The objective of this work was to compare fetal and placental measurements at Days 34, 50, and 64 for bovine clones and control pregnancies to determine whether these could be predictive of a further development of LOS. Twenty-five clone and 32 control (28 AI and 4 IVF) singleton Holstein pregnancies were used for US with a 5-MHz rectal probe. Clones originated from adult skin fibroblasts (three genotypes), used after serum starvation, as previously published (Vignon et al., 1998). CRL and head length (HL), orbital diameter (OD), vesicle width (VW), and length and width of the placentome closest to the fetus were measured at each stage. CRL were also recorded from six clone, six IVF and five AI, Day 34 and Day 60 fetuses recovered at slaughter. Maternal clinical parameters, US images with increased fetal fluids, excessive weight, and clinical features at surgical removal or birth were used to diagnose LOS. Data were analysed using multiple regression analysis with SAS software. There were no differences between IVF and AI fetal and placental measurements by ultrasound and the two groups were pooled as a single control group. LOS was diagnosed in 29% of the clones. CRL and HL were significantly smaller in clones at all stages (CRL: 10.2±3.8mm, n=5 v. 13.6±2.4mm, n=7, at retrieval at Day 34; 31.8±5.6mm v. 36.0±4.6 in controls at Day 50, by US, and 48.6±11.4 v. 63.3±4.9 at Day 64, by US, P<0.05, in clones and controls, respectively) but there was no difference between the clones developing LOS and those that did not. There was no difference for OD or VW at any stage. Placentome width but not length was statistically smaller in clones compared to controls (4.8±1.5 v. 7.2±1.7mm at Day 50 and 5.7±2.0 v. 9.4±4.2 at Day 64, in clones and controls, respectively, P<0.05). There were only two clones retrieved at Day 64, one degenerated and the other similar to controls. Placentome development was delayed. These data clearly show that although a large proportion of clones develop LOS in late gestation, they are actually smaller than controls in early pregnancy, whether or not they will develop LOS. This is probably due to delayed or abnormal early placental development, which can be detected by US. US cannot be used, however, to predict further development of LOS, and other means such as maternal plasma PSP60 concentrations (Heyman et al., 2002) must be used.
329PREGNANCY RATE IN EWES INSEMINATED WITH SEXED SEMEN IN
ARGENTINA
Authors: Cattaneo L. , Martín P. , Caballero J.O. , Calvi M. , Vietri B. , Puyó E. , Medina M.
Published: February 2004

Offspring of many species have been born from flow cytometrically sex-sorted sperm since its introduction in 1989 (LA Jonhson et al.; 1999, Theriogenology). Births of lambs after insemination of ewes with low doses of sexed semen were first reported by Hollinshead et al. (Rep. Fert. Dev. 2002). A field trial was carried out in Patagonia Argentina comparing the use of low doses of sexed and non-sexed frozen semen. This study is aimed at comparing pregnancy rates in ewes inseminated with sorted and nonsorted frozen-thawed sperm. Ejaculates from two Merino rams were diluted with modified tirode solution (Shenk et al., 1999, Theriogenology), stained with Hoescht 33342, and finally incubated at 35°C for 40 minutes. The addition of 10% ram seminal plasma to the sample was done to avoid sperm agglutination caused by dilution effect (Mann, 1964). Sorting was performed with a high speed flow cytometer (MOFLO®). Up to 8 million of sexed spermatozoa were collected in 15-mL conical tubes containing modified tirode solution plus 10% ram seminal plasma, centrifuged at 600g 12min and resuspended with test yolk media plus 11% skimmed milk and 5% glycerol. After being refrigerated at 4°C for at least 1.5h, sexed and nonsexed semen were packaged in 0.25-mL straws at a concentration of 4 to 5 total million sperm and then frozen in an automatic freezing machine (IMV®). One hundred and eighty-three ewes and hoggets were laparoscopically inseminated by the same technician with eight to ten million sorted and nonsorted frozen-thawed semen 14h after heat detection. Pregnancy diagnosis was performed by ultrasound at day 30 post-insemination. Data were analized by Chi-square. There was significant difference in the overall pregnancy rate between sorted and nonsorted sperm, although, for one of the rams that difference was not significant. There was, also, no significant difference in pregnancy rate between ram 1 and 2 in ewes inseminated with either sexed or nonsexed semen (Table 1). These results indicate that the different performance of sorted compared to non sorted sperm is mainly explained by the lower results achieved with semen from ram 2. According to this conclusion, further studies should be done in order to evaluate the degree to which the sorting process affects the sperm fertilizing capability in different rams. This research was supported by Fundación Margarita Perez Companc.

Table 1
33DEVELOPMENT OF BOVINE EMBRYOS CLONED WITH FETAL FIBROBLASTS ARRESTED
AT G0/G1 PHASE BY DIFFERENT TREATMENTS
Authors: Cho S.R. , Son W.J. , Park C.S. , Choe S.Y. , Rho G.J.
Published: February 2004

Numerous factors have an effect on the development of cloned embryos, and one of the most important might be the synchronization between donor nuclei and recipient ooplasts. The objective of this study was to examine the effect of donor cell treatments for G0/G1 synchronization and the donor cell type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on Day 50 of gestation and adult ear skin biopsies. The cells were used for assessements of cell cycle and apoptosis, and for nuclear transfer. Cells were randomly allocated into 3 experimental treatment groups after 6–8 passages: Group 1 (confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent; Group 2 (serum-starvation), cells were cultured in DMEM supplemented with 0.5% FBS for 5 days; Group 3 (Roscovitine), cells were cultured in DMEM supplemented with 10% FBS and 30μM Roscovitine for 12h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1, respectively. At 19h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6kV/cm, 60μs) delivered by a BTX 200. After activation with the combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1, 5h), the eggs were cultured in CR1aa medium for 3 days and additionally cultured in CR1aa medium supplemented with 30mgmL−1 BSA for 5 days at 39°C in a humidified atmosphere of 5% CO2 in air. Differences between groups were analyzed using one-way ANOVA after arc-sine transformation of the proportional data. There were no significantly differences in the incidence of cells arrested at G0/G1 for fetal fibroblasts cultured in the three treatment groups (87%, 83% and 80%; confluent, serum starvation and Roscovitine, respectively). More cells were apoptotic in Group 2 compared to the cells in Groups 1 and 3 (12% v. 6 and 6%, respectively) (P<0.05). Blastocyst development of cloned embryos was significantly (P<0.05) higher when fetal fibroblasts from Group 1 were used, compared to Groups 2 and 3 (35.1%, v. 31 and 29.7%, respectively). Similar results were observed in the use of ear skin fibroblasts as nuclear transfer donor cells (32.7%, v. 24 and 24%, respectively). These results suggest that fetal fibroblasts can be effectively synchronized at G0/G1 by three different treatments, including growth to confluence, serum-starvation and Roscovitine treatment. However, based on blastocyst development and levels of apoptosis, the use of confluent fetal fibroblasts as donor cells is more effective than using cells synchronized by serum-starvation or Roscovitine treatment in the production of cloned bovine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 30012-05-3-SB010 and Cho-A Pharm. Co. LTD.]
330THE EFFECT OF SHEATH FLUID ON THE QUALITY OF SEX-SORTED RAM SPERMATOZOA
Authors: de Graaf S.P. , Gillan L. , Evans G. , Maxwell W.M.C. , O¿Brien J.K.
Published: February 2004

The fertility of sexed frozen-thawed ram spermatozoa at low AI doses remains below commercially acceptable levels (Maxwell WMC et al. 2003 Theriogenology 59, 511 abst). The aim of the present study was to determine if the use of an artificial seminal plasma sheath fluid could attenuate the stress of dilution during flow cytometry and thus improve the functional capacity of sexed ram spermatozoa. Semen was collected from 3 Merino rams, processed for sex-sorting (Hollinshead FK et al. 2002 Reprod. Fertil. Dev. 14, 503–508), and then allocated to 2 treatment groups according to the sheath fluid used within the flow cytometer: (i) a tris-citrate-fructose diluent (TRIS), or (ii) an artificial seminal plasma diluent (ASP) (ODonnell JM 1969 J. Reprod. Fert. 19, 207–209). Spermatozoa were sorted using a modified high speed cell sorter (SX MoFlo®, DakoCytomation, Fort Collins, USA) without separation of X and Y bearing gametes, and then frozen and thawed using established protocols (Hollinshead FK et al. 2002). Motility characteristics (HTM-IVOS; Hamilton-Thorne, Beverly, USA) and acrosome integrity (FITC-PNA) were assessed throughout. Statistical analyses were conducted by ANOVA. Results indicate that in all but one instance (VSL pre-freeze), TRIS spermatozoa exhibited higher (P<0.05) total motility (TM), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) compared to ASP spermatozoa (Table 1). Conversely, ASP treatments displayed enhanced straightness (STR) and linearity (LIN) pre-freezing and immediately post-thaw. In addition, the percentage of hyperactivated (HA) spermatozoa prior to freezing (± SEM) was lower (P<0.05) for ASP (3±1.1) than for TRIS (20±3.4), although post-thaw the differences were NS. Acrosome integrity remained similar (P>0.05) among treatments before and after thawing (range: 92.3–95.9% intact). In conclusion, the ASP sheath fluid resulted in decreased TM, velocity and ALH of sex-sorted frozen-thawed ram spermatozoa, a clear indication of the superiority of TRIS sheath fluid. The partial improvement in STR, LIN and HA that the ASP affords sex-sorted ram spermatozoa does not offset the aforementioned negatives, suggesting ASP is unsatisfactory for use as a sheath fluid during sperm sorting. Research supported by XY, Inc.

Table 1
The effect of sheath fluid on motility characteristics (±SEM)
336EFFECT OF AN INDIVIDUALLY ADAPTED SUPERSTIMULATION PROTOCOL FOR OPU IN COWS
Authors: De Roover R. , Genicot G. , Leonard S. , Massip A. , Dessy F.
Published: February 2004

The aim of this experiment was to apply an ovarian superstimulation protocol to OPU, with attention to the individual responses of each cow, and to show effects on follicle numbers and diameters. Ten non-lactating dairy cows were stimulated with pFSH and submitted to OPU-IVF 6 times at 2-week intervals. On Day 0 of each 2-week period, the dominant follicle was punctured and a progestin ear implant was inserted. Starting on Day 2, animals were treated with 6 equal doses of pFSH, administered twice daily over 3 days. On Day 7, 48h after the last pFSH injection, follicle diameters were measured by ultrasound and animals were submitted to OPU (OPU-IVF results are not shown here). The administered dose of pFSH for the first OPU session was 30mg per animal (stimufol, ULg-FMV, Liège, Belgium). In the following OPU sessions, pFSH doses were individually adapted to the percentage of follicles larger than 11mm, present during the previous OPU session. If the number of follicles larger than 11mm was between 1% and 20%, total dose remained unchanged;; if between 20% and 40%, total dose was reduced by 5mg; and if more than 40%, total dose was reduced by 10mg. If no follicles larger than 11mm (0%) were present, total dose was increased by 5mg. Only one animal received the initial pFSH dose for all 6 OPU sessions for a total of 180mg. All other animals received more or less than 180mg, ranging from 135 to 230mg. For interpretation of the effects of pFSH dose on follicular diameter, data were grouped in pairs (before and after changing the dose of pFSH). This resulted in 3 groups: Fixed pFSH dose—pFSH dose was unchanged in the 2 sessions (of that pair); reduced pFSH dose—pFSH dose was reduced in the second session (of that pair); and increased pFSH dose—pFSH dose was increased in the second session (of that pair). No changes in response were found when pFSH dose remained unchanged. However, the number of small-sized follicles increased and the number of large-sized follicles decreased when dose of pFSH was reduced. The number of larger-sized follicles increased when pFSH dose was increased. The total number of follicles remained unchanged regardless of pFSH dose. Overall, the mean number of punctured follicles per session was 11.9±7.7 with 16% of the follicles over 11mm. In conclusion, subtle changes in dose of pFSH influenced follicle sizes but not follicle numbers.

Table 1
Effect of fixed, reduced or increased doses of pFSH on follicular diameters (mean number±SD)