bluuebunny/crossref_metadata_2025 huggingface dataset:
319IN VITRO MATURATION OF EQUINE OOCYTES IN A COMPLETELY DEFINED MEDIUM
SUPPLEMENTED WITH PROGESTERONE
Authors: Merlo B. , Iacono E. , Prati F. , Mari G.
Published: February 2004

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500ngmL−1) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1IUmL−1 (Pluset, Laboratorios Calier, Barcelona, Spain), EGF* 50ngmL−1, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytesmL−1): (1) MSOF+FCS 10% (MSOF-FCS), (2) MSOF+progesterone 100ngmL−1 (MSOF-P4), (3) MSOF. After 24h of culture in 5% CO2 in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10μgmL−1 in PBS) for 30min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P<0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P<0.05) increases the nuclear maturation rate at 24h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4±19.3ngmL−1, healthy preovulatory follicle 1094.3±170.9ngmL−1; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system.*All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated.

Table 1
Maturation of equine oocytes in different media
320EFFECT OF CYSTEAMINE DURING IN VITRO MATURATION ON FURTHER EMBRYONIC
DEVELOPMENT AND POSTTHAW SURVIVAL OF IVP BOVINE EMBRYOS
Authors: Merton J.S. , Gerritsen M. , Langenbarg D. , Vermeulen Z.L. , Otter T. , Mullaart E. , Landman B. , Knijn H.M.
Published: February 2004

The uptake of cysteamine by immature oocytes may facilitate the synthesis of glutathione (GSH) during in vitro maturation, as reported by Matos et al. (1995 Mol. Reprod. Dev. 42 432–436). GSH plays an important protective role in relation to reactive oxygen species generated by normal oxidative metabolism. This study investigated the effects of the presence of cysteamine during in vitro maturation on subsequent in vitro embryonic development and postthaw in vitro survival. Immature Cumulus-Oocyte-Complexes (COCs) were recovered from ovaries 6 to 8h after slaughter. COCs were matured in vitro for 22 to 24h in TCM199/FCS/LH/FSH supplemented either with or without cysteamine (0.1mM), Subsequently, matured oocytes were fertilized with frozen-thawed Percoll-separated semen and further cultured for seven days in SOFaaBSA. Morulae grade 1 (IETS) and blastocysts grades 1 and 2 (IETS) were frozen on Day 7 in 10% Glycerol using a conventional slow freezing procedure (Wagtendonk-de Leeuw et al. 1995 Cryobiology;; 32 157–167). In vitro survival was measured by rates of blastocyst formation and reexpansion at 24h and hatching/ed blastocysts at 72h in SOFaaBSA supplemented with 5% FCS. Results were analyzed by Chi-square analyses. The presence of cysteamine during in vitro maturation significantly affected the embryo production rate (19.4% and 24.0% for control and cysteamine at Day 7, respectively). The higher number of embryos at Day 7 was totally due to an increased number of blastocysts (Table 1); however, the distribution of embryos among the different quality grades was not affected. Addition of cysteamine did not affect the post thaw survival of the frozen/thawed embryos (85% v. 91% reexpansion and 33% v. 34% hatching/ed for control v. cysteamine, respectively). These results show that the presence of cysteamine during in vitro maturation, does affect further in vitro embryonic development, resulting in a higher embryo production rate. Embryo quality, expressed in morphological grades and postthaw survival rates, were not affected. A field trial will be conducted in order to confirm these results with ovum pick up-derived oocytes.

Table 1
Effect of cysteamine during in vitro maturation on subsequent in vitro embryonic development of IVP bovine embryos (number of replicates: 5)
322MEIOTIC COMPETENCE OF CANINE OOCYTES EMBEDDED IN COLLAGEN GELS
Authors: Otoi T. , Yuge M. , Murakami M.-k. , Karja N.W.K. , Wongsrikeao P. , Agung B. , Murakami M.
Published: February 2004

The low meiotic competence of canine oocytes cultured in vitro is a major obstacle to the in vitro production of canine embryos. The objectives of the present study were to examine meiotic competence of oocytes embedded in collagen gels and to investigate the effects of timed exposure of the oocytes embedded in collagen gels to hormone supplements on the nuclear maturation. Ovaries were collected from 17 bitches at various stages of the estrous cycles by ovariohysterectomy following anesthesia at local veterinary practices. Only non-degenerate COCs were collected and then suspended in TCM-199 supplemented with 5% fetal calf serum. Only oocytes with diameter >110μm were selected and used for this study. In the first experiment, the effect of embedding in collagen gels of COCs on their meiotic competence was tested. Half of selected oocytes were embedded in 0.3mL of collagen gels (3 to 4 COCs per gel) as described by Yamamoto et al. (Yamamoto K et al., 1999 Theriogenology 52, 81–89). The COCs with or without collagen gels were cultured in a dish containing 2.5mL of TCM-199 supplemented with 0.1IUmL−1 HMG and 10IUmL−1 hCG (3 to 4 COCs per dish) for 72h at 38.5°C in a humidified atmosphere of 5% CO2 in air. In the second experiment, the effect of removal of hormonal supplements from maturation medium on nuclear maturation in vitro was examined. At 24 and 48h after the start of culture, the COCs embedded in collagen gels were cultured in TCM-199 without HMG and hCG for 48 and 24h, respectively. As a control, the COCs embedded in collagen gels were cultured with hormone supplement for 72h. After 72 of maturation culture, the oocytes were fixed, stained with Hoechst 33342 and examined for the meiotic stage of the oocytes using a fluorescence microscope. Data were analyzed by ANOVA. The proportion of oocytes that resumed meiosis was significantly higher (P<0.05) in the COCs with collagen gels than in the control COCs without collagen gels (50.6 v. 26.5%). Significantly more oocytes reached metaphase I to metaphase II stage (MI/II) in the collagen gels culture than in the control culture (P<0.05; 27.4 v. 8.3%). The proportion of collagen embedded-oocytes that resumed meiosis was significantly higher (P<0.05) in COCs cultured with hormone supplements for 24h than in COCs cultured for 48h (59.1 v. 30.4%) but not different from COCs exposed for 72h (41.9%). Moreover, there were no significant differences of MI/MII rates (22 to 24%) among the three treatment groups. These observations indicate that embedding of COCs in collagen gels enhances the meiotic competence of canine oocytes, but removal of hormone supplement from maturation medium does not improve the ability of the oocytes to reach MI/MII stage.
325RETINOID-DEPENDENT POLY(A) MRNA CONTENTS IN BOVINE OOCYTES
PREMATURED AND/OR MATURED IN VITRO
Authors: Royo L.J. , Rodriguez A. , Gutierrez-Adan A. , Diez C. , Moran E. , Alvarez I. , Goyache F. , Gomez E.
Published: February 2004

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24h in DM with 25μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E2 for 24h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO2 in air and high humidity. Within experiments, COCs were cultured with 5nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (v,x,y,zP<0.01; a,bP<0.05), and poly(A) mRNA (pg oocyte−1) was expressed as LSM±SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7±4.8x) and untreated (95.5±4.8y) oocytes, as compared to 1% ethanol (72.2±4.8z) and immature (71.5±4.8z) oocytes. After IVM, untreated oocytes (23.0±2.2v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2±2.2y) basically equalled that in 1% ethanol (35.2±2.2y), while 3% (44.5±2.2yz) and 5% ethanol (52.0±2.2z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5±4.8x). After prematuration+maturation, poly(A) mRNA values were 34.2±2.2v (untreated+untreated), 36.5±2.2v (9-cis-RA+untreated), 49.5±2.2xa (untreated+9-cis-RA), 41.0±2.2vxb (9-cis-RA+9-cis-RA) and 59.0±2.2y (untreated+1% ethanol). Levels of poly(A) mRNA from prematured+matured oocytes were again lower than in immature (71.5±4.8x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).
326FOLLICULAR SIZE, BUT NOT STAGE OF REPRODUCTION OR SEASON, INFLUENCES
MEIOTIC MATURATION OF DOMESTIC DOG OOCYTES
Authors: Songsasen N. , Spindler R. , Wildt D.E.
Published: February 2004

The current in vitro maturation system (IVM) for dog oocytes is inefficient. On the average, only 15% of ovarian oocytes complete nuclear maturation in vitro. For unknown reasons, the ability of oocytes to develop to the metaphase II stage (MII) varies markedly among bitches (Songsasen et al., 2002, Mol. Reprod. Dev. 62, 407–415). The objective of this study was to identify the cause(s) underlying these significant variations in nuclear maturation. Initially, we retrospectively analyzed data obtained during the past 3 years;; 1661 oocytes were obtained from 74 bitches where stage of reproduction for the donor was known based on ovarian morphology. Oocytes were cultured in TCM 199+0.1% polyvinyl alcohol at 38.5°C in 5% CO2 in humidified air under various experimental conditions. Analysis of variance (ANOVA) was performed to compare differences in meiotic competence of oocytes obtained at various reproductive stages and during different seasons. Stage of reproduction did not influence meiotic abilities of oocytes. Percentages of oocytes obtained during proestrus/estrus (n=468 oocytes), diestrus/metestrus (n=333), anestrus (n=331) or prepuberty (6–8 months of age, n=479) and developing to MII were 17.9±2.9%, (mean±SEM), 24.0±6.0%, 20.8±4.7%, and 17.8±5.2%, respectively (P>0.05). A similar analysis across seasons (spring, summer, fall, winter) also indicated no influence of time of year on nuclear maturation (P>0.05). Because there is a known strong link between follicular growth and meiotic competence of goat oocytes (De Smedt et al., 1994 J. Exp. Zool. 269, 128–139), we also examined the impact of follicular size on nuclear maturation. The cortex of ovaries from 15 bitches was horizontally dissected (5mm thickness) so follicles could be observed and divided into three classes: (1) <0.5mm diameter (n=60); (2) ≥0.5 to <1mm (n=110); and (3) 1–2mm (n=72). Follicles were separated according to these size classes;; oocytes were recovered and cultured in TCM 199+0.25mM pyruvate, 2mM glutamine, 25mM β-mercaptoethanol, 10ng/mL epidermal growth factor (Basal TCM) supplemented with 0.5IU/mL equine chorionic gonadotropin for 1h. Oocytes then were cultured in Basal TCM for 48h before staining with 1% orcein to assess nuclear status. Follicular size influenced meiotic competence of the oocytes (ANOVA, P<0.05). Mean percentages of MII oocytes were 14.2±7.2, 15.6±4.5, and 30.9±8.2, for oocytes recovered from <0.5-mm, ≥0.5 to <1-mm and 1–2-mm diameter follicles, respectively. This study revealed that stage of reproduction and season have no impact on in vitro nuclear maturation of the dog oocyte. However, the findings demonstrate that dog oocytes acquire meiotic competency during follicular development. Because the source of most dog oocytes for IVM are small follicles, results suggest that oocytes may be incapable of completing nuclear maturation under in vitro conditions that are designed for fully-grown oocytes.
328MEIOTIC COMPETENCE AND DNA FRAGMENTATION OF PORCINE OOCYTES FROM
OVARIES STORED IN VARIOUS TEMPERATURES
Authors: Wongsrikeao P. , Karja N.W.K. , Budiyanto A. , Mtango N.R. , Murakami M. , Nii M. , Otoi T.
Published: February 2004

The aim of this study was to investigate the effects of storage of porcine ovaries at different temperatures before oocyte collection on the nuclear maturation and DNA fragmentation of cumulus-oocyte complexes (COCs). Ovaries were collected at a local abattoir and randomly kept in physiological saline at 4°C, 15°C, 25°C and 35°C. Ovaries were stored for 6 hours prior to follicle aspiration. After storage at each temperature (about 80 oocytes each group), COCs were fixed immediately after aspiration and stained by the terminal deoxynucleotidyl transferase (TdT) nick-end labeling (TUNEL) method to examine the DNA fragmentation under fluorescein microscope. To investigate meiotic competence of the oocytes, some COCs of each treatment group (about 100 oocytes each group) were matured in vitro for 45 hours in a modified North Carolina State University (NCSU)-37 solution supplemented with 10% (v/v) porcine follicular fluid, 0.6mM cysteine, 10IUmL−1 eCG and 10IUmL−1 hCG. After maturation culture, the cumulus cells were removed from COCs and fixed in acetic acid-ethanol (1:3, v/v) for 48–72h. The fixed oocytes were stained with acetic-orcein (1% orcein in 45% acetic acid) and examined under a phase-contrast microscope. Data were subjected to arc-sin transform before analyzing by ANOVA. The proportions of oocytes with DNA fragmentation increased with increasing storage temperature of ovaries (25.2% in 4°C, 31.8% in 15°C, 37.4% in 25°C and 54.7% in 35°C, respectively). There was no significant difference between the proportions of germinal vesicle breakdown (GVBD) of 25°C and 35°C storage groups (74.7 and 83.6%, respectively), but the proportions of 25°C and 35°C storage groups were significantly higher (P<0.05) than those of 4°C and 15°C storage groups (58.1 and 59.6%, respectively). The proportions of oocytes reaching metaphase II (MII) was significantly higher (P<0.05) in the 25°C storage group than in other groups (48.0% in 25°C v. 0% in 4°C, 0% in 15°C and 40.1% in 35°C). Moreover, none of oocytes in 4°C and 15°C storage groups reached MII. These results indicate that 25°C is the most suitable temperature for long-term storage of ovaries to maintain meiotic competence and prevent DNA fragmentation of porcine oocytes.
332IN VIVO DEVELOPMENTAL CAPACITY OF IN VITRO-PRODUCED
EMBRYOS DERIVED FROM SEX-SORTED AND RE-CRYOPRESERVED FROZEN-THAWED RAM SPERM
Authors: O'Brien J.K. , Hollinshead F.K. , Evans G. , Maxwell W.M.C.
Published: February 2004

The ability to sort and re-freeze frozen-thawed sperm would significantly increase the potential application of sperm sexing technology to species management. Frozen-thawed, sorted, re-frozen and then thawed ram sperm appear fully functional in vitro with blastocyst production greater than that for frozen-thawed, non-sorted sperm (Hollinshead FK et al. 2003 Theriogenology 59, 209 abst). The aim of this study was to evaluate the in vivo capacity of in vitro-produced embryos derived from frozen-thawed sperm after sorting and a second cryopreservation/thawing step. Frozen semen from 2 rams (n=3 ejaculates per ram) was used throughout. Post-thaw sperm treatments comprised (i) non-sorted (Control); (ii) sorted (Froz-Sort) and (iii) sorted, then re-frozen (Froz-Sort-Froz). X and Y sperm were separated using a high-speed sorter (SX MoFlo®, DakoCytomation, Fort Collins, CO, USA) after incubation with Hoechst 33342 and food dye to eliminate nonviable sperm. Reanalysis revealed high levels (mean±SEM) of purity for X- and Y-enriched samples for all treatments (89±1.2%). At Day 6 post-insemination, 2 embryos (blastocyst stage or greater) were transferred per recipient. Data were analyzed by chi-square and Fisher Exact Test. In vivo embryo survival was similar across sperm treatments (28/64, 43.8% overall) and 20 of 23 (87.0%) sexed lambs were of the predicted sex (Table 1). These results demonstrate high in vivo developmental capacity of in vitro-produced sexed embryos derived from frozen-thawed ram sperm after sorting and a second cryopreservation/thawing step, and increase the potential application of sperm sexing technology. Research support by XY, Inc., Australian Research Council and Zoological Parks Board of NSW.

Table 1
In vivo survival of transferred in vitro produced embryos derived from frozen-thawed non-sorted (Control), frozen-thawed and sorted (Froz-Sort) and frozen-thawed, sorted, then frozen-thawed (Froz-Sort-Froz) ram sperm.
335EFFECT OF PATERNAL LINE OF DONOR ON SUPEROVULATORY RESPONSE AND EMBRYO
QUALITY IN JAPANESE BLACK HEIFERS
Authors: Asada M. , Hashiyada Y. , Konishi K.
Published: February 2004

The objective of this study was to investigate the effect of paternal line of donor on superovulatory response and embryo quality in Japanese Black cattle. Japanese Black cattle have paternal lines that can be classified into the following, according to meat quality and the growth rate: ‘high marbling meat type’, ‘high growth rate type’ and ‘high marbling meat and high growth rate (intermediate type)’. We hypothesized that paternal line of donor may affect superovulatory response. One hundred and forty superstimulations were performed on 56 Japanese Black heifers, and data on superovulatory response and embryo collection were analyzed. Japanese Black donors used in this study were from the following 4 paternal lines: Kedaka (large-framed with high growth rate;; n=9, 30 times), Fujiyoshi (intermediate type;; n=22, 47 times), Shigekane (high marbling meat type;; n=13, 40 times), and Tajiri (high marbling meat type and small-frame with slow growth rate;; n=12, 23 times). Donors were synchronized using a CIDR-B (EAZI-BREED, InterAg, NZ) for 8 to 12 days, and 500μg PGF analog (Resipron-C; Teikoku Zouki, Japan or Clopromate-C; Sumitomo, Japan) administered at CIDR removal. Superstimulation was initiated on Day 10 (Day 0=day of estrus) of the synchronized cycle;; FSH (Antrin R10;; Denka, Japan) was administered twice daily for 4 days with decreasing doses for a total of 28 Armour units FSH. PGF analog was administered in the morning on the last day of FSH treatment. Donors were given 50 or 100μg GnRH analog (Sporunen, Denka, or Conceral, Takeda Schering prau, Japan) at the time of estrus detection, and were inseminated 12h after the onset of estrus. Embryos were recovered on Day 7 after AI. The numbers of CL and follicles were recorded with transrectal ultrasonography immediately after embryo recovery. Embryos were classified according to the IETS Manual. Data were analyzed by Kruskal-Wallis test combined with Scheffe’s multiple comparison test. There were no significant differences in CL numbers among paternal lines: Kedaka (9.5±4.2), Fujiyoshi (8.5±5.1), Shigekane (7.8±5.0) and Tajiri (8.6±4.1). Mean number of recovered ova/embryos per donor was significantly (P<0.05) higher in the Kedaka line (8.4±5.1) than in the Fujiyoshi (4.9±4.1) and Shigekane (5.5±7.0) lines. The percentages of viable embryos was greater (P<0.05) in the Fujiyoshi (73.7%) and Shigekane lines (62.8%) than in the Tajiri line (50.9%), whereas the percentage of unfertilized oocytes was significantly (P<0.05) higher in the Tajiri line (41.5%) than in the Fujiyoshi (17.5%) or Shigekane (26.1%) lines. The percentages of freezeable and transferable embryos were not significantly different among paternal lines. Data suggest that the paternal line had an effect on the superstimulatory response in Japanese Black heifers, and especially the number and percentage of viable embryos per donor.
341APOPTOSIS IN SOMATIC CELL CLONED AND EGFP TRANSGENIC BOVINE EMBRYOS
Authors: Lee S.L. , Choe S.Y. , Rho G.J.
Published: February 2004

The overall success rate achieved by cloning techniques to date is low, mainly due to deficiencies in nuclear reprogramming, gene expression and DNA fragmentation, which result in early and late embryonic losses. This study was carried out to compare the incidences of DNA fragmentation during development of IVF, parthenote (PT), nuclear transfer (NT) and transgenic-cloned (TG) embryos. Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) with propidium iodide counterstaining was used for determination of DNA fragmentation and total cell number. Donor cells were fetal fibroblasts with or without transfection with EGFP, and cultured in DMEM+15% FCS until confluent, for up to 5 days. At 19h post maturation (hpm), enucleated oocytes were reconstructed with donor cells and activated at 24hpm with the combination of ionomycin (5μM, 5min) and cycloheximide (10μg/mL, 5h) after electric fusion by a single DC pulse (1.6KV/cm, 60μs) delivered by a BTX 200. Parthenotes were produced by the same activation protocol at 24hpm. The eggs and control IVF embryos were cultured in CR1aa at 39°C in a humidified atmosphere of 5% CO2 in air. Differences among groups were analyzed using one-way ANOVA after arc-sine transformation of proportional data. Embryos at the 8-cell stage in all treatments, IVF, PT, NT and TG, showed DNA fragmentation. The apoptotic cell index (total number of apoptotic nuclei/total number of nuclei) of Day 7 blastocysts was significantly (P<0.05) higher in TG and NT embryos (17/91, 18.6±4.0% and 13/94, 13.8±4.7%, respectively) compared to IVF and PT embryos (9/122, 7.4±3.4% and 8/93, 8.6±2.9%, respectively). TUNEL positive cells were detected in almost all blastocysts at Day 7 and were mainly observed in the ICM. The DNA fragmentation ratio of the ICM in the blastosysts at Day 7 (number of apoptotic nuclei in the ICM/total number of apoptotic nuclei in the blastocyst) was significantly (P<0.05) higher in TG embryos (64.7±21.4%) than in IVF (44.4±28.0%), PT (50.0±18.6%) or NT (53.0±32.5%) embryos. These results indicate a higher occurrence of DNA fragmentation observed in NT and TG embryos when compared to IVF and PT embryos. In addition, ICM of TG blastocysts revealed a high DNA fragmentation ratio, which may be related to early embryonic loss after transfer of the resulting embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010 and Cho-A Pharm. Co. LTD.]
5MPF AND MAP KINASES IN OVINE OOCYTES: EFFECTS OF ENUCLEATION AND CAFFEINE ON ACTIVITY AND DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS
Authors: Lee J-H. , Campbell K.H.S.
Published: February 2004

In nuclear transfer (NT) embryos, exposure of the donor chromatin to the MII cytoplasm results in premature chromatin condensation (PCC) which may be beneficial for nuclear reprogramming (Campbell KHS and Alberio R 2003 Reprod. Suppl. 61, 477–494). Following enucleation, maturation promoting factor (MPF) activity in murine oocytes is primarily associated with the meiotic spindle. This reduced MPF activity in the cytoplast may result in decreased PCC and reprogramming. Conversely, increasing cytoplast MPF activity may increase reprogramming. The aims of this study were to perform quantitative analysis of MPF and MAPK activities in ovine oocytes: 1. at anaphase/telophase I (A/TI) or MII; 2. following enucleation; 3. following treatment with caffeine (an inhibitor of Myt1/Wee1 activity). The development of ovine NT embryos reconstructed using caffeine-treated oocytes as cytoplast recipients was then determined. Oocytes were matured in TCM 199, 10% FBS, 5μgmL−1 FSH, 5μgmL−1 LH,1μgmL−1 estradiol, 0.3mM sodium pyruvate and 100μM cysteamine. 15h post-onset of maturation (hpm) oocytes were stripped of cumulus cells and enucleated in HSOF containing 5μgmL−1 Hoechst 33342 and 7.5μgmL−1 cytochalasin B (CB). Control oocytes were sham-enucleated by removing an equal volume of cytoplasm. Oocytes were cultured in SOF±10mM caffeine. Groups of 10 oocytes were sampled and analyzed for MPF and MAPK activities as previously described (Ye JP et al., 2003 Reproduction 125, 645–656). For NT, primary foetal fibroblasts were quiesced in DMEM containing 0.1% FBS for 2–3 days. Cell fusion was induced with two DC pulses of 25VcM-1 for 80μs. 3 methods of NT were compared: A. fusion 20hpm, activation 21hpm; B. fusion 24hpm, activation 25hpm; C. 10mM caffeine 18–24hpm, fusion 24hpm, activation 25hpm. All oocytes were activated in HSOF containing 5μgmL−1 calcium ionophore (A23187), cultured in SOF with 10μgmL−1 of cycloheximide and 7.5μgmL−1 CB for 5h, and then transferred to mSOFaaBSA medium, all at 5% CO2, 5% O2 and 90% N2 at 39°C. On Day 2 cleavage was assessed and 10% FBS added to the medium. Development to blastocyst was assessed on Day 7. All data were analyzed by chi-square test. Both MPF and MAP kinase activities were increased at MII compared to A/TI (P<0.05). There were no differences in activities of both kinases between intact and enucleated oocytes. Following enucleation, both kinase activities were identical in all groups, reaching maximum activities 24hpm followed by a slow decline. Caffeine increased the activity of both kinases (MPF in particular) in all groups. Following 5 replicates (total oocytes 145, 143, 144 for NT methods A,B,C, respectively), no significant differences were observed between fusion (82.1%, 67.8%, 67.4%), cleavage (90.8%, 88.7%, 89.7%) or development to blastocyst (20.2%, 18.6%, 25.8%). Analysis of total cell numbers on limited numbers of blastocysts (7, 6, 7) were NS(70.9±38.5, 69.3±25.4, 93.3±17.8).
53MODIFIED AIR-DRY METHOD REVEALS THAT DIPLOID CELLS ARE PREDOMINANT IN
CLONED BOVINE EMBRYOS
Authors: Li G.-P. , Bunch T.D.
Published: February 2004

Chromosomal analysis is important for evaluating embryo quality. Reports on the cytogenetic analyses of bovine nuclear transfer (NT) embryos, however, have varied according to methods used (Mohamed NMS et al. 2000 J. Vet. Med. Sci. 62, 339–342; Arat S et al. 2002 Biol. Reprod. 66, 1768–1774; Booth PJ et al. 2003 Biol. Reprod. 68, 922–928). An improved air-dry method was developed in this study to specifically investigate the chromosomal composition of bovine NT embryos. NT embryos were obtained by transferring cumulus cells into enucleated oocytes, the fused embryos were then co-cultured in CR1aa with bovine cumulus cells. Day 5 embryos were cultured in the presence of 0.01μgmL−1 colcemid for 18h (classical method is 5h) and then in 1% sodium citrate for 3 to 5min (classical method is 15 to 30min), and fixed on slides, total of 55 embryos were analyzed. The categories for the chromosome composition were classified as follows: diploid (2n), triploid (3n), tetraploid (4n), mixoploid (including 2n/3n, 2n/4n, and 2n/3n/4n). Any embryo that did not show an interpretable metaphase spread due to gross overspreading or clumped chromosomes was not classified. The mitotic index (number of metaphase plates/total cell number of analyzed embryo) was 41.1% (700/1701), which is significantly higher (P<0.001) than that obtained by the classical method (11.9%, 50/419). Of 700 metaphase plates observed, 608 (86.9%) were diploid. Triploidy and tetraploidy were 49 (7%) and 42 (6%), respectively. There were 72.7% (40/55) of the embryos diploid, and 27.3% (15/55) polyploid or mixploid, which included 3n (1/15), 4n (1/15), 2n/3n (5/15), 2n/4n (3/15), and 2n/3n/4n (4/15). Of the 15 non-diploid embryos, 64% of the total cells were diploid. The results of this study indicate that diploidy is the predominant state in bovine Day 5 NT embryos.
56CARDIOMYOCYTES FOR THE STUDY OF DEDIFFERENTIATION IN BOVINE NUCLEAR
TRANSFER
Authors: Lucas-Hahn A. , Schwarzer M. , Lemme E. , Schindler L. , Niemann H.
Published: February 2004

Nuclear transfer facilitates the study of the dedifferentiation process of differentiated somatic cells. Cardiomyocytes are a good model of terminally differentiated cells showing a unique gene expression pattern of cardiac marker genes. The purpose of this study was to test bovine cardiomyocytes as donor cells in nuclear transfer. Cardiomyocytes were isolated from fetal heart muscle (3–5 months of gestation), which were obtained at the abbatoir and immediately perfused with cold Custodiol (Dr. Franz Köhler Chemie, Germany) to reduce metabolism and protect the cells against ischaemia. Subsequently, hearts were perfused with collagenase in Krebs-Henseleit buffer (KHB) to dissociate the tissue and isolate single elongated, contractile cells. For nuclear transfer and fusion the cardiomyocytes were rounded up by exposure to increasing calcium concentrations (2.5–200μM) in the culture medium before the cells were incubated in suspension for 46–48 hours in MEM medium plus 10% FCS. Nuclear transfer was performed as described earlier (Lucas-Hahn et al., 2002, Theriogenology 57, 433). As a control, adult female fibroblasts were employed. Fusion rate, cleavage (day 3 of in vitro culture) and development up to the morula/blastocyst (day 7 of in vitro culture) were recorded and statistically analysed with Student’s t-test. A total of 243 nuclear transfer complexes with cardiomyocytes and 127 with fibroblasts were produced. Fusion rates for cardiomyocyte complexes were significantly (P<0.001) lower (28.8%) compared to fibroblasts (84.3%). Cleavage rates were 48.1% for cardiomyocytes and 62.8% for the fibroblast-derived embryos. The developmental capacity to the morula/blastocyst was significantly (P<0.01) reduced for cardiomyocyte (9.4%) compared with the fibroblast-derived (32.4%) reconstructed embryos. Most of the Day 7 embryos were frozen for investigation of gene expression patterns of cardiac marker genes. Staining with Hoechst 33342 for counting total cell numbers revealed that 87.3±20.9 blastocysts were derived from fibroblasts and 100 blastocysts from cardiomyocytes. These results indicate that nuclear transfer with terminally differentiated cardiomyocytes is possible, although with reduced rates. Studies are underway to analyze the gene expression of cardiac marker genes in reconstructed embryos to gain insight into dedifferentiation after nuclear transfer using cardiomyocytes as a model. This study was supported by Deutsche Forschungsgemeinschaft (DFG; Ni 256/16-1)
6ACETYLATION OF HISTONE H4 LYSINE-5 AND LYSINE-8 DURING DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Maalouf W.E. , Alberio R. , Campbell K.H.S.
Published: February 2004

The oocyte is remarkable in its ability to remodel the parental genomes following fertilization and to reprogram somatic nuclei as in nuclear transfer. While significant research has been carried out on DNA methylation patterns in the early embryo, increased interest in histone acetylation is more recent. The objective of this study was to characterize the pattern of acetylation of histone H4 lysine-5 (H4L5) and lysine-8 (H4L8) in the early pre-implantation bovine embryo. Bovine embryos were produced as previously described (Fouladi Nashta AA et al., 1998 Biol. Rep. 59, 255–262) and collected at different developmental stages, 1-cell (20h), 2-cell (30h), 4- and 8-cell (Day 2), 16-cell (Day 4), and blastocyst (Days 7–8) with an average of 6 embryos per group in two replicates. Embryos were fixed in 2.5% paraformaldehyde, 15min at room temperature (RT), stained with polyclonal rabbit antibodies against H4L5 (1:800) and H4L8 (1:600) residues (Serotec, UK) at 4°C overnight. A polyclonal swine anti-rabbit (1:200; Dako, Denmark) was used as secondary antibody for 40min at RT. Images were examined using a fluorescent microscope (Leica DMR, Germany). Image analysis and quantification were performed using Simple PCI software (Compix Imaging Systems, USA). Changes in intensities within and between different embryo stages were recorded as a ratio of red stain to blue counterstain. Data were corrected for confounding area and absorbance and analysed using a multivariate linear regression model. The intensity of staining for H4L5 appeared higher in 8-cell embryos than 2- and 4-cell embryos but not to a significant level (P≥0.05); 8-cell embryos also appeared higher in stain intensity than 16-cell but of borderline significance (P=0.073). Staining intensity decreased between the 8-cell and blastocyst stage (P≤0.05). In contrast, the intensity of acetylation staining for H4L8 residue decreased slightly between the 1- and 4-cell stages and then decreased significantly between the 4- and 8-cell stages (P≤0.05), increasing significantly by the 16-cell stage (P≤0.05). A significant decrease in staining intensity was observed at the blastocyst stage (P≤0.05). In blastocyst-stage embryos both lysine-5 and lysine-8 showed a differential staining of inner cell mass (ICM) and trophectoderm (TE) cells. ICM cells showed intense staining and TE cells stained very weakly. The intensity results presented are cumulative of ICM and TE intensities, which explains the overall low levels of acetylation in blastocysts when compared to the earlier stages. Acetylation of H4L5 starts high in 1-cell embryo, as it is necessary for protamine replacement (Adenot et al., 1997 Development 124, 4615–4625), decreases when methylation is high and increases when methylation is low (as in the 8-cell stage which corresponds with zygotic gene activation). Acetylation of H4L8 decreases between the 1-and 8-cell stages; however, its association with changes in DNA methylation has yet to be determined.
60EPIGENETIC CHARACTERISTICS OF BOVINE AND CAPRINE EMBRYOS AND DONOR
CELLS USED FOR NUCLEAR TRANSFER
Authors: Memili E. , Behboodi E. , Meade H.M. , Echelard Y.
Published: February 2004

The molecular aspects of epigenetic events taking place in nuclear transfer (NT)-derived embryos are not well defined, but DNA methylation is known to be involved. One leading hypothesis is that the significant losses that occur during both pre- and post-implantation development are in great part due to improper epigenetic reprogramming. Aims of this study were to perform comparative quantitative analyses of the overall DNA methylation of bovine (both IVF- and NT-derived) and caprine IVF-derived preimplantation embryos as well as of donor cells used for NT. Caprine IVF was performed according to Blash S et al. (2001 Theriogenology 54, 899–905). Bovine and caprine 8- to 16-cell embryos were harvested on Day 4 post-insemination (dpi). Caprine donor cells (two adult fibroblast cell lines, T75-514 and F638) at the G1 phase of the cell cycle were prepared as previously described (Memili E et al. 2003 Theriogenology 59, 274 abst). Bovine cumulus cells were serum starved for four days prior to use and NT were performed as previously described (Echelard Y et al. 2002 Theriogenology 57, 779 abst). Embryos and the donor cells were vacuum-fixed onto 10-μm filters, and DNA methylation was determined by immunoassaying with a well-defined anti-5-methyl-cytosine antibody (Dean W et al. 2001 Prod Natl Acad Sci 98(24), 13734–13738) and fluorescent labeled anti-mouse IgG as secondary antibody following the protocol of Shi and Haaf (2002Mol Reprod Dev 63, 329–334). Fluorescent imaging was performed by epifluorescence microscopy. The methylation-specific signal was recorded digitally with a high-resolution charge-coupled devise camera, followed by analysis with the MetaMorph™ imaging software (Universal Imaging Corporation, Downingtown, PA, USA). These experiments showed a high level of heterogeneity in the methylation levels of bovine and caprine donor cells. However, caprine 8- to 16-cell IVF embryos exhibited similar levels of DNA methylation to their bovine IVF counterparts. Conversely, when the DNA methylation level of 97 bovine IVF nuclei was compared to that of 55 bovine NT embryonic nuclei, significant differences were found. Levels of signal intensities per nucleus were almost 9-fold greater for NT embryos (133 v. 15), a significant difference (P<0.01). In conclusion, donor cell populations with heterogeneous DNA methylation and incomplete reprogramming of DNA methylation in NT embryos are likely to be the underlying reasons for low level of successful NT embryonic and fetal development. Thus, designing NT protocols supporting better reprogramming may result in improvement since, in order for a successful embryonic development after NT, DNA methylation needs to be reprogrammed from a somatic cell pattern to an early embryonic pattern. Furthermore, similar levels of DNA methylation between caprine and bovine IVF embryos may be an indication of a similar pattern of embryonic gene expression between these species.
64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO
RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT
Authors: Park H.S. , Lee M.Y. , Hong S.P. , Jin J.I. , Park J.K. , Lee J.S. , Sohn S.H. , Jung J.Y.
Published: February 2004

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P<0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P<0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P<0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro.

Table 1
Effect of different oocyte source on in vitro development following caprine SCNT
69DOLLY: A FINAL REPORT
Authors: Rhind S. , Cui W. , King T. , Ritchie W. , Wylie D. , Wilmut I.
Published: February 2004

Dolly, the first animal to be derived by transfer of a nucleus from an adult somatic cell was euthanized on February 14, 2003, because of the presence of a virally-induced lung tumor resulting in progressive decline in respiratory function. We have carried out full gross and histopathological analysis of tissues and additionally re-examined the length of her telomeres. Dolly was derived from an oocyte recovered from a Scottish Blackface ewe and the nucleus of a cell cultured from mammary tissue of a 6-year-old Finn Dorset ewe. The culture had been through 27 population doublings. Dolly was 5 years and 7 months old at the time of her death. A detailed comparison was made between Dolly and four other ewes of similar breed and age produced by natural mating. Full post mortem examination was carried out and a range of tissues examined histologically. Major findings were the presence of extensive tumor infiltrates in both lungs and bilateral severe stifle arthritis in Dolly. Histopathology confirmed the presence of extensive sheep pulmonary adenomatosis/SPA (syn. ovine pulmonary adeoncarcinoma/OPA) lesions in the lung. This infection is caused by Jaagsiekte sheep retrovirus (JSRV) and is transmitted in respiratory droplets. Other animals in the barn had previously developed SPA. The risk of transmission of infection may have been increased by the fact that the animals were housed in close proximity in a barn. There is currently no reliable diagnostic test before the tumor develops and it is probable that Dolly was infected before we were aware of the presence of the infection in the group of sheep. There is no treatment for the disease. There is no reason to think that Dolly was more vulnerable to infection because she was a clone. The osteoarthritis was first observed during the autumn of 2001 when radiographs of the left stifle revealed osteophytes and osteophytosis associated with the patella, distal femur and proximal tibia. Radiographs of the right stifle were normal at that stage. A regime of anti-inflammatory drugs enabled the ewe to live a normal life. None of the other cloned sheep at the Institute have shown any symptoms of arthritis. Arthritis has been described in a cloned bull, but has not been described in any other cloned sheep. Telomeres in kidney tissue taken from Dolly were found to be shorter than those in kidney of the four control sheep (Dolly’s=15.6kb v. controls=17.9±0.27kb). This confirms the observation that her telomeres were shorter at 1 year of age than those of age-matched controls (Dolly 19.14kb v. controls 24kb). It is not appropriate to contrast these measurements, as they were not made in a single experiment. Dolly was fertile and delivered a total of 6 healthy lambs in three pregnancies. During routine husbandry there were no unusual findings apart from the development of the arthritis. Since the birth of Dolly, experiments in several species have revealed a range of abnormal phenotypes associated with unusual patterns of gene expression.
70PRODUCTION OF CLONES BY FIBROBLAST NUCLEAR TRANSFER FROM
AN X-AUTOSOME TRANSLOCATION CARRIER COW
Authors: Rho G.J. , Kasimanickam R. , Johnson W.H. , Semple E. , Betts D.H. , Basrur P.K. , King W.A.
Published: February 2004

Poor reproductive outcome associated with chromosome anomalies is well documented in humans and domestic animals. In cattle, female carriers of Robertsonian and X-autosome translocations tend to be repeat breeders, probably due to synaptic difficulties during meiotic prophase of gametogenesis. Although viable offspring have been obtained through somatic cell nuclear transfer (NT) using adult or fetal cells from normal animals in various species, there have been no reports to date on the application of this technology to translocation carriers. Since NT circumvents meiotic problems encountered by translocation carriers, we used this approach to generate cloned embryos, fetuses and calves from a subfertile Limousin-Jersey crossbred cow previously identified as a carrier of an X-autosome translocation. Primary cultures were established from ear skin biopsies, and used at the 5th or 6th passage for NT. Recipient oocytes were enucleated at 19h post-maturation (hpm), fused with individual fibroblasts by a single electrical pulse (1.4KVcm−1, 40μs) and activated at 24hpm with a combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1). Reconstructed eggs were cultured in SOF at 39°C in a humidified low oxygen atmosphere. In 33 runs involving 2470 oocyte-donor cell complexes, cleavage and blastocyst rates were 88% (2173/2470) and 36% (889/2470), respectively. Two or three blastocysts (Day 7±1) were transferred into each recipient, previously synchronized with a combination of CIDR, GnRH and PGF2α. Ultrasonography was performed at Days 28 to 60 and at Days 90 and 150. Pregnancy was confirmed on Day 28 in 10 of a total of 22 recipients, 2 of which were later found to be carrying twin fetuses. Of 60 embryos transferred, 11 (18.3% of embryos) survived to Day 42, 6 (10%) to Day 60, and 4 (6.6%) to Day 90. A Day-94 fetus was surgically retrieved to examine the synaptic pattern of meiocytes in fetal ovaries. The fetus and internal organs were normal in appearance, and of normal size (16.5cm C-R length). The X-autosome translocation was confirmed in blood cultures, and synaptic anomalies involving chromosome 23 and the X chromosome were detected in fetal ovaries. Another clone was delivered by C-section at 276 days but died within 1h of delivery, while one singleton pregnancy is still ongoing at >200 days. These results demonstrate that NT can be used to produce embryos, fetuses and offspring from an X-autosome translocation carrier, with the potential to facilitate study of synaptic behaviour of female germ cells, and X-inactivation in different cell lineages of cloned blastocysts, and to generate individuals with otherwise poor reproductive prospects. [Supported by NSERC, Canada and OMAFRA]