bluuebunny/crossref_metadata_2025 huggingface dataset:
171 NORMAL REPROGRAMMING OF IMPRINTING IN PARTHENOGENETIC FEMALE GERM CELLS
Authors: Horii T. , Nagao Y. , Kimura M. , Hatada I.
Published: December 2004

Mammalian parthenotes cannot develop normally to term. Mouse parthenogenetic embryos die by Day 10 of gestation. On the other hand, viable parthenogenetic chimeras were produced by normal host embryos, although parthenogenetic cells were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. This can be explained by the aberrant expressions of imprinted genes in parthenogenetic cells. In female mice, erasure of imprints occurs around the time that primordial germ cells enter the gonad, and establishment of imprints occurs in the postnatal growth phase of oogenesis. In this study, we investigated whether aberrant imprints in parthenogenetic embryonic stem (PgES) cells can be erased through the germline. Diploid parthenogenetic embryos were produced by activation of (CBA × C57BL/6-EGFP) F1 mouse superovulated unfertilized oocytes by exposure to Sr2+ and cytochalasin B. Ten parthenogenetic blastocysts were plated and three PgES cell lines were isolated. Chimeras were made by injecting 10–15 PgES cells into ICR(CD-1) mouse blastocysts. Chimeras and chimeric tissues were detected by fluorescent microscopy. In all, 173 chimeric blastocysts were transferred to 9 recipient females, and 101 live pups containing 9 female and 21 male chimeras were born. No significant growth retardation was apparent in PgES chimeras, irrespective of their degree of chimerism. In 5 male chimeras killed at 1 day postpartum (dpp), PgES cells showed a restricted tissue contribution. The contribution to lung, liver, and intestine was considerably lower than in the other tissues such as brain, heart, spleen, and kidney. PgES derived or host embryo derived non-growing oocytes were isolated from dissociated ovaries of female chimeras at 1 dpp under fluorescent microscopy. Methylation imprints in non-growing oocytes were analyzed for maternally methylated imprinted genes Peg1, Snrpn, and Igf2r by the combined bisulfite restriction analysis (COBRA). In normal oocytes, imprints are expected to be erased and these genes are unmethylated at this stage. We observed that these genes were unmethylated in both PgES derived and host embryo derived non-growing oocytes. These results suggest that aberrant imprints in PgES cells can also be erased normally through the germline.
20 EFFECTS OF DOSE OF ESTRADIOL BENZOATE AND PROGESTERONE IN PROSTAGLANDIN-TREATED BEEF HEIFERS
Authors: Martínez M. , Caccia M. , Colazo M. , Bó G. , Kastelic J. , Mapletoft R.
Published: December 2004

Estradiol and progesterone have been used to synchronize follicular wave emergence and ovulation in a two-dose prostaglandin (PGF)-based synchronization program (Martínez et al. 2004 Theriogenology 62, 363–372). However, it was observed that some heifers displayed estrus prior to the second PGF, suggesting that premature luteolysis may have occurred. An experiment was designed to determine the effects of dose of estradiol benzoate (EB) and/or progesterone (P) on follicular and luteal dynamics in a two dose PGF-based protocol in beef heifers. In two replicates, beef heifers (n = 28; Simmental, Hereford, and Charolais crosses, 350 to 450 kg) received 500 μg cloprostenol (Schering-Plough Animal Health, Pointe-Claire, PQ, Canada) on Day −7. On Day 0, heifers were randomly allocated to nine treatment groups to receive 0, 1, or 2 mg of EB and 0, 50, or 100 mg of P i.m. in canola oil in a 3 × 3 factorial design. A second PGF treatment was administered on Day 14. Ultrasonography was done once daily from Days −5 to 9, and every 12 h thereafter until ovulation. Blood samples were collected at 12-hour intervals from Day 0 to 5 for estradiol and FSH concentrations, and every 24 h for progesterone. The effects of EB and P and their interaction on corpus luteum (CL), follicles, and hormone profiles were analyzed by analysis of variance, and means were compared by LSD or Tukey's test. All variables were normally distributed (Wilk-Shapiro test and rankit plots). The day of follicular wave at the time of treatment tended to vary among groups (P = 0.08) and the diameter of the dominant follicle also differed (P < 0.05). The interval from treatment to wave emergence was shorter (P < 0.05) in heifers that received 2 mg EB (4.6 ± 0.3 d) than in those that did not receive EB (5.9 ± 0.6 d), while the 1 mg EB group (5.1 ± 0.6 d) was intermediate. The interval to wave emergence in the 2 mg EB group was the least variable (P < 0.05). There was no effect of EB (P = 0.72) on the diameter of the CL at the time of the second PGF, but there was an effect of P treatment (P = 0.01). The variability of the interval from the second PGF to ovulation may have been influenced by treatment group but was statistically not significant (P < 0.1). There was an effect of time (P < 0.01) on plasma progesterone concentrations and a P × time interaction (P = 0.06) can also be assumed. Estradiol concentrations were affected by EB dose, time, and EB × time interaction (all P < 0.01). FSH concentrations were modified by time (P < 0.01). In summary, treatment with EB 7 days after a single injection of PGF affected follicular development, while P treatment at that time appeared to influence CL function.
224 TETRACYCLINE-INDUCIBLE GENE EXPRESSION WITH RETROVIRUS VECTOR
Authors: Koo B.C. , Kwon M.S. , Jeon I. , Chang W. , Kim T.
Published: December 2004

One of the critical problems to be solved in transgenic animal production is non-controllable constitutive expression of the foreign gene, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP (green fluorescent protein) gene under the control of tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence was also introduced into the retrovirus vector downstream of either the GFP gene or the sequence encoding rtTA (reverse tetracycline-controlled transactivator). Transformed cells derived from porcine fetus were cultured in the medium supplemented with or without doxycycline (tetracycline derivative) for 48 h, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at the 3′ side of the GFP gene, while tighter expression control (up to 20-fold) was obtained from the vector in which the WPRE sequence was placed at the 3′ side of the rtTA sequence. Encouraged with these data, we substituted the hPTH (human parathyroid hormone) gene for the GFP gene in the retrovirus vector. The porcine fetal fibroblast cells transformed by the modified retrovirus vector secreted hPTH into the medium under the tight control of doxycycline as observed in GFP expression. The resulting porcine cells secreting hPTH will be used in nuclear transfer experiment.
This study was financially supported by the National Livestock Research Institute RDA (Suwon 441-350, Korea), ARPC (Agriculture R & D Promotion center, 2002–2005), and by grant No. R11-2002-100-01000-0 from the ERC program of the Korea Science & Engineering Foundation.
236 SPATIAL GENE EXPRESSION PATTERNS OF Dnmt1, Dnmt3a, AND Hdac2 IN PREIMPLANTATION EMBRYOS
Authors: Wrenzycki C. , Herrmann D. , Lucas-Hahn A. , Korsawe K. , Lemme E. , Niemann H.
Published: December 2004

In mammals, DNA methylation and the modification of histones account for the major epigenetic alterations. Usually DNA methylation is associated with transcriptional silencing, which is catalyzed by two important classes of DNA methyltransferases. DNA methyltransferase 1 (Dnmt1), a maintenance enzyme, methylates hemi-methylated DNA after DNA replication. Dnmt3a and Dnmt3b are required for de novo methylation in vivo and for establishing new DNA methylation marks during development. In addition to DNA methylation, post-translational modifications of the amino termini of core histones are thought to affect the expression or repression of transcription. Histone deacetylation catalyzsed by histone deacetylases (Hdac) often results in transcriptional repression. Perturbated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases introduced by assisted reproduction technologies. The objective of the present study was to determine the relative abundance of Dnmt1, Dnmt3a, and Hdac2 transcripts in ICM and TE cells of pre-implantation bovine embryos of different origin. Embryos were generated with standard protocols of in vitro production (IVP) and parthenogenetic activation using SOF medium supplemented with BSA (SOF d7/d8, Parth d7/d8) or TCM medium supplemented with estrus cow serum (TCM d7/d8; Wrenzycki et al. 2001 Hum. Reprod. 16, 893–901; Wrenzycki et al. 2002 Biol. Reprod. 66, 127–134). Expanded blastocysts were collected at Days 7 and Day 8 (d7/d8) of culture (Day 0 = IVF). In vivo-generated blastocysts collected from superovulated animals were included as controls (Wrenzycki et al. 1996 J. Reprod. Fert. 108, 17–24). A highly sensitive RT-PCR assay (Wrenzycki et al. 2003 Biol. Reprod. 68, 2073–2080) was used to determine the relative abundance (RA) of gene transcripts in isolated ICM and TE cells. The allocation of ICM and TE cells was analyzed using a differential staining technique (Eckert and Niemann 1998 Mol. Hum. Reprod. 4, 957–965) to calculate the RA of each transcript on a per cell basis. RT-PCR assays were repeated at least six times. Significant differences in the RA of Dnmt1 (In vivo, TCM d7, Parth d7/d8, SOF d8), Dnmt3a (In vivo, SOF d7), and Hdac2 (TCM d7) transcripts were detected between ICM and TE cells. No differences were detected for all transcripts in ICM cells of embryos collected at either Day 7 or Day 8. In TE cells, the RA of Dnmt1 transcripts was significantly reduced or increased in embryos generated in SOF or TCM medium at Day 7, respectively, and the RA of Hdac2 transcripts was significantly higher in embryos generated in TCM medium at Day 8. These results suggest that in vitro culture alters the spatial expression pattern of genes related to epigenetic modifications in the cell lineages critical for a normal embryonic and fetal development.
This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).
315 AN ATTEMPT AT INDUCING DIFFERENTIATION INTO ROUND SPERMATIDS OF RAT SPERMATOGONIA BY CO-CULTURING WITH SERTOLI CELLS
Authors: Iwanami Y. , Kobayashi T. , Kato M. , Hirabayashi M. , Hochi S.
Published: December 2004

Mammalian spermatogenesis is a complex process of germ cell development at the seminiferous tubules whereby diploid spermatogonia proliferate and differentiate into haploid spermatozoa via round and elongating spermatids in close association with somatic Sertoli cells. In the present study, the potential of rat spermatogonia to undergo meiosis during co-culture with Sertoli cells was assessed. The type-A spermatogonia and Sertoli cells were prepared from Day 7 heterozygous transgenic male rats carrying EGFP DNA, and co-cultured on the dishes (coated; BD Falcon™ 35-3801, or non-coated: BD Falcon™ 35-1008, 4 × 106 cells/4-mL dish) at 37°C for 3 days and at 34°C for a subsequent 7 days in 5% CO2 in air. The culture medium was DMEM medium supplemented with 10% fetal bovine serum, growth factors (10 ng/mL EGF and 10 ng/mL IGF) and various hormones (500 ng/mL FSH, 133 μIU/mL hGH, 5 μg/mL insulin, 0.1 μM testosterone and 0.1 μM dihydrotestosterone). During culture, appearance of round spermatid-like cells (ca. 15 μm in cellular diameter and 7–8 μm in nuclear diameter) was traced. The ploidy of the cells was also analyzed by flow cytometry (FCM). At the end of culture, the proportion of EGFP DNA-bearing cells in the total cultured cell population was examined under UV light at 365 nm. Thereafter, continuation of the spermatid-like cells to full-term development was examined by ooplasmic microinjection (Kato et al. 2004 Contemp. Top. Lab. Anim. Sci. 43/2, 13). Briefly, oocytes from the Sprague-Dawley rats were denuded, activated with two direct-current pulses at 100 V/mm for 99 μs and held in 2 mM 6-dimethylaminopurine for 20 min. The nuclei of spermatid-like cells were microinjected into the oocytes by using a piezo impact driving unit, and the injected oocytes after 24 h culture were transferred into recipients. Round spermatid-like cells were first observed at the 5th day of culture on both dishes, but the proportion of spermatid-like cells on the coated dish was higher than that on the non-coated dish. The FCM analysis showed that a single peak of haploid cells was detected in the cell population cultured on the coated dish at the 5th day of culture, while no haploid peak was detected on the non-coated dish. The cultured cells exhibited two distinct patterns of EGFP fluorescence, with a proportion of EGFP-positive cells at 53.5% (total 1,000 counts). The microinsemination into 263 oocytes resulted in the production of 27 oocytes with two pronuclei (10.3%) and 15 cleaved oocytes (5.7%). However, the oviductal transfer of 143 microinseminated oocytes resulted in only 8 implantation sites without viable offspring (5.6%). These results indicated that rat type-A spermatogonial cells seemed to undergo meiosis, but the potential of the cultured spermatid-like cells to participate into full-term development was questionable.
325 INSULIN-LIKE GROWTH FACTOR-I CONCENTRATIONS IN OVIDUCT FLUID OF SUPEROVULATED EWES DURING THE PERI-OVULAR PERIOD
Authors: Kakar M.A. , Maddocks S. , Lorimer M.F. , Kleemann D.O. , Walker S.K.
Published: December 2004

This study examined the concentrations of insulin-like growth factor-1 (IGF-1) in oviduct fluid during the peri-ovular period as a reference for the establishment of optimal in vitro culture conditions for sheep embryos. Six mature ewes (4–5 years, 58–67 kg) of comparable body condition were fed a standard diet for two weeks before the start of fluid collection. Ewes were superovulated using conventional treatment involving a progestagen, FSH, and GnRH treatment. Oviducts were catheterized four days (which is sufficient time to recover from surgery) before collection of oviductal fluid, which started one day (Day 1) before the time of ovulation (Day 0) and continued until five days later (Day 5). Oviductal fluid was acidified by diluting into 0.8 M acetic acid/0.2 M trimethylamine, pH 2.8, mixed, and incubated to dissociate IGFs from IGF-binding proteins (IGFBPs). Following incubation, acidified fluid was centrifuged at 10,000g through a 0.1-mm Micro-spin centrifuge filter; the filtrate transferred to glass high-performance liquid chromatography (HPLC) vials. IGFs and IGFBPs were separated from one another by high-performance size-exclusion liquid chromatography using a Protein-Pak 125 column (Waters Corporation, Milford, MA, USA) and 0.2 M acetic acid, 0.05 M trimethylamine, pH 2.8, at a flow rate of 1 mL/min. Oviductal fluid IGF-I was collected in a single 2-mL fraction directly from the HPLC and its concentration measured by an IGF-I-specific enzyme-linked immunosorbent assay (Diagnostic Systems Laboratories, Inc. Webster, TX, USA). The data were analyzed by analysis of variance. The non-superovulation group had significantly higher concentrations of oviductal IGF-I compared with the superovulation group. In the superovulated group, there was, however, a significant effect of day on the oviductal fluid IGF-I concentration (P < 0.01) such that the concentrations of IGF-I first increased for three days and then decreased for the remaining four days. In the non-superovulation group, there was no significant two-way interaction between ovulation and day. It can be concluded that the levels of IGF-I increase over time and then decrease.
Authors express thanks to the help of Jenn Skye and Hemish Turretfield Research Station SA.
47 CHROMATIN AND CYTOSKELETAL REORGANIZATION OF RABBIT OOCYTES AFTER CUMULUS CELL NUCLEAR TRANSFER
Authors: Liu C.-T. , Lin T.-A. , Tang P.-C. , Ju J.-C.
Published: December 2004

It has been reported that a preincubation of the reconstructed oocytes prior to activation treatment might enhance the efficiency of nuclear reprogramming after somatic cell nuclear transfer in many species. The objectives of this study were to evaluate chromatin and cytoskeletal reorganization of nuclear-transferred rabbit oocytes before activation treatment. Sexually mature New Zealand White rabbits (6-month-old) were superovulated by six consecutive injections of FSH (0.4 mg) at 12-h intervals, followed by a single hCG (200-IU) injection. Mature oocytes were collected by flushing the oviducts 14–15 h post-hCG treatment. Cumulus cells were introduced into enucleated oocytes by piezo-driven microinjection. The reconstructed oocytes were randomly allocated to two treatment groups, with or without electric pulses (EP, 2 kV cm−1), and the progressive changes of the chromatin and cytoskeleton were examined by immunocytochemical staining at 0, 2, and 4 h after EP. The oocytes (n = 59) without successful enucleation served as the control, in which 30 out of 59 oocytes were electrically pulsed. After immunocytochemical staining, the proportions of reconstructed oocytes possessing dense chromatin decreased from 0 to 4 h (40-0% vs. 82-9%, respectively) in both groups, but only the oocytes without EP showed a significant reduction (P < 0.05). In contrast, the proportions of reconstructed oocytes with spread chromatin increased significantly from 0 to 4 h in both groups (20–95% vs. 5–82%, respectively; P < 0.05). The oocytes at 0 h after EP had the greatest percentage (35%) of normal chromosome alignment on the spindle compared to the other EP groups. The EP treatment also caused significant increase of the cytasters in both the reconstructed (5 to 45%, P < 0.05) and the control oocytes (20 to 78%) at 0 h. These changes were recovered at 2 h after EP, but the proportions of cytasters in the reconstructed oocytes increased to 59% in the non-EP group and 81% in EP group at 4 h after EP. The percentages of reconstructed oocytes with multiple spindles increased from 0 to 27% and 43% in EP and non-EP groups, respectively. These results demonstrated that EP changed the proportions of chromatin configurations from the condensed form to normal alignment on the metaphase plate. The extended preincubation period for the reconstructed oocytes prior to activation treatment might increase the incidences of chromatin and spindle abnormalities of the reconstructed rabbit embryos.
53 CELL CYCLE SYNCHRONIZATION OF DONOR CELLS AT G1 PHASE AND DEVELOPMENTAL ABILITY OF NUCLEAR TRANSFER EMBRYOS IN MINIATURE PIGS
Authors: Miyamoto K. , Hoshino Y. , Nagao Y. , Minami N. , Yamada M. , Imai H.
Published: December 2004

The cell cycle of donor cells is one of the essential factors for the success of somatic cell nuclear transfer (SCNT), and G0/G1-phase cells have been widely used as donor cells. However, cells synchronized at the G0/G1-phase also have a population of cells with cell cycles other than G0/G1-phase, and we cannot precisely know the cell cycle of donor cells being used for SCNT. In this experiment, we reconstructed SCNT embryos from donor cells that were synchronized at the G1-phase or at the G0/G1-phase and compared their developmental ability in two different culture media. Immature oocytes were isolated from ovaries collected from domestic gilts at a local slaughterhouse and were co-cultured with follicle shells for in vitro maturation (Hoshino et al. 2003 Theriogenology 59, 260). Donor cells were collected from fibroblast cells of miniature Potbelly pigs. Cells synchronized at the G1-phase were prepared shortly after dividing M-phase cells that had been synchronized using 2-methoxyestradiol, as described by Urakawa et al. (2004 Theriogenology 62, 714–728). The G0/G1 cells were also prepared from a fully confluent culture of cells. Donor cells were fused with enucleated oocytes and simultaneously activated by two electric pulses. Reconstructed embryos were cultured in two different media [Whitten and Biggers medium supplemented with 0.5 mg/mL hyaluronic acid sodium salt (WM) and porcine zygote medium-3 (PZM-3, Yoshioka et al. 2002 Biol. Reprod. 66, 112–119)] under 5% CO2 in air. Cleavage rate and development rate to the blastocyst stage were assessed after 48 and 168 hr of culture, respectively. The results are summarized in Table 1. Developmental rate to the blastocyst stage of SCNT embryos reconstructed from G1 cells and cultured in PZM-3 (40%) was significantly higher than that of embryos cultured in WM (25%). The SCNT embryos of the G1 cells showed significantly lower cleavage rate (51%) than that of the G0/G1 cells (69%). However, the developmental rates to the blastocyst stage per cleaved embryo in WM were significantly higher in G1 cells (50%) compared with G0/G1 cells (32%). In addition, the total cell number of the SCNT blastocysts was comparable between the cultures in WM (58 ± 4) and PZM-3 (46 ± 5), although the ratio of inner cell mass cells to the total cell number was significantly higher in PZM-3 (32%) compared with WM (14%). These results suggest that PZM-3 may fit with the culture of SCNT embryos, and that the G1 synchronized cells could be stably reprogrammed for early embryonic development in SCNT embryos and be useful as donor cells for analyzing the processes of nuclear reprogramming.

Table 1.
Development of nuclear transfer embryos reconstructed from G1 or G0/G1 cells in different culture media
56 SOMATIC CELL NUCLEAR TRANSFER IN NON-HUMAN PRIMATES: THE POSSIBILITY OF USING OOCYTES MATURED IN VITRO FOR UP TO 3 DAYS AS HOST OOPLASTS
Authors: Uoc N.T. , Bavister B.D. , Hanh N.V. , Bui L.C. , Thanh N.T. , Duc N.H. , Huu Q.X. , Linh N.V. , Bui X.N.
Published: December 2004

Production of cloned nonhuman primate embryos has been reported using mature oocytes obtained from donors treated in vivo with a high dose of recombinant human FSH (r-hFSH, 35 IU per day for 10 days). The disadvantages of this approach are the high cost of hormones and the need to use the oocytes shortly after collection. Our study aimed to investigate the possibility of using initial in vivo treatment with a reduced FSH dose followed by in vitro culture for long periods of up to 3 days to produce mature monkey oocytes as host ooplasts for somatic cell nuclear transfer (SCNT). Adult female long-tailed Macaque (Macaca fascicularis) monkeys were treated with r-hFSH (Serono, Aubonne, Switzerland, 35 IU per day, i.m.) either for 10 days with an injection of hCG (1000 IU, i/m) 34 h before oocyte collection (G.I) or with only r-hFSH for 7 days (G.II). Cumulus oocyte complexes (COCs) were collected by follicular aspiration and then cultured in TCM-199 medium (GIBCO) supplemented with estradiol-17β, FSH, LH, and 10% FCS at 39°C in an incubator with 5% CO2 in air. The maturation rate based on the level of cumulus expansion and the presence of the first polar body was recorded at the moment of collection and during 24 h, 48 h, and 72 h of in vitro maturation (IVM). For SCNT, the mature Metaphase II oocytes were separated from cumulus cells and selected for enucleation in the presence of cytochalasin B (Sigma, St. Louis, MO, USA). Skin fibroblasts obtained from adult monkeys were cultured in DMEM+ 10% FCS and induced to quiescence in DMEM 0% FCS 2 days before use. A single cell was transferred under the zona of each enucleated oocyte. Couplets were fused with two direct current (DC) pulses of 220 V/mm for 25 μs in Zimmerman medium. Fused oocytes were cultured in medium containing cyclohexamide for 6 h before placing them into monkey culture medium (Cook, Brisbane, Australia). The average number of oocytes collected per animal were 21.2 (n = 18) and 18.6 (n = 12) for the G.I and G.II treatments, respectively. For G.I, the rate of COCs with fully expanded cumulus was 42% at collection and was maximal (80%) at Day 1 of IVM. For G.II, fully expanded cumulus was not observed at the time of collection and during the first 2 days of IVM, but 75% of COCs had full cumulus expansion by Day 3 of IVM. The rates of intact and fused oocytes were 50.3% for G.I and 55.4% for G.II. From the fused oocytes, 67.8% and 64.4% developed to the 4- to 8-cell stages at Days 2–3 after nuclear transfer for G.I and G.II, respectively. From these data, it can be concluded that this approach can be applied to optimize production of mature oocytes for non-human primate SCNT and ART (assisted reproductive technologies) programs.
This work was supported by AIRE-Development.
69 PRODUCTION OF CLONED PIGS BY NUCLEAR TRANSFER OF PREADIPOCYTES
Authors: Tomii R. , Kurome M. , Ueda H. , Ueno S. , Hiruma K. , Kano K. , Nagashima H.
Published: December 2004

Since the first success in producing cloned pigs, donor cells have been limited to fetal fibroblasts and a few other cell types. The aim of the present study was to determine if porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (NT) in pigs. Preadipocytes established from subcutaneous adipose tissue of a male adult pig were used as nuclear donor cells. Cell cycle synchronization was carried out by serum starvation (5 days), confluency (5 days), roscovitine treatment (15 μM, 2 days), or differentiation induction by 0.5 mM 3-Isobutyl-1-methylxanthine, 0.25 μM dexamethasone, and 5 μg/mL insulin (5 days). Cell cycle synchronization and apoptosis of the donor cells were examined by flow cytometry and Annexin V staining and TUNEL. IVM oocytes were obtained from abattoir ovaries and matured in NCSU23. Donor cells were fused with the enucleated recipient oocytes by a single DC pulse of 200 V/mm for 10 μs in 0.28 M mannitol + 0.15 mM MgSO4. Reconstructed embryos were electrically activated at 1–1.5 h after the NT, followed by cytochalasin B treatment for 3 h. Development of the NT embryos was assessed by fixation/staining at 3 h after NT, culture for 7 days in NCSU23, and transfer to the oviducts of estrus-synchronized recipient gilts. The cells immediately entered the G0 phase by differentiation induction (92.5 ± 0.4%), with higher efficiency of synchronization than for the other methods (roscovitine: 80.3 ± 0.2%; confluency: 79.9 ± 0.3%, P < 0.05) except for serum starvation (89.8 ± 0.6%). The proportion of apoptotic cells in the differentiation group was significantly lower than the other groups (Annexin V: 7.7% vs. 15.7 to 19.3%, TUNEL: 8.3% vs. 12.8 to 14.0%, P < 0.05). Incidence of premature chromosome condensation following NT (88.0%) was as high as that observed after NT with fetal fibroblasts previously (data not shown). In vitro developmental rates of the NT embryos did not differ significantly among the cell cycle synchronization methods of the donor cells (7.2 to 10.8%). Cell number of the blastocysts was highest in the differentiation group (49.0 vs. 30.2 to 41.9, P < 0.05). Transfer of 1004 cloned embryos of the serum starvation group to 5 recipients resulted in the production of 4 live and 1 stillborn piglets from 1 recipient. Transfer of cloned embryos reconstructed of donor cells treated by differentiation induction is currently underway. These data demonstrate that preadipocytes collected from an adult pig are promising nuclear donor cells for pig cloning.
This study was supported by PROBRAIN.
71 DEVELOPMENTAL DELAY OF PRE-IMPLANTATION OVINE IN VITRO CULTURED AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Tveden-Nyborg P. , Peura T. , Hartwich K. , Maddox-Hyttel P.
Published: December 2004

Despite advances in the production of somatic cell nuclear transfer (SCNT) embryos, significant embryo losses are persistent, particularly around implantation. Malformations of the placenta and in a variety of organs are seen, and have been linked to deviant epigenetic reprogramming. The aim of the present study was to examine the formation of germ layers, which are prerequisites for formation of the embryo proper and placenta, in invivo-derived (in vivo), partly in vitro-cultured (IVC), and SCNT ovine embryos. Embryos were derived as follows: In vivo embryos (n = 27) were flushed from the uterus on Days 7, 9, 11, and 13. For IVC embryos (n = 22) in vivo zygotes were flushed, followed by culture in the presence of 20% human serum, transfer to the uterus on Day 6, and flushing as in vivo embryos. SCNT embryos (n = 41) were produced by fusion of serum starved granulosa cells with enucleated oocytes, followed by activation, culture in SOF, transfer to the uterus on Day 6, and flushing as described for in vivo embryos. Recovered embryos were processed for light microscopy (LM) and transmission electron microscopy (TEM), and paraffin sections were immunohistochemically labelled for the germ layers: alpha-1-fetoprotein for potential endoderm, cytokeratin-8 for potential ectoderm, and vimentin for potential mesoderm. A consistent delay of the IVC and particularly the SCNT embryos was noted throughout all time points: On Days 7 and 9, differentiation of the inner cell mass into hypoblast and epiblast was evident in 7 out of 12 in vivo embryos, whereas this phenomenon was less prominent or absent in 9 out of 13 IVC and 13 out of 15 SCNT embryos. Furthermore, 6 of the IVC and 12 of the SCNT embryos lacked an identifiable embryonic disc. On Day 11, half of the in vivo embryos had initiated gastrulation, evidenced by localization of endoderm and mesoderm precursor cells between the hypoblast and the epiblast. This feature was noted in only a single IVC and in none of the SCNT embryos. On Day 13, all in vivo embryos had completed gastrulation including the formation of somatic and visceral mesoderm. This feature was noted in only 1 out of 3 IVC and in none of the SCNT embryos. Likewise, amniotic folds were seen in one third of the in vivo embryos at this stage, but not observed in any IVC or SCNT embryos. The immunohistochemical markers displayed the same cell lineage localization in all three groups of embryos, but a developmental delay in the IVC and in particular the SCNT embryos was evident. In conclusion, ovine IVC and SCNT embryos develop at a slower rate than in vivo embryos at least up until Day 13 of gestation.
89 EVALUATION OF A CUSHIONED CENTRIFUGATION TECHNIQUE FOR PROCESSING BOAR SEMEN FOR FREEZING
Authors: Matas C. , Gadea J. , Decuadro-Hansen G.
Published: December 2004

Boar semen freezing procedures include the use of centrifugation to concentrate sperm and remove seminal plasma prior to dilution in freezing extender. The centrifugation techniques employed have necessarily been a compromise between the need to recover as many spermatozoa as possible after centrifugation and the damage caused by pelleting the sperm. The use of an inert, dense, and isotonic solution as a cushion in the bottom of the tube leads to the use of higher-speed centrifugation to ensure maximum sperm recovery. However, it is necessary to know the viability and functionality of the samples after the thawing process. The aim of this work was to evaluate the effect of cushion-technique centrifugation on the in vitro sperm viability and the penetrating capacity after thawing. Sperm-rich fractions from five fertile boars were diluted and cooled to 15°C before centrifugation. Two centrifugation regimes were used: 800g for 10 min called the “standard method” (SM) (Westendorf P etal. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267) and 1000g for 20 min on an iodixanol isotonic solution 60% w/v gradient (Sigma Chemical Co., St. Louis, MO, USA) called the “cushion method” (CM). Spermatozoa were diluted in lactose/egg-yolk extender, cooled to 5°C over 2 h and then frozen with glycerol and Equex by classic methodology (Westendorf P et al. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267). Frozen sperm samples were thawed in a circulating water bath at 38°C for 30 s. To detect increases in plasma membrane lipid packing disorder and viability, frozen-thawed samples of sperm were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378–391) and evaluated by flow cytometry. In vitro penetration ability was assessed using the homologous in vitro penetration (hIVP) test with immature oocytes (Gadea and Matas 2000 Theriogenology 54, 1343–1357). ANOVA analysis revealed that centrifugation by CM showed higher values of intact viable spermatozoa than SM centrifugation (60.21 v. 54.68%, P < 0.05). The in vitro penetration assay showed no differences in penetration rate or mean number of sperm penetrated per oocyte. However, significant boar and interaction effects were found (P < 0.01). These results indicated that different effects of the treatment were found for every boar. In conclusion, the cushioned centrifugation method gives a simple means of processing porcine semen for freezing more efficiently without loss of fertilizing capacity.
This work was supported by AGL-2003-03144.
95 EFFECT OF ESTROUS COW SERUM ON SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS AFTER SLOW FREEZING OR VITRIFICATION
Authors: Mucci N. , Aller J. , Ross P. , Kaiser G. , Cabodevila J. , Hozbor F. , Alberio R.
Published: December 2004

Until now, the major obstacle associated with the extensive use of in vitro-produced bovine embryos is the lack of suitable methods to cryopreserve them. At least two approaches exist for overcoming this problem. One is to adjust cryopreservation methods to the requirements of these embryos, and the other is to improve embryo quality by using an appropriate in vitro environment for embryo production. The objective of this study was to determine the effect of estrous cow serum (ECS) during in vitro culture on embryo survival after cryopreservation by slow freezing or vitrification. Cumulus-oocytes complexes were in vitro-matured and fertilized as previously described (Ferre et al. 2003 Theriogenology 59, 301 abst). Presumptive zygotes were denuded from cumulus cells and cultured in groups of 50 in 400 μL drops of CR1aa medium. Seventy-two hour post-insemination (PI) embryos were randomly separated into three groups. Each group was then cultured in CR1aa + 5% ECS (without BSA; CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA), or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). Embryos were cultured under 38.5°C, 5% CO2, 5% O2, and 90% N2. At 7.5 days PI, blastocysts from each group were double stained using propidium iodide and bisbenzimide (Hoechst 33342) to determine damaged cells and total cell number. The remaining embryos were randomly cryopreserved by freezing (1.5 M ethylene glycol; cooled at 0.5°C/min to −35°C) or vitrification (open pulled straw, Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). After thawing or warming, embryos were cultured in CR1-ECS-BSA to evaluate embryo survival (hatching rate). Data were analyzed by χ2, ANOVA and Student's t-test (SAS Institute, Inc., Cary, NC, USA). Total cell number was higher in embryos cultured in CR1-ECS than in CR1-BSA or CR1-ECS-BSA (CR1-ECS: 142.1 ± 4.7, n = 23 vs. CR1-BSA 124.7 ± 4.9, n = 21, and CR1-ECS-BSA 125.8 ± 4.5, n = 25; t-test, P < 0.05). No differences were found in percent of damaged cells (CR1-ECS: 0.7%; CR1-BSA: 1.8%; CR1-ECS-BSA: 0.7%). Blastocyst survival after thawing was affected by cryopreservation methods and culture media (P < 0.01, Table 1). No interaction was found between both factors. In conclusion, under our experimental conditions elimination of ECS from CR1aa medium improves embryo cryotolerance. Vitrification allows for higher survival rates, regardless of the presence of serum during embryo culture.

Table 1.
Effect of cryopreservation method and serum supplementation during embryo culture on survival rate of in vitro-produced bovine embryos
Structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus S2 fusion protein
Authors: Supekar Vinit M. , Bruckmann Chiara , Ingallinella Paolo , Bianchi Elisabetta , Pessi Antonello , Carfí Andrea
Published: December 2004

A coronavirus (CoV) has recently been identified as the causative agent of the severe acute respiratory syndrome (SARS) in humans. CoVs enter target cells through fusion of viral and cellular membranes mediated by the viral envelope glycoprotein S. We have determined by x-ray crystallography the structure of a proteolytically stable core fragment from the heptad repeat (HR) regions HR1 and HR2 of the SARS-CoV S protein. We have also determined the structure of an HR1-HR2 S core fragment, containing a shorter HR1 peptide and a C-terminally longer HR2 peptide that extends up to the transmembrane region. In these structures, three HR1 helices form a parallel coiled-coil trimer, whereas three HR2 peptides pack in an oblique and antiparallel fashion into the coiled-coil hydrophobic grooves, adopting mixed extended and α-helical conformations as in postfusion paramyxoviruses F proteins structures. Our structure positions a previously proposed internal fusion peptide adjacent to the N-terminus of HR1. Peptides from the HR2 region of SARS-CoV S have been shown to inhibit viral entry and infection
in vitro
. The structures presented here can thus open the path to the design of small-molecule inhibitors of viral entry and candidate vaccine antigens against this virus.
Chromosomal locus rearrangements are a rapid response to formation of the allotetraploid
Arabidopsis suecica
genome
Authors: Pontes Olga , Neves Nuno , Silva Manuela , Lewis Michelle S. , Madlung Andreas , Comai Luca , Viegas Wanda , Pikaard Craig S.
Published: December 2004

Allopolyploidy is a significant evolutionary process, resulting in new species with diploid or greater chromosome complements derived from two or more progenitor species. We examined the chromosomal consequences of genomic merger in
Arabidopsis suecica
, the allotetraploid hybrid of
Arabidopsis thaliana
and
Arabidopsis arenosa
. Fluorescence
in situ
hybridization with centromere, nucleolus organizer region (NOR), and 5S rRNA gene probes reveals the expected numbers of progenitor chromosomes in natural
A
.
suecica
, but one pair of
A
.
thaliana
NORs and one pair of
A
.
arenosa
-derived 5S gene loci are missing. Similarly, in newly formed synthetic
A
.
suecica
-like allotetraploids, pairs of
A
.
thaliana
NORs are gained
de novo
, lost, and/or transposed to
A
.
arenosa
chromosomes, with genotypic differences apparent between F
3
siblings of the same F
2
parent and between independent lines. Likewise, pairs of
A
.
arenosa
5S genes are lost and novel linkages between 5S loci and NORs arise in synthetic allotetraploids. By contrast, the expected numbers of
A
.
arenosa
-derived NORs and
A
.
thaliana
-derived 5S loci are found in both natural and synthetic
A
.
suecica
. Collectively, these observations suggest that some, but not all, loci are unstable in newly formed
A
.
suecica
allotetraploids and can participate in a variety of alternative rearrangements, some of which resemble chromosomal changes found in nature.
Regulation of fibronectin splicing in sinusoidal endothelial cells from normal or injured liver
Authors: Chang Ming-Ling , Chen Jeng-Chang , Alonso Claudio R. , Kornblihtt Alberto R. , Bissell D. Montgomery
Published: December 2004

Fn containing an extra type III domain (EIIIA in the rat, ED1 or EDA in humans) is commonly termed “fetal” fibronectin, but it is prominent during the injury response of adult tissues and mediates important early events in the response. This form is particularly apparent in acute liver injury, where it has been shown that sinusoidal endothelial cells produce EIIIA-fibronectin. This fibronectin isoform arises by alternative splicing of the primary transcript. In the present experiments, we have studied the regulation of fibronectin splicing in primary sinusoidal endothelial cells by transfecting a minigene containing the EIIIA exon and its flanking introns, driven by various promoters. The results indicate that fibronectin splicing in endothelial cells from normal liver is in part promoter-dependent. However, in cells from injured liver in which expression of both total and EIIIA-fibronectin is strikingly increased, promoter effects disappear. Because fibronectin splicing is known to be regulated in part by TGFβ, we also examined the effect of a soluble inhibitor of the TGFβ type 2 receptor. This agent had no effect on splicing by normal endothelial cells. By contrast, for endothelial cells from the injured liver, the splicing pattern reverted to that of normal cells, i.e., it became promoter-dependent. We conclude that, in the setting of injury
in vivo
, TGFβ overrides the promoter dependence of fibronectin splicing in normal cells. The data suggest that TGFβ modifies the spliceosome, if not through its known signaling intermediates, then through the products of genes regulated by this cytokine.
Slope aspect affects the seed mass spectrum of grassland vegetation
Authors: Csontos Péter , Tamás Júlia , Podani János
Published: December 2004

Seed mass distribution in grassland communities of slopes of contrasting aspect was analysed in dolomite regions of the Pannonian Basin. Species frequencies were obtained for four pairs of data sets, which originated from corresponding south- and north-facing dolomite grasslands, thus forming four independent case studies. The data sets comprised 5–15 sample plots and 51–114 (average 85) species. The species were classified using an eight-class system reflecting their mean seed mass (MSM) records (class 1 being the lowest, MSM ≤ 0.2 mg; class 8 being the highest, MSM > 50 mg). Seed mass class distributions derived from slopes of contrasting aspect showed strong significant differences in chi-square tests for trend for all the four case studies. Small-seeded species (classes 1 and 2) showed a positive balance for the south-facing slopes, whereas large-seeded species (classes 5, 6, 7 and 8) were more frequent on north-facing slopes. Species with intermediate seed mass (classes 3 and 4) were not distinctive between the slopes. These results represent strong evidence of increased seed mass in the vegetation of north-facing grasslands, when compared to their south-facing counterparts. Among the phenomena potentially responsible for the new findings, we discuss the roles of microclimatic effects (especially drought stress and light regime), grass litter, interspecific competition and seed predation.
Perturbation Analysis of the Voltage-sensitive Conformational Changes of the Na+/Glucose Cotransporter
Authors: Loo Donald D.F. , Hirayama Bruce A. , Cha Albert , Bezanilla Francisco , Wright Ernest M.
Published: December 2004

Conformational changes of the human Na+/glucose cotransporter (hSGLT1) were studied using voltage-jump methods. The cotransporter was expressed in Xenopus laevis oocytes, and SGLT1 charge movements were measured in the micro- to millisecond time scale using the cut-open oocyte preparation and in the millisecond to second time scale using the two-electrode voltage clamp method. Simultaneous charge and fluorescence changes were studied using tetramethylrhodamine-6-maleimide-labeled hSGLT1 Q457C. In 100 mM external [Na+], depolarizing voltage steps evoked a charge movement that rose initially to a peak (with time constant τ = 0.17 ms) before decaying to steady state with two time constants (τ = 2–30 and 25–150 ms). The time to peak (0.9 ms) decreased with [Na+], and was not observed in 0 mM [Na+]. In absence of Na+, charge movement decayed monotonically to steady state with three time constants (0.2, 2, and 150 ms). Charge movement was accompanied by fluorescence changes with similar time courses, indicating that global conformational changes monitored by charge movement are reflected by local environmental changes at or near Q457C. Our results indicate that the major voltage-dependent step of the Na+/glucose transport cycle is the return of the empty carrier from inward to outward facing conformations. Finally, we observed subtle differences between time constants for charge movement and for optical changes, suggesting that optical recordings can be used to monitor local conformational changes that underlie the global conformational changes of cotransporters.
Functional Roles of Nonconserved Structural Segments in CFTR's NH2-terminal Nucleotide Binding Domain
Authors: Csanády László , Chan Kim W. , Nairn Angus C. , Gadsby David C.
Published: December 2004

The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282–293) identified two regions absent from structures of all other NBDs determined so far, a “regulatory insertion” (residues 404–435) and a “regulatory extension” (residues 639–670), both positioned to impede formation of the putative NBD1–NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1–414 with residues 433–1480, or residues 1–633 with 668–1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.
A visit to mars to discover progressive automation
Authors: Wittenberg Gunter
Published: December 2004

Examines how Mars Electronics International Ltd of Wokingham, expanded
into transaction electronics [the handling of money] investing
heavily in equipment for the automatic assembly of printed circuit boards and
subsequently investigating ways of minimizing work in progress and lead time,
with the eventual aim of producing single boards with zero change‐over
time and no loss of efficiency. Discusses how MEI have progressed from bench
assembly to fully automated robotics assembly via an interim flexible guided
manual assembly line for small batch production. Concludes that with its wide
range of assembly automation experience, MEI has learnt the importance of
flexibility in a system and predicts that in the future attention will focus
on “machines built around people” with concern for access,
servicing and the environment.