bluuebunny/crossref_metadata_2025 huggingface dataset:
257 DETECTION OF MICROTUBULES BY POLARIZED LIGHT MICROSCOPY IN SHEEP AND GOAT OOCYTES
Authors: Caamaño J. N. , Catalá M. , Romaguera R. , Diez C. , Muñoz M. , Martín D. , Morató R. , Carrocera S. , Mogas T. , Paramio M. T. , Gomez E.
Published: August 2011

The meiotic spindle in the oocyte is composed of microtubules and plays a key role in normal chromosome alignment and segregation during meiosis. In oocytes from farm animals, the meiotic spindle cannot be detected by conventional light microscopy due to the characteristic of their cytoplasm. Conventional methods to image the meiotic spindle rely on fixation of the oocytes. Polarized light microscopy (PLM) allows noninvasive evaluation of the meiotic spindle of metaphase oocytes. The aim of this study was to assess the efficiency of polarized light microscopy to detect microtubule-polymerized protein within in vitro matured prepubertal sheep and goat oocytes. We carried out 2 studies. In the first one, cumulus–oocyte complexes from slaughterhouse sheep ovaries were matured in vitro for 27 h. After in vitro maturation, oocytes (n = 77) were denuded of cumulus cells and placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein, which could be associated with the forming of a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by (Morató et al. 2008 Mol. Reprod. Dev. 75, 191–201). The experiment was replicated 4 times. The correlation analysis was performed using the Proc Corr procedure of SAS. There was a positive correlation (r = 0.87; P < 0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A positive PLM signal was detected in 87.0% of the oocytes, and 69.0% of the oocytes reached the metaphase II (MII) stage after in vitro maturation. A barrel-shaped spindle was observed in 77.3% of the MII oocytes. In the second study, we performed a similar experiment but used goat oocytes. A total of 78 oocytes were used, and PLM and immunostaining were performed in each individual oocyte as it was described with sheep oocytes. There was also a positive correlation (r = 1; P < 0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein. A positive PLM signal was detected in 98.7% of the oocytes, and 80.7% of the oocytes reached the MII stage after in vitro maturation. A barrel-shaped spindle was observed in 92.0% of the MII oocytes. These results indicate that PLM is an efficient system to detect polymerized protein in in vitro matured sheep and goat oocytes.

This work was supported by the following grant: INIA: RZ2007-00013-00-00. M. Muñoz and D. Martín are sponsored by RYC08-03454 and PTA2007-0268-I, respectively.
26 EFFECT OF TREATMENT OF BOVINE DONOR CELLS WITH MOUSE EMBRYONIC STEM CELL EXTRACT ON THE DEVELOPMENT OF EMBRYOS AFTER NUCLEAR TRANSFER
Authors: Akagi S. , Mizutani E. , Inaba Y. , Kaneda M. , Somfai T. , Haraguchi S. , Watanabe S. , Hashiyada Y. , Matsukawa K.
Published: August 2011

The efficiency of somatic cell cloning is very low, probably because of incomplete reprogramming of the somatic cell nucleus. In recent studies, it is suggested that transient exposure of donor somatic cells to mouse embryonic stem cell (ESC) extract enhances pluripotency of the cells in vitro (Bru et al. 2008 Exp. Cell Res. 314, 1634–1642; Xu et al. 2009 Anat. Rec. 292, 1229–1234). In the present study, we examined the effect of treatment of donor cells with mouse ESC extract on the in vitro development of bovine NT embryos. First, in order to examine effect of treatment of donor cells with streptolysin O (SLO), which reversibly permeabilizes the plasma membrane, we compared the in vitro development of NT embryos using donor cells treated with 5 μg mL–1 SLO (SLO group) and untreated donor cells (control group). As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used. Fibroblasts were treated with 5 μg mL–1 SLO for 45 min, and then incubated for resealing in DMEM including 2 mM CaCl2 for 60 min. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). After in vitro culture for 8 days, blastocyst formation and cell number of blastocysts were examined. There were no significant differences between SLO and control groups in the fusion rate (80% and 72%, respectively), cleavage rate (60% and 65%, respectively), developmental rate to the blastocyst stage of NT embryos (31% and 28%, respectively), and blastocyst cell number (127 ± 6 and 112 ± 14, respectively). These results suggest that SLO treatment of donor cells has no negative effect on the in vitro development of NT embryos. Next, we examined the in vitro developmental ability of NT embryos using donor cells treated with mouse ESC extract (ES extract group). After SLO treatment for 45 min, permeabilized fibroblast cells were treated with mouse ESC extract for 45 min, and then incubated in DMEM including 2 mM CaCl2 for 60 min, and used for producing NT embryos. There were no differences between ES extract and control groups in the fusion rate (68% and 69%, respectively), cleavage rate (86.7% and 80.6%, respectively), and developmental rate to the blastocyst stage of NT embryos (39.8% and 43.5%, respectively). The cell number of NT embryos at the blastocyst stage in ES extract group (201 ± 30) was significantly (t-test; P < 0.05) higher than that in control group (140 ± 14). In conclusion, treatment of bovine donor cell with mouse ESC extract did not affect the in vitro developmental ability of NT embryos, but improved the quality of blastocysts.
260 IN VITRO DEVELOPMENTAL COMPETENCE OF PREPUBERTAL GOAT OOCYTES CULTURED IN GROWTH MEDIUM
Authors: Hammami S. , Romaguera R. , Roura M. , Catalá M. G. , Morató R. , Mogas T. , Paramio M. T. , Izquierdo D.
Published: August 2011

The prepubertal goat ovary presents a large number of small oocytes with a compromised competence to develop up to blastocyst stage. In pigs (Wu et al. 2006), using growth medium (GM) composed by low hormone concentrations, ascorbic acid, and insulin transferrin selenium (ITS) during the first 24 h of in vitro maturation (IVM) improved embryo development of small oocytes. The aim of this study was to test the GM in small prepubertal goat oocytes in order to increase blastocyst yield. The cumulus–oocyte complexes (COC) were recovered from prepubertal (1–2 months old) goat ovaries by slicing. The COC with a compact cumulus and homogeneous cytoplasm were selected and classified into 2 categories based on oocyte diameter: <125 μm and ≥125 μm. The ≥125 μm oocytes were matured in groups of 25 to 30 COC/100 μL drops of conventional IVM medium covered with mineral oil for 24 h (Treatment A). This medium was TCM-199 supplemented with 10% donor bovine serum, 10 μg mL–1 FSH, 10 μg mL–1 LH, 1 μg mL–1 17β-oestradiol, and 100 μM cysteamine. The <125 μm oocytes were distributed into 3 experimental groups: Treatment B, COC matured in the conventional IVM medium; Treatment C, COC cultured in GM (TCM-199, 10% donor bovine serum, 0.04 μg mL–1 FSH, 0.04 μg mL–1 LH, 0.004 μg mL–1 17β-oestradiol, 100 μM cysteamine, 100 μg mL–1 ascorbic acid, and 5 μL mL–1 ITS) for 12 h before placement for other 12 h in the conventional IVM medium, all drops of growth or maturation medium were covered with mineral oil; Treatment D, COC cultured during the first 12 h in GM and other 12 h into the conventional medium supplemented with 100 μg mL–1 ascorbic acid and 5 μL mL–1 ITS. After IVM, oocytes were fertilized for 24 h with a sperm concentration of 4 × 106 spz mL–1. Presumptive zygotes were cultured in SOF for 9 days. The cleavage rate was evaluated at 48 h post-insemination and blastocyst percentages at the final in vitro embryo culture (treatments A, B, C: 5 replicates; treatment D: 4 replicates). The results are shown in the Table 1. Cleavage and embryo development did not show different results when we compared small oocytes matured in GM to those matured in conventional IVM medium. However, the biggest oocytes (≥125 μm) showed the highest percentage of blastocyst development. The current study shows that the culture of small prepubertal goat oocytes in GM does not improve blastocyst yield.

Table 1.Effect of growth medium on embryo development of small oocytes (<125 μm) from prepubertal goats
261 INFLUENCE OF IN VITRO MATURATION ON EPIGENETIC MARKS AND GENE EXPRESSION IN BOVINE OOCYTES
Authors: Heinzmann J. , Hansmann T. , Wrenzycki C. , Zechner U. , Haaf T. , Niemann H.
Published: August 2011

In cattle, in vitro maturation (IVM) of oocytes is an integral part of assisted reproduction technology. However, only 30% of in vitro matured bovine oocytes develop to the blastocyst stage after fertilization (compared with 60% for in vivo matured oocytes), indicating critical involvement of maturation conditions in the developmental competence of oocytes. Oocytes for IVF and intracytoplasmic sperm injection in humans are typically allowed to mature in vivo after superovulation because IVM is not considered to be a safe medical procedure. Several studies have shown that assisted reproduction technology involving prolonged in vitro culture of human and ruminant embryos can be associated with increased risk of fetal or placental abnormalities due to aberrant DNA methylation of imprinted and non-imprinted genes. Similarities between the bovine large offspring syndrome and imprinting-related human Beckwith–Wiedemann syndrome and the general similarity of bovine and human pre-implantation development make bovine oocyte maturation and embryonic development an increasingly accepted model of human development. Differentially methylated regions and imprinting control regions for the bovine paternally imprinted gene H19 and the maternally imprinted genes PEG3 and SNRPN were identified and characterised in this study. The DNA methylation profiles of bovine oocytes could be determined by bisulfite treatment of DNA from pools of 10 oocytes, but no significant differences were observed between IVM in TCM medium with 20% O2, in SOF medium with 5% O2, or after in vivo maturation. In contrast, quantitative PCR analysis of single oocyte preparations (n ≥ 8) revealed significant differences between these groups in the expression of the 3 genes. We then investigated the expression of genes involved in other critical processes in the developing oocyte and early embryo by quantitative PCR, including SLC2A8 (glucose transport), GDF9 (growth factor), PRDX1 (antioxidant and intercellular communication), DNMT1a/b (maintenance of methylation), and DNMT3a/b (de novo methylation). We also studied IGF2R, an imprinted gene implicated in large offspring syndrome. We observed significant differences in the expression of several of these genes. Interestingly, the expression of DNMT3a and DNMT3b was significantly higher in in vitro matured oocytes than in in vivo matured oocytes and could result in the above-mentioned aberrant methylation patterns established later in embryonic development.

This work was funded by DFG (FOR1041).
263 REGULATION OF SEVERAL TRANSCRIPTS DURING IN VITRO MATURATION IN PORCINE OOCYTES COLLECTED FROM DIFFERENT SIZE FOLLICLES
Authors: Izquierdo-Rico M. J. , Romar R. , Kohata C. , Funahashi H.
Published: August 2011

Oocyte-specific transcripts play important roles in oocyte maturation, fertilization, and early embryonic development. Currently, oocytes from medium-size follicles have been used for different assisted reproductive techniques after in vitro maturation (IVM). The aim of this study was to compare the mRNA expression level in porcine oocytes collected from medium (3–6 mm) and small (<2 mm) size follicles. Genes were selected based on their described maternal effect (NALP9, HSF1), their identification as markers of oocyte maturation (AURK-A, AURK-B, MOS, and C-mos), their involvement in fertilization (ZP3, ZP4), and anti-apoptotic effect (Bcl-2). All transcripts were studied in oocytes just after collection [germinal vesicle (GV) stage] and after in vitro maturation (IVM; metaphase II stage). To ensure nuclear stage of immature oocytes, oocytes were mechanically denuded just after collection, centrifuged (10 000 rpm, 5 min, RT), and observed under the microscope (60×). Those oocytes with clear nucleolus and evident nuclear membrane were selected and stored (n = 10) until study. For metaphase II oocytes, only those exhibiting the extrusion of first polar body after IVM (n = 10) were selected. Total RNA was extracted from the pool of 10 immature and mature oocytes. One picogram of luciferase mRNA per oocyte was added as an exogenous standard. Total RNA was extracted from oocytes and cDNA was obtained and used as a template for quantitative PCR to analyse the level of different transcripts. The whole process was replicated 4 times. Data were normalized to the luciferase RNA and analysed by one-way ANOVA with maturational stage (GV or metaphase II) and follicle size (small or medium) as fixed factors. Results show that all transcripts were significantly decreased during IVM (P < 0.05). Therefore, after IVM, NALP9, AURK-A, MOS, C-mos, ZP3, ZP4, and Bcl-2 transcripts were significantly reduced in matured oocytes compared with immature ones irrespective of follicle diameter. Transcripts of AURKAB and HSF1 decreased after IVM in oocytes from medium follicles or small follicles, respectively. A significant effect of follicular size was only detected in MOS transcripts in GV-stage oocytes because those collected from middle follicles had a higher amount than the ones from small follicles (Table 1). These results suggest that the variations in the maternal store of RNA during IVM are not related with follicle diameter for the studied genes. Further investigations are necessary to determinate the developmental competence of oocytes that came from different types of follicles (small and medium follicles).

Table 1.Variation of transcripts during in vitro maturation in porcine oocytes collected from small and medium follicles

This study was supported by Okayama University. R. Romar was given a grant by JSPS (Ref. S-09210).
274 EFFECT OF HOLDING ASPIRATED FLUID FROM IMMATURE EQUINE FOLLICLES ON OOCYTE MATURATION AND BLASTOCYST PRODUCTION AFTER INTRACYTOPLASMIC SPERM INJECTION
Authors: Velez I. C. , Norris J. D. , Choi Y. H. , Loux S. , Hinrichs K.
Published: August 2011

Transvaginal ultrasound-guided follicle aspiration (TVA) is the most effective way to recover multiple immature oocytes from live mares. Because of the tight attachment of the equine immature oocyte to the follicle wall, TVA in this species is time consuming, taking up to 1 h per horse. Thus, it may be difficult to search follicular aspirates immediately after recovery. In 2009 in a series of 6 replicates, we observed a blastocyst development rate of 32% (13/41) after intracytoplasmic sperm injection of oocytes collected by TVA and held for ∼1.5 h at ambient temperature (26 to 32°C) before isolation from aspirated fluid (unpublished data). Therefore, in the present study we compared the effects of immediate oocyte isolation v. holding the follicular aspirate before oocyte isolation on oocyte maturation and blastocyst rates. Ten mares were used for this study; TVA was performed on each mare every 14 days, for 4 aspiration sessions per mare. Collected aspiration fluid was either processed immediately or held for 2 h at 32°C before processing. At each aspiration, one ovary was randomly assigned to each treatment [immediate (Imm) or 2-h holding (2-H)]. Follicle aspiration was performed as previously described (Jacobson et al. 2010 Theriogenology 73, 1116–1126) using M199 with Hank’s salts to flush the follicle lumen up to 8 times per follicle. Oocytes were recovered from the aspirates by filtration. Overall, 325 follicles were aspirated and 140 oocytes were obtained (43% recovery). The proportion of degenerating oocytes was not significantly different between treatments (1/68 and 0/72 for Imm and 2-H, respectively). Oocytes were held overnight in modified M199 as previously described (Choi et al. 2006 Theriogenology 66, 955–963) before maturation culture. After 30 h of maturation culture, there was no significant difference in maturation rates between treatments [75% (50/67) and 65% (46/71) for Imm and 2-H, respectively]. Fertilization was performed by intracytoplasmic sperm injection, and injected oocytes were cultured 7 to 10 days, as previously described (Choi et al. 2006). The rates of blastocyst development per injected oocyte were 23% (11/47) for Imm and 16% (7/44) for 2-H; these were not significantly different (P > 0.3). The reason for the discrepancy in blastocyst rates for held oocytes in this study (16%) compared with our 2009 observations (32%) is unclear; this could be a factor of the time held (∼1.5 v. 2 h), the temperature (26–32°C v. 32°C), or minor changes in protocol between the 2 years. From these results, we conclude that holding oocytes in the follicular aspirate for up to 2 h following collection may be performed when necessary without significantly affecting the rate of subsequent blastocyst development.

This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.
280 BOVINE SPERM DNA FRAGMENTATION RATES AND EXTENDER pH: A DYNAMIC EXPERIMENTAL APPROACH
Authors: González-Marín C. , Travis A. L. , Kjelland M. E. , Gosálvez J. , López-Fernández C. , Lenz R. W. , Moreno J. F.
Published: August 2011

Flow cytometry technology for the sorting of X- and Y-chromosome bearing sperm is currently utilised in research and for commercial applications. During sample preparation before the sex-sorting process, sperm go through different pH treatments that may affect their quality. The hypothesis to test in this study is that sperm DNA damage could occur due to differences in the pH of sperm extenders. Bull semen doses from 15 dairy and 15 beef bulls (2 ejaculates per individual), ranging in age between 13 and 96 months, were randomly selected from a bull stud in Texas (Sexing Technologies, Navasota, TX, USA). Each semen sample was divided into 4 separate aliquots. One neat semen sample was kept as a control and the other 3 aliquots were treated with 16 μL of 8.1 mM Hoechst 33342 (Molecular Probes, Eugene, OR, USA). A calculated amount of modified Tyrode’s albumin lactate pyruvate (Clear TALP), pH 7.4, was added based on neat ejaculate concentration. For the separation of live and dead sperm during the sex-sorting process, 2 mL of red TALP (Red Food Dye FD&C #40; Sensient Technologies Corp., Milwaukee, WI, USA) at 3 different pH treatment levels (5.5, 6.4, and 7.4) were added to the sperm samples. The dynamics of sperm DNA fragmentation were assessed immediately following the addition of red TALP treatment and after 24 h of incubation at 34°C. Sperm DNA fragmentation was measured using the commercial variant of the sperm chromatin dispersion test, the bull Sperm-Halomax® kit (Halotech DNA, Madrid, Spain), and counting 300 sperm cells under fluorescence microscopy. Analysis of variance was used to determine if there were statistical differences (α = 0.05) among mean values of the groups (SPSS v.17.0 for Windows, SPSS Inc., Chicago, IL, USA). Bonferroni post hoc tests were utilised to determine the pairwise directional differences between groups. Differences in DNA fragmentation were not observed at 0 h (P > 0.05); however, at 24 h this value was significantly higher (P < 0.05) in semen samples treated with red TALP, pH 6.4, v. samples treated with red TALP, pH 7.4, (44.0 ± 24.9% v. 34.8 ± 24.9%), and even higher when using red TALP pH 5.5 (50.8 ± 23.8%). Further experiments should be designed to demonstrate that the DNA molecule could be affected by pH fluctuations, which may be important in refining the sex-sorting process. Notably, the undiluted neat semen controls had greater levels of DNA fragmentation overall (57.5 ± 29.4%), suggesting a negative effect of high sperm concentrations and seminal plasma.

The authors thank Mike Evans, Eddy Valenzuela, David Del Olmo, Jamie Jeffers, and the staff at Sexing Technologies for their technical assistance. This research was funded by Sexing Technologies and XY Inc.
281 EVALUATION OF IN VITRO EMBRYO PRODUCTION EFFICIENCY USING SEXED BULL SPERM SORTED WITH TWO TYPES OF CELL SORTER
Authors: Hayakawa H. , Hirata T.-I.
Published: August 2011

Cell sorting is an important part of the sperm sexing process. The objective of this study was to compare the efficiency of in vitro embryo production using sexed frozen–thawed bull sperm sorted with 2 types of cell sorter. Ejaculates from 2 Bos taurus (Holstein, 5 years old) bulls underwent conventional processing (control) or sorting for X chromosome bearing sperm using MoFlo® SX (SX, Dako, Fort Collins, CO, USA) or MoFlo® XDP-SX (XDP, Beckman Coulter, Fullerton, CA, USA) following XY™ sperm-sorting protocols. Processed sperm samples were cryopreserved in 0.5-mL plastic straws. Cumulus–oocyte complexes obtained from abattoir-derived ovaries were matured for 20 h in HEPES–TCM-199 (Lu and Seidel 2004 Theriogenology 62, 819–830) and randomly assigned to each of 3 sperm treatment groups. Thawed sperm were centrifuged for 20 min at 448 × g through an ISolate® (Irvine Scientific, Santa Ana, CA, USA) gradient (45:90%). Sperm pellets were washed in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) by centrifugation for 5 min at 252 × g. Oocytes were co-incubated with washed sperm (5 to 10 × 106 sperm mL–1) in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) for 8 h at 38.5°C in 5% CO2 and 95% air (Day 0). Presumptive zygotes were cultured for 90 h in CDM-1 (Lu and Seidel 2004 Theriogenology 62, 819–830) and then washed and cultured in IVD101 (Hoshi 2003 Theriogenology 59, 675–685) at 38.5°C in 5% CO2, 5% O2, and 90% N2. Cleavage rates on Day 2 and blastocyst rates on Day 7 to 9 were recorded after insemination. Two-way ANOVA was used for data analysis, followed by Fisher’s PLSD test. Experiments were replicated 4 times for bull A (total of 1 350 oocytes used) and 5 times for bull B (total of 1 529 oocytes used). The data are summarised in Table 1. No interaction was observed between the treatments and bulls. Cleavage rates were not significantly different in the 3 treatment groups. However, blastocyst rates were significantly lower in both SX (P < 0.001) and XDP (P < 0.002) groups than in control groups for both bulls but not different between SX and XDP (P > 0.8). Bull B showed significantly poorer results than bull A regarding both cleavage (P < 0.003) and blastocyst (P < 0.02) rates. MoFlo® SX (analogue processing) has been used for a decade, and XDP (digital processing) is the replacement model with its accelerated sorting speed. The current results indicated that the in vitro embryo production efficiency did not differ between sperm sorted with either SX or XDP. We suggest that sperm can be sorted using XDP without compromising sperm health.

Table 1.Cleavage and blastocyst rates after IVF with 2 Holstein bulls for three sperm treatments
282 IDENTIFICATION OF SRY AND STEROIDOGENIC FACTOR-1 (SF1) GENES IN CANINE XY MALE-TO-FEMALE SEX DEVELOPMENTAL DISORDER
Authors: Jang G. H. , Jeong Y. H. , Hwang I. S. , Jeong Y. W. , Hyun S. H. , Kim Y. W. , Shin T. , Jeung E. B. , Hwang W. S.
Published: August 2011

Cloning by the process of somatic cell nuclear transfer (SCNT) has been achieved in a variety of mammalian species and has many promising applications. In this process, however, due to reasons beyond current scientific understanding, many results contrary to expectation have also been produced. For instance, abnormal sex development such as demasculinization has been observed in 1 of 6 healthy German shepherd offspring produced with SCNT (1 normal donor (not cloned), 5 cloned but normally developed progenies, 1 cloned sex reversed progeny, and 1 recloned sex reversed progeny from 1 cloned sex reversed progeny). Sex-determining region Y (SRY) is one of the most basic and crucial genes that initiate male sex determination in many mammals. Steroidogenic factor-1 (SF1, NR5A1), which is closely related to SRY, also regulates several genes involved in sex determination. Numerous studies have reported that reduced or deleted SRY gene expression as well as SF1 gene mutations can produced XY sex reversal. To verify the hypothetical association between phenotypic disorder of sex determination and genetic modification by SCNT, we extracted genomic DNA from tissues of normal progeny (not cloned), primary cultured cells of cloned but normally developed progeny, cloned sex reversed progeny, and recloned sex reversed progeny at the age of 1 year and carried out PCR with produced primers based on available SRY and SF1 gene information (SRY gene from AF107021 in GenBank; SF1 gene from ENSCAFG00000023086 in Ensembl). The cloned PCR products were subcloned into T-vector for sequence analysis, which showed no mutation in genetic sequences of SRY and SF1. Taken together, in our case of abnormal sex determination, there was no apparent association between phenotypic sex determination disorder and SRY/SF1 gene mutation. Other sex reversal and related mutation studies have pointed to a wide range of signal networks that include Sox9 and so forth. Further studies should be focused on these other genes in the signal network.
284 IN VITRO FUNCTION OF FROZEN - THAWED BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) SPERM UNDERGOING SORTING AND RECRYOPRESERVATION
Authors: Montano G. A. , Kraemer D. C. , Love C. C. , Robeck T. R. , O'Brien J. K.
Published: August 2011

Artificial insemination (AI) using sex-selected sperm of bottlenose dolphins is currently used for the reproductive and social management of captive populations, but distance of males to the sorting facility represents a limitation of the procedure. Sorting and recryopreservation of previously frozen–thawed (FSF) sperm would facilitate the global application of this technology. Although a calf has been produced using FSF sperm (O’Brien et al. 2009 Theriogenology 71, 98–107), a comprehensive examination of the in vitro quality of such samples is needed. The objective was to compare the in vitro quality of nonsorted (CNTR) and sorted (FSF) dolphin sperm before and after recryopreservation using straw (STR) and directional freezing (DF) methods. At all assessment intervals, sperm were evaluated for 1) motility parameters with computer-assisted sperm analysis (CASA); 2) plasma membrane integrity (viability) and acrosome integrity using propidium iodide/fluorescein isothiocyanate-labeled peanut agglutinin (PI/FITC-PNA) staining and 3) DNA denaturation using the sperm chromatin structure assay (SCSA). Semen from 3 ejaculates × 3 males was cryopreserved by DF. After thawing, samples were divided into CNTR and FSF. The CNTR sperm were recryopreserved using STR and DF methods with assessments performed after the first thaw (PT1) and before recryopreservation (PF2). The FSF sperm were prepared for sorting using a density gradient centrifugation (DGC) method, stained with Hoechst 33342, sorted (SX MoFlo®, Dako, Fort Collins, CO, USA), then recryopreserved using STR and DF methods. The FSF sperm were assessed post-PT1, post-DGC, post-stain, post-sort, and at PF2. After the second thaw (PT2), CNTR and FSF samples were diluted (1:0.1, vol/vol) with Androhep Enduraguard™ (AE; Minitube of America, Verona, WI, USA), incubated at room temperature, and assessed at 0, 6, 12, 18, and 24 h PT2. The PT1 samples retained high proportions of their PF1 total motility (TM) and progressive motility (PM) (mean ± SD; 87.9 ± 7.3% and 92.2 ± 5.9%, respectively). The FSF sperm had improved (ANOVA; P < 0.05) motility (TM, PM, VAP, VCL, VSL) and viability at PF2 compared with PF1. The FSF sperm recryopreserved using DF had higher (P < 0.05) motility over the 24-h post-thaw incubation period compared with STR. The CNTR sperm DNA fragmentation remained unchanged throughout the process. The DNA fragmentation of FSF samples increased after staining (P < 0.05), then decreased during the PT2 incubation period, stabilising at lower values (P < 0.05) than CNTR from 6 to 24 h PT2. This unusual pattern indicates a possible interaction between Hoechst 33342 and acridine orange. After recryopreservation, the viability of FSF sperm was higher (P < 0.05) than that of CNTR sperm. Results indicate that bottlenose dolphin sperm undergoing cryopreservation, sorting, and recryopreservation are of adequate quality for use in AI.
285 THE EFFICIENCY OF INTRACYTOPLASMIC SPERM INJECTION v. LASER-ASSISTED IN VITRO FERTILIZATION WITH FROZEN SPERM FOR RECOVERY TRANSGENIC C57BL/6 MOUSE STRAINS
Authors: Carstea A. C. , Polgar Z. , Kovacs L. , Dinnyes A.
Published: August 2011

The progress of molecular genetics generated thousands of new transgenic strains of mice which also requires their economic and safe maintenance in the form of genetic banks. Sperm freezing would be one of the easiest options; however, cryosensitivity of sperm in mice strains is prone to variation. In this study, we examined the efficiency of laser-assisted zona drilling in vitro fertilization (ZD-IVF) v. intracytoplasmic sperm injection (ICSI) for attempting to recover two transgenic (UBI-GFP/BL6 and B6;129P2- Hvcn1) and one mutant (C57BL/6J-Tyrc-2J) lines. The sperm was frozen with 18% raffinose and 3% skim milk (Nakagata 2000 Mamm Genome 11, 572–576). Data of the replicates was analysed by chi-square method. The motility rates after thawing of cryopreserved sperm were 10% for UBI-GFP/BL6, 30% C57BL/6J-Tyrc-2J and 50% for B6;129P2-Hvcn1 strains. Regular IVF attempts in the UBI-GFP/BL6 and the mutant strain resulted in very few embryos and no pups (data not shown). Following ZD-IVF, the 2-cell stage rates were 7/60 (12%) for UBI-GFP/BL6, 18/60 (30%) for C57BL/6J-Tyrc-2J, and 34/60 (56%) for B6;129P2- Hvcn1. After ICSI, 66–74% of the oocytes survived the procedure and their development to 2-cells stage were 12/20 (60%) (UBI-GFP/BL6), 25/39 (64%) (C57BL/6J-Tyrc-2J) and 26/37 (70%) (B6;129P2-Hvcn1). The number of 2-cell stage embryos produced by ICSI was significantly (P < 0.05) increased compared with those produced following ZD-IVF in case of UBI-GFP/BL6 and C57BL/6J-Tyrc-2J strains. The 2-cell stage embryos were transferred into recipients and the newborn rates from ZD-IVF v. ICSI embryos were 0% v. 17% (UBI-GFP/BL6), 28% v. 36% (C57BL/6J-Tyrc-2J) and 12% v. 12% (B6;129P2- Hvcn1), respectively; none of them were significantly different. In conclusion, when using cryopreserved sperm, the post-thaw motility is an important indicator for the selection of the rederivation method of cryopreserved transgenic mouse strains; while ZD-IVF, an easier method to perform, is suitable for the higher motility samples, ICSI could be strongly recommended for those showing low motility.

This work was financed by EU FP6: CLONET (MRTN-CT-2006-035468), TEAMOHOLIC (MEXT-CT-2003-509582); EU FP7: RESOLVE (FP7-HEALTH-F4-2008-202047), RabPStem (PERG07-GA-2010-268422), and NKFP_07_1-ES2HEART-HU (OM-00202-2007).
286 PRODUCTION OF NORMAL MICE USING LONG-TERM PRESERVED MOUSE SPERMATOZOA WITHOUT FREEZING
Authors: Li C. , Mizutani E. , Ono T. , Wakayama T.
Published: August 2011

Mammalian spermatozoa preservation now plays an important role in fertility treatments, generating hybrid animals and protecting endangered and extinct species. To date, the most common method of sperm preservation is freezing in liquid nitrogen (LN2). However, this method requires constant supplementation of LN2 and also presents some safety issues involved in transporting LN2. Here we describe a new sperm preservation method that does not involve freezing. Mouse spermatozoa were cultured in four basic media (HEPES–Chatot-Ziomele-Barister’s medium (HCZB), KSOM, K+-rich nuclear isolation medium (NIM), and PBS) with or without 10% BSA or 15% Ficoll as a cryoprotectant, and preserved in a refrigerator for up to 6 months. These preserved sperm were then injected into fresh oocytes and cultured to the blastocyst stage in vitro or transferred into recipient females to demonstrate their genetic integrity. Oocytes injected with 1-month-preserved spermatozoa in NIM and PBS showed significantly higher blastocyst rates (22.8% and 18.9%) than those in HEPES-CZB and KSOM (1-way ANOVA, P < 0.05). In embryos with 3-month-preserved spermatozoa in NIM or PBS with BSA or Ficoll, 5.3–24.0%; P < 0.05 of embryos, (n = 1056) developed to the blastocyst stage, and the developmental ratio was not decreased even for 6-month preservation (13.6–18.2%; P > 0.05). Surprisingly, 18 pups were obtained using spermatozoa stored in those mediums for 6 months. Moreover, this new method allowed easy production of healthy offspring even after transporting spermatozoa between two countries by aircraft at room temperature without any protection. In conclusion, this method allows for easy long-term preservation of mouse spermatozoa in a simple, modified medium at refrigerator temperature with very low cost and wide application.
289 DERIVATION OF MOUSE EMBRYONIC STEM CELL FROM C57BL/6/EGFP STRAIN WITH FETAL CALF SERUM AND KNOCKOUT SERUM REPLACEMENT® SUPPLEMENTATION MEDIUM
Authors: Campanha B. C. S. , Oliveira C. S. , Souza D. M. , Godoi C. P. , Fernandes H. , Ribeiro-Paes J. T. , Garcia J. M. , Nogueira M. F. G.
Published: August 2011

Embryonic stem cells (ESC) have been used in attempts to obtain specific tissues or even individuals. Embryonic stem cells are pluripotent, allowing the differentiation of cell types from 3 germ layers. The establishment of a stable lineage of ESC is a valuable tool; however, some strains of mice are less permissive to ESC derivation or generation of chimeric animals (e.g. C57BL/6). Supplementation of culture medium with FCS, in the ESC derivation, may influence the potentiality to derivation or use of these strains in tetraploid complementation assays (Sato et al. 2009 Tsukuba Res. Inst. 47, 414–422). Thus, its replacement was carried out using knockout serum replacement (KSR®) to minimize the deleterious action of serum (Wang et al. 2007 Inst. of Biotech. 23, 269–272). Embryos were obtained from 5 females of lineage C57BL6/EGFP, aged between 21 and 30 days and weighing ∼35 g, and superstimulated according (Mancini et al. 2008 Transg. Res. 17, 1015). The animals were placed for mating with fertile males of the same strain in a proportion of one to one (male:female). The copulation was confirmed by plug vaginal (0.5 days postcopulation). Embryo recovery was performed 3.5 to 4.0 days postcopulation to obtain expanded (EB) or hatched blastocysts (HB). Zona pellucida was removed from EB with the aid of pronase solution, and the whole embryos (n = 8) were placed on a 4-well dish pretreated with pig skin gelatin 0.1%, under murine fibroblast primary in DMEM medium supplemented with 7.5% FCS and 7.5% KSR®, 10 mM βmercaptoetanol, 1 mM sodium pyruvate, 2 mM L-glutamine, and 83.4 mg mL–1 amikacin for 24 h. After this period, the medium was replaced by DMEM supplemented with 15% KSR®. The colonies began to grow between 3 and 6 days after in vitro culture of the embryos. Once established, the colony was picked and placed into new plates containing murine fibroblast primary every 48 to 72 h. After 14 days, the derivation was confirmed with some proved pluripotency markers by immunofluorescence (Oct3/4, SSEA-1, and Nanog) and karyotyping for ploidy detection. The reaction was positive for all tested markers in addition to the detection of the endogenous fluorescence from EGFP protein itself (C57BL/6EGFP origin). It was concluded that ESC derivation with partial serum replacement and using a less permissive strain such as C57BL/6EGFP is feasible, although with a reduced success rate (12.5%; i.e. 1 lineage – named BCM04 – from 8 attempts).

Fellowships and grants were received from FAPESP, Brazil: 09/15919-4 (BCSC), 09/16254-6 (DMS), 09/17605-7 (CPG), 06/06491-2 (MFGN), and 07/07705-9 (MFGN).
29 PROTEOMIC ANALYSIS OF CLONED TERM PLACENTA DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER CATS
Authors: Bang J. I. , Bae D. W. , Kwon Y. S. , Cho S. J. , Deb G. K. , Ha A. N. , Kong I. K.
Published: August 2011

The normal placenta formation and development affect subsequent survival and development of fetus. Somatic cell nuclear transfer (SCNT) is still associated with functional and structural abnormalities of placentation in cloned pregnancy. Moreover, the molecular basis of placenta derived from cloned embryos is poorly understood. To determine the abnormal expression of proteins of cloned term placenta (CTP; n = 3) compared with control placenta (n = 3), a proteomic analysis was performed using 2-DE, MALDI-TOF MS, and MALDI-TOF/TOF MS/MS. The cloned kittens were killed 1 month after Caesarean section. The results showed that 42 proteins were significantly up/down expressed in the CTP (P < 0.05). In CTP, thirty proteins were up-regulated, such as apoptosis-related cathepsin D (CD), annexin A1, and heat shock protein 27, and 12 proteins were down-regulated, such as prohibitin (PHB). Abnormal expression of CD and PHB may be related to induced reactive oxygen species (ROS) resulting in promoted cellular senescence and apoptosis. The expression patterns of CD and PHB were validated by Western blotting and immunofluorescence assay. Additionally, we also confirmed the abnormal expression of SOD (up-regulated) and CAT (down-regulated) enzymes in the CTP. Increased generation of ROS in the CTP was related to decreased mitochondrial membrane potential as detected in placenta tissues by MitoTracker green FM (MTG; 1 μM, Invitrogen, Carlsbad, CA). ROS-induced mitochondrial damage accelerated telomere loss [as detected by IQ-fluorescent in situ hybridization (FISH)] compared with control. ROS is an important modulator of telomere loss and that telomere-driven cellular senescence is primarily a stress responsive. Thus, the data suggest that combined abnormal proteins expression were associated with hindered development of cloned placenta and viability of fetus. Therefore, if we can solve this problem, the efficiency of SCNT and transgenic cat production will be increased, which in turn, may be applied as a potential means of restoration of endangered animals.

This work was partly supported by the BK21 program and the KOSEF (10525010001–05N2501–00110).
292 GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS FROM VARIOUS GENETIC BACKGROUND BY SLEEPING BEAUTY TRANSPOSON-MEDIATED GENE TRANSFER
Authors: Muenthaisong S. , Ujhelly O. , Varga E. , Carstea A. C. , Ivics Z. , Pirity K. M. , Dinnyes A.
Published: August 2011

Induced pluripotent stem (iPS) cell technology allows the reprogramming of somatic cells to a pluripotent state; however, it requires viral gene transduction and permanent existence of the exogenous genes in the genome, which is a potential risk for abnormalities in the derived iPS cells. Recently, there was report that iPS cells have been made with piggyBack transposon. Here, we first reported that nonviral transfection of a Sleeping Beauty transposon, which comprises c-Myc, Klf-4, Oct3/4 (Pou5f1), and Sox-2, can reprogram mouse fibroblasts from 3 different genetic backgrounds: ICR (outbred), C57BL/6 (inbred), and F1 hybrid (C57BL/6 × DBA/2J), with parallel robust expression of all exogenous (c-Myc, Klf-4, Oct3/4, and Sox-2) and endogenous (e.g. Nanog) pluripotency genes. The iPS cells were cultured under standard conditions with promotion of differentiate by withdrawal of leukemia inhibitory factor. We chose 6 cloned of each line that exhibited characteristics typical for undifferentiated embryonic stem (ES) cell: ES-cell-like morphology, alkaline phosphatase positivity, and gene expression pattern [quantitative real-time PCR and immunofluorescence of ES cell markers (e.g. Oct-4, SSEA1, Nanog]. Furthermore, cells were able to form embryoid bodies and beat rhythmically and expressed cardiac markers assayed by immunofluorescence (e.g. cardiac Troponin T, desmin). In vivo testing of iPS cell lines for their developmental potential (diploid and tetraploid embryo complementation assay) is currently underway. The iPS cell lines generated from the ICR strain appeared the earliest in time (ICR-d11, F1 day-2 and Bl6-d12), with higher efficiency than colonies from the other 2 backgrounds. The differentiation potential of the iPS lines derived from the 3 genetic backgrounds was similar. Interestingly, the ICR-iPS lines had higher differentiation potential than did the ICR-ES cell lines: the rate of embryoid bodies forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. Our results suggest that the iPS technology provide a new tool to generate pluripotent stem cells from genetic backgrounds where good-quality ES cell generation is difficult. These studies provide new insights into virus-free iPS technology and contribute to defining future cell-based therapies, drug screening methods, and production of transgenic animals with genetically modified iPS cells.

This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205, and NKTH/KPI (Jedlik NKFP_07_1-ES2HEART-HU OM-00202-2007).
293 MASS PRODUCTION OF Nkx2.5-POSITIVE CARDIAC PROGENITOR CELLS DERIVED FROM MOUSE EMBRYONIC STEM CELLS IN SLOW-TURNING LATERAL VESSEL FOR CELL TRANSPLANTATION AND DRUG TESTING
Authors: Rungarunlert S. , Klincumhom N. , Nemes C. , Techakumphu M. , Pirity M. K. , Dinnyes A.
Published: August 2011

Regenerative cell therapy against cardiovascular disease would require mass production and purification of specific cell types before transplantation. To enable large-scale production of embryonic stem (ES)-derived pure cardiomyocytes, we developed an animal model for a single-step scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled slow-turning lateral vessel (STLV, Synthecon, Inc., Houston, TX, USA) bioreactor following inoculation with a single cell suspension of mouse ES cells. To enhance the yield of cardiac progenitor cells, mouse ES cells (HM1; 129Sv/Ola, Magin et al. 1992 Nucl. Acids Res. 20, 3795–3796) were targeted with the cardiac-specific mouse Nkx2.5 promoter driven enhanced fluorescent green protein (EGFP). Among 15 targeted colonies, which were characterised based on morphology, the ability to form EB, EGFP expression, and in vitro differentiation ability toward cardiomyocytes, 3 lines were further evaluated for the efficiency of cardiomyocyte production. The 3 lines were cultured in STLV bioreactor and compared with classical hanging drop (HD) and static suspension culture methods. Embryonic bodies at day 3 to 8 were collected and analysed by using fluorescence-activated cell sorting for markers of pluripotency (e.g. Oct-4, SSEA1, Nanog) and cardiac (e.g. Nkx2.5, Troponin T) lineage commitments. Data was analysed by one-way ANOVA and t-tests. The results showed that both level and kinetics of Nkx2.5 expression was dependent on culture conditions. The STLV and static suspension culture methods produced higher rates of Nkx2.5-positive cells on day 5 than that of HD (70 and 54 v. 30%, respectively). The STLV method produced a highly uniform population of efficiently differentiating EB in large quantities and resulted in the highest, 108 yield of cardiomyocytes in a single 110-mL STLV on day 4. In conclusion, the STLV method provides a technological platform for controlled large-scale generation of ES-cell-derived cardiomyocytes for clinical and industrial applications. In vivo transplantation tests of cardiomyocytes produced via STLV are currently underway.

This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205 and NKTH/KPI (NKFP_07_1-ES2HEART-HU OM-00202-2007), CHE-TRF senior scholarship, No. RTA 5080010 (M.T.), and the Thailand Commission on Higher Education [CHE-PhD-SW-2005-100 (S.R.), CHE-PhD-SW-RG-2007 (N.K.)].
294 PRODUCTION AND CHARACTERIZATION OF PUTATIVE NUCLEAR TRANSFER EMBRYONIC STEM CELLS DERIVED FROM HANDMADE CLONED EMBRYOS USING EMBRYONIC STEM CELLS, LYMPHOCYTES, AND ADULT FIBROBLAST CELLS AS DONOR CELLS IN GOAT
Authors: Dutta R. , Malakar D. , Khate K. , Akshay J.
Published: August 2011

The handmade cloning technique has been a relatively recent addition in the field of nuclear transfer. In the present study, attempts were made to efficiently derive stem cells from handmade cloned (HMC) embryos in goat using adult fibroblast cells, embryonic stem (ES) cells, and lymphocytes as donor cells, and to characterise the derived putative nuclear transfer ES (ntES) cells for their stemness. Efficiency of the donor cells for nuclear transfer was also compared, and an overall cleavage and morula formation rates of 62.44 ± 3.9% and 35.30 ± 3.86%, 75.45 ± 3.92% and 45.84 ± 3.86%, and 56.38 ± 3.92% and 29.09 ± 3.86% were obtained from adult fibroblasts, ES cells, and lymphocytes, respectively. A significant difference was found between ES cells and the other 2 donor cells in terms of cleavage and morula formation. However, no such difference existed between fibroblasts and lymphocyte donor cells. Stem cell colonies were successfully derived from HMC embryos obtained from all 3 different donor cells. The rate of primary colony formation was 61.66 ± 4.62% for fibroblast-donor-cell-derived embryos. This rate was 59.91 ± 4.62% for ES-donor-cell-derived embryos and 62.49 ± 4.62% for lymphocyte-donor-cell-derived embryos. The putative ntES colonies were positively characterised for TRA-1-60, TRA-1-81, SSEA-1, SSEA-4, OCT-4, SOX-2, and Nanog by immunocytochemistry and RT-PCR. Results indicated that ES cells had better efficiency as donor cells in cloned embryo production than did adult fibroblasts and lymphocytes. The finding also suggested that terminally differentiated cell-like lymphocytes can also be reprogrammed. Moreover, there was no difference between the different donor-cell-derived HMC embryos in terms of ntES cell derivation. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. This could be of substantial significance because patient-specific ntES cells have proven therapeutic significance.

The authors acknowledge N.D.R.I for the financial and infrastructural assistance.