bluuebunny/crossref_metadata_2025 huggingface dataset:
66 HISTIOTROPHIC NUTRITION AND IRON TRANSFER ACROSS THE MATERNO-FETAL INTERFACE IN NUCLEAR TRANSFER-DERIVED SOMATIC CATTLE CLONES
Authors: Pereira F. , Meirelles F. , Braga F. , Visintin J. , Rumpf R. , Garcia J. , Kfoury Júnior J. R. , Oliveira L. , Monteiro J. , Papa P. , Carvalho A. F. , Neto A. , Ambrósio C. E. , Bazer F. , Miglino M. A.
Published: December 2006

The histiotrophic nutrition by the endometrial glands and the materno-fetal interface in the cloned cattle placenta were analyzed in order to investigate the iron transfer. Placentomes and intercaruncular region samples were recovered at term Caesarean delivery from 14 cloned cattle and 10 controls, fixed in 4% paraformaldehyde and 10% formaldehyde in PBS, processed and stained for light microscopy (hematoxylin-eosin, picrosirius, and Masson's trichrome), histochemistry [Perls, acid phosphatase and periodic acid Schiff (PAS) reactions], and immunohistochemistry (with rabbit anti-pig uteroferrin antibody because the uteroferrin is an iron transporter protein). In the controls we verified blood extravasations in the materno-fetal interface between the uterine and the trophoblast epithelium characterized by hemophagous areas with consequent erythrophagocytosis by the adjacent trophoblast. This content presented extravasated erythrocytes, plasm, cell debris, and cells in a probable apoptotic process. The Perls histochemical reactions that exposed the ferric iron in the placentomes were positive, as was the uteroferrin immunohistochemistry in the trophoblast cytoplasm and in other deep points in the placentomes. The histochemical reactions, demonstrating the acid phosphatase enzyme that detects the phagocytic activity, were positive in the mesenchyme and trophoblast, with a weak stain in an endometrial stroma. In the top of fetal villi, mainly in the binucleate cells, we visualized accumulations of PAS-positive secretions, indicating the presence of mucoid material. The uterine gland epithelium was columnar-type and in the gland lumina there were cell debris and PAS-positive mucoid secretions. We confirmed the reactivity of the uterine glands to the acid phosphatase enzyme and to the Perls reaction in the epithelium and in the gland lumina. The uteroferrin immunohistochemistry showed a strong stain in the cytoplasm of the endometrial glands cells and in the lumina. In the NT bovine placentae, the blood extravasations between uterine and trophoblast epithelium were aberrant. There was also the remodeling of the maternal connective tissue (endometrial stroma) in this area. We also demonstrated phagocytic uptake of uteroferrin by the trophoblast, although the histochemical and immunohistochemical reactions were weak in the trophoblast of the placentomes and in the endometrial glands of the intercaruncular region, when compared with the controls. The results obtained by the histochemistry and immunohistochemistry indicated that these sites of transfer substances from mother to fetus are very important in providing adequate nutrition to the fetus, key to a successful pregnancy in NT bovines.
This work was funded by FAPESP, Brazil.
71 WHAT IS THE UTERINE RESPONSE IN A CLONED BOVINE PREGNANCY?
Authors: Santos T. C. , Pereira F. T. V. , Assis Neto A. C. , Ambrósio C. E. , Meirelles F. V. , Garcia J. M. , Maria D. A. , F. Carvalho A. , Miglino M. A.
Published: December 2006

Bovine has a synepitheliochorial placenta and characteristically there is no invasion of the trophoblast, but there is migration of the binucleate trophoblast giant cells into the maternal endometrium. The feto-maternal interface occurs in placentome where a tridimensional organization permits interactions between maternal epithelium and trophoblast, and in the intercaruncular area it is possible to observe a few mini-placentomes and the uterine glands opening. The objective of the present investigation was to study the morphological aspects of the uterus in bovine that had a cloned cattle gestations to understand the differences with natural gestation. The uterus and fetal membranes from natural and cloned cattle gestations were collected, fixed in 10% formaldehyde, processed, and stained for light microscopy and immunohistochemistry. The morphological differences observed in the surrogate uterus were: extensive areas without placentome, hemorrhagic uterine areas, caruncular fusion giving a reduced number of caruncules, increase in size and weight (megacaruncules), and a significant number of mini-caruncules giving miniplacentomes (diameter < 1 cm). In particular the mini-placentome showed functional trophoblastic cells with PAS+ granules in the binucleate trophoblast giant cells and an intense subepithelial capillary organization in maternal and fetal sides. The normal and clone placentomal cell populations were analyzed throughout pregnancy. The population of tetraploid and diploid trophoblastic cells was stained; detached cell cycle and DNA content was measured in FL2 using a FACscalibur flow cytometric system. We determined the percentage of cells in apoptosis (sub-G1), quiescent cells (G0/G1), synthesis (S), and proliferative cells (G2/M) with the aid of ModFit software. In addition, a cell cycle differential analysis was performed, and the tetraploid population presented statistical differences in cell cycle phases and populations relative to the apoptosis rate for the first (7.5 � 3.1%), second (15.2 � 5.0%) and third (17.3 � 4.3%) trimesters. The number of apoptotic cells increased significantly during pregnancy stages. The results showed that first trimester presented the majority of its cells in the G0-G1 phase, starting the cell cycle. On the other hand, the second and third trimesters presented the majority of their cells in the G2-M phase, ending the cell cycle. The relationship between cell cycle phases/rate of apoptosis in mononucleate cells, days of normal and cloned pregnancy, the number of binucleate cells, and their metabolic activity as well as their developmental kinetics could be important data in several studies that involve placental development in natural pregnancy or that derived from laboratory-manipulated embryos.
This work was supported by FAPESP and CNPq.
73 PROTEOMIC ANALYSIS OF THE MAJOR CELLULAR PROTEINS OF BOVINE TROPHECTODERM CELL LINES DERIVED FROM IVP, PARTHENOGENETIC, AND NUCLEAR TRANSFER EMBRYOS: REDUCED EXPRESSION OF ANNEXIN I IN NUCLEAR TRANSFER-DERIVED CELL LINES
Authors: Talbot N. , Powell A. , Caperna T. , Garrett W.
Published: December 2006

Trophectoderm cell lines were established from 8-day in vitro cultured bovine embryos that were derived from the fertilization (IVF), somatic cell nuclear transfer (NT), or parthenogenetic activation (P) of in vitro-matured oocytes and from five 8-day-old in vivo (V) embryos. The most abundant cellular proteins of 2 V-, 16 NT-, 12 P-, and 13 IVF-derived cell lines were compared by 2-D gel electrophoresis and mass spectrometry; that is, the unaltered thiourea/urea extract of each cell culture was analyzed. One-hundred and eighteen in common protein spots were examined, and 95% were identified with significant scores from protein and gene database searches. Of the proteins detected and identified, actin and cytokeratin-8 were found to be the most abundant. Other prominently expressed cellular proteins were metabolic enzymes such as aldose reductase, phosphoglycerate mutase, enolase, triosephosphate isomerase, cytoskeletal interacting proteins transgelin and stratifin, antioxidant proteins peroxiredoxin 1 and anti-oxidant protein 2, and the calcium-dependent lipid-binding proteins annexin I and II. In comparative analysis of the 2-D gels, the NT-derived trophectoderm showed diminished annexin I expression in comparison to the IVF-derived trophectoderm. Since annexin I is abundantly expressed in the placenta and has functions important to the maintenance of placentation, the down-regulation of annexin I in the cultured NT trophectoderm may be related to the frequent failures of NT pregnancies.
74 EXAMINATION OF ABNORMAL CELL DIVISION AND CHROMOSOME ABERRATION IN PIG PARTHENOTES AND CLONED EMBRYOS
Authors: Uhm S. J. , Kim M. S. , Gupta M. K. , Lee H. Y. , Park S. J. , Park C. K. , Chung H. M. , Kim Y. B. , Chung K. S. , Lee H. T.
Published: December 2006

Blastomere fragmentation is commonly observed in pig embryos and is associated with reduced blastocyst and pregnancy rates. This study examined the effect of the frequency of abnormal cell division and chromosome aberration on the embryonic developmental ability of pig parthenotes and nuclear transferred (NT) embryos. Pig immature oocytes cultured in TCM-199 supplemented with 10% pig follicular fluid, 0.2 mM pyruvate, 10 ng/mL epidermal growth factor (EGF), 5 �g/mL Folltropin V, 1 �g/mL estradiol-17�, and 25 �g/mL gentamycin for 44 h. Cumulus cells from matured oocytes were removed by vortexing for 1 min in TL-HEPES medium containing 0.1% hyarunonidase. Denuded oocytes were enucleated using 20 um micropipette in TCM-HEPES medium containing 7.5 �g/mL cytochalasin B (CB) and 10% fetal bovine serum, and were reconstructed with fetal fibroblasts by electrofusion (two DC pulses of 2.0 kV/cm for 30 �s). For production of parthenotes and reconstructed embryos, denuded oocytes were activated by a DC pulse of 1.0 kV/cm for 30 �s and then cultured for 4 h in NCSU23 with 10 �g/mL CB and 0.4% bovine serum albumin for inhibition of polar body extrusion. Subsequently, these oocytes were cultured in 50 �L of NCSU23 containing 0.4% BSA for 7 days at 39�C in a humidified atmosphere of 5% CO2 in air. The frequency of chromosome aberrations was evaluated using fluorescent in situ hybridization technique with a porcine chromosome-1 submetacentric specific probe. Data were analyzed by Student's t-test and ANOVA using SAS software as appropriate (SAS Institute, Inc., Cary, NC, USA). Parthenotes and NT embryos showed similiar cleavage rates (61.4 and 62.9%), but the blastocyst rate of parthenotes (18.4%) was significantly higher (P < 0.05) than that of NT embryos (10.4%). The frequency of chromosome aberration in NT embryos (39.8%) at the 4-cell stage on Day 3 of culture was significantly higher (P < 0.05) than that of parthenotes (21.9%). The percentage of fragmentation was significantly higher (P < 0.05) in NT embryos (51.7%) than in parthenotes (27.1%). Furthermore, the developmental rates of non-fragmented parthenotes (40.0%) and NT (22.9%) embryos to the blastocyst stage were significantly higher (P < 0.05) than those of fragmented parthenote and NT embryos (17.3 and 5.9% respectively). The total cell number of non-fragmented parthenote and NT embryos (34.4 � 10.0 and 29.7 � 7.5) were significantly higher (P < 0.05) than those of fragmented parthnote and NT embryos (22.3 � 9.6 and 18.4 � 6.2 respectively). Therefore, these results indicate that chromosomal abnormality and embryonic fragmentation could be associated with reduced developmental ability in pig NT embryos.
This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea.
78 EFFECTS OF DIFFERENT ACTIVATION PROTOCOLS ON ACTIN FILAMENT DISTRIBUTION AND IN VITRO DEVELOPMENT OF MINIATURE PIG NT EMBRYOS
Authors: Yamanaka K. , Sugimura S. , Wakai T. , Shoji T. , Sasada H. , Sato E.
Published: December 2006

In the process of producing reconstructed oocytes nuclear transfer (NT) embryos by somatic cell nuclear transfer, in vitro-matured oocytes can be used as recipient ones. It, however, has been well documented that after IVF porcine embryos derived from in vitro-matured oocytes have a small number of cells and low viability compared from those in vivo. As one possible reason, abnormal actin filament distribution has been detected in abnormal embryo cleavage and small cell numbers (Wang et al. 1999 Biol. Reprod. 60, 1020-1028). Artificial activation, which is necessary for development of NT embryos, can affect actin filament distribution of porcine oocytes matured in vitro, resulting in fragmentation (Kawahara et al. 2002 Theriogenology 58, 1081-1095). In the present study, we investigated effects of different activation protocols on actin filament distribution and in vitro development of miniature pig NT embryos. Porcine oocytes collected from ovaries were matured in vitro for 40 to 44 h in NCSU-23. First, we compared different activation protocols in development rates to blastocysts of oocytes activated. We used three activation methods (15 �M ionomycin treatment for 20 min (I), double DC pulses of 1.2 kV/cm for 60 ms in intervals of 5 s (E), and 5 mg/mL cycloheximide treatment for 5 h (C)) to prepare seven activation protocols (I, E, C, I + C, I + E, E + C, and I + E + C). Second, we examined effects of different activation protocols on actin filament distribution and subsequent development of NT embryos activated by the different activation protocols. Matured oocytes were enucleated, and fused with miniature pig fetal fibroblasts in calcium-free medium; approximately 3 h later, the resultant NT embryos were activated with three activation protocols (E, I + C, or I + E + C). All data were analyzed by chi-square test. The developmental rates to blastocysts in the I, E, C, I + C, I + E, E + C, and I + E + C groups were 5.6, 11.1, 0.0, 36.1, 20.7, 14.6, and 24.7%, respectively, showing that the rate in oocytes activated with I + C was significantly higher (P < 0.05) than the rates in oocytes activated by other treatments. In NT embryos, the developmental rates to blastocysts in the E, I + C, or I + E + C groups were 4.1, 14.3, and 4.6%, respectively, showing that the rate in NT embryos activated with I + C was significantly higher (P < 0.05) than the rate in NT embryos activated with other treatments. The abnormal rate of actin filament distribution in NT embryos activated with E or I + E + C was significantly higher (P < 0.05) than that in NT embryos activated with I + C (26.7% or 33.3% vs. 6.7%). The present results suggest that in miniature pig NT embryos an activation protocol by ionomycin combined with cycloheximide treatments may avoid physical damage to actin filaments with the resultant improvement of subsequent development.
79 THE AMOUNT OF TELOMERIC DNA IN CLONED CATTLE AND THEIR CALVES IS LESS THAN THAT OF AGE MATCHED CATTLE
Authors: Yang B. C. , Im G. S. , Kim Y. H. , Choi J. W. , Park Y. S. , Hwang H. W. , Joo S. C. , Kim D. H. , Ko Y. G. , Seong breakH. H. , Cho E. J. , Sohn S. H. , Yang B. S.
Published: December 2006

A telomere is a structure consisting of tandem repeats sequences of (TTAGGG)n at the end of the eukaryotic chromosome. Telomere lengths in animals vary by species, age, and tissues, as well as environment. This experiment concentrated on the amount of telomeric DNA in cloned cattle, their calves, and age-matched normal cattle. Using somatic cell nuclear transfer (SCNT), we had obtained 16 cloned Korean Native cows derived from ear skin fibroblasts and two cloned bulls from fetal fibroblasts. In addition, four female calves were produced from each cloned cow by artificial insemination. Control cattle selected to have matched age and the same raising place served as counter-parts of cloned the cattle in this study. The lymphocytes of all cloned cattle, their calves, and the age-matched controls were examined for telomere quantity. The amount of telomeric DNA was analyzed by quantitative fluorescence after in situ hybridization (Q-FISH) with a human telomeric DNA repeat probe. A minimum of 100 interphase nuclei from each set of harvests was studied to determine the mean and medium percentages of telomeric DNA using the MetaMorph Imaging System (Universal Imaging Co., West Chester, PA, USA). The amount of telomeric DNA obtained was found to decrease in cloned and control animals during growth. The amounts of telomeric DNA in cloned cattle from both ear skin fibroblasts (n = 16) and fetal fibroblasts (n = 2) was less than that of age-matched controls (P < 0.01). Surprisingly, the amount of telomeric DNA of calves from cloned cattle was also lower than that of age matched controls (n = 4, P < 0.01). The results showed a remarkable difference in the amount of telomeric DNA between SCNT cloned cattle and normal cattle. In conclusion, the telomeres of cloned animal and their calves are significantly shorter than those of normal cattle. Moreover, the short telomeres in calves could be inherited from their cloned mothers.
82 IN VITRO SURVIVAL OF PORCINE BLASTOCYSTS VITRIFIED USING THE CRYOLOGIC VITRIFICATION METHOD
Authors: Beebe L. , McIlfatrick S. , Ashman R. , Nottle M.
Published: December 2006

Porcine embryo cryopreservation is an important technology for the storage and transport of valuable genetic material. With many of the current vitrification and storage systems, such as the open pulled straws and microdrops, there is direct contact between the medium containing the embryos and the liquid nitrogen. This represents a possible contamination risk. One system with which there is no direct contact between the embryos and liquid nitrogen during the vitrification process is the Cryologic Vitrification System (CVM; Lindemans et al. Reprod. Fertil. Dev. 16, 174) which uses solid surface vitrification. Microdrops of vitrification medium containing the embryos are placed in contact with a metal block that has been precooled by partial submersion in liquid nitrogen, resulting in very rapid cooling rates. Blastocysts were collected surgically on day 5 of pregnancy from mature sows, and the embryos were randomly divided into two groups; each group was then vitrified and warmed with either of two previously published protocols except that the CVM replaced the open pulled straws plunged into liquid nitrogen in both protocols. The first method (OPS/CVM) was based on the open pulled straw method (Cuello et al. Theriogenology 61, 843-850), and used DMSO and ethylene glycol as cryoprotectants and TCM-199 as the basic medium. The second method (EG/CVM) used HEPES-buffered NCSU23 as the basic medium; the blastocysts were centrifuged prior to vitrification in ethylene glycol and polyvinylpyrrolidone (PVP) and the zona pellucida was removed immediately after warming (Cameron et al. Theriogenology 61, 1533-1543). Embryos were then cultured in NCSU23 +10% fetal bovine serum for 48 h at 38.5�C in an humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Embryos that had reformed the blastocoel and continued to expand were considered to have survived. These were stained with Hoechst 33342 and the nuclei counted using fluorescence microscopy. There was no difference between the OPS/CVM or EG/CVM methods in either the survival rates (27/29; 93%, and 24/27; 89%, respectively) or the number of cells (mean � SEM; 109 � 6 and 112 � 6, respectively). The survival rates are comparable to previously published rates using these two methods and open pulled straws. These data suggest that the CVM can successfully replace the open pulled straws in these two protocols. However, transfer of vitrified and warmed embryos into recipients would be needed to confirm the viability of the surviving embryos.
83 VITRIFICATION OF IMMATURE AND IN VITRO-MATURED HORSE OOCYTES
Authors: Bogliolo L. , Ariu F. , Rosati I. , Zedda M. T. , Pau S. , Naitana S. , Leoni G. , Kuwayama M. , Ledda S.
Published: December 2006

Few attempts have been carried out to cryopreserve equine oocytes, and an effective cryopreservation protocol is not defined yet. Studies were conducted to compare the viability of immature and in vitro-matured horse oocytes vitrified by the minimal volume cooling (MVC) cryotop vitrification method (Kuwayama et al. 2005 Reprod. BioMed. Online 11, 300–308). Oocytes were recovered from slaughterhouse ovaries and divided, on the basis of the morphology of cumulus cells, into cumulus-expanded (CE) and cumulus-compacted (CC) oocytes. Groups of CC and CE oocytes were vitrified immediately after recovery [germinal vesicle (GV) stage] or matured in vitro (IVM) and cryopreserved at the MII stage as follows: oocytes were incubated 30 min in TCM-199 + 20% FCS + 10% ethylene glycol (EG) + 10% DMSO, followed by 20 min in TCM-199 + 20% FCS + 20% EG + 20% DMSO + 0.25 M sucrose, loaded in cryotops (2 µL), and plunged into liquid nitrogen. Warming was performed at 38.5°C by washing the oocytes in TCM-199 + 20% FCS with decreasing sucrose concentrations (1.25 M, 0.62 M, 0.31 M). After warming oocytes cryopreserved at the GV stage were matured in vitro for 24 h (CE) or 36 h (CC) in TCM-199 + 10% FCS + FSH, LH each at (0.1 UI/mL) + cysteamine, fixed, and stained with glycerol-Hoechst 33342 to assess nuclear maturation. Oocytes vitrified at the MII stage were in vitro cultured for 2 h to evaluate their morphological survival on the basis of the presence of an intact zona pellucida and membrane. Nonvitrified oocytes undergoing the same maturation protocol were used as controls. Results (Table 1) indicated that the survival rate of oocytes vitrified at the GV stage, after IVM, was similar between CE and CC oocytes (43.6% vs 42.6%). Significantly (P < 0.01) higher numbers of vitrified CE MII oocytes (52.9%) survived, compared to CC (34.8%), after 2-h culture. The percentages of viable MII oocytes from CE and CC GV vitrified oocytes were 43.6% and 40.9% respectively and were comparable to those from vitrified MII oocytes (CE, 52.9%; CC, 34.8%) and control oocytes (CE, 56.4%; CC, 53.3%). In conclusion, the results of this study showed that vitrification by the MCV Cryotop method of horse oocytes at either the GV or the MII stage allows a similar number of viable mature oocytes to be recovered.

Table 1.
Maturation and survival rates of immature and mature equine oocytes vitrified by the MCV Cryotop method
86 MOTILITY AND VIABILITY OF BULL SEMEN DURING 24 HOURS OF STORAGE AT 4°C
Authors: Carini M. E. , Cavia R. , Larraburu G. , Brogliatti G. M.
Published: December 2006

Currently, cryopreservation process of fresh bull semen is carried out between 3 and 6 hours of refrigeration at 4°C post-collection (Hafez, 1989). However, it is sometimes difficult when the cryopreservation process is not available at the site of collection. The objective of this study was to determine seminal motility and viability in samples maintained at 4°C during 24 hours. A total of 98 ejaculates from 23 adult bulls (Angus, Brangus, Braford and Hereford) were collected and diluted in a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) and stored at 4°C. Parameters of velocity average path (VAP, µm/s), velocity straight line (VSL, μm/s), amplitude lateral head (ALH, μm), linearity (LIN, %), percentage of rapid cells (RAPID, %), percentage of slow and static cells (SL/ST, %), and viability (VIA, %) were determined by Computer Assisted Semen Analysis (CASA, HTM-ceros 12.1, Berkeley, CA, USA). Measurements were done at 6, 9, 12, and 24 h. The obtained results were analyzed statistically with one-way ANOVA and Dunnet Multiple Comparison Test and are summarized in Table 1. There were no significant differences (P > 0.05) in the VAP, RAPID, or SL/ST during 24 h of storage at 4°C. Measurements were significantly different (P < 0.01) for VSL and VIA at 24 h. Measurements of ALH were increased from 12 h (P < 0.01) and consequently, LIN decreased at the same time (P < 0.01). These results suggest that there are no differences in velocity, except in VSL at the end of the storage time. The type of movement of the spermatozoa change, because ALH increases and the trajectory loses linearity. A decrease in viability suggests that from 24 h of storage, the membrane of the spermatozoa becomes more susceptible. More research needs to be done to evaluate the competence of this time-storage semen in the artificial insemination trial.

Table 1.
Parameters of motility and viability of semen maintained at 4°C during 24 h

This research was supported by Centro Genético Bovino de EOLIA S.A.
9 USE OF SEXED SEMEN FOR ARTIFICIAL INSEMINATION IN HOLSTEIN HEIFERS IN MEXICO: PRELIMINARY RESULTS
Authors: Barba A. , Ducolomb Y. C. , Romo S.
Published: December 2006

Nowadays, X- and Y- sperm can be sorted reliably by flow cytometry/cell sorting. However, this new technology is rarely being adopted by dairy farmers in Mexico and in developing countries, mainly due to the higher price of sexed semen straws and to a lack of information and knowledge of the results that can be obtained with its use under field conditions. In 2004, a field experiment started in a dairy farm in Tizayuca, State of Hidalgo, Mexico, aimed to examine fertility and sex ratios after artificial insemination (AI) in heifers under field conditions. Semen from two Holstein bulls was used for the experiment. The semen was sexed and frozen by XY Mexico (subsidiary of XY, Inc., Fort Collins, CO, USA). Only the X fraction of spermatozoa was sorted and frozen in 0.25 cc plastic straws with 2.0 � 106 spermatozoa/straw. Sixty Holstein heifers averaging 14 months of age were artificially inseminated 12 hours after naturally occurring standing heat, using one straw of sexed semen, from June to October 2004. Fertility data of all inseminations were collected by rectal palpation from Days 45 to 60 after AI. A total of 30 heifers were pregnant, indicating a fertility of 50% froma single service. Sex of the calves was confirmed after calving between March and July 2005. From a total of 30 calves born after normal pregnancy, 27 were females (90%) and 3 were males (10%). In contrast, the pregnancy rate obtained in 60 heifers inseminated with non-sexed semen from the same two bulls was 68% (41 pregnant). From this group of heifers, 20 female (49%) and 21 male (51%) calves were born. Chi-square analysis indicated that pregnancy rates and sex ratios between the two groups were significantly different (P < 0.05). This preliminary information indicates that there is an advantage in the sex ratio obtained when using sexed semen for AI in Holstein heifers. This is especially important at a time when importation of replacement heifers to Mexico from Canada and the USA is not an option. However, more experiments are needed to confirm the results obtained in this preliminary study.
Sexed semen and Holstein heifers were provided by Rancho El Nacimiento, Establo 196, Tizayuca, State of Hidalgo, M�xico.
90 SUCCESSFUL VITRIFICATION OF PARTHENOGENETIC PORCINE BLASTOCYSTS PRODUCED FROM DELIPATED IN VITRO-MATURED OOCYTES
Authors: Du Y. , Kragh P. M. , Zhang X. , Yang H. , Vajta G. , Bolund L.
Published: December 2006

Cryopreservation of cloned porcine embryos may improve the output of somatic cell cloning considerably by alleviating logistic problems. However, the high lipid content of porcine oocytes and embryos compromises their cryotolerance. Recently a noninvasive procedure was published for delipation of porcine embryos with centrifugation but without subsequent micromanipulation (Esaki et al. 2004 Biol. Reprod. 71, 432-436). This method was applied to our present work with few modifications to compare the cryosurvival of porcine blastocysts produced from delipated vs. intact oocytes with parthenogenetic activation. In four replicates, a total of 192 oocytes were used for the experiments. After in vitro maturation for 44 h, cumulus cells were removed and oocytes were randomly distributed into two groups. For delipation, oocytes were digested with 1 mg/mL pronase in the presence of 50% cattle serum (CS) for 3 min, and washed in HEPES-buffered TCM-199 medium supplemented with 20% CS. Subsequently, 40-50 oocytes were centrifuged (12 000g, 20 min) in HEPES-buffered TCM-199 medium supplemented with 2% CS, 3 mg/mL polyvinyl alcohol and 7.5 �g/mL cytochalasin B (CB). Zonae pellucidae of both centrifuged and intact oocytes were removed completely by further digestion in 2 mg/mL pronase solution. For activation, a single direct current of 85 kV/cm for 80 �s was applied to both groups, followed by 4-h treatment with 5 �g/mL CB and 10 �g/mL cycloheximide. All embryos were then cultured in modified NCSU37 medium. Day 7 blastocysts were vitrified and warmed by using the Cryotop technique (Kuwayama et al. 2005 RBM Online 11, 300-308) at 38.5�C. Survival of vitrified blastocysts was determined according to re-expansion rates after 24 h recovery in culture medium supplemented with 10% CS. Cell numbers of reexpanded blastocysts from both groups were determined after Hoechst staining. Results were compared by ANOVA. Partial zona digestion and centrifugation resulted in successful delipation in 173/192 (90%) of oocytes. The development to blastocysts was not different between delipated and intact oocytes (28 � 7% vs. 28 � 5%, respectively; P > 0.05). However, survival rates of blastocysts derived from delipated oocytes were significantly higher than those developed from intact oocytes (85 � 6% vs. 32 � 7%, respectively; P < 0.01). There was no difference in average cell number of re-expanded blastocysts derived from either delipated or intact oocytes (36 � 7 vs. 38 � 9, respectively; P > 0.05). Our results prove that the simple delipation technique does not hamper the in vitro developmental competence of activated porcine oocytes, and improves the cryosurvival of the derived blastocysts without significant loss in cell number. Future investigations are required to prove the value of the method in an analogue system with blastocysts produced by somatic cell nuclear transfer.
97 DIFFERENCES IN SPERM CHROMATIN STRUCTURE AMONG GOOD AND BAD BOAR SPERM FREEZERS
Authors: Hernández M. , Roca J. , Ballester J. , Vázquez J. M. , Martínez E. A. , Johannisson A. , Saravia F. , Rodríguez-Martínez H.
Published: December 2006

Inter- and intra-boar differences in sperm freezability are observed independent of the sperm quality before freezing, the breed, or the genetic line. This study aimed to determine whether boars with different post-thaw sperm quality also show differences in sperm DNA integrity. Sperm-rich fractions (3 to 10 ejaculates per boar) from 19 fertile mature boars were extended in Beltsville thawing solution (BTS) and cooled to 17�C for 16 h. Then, samples were centrifuged at 2400g for 3 min, extended in freezing extender (lactose/egg yolk/glycerol/Equex STM; Nova Chemical Sales, Inc., Scituate, MA, USA) to a final concentration of 1 � 109 spermatozoa/mL, dispensed into 0.5 mL straws, and frozen in a programmable cell freezer at a rate of -20�C min. Thawing was carried out in a water bath at 37�C for 20 s. Frozen-thawed spermatozoa were evaluated for progressive sperm motility (PSM) using a computer-assisted sperm analysis (CASA) system, and sperm viability (PMI) using flow cytometry. All data generated were used for a multivariate pattern analysis (PATN; CSIRO, Canberra, Australia) which objectively classified all boars into two groups, categorized as good (n = 10; >60% PSM and PMI) or bad (n = 9; <40% PSM and PMI) based on their sperm freezability. Post-thaw sperm quality was consistent for different ejaculates within boars (P < 0.05). The DNA-integrity of frozen-thawed spermatozoa was evaluated according to the sperm chromatin structure assay (SCSA; Evenson et al. 1980 Science 210, 1131-1133). All SCSA variables (X mean, DNA fragmentation index (DFI), and the standard deviation of the DFI), were significantly higher for bad freezers (P < 0.001). The percentage of spermatozoa with abnormal chromatin structure ranged from 1.06 to 3.42% for good and 3.06 to 6.04% for bad freezers. Although these differences exist between good and bad sperm freezers, and can only to some extent be the product of cryopreservation, the levels of affected spermatozoa can not explain the differences on post-thaw sperm survival seen in the two categories of sires.
This work was supported by CICYT, AGL05-0471 (Spain), SLF and Formas (Sweden).
99 EFFECT OF CYSTEAMINE ON SURVIVAL OF BOVINE AND OVINE OOCYTES VITRIFIED USING THE MINIMUM VOLUME COOLING (MVC) CRYOTOP METHOD
Authors: Kelly J. , Kleemann D. , Kuwayama M. , Walker S.
Published: December 2006

The addition of cysteamine to maturation (IVM) media increases glutathione (GSH) synthesis in bovine oocytes and improves embryo development and quality (de Matos et al. 1995 Mol. Reprod. Dev. 42, 432–436). This study assesses the effect of adding cysteamine to IVM media on the survival of bovine and ovine oocytes following vitrification using the MVC cryotop method (Kuwayama and Kato 2000 J. Assist. Reprod. Genet. 17, 477 abst.). Abattoir-sourced bovine and ovine cumulus–oocyte complexes (COC) were matured in IVM media with or without 100 μM cysteamine for 24 h. After maturation, the COC were partially denuded and a proportion vitrified. Oocytes were equilibrated with 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO) for 30 s and then exposed to 20% EG, 20% DMSO, 0.5 M sucrose, and 20% FCS for 15 s. Oocytes were loaded onto a MVC plate (Cryotop, Kitazato Supply, Tokyo), and plunged into liquid nitrogen. After 5 days, oocytes were thawed directly into 1.25 M sucrose solution at 38.5°C, followed by stepwise dilution of the cryoprotectants. Ova were subsequently fertilized (Day 0) and cultured in modified SOF. Oocyte survival was assessed by cleavage and development to Day 8 compared with the development of fresh oocytes. Main effects, interactions and individual treatment differences were tested using procedure CATMOD in SAS. Cleavage rate was higher (P < 0.001) for fresh oocytes than for vitrified oocytes and it increased (P < 0.05) only for fresh ovine oocytes when cysteamine was added to maturation media. Blastocyst development was influenced by a significant (P < 0.001) interaction between species and whether or not oocytes were vitrified. This interaction occurred because cysteamine improved blastocyst rate in fresh ovine and vitrified bovine oocytes but not in other treatments. These results show that bovine oocytes (38.3% blastocyst rate) can be vitrified successfully when maturation occurs in the presence of cysteamine; however, a comparable result did not occur in ovine oocytes (10.6% blastocyst rate) despite >70% cleavage.

Table
Bcl10 plays a critical role in NF-κB activation induced by G protein-coupled receptors
Authors: Wang Donghai , You Yun , Lin Pei-Chun , Xue Liquan , Morris Stephan W. , Zeng Hu , Wen Renren , Lin Xin
Published: December 2006

G protein-coupled receptors (GPCRs) play pivotal roles in cell proliferation, differentiation, and survival. Although many studies indicate that the stimulation of GPCRs leads to NF-κB activation, the molecular mechanism by which GPCRs induced NF-κB activation remains largely unknown. Bcl10 is an essential adaptor molecule connecting antigen receptor signaling cascades to NF-κB activation in lymphocytes. However, the function of Bcl10 in nonlymphoid cells remains to be determined. In this study, we demonstrated that the deficiency of Bcl10 resulted in the defect in NF-κB activation induced by either expressing the constitutively active mutant of G protein or stimulation of cells with lysophosphatidic acid or endothelin-1, which activate their GPCR. In contrast, TNF-α-, LPS-, and integrin-induced NF-κB activation was not affected in Bcl10-deficient cells. Together, our results provide genetic evidence showing that Bcl10 is a key signaling component mediating NF-κB activation induced by GPCRs in nonlymphoid cells.
A PDZ domain recapitulates a unifying mechanism for protein folding
Authors: Gianni Stefano , Geierhaas Christian D. , Calosci Nicoletta , Jemth Per , Vuister Geerten W. , Travaglini-Allocatelli Carlo , Vendruscolo Michele , Brunori Maurizio
Published: December 2006

A unifying view has been recently proposed according to which the classical diffusion–collision and nucleation–condensation models may represent two extreme manifestations of an underlying common mechanism for the folding of small globular proteins. We report here the characterization of the folding process of the PDZ domain, a protein that recapitulates the three canonical steps involved in this unifying mechanism, namely: (
i
) the early formation of a weak nucleus that determines the native-like topology of a large portion of the structure, (
ii
) a global collapse of the entire polypeptide chain, and (
iii
) the consolidation of the remaining partially structured regions to achieve the native state conformation. These steps, which are clearly detectable in the PDZ domain investigated here, may be difficult to distinguish experimentally in other proteins, which would thus appear to follow one of the two limiting mechanisms. The analysis of the (un)folding kinetics for other three-state proteins (when available) appears consistent with the predictions ensuing from this unifying mechanism, thus providing a powerful validation of its general nature.
Site-specific collapse dynamics guide the formation of the cytochrome
c′
four-helix bundle
Authors: Kimura Tetsunari , Lee Jennifer C. , Gray Harry B. , Winkler Jay R.
Published: December 2006

The evolution of tryptophan-to-heme (W/heme) distance distributions extracted from analysis of fluorescence energy transfer kinetics during the refolding of
Rhodopseudomonas palustris
cytochrome
c′
reveals dramatic differences between two variants [W32 (Q1A/F32W/W72F) and W72 (Q1A)]. Both W32/heme and W72/heme distance distributions measured at the earliest time point attainable with a continuous-flow mixer (150 μs) confirm that the polypeptide ensemble is not uniformly collapsed and that native structure is not formed. Time-resolved fluorescence spectra indicate that W32 is sequestered from the aqueous solution during the first 700 μs of folding, whereas W72 remains exposed to solvent. The first moment of the W32/heme distance distribution evolves to its native value faster than that of W72, suggesting that the approach of W32 to the heme precedes that of W72.
Mutations in the connection domain of HIV-1 reverse transcriptase increase 3′-azido-3′-deoxythymidine resistance
Authors: Nikolenko Galina N. , Delviks-Frankenberry Krista A. , Palmer Sarah , Maldarelli Frank , Fivash Matthew J. , Coffin John M. , Pathak Vinay K.
Published: December 2006

We previously proposed that a balance between nucleotide excision and template RNA degradation plays an important role in nucleoside reverse transcriptase inhibitor (NRTI) resistance. To explore the predictions of this concept, we analyzed the role of patient-derived C-terminal domains of HIV-1 reverse transcriptase (RT) in NRTI resistance. We found that when the polymerase domain contained previously described thymidine analog resistance mutations, mutations in the connection domain increased resistance to 3′-azido-3′-deoxythymidine (AZT) from 11-fold to as much as 536-fold over wild-type RT. Mutational analysis showed that amino acid substitutions E312Q, G335C/D, N348I, A360I/V, V365I, and A376S were associated strongly with the observed increase in AZT resistance; several of these mutations also decreased RT template switching, suggesting that they alter the predicted balance between nucleotide excision and template RNA degradation. These results indicate that mutations in the C-terminal domain of RT significantly enhance clinical NRTI resistance and should be considered in genotypic and phenotypic drug resistance studies.
Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function
Authors: Treynor Thomas P. , Vizcarra Christina L. , Nedelcu Daniel , Mayo Stephen L.
Published: December 2006

To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made
in vivo
. Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods.
The Low Lying Electronic States of GeO
Authors: A Lagerqvist  , I Renhorn
Published: December 2006

TheA1Π-X1Σ+band system in74Ge16O and74Ge18O is analyzed and the local perturbations of theA1Π state are assigned. Four new electronic states are reported, namely thed3Δ,e3Σ-,C1Σ-andD1Δ states. Vibrational numbering is determined from the isotopic shifts. The vibrational variations of the perturbation matrix elements are shown to be in agreement with single configuration arguments. The electronic spin-orbit parametera+and the Coriolis parameterbare determined. The molecular constants for the eight lowest electronic states are reported. Semi-empirical and isovalent relationships between spin-orbit and spin-spin constants are also discussed.

The equilibrium constants for theA1Π state and the four perturbing states are in cm-1:

StateTeωeBeA1Π37 763.1648.80.414 32D1Δ32 7155910.362 2C1Σ-32 4755910.362 2e3Σ-32 2146000.362 2d3Δ130 4925970.362 2
The A2Π-X2Σ+Emission System of Cadmium Deuteride
Authors: Walter J Balfour
Published: December 2006

The arc emission spectrum of CdD has been photographed at high resolution and seven bands and two sub-bands of theA2Π-X2Σ+system rotationally analyzed. The principal114CdD molecular constants are, in cm-1:

TeωeωexeBeαe105DeX2Σ+:01026.1224.432.75050.0838.0A2Π:22620.531249.6320.293.07790.0677.4

The dependences on vibration and rotation of the spin-splitting in theX2Σ+state and the spin-orbit coupling and Λ-type doubling in theA2Π state have been studied. 𝒜0= 1025.9 + 9.8 × 10-3J(J+ 1) cm-1.
Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts
Authors: Wang Hongyan , Somers Gregory W. , Bashirullah Arash , Heberlein Ulrike , Yu Fengwei , Chia William
Published: December 2006

The choice of self-renewal versus differentiation is a fundamental issue in stem cell and cancer biology. Neural progenitors of the Drosophila post-embryonic brain, larval neuroblasts (NBs), divide asymmetrically in a stem cell-like fashion to generate a self-renewing NB and a Ganglion Mother Cell (GMC), which divides terminally to produce two differentiating neuronal/glial daughters. Here we show that Aurora-A (AurA) acts as a tumor suppressor by suppressing NB self-renewal and promoting neuronal differentiation. In aurA loss-of-function mutants, supernumerary NBs are produced at the expense of neurons. AurA suppresses tumor formation by asymmetrically localizing atypical protein kinase C (aPKC), an NB proliferation factor. Numb, which also acts as a tumor suppressor in larval brains, is a major downstream target of AurA and aPKC. Notch activity is up-regulated in aurA and numb larval brains, and Notch signaling is necessary and sufficient to promote NB self-renewal and suppress differentiation in larval brains. Our data suggest that AurA, aPKC, Numb, and Notch function in a pathway that involved a series of negative genetic interactions. We have identified a novel mechanism for controlling the balance between self-renewal and neuronal differentiation during the asymmetric division of Drosophila larval NBs.
Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation
Authors: Lee Cheng-Yu , Andersen Ryan O. , Cabernard Clemens , Manning Laurina , Tran Khoa D. , Lanskey Marcus J. , Bashirullah Arash , Doe Chris Q.
Published: December 2006

Regulation of stem cell self-renewal versus differentiation is critical for embryonic development and adult tissue homeostasis. Drosophila larval neuroblasts divide asymmetrically to self-renew, and are a model system for studying stem cell self-renewal. Here we identify three mutations showing increased brain neuroblast numbers that map to the aurora-A gene, which encodes a conserved kinase implicated in human cancer. Clonal analysis and time-lapse imaging in aurora-A mutants show single neuroblasts generate multiple neuroblasts (ectopic self-renewal). This phenotype is due to two independent neuroblast defects: abnormal atypical protein kinase C (aPKC)/Numb cortical polarity and failure to align the mitotic spindle with the cortical polarity axis. numb mutant clones have ectopic neuroblasts, and Numb overexpression partially suppresses aurora-A neuroblast overgrowth (but not spindle misalignment). Conversely, mutations that disrupt spindle alignment but not cortical polarity have increased neuroblasts. We conclude that Aurora-A and Numb are novel inhibitors of neuroblast self-renewal and that spindle orientation regulates neuroblast self-renewal.
Muscle cachexia is regulated by a p53–PW1/Peg3-dependent pathway
Authors: Schwarzkopf Martina , Coletti Dario , Sassoon David , Marazzi Giovanna
Published: December 2006

Muscle wasting (cachexia) is an incurable complication associated with chronic infection and cancers that leads to an overall poor prognosis for recovery. Tumor necrosis factor-α (TNFα) is a key inflammatory cytokine associated with cachexia. TNFα inhibits myogenic differentiation and skeletal muscle regeneration through downstream effectors of the p53 cell death pathway including PW1/Peg3, bax, and caspases. We report that p53 is required for the TNFα-mediated inhibition of myogenesis in vitro and contributes to muscle wasting in response to tumor load in vivo. We further demonstrate that PW1 and p53 participate in a positive feedback regulatory loop in vitro. Consistent with this observation, we find that the number of PW1-expressing stem cells in skeletal muscle declines significantly in p53 nullizygous mice. Furthermore, gene transfer of a dominant-negative form of PW1 into muscle tissue in vivo blocks myofiber atrophy in response to tumor load. Taken together, these results show a novel role for p53 in mediating muscle stem cell behavior and muscle atrophy, and point to new targets for the therapeutic treatment of muscle wasting.
Domestic violence and child protection: exploring the role of perpetrator risk assessments
Authors: Humphreys Cathy
Published: December 2006

This article explores the issue of severity in relation to domestic violence and provides a number of reasons for the necessary engagement by workers with such a contentious issue. The specific role that the assessment of the risks posed by the perpetrator which has now developed in some police forces in the United Kingdom is examined, and its relevance to child welfare intervention discussed. A range of factors are identified that heighten the risks of increased violence. These include prior sexual assault; stalking and controlling behaviour; substance misuse and mental‐health problems; separation and child contact disputes; pregnancy; escalation including the use of weapons and psychological abuse; attempts and threats to kill; child abuse; isolation and barriers to help‐seeking. The ways in which perpetrator risk assessment can be used to inform the filtering of referrals to the statutory child care agency, enhance multi‐agency working, provide a structure for the assessment of the perpetrator, enhance partnership‐working with survivors (usually women) and inform the protection strategies for workers are explored.