Selection for increased adult body weight in mouse lines with and without the rat growth hormone transgene

SUMMARY: Four lines of mice with and without the rat growth hormone (rGH) transgene were developed to measure responses to selection for increased 42-day body weight and evaluate fitness of mice with and without the rGH transgene. Each line contained selected and unselected (control) sublines. At the last three generations of selection (Generations 12-14), selected sublines differed from unselected controls by 3.8 to 4.7 g (14.8 to 19.8%) in 42-day weight, -0.5 to -8.3% in fertility, and 0.5 to 1.6 in litter size at birth. The origin of the lines (W: previously selected for 42-day weight and C: unselected) affected 42-day weight, i. e. 42-day weight of mice originating from W was significantly (P < 0.01) heavier than that of mice originating from C. Responses to selection, as measured by the deviation of the selected subline from the control, continued to be positive over 14 generations. Realized heritability of 42-day weight ranged from 0.30 to 0.42. The rGH transgene that increased 63-day weight by 54% was not found at Generation 12. The unexpected loss of rGH transgene was due to poor fitness of mice with the rGH transgene. Mice with the transgene had lower fertility rate than those without the transgene (50.0 to 73.7% vs. 95.0%), smaller litter size (6.8 to 7.8 vs. 8.6) and poorer survival of the progeny (69.2 to 74.5% vs. 88.3%). Based on these data, selective advantage/disadvantage of the rGH transgene in the fitness traits was estimated quantitatively. The results from the study on growth and reproductive traits suggest that desirable effects of gene transfer on a specific trait (42- and 63-day weight in the present study) might be offset by undesirable effects on other traits (e. g., reproduction and survival) in some cases of transgenic animals. ZUSAMMENFASSUNG: Selektion auf hohes adultes Gewicht in M?uselinien mit und ohne Rattenwachstumshormon-Transgenen Vier M?uselinien mit und ohne das Rattenwachstumshormon (rGH) Transgen wurden zur Messung des Selektionserfolges auf gesteigertes 42-Tage-K?rpergewicht entwickelt, um auch Fitne? zu prüfen. Jede Linie bestand aus einer selektierten und aus einer unselektierten (Kontroll-)Unterlinie. In den drei letzten Selektionsgenerationen (Generationen 12-14) unterschieden sich die selektierten Unterlinien um 3,8 bis 4,7 g (14,8 bis 19,8%) im 42-Tage-Gewicht, -0,5 bis -8,3% in Fruchtbarkeit und 0,5 bis 1,6 in Wurfgr??e bei Geburt. Der Ursprung der Linien (W: früher auf 42-Tage-Gewicht selektiert, C: unselektiert) beeinflu?te das 42-Tage-Gewicht. M?use aus der ersten waren signifikant (p < 0,01) schwerer als jede aus C. Selektionserfolg gemessen als Abweichung von der Kontrolle war über 14 Generationen positiv, realisierte Heritabilit?t variierte von 0,3 bis 0,42. Das rGH-Transgen steigerte das 63-Tage-Gewicht um 54%, aber war in der Generation 12 nicht mehr vorhanden. Der unerwartete Verlust des RGH-Transgens war auf schlechte Fitne? der besitzenden M?use zurückzuführen. Transgene M?use hatten geringere Fruchtbarkeit als jene ohne das Transgen (50 bis 73,7% im Vergleich zu 95%), kleinere Wurfgr??e (6,8 bis 7,8 gegenüber 8,6) und schlechtere überlebensrate der Nachkommen (69,2 bis 74,5% im Vergleich zu 88,3%). Die Ergebnisse erlauben die quantitative Sch?tzung der selektiven Vorteile/Nachteile des rGH-Transgens im Bezug auf Fitne?. Erwünschte Wirkungen von Gentransfer auf spezifische Merkmale (41- und 63-Tage-Gewicht in dieser Untersuchung) k?nnen durch unerwünschte Wirkungen von Transgenen auf andere Merkmale (Reproduktion und überleben) neutralisiert werden.     

Expression of prostaglandin F(2α) (PGF(2α)) receptor and its isoforms in the bovine corpus luteum during the estrous cycle and PGF(2α)-induced luteolysis

Prostaglandin F(2α) (PGF(2α)) induces luteolysis via a specific receptor, PTGFR. Although PTGFR mRNA expression in the bovine corpus luteum (CL) has been studied previously, changes in PTGFR protein and its localization are not fully understood during the life span of the CL. In addition to full-length PTGFR, several types of PTGFR isoforms, such as PTGFRα (type I) and PTGFRζ (type II), were reported in the bovine CL, suggesting isoform-specific luteal action. Full-length PTGFR mRNA in the bovine CL increased from the early to the mid-luteal phase and decreased during luteolysis, whereas PTGFR protein remained stable. PTGFR protein was localized to both luteal and endothelial cells and was expressed similarly during the life span of the CL. Like full-length PTGFR mRNA, PTGFRα and PTGFRζ mRNA also increased from the early to mid-luteal phases, and mRNA of PTGFRζ, but not PTGFRα, decreased in the regressing CL. During PGF(2α)-induced luteolysis, the mRNAs of full-length PTGFR, PTGFR,α and PTGFRζ decreased rapidly (from 5 or 15 min after PGF(2α) injection), but PTGFR protein decreased only 12 h later. Silencing full-length PTGFR using small interfering RNA prevented PGF(2α)-stimulated cyclooxygenase-2 (PTGS2) mRNA induction. By contrast, PGF(2α) could stimulate vascular endothelial growth factor A (VEGFA) mRNA even when full-length PTGFR was knocked down, thus suggesting that PGF(2α) may stimulate PTGS2 via full-length PTGFR, whereas VEGFA is stimulated via other PTGFR isoforms. Collectively, PTGFR protein was expressed continually in the bovine CL during the estrous cycle, implying that PGF(2α) could function throughout this period. Additionally, the bovine CL expresses different PTGFR isoforms, and thus PGF(2α) may have different effects when acting via full-length PTGFR or via PTGFR isoforms.     

Effects of Chemical Activation Treatment on Development of Swamp Buffalo (Bubalus bubalis) Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Injection

The objective of this study was to optimize the activation protocol for buffalo oocytes after intracytoplasmic sperm injection (ICSI). The release of the second polar body (PB) at 3, 6 and 9 h after ICSI of in‐vitro matured oocytes activated either with 5 μm ionomycin (Io) or with 7% ethanol (EtOH) was preliminary examined. The highest rate of second PB extrusion occurred at 3 h of activation, and the second PB extrusion in EtOH group was significantly higher than that in Io group. Oocytes that extruded the second PB were selected and cultured either with 1.9 mm 6‐dimethylaminopurine (6‐DMAP) for 3 h or with 10 μg/ml cycloheximide (CHX) for 5 h. Significantly higher rate of oocytes formed 2 pronuclei in EtOH combined with CHX (EtOH + CHX) (62%) group compared to those of Io + CHX (42%) and EtOH + 6‐DMAP (48%) groups (p < 0.01) whereas Io + 6‐DMAP group showed intermediate value (58%). Significantly higher blastocyst formation rates were obtained in Io + 6‐DMAP (29%) and EtOH + CHX (24%) groups than in Io + CHX (6%) and EtOH + 6‐DMAP (17%) groups. Our results indicate that buffalo ICSI oocytes are effectively activated by combination treatment of Io with 6‐DMAP and EtOH with CHX resulting in the highest cleavage and blastocyst formation rates.     

Reactivity and prevalence of neutralizing antibodies against Japanese strains of bovine viral diarrhea virus subgenotypes

Bovine viral diarrhea virus (BVDV) field isolates show genetic and antigenic diversity. At least 14 subgenotypes of BVDV-1 and 4 of BVDV-2 have been identified in Artiodactyla worldwide. Of these, 6 subgenotypes of BVDV-1 and 1 of BVDV-2 have been isolated in Japan. Previously, we reported that each subgenotype virus expresses different antigenic characteristics. Here we investigated the reactivity of neutralizing antibodies against representative strains of Japanese BVDV subgenotypes using sera from 266 beef cattle to estimate the prevalence of this epidemic virus among cattle in Japan. Antibody titers at concentrations at least 4-fold higher than antibodies against other subgenotype viruses were considered subgenotype specific. Subgenotype-specific antibodies were detected from 117 (80.7%) of 145 sera samples (69.7% against BVDV-1a, 1.4% against BVDV-1b, 8.3% against BVDV-1c, and 1.4% against BVDV-2a). The results suggest that neutralization tests are useful in estimating currently epidemic subgenotypes of BVDV in the field.     

Parthenogenetic activation and subsequent development of porcine oocytes activated by a combined electric pulse and butyrolactone I treatment

This study was designed to evaluate the parthenogenetic activation of porcine oocytes matured in vitro for a varied period after combined electric pulse (EP; 1500 V/cm, 100 microsec) and Butyrolactone I (BL I). After 36 h of maturation culture, the rates of activated oocytes and oocytes with two pronuclei were significantly lower than those of oocytes cultured for 42 and 48 h after EP. However, when treated by a combined EP and BL I (150 microM), these rates increased to the same level as 42 and 48 h oocytes. When oocytes cultured for 48 h and activated by a combined EP and BL I treatment were subsequently cultured in mNCSU37 medium, the rates of embryos cleaved and developed to the blastocyst stage were significantly higher than those in Whitten's medium. In contrast, when activated oocytes were cultured in mNCSU37 medium under two oxygen environments (5% vs 20% O(2)), there was no difference in the rates of cleavage, blastocyst formation and nuclear numbers per blastocyst. Our results demonstrated that the combined EP and BL I treatment of porcine oocytes matured in vitro is capable of producing high rates of good quality blastocysts when cultured in a suitable in vitro condition.     

Genetic Analyses of an H3N8 Influenza Virus Isolate, Causative Strain of the Outbreak of Equine Influenza at the Kanazawa Racecourse in Japan in 2007

In August 2007, an outbreak of equine influenza occurred among vaccinated racehorses with Japanese commercial equine influenza vaccine at Kanazawa Racecourse in Ishikawa prefecture in Japan. Apparent symptoms were pyrexia (38.2-41.0 degrees C) and nasal discharge with or without coughing, although approximately half of the infected horses were subclinical. All horses had been shot with a vaccine that contained two inactivated H3N8 influenza virus strains [A/equine/La Plata/93 (La Plata/93) of American lineage and A/equine/Avesta/93 (Avesta/93) of European lineage] and an H7N7 strain (A/equine/Newmarket/1/77). Influenza virus, A/equine/Kanazawa/1/2007 (H3N8) (Kanazawa/07), was isolated from one of the nasal swab samples of diseased horses. Phylogenetic analysis indicated that Kanazawa/07 was classified into the American sublineage Florida. In addition, four amino acid substitutions were found in the antigenic sites B and E in the HA1 subunit protein of Kanazawa/07 in comparison with that of La Plata/93. Hemagglutination-inhibition (HI) test using 16 serum samples from recovering horses revealed that 1.4- to 8-fold difference in titers between Kanazawa/07 and either of the vaccine strains. The present findings suggest that Japanese commercial inactivated vaccine contributed to reducing the morbidity rate and manifestation of the clinical signs of horses infected with Kanazawa/07 that may be antigenically different from the vaccine strains.     

Attempt at intracytoplasmic sperm injection of in vitro matured oocytes in common minke whales (Balaenoptera acutorostrata) captured during the Kushiro Coast Survey

The present study was conducted during the Kushiro Coast Survey in an attempt to produce common minke whale embryos. In Experiment 1, we attempted to determine the appropriate culture duration (30 or 40 h) for in vitro maturation (IVM) of immature oocytes using the Well of the Well method. In Experiment 2, and intracytoplasmic sperm injection (ICSI) was applied to matured oocytes from prepubertal and adult common minke whales after IVM culture (40 or 48 h), and then their embryonic development was assessed. In Experiment 1, the maturation rate of oocytes cultured for 40 h (30.4%) was significantly higher than that of oocytes cultured for 30 h (6.8%; P<0.01). In Experiment 2, a total of 35 and 46 immature oocytes derived from adult (n=2) and prepubertal (n=6) minke whales, respectively, were cultured for 40 or 48 h. The maturation rate in the oocytes from the adult whales (34.2%) tended to be higher than that of the oocytes from the prepubertal whales (19.6%), but there was no significant difference. Following ICSI, 3 out of the 10 inseminated and cultured oocytes from the adult whales cleaved (2-, 8-, and 16-cell stages); all of these oocytes had been matured for 40 in culture. However, these oocytes did not develop to further stages. Only one of the 6 oocytes derived from the prepubertal whales, IVM cultured for 40 h and inseminated, developed to the 4-cell stage. The present results indicate that a 40 h IVM culture produces significantly higher rates of in vitro maturation than a 30 h IVM culture for common minke whale oocytes. Following ICSI, some oocytes cleaved to the 16-cell stage, but no further development was observed.     

Anti-inflammatory potency of oral disulfiram compared with dexamethasone on endotoxin-induced uveitis in rats

To investigate potency of oral disulfiram (DSF) compared with that of dexamethasone (Dexa), on endotoxin-induced uveitis (EIU) in rats. The oral administration with 750 mg/kg DSF suppressed the number of inflammatory cells, protein concentration, and levels of tumor necrosis factor (TNF)-α, Nitric oxide (NO) and prostaglandin (PG) E2 in the aqueous humor and improved the histiologic status of the ocular tissue at 24 hr after lipopolysaccharide (LPS) injection. The anti-inflammatory potency of DSF oral administration was as strong as that observed with 0.5 mg/kg Dexa in the present study. The results suggest that DSF might pave the way for a novel therapeutic agent for the management of uveitis.     

An expanded palette of genetically encoded Ca²⁺ indicators

Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.