Nuclear Replacement of In Vitro‐Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method

The objective of the present study was to establish a method for nuclear replacement in metaphase‐II (M‐II) stage porcine oocytes. Karyoplasts containing M‐II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro‐matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona‐free M‐II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2–77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2–32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0–15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri‐Fusion) is an effective method for producing M‐II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.     

Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro

Fifty-four polyphenols isolated from tea leaves were evaluated for their inhibitory activities against pancreatic lipase, the key enzyme of lipid absorption in the gut. (-)-Epigallocatechin 3-O-gallate (EGCG), which is one of major polyphenols in green tea, showed lipase inhibition with an IC50 of 0.349 microM. Moreover, flavan-3-ol digallate esters, such as (-)-epigallocatechin-3,5-digallate, showed higher activities of inhibition on lipase with an IC50 of 0.098 microM. On the other hand, nonesterified flavan-3-ols, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin, showed zero and/or the lowest activities against pancreatic lipase (IC50 > 20 microM). These data suggested that the presence of galloyl moieties within the structure was required for enhancement of pancreatic lipase inhibition. It is well-known that flavan-3-ols are polymerized by polyphenol oxidase and/or heating in a manufacturing process of oolong tea. Oolonghomobisflavans A and B and oolongtheanin 3'-O-gallate, which are typical in oolong tea leaves, showed strong inhibitory activities with IC50 values of 0.048, 0.108, and 0.068 microM, respectively, even higher than that of EGCG. The oolong tea polymerized polyphenols (OTPP) were prepared for the assay from oolong tea extract, from which the preparation effectively subtracted the zero and/or less-active monomeric flavan-3-ols by preparative high-performance liquid chromatography. The weight-average molecular weight (Mw) and number-average molecular-weight (Mn) values of OTPP were 2017 and 903, respectively, by using gel permeation choromatography. OTPP showed a 5-fold stronger inhibition against pancreatic lipase (IC50 = 0.28 microg/mL) by comparison with that of the tannase-treated OTPP (IC50 = 1.38 microg/mL). These data suggested that the presence of galloyl moieties within their chemical structures and/or the polymerization of flavan-3-ols were required for enhancement of pancreatic lipase inhibition.     

Endocrine status and development of porcine testicular tissues in host mice

Clarification of the endocrine status of host mice provides us with basic knowledge with which we can manipulate the growth and function of xenografted testicular tissues. We investigated the hormonal profiles of castrated mice grafted with porcine immature testicular tissues from 30 to 210 or more days after grafting (day 0=castration and grafting). The serum follicle stimulating hormone (FSH) concentrations of the host mice declined (P<0.05) from day 60 compared with those of the castrated, ungrafted mice. The serum inhibin and testosterone levels were higher (P<0.05) than those in the castrated, ungrafted mice from days 30 and 90 days, respectively. The inhibin levels further increased (P<0.05) from day 90, during which time the levels were higher (P<0.05) than those in the intact male mice. In the grafts, formation of lumens occurred in the seminiferous cords on day 90 and spermatozoa appeared in the lumens from day 120. However, spermatogenesis in the grafts did not reach the qualitatively normal levels observed in adult boars. The intensity of the immune reaction to inhibin alpha subunits in the Sertoli cells of the grafts decreased with differentiation of the seminiferous tubules. The present findings indicate that a feedback loop was established between the mouse hypothalamo-pituitary axis and the grafted porcine tissues from 60 days post-grafting. The results also indicate that the serum inhibin levels in the host mice remained high even after the appearance of lumens in the seminiferous tubules of the grafted tissues; this is strikingly different to the situation in normal male animals, in which the serum inhibin levels decline at around the time of tubular differentiation. The lack of efferent ducts in the tubules of the grafted tissues probably caused the accumulation of inhibin to be released into the lumens, resulting in high concentrations of circulating inhibin. These high levels of inhibin may directly affect spermatogenic activity and suppress FSH secretion.     

Epidemiological study on <Emphasis Type="Italic">Lactococcus garvieae</Emphasis> isolates from fish in Japan

In Japan, Lactococcus garvieae infection has been the main fish disease in aquaculture. Although commercial oral and injectable vaccines have been used to prevent L. garvieae infection in Japan, L. garvieae has been isolated not only from unvaccinated fish but also from vaccinated fish in which immunity induced by vaccination had diminished. In order to obtain epidemiological information on this fish pathogen, we conducted biased sinusoidal field gel electrophoresis (BSFGE) pattern analysis and phage typing of L. garvieae isolates (n = 427) from fish in Japan. These isolates were obtained from 13 different fish species between 1980 and 2007. In the BSFGE analysis, L. garvieae isolates were classified into 17 groups (S1–S17) based on the SmaI digestion patterns and into four groups (A1–A4) based on the ApaI digestion patterns. Phage typing revealed five different phage susceptibility profiles (A–E) in L. garvieae isolates. Since 2005, comparisons of the results of phage typing and BSFGE have indicated the presence of a novel genotype (S16/A4) with phage type E. All the strains belonging to this type showed lincomycin sensitivity.     

Detection of Cryptosporidium muris type oocysts from beef cattle in a farm and from domestic and wild animals in and around the farm

Cryptosporidium muris type oocysts were detected from 21 of 516 beef cattle in a farm. Then we surveyed Cryptosporidium oocysts in 348 beef and dairy cattle, 500 pigs, 101 dogs, 38 wild animals and 11 zoo-kept animals in and around the farm. Oocysts were detected from only 2 of 25 Japanese field mice, Apodemus speciosus in the same farm. Gene analysis suggested that the oocysts were different from the C. muris type bovine isolate.     

Time-course comparison of pulmonary inflammation induced by intratracheal instillation of four different nickel oxide nanoparticles in male Fischer rats

Occupational exposure to nickel oxide (NiO) is an important cause of respiratory tract cancer. Toxicity is known to be associated with the dissociated component, i.e. nickel (II) ions. To address the relationship between physicochemical properties, including solubility in artificial lysosomal fluid, of NiO and time-course changes in the pulmonary response, we conducted an intratracheal instillation study in male Fischer rats using four different well-characterized NiO products, US3352 (NiO A), NovaWireNi01 (NiO B), I small particle (NiO C), and 637130 (NiO D). The NiOs were suspended in purified water and instilled once intratracheally into male F344 rats (12 weeks old) at 0 (vehicle control), 0.67, 2, and 6 mg/kg body weight. The animals were euthanized on days 3, 28, or 91 after instillation, and blood analysis, bronchoalveolar lavage fluid (BALF) testing, and histopathological examination were performed. The most soluble product, NiO B, caused the most severe systemic toxicity, leading to a high mortality rate, but the response was transient and surviving animals recovered. The second-most-soluble material, NiO D, and the third, NiO A, caused evident pulmonary inflammation, and the responses persisted for at least 91 days with collagen proliferation. In contrast, NiO C induced barely detectable inflammation in the BALF examination, and no marked changes were noted on histopathology. These results indicate that the early phase toxic potential of NiO products, but not the persistence of pulmonary inflammation, is associated with their solubility.