Inhibition of tumorigenesis in ApcMin/+ mice by a combination of (-)-epigallocatechin-3-gallate and fish oil

The effect of a combination of (-)-epigallocatechin-3-gallate (EGCG) with fish oil on intestinal tumorigenesis in Apc (Min/+) mice fed a high-fat diet was investigated in the present study. The combined treatment of EGCG and fish oil for 9 weeks reduced the tumor number by 53% as compared to controls while neither agent alone had a significant effect. Apoptosis was significantly increased in all treatment groups. beta-Catenin nuclear positivity in adenomas from the combination group was lower than control mice, implicating the modulation of Wnt signaling by the combination. Fish oil and the combination significantly reduced prostaglandin E 2 (PGE 2) levels in small intestinal tumors as compared to controls, suggesting modulation of aberrant arachidonic acid metabolism by fish oil. Akt phosphorylation in adenomas was significantly reduced in all treatment groups, which may have contributed to the observed increase in apoptosis. The results indicate that a combination of low doses of EGCG and fish oil can inhibit tumor multiplicity in Apc (Min/+) mice.     

Soybean mosaic virus Helper Component-Protease Alters Leaf Morphology and Reduces Seed Production in Transgenic Soybean Plants

ABSTRACT Transgenic soybean (Glycine max) plants expressing Soybean mosaic virus (SMV) helper component-protease (HC-Pro) showed altered vegetative and reproductive phenotypes and responses to SMV infection. When inoculated with SMV, transgenic plants expressing the lowest level of HC-Pro mRNA and those transformed with the vector alone initially showed mild SMV symptoms. Plants that accumulated the highest level of SMV HC-Pro mRNA showed very severe SMV symptoms initially, but after 2 weeks symptoms disappeared, and SMV titers were greatly reduced. Analysis of SMV RNA abundance over time with region-specific probes showed that the HC-Pro region of the SMV genome was degraded before the coat protein region. Transgenic soybean plants that expressed SMV HC-Pro showed dose-dependent alterations in unifoliate leaf morphologies and seed production where plants expressing the highest levels of HC-Pro had the most deformed leaves and the lowest seed production. Accumulation of microRNAs (miRNAs) and mRNAs putatively targeted by miRNAs was analyzed in leaves and flowers of healthy, HC-Pro-transgenic, and SMV-infected plants. Neither expression of SMV HC-Pro nor SMV infection produced greater than twofold changes in accumulation of six miRNAs. In contrast, SMV infection was associated with twofold or greater increases in the accumulation of four of seven miRNA-targeted mRNAs tested.     

Influence of Temperament Score and Handling Facility on Stress,Reproductive Hormone Concentrations,and Fixed Time AI Pregnancy Rates in Beef Heifers

The objectives were (i) to evaluate the effect of temperament, determined by modified 2‐point chute exit and gait score, on artificial insemination (AI) pregnancy rates in beef heifers following fixed time AI and (ii) to determine the effect of temperament on cortisol, substance‐P, prolactin and progesterone at initiation of synchronization and at the time of AI. Angus beef heifers (n = 967) at eight locations were included in this study. At the initiation of synchronization (Day 0 = initiation of synchronization), all heifers received a body condition score (BCS), and temperament score (0 = calm; slow exit and walk or 1 = excitable; fast exit or jump or trot or run). Blood samples were collected from a sub‐population of heifers (n = 86) at both synchronization initiation and the time of AI to determine the differences in serum progesterone, cortisol, prolactin and substance‐P concentrations between temperament groups. Heifers were synchronized with 5‐day CO‐Synch+ controlled internal drug release (CIDR) protocol and were inseminated at 56 h after CIDR removal. Heifers were examined for pregnancy by ultrasound 70 days after AI to determine AI pregnancy. Controlling for synchronization treatment (p = 0.03), facility design (p = 0.05), and cattle handling facility design by temperament score interaction (p = 0.02), the AI pregnancy differed between heifers with excitable and calm temperament (51.9% vs 60.3%; p = 0.01). The alley‐way with acute bends and turns, and long straight alley‐way had lower AI pregnancy rate than did the semicircular alley‐way (53.5%, 56.3% and 67.0% respectively; p = 0.05). The serum hormone concentrations differed significantly between different types of cattle handling facility (p < 0.05). The cattle handling facility design by temperament group interactions significantly influenced progesterone (p = 0.01), cortisol (p = 0.01), prolactin (p = 0.02) and substance‐P (p = 0.04) both at the initiation of synchronization and at the time of AI. Inter‐ and intra‐rater agreement for temperament scoring were moderate and good (Kappa = 0.596 ± 0.07 and 0.797 ± 0.11) respectively. The predictive value for calm and pregnant to AI was 0.87, and excited and non‐pregnant to AI was 0.76. In conclusion, the modified 2‐point temperament scoring method can be used to identify heifers with excitable temperament. Heifers with excitable temperament had lower AI pregnancy. Further, cattle handling facility design influenced the temperament and AI pregnancy.     

Determination of Feeder Cell‐Based Cellular Niches Supporting the Colonization and Maintenance of Spermatogonial Stem Cells from Prepubertal Domestic Cat Testes

Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long‐term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC‐specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome‐shaped morphology, AP activity and expression of SSC‐specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats.     

In vitro modulation of reactive oxygen and nitrogen intermediate (ROI/RNI) production in Crassostrea gigas hemocytes

Bivalve hemocyte competence has been measured by quantifying functional characteristics, including reactive oxygen intermediate (ROI) production after activation with zymosan or phorbol myristate acetate (PMA). However, untreated oyster hemocytes also produce ROI and RNI (reactive nitrogen intermediates) after bleeding even if not stimulated by zymosan or PMA. Extensive investigation of this parameter by flow cytometry showed that, in vitro, ROI/RNI production by untreated hemocytes maintained in seawater appeared to be independent of both bacterial burden in the serum and non-self particle phagocytosis. ROI/RNI production in granulocytes was higher than in hyalinocytes and could be intensified when activated by zymosan but not by PMA. Both cell types used NADPH-oxidase- and NO-synthase-like pathways to produce these molecules; the NO-synthase pathway seemed relatively more dominant in hyalinocytes and NADPH-oxidase appeared more effective in granulocytes. These results provide new insights for interpreting the modulation of ROI/RNI production by untreated hemocytes shown by other studies, relative to environmental conditions or physiological status of the oysters.     

Steroidogenesis during induced oocyte maturation and ovulation in the African catfish,Clarias gariepinus

Changes in ovarian steroidogenesis accompanying oocyte maturation and ovulation were studied in the African catfish,Clarias gariepinus. Laboratory-reared females with postvitellogenic ovaries were treated with pimozide and LHRH-analogue. The plasma gonadotropin levels were determined by means of a homologous radioimmunoassay, the condition of the ovaries was studied by histological examination of the follicles, and the steroidogenetic capacity of the ovaries was analyzed byin vitro incubation of tissue fragments for 3 h with [³H]-pregnenolone and [³H]androstenedione as precursors. Data were collected at regular intervals between 0 and 16 h after pimozide-LHRH analogue administration.Until 4 h after the beginning of the experiments the plasma gonadotropin levels did not rise above 25 ng/ml, the ovaries remained in the stage of postvitellogenesis, and testosterone was the main end product of steroidogenesis. Four hours later the gonadotropin concentration in the blood had risen to more than 150 ng/ml, and the ovaries had entered the stage of germinal vesicle migration. At the same time steroidogenesis shifted towards the production of 17,20-dihydroxy-4-pregnen-3-one, 5-pregnane-3, 17-diol-20-one, 5-pregnane-3,6,17-triol-20-one, 5-pregnane-3,17,20-triol and 5-pregnane-3,6,17,20-tetrol. During the subsequent stage of germinal vesicle breakdown the plasma gonadotropin level remained high, and the synthesis of the C₂₁-steroids showed a further increase. Simultaneously, the production of some C₁₉-steroid glucuronides was enhanced. The preovulation and especially the postovulation stages were accompanied by a gradual decrease in steroidogenic capacity of the ovaries, even though the plasma gonadotropin level remained high. It is concluded that the prematuration surge of gonadotropin influences the activity of enzymes involved in steroidogenesis, leading to a reduced C_17,20-lyase and to an augmented activity of the enzymes 20-hydroxysteroid dehydrogenase (HSD), 5-reductase, 3-HSD, 6-hydroxylase and UDP-glucuronosyltransferase. During ovulation the activity of all steroidogenic enzymes, including such key enzymes as 3-HSD and 17-hydroxylase, gradually decreases.Not only 17,20-dihydroxy-4-pregnen-3-one, but also the 5-reduced pregnanes may be involved in inducing oocyte maturation and/or ovulation. The very polar triol and tetrol products may function, together with the steroid glucuronides as sex pheromones.A preliminary account of these results was presented at the XIII Conference of European Comparative Endocrinologists, Belgrade, September 7–12, 1986     

Pubertal development of male African catfish,Clarias gariepinus. In vitro steroidogenesis by testis and interrenal tissue and plasma levels of sexual steroids

In fish, sex steroids initiate and/or accelerate the maturation of the brain-pituitary-gonad axis. In order to obtain information on the steroid milieu during the pubertal development of male African catfish, we have monitored the conversion of [³H]-pregnenolone and [³H]-androstenedione by testis and [³H]-pregnenolone by interrenal tissue fragmentsin vitro. Pubertal development occurs between two and six months of age. Testicular development proceeds through four main stages that are characterised histologically by the presence of spermatogonia (stage I), spermatogonia and spermatocytes (stage II), spermatogonia, spermatocytes and spermatids (stage III), and all germ cells including spermatozoa (stage IV). 11β-Hydroxyandrostenedione and cortisol were the main products of testes and interrenal tissue, respectively, in all stages of the pubertal development; a change in the steroidogenic pattern was not observed during this period. The interrenal tissue displayed no significant conversion of [³H]-pregnenolone to 11-oxygenated androgens. Blood plasma was analyzed for the presence of five androgens; testosterone, 11β-hydroxytestosterone, 11β-hydroxyandrostenedione, androstenetrione, and 11-ketotestosterone. 11-Ketotestosterone was the quantitatively dominating androgen in the circulation at all stages of development, which was more pronounced in stages III and IV. The obvious differences between thein vitro andin vivo results, namely 11β-hydroxyandrostenedione being the main testicular productvs. 11-ketotestosterone dominating in the blood, may indicate that 11β-hydroxyandrostenedione is converted to 11-ketotestosterone at extratesticular sites.     

In vitro steroidogenesis by gonads of the Russian sturgeon, Acipenser gueldenstaedti Brandt

Ovaries and testes from the Russian sturgeon at different stages of sexual maturity were incubated with ;sup3;H-androstenedione or ³H-pregnenolone. The major metabolites of androstenedione in both sexes were testosterone and 5- and 5ß-androstanediols, but no evidence was found for the gonadal production of 11-oxygenated androgens. Pregnenolone was converted to 17-hydroxyprogesterone, androstenedione and testosterone together with reduced metabolites. 11-Oxygenated androgens were found in female serum by gas chromatography-mass spectrometry (GC-MS), in serum of both sexes by radioimmunoassay (RIA), and was detectable by RIA in interrenal but not gonadal tissue. The results suggest that sturgeon may differ from teleosts in that 11-oxygenation may take place in extragonadal tissues.     

Monitoring distances travelled by horses using GPS tracking collars

Objective The aims of this work were to (1) develop a low-cost equine movement tracking collar based on readily available components, (2) conduct preliminary studies assessing the effects of both paddock size and internal fence design on the movements of domestic horses, with and without foals at foot, and (3) describe distances moved by mares and their foals. Additional monitoring of free-ranging feral horses was conducted to allow preliminary comparisons with the movement of confined domestic horses. Procedures A lightweight global positioning system (GPS) data logger modified from a personal/vehicle tracker and mounted on a collar was used to monitor the movement of domestic horses in a range of paddock sizes and internal fence designs for 6.5-day periods. Results In the paddocks used (0.8–16 ha), groups of domestic horses exhibited a logarithmic response in mean daily distance travelled as a function of increasing paddock size, tending asymptotically towards approximately 7.5 km/day. The distance moved by newborn foals was similar to their dams, with total distance travelled also dependent on paddock size. Without altering available paddock area, paddock design, with the exception of a spiral design, did not significantly affect mean daily distance travelled. Feral horses (17.9 km/day) travelled substantially greater mean daily distances than domestic horses (7.2 km/day in 16-ha paddock), even when allowing for larger paddock size. Conclusions Horses kept in stables or small yards and paddocks are quite sedentary in comparison with their feral relatives. For a given paddock area, most designs did not significantly affect mean daily distance travelled.