West Nile virus envelope protein: role in diagnosis and immunity.

The role of antibodies to the West Nile virus envelope (E) protein in serodiagnosis and protection was examined. The E protein was expressed and purified in recombinant form. Antibodies to the E protein were detected in patients with West Nile virus infection. Passive immunization with rabbit anti-E protein sera also partially protected mice from challenge with West Nile virus. The humoral response to the West Nile virus E protein is therefore useful as an aid in the diagnosis and may also play a role in immunity to infection.

Establishment and characterization of a caprine mammary epithelial cell line (CMEC).

We describe the establishment of a continuous, nontransformed cell line obtained from primary culture of a lactating (114 days postparturition) Anglo-Nubian (Capra hircus) goat mammary gland biopsy. These cells (CMEC), have been cultured in the presence of supraphysiologic concentrations of insulin and hydrocortisone for more than 560 population doublings (over 80 passages) without any sign of senescence while maintaining a normal/near-normal diploid chromosome modal number of 2n = 60 and are responsive to contact inhibition of proliferation. Cytoskeletal analysis indicates that CMECs are epithelial, without detectable fibroblastic or myoepithelial cells. When grown at low density on plastic substratum, the cells tend to form island monolayer aggregates with the characteristic cobblestone morphology of epithelial cells. With increasing density, the cells organize into lumen-like structures with various morphology consisting of large and small vacuolized and nonvacuolized cells. Postconfluent cultures form epithelial raised dome-like structures, implying a process of contact-induced differentiation. This is corroborated by positive immunocytochemistry to lactation-specific proteins: beta-casein and alpha-lactalbumin, which were predominantly expressed in dome-forming cells. We also observed an overall modulation of cytokeratin 18/19 expression associated with number of days post subculture and with the expression of lactation-specific proteins. Postconfluent cultures which contain lactation-specific, antibody-reactive, dome-like structures showed a decreased expression of keratin 18 and no (null) expression for keratin 19. Lastly, cells cultured within a collagen matrix show morphological differentiation with the organization of branching duct-like and acini-like structures. This study suggests that CMECs are a useful in vitro model for study of mammary gland development and differentiation, in particular, direct modulation of epithelial cells grown on plastic substratum or extracellular matrix without the influence of stromal elements or the necessity and variability associated with primary cell culture or tissue explants.

Bovine corpus luteum function after removal of granulosa cells from the preovulatory follicle.

Experiments were conducted to determine the effects of removing granulosa cells from bovine preovulatory follicles on subsequent corpus luteum (CL) function. Holstein heifers were assigned to three groups: untreated controls (n = 6), removal of granulosa cells (n = 9) and removal and return of granulosa cells (n = 7). Surgery was performed 18-24 hr after the onset of estrus and in all cases after the preovulatory luteinizing hormone (LH) surge. Jugular venous blood was collected and estrous activity monitored twice daily. Corpora lutea were formed in six heifers in each group. Concentrations of plasma progesterone were reduced (P less than 0.05) on Days 7-17 in heifers from which granulosa cells were removed when compared to the other two groups. There were no differences in the lengths of the estrous cycles nor concentrations of LH in the three groups. In summary, these experiments support the concept that granulosa cells make a substantial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. These experiments also establish the fact that granulosa cells develop into functional luteal cells after their removal and return to the preovulatory follicle.

Effect of dietary monensin on ovarian response following gonadotropin treatment in prepuberal heifers.

Twenty prepuberal Charolais X Brahman-Hereford heifers were randomly assigned to be fed a concentrate containing either 0 mg (C) or 200 mg (M) monensin sodium/head/day. Coastal bermudagrass hay was fed ad libitum. Average daily gain was similar for the two groups. Each heifer received 1 mg of porcine follicle stimulating hormone (FSH-P) (Armour) at 0800 and 2000 hr on days 22 through 26 (10 mg total) and 2,500 IU human chorionic gonadotropin (HCG) on day 27. Flank laparotomy was performed on day 30, for examination of ovaries, and ovariectomy was performed on day 37. The average ovarian size +/- standard error at day 15 ws 3,730 +/- 66 mm3 and 1,848 +/- 55 mm3 for groups M and C, respectively (P < .025), as measured by rectal palpation. Numbers of ovulation sites measured on day 30 were 9.1 +/- 2.2 and 4.9 +/- 1.8 per heifer for groups M and C, respectively (P < .01). After ovariectomy on day 37, heifers fed M were found to have greater ovarian weight (P < .05), more corpora lutea (CL) (P < .05), greater total luteal weight (P < .05), more follicles (P < .01) and greater weight of follicular fluid (P < .05) and stroma (P < .025) than controls. CL were analyzed for progesterone content by spectrophotometric procedures. Heifers fed M had slightly larger CL (P < .10) with progesterone concentrations similar to those in CL from controls. This resulted in more luteal progesterone per CL and more luteal progesterone per heifer in the M heifers than in the controls. Prepuberal heifers fed M, which caused the expected shifts in rumen fermentation and volatile fatty acid production, exhibited an enhanced ovarian response to gonadotropins compared to that exhibited by controls.

Long chain polyunsaturated fatty acids and bovine luteal cell function.

Experiments were conducted to determine the effects of exogenous polyunsaturated fatty acids (PUFA) on the production of progesterone and prostanoids by dispersed bovine luteal cells and to characterize endogenous luteal fatty acids throughout the estrous cycle. The addition of eicosapentaenoic acid (20:5, n3) resulted in a dose-dependent reduction of progesterone production and an increase in production of prostaglandin (PG) F2 alpha (PGF2 alpha) (p < 0.05). Nordihydroguaiaretic acid abolished the inhibitory effects of 20:5, n3 on progesterone production, while indomethacin did not alter these effects. The addition of 10 micrograms docosahexaenoic acid (22:6, n3) resulted in a suppression of progesterone synthesis (p < 0.05) and reduced PGF2 alpha synthesis. The addition of 0.1, 1, and 10 micrograms docosatetraenoic acid (22:4, n6) inhibited basal progesterone production, whereas only the highest dose decreased LH-stimulated synthesis of progesterone. The addition of 22:4, n6 resulted in increased PGF2 alpha synthesis (p < 0.05) and in lowered synthesis of prostacyclin (p < 0.05). Variations in luteal fatty acids were confirmed by an experiment in which endogenous fatty acids were characterized throughout the estrous cycle. The percentages and ratios of PUFA were altered throughout the estrous cycle. In summary, PUFA have potent inhibitory effects on the production of progesterone and PGI2 in vitro and may play a role in bovine luteal cell function by mechanisms yet to be determined.

Function and lifespan of corpora lutea in ewes treated with exogenous oxytocin.

Oxytocin was administered to Dorset and Shropshire ewes in one experiment and to Dorset ewes in a further 4 experiments. In Exp. 1, concentrations of plasma progesterone and lengths of the oestrous cycle in ewes given oxytocin subcutaneously twice a day on Days 0-3, 2-5, 4-7, 6-9, 8-11, 10-13, 12-15 or 14-17 were similar to those of control ewes. In Exp. 2, intraluteal infusions of oxytocin from Day 2 to Day 9 after oestrus had no effect on concentration of progesterone, weight of CL collected on Day 9 or length of the oestrous cycle. In Exp. 3, intraluteal infusions of oxytocin on Days 10-15 after oestrus had no effect on weight of CL collected on Day 15. In Exp. 4, s.c. injections of oxytocin on Days 3-6 after oestrus had no effect on weight of CL collected on Day 9, concentrations of progesterone or length of the oestrous cycle. In Exp. 5, s.c. injections of oxytocin twice a day did not affect the maintenance and outcome of pregnancy in lactating and nonlactating ewes. Exogenous oxytocin, therefore, does not appear to affect luteal function at any stage of the ovine oestrous cycle although oxytocin has been reported by others to alter ovine CL function.