Use of phosphatidylcholine to improve the function of turkey semen stored at 4°C for 24 hours.

The ability to store turkey semen for 24 h in vitro without a significant loss in fertility would benefit the commercial turkey industry. We investigated the use of exogenous phosphatidylcholine to improve the viability, mobility, hydrolyzing ability, and fertility of stored turkey sperm. For experiment 1, semen was diluted 1:1 with extender containing 0.0 (control), 0.5, 2.5, or 10.0 mg/mL of phosphatidylcholine labeled with a fluorochrome and maintained under aerobic conditions for 24 h at 4°C. Semen aliquots were removed at 30-min intervals during the first 4 h and at 1-h intervals from 8 to 24 h of storage for fluorometric evaluation by flow cytometry. Turkey sperm incorporated labeled phosphatidylcholine in a dose-dependent manner during the first 12 h of storage (P<0.05). At 24 h of storage, phosphatidylcholine uptake increased 7.8-fold for the 0.5-mg treatment, 9.2-fold for the 2.5-mg treatment, and 6.7-fold for the 10-mg labeled phosphatidylcholine treatment. For experiment 2, turkey semen was diluted and stored as for experiment 1 except the phosphatidylcholine was unlabeled. After 24 h, the viability, mobility, hydrolyzing ability, and fertility of turkey sperm was assessed. Supplemental phosphatidylcholine did not improve (P>0.05) the viability or mobility of stored sperm. At 2.5 mg/mL, phosphatidylcholine improved the hydrolyzing ability of stored sperm compared with control or other phosphatidylcholine treatments (P<0.05). The mean fertility rate of eggs from hens inseminated with control semen was 33.5%±4.5. Use of 0.5, 2.5, or 10.0 mg phosphatidylcholine/mL improved (P<0.05) the fertility rates during the first 11 wk of egg production; higher fertility rates occurred with 2.5 mg phosphatidylcholine/mL compared with other phosphatidylcholine treatments for 5 of those 11 wk (P<0.05). We conclude that supplemental phosphatidylcholine appears to counteract the damaging effects of lipid peroxidation or enzymatic degradation during in vitro storage by providing exogenous phospholipids for incorporation into the turkey sperm plasma membrane.

Vibrio cholerae ACE stimulates Ca(2+)-dependent Cl(-)/HCO(3)(-) secretion in T84 cells in vitro.

ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current (I(sc)) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in I(sc) and potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl(-) secretory cell. ACE added to the apical surface alone stimulated a rapid increase in I(sc) and PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl(-) and HCO(3)(-). ACE acted synergistically with the Ca(2+)-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca(2+) but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca(2+) as a second messenger and that this toxin stimulates a novel Ca(2+)-dependent synergy.

Production of Vibrio cholerae accessory cholera enterotoxin (Ace) in the yeast Pichia pastoris.

Accessory cholera enterotoxin (Ace) is a recently identified toxin of Vibrio cholerae. Preliminary studies using crude toxin extracts in animal models indicate that Ace increases transcellular ion transport, which is proposed to contribute to diarrhea in cholera. The lack of purified toxin has hindered elucidation of the mechanism of action of Ace. In this study, ace was cloned and was expressed in and secreted by the methylotrophic yeast Pichia pastoris. Secreted toxin constituted 50% of the total supernatant protein from Pichia pastoris. Presumed monomer and dimer forms with molecular masses of 9 and 18 kDa, respectively, were observed. The 18-kDa form predominated. Biological activity was assayed by studying ion fluxes across epithelial membranes in Ussing chambers. Among the characteristics of Ace was the unusual property of staining with silver but not Coomassie blue stain. To our knowledge this is the first report of a biologically active bacterial toxin produced with the P. pastoris system. The purified protein may now be used in studies of the mechanism of action of Ace in physiologic systems.