Circadian rhythm of acidification in insect vas deferens regulated by rhythmic expression of vacuolar H+-ATPase
Piotr Bebas1,2,
Bronislaw Cymborowski2 and
Jadwiga M. Giebultowicz1,*
1 Department of Entomology and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR 97330, USA and
2 Department of Invertebrate Physiology, Warsaw University, 02-096 Warsaw, Poland
View larger version (24K):
[in a new window]
|
Fig. 1. Schematic representation of the rhythm of sperm release in moths. Sperm bundles (SB) descend from the testis (T) into the upper vas deferens (UVD) during a period of a few hours before and after lights-off. Sperm bundles are retained in the UVD lumen during the night and then transferred to seminal vesicles (SV) within a few hours after lights-on. During each daily cycle, the sperm bundles closest to the base of the testis are released in this stepwise fashion. Black and white bars represent, respectively, the dark and light phases of the 16 h:8 h L:D cycle.
|
|
View larger version (13K):
[in a new window]
|
Fig. 2. Pattern of pH changes in the upper vas deferens (UVD) lumen of Spodoptera littoralis. (A) Under a 16 h:8 h L:D cycle, rhythmic changes are detected in the luminal pH, with significant acidification of the UVD occurring in the middle of the dark phase. (B) The robust pH rhythm continues on the second day in constant darkness. (C) The rhythm is abolished in constant light. Each data point represents the pH measured in 10 individuals. Values are means ± S.E.M. The horizontal bars represent the day (open), night (filled) and former day (hatched) portions of the photo-regime.
|
|
View larger version (93K):
[in a new window]
|
Fig. 3. Expression of the B-subunit of V-ATPase in the vas deferens of Spodoptera littoralis. (A) Western blot analysis of B-subunit levels (arrow) in the upper vas deferens (UVD) during a 16 h:8 h L:D cycle (LD) at the Zeitgeber times indicated above the blot. The protein levels (arrow) show daily oscillations. The lane marked 20C was treated with antigen-preabsorbed antibody. Non-specific protein (arrowhead) served as a loading control. (B) UVDs from moths kept in constant darkness (DD) maintained daily oscillations in B-subunit levels, with a lower level during former daytime at circadian time 8 (Ct8) and a higher level during former nighttime at Ct20. Western blots were repeated three times with similar results. (C–E) Immunostaining for the B-subunit of V-ATPase in longitudinal sections of UVDs dissected every 4 h in LD (C), DD (D) or constant light (LL) conditions (E). Each picture shows representative columnar epithelial cells with their apical sides on the left and blue-gray elongated nuclei. Red V-ATPase signals appear to be concentrated in small vesicle-like structures. A robust increase in the amount of V-ATPase at Zt16–20 is evident in LD and in the second cycle in DD conditions; however, the amount of protein does not show a daily rhythm in LL conditions. The experiment was performed on six males per time point in each photoregime with similar results. No staining was detected in control tissues treated with antigen-preabsorbed antibody. Scale bar, 20 µm. Horizontal bars represent day (open), night (filled) and former day (hatched) portions of the photo-regime.
|
|
View larger version (79K):
[in a new window]
|
Fig. 4. Localization of V-ATPase in the upper vas deferens (UVD) of intact, solvent-injected and colchicine-injected moths. Injections were delivered at Zeitgeber time 8 (Zt8), when moths have low levels of V-ATPase. Colchicine inhibited apical accumulation of V-ATPase in UVDs dissected at Zt12 and Zt16. V-ATPase was detected with an antibody against its B-subunit followed by Texas-Red-conjugated secondary antibody. The images shown are representative of six UVDs processed after each treatment at each time point. Scale bar, 20 µm.
|
|
View larger version (14K):
[in a new window]
|
Fig. 5. Effects of colchicine on luminal pH. The columns show luminal pH in the upper vas deferens (UVD) of intact moths at Zeitgeber time 8 (Zt8-I), and in moths that were injected at Zt8 and evaluated at Zt12 after colchicine injection (Zt12-C) or solvent injection (Zt12-S). pH remained high in colchicine-injected moths, but declined significantly (Student’s t-test; P<0.05) in solvent-injected moths. Each column represents the pH (mean ± S.E.M.) for 5–6 individuals per treatment.
|
|
View larger version (14K):
[in a new window]
|
Fig. 6. Effects of bafilomycin (BA) on the pH in the lumen of the upper vas deferens (UVD). UVDs were injected at Zeitgeber time 8 (Zt8) with bafilomycin or 6 % dimethylsulfoxide (Solv.) or left untreated (Intact). The pH was measured at Zt8 in groups of control moths and at Zt16 in injected moths. Bafilomycin prevented the decline in pH relative to the beginning of the experiment at Zt8, while solvent-injected and intact UVDs showed a statistically significant (Student’s t-test; P<0.01) decline in pH. Each column represents the pH (mean ± S.E.M.) for eight individuals per treatment.
|
|
© The Company of Biologists Ltd 2002
