Figure 4

Enhanced muscle differentiation and increased innervation observed post-mating. (A-F) Ultrastructure of muscle in different regions of oviduct (unmated vs. mated). (A and B) Lateral oviducts: muscle fibers that vary in electron density are observed in this region. Light (M) and dark (*) muscle fibers may represent different stages of differentiation. Bar = 2 μm. Epithelium (EP); (C and D) Upper common oviduct: Z-bodies (arrows) are more organized in mated (D) than unmated muscles (C). Bar = 0.5 μm. (E and F) Lower oviducts in both unmated and mated: muscle fibers in the lower oviduct are more electron dense than muscle fibers in upper oviduct. An increased density of z-bodies (arrows) and myofilaments that run between the z-bodies was observed. Muscle fibers are surrounded by a well developed basal lamina (arrowheads). Bar = 0.5 μm. (G and H) Confocal images of immunolabeled type I and II boutons innervating the oviduct. Common oviducts of 3-day-old unmated (UM₃) (G) and mated females (M₃) (H), were stained with anti-DLG (green) and anti-HRP (red); Bar = 20 μm. Insets show high magnification of labeled type I (green) and II (red) boutons; bar = 5 μm. (I) Quantitation of number of immunolabeled boutons per unit area in lateral oviducts (LO) and common oviduct (CO). Anti-HRP labels both type I & II boutons, whereas anti-DLG only labels type I boutons. We found a significant increase in the number of HRP labeled boutons (lateral oviducts, p < 2.53E-05; common oviduct, p < 0.005) but not in the number of DLG labeled boutons, indicating that mating induces an increase in type II innervation. As a control, we also included 5-day-old unmated females (UM₅) which showed the same innervation pattern as in UM₃ oviducts. For each treatment oviducts were obtained from 3 biological replicate; in each replicate for each treatment n = 10 oviducts. See also additional file 3.