QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 23 June 2005
doi: 10.1242/dev.01916

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kosinski, M. Articles by Greenstein, D. Search for Related Content PubMed PubMed Citation Articles by Kosinski, M. Articles by Greenstein, D.

C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes

¹ Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, 465 21st Avenue South, Nashville, TN 37232, USA
² Electron Microscopy Laboratory, University of California, Berkeley, 26 Giannini Hall, Berkeley, CA 94720-3330, USA
³ Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 465 21st Avenue South, Nashville, TN 37232, USA

View larger version (73K): [in a new window]   Fig. 1. Anatomy of MSP signaling. (A) Diagram of the hermaphrodite reproductive tract. Oocytes undergo meiotic maturation in an assembly line fashion in response to MSP signaling. At ovulation, the distal constriction of the spermatheca dilates, the oocyte enters and is fertilized. (B) Electron micrograph of the spermatheca. Spermatozoa are unable to enter the proximal gonad because the constriction of the distal spermatheca provides a barrier. Some spermatozoa enter the uterus with embryos, and must then crawl back into the spermatheca so they can fertilize oocytes. (C) Spermiogenesis is the process during which non-motile spermatids become fertilization-competent motile spermatozoa with a pseudopod. Spermiogenesis occurs when spermatids enter the spermatheca during the first ovulation in hermaphrodites, or as they enter the uterus during mating. In A, a few spermatids are shown remaining in the gonad arm, as is typically seen on the first two days of adulthood. These spermatids will enter the spermatheca in the next few ovulations.

View larger version (68K): [in a new window]   Fig. 2. Evidence that spermatozoa release MSP. (A) Western blot. Males and mated females contain MSP, but unmated females do not. Minor N- and C-terminal fragments (*N and *C) result from scission of MSP during the boiling step of lysate preparation (data not shown). The male lysate was overexposed to visualize *N (center lane, 15 seconds exposure time). (B,C) Detection of MSP (red) in the proximal gonad arm of mated females (left panels in B). MSP extends beyond the distal constriction (arrowhead) of the spermatheca (sp). A sharp boundary in staining intensity is observed between the –1 and –2 oocytes (arrow). DNA (blue) is shown in the merged images (lower panels in B). No MSP staining is seen in unmated females (right panels in B). The unmated control was overexposed to visualize the outline of the gonad. The relative fluorescence intensity of the MSP signal is shown in C. (D) The distance that the MSP signal extends from spermatozoa in mated females (*P<0.001, error bars represent s.d.). (E) The relative intensity of the MSP signal (fold above background) in the proximal gonad. *P<0.02, when compared to all the other measurements shown. P>0.15, when compared with the other emo-1(oz1) mated female values, but P<0.05, when compared with the unmated female controls. (F) Punctate distribution of extracellular MSP. Projections of confocal 3D data stacks from mated females prepared by gonad dissection (left panel, MSP is red) or whole-mount fixation (right panel, MSP is pink and DNA is red). Large MSP puncta (arrows) are outside spermatozoa (s) in both the spermatheca (left panel, sp) and the uterus (u). More diffuse MSP fills the spermatheca (left panel) and extracellular spaces surrounding embryos (e, right panel). No MSP is observed in the distal gonad (dg). (G) MSP puncta (arrows) in close proximity to spermatozoa (s) in the uterus, detected by wide-field microscopy. Note the extended pseudopod (ps, bottom panels) and the sperm DNA (blue). Scale bars: B,C, 10 µm; F, 5 µm; G, 10 µm.

View larger version (47K): [in a new window]   Fig. 3. Localization of exported MSP and specificity of release. (A) Localization of MSP at the surface of the –1 oocyte. Single confocal sections at the indicated level of a 3D data stack through a mated female gonad stained for MSP (red) and RME-2 yolk receptor (green). No spermatozoa were seen in the indicated region (line) of the spermatheca (sp), thus the staining observed is extracellular to spermatozoa. A projection of the entire stack is presented as Movie 3 in the supplementary material. (B) Single angle views of a 3D reconstruction of the data stack represented in A. The image is cut to show surface and interior views of the –1 oocyte, at the indicated angles. Overlap between the MSP (red) and RME-2 (green) signals is yellow. Note the oocyte surface is slightly compressed where it abuts the spermatheca. The entire reconstruction is presented as Movie 4 in the supplementary material. (C) Intact and viable spermatozoa release MSP, as shown by a mated female stained for DNA (blue), MitoTracker (red) and MSP (green). Note, the MitoTracker staining is limited to the spermatozoa (s), but the MSP staining extends at least 50 µm from the most distal spermatozoa (arrowhead). (D) MSP localizes to extracellular puncta and apparent buds at the spermatozoa surface. Projections of confocal 3D data stacks from mated females stained in wholemount for MSP (red) and MSD proteins (green), with overlap in yellow. Images are superimposed on the DIC channel, showing spermatozoa (s) in the uterus. Puncta (arrows) and surface blebs (arrowheads) contain MSP, but not MSD proteins. A 3D projection of similar data is presented in Movie 5 in the supplementary material. (E) Budding generates MSP puncta. Single angle views of a 3D reconstruction of MSP (red) and MSD (green) staining, with overlap in yellow. The image is cut to show interior and surface views of the spermatozoa. Apparent sites of budding contain MSP but not MSD. The entire reconstruction is presented as Movie 6 in the supplementary material. Scale bars: 20 µm.

View larger version (171K): [in a new window]   Fig. 4. Detection of a new class of vesicle by electron microscopy. (A) Low-power views of vesicles (arrows) in the extracellular space near spermatozoa (s) in the spermatheca (left), in the spermathecal-uterine junction region (middle), and in an extracellular space of the uterus formed by close packing of embryos (e; right). Pseudopods (ps) and an apical junction between spermathecal cells (arrowhead) are indicated. (B) Serial-section analysis of two vesicles in the spermatheca. Inset is a magnified view. Sections are 75 nm thick. (C) The vesicles possess two concentric lipid bilayers. Vesicles were tilted through the indicated angles in the EM beam to visualize the individual leaflets of the inner and outer membranes. (D) Tilting of a vesicle to visualize its scalloped appearance. (E) No MSP vesicles are observed in the spermathecal lumen (sl) of a fog-2(q71) female, instead the lumen is filled with material that resembles yolk lipoprotein particles. Scale bars: in A, 500 nm; in B, 125 nm; in C,D, 100 nm; in E, 500 nm.

View larger version (140K): [in a new window]   Fig. 5. Vesicles contain MSP and form by budding. (A) Detection of MSP by immunoEM. Low-power view of two spermatozoa in the spermathecal-uterine junction region of an adult hermaphrodite. Intense labeling in pseudopods (ps). (B) Detection of MSP at the plasma membrane of the spermatozoa cell body. (Left) MSP labeling excluded from cellular organelles, including mitochondria (m), membranous organelles (mo), and the nucleus (n). Inset is a magnified view showing MSP associated with the plasma membrane. (Middle) MSP associated with the plasma membrane and a protrusion (arrow), magnified in the inset. Note free labeling in the extracellular space (bracket). (Right) MSP is not detected in distal germ cells. (C) MSP is contained within the vesicles; a gallery of seven vesicles (the lower right two panels are views of the same vesicle in non-adjacent sections) is shown. MSP is located in the annulus between the inner and outer membranes. (D) Vesicle budding from spermatids. Shown are non-adjacent sections of two different spermatids in the hermaphrodite gonad. Views 1 and 1', and 2 and 2', are corresponding pairs of non-adjacent sections. The budding vesicles contain MSP in both views. Vesicles connect to the cell body by a stalk (thin arrows), and the plasma membrane at the budding site appears to be intact (thick arrow). MSP is enriched in a cross-sectional view at the base of the budding projection (arrowheads in lower panels). (E) Section of epon-embedded material showing a lipid whorl deposit in an extracellular space of the spermatheca between a portion of two spermatozoa. (F) MSP associated with a lipid whorl structure in the extracellular space of the spermatheca. Scale bar: in A, 500 nm; in B, 500 nm (inset, 125 nm); in C,D, 100 nm; in E, 500 nm; in F, 100 nm.

View larger version (55K): [in a new window]   Fig. 6. Spermatids provide a long-acting MSP signal. (A) Time-course analysis of MAPK activation in the wild type and spe-8(hc50) mutants. The percentage of gonad arms with activated MAPK was measured by staining dissected gonads at the indicated times of adulthood. (B) Time course of total MSP levels analysed by western blots of MSP in spe-8(hc50) and the wild type (10 animals/lane). The number of spermatids and spermatozoa were counted at each time point. The data represents the average of three trials. (C) Detection of MSP puncta (arrows) located near spermatids (sd) in the proximal gonad arm of spe-8(hc50) and wild-type hermaphrodites. MSP (red) and DNA (blue) were detected. MSP is detected in the spermatheca (sp) of spe-8(hc50) hermaphrodites, but no spermatids are observed. (D) Projection of a confocal 3D-data stack showing MSP puncta (arrow) distributed widely in the proximal gonad, far from the single spe-8(hc50) spermatid (sd) that can be seen. (E) MSP perdures in spe-8(hc50) mutants. MSP (red) staining is observed, but spermatids are not, confirmed by viewing the DNA (blue) signal in multiple focal planes. (F) Western blot of MSP in C. elegans (C.e), and the Cephalobid nematodes Acrobeloides maximus (A.m) and Zeldia punctata (Z.p). (G) Detection of MSP puncta (arrows) in the A. maximus gonad. Only the distal arm is shown. Scale bars: in C, 20 µm; in D, 10 µm; in E,G, 20 µm.

View larger version (169K): [in a new window]   Fig. 7. Production of MSP vesicles in the spermiogenesis-defective spe-8(hc50) mutant. (A) Low-power view of the spermathecal-uterine junction region. MSP vesicles (arrows) are abundant in extracellular spaces of this region. (B) High-magnfication view of MSP vesicles located in the region shown in A, from an adjacent section. (C) MSP vesicle in the spermathecal lumen surrounded by cytoplasmic debris from a lysed oocyte (arrowheads). Note, the inner ring of the MSP vesicle contains material (arrows) similar to the oocyte cytoplasmic contents. (D-I) Protrusions (arrows) from the cell body of spermatids located in the gonad arm. (F,I) Detection of MSP in protrusions. Scale bars: in A, 500 nm; in B-I, 100 nm.