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A Sperm Cytoskeletal Protein That Signals Oocyte Meiotic Maturation and Ovulation

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Science  16 Mar 2001:
Vol. 291, Issue 5511, pp. 2144-2147
DOI: 10.1126/science.1057586

Abstract

Caenorhabditis elegans oocytes, like those of most animals, arrest during meiotic prophase. Sperm promote the resumption of meiosis (maturation) and contraction of smooth muscle–like gonadal sheath cells, which are required for ovulation. We show that the major sperm cytoskeletal protein (MSP) is a bipartite signal for oocyte maturation and sheath contraction. MSP also functions in sperm locomotion, playing a role analogous to actin. Thus, during evolution, MSP has acquired extracellular signaling and intracellular cytoskeletal functions for reproduction. Proteins with MSP-like domains are found in plants, fungi, and other animals, suggesting that related signaling functions may exist in other phyla.

In sexually reproducing metazoans, oocyte meiotic cell cycle progression is coordinated with ovulation and fertilization to ensure fusion of haploid gamete nuclei. In many animals, sperm trigger the resumption of meiosis in arrested oocytes, but the underlying mechanisms are not clear. During C. elegans reproduction, sperm promote oocyte meiotic maturation (M-phase entry) and gonadal sheath cell contraction, which act in concert to facilitate ovulation (1). Fertilization then occurs as ovulating oocytes enter a sperm storage compartment called the spermatheca (Fig. 1A).Caenorhabditis elegans sperm are separated from oocytes and sheath cells by a valve-like constriction of the distal spermatheca. Therefore, we reasoned that sperm likely secrete factors that promote both oocyte maturation and sheath contraction. To identify the sperm signals, we developed an in vivo bioassay by microinjecting sperm-conditioned medium (SCM) (2) into the uterus offog-2(q71) females (3) (Fig. 1A). The oocyte maturation and sheath contraction rates are very low in these mutants (1), which lack sperm due to a defect in germline sex determination (3). Microinjection of SCM intofog-2 females causes robust increases in the oocyte maturation and sheath cell contraction rates, as visualized by time-lapse video microscopy [Web movies (4)]. Sheath cells also respond with an increased contraction intensity, as measured by their lateral displacement. No activity is observed after the microinjection of bacterial extracts, female extracts, 1-methyladenine, serotonin, oxytocin, or M9 buffer.

Figure 1

Purification of the sperm signal. (A) Microinjection of SCM into the uterus of fog-2 females. The gonad has two arms, and the germ line is shown in the anterior arm on the left, with the sheath encasing the germ line shown on the right. (B) SDS-PAGE analysis of lysed sperm (lane 1) and SCM (lane 2) showing enrichment of ∼15-kD protein in SCM. (C) HPLC fractionation of SCM using C4 and C18 columns. The oocyte maturation- and sheath contraction-inducing activities co-purified in a single peak of activity. (D) Mass spectrometry. The active fractions contained two peaks, a singly charged species (MH+, MW = 14,121 ± 1 dalton) and a doubly charged species (MH2 2+). The inset shows a higher resolution view of the MH+ peak, which consisted of a MW = 14,121 ± 1 dalton peak and a 14,1475 ± 1 dalton peak. Peak 1 corresponds to MSP-142 whereas peak 2 corresponds to MSP-3.

The bioactive factors present in SCM are heat-resistant (100°C, 20 min) and sensitive to proteinase K digestion, suggesting that they are proteinaceous. Comparison of SCM to sperm lysates by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) indicates that SCM is highly enriched with a single protein (Fig. 1B). The bioactive factors were purified with reversed-phase high-performance liquid chromatography (HPLC) using C4 and C18 columns (5). Collected fractions were dialyzed in M9 buffer and assayed individually. Single peaks of maturation- and contraction-inducing activity elute from both columns (Fig. 1C). The identical separation characteristics of both activities on two columns with gradient elution indicates that both activities are likely contained in the same protein or protein complex. MALDI-TOF mass spectrometry shows that a polypeptide of 14,121 ± 1 dalton is present in the active fractions (Fig. 1D). Tryptic peptide mapping and sequencing representative fragments with post source decay mass spectrometry identify the bioactive polypeptide as the major sperm protein (MSP) (6). Caenorhabditis elegansMSP variants differing by one to four amino acids are encoded by a multigene family of approximately 40 genes (7). Closer analysis of the mass spectra reveals that several isoforms with similar molecular weights are present. Two of the major peaks match the calculated molecular weights of MSP-3 and MSP-142 (Fig. 1D). Nanomolar concentrations of SCM-purified MSP cause dramatic increases in the oocyte maturation and sheath cell contraction rates when microinjected into the uterus of fog-2 females (Fig. 2). MSP, purified from sperm lysed with glass beads, produces identical signaling results and is indistinguishable from SCM-purified MSP by MALDI-TOF (8).

Figure 2

MSP promotes oocyte maturation and sheath cell contraction. Caenorhabditis elegans fog-2(q71) orC. remanei females injected with MSP purified from sperm (Sperm MSP), 6His-MSP-77, and 6His-MSP-38 (two MSP isoforms differing by three amino acids) at different concentrations, or a buffer control. (A) Oocyte maturation rate per gonad arm. (B) Basal sheath contraction rate. (C) Maximal displacement of the sheath during contractions. Error bars indicate SD. The standard deviation of 100 nM sperm-purified MSP in (C) is 3.0. The values indicated by asterisks are significantly different (P< 0.01) than the buffer control by a two-sample t test. Values for n indicate the number analyzed. See Web video (4) for representative results.

To verify that MSP is the signal for oocyte maturation and sheath contraction, we expressed and purified two MSP isoforms, MSP-77 and MSP-38, from Escherichia coli (9). Both isoforms, which differ by three amino acids, promote oocyte maturation and sheath contraction at rates equivalent to MSP purified from sperm (Fig. 2). The optimal injected MSP concentration in the bioassay is 100 nM (Fig. 2, A through C), although the effective concentration in the female reproductive tract is likely much less because of dilution. Tonic sheath cell hypercontraction often results when concentrations of 200 or 400 nM are injected (Fig. 2B).

To test the hypothesis that MSP is the endogenous signal in wild-type animals, we injected antibodies to MSP into the uterus of adult hermaphrodites (10). This results in a reduction in the ovulation rate relative to control antibody injections [1.1 ± 0.3 ovulations per gonad arm per hour (n = 28) for antibodies to MSP versus 2.2 ± 0.3 ovulations per gonad arm per hour (n = 31) for control antibodies; P< 0.001]. Analysis of injected hermaphrodites using video microscopy indicates that the ovulation defect is due to a reduction in the oocyte maturation rate.

Sensing sperm availability is critical for reproduction in male-female nematode species, like Caenorhabditis remanei. The signal for ovulation in the genus Caenorhabditis is evolutionarily conserved because sperm from one species can promote ovulation in other species (11). Therefore, we tested whether C. elegans MSP could promote oocyte maturation and sheath contraction in C. remanei females. The injection of MSP-77 causes significant increases in the oocyte maturation and sheath contraction rates relative to the injection of buffer alone (Fig. 2).

These results strongly support the hypothesis that MSP is the signal that facilitates ovulation in C. elegans by promoting both oocyte maturation and sheath cell contraction. To determine how MSP signals these two responses, we analyzed MSP deletion mutants. The COOH-terminal 20 amino acids of MSP are highly conserved among diverse nematodes (Fig. 3A). An MSP mutant lacking this COOH-terminal region [MSP(1 to 106)] promotes oocyte maturation, but not sheath cell contraction (Fig. 3, B through D). By contrast, a peptide (12) corresponding to the conserved COOH-terminal region [MSP(106 to 126)] promotes sheath cell contraction, but not oocyte maturation (Fig. 3, B through D). These results suggest that MSP has two separable signaling functions and likely activates distinct signal transduction pathways in oocytes and sheath cells.

Figure 3

Signaling activity of MSP deletion mutants. (A) Alignment of COOH-terminal MSP sequences (residues 106 to 126) from Ascaris suum (As) (GenBank accession numbers P27439 and P27440), the potato cyst nematodeGlobodera rostochiensis (Gr) (P53021, P53022, and AAA29148),C. elegans (Ce) (P53017 and P53019), and Onchocerca volvulus (Ov) (P13262 and P13263), which causes river blindness. (B through D) Caenorhabditis elegans fog-2(q71) females were injected with 100 nM MSP purified from sperm (Sperm MSP), 100 nM 6His-MSP-77, 100 nM MSP(1 to 106), 80 nM MSP- (106 to 126), or a buffer control. (B) Oocyte maturation rate per gonad arm. (C) Basal sheath contraction rate. (D) Maximal displacement of the sheath during contractions. Error bars indicate standard deviation. The standard deviation of 100 nM sperm-purified MSP in (D) is 3.0. The values indicated by asterisks are significantly different (P < 0.01) than the buffer control by a two-samplet test. Values for n indicate the number analyzed.

Mitogen-activated protein kinase (MAPK) activation is a critical biochemical step in oocyte maturation in vertebrates (13). To determine if MAPK is activated in C. elegans oocytes during maturation, we stained dissected gonadal preparations of mated and unmated fog-2 females with a monoclonal antibody against the activated, diphosphorylated form of MAPK (MAPK-YT) (14). This antibody stains the two to three most proximal oocytes only in the presence of sperm (Fig. 4, A and C). The MAPK-YT antibody only recognizes mpk-1 MAP kinase gene products because no staining is observed in gonads from mpk-1(ga117) homozygotes (Fig. 4, I and J), a likely protein null (15). To determine whether MSP signaling is sufficient to activate MAP kinase in oocytes, we injected MSP into the uterus of females and stained with the MAPK-YT antibody (Fig. 4, E and G). This results in MAPK-YT staining in oocytes, indicating that MSP signaling activates the conserved MAP kinase cascade.

Figure 4

MSP activates MAP kinase in oocytes. Activated MAP kinase (red) was observed in females that were mated (A) or injected with 100 nM 6-His-MSP-77 (Eand G), but not in unmated females, which lack sperm (C). No staining is observed in mpk-1(ga117)hermaphrodites (I). DNA (blue) was visualized with 4′,6′-diamidino-2-phenylindole (DAPI) (B,D, F, H, and J). Arrowheads indicate oocytes that stain strongly and arrows indicate weakly staining oocytes. Proximal is to the right.

X-ray crystallography reveals that MSP folds into an immunoglobulin-like seven-stranded β sandwich, termed here the MSP domain, which is structurally related to the NH2-terminal domain of the bacterial chaperonin, PapD (16). BLAST searches indicate that transmembrane proteins containing NH2-terminal MSP-like domains are found in fungi, plants, and animals. These proteins, which are called VAMP-associated proteins (VAPs), appear to have diverse functions in somatic cells, including neurotransmitter release (17) and vesicle transport (18). The role of VAPs in germ cells is not known, but solubleN-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins, which can interact with VAPs during neurosecretion (17), have been implicated in fertilization in mammals (19) and sea urchins (20). To infer the evolutionary history of the sperm-specific MSPs and the more broadly expressed VAPs, we performed phylogenetic analyses using distance and parsimony methods. These results indicate that the VAP and MSP families form statistically well-supported, distinct monophyletic groups [Web fig. 1, (4)], suggesting that the MSP domain predates nematodes. However, further interpretation of these phylogenetic analyses is complicated by two considerations. First, clear MSP orthologs have not been identified outside of nematodes. Second, some proteins involved in sexual reproduction have been observed to undergo rapid evolution (21), which could eclipse evolutionary relationships. Thus, the current data do not allow us to distinguish between two alternative models. One possibility is that MSP arose from VAP during the evolution of the nematode reproductive system and diverged, acquiring one or multiple new functions. Alternatively, MSP was present in the common ancestor of many animals and was lost in some lineages or remains to be identified. MSP signaling functions could be derived and unique to MSP, or ancestral and shared among MSP and some VAP homologs. In any event, the exceptionally high degree of conservation in nematodes makes MSP an attractive anti-helminthic drug target.

Nematode sperm use a pseudopod to move over short distances by crawling (22). MSP is the most abundant protein in sperm (23) and forms self-assembling filaments in the pseudopod (24). Unlike flagellar sperm found in many animals, nematode sperm contain essentially no actin (25) and crawling is thought to be dependent on MSP function (26). The mechanism by which MSP signals are delivered to oocytes and sheath cells is not currently understood and may be novel. MSP does not have a signal sequence nor do C. elegans sperm have ribosomes, an endoplasmic reticulum, or a Golgi system (22). Pseudopod formation is not required for MSP signaling because spe-4 spermatocytes (27), which fail to form pseudopods, are still capable of promoting oocyte maturation and sheath contraction (1).

These results, taken together with previous studies (24), indicate that MSP has acquired both extracellular signaling and intracellular cytoskeletal functions during evolution. MSP appears to perform these functions by mediating multiple protein-protein interactions using its single immunoglobulin-like fold (16, 28). The presence of MSP-like domains in yeast, plants, and animals suggests that some of these functions have been conserved during the evolution of multicellular organisms.

  • * To whom correspondence should be addressed. E-mail: david.greenstein{at}mcmail.vanderbilt.edu

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