Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

Freeze fracture and freeze etching.

Freeze fracture depends on the property of frozen tissues or cells, when cracked open, to split along the hydrophobic interior of membranes, thus revealing broad panoramas of membrane interior. These large panoramas reveal the three-dimensional contours of membranes making the methods well suited to studying changes in membrane architecture. Freshly split membrane faces are visualized by platinum or tungsten shadowing and carbon backing to form a replica that is then cleaned of tissue and imaged by TEM. Etching, i.e., removal of ice from the frozen fractured specimen by sublimation prior to shadowing, can also reveal the true surfaces of the membrane as well as the extracellular matrix and cytoskeletal networks that contact the membranes. Since the resolution of detail in the metal replicas formed is 1-2 nm, these methods can also be used to visualize macromolecules or macromolecular assemblies either in situ or displayed on a mica surface. These methods are available for either specimens that have been chemically fixed or specimens that have been rapidly frozen without chemical intervention.

Sperm guidance to the egg finds calcium at the helm.

Sperm respond to multiple cues during guidance to the egg including chemical attractants, temperature, and fluid flow. Of these, sperm chemotaxis has been studied most extensively-over 100 years-but only recently has it started to be understood at the molecular level. The long gestation in this understanding has largely been due to technical limitations that include the detection of calcium signal dynamics in a relatively small structure-the flagellum, measurement of actual chemoattractant gradients, the fact that only subpopulations of sperm respond at any given time, and the diversity in swimming behaviors that sperm exhibit from different species. Today, measurements of flagellar calcium signals on a fast time scale, discovery of the ion channels and organelles that may regulate these signals, and better understanding and quantitation of sperm swimming behaviors involved have given more certainty to our understanding of sperm directional swimming and its control by characteristic, calcium-directed asymmetric flagellar bends. Future research will need to apply these technical advances to other forms of sperm guidance such as thermotaxis and rheotaxis as well as gaining an understanding of how the flagellar apparatus is controlled by calcium.

Allurin, an amphibian sperm chemoattractant having implications for mammalian sperm physiology.

Eggs of many species are surrounded by extracellular coats that emit ligands to which conspecific sperm respond by undergoing chemotaxis and changes in metabolism, motility, and acrosomal status in preparation for fertilization. Here we review methods used to measure sperm chemotaxis and focus on recent studies of allurin, a 21-kDa protein belonging to the Cysteine-RIch Secretory Protein (CRISP) family that has chemoattraction activity for both amphibian and mammalian sperm. Allurin is unique in being the first extensively characterized Crisp protein found in the female reproductive tract and is the product of a newly discovered amphibian gene within a gene cluster that has been largely conserved in mammals. Study of its expression, function, and tertiary structure could lead to new insights in the role of Crisp proteins in sperm physiology.

Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family.

Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm-egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.

Egg jelly proteins stimulate directed motility in Xenopus laevis sperm.

Previously we have shown that extracts from Xenopus egg jelly (egg water) increase the passage of sperm through a porous membrane in a dose-dependent manner. Although this assay has shown that sperm accumulation occurs only in the presence of an egg water gradient, it has not revealed the dynamic features of how Xenopus sperm swim in such gradients. Here, we use video microscopic observations to trace sperm trajectories in a Zigmond chamber. Our results show that Xenopus sperm swim in linear and gently curving paths and only infrequently perform turns. In the presence of an egg water gradient, however, the percent of sperm swimming up the gradient axis and the net distance traveled by each sperm along this axis was increased significantly. There was no change in curvilinear velocity. Rather, the orientation of sperm travel was shifted to more closely match that of the gradient axis. In addition, using a porous filter assay, we demonstrate that the egg water protein allurin, in both purified and recombinant forms, stimulates directed motility of sperm. Finally, we use Oregon Green 488-conjugated allurin to show that this protein binds primarily to the sperm midpiece; binding of allurin to the entire head was observed in a minor subpopulation of sperm. Dose dependence of allurin binding occurred over the 0-1 µg/ml range and correlated well with previously published dose-dependent sperm attraction data. Binding was rapid with a half-time of about 10 sec. These data suggest that egg water proteins bind to sperm and modify sperm-orienting behavior.

Swimming of Xenopus laevis sperm exhibits multiple gears and its duration is extended by egg jelly constituents.

The motility of Xenopus sperm is initiated by the osmotic shock experienced when these cells are ejaculated into low-salinity pond water. Motility is brief and is required for the sperm to penetrate the jelly layers and fertilize the egg. In this study we demonstrate that extracts of egg jelly contain factors that extend the period of sperm motility as well as providing a chemoattractant activity as previously reported. Both activities are partially dependent on extracellular calcium. Time-lapse and video microscopy show that after activation of motility the number of motile sperm decreases rapidly, with a half-time of about 2 min. Addition of 10% v/v egg jelly extract ("egg water") increased the number of motile sperm 2-fold over controls at 20 s and about 4- to 10-fold over controls at 10 min after initiation of motility. Extension of motility lifetime was not mediated by a nonspecific protein or by allurin, the egg-water protein that has chemoattractant activity. The helical path of Xenopus sperm exhibited tight coupling between rotational and forward velocities in egg jelly, but coupling changed rapidly from moment to moment in low-salinity buffer. Our observations suggest that jelly-derived factors regulate both the longevity and directionality of sperm propulsion.

Two types of assays for detecting frog sperm chemoattraction.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm. Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg. Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 µm diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer. The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Purification and multimer formation of allurin, a sperm chemoattractant from Xenopus laevis egg jelly.

Allurin, a sperm chemoattractant isolated from Xenopus laevis egg jelly, can be purified in one step from an extract of diffusible jelly proteins ("egg water") using a FPLC or HPLC anion exchange column and a multi-step NaCl gradient. Allurin homomultimers were detected by Western blotting with antibodies prepared against the purified protein or peptides within the protein. Allurin multimers were stable and resisted dissociation by SDS and beta-mercaptoethanol. Alkylation of allurin provided evidence for two free sulfhydryl groups but did not eliminate multimer formation, suggesting that intermolecular disulfide bond formation is not required for allurin aggregation. Concentration of egg water was accompanied by a reduction of chemoattractant activity that could not be fully accounted for by homomultimer formation. Rather, the presence of a multiphasic dose-activity curve upon partial purification and formation of hetero-allurin complexes during concentration suggested that egg water may contain allurin-binding proteins that reduce multimer formation and activity.

Crisp proteins and sperm chemotaxis: discovery in amphibians and explorations in mammals.

Crisp proteins appear to play multiple roles in the life history of sperm. One of these roles is to act as a sperm chemoattractant. Allurin, a 21 kDa Crisp protein rapidly released from the egg jelly of at least two frogs, X. laevis and X. tropicalis, elicits directed motility in both homospecific and heterospecific sperm. In X. tropicalis, allurin is coded for by the newly documented Crisp A gene. Recently, the observation that allurin can also elicit chemotaxis in mouse sperm raises the question of whether allurin-like proteins might act as sperm chemoattractants in mammals. Although an allurin gene has yet to be documented in mammals, Crisp proteins truncated post-translationally appear to exist in both the male and female reproductive tract of mammals.

Xenopus tropicalis allurin: expression, purification, and characterization of a sperm chemoattractant that exhibits cross-species activity.

Previously we reported the identification of the first vertebrate sperm chemoattractant, allurin, in the frog Xenopus laevis (Xl) and demonstrated that it was a member of the CRISP family of proteins. Here we report identification, purification, and characterization of Xenopus tropicalis (Xt) allurin, a homologous protein in X. tropicalis. "Egg water" as well as purified allurin from both species exhibit efficient cross-species sperm chemoattractant activity. Western blots show that Xt egg water contains a single anti-allurin cross-reactive protein whose molecular weight (20,497 Da by MALDI MS) agrees well with the molecular weight of the hypothetical gene product for a newly recognized "Crisp A" gene in the X. tropicalis genome. A recombinant form of the protein, expressed in 3T3 cells, exhibits chemoattraction for both Xt and Xl sperm and cross reacts with anti-allurin antibodies. Examination of Crisp protein expression in the Xt oviduct using RT-PCR showed that of five documented Xt Crisp genes (Crisps 2, 3, LD1, LD2 and A) only Crisp A was expressed. In contrast, Crisp 2, Crisp 3, Crisp LD1, and Crisp LD2, but not Crisp A, were all found to be expressed in the Xt testes while subsets of Crisp proteins where expressed in the Xt ovary. These data suggest that Crisp proteins in amphibians may play multiple roles in sperm production, maturation and guidance just as they are thought to in mammals indicating that Crisp protein involvement in reproduction may not be limited to mammals.

Phorbol Myristate Acetate Induces the Phosphorylation of Plasma Membrane-Associated Proteins in Sea Urchin Eggs: (Protein phosphorylation/protein kinase C/egg activation).

Immediately after fertilization sea urchin eggs undergo an increase in cytoplasmic pH from 6.8 to 7.2. This pH change occurs by activation of a Na+ /H+ antiporter, and is a necessary signal for later steps in metabolic activation of development. Activators of protein kinase C such as phorbol myristate acetate (PMA) and diacylglycerol produce a similar pH increase in eggs. Phosphorylation of the antiporter or a regulatory protein may be a step in activating Na+ /H+ exchange. Here we show that treatment of sea urchin eggs (S. purpuratus) with PMA results in increased phosphorylation of over a dozen proteins. Of these, three proteins of Mr=240, 92 and 80 kD are located in the egg cortex; under-representation of these bands in isolated cortical granules suggests that they are plasma membrane-associated. Phosphorylation of the 92 kD band is concentration-dependent over a range of 10 to 1000 nM PMA and occurs over a time-course of 1 to 3 min. Phosphoamino acid analysis indicates that phosphorylation is on serine residues. Phosphorylation appeares to be mediated by protein kinase C since the inactive PMA analogue, 4α-phorbol 12, 13-didecanoate, does not induce phosphorylation nor does experimental alkalinization of the egg cytoplasm.