Allurin, a 21 kD sperm chemoattractant, is rapidly released from the outermost jelly layer of the Xenopus egg by diffusion and medium convection

Allurin, a 21 kD protein from Xenopus laevis egg jelly, has been demonstrated to attract sperm by video microscopy and by quantitative chemotaxis chamber assays. Here, we use immunocytochemistry to demonstrate that this sperm chemoattractant is located in the outermost layer of egg jelly (J3) and is rapidly released into the surrounding medium. SDS-PAGE analysis and Western blotting confirm the appearance of allurin in the medium within 1.5 min and separation of proteins in the medium by anion exchange FPLC, shows that nearly half of the allurin released over a 12-hr period is discharged in the first 5 min. The kinetics of allurin release from J3 and its appearance in the medium were quantitatively accounted for, by computer simulation of mathematical diffusion and convection models. Comparison of simulation data to quantitative measurements of allurin appearance in the medium suggests that allurin, although larger than most chemoattractants, is effectively dispersed by a combination of diffusion and medium mixing at the jelly surface during spawning. Our model further predicts that the innermost jelly layer, J1, is less permeable to allurin than the other layers, allowing it to act as a "reflector" to speed up allurin discharge.     

The outermost layer of egg-jelly is crucial to successful fertilization in the newt, Cynops pyrrhogaster

The significance of egg-jelly layers in internal fertilization was evaluated in the newt, Cynops pyrrhogaster. In this species, six egg-jelly layers, J1, J2, J3, J4, J5 and the outermost J6 layers, are accumulated on the surface of the fertilizable eggs in pars convoluta of the oviduct. When a large number of sperm (about 6 x 10(5)) were placed on eggs having different numbers of jelly layers, all the eggs were fully fertilized, although many of the eggs developed abnormally. Upon insemination using about 600 sperm, only eggs with the full set of jelly layers were fertilized at a high rate with normal development. Since around 300 (the range of 48-1,192) sperm were observed on and in the egg-jelly in naturally spawned eggs, we conclude that the J6 layer must be present on the outermost surface of the egg-jelly for successful internal fertilization of the newt. Previous studies have suggested that the J6 layer is a prerequisite for the initiation of sperm motility and the acrosome reaction. In the present study, the fertilization rate decreased in eggs with a full set of jelly layers when inseminated using acrosome-reacted and motile sperm. However, the fertilization rate was high when motile sperm with intact acrosome was used. These results suggest that induction of the sperm acrosome reaction in the J6 layer is an important step in the internal fertilization of the newt.     

Oviduct histochemistry and site of synthesis of a 29.7 kDa jelly coat glycoprotein in the anuran Lepidobatrachus laevis

The oviduct of the anuran Lepidobatrachus laevis contains three morphological regions, each of which contains a histochemically distinct luminal mucosa. In the pars recta, the most anterior portion of the oviduct, there are periodic acid-Schiff base (PAS)-positive simple glands and epithelia. In the pars convoluta, there are alcian blue-positive, combined alcian blue- and PAS-positive and PAS-positive gland types. The most posterior region, the pars uterina, contains alcian blue-positive and alcian blue-negative epithelial cells. Previous work has shown that solubilized egg jelly contains a major 29.7 kDa glycoprotein subunit that was detected in oviduct tissue extracts from the pars convoluta in the present study. Rabbit antisera to the 29.7 kDa egg jelly glycoprotein of L. laevis reacted with the major pars convoluta glycoprotein and there were no immunoreactive components in the pars uterina. The slight immunoreactivity detected at 29.0–37.0 kDa in pars recta extracts is not believed to be the jelly molecule, based on low immunoreactivity and subunit molecular weight measurements. We conclude that the synthesis of the 29.7 kDa egg jelly glycoprotein is restricted to the pars convoluta region of the oviduct.     

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.     

Immunoelectron Microscopic Demonstration of the Pre-fertilization Layer in Xenopus eggs

The formation of the fertilization (F) layer in Xenopus laevis was studied by immunoelectron microscopic protein A-gold technique, employing an antiserum specific to the secretory granules in the bottom cells at the posterior portion of the pars recta of the oviduct (pars recta 2). In unfertilized eggs, the gold particles revealing the antigenic sites were specifically present over the pre-fertilization (PF) layer located between the vitelline coat and the jelly layer. Upon transformation of the PF layer into the F layer, the labeling became confined to the region on the outer surface of the F layer, suggesting the loss of antigenic site in the inner part of newly-formed F layer. These results provide an ultrastructural basis for the view that the PF layer is supplied by specific cells in the oviducal pars recta 2.     

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.     

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.     

The jelly envelopes and fertilization of eggs of the newt, Notophthalmus viridescens

Fertilization in Notophthalmus viridescens is internal and involves passage of the sperm through five layers of egg jelly (J5-J1, from outermost to innermost), each of which is secreted by a discrete region of the oviduct. Polyspermy is normal. Passage of the sperm through the jelly and into the egg was studied by a technique of artificial insemination similar to natural insemination, in that undiluted fluid from the vas deferens was applied directly to eggs with various layers of jelly present, followed by flooding with water three to five minutes later. In general, successful fertilization increased as the number of jelly layers increased; jellyless coelomic eggs were not fertilizable. Sperm passage through the jelly and into the egg usually occurs within one to three minutes. Upon hydration of the jelly, barriers to sperm penetration develop in layers J5 and J3. Changes in the egg jelly thus seem to be involved in the restriction of polyspermy to a low level.     

Profiling the morphological distribution of O-linked oligosaccharides

The morphological distribution of oligosaccharides is determined in the egg jelly surrounding Xenopus laevis eggs. This biological system is used to illustrate a method for readily identifying and quantifying oligosaccharides in specific tissues. The extracellular matrix surrounding X. laevis eggs consists of a vitelline envelope and a jelly coat. The jelly coat contains three morphologically distinct layers designated J1, J2, and J3 from the innermost to the outermost and is composed of 9-11 distinct glycoproteins. Each jelly layer is known to have specific functions in the fertilization of the egg. We developed a rapid method to separate and identify the oligosaccharides from X. laevis egg jelly layers. Identification was based on the retention times in high-performance liquid chromatography (porous graphitized carbon column), exact masses, and tandem mass spectrometry. Over 40 neutral and 30 sulfated oligosaccharides were observed in the three jelly layers. Neutral oligosaccharide structures from different jelly layers were both unique and overlapping, while sulfated oligosaccharides were detected only in layers J1 and J2. Neutral oligosaccharides unique to jelly layer J3 and the combined layers J1+J2 had similar core structures and similar residues. However, differences between these two sets of unique oligosaccharides were also observed and were primarily due to the branching carbohydrate moieties rather than the core structures.     

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.     

Ultrastructure function and regulation of the oviduct of the Rana ridibunda (author's transl)]

An investigation has been carried out into the structure, ultrastructure, function and of the oviduct on the adult female of Rana ridibunda. The most important part of the oviduct comprises tubulary glands and a luminal epithelium which is composed of ciliated cells and vesicular cells. The discharge processes of secretory substances were studied. Injection of the mature females with estrogens and progesterone have show that progesterone was the most effective in provoking jelly release. It is probably that in Rana ridibunda the pituitary hormones act on the follicle cells of ripe oocytes, causing them to secrete a progesterone-like hormone which provodes the maturation of the oocytel and jelly release from the oviducal glands.     

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.     

Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer

Resact, a peptide of known sequence isolated from the jelly layer of Arbacia punctulata eggs, is a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for millimolar external calcium. A. punctulata spermatozoa do not respond to speract, a peptide isolated from the jelly layer of Strongylocentrotus purpuratus eggs. This is the first report of animal sperm chemotaxis in response to a defined egg-derived molecule.     

Complex secretory products in the oviduct of the plethodontid salamander Bolitoglossa dofleini (Amphibia, Urodela)

The ultrastructure of the secretory granules in the cells of the subdivisions of the oviduct in the neotropical plethodontid salamander Bolitoglossa dofleini was studied by transmission electron microscopy. In addition, we applied the cationic dye Cuprolinic Blue (CB) at different electrolyte concentrations to demonstrate proteoglycans, and the pyrogallol red-copper (PR-C) method to stain proteins at the ultrastructural level. The entire oviduct is lined by a simple epithelium that contains ciliated and microvillous cells in the first subdivision, the aglandular pars recta; microvillous cells show a moderate secretory activity. The following pars convoluta is differentiated into five glandular subdivisions and the aglandular “uterine portion”. Especially in the glandular parts, the epithelium is arranged in longitudinal folds. At their crests ciliated and microvillous cells similar to those in the pars recta occur. Gland cells are crowded with secretory granules that differ in their structural complexity (with and without electron-dense spheres or masses; elaborated, homogeneous or granular matrix; spherical; distorted) along the various subdivisions. Further, as suggested by the CB-technique, the cranial subdivisions contain large amounts of sulphated proteoglycans that decrease in the caudal direction. Carboxylated proteglycans appear to be present in all subdivisions examined. Electron-dense spheres of secretory granules are largely free of CB-precipitates, but stain more or less intensely with PR-C. The ultrastructure of the pars recta, and especially the “uterine portion” indicates transporting capability. The epithelial cells of the “uterus” have coated pits and a considerable amount of lysosome-like bodies.     

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. Mol. Reprod. Dev. 78:450–462, 2011. © 2011 Wiley‐Liss, Inc.     

Oviducal Contribution to Alteration of the Vitelline Coat in the Frog, Rana japonica. An Electron Microscopic Study

The vitelline coat (VC) surrounding coelomic eggs of the frog, Rana japonica , comprises bundles of filaments running both parallel and perpendicular to the egg surface. The coat gives little or no staining reaction with PA-CrA-Silver methenamine. In contrast, in the VC of uterine eggs the filament bundles are less conspicuous. and the interstices between the filament bundles stain strongly for carbohydrate. This alteration occurs during passage of the eggs down the first 1/20 th of the oviduct, the pars recta. The epithelium of the p. recta contains secretory cells, which contain electron-dense granules distinct from those in the jelly-secreting cells in more caudal portions of the oviduct. Treatment of coelomic eggs with an extract of p. recta followed by exposure to a sperm suspension resulted in marked swelling and softening of the VC. These results indicate that the contents of the granules secreted from the epithelial cells in the p. recta are deposited in the VC to increase its susceptibility to a fertilizing sperm.     

绿蟾蜍繁殖期间输卵管变化的组织学研究

本文研究了绿蟾蜍繁殖期间输卵管组织结构变化的规律。产卵前，输卵管壁最厚，固有层中的单管状腺充满胶质；产卵时，粘膜层形成细长的皱襞，固有层中的腺体分泌胶质，形成包裹卵子的卵胶膜；产卵后，输卵管壁变薄，腺体细胞缩小，但腺体间的结缔组织隔膜增厚，结缔组织可能对腺体细胞的修复起重要作用。     

Analyses of Oviductal Pars Recta-Induced Fertilizability of Coelomic Eggs in Xenopus laevis

The acquisition of fertilizability in coelomic eggs of Xenopus laevis has been shown to be correlated with the physical, biochemical, and ultrastructural alterations of the egg envelope [coelomic envelope (CE)] induced during the passage of eggs through the pars recta portion of the oviduct. However, no direct evidence that the pars recta renders eggs fertilizable has yet been presented. In this study, we show that coelomic eggs are highly fertilizable when they are incubated with continuous shaking for 4 h at 15 degrees C in pars recta extract (PRE) derived from females prestimulated by pregnant mare serum gonadotropin. The PRE from pituitary-stimulated Bufo japonicus was as potent as homologous PRE in rendering Xenopus eggs fertilizable. Incubation of coelomic eggs in PRE for 30 min induced a dramatic increase in the rates of sperm binding to the envelope to a level equivalent to that exhibited by the envelope from uterine eggs (VEs). The CE-to-VE ultrastructural conversion and a 43k-to-41k hydrolysis of the envelope glycoprotein component started 5 min after, and were completed by 15 min after, the start of incubation in PRE and were accompanied by an exposure of a new N-terminal sequence typical to gp41. Thus, the biochemical and ultrastructural conversions and the sperm-binding activity of the envelope induced by PREs, although being prerequisite, were not sufficient to render coelomic eggs fully accessible to fertilizing sperm.     

Allatostatin in ovaries, oviducts, and young embryos in the cockroach Diploptera punctata

The quantity and localization of -Phe-Gly-Leu-amide allatostatins (-F-G-L-amide AST) was determined by ELISA and immunohistochemistry in ovaries and oviducts and in pre-dorsal closure embryos. AST in the cytoplasm of basal oocytes gradually increased from 4 to 35 fmol/ovary pair from the start (day 2) to the completion of vitellogenesis (day 6), then rapidly increased to 121 fmol/ovary pair during choriogenesis. In oviducts, AST-immunoreactivity was found in nerves to the muscle layer and in epithelial cells. AST-immunoreactivity in oviduct epithelial cells increased during vitellogenesis. A marked increase in quantity of AST in oviduct tissue between completion of chorion formation and immediately after ovulation appears to result from AST released from oocytes as they travel down the oviducts because AST content of newly ovulated eggs was 40% lower than late stage chorionated oocytes, and these oocytes released AST when incubated in saline. AST in embryos, localized in yolk cells, decreased as embryos approached dorsal closure. That this material in ovaries and embryos is AST was confirmed by its ability to inhibit JH synthesis in vitro and identification by MALDI-TOF mass spectrometry of a peptide with a mass corresponding to that of a Diploptera punctata AST. These findings indicate likely novel functions for ASTs: facilitation of ovulation and utilization of yolk.     

Maternal energy investment in eggs and jelly coats surrounding eggs of the echinoid Arbacia punctulata

In free-spawning marine invertebrates, the amount of maternal energy that is invested in each egg has profound implications for all life-history stages of the offspring. The eggs of echinoids are freely spawned into the water and are surrounded by several structurally complex extracellular layers. These extracellular layers, or jelly coats, do not contribute energy to embryonic development but must impose an energy cost on the production of each egg. The investment of maternal energy reserves in the jelly coats of echinoid eggs may have important implications for the number of eggs that can be produced (i.e., fecundity) and the amount of energy that can be invested in each egg. We estimated the degree to which maternal energy is invested in the jelly coats surrounding eggs of the echinoid Arbacia punctulata. Estimates were derived from measurements of the amount of energy contained in the combined eggs and jelly coats, and in the eggs alone. The amount of energy contained in A. punctulata eggs ranged from 2.70 to 5.53 x 10(-4) J egg(-1). The amount of energy contained in the jelly coats ranged from 0.13 to 0.48 x 10(-4) J jelly coat(-1). The mean concentration of energy in the eggs was 2.15 mm(-3) and 0.29 J mm(-3) in the jelly coats. These results indicate that between 3% and 11% (mean = 7%) of the total energy invested in each A. punctulata egg is partitioned to the jelly coat alone. A significant positive relationship was found between the volumes of the jelly coats and the amount of energy they contained. Based on this relationship and an analysis of differences in the size of jelly coats between echinoid species, we suggest that the degree to which energy is invested in jelly coats may vary among echinoid species and is therefore likely to be an important life-history characteristic of these organisms.