Modelling human sperm-egg interactions in vitro: signal transduction pathways regulating the acrosome reaction

Recent advances in characterizing sperm surface receptors and ion channels, when combined with the rapidly expanding knowledge of interactions among second messenger systems in somatic cells, permit formulation of a tentative molecular mechanism for the regulation of the human sperm acrosome reaction. As spermatozoa pass through the cumulus mass, progesterone binds to its sperm surface receptor, alkalinizes the sperm head cytosol and potentiates changes in intracellular ionized calcium. Primary binding of spermatozoa to egg involves receptors for mannosyl, N-acetylglucosaminyl and, possibly, fucosyl residues of the glycosylated zona protein, ZP3. These receptors aggregate on multivalent ligand binding, migrate to the equatorial region along an actin filament network formed between the plasma and acrosomal membranes during capacitation, and activate a G protein/protein kinase A/protein kinase C second messenger system and a secondary proteolysis signal. Binding of a receptor tyrosine kinase to ZP3 amino acid residues simultaneous with the sugar recognition event triggers tyrosine phosphorylation signalling. All signals combine to open a voltage-dependent calcium channel. The resulting elevated calcium signal depolymerizes the inter-membrane actin network and activates phospholipases, leading to an acrosome reaction.      

Carbohydrates and fertilization: an overview

Initial sperm-egg binding in mammals involves recognition of glycosylated proteins of egg zonae by glycosylated proteins on sperm surfaces. Egg zona protein structure is relatively simple, and has been strongly conserved. Species specificity must reside in the carbohydrate modifications on the egg surface, and in the co-ordinated assembly of a unique cohort of sperm proteins at capacitation. Fruitful advances have been made along four lines. Oligosaccharide structures capable of binding spermatozoa have been dissected by in-vitro synthesis and binding experiments, informed by the general advance of knowledge of protein glycosylation processes. Site-specific mutagenesis of zona proteins and their expression in tissue culture have identified glycosylation sites involved in species-specific sperm binding. Antibody and lectin labelling studies show a continuing process of remodeling of glycosylated sperm surface epitopes within a set of stable compartments during epididymal transit and capacitation of spermatozoa. Characterization of sperm-egg binding proteins from a variety of mammalian species shows that a different set of effectors induce acrosome reactions in each species, with each set including one or more sugar-recognizing proteins. Sequencing of some of these effectors suggests that each group may form a supermolecular complex to induce a species-specific acrosome reaction, with the functional activities distributed in a species limited or non-limited manner among the individual proteins.      

Galactosyltransferase function during mammalian fertilization

Gamete recognition has been studied extensively in the mouse. In this system, it is generally believed that sperm bind to a class of O-linked oligosaccharides on the zona pellucida glycoprotein, ZP3. The best characterized sperm receptor for ZP3 is beta1, 4-galactosyltransferase (GalT), which functions in a lectin-like capacity by binding to N-terminal N-acetylglucosamine residues on ZP3 oligosaccharides. Multivalent oligosaccharides on ZP3, as well as synthetic polymers terminating in N-acetylglucosamine aggregate GalT, leading to activation of a heterotrimeric G protein cascade and culminating in the acrosome reaction. Following fertilization, cortical granules release N-acetylglucosaminidase, which removes the binding site for sperm GalT and facilitates the zona block to polyspermic binding. Genetic manipulation of GalT expression has confirmed its function as a ZP3 receptor. Overexpressing GalT on sperm leads to increased binding of ZP3, increased G protein activation, and precocious acrosome reactions. In contrast, sperm from mice made null for GalT by homologous recombination are refractory to ZP3, in that they are unable to bind soluble ZP3 and fail to undergo the acrosome reaction in response to zona glycoproteins. Surprisingly, GalT null sperm still bind to the zona and achieve low rates of fertilization in vitro. This then suggests that sperm-egg binding involves receptor-ligand interactions independent of GalT and ZP3. The current model suggests that GalT functions as the ZP3 receptor that is responsible for inducing the acrosome reaction, whereas initial sperm-zona binding is dictated by other sperm surface receptors. Consistent with this, at least three other zona pellucida monosaccharides have been implicated in sperm binding, and novel sperm surface glycoproteins have been suggested to function in gamete binding. A large scaffolding protein has been identified that associates with the GalT cytoplasmic domain and may be responsible for orchestrating its signal transduction capacities that lead to the acrosome reaction.     

An electron microscopic study of sperm penetration into the rabbit egg after natural mating

The ultrastructure of fertilization has been studied in rabbit eggs recovered 11 to 15 hours after natural mating. Many sperm passing between the granulosa cells had undergone the acrosomal reaction, but this was not invariable, and, occasionally, intact sperm were present close to the zona pellucida. The cells of the corona radiata sometimes develop pseudopodial processes at the abovular surface and can ingest sperm after natural mating. The bulk of the content of the acrosome and the vesiculated elements formed during the acrosomal reaction, are lost before the sperm penetrates the zona pellucida, at which point the naked inner membrane of the acrosome is brought into intimate apposition with the zona. As the sperm cleaves a path through the zona pellucida, the posterior equatorial segment of the acrosome remains intact, and later persists as such in perivitelline sperm and quite possibly after incorporation of the sperm head into the vitellus. Sperm head entry into the vitellus is a two-fold process. The fertilizing sperm invariably fuses first with the vitelline membrane over the midposterior region of the head, whereas the rostral or acrosomal portion is drawn into the vitellus while encased by a flattened vesicle; this vesicle is comprised by the persistent inner membrane of the acrosome and externally by vitelline membrane sequestered from the egg surface. Soon after exposure to ooplasm, the sperm nucleus begins to decondense at a variable rate into a web of electron-dense strands; this process begins in the midposterior region, and then extends rostrally and caudally. At the same time the encasing membranes are reflected away from the anterior region of the nucleus, exposing subacrosomal material to the ooplasm. At this point the perforatorium remains, but this and the associated membranes are presumed to disintegrate eventually within the egg. After decondensation of the nucleus is complete, the faintly staining chromatin becomes enveloped by a series of compressed vesicles which together will form the porous limiting membrane of the male pronucleus. The last region to be incorporated is the sperm tail, the plasma membrane of which is lost as organelles of the tail pass into the ooplasm. During its incorporation, the midpiece engenders some reaction at the egg surface, and the mainpiece sometimes becomes fused with surface processes before it enters the body of the egg. The midpiece then commonly disintegrates, with dispersion of the mitochondrial sheath, whereas the mainpiece usually remains essentially intact until the time of syngamy or beyond.     

Molecular aspects of mammalian fertilization

The initial communication between the gametes is a molecular,receptor-mediated process that takes place at the surface ofthe egg coat. The zona pellucida plays a central role in thisprocess such that, on the one hand, spermatozoa may bind toit and, on the other hand, it prevents polyspermy. In the mouse,ZP3, a glycoprotein of the zona pellucida with a mol. wt of84 kd, serves as a sperm receptor. Only a relatively small partof ZP3, namely certain O-linked carbohydrate side chains, isinvolved in the process of binding. These oligosaccharides probablybecome bound to enzymes associated with the plasma membraneof the sperm head and thus form an enzyme-substrate complex.A terminal -galactose has been found to be one of the decisivesugar molecules and, moreover, the critical chemical group.After sperm binding to the zona pellucida has taken place, thepolypeptide chain of ZP3 initiates the acrosome reaction inthe sperm head. In the mouse, numerous binding proteins havebeen detected in the sperm plasma membrane: these are enzymessuch as glycosyl transferases, proteinases, and glycosidases.A galactosyl transferase has been found on the surface of themouse sperm that binds specifically to N-acetylglucosamine inthe mouse zona pellucida. It is therefore apparent that carbohydrate-bindingproteins on the sperm surface mediate gamete recognition throughtheir high affinity and specificity for complex glycoconjugatesin the egg coat. In fact, it is not at all surprising that complementarycell-surface protein and glycoconjugates are involved in fertilization,since many somatic cells exhibit a similar mechanism of cellrecognition.     

Human sperm non-nuclear progesterone receptor expression is a novel marker for fertilization outcome

In a prospective, blind study, we have examined the relationship among the expression of human sperm surface progesterone receptors, the ability to undergo a mannose-stimulated acrosome reaction and the rate of fertilization in vitro. Individual aliquots of motile spermatozoa were surface-labelled with progesterone and/or mannose-fluoresceinated ligands. Spontaneous acrosome loss and the increase in acrosome reactions following exposure of spermatozoa to mannose ligands were assessed using rhodaminated Pisum sativum agglutinin. Progesterone fluoresceinated ligand binding was observed to occur in two patterns: (i) a uniform distribution of labelling over the acrosome cap (pattern II), and (ii) labelling limited to the equatorial and postacrosomal regions of the human sperm head (pattern III). A conversion of pattern II to pattern III binding was observed and was associated with the acrosome reaction. Pattern III binding was highly correlated with both fertilization potential and the ability to undergo a mannose-stimulated acrosome reaction (P < 0.001). In contrast, normal sperm mannose receptor expression was seen in five men whose abnormal progesterone receptor expression/function and inability to acrosome react after mannose treatment were correlated with their reduced fertility in vitro. In conclusion, surface progesterone receptor aggregation enhances the mannose ligand-stimulated acrosome reaction. Such detection of defective sperm surface progesterone receptor expression/function may be useful in the evaluation and management of male infertility.      

A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona spellucida: lack of relationship with ionophore A23187-induced acrosome reaction

Acrosome reactions induced by the calcium ionophore A23187 [GenBank] andzona pellucid a (ZP) were studied. Sperm samples were obtainedfrom fertile men or men with normal semen analysis and normalsperm-ZP binding. Oocytes were obtained, with the consent ofthe patients, after the failure of fertilization in vitro. Motilespermatozoa selected by a swim-up technique were incubated with10 µM A23187 [GenBank] for 1 h, four oocytes for 2 h or solubilizedZP (4 ZP/µl) for 2 h. Spermatozoa bound to the ZP weredislodged and collected in a small volume of phosphate-bufferedsaline by aspirating the oocytes with a glass pipette with aninner diameter (120 µm) slightly smaller than the diameterof the oocyte. The acrosome status of the spermatozoa was determinedusing fluorescein-labelled Pisum sativum agglutinin. The proportionof spermatozoa undergoing the acrosome reaction on the ZP at2 h varied over a wide range (5–99%), but the agreementbetween results for the same semen sample exposed to differentgroups of oocytes was good: the standard deviations of the differencesbeing 9%. Pre-incubation of spermatozoa for 2 h did not increasethe ZP-induced acrosome reaction. Re-incubation of ZP with thesame sperm suspension for 2 h after removing ZP-bound spermatozoafrom the first 2 h incubation produced a significantly lowerZP-induced acrosome reaction in the second incubation (22 ±16%) than in the first incubation (30 ± 14%; P < 0.001,n = 20). There was no significant difference in the ZP-inducedacrosome reaction with oocytes with ZP which had or had notbeen penetrated by spermatozoa during the in-vitro fertilizationinsemination. Pre-incubation of spermatozoa with solubilizedZP blocked sperm-ZP binding. However, the acrosome reactioninduced by solubilized ZP (4 ZP/µl) was significantlylower than the acrosome reaction induced by intact ZP (10 ±5 and 30 ± 13% respectively, n = 11, P < 0.001), butthere was a high correlation (Spearman r = 0.822, P < 0.01)between the results. On the other hand, although the averageof the acrosome reaction was similar for A23187 [GenBank] (42%) and forZP (43%), there was no significant correlation between the resultsfor the two stimuli (n = 60). In conclusion, a useful methodfor assessing the ZP-induced acrosome reaction has been developedusing oocytes which failed to fertilize in vitro. The lack ofa relationship between the results of the chemical (A23187 [GenBank] )and physiological (ZP) stimali for the acrosome reaction inthe same subjects questions the biological basis of using A23187 [GenBank] for tests of sperm function. Solubilized human ZP in a concentrationthat blocks sperm-ZP binding is a less efficient inducer ofthe acrosome reaction than is intact ZP. It is possible thatthe three-dimensional structure of the ZP is important for inductionof the acrosome reaction or that spermatozoa which bind to theZP are more likely to acrosome react Assessment of the physiologicalacrosome reaction for diagnosis of sperm defects which interferewith the fertilization process should be concentrated on thespermatozoa which are capable of binding to the ZP.     

The human sperm acrosome reaction: physiology and regulatory mechanisms. An update

The acrosome reaction is a crucial step during gamete interactionin all species, including man. It allows spermatozoa to penetratethe zona pellucida and fuse with the oocyte membrane. Spermatozoaunable to undergo the acrosome reaction will not fertilize intactoocytes. This article concentrates on the characteristics andregulatory mechanisms of the acrosome reaction in human spermatozoa.During recent years, various entities found in the vicinityof the ovulated oocyte have been identified as stimulators ofthe acrosome reaction, of which zona protein is considered theprime physiological inducer in vivo. The steroid hormone progesteronehas been shown to evoke critical responses in sperm cells leadingto the acrosome reaction. Calcium has also been shown to playa central role during the acrosome reaction. Calcium flux isinduced specifically by progesterone in capacitated and uncapacitatedsperm cells, whereas only capacitated spermatozoa are able tosubsequently complete the acrosome reaction. Progesterone aswell as zona protein has been shown to evoke crucial responseswithin human spermatozoa, shedding light on the cascade of intracellularsignalling events leading to the completion of the acrosomereaction. Furthermore, chemical agents which bring about thereaction in vitro, such as the ionophores ionomycin or A23187 [GenBank] ,have been used to shed light on its regulatory mechanisms. Anumber of molecules have been postulated to regulate the acrosomereaction in mammals, for example a galactosyl-transferase anda sperm protein tyrosine kinase. In addition, a novel protein,termed SAA-1, that was first detected on human spermatozoa isdiscussed with respect to its potential role as a regulatoryprotein closely involved in the initiation of the acrosome reaction.     

Antifertility Characteristics of the N‐terminal Region of Mouse Equatorial Segment Protein

To investigate antifertility characteristics of the equatorial segment protein (ESP) and its potential immunocontraceptive effect, three partially overlapping cDNA fragments P1/P2/P3, together covering the entire mouse ESP, were cloned, expressed, and purified. The roles of P1/P2/P3 in fertility were investigated through in vitro fertilization and mouse mating test. Antibodies against P1/P2 significantly reduced the rates of fertilization in vitro in the zona‐intact experiments. Coincubation of zona‐free mouse oocytes with capacitated mouse spermatozoa in the presence of antibodies against P1/P2 also inhibited sperm‐oolemma binding and fusion, while anti‐P3 antibody virtually had no effect on in vitro fertilization at the same concentration. Immunization of female BALB/c mice with N‐terminal of mouse ESP (recombinant P1 and P2) resulted in a significant decrease in the fertility rate as well as the litter size. Double immunofluorescence staining showed that mouse ESP protein was localized to the equatorial segment of acrosome of mouse sperm, and was exposed and surface‐accessible after acrosome reaction. Mouse ESP was also demonstrated to have complementary binding sites on the mouse egg plasma membrane by indirect immunofluorescence assay. These findings suggest that the N‐terminal of mouse ESP could play an important role in fertility and might be a vaccine candidate for contraception. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Ion channels in sperm physiology

Fertilization is a matter of life or death. In animals of sexual reproduction, the appropriate communication between mature and competent male and female gametes determines the generation of a new individual. Ion channels are key elements in the dialogue between sperm, its environment, and the egg. Components from the outer layer of the egg induce ion permeability changes in sperm that regulate sperm motility, chemotaxis, and the acrosome reaction. Sperm are tiny differentiated terminal cells unable to synthesize protein and difficult to study electrophysiologically. Thus understanding how sperm ion channels participate in fertilization requires combining planar bilayer techniques, in vivo measurements of membrane potential, intracellular Ca2+ and intracellular pH using fluorescent probes, patch-clamp recordings, and molecular cloning and heterologous expression. Spermatogenic cells are larger than sperm and synthesize the ion channels that will end up in mature sperm. Correlating the presence and cellular distribution of various ion channels with their functional status at different stages of spermatogenesis is contributing to understand their participation in differentiation and in sperm physiology. The multi-faceted approach being used to unravel sperm ion channel function and regulation is yielding valuable information about the finely orchestrated events that lead to sperm activation, induction of the acrosome reaction, and in the end to the miracle of life.     

Identification of phosphatases on the membranes of guinea pig sperm

Guinea pig epididymal sperm, incubated for ATPases at pH 7.0 or pH 9.0, localize reaction product on both the periacrosomal segment of the plasmalemma and the outer acrosome membrane. In other species, e.g., rabbit, Ca⁺⁺-ATPase is identified with the outer acrosome membrane. It may transport Ca⁺⁺ into the acrosome for activation of enzymes released during the acrosome reaction. The neutral ATPase is demonstrable on the periacrosomal plasmalem-ma and possibly modifies Ca⁺⁺ concentration in the fluid around the acrosome. In guinea pig sperm, Ca⁺⁺-ATPase is sensitive to centrifugation or washing of sperm which indicates that the ductal fluid has unusual properties for preservation of the acrosome. Inhibition of the enzyme by these treatments suggests that conditions on the plasmalemmal surface affect the acrosome membrane. Inability to separate reaction product on the plasmalemma from that on the acrosome membrane may be due to migration of reaction product across the periacrosomal space. However, the ATPases are elicited in the guinea pig under the same conditions as in other species. The pH 9.0 enzyme requires Ca⁺⁺ while the enzyme at pH 7.0 has no ion specificities. Demonstration of these enzymes indicates that mechanisms of acrosome activation, similar to those in other sperm, are relevant to the guinea pig.     

Human sperm capacitation and the acrosome reaction.

A model is presented that describes the mechanism of human sperm capacitation and the acrosome reaction. The processes of capacitation and the acrosome reaction are proposed to function in control of the activation/release of acrosomal enzyme(s) involved in sperm penetration through the zona pellucida. During capacitation, the sperm head membranes are biochemically modified, allowing the acrosome reaction to take place when the spermatozoon approaches or reaches the zona pellucida, resulting in the localized activation and release of the appropriate enzyme(s). Further, capacitation is presented as a continuing process that occurs during sperm transport through the female genital tract and is physiologically not completed until the spermatozoon reaches the oocyte (unless the spermatozoa are kept at a particular genital tract site for prolonged periods). The biochemical alterations that occur during capacitation are discussed. It is suggested that extensive modifications in the lipid bilayer structure, e.g. in the cholesterol or phospholipid content, are not part of capacitation because such changes would prematurely destabilize the membranes. Rather, such changes occur during the acrosome reaction. It is also proposed that the human sperm acrosome reaction has many similarities to the somatic cell exocytotic events which occur during the regulated pathway of secretion. One or more oocyte stimuli result in the activation of protein kinases, likely (but not necessarily) via activation of G-protein coupled receptors on the sperm plasma membrane and the formation of second messengers. The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction. The role of other enzyme systems such as those involved in ion transport, proteolysis, phospholipid metabolism (including that of arachidonic acid) and other metabolic events, is discussed. Calcium ion influx as initiator of the acrosome reaction is reconsidered. The proposed model also takes into consideration the structural events of membrane fusion.     

The early egg of Austramphilina elongata (Cestodaria)

Austramphilina elongata Johnston, 1931 were collected from the freshwater turtle Chelondina longicollis taken in New South Wales, Australia. The morphology and histochemistry of the oogenotop is described with emphasis on egg shell formation. The vitelline gland granules compose most of the early egg shell; the granules stain positively with PAS, bromophenol blue and Heidenhains iron haematoxylin. There is a weak reaction for phenolase. The Mehlis gland secretory granules are PAS-positive. The first formed egg shells stain positively with PAS, bromophenol blue and Heidenhains iron haematoxylin; there is a weak phenolase reaction. The significance of these reactions is discussed.Abbreviations E embryo - FC fertilization canal - Ma macromere - Mi micromere - MG mehlis gland - N nucleus - OD oviduct - Ov ovary - S shell - SD sperm duct - SR seminal receptacle - St stereocilia - U uterus - UG uterine granules - VC vitelline cell - VD vitelline duct - VG vitelline granule - Z zygote     

Testis-specific antigen (TSA-1) is expressed in murine sperm and its antibodies inhibit fertilization

PROBLEM: We recently cloned and sequenced a sperm-specific antigen, designated as testis-specific antigen-1 (TSA-1), from human testis. The present study was conducted to examine its expression and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects. METHOD OF STUDY: The antibodies (Ab) were raised against purified human rTSA-1 in virgin female rabbits. The rTSA-1 was run in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gel containing the approximately 18 kDa band was cut, minced and used for immunization to obtain the specific Ab. The immunoglobulins from preimmune bleed and from animals injected with adjuvant alone served as control. These Ab were analysed in enzyme-linked immunosorbent assay (ELISA), Western blot procedure, immunoprecipitation procedure, immunocytochemical technique (ICT), immunobead binding technique (IBT), acrosome reaction and sperm-zona binding assay. RESULTS: Active immunization of female rabbits with purified rTSA-1 protein of 18 kDa, produced high titer Ab against the recombinant antigen. These Ab to rTSA-1 were used in the present study. In Western blot procedure, rTSA-1 Ab recognized a specific protein band of approximately 24 +/- 3 kDa in murine sperm extract, the band similar to found in human sperm extract. In the immunoprecipitation procedure, rTSA-1 Ab immunoprecipitated the protein band of similar size from extracts of murine sperm and murine testis. The ICT and the IBT studies revealed the subcellular localization of TSA-1 on the surface of acrosome and tail regions of the non-capacitated and capacitated murine sperm cells. In functional bioassays, rTSA-1 Ab inhibited the acrosome reaction and sperm-egg binding in vitro. CONCLUSIONS: These data indicate that the TSA-1 is expressed in murine sperm and may have a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggests that the mouse can provide a suitable model to examine the immunocontraceptive effects of TSA-1 in actively immunized animals.     

Molecular cloning and characterization of a receptor for activated protein kinase C1 (RACK1) from Chinese white shrimp; Fenneropenaeus chinensis

Receptor for activated C kinase 1 (RACK1), which has seven tandem WD40 domains, is a scaffolding protein. RACK1 plays different roles by binding to different partner proteins. It is involved in hormone signaling and development, and now some evidence indicates it may have a role in innate immunity. In this paper, RACK1 cDNA from Chinese white shrimp (FcRACK1) was identified. The full length of the FcRACK1 gene is 1037 bp, including a 30 bp 5′UTR, a 957 bp ORF encoding a 318 amino acid protein, and a 50 bp 3′UTR with the polyadenylation sequence AATAAA and a poly (A) tail. The FcRACK1 protein is characterized by seven WD40 repeat domains; the ending two amino acids of each WD40 domain are WK, WD, WN, WS, WD, WD, and WQ, respectively. The length of each domain is between 30 and 44 amino acids. Multiple alignments of RACK1s showed that RACK1s are highly conserved. RT-PCR showed that FcRACK1 could be detected in hemocytes, the heart, hepatopancreas, gills, stomach, intestine, and ovary. FcRACK1 in hemocytes was down-regulated after a 2 h WSSV challenge, and FcRACK1 in gills was up-regulated after a 2 h Vibrio challenge. FcRACK1 in ovary went down after a 12 h Vibrio challenge and then up-regulated at 24 h. FcRACK1 in ovary was first down-regulated at 2 h after a WSSV challenge and then up-regulated to the highest level at 6 h. It finally went down from 12 to 24 h. In hepatopancreas, FcRACK1 was also up-regulated by microbe challenge. Our results indicated its probable role in shrimp innate immunity.     

Fertilization and early embryology: Blocking of human fertilization by carbohydrates

The zona penetration test and triple stain technique were usedto elucidate the blocking effects of carbohydrates on humanfertilization and their mechanisms. In the presence of D-mannoseor D-fructose (final concentration, 50 mmol/1), sperm penetrationthrough the human zona pellucida was completely blocked. Thetriple stain technique revealed that D-fructose (50 mmol/1)significantly (P < 0.01) suppressed the acrosome reactionof human spermatozoa, while D-mannose did not show a suppressiveeffect on the acrosome reaction. These results reinforce ourhypothesis proposed previously, that a mandatory step in humanfertilization is the binding of a D-mannose-binding constituentof the sperm surface to a D-mannose residue in the sperm receptorsite on the zona pellucida. In addition, D-fructose may playan important role as an acrosome stabilizing factor in seminalfluid.     

The proteasome-ubiquitin pathway in the Schistosoma mansoni egg has development- and morphology-specific characteristics

Schistosoma mansoni eggs, consisting of an ovum surrounded by nutritive vitelline cells packaged in a tanned protein shell, are produced by paired worms residing in the mesenteric veins of the human host. The vitelline cells are degraded as the larval miracidium matures, the fully developed egg either crossing the gut wall to escape the host or becoming lodged in the host's tissues where it dies and disintegrates, inducing a potentially pathological immune response. Thus, the egg is central to both the transmission of the parasite and the aetiology of the disease. Here we present the first study investigating protein turnover in the egg. We establish that the ubiquitin-proteasome pathway (UPP) changes with egg development and furthermore, that the morphological components of the fully developed egg (the miracidium and the subshell envelope) also exhibit different proteasome subunit expression profiles. We conclude that the UPP is responsible not only for degrading the vitelline cells but is also more highly developed in the envelope than in the miracidium. The envelope is involved in the defence of the miracidium and produces the proteins that the egg secretes, presumably to facilitate its escape from the host, so the UPP probably has a multi-faceted role in the egg's biology.     

Co-Expression of Mannose-Ligand and Non-Nuclear Progesterone Receptors on Motile Human Sperm Identifies an Acrosome-Reaction Inducible Subpopulation

PROBLEM : To determine whether surface expression of receptors for progesterone and mannose can be used to identify spermatozoa likely to undergo an acrosome reaction after zona binding and to compare the reactivity of these receptors with naturally occurring sperm head-directed anti-sperm antibodies (ASAs). METHOD : Progesterone binding sites on the surface of fresh and capacitated motile human sperm in relation to acrosome status were visualized using a cell-impermeant progesterone. Free progesterone and/or mannose ligands were compared for percent sperm binding and ability to induce an acrosome reaction. Western blots of sperm proteins localized to the plasma membrane and surface proteins precipitated following passive transfer of serum ASAs were probed with progesterone-horseradish peroxidase. The effects of the same ASAs on ligand binding and on the induced acrosome reaction were examined. RESULTS : The two receptors are located in close proximity on a subset of capacitated motile sperm and are coordinately cleared from the plasma membrane overlying the acrosomal cap prior to exocytosis. The surface appearance of functional binding sites for each ligand, however, is regulated by different mechanisms and the progesterone receptor alone is specifically precipitated by ASAs. Passive transfer of ASAs to capacitated sperm selectively inhibits the progesterone-stimulated acrosome reaction but not the ionomycin-induced acrosome reaction or the ability of sperm to bind mannose ligands. CONCLUSIONS : Sperm from fertile donors incubated under capacitating conditions in vitro can be subdivided into acrosome reaction inducible and noninducible subpopulations on the basis of the co-expression or total absence of these receptors. The combined data indicate that reaction of sperm surface progesterone receptors with ASAs contributes to the acrosome reaction insufficiency observed in anti-sperm immune infertility.