Andrology: Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro

It is believed that during the process of human fertilization,acrosome-intact spermatozoa bind to the surface of the zonapellucida which triggers the acrosome reaction and the enzymesreleased facilitate sperm penetration through the zona pellucida.We describe here reduced frequency of the acrosome reactionon the zona pellucida as a cause of infertility in 10 coupleswith long durations of infertility (average 6 years) and low(<15%, n= 3) or zero (n= 7) fertilization rates in vitro.Sperm concentration, motility, velocity (Hamilton-Thorn), morphologyand DNA normality were within the normal range in all the patients.Electron microscopy of spermatozoa did not reveal any specificultrastructural defects. All couples were negative for antispermantibodies by immunobead tests. Oocytes from other patientswhich failed to fertilize in in-vitro fertilization and normaldonor spermatozoa were used as controls for sperm-zona pellucidabinding and penetration experiments. Acrosome status of spermatozoabound to the zona pellucida was assessed with a fluorescentlectin and electron microscopy. The mean number of spermatozoabound to the zona pellucida was not significantly differentbetween patients and controls. However, the acrosome reactionof spermatozoa bound to the zona pellucida after 2 h incubationwas significantly lower (P< 0.001) in the patients (mean5%, range 0–16) than in the controls (mean 68%, range44–96). No zona pellucida (out of 40) was penetrated bypatient spermatozoa whereas most (39/40) zonae were penetratedby control spermatozoa (average 27 spermatozoa/four zonae pellucidae).The spontaneous acrosome reaction of spermatozoa in inseminationmedium was not different between patients (4%) and controls(3%), the acrosome reaction induced by calcium ionophore waslow (21 and 43% respectively) in six of the eight patients examined.In conclusion, these patients have spermatozoa with a disorderof the zona pellucida-induced acrosome reaction that resultsin failure of sperm-zona pellucida penetration and explainstheir infertility.     

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.     

Fertilization and early embryology: Human follicular fluid inhibits the binding of human spermatozoa to zona pellucidain vitro

The effect of human follicular fluid on human zona pellucidabinding of spermatozoa was investigated using the hemizona bindingassay (HZA). This effect was compared to that of progesterone,a known component of human follicular fluid. Exposure of spermatozoato 25% pooled human follicular fluid for 1 h significantly reducedthe number of spermatozoa bound to zona pellucida when comparedto those without human follicular fluid treatment (149.1 ±30.7 versus 177.1 ± 33.8, P 0.01). The same phenomenonwas observed after 3 h of treatment The corresponding numbersof bound spermatozoa were 140.4 ± 19.1 and 200.2 ±23.4 (P 0.0001). Progesterone (1.0µg/ml) stimulated thezona pellucida-binding capacity of spermatozoa significantlyunder the same conditions (P 0.01). The numbers of bound spermatozoaafter 1 and 3 h progesterone treatment were 235.5 ± 44.7(control, 168.1 ± 32.9) and 204.3 ± 27.4 (control,162.3 ± 20.1) respectively. HZA comparing the effectsof human follicular fluid and progesterone at concentrationsequivalent to those found in human follicular fluid using matchinghemizonae confirmed the inhibitory effect of human follicularfluid on sperm binding to zona pellucida (80.4 ± 28.4versus 149.8 ± 35.2, P 0.05). This inhibitory effectwas also found in another eight individual human follicularfluid samples. Both human follicular fluid and progesteronedid not affect the motility and viability of the treated spermatozoawhen compared to the controls with the same incubation period.Although more spermatozoa underwent the acrosome reaction after1 and 3 h of human follicular fluid treatment than in the control,the extent was comparable to those after progesterone treatmentThese results suggested that human follicular fluid inhibitedthe zona pellucida-binding capacity of spermatozoa in vitro.This inhibitory effect of human follicular fluid was not mediatedby progesterone, and did not result from the effects of humanfollicular fluid on sperm motility, viability and acrosome reaction.     

Acrosome status and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro

It is known that only acrosome-reacted spermatozoa can fusewith the oolemma during normal fertilization with zona pellucida-intactoocytes. The aim of this study was to determine if the oolemmaof human zona pellucida-free oocytes selectively binds spermatozoawith normal morphology and a reacted acrosome. Oocytes thatfailed to fertilize in vitro because of severe sperm defectswere used. The zona pellucida was removed with acidic (pH 2–3)saline. Sperm samples were obtained from normal fertile donorsand normozoospermic men. Motile spermatozoa were selected witha swim-up technique and 2x10⁶/ml incubated with oocytes. Theresults from 23 experiments showed that at 2 h there was a significantlyhigher mean percentage of acrosomereacted spermatozoa boundto the zona pellucida (mean ±SD, 42±22) than inthe insemination medium (27 ± 12). In contrast, all spermatozoabound to the oolemma at 2h were acrosome reacted. Furthermore,each fresh zona pellucida had>100 spermatozoa bound comparedwith an average of 28 (range 4–81) spermatozoa bound perzona pellucida-free oocyte. There was no significant differencein the zona pellucida-induced acrosome reaction between fresh(45 ± 21)and salt-stored (35 ± 22) zonae pellucidae.The percentage with normal morphology was significantly higherfor spermatozoa bound to the zona pellucida (84 ± 13)and oolemma of zona pellucida-free oocytes (71 ± 25)than for spermatozoa in insemination medium (39 ± 11)(P<0.01). Extending the time of incubation of spermatozoawith zona pellucida-intact oocytes increased the proportionof spermatozoa undergoing the acrosome reaction (n=6, 2 h, 41± 23; 3 h, 53 ± 31; 4 h, 61 ± 34). However,there was a large variation in the percentage of acrosome reactionsamong spermatozoa bound to the zona pellucida between individualsperm samples. In conclusion, only acrosome-reacted spermatozoacan bind to the oolemma of zona pellucida-free oocytes. Bothfresh and salt-stored human zonae pellucidae are equally effectivein inducing the acrosome reaction. Both the zona pellucida andthe oolemma of zona pellucida-free oocytes selectively bindspermatozoa with normal morphology     

Regulators of sperm function: Effect of gastrin-releasing peptide on sperm functions

Male infertility can be related to defects in motility, capacitation,acrosome reaction, binding and penetration of the zona pellucida.While different in-vitro techniques (such as micromanipulationwhich is complicated and expensive) are available for the treatmentof male infertility, several pharmacological agents have beenshown to increase fertilizing capacity under accurate experimentalconditions. Gastrin-releasing peptide (GRP, the mammalian homologueof the amphibian skin peptide bombesin) is present in the reproductivetract and expressed by the pregnant ovine endometrium priorto attachment and throughout the pregnancy. A bombesin-likepeptide resulting from alternate splicing of the GRP gene intestis has been detected in primates. In this study, we havetested the ability of GRP to enhance human sperm functions suchas motility, capacitation, zona binding and acrosome reaction.Analysis of sperm motility was performed with the ATS 20 computer-assistedsemen analysis (CASA) system. Zona binding was analysed usingintact human unfertilized oocytes and selective labelling ofspermatozoa with two fluorochromes. Our results did not showany positive effect of GRP on these parameters under our experimentalconditions. However, when GRP at the concentration of 100 nMwas added after ionophore treatment, the percentage of reactedcells increased significantly (P <0.05) compared with situationswhere each agent was used alone. This led us to suppose thatthe role of bombesin in the different stages of fertilizationmight not exclude other unknown factors. acrosome reaction/gastrin-releasing peptide (GRP)/human male infertility/sperm motility/zona pellucida binding     

Artificial induction of the acrosome reaction in human spermatozoa

This study investigated the use of human follicular fluid andpentoxifylline as inducers of the human sperm acrosome reactionin vitro. Motile sperm suspensions were prepared using a discontinuousPercoll gradient, preincubated for 3 h, divided into aliquotsand exposed to various concentrations of non-heart-inactivatedfollicular fluid for 1 and 24 h and pentoxifylline for 30 min.Detection of the acrosome reaction involved the combined useof a fluorescent vital stain, H33258 [GenBank] , and fluorescein isothiocyanate-conjugatedpeanut agglutinin (FITC-PNA). A short (1 h) exposure to follicularfluid at concentrations of 50% or more, did not compromise spermmotility and significantly increased the proportion of spermatozoahaving completed the acrosome reaction. Similarly, a 30 minexposure to pentoxifylline also significantly increased theproportion of spermatozoa having completed the acrosome reaction.     

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.     

Fertilization and early embryology: Does zona pellucida thickness influence the fertilization rate?

In human in-vitro fertilization (IVF), the cumulus oophorusis routinely removed to assess fertilization and hence the thicknessof the zona pellucida is measurable. This study aimed to measurethe thickness of the zona pellucida and to assess its influenceon fertilization rate in IVF programmes. The zona pellucidathickness varies from 10 to 31 µm with a mean of 17.5µm. One-way analysis of variance revealed that in IVFtrials performed with normal semen, the zona pellucida of fertilizedoocytes (16.6 ± 3.2 µm) was significantly thinnerthan the zona pellucida of unfertilized oocytes (18.9 ±4.0 µm; P < 0.001). As measured on micro-injected oocytes,the zona pellucida thickness did not change between ovulationand 16–20 h after fertilization. Zona pellucida thicknesswas not related to ooplasm diameter. In conclusion, zona pellucidathickness appears to be an additional factor that should betaken into account when interpreting the fertilization rate.Zona pellucida thickness influences sperm penetration, evenwhen the spermatozoa are considered normal. From a clinicalpoint of view, a thick zona pellucida (22 µm) could bean indicator for the use of micro-injection procedures.     

Zona-opening of hamster oocytes: a comparative study using macro- and micro-manipulation methods

The present study compared a new macro-manipulation techniquefor zona-opening of hamster oocytes with existing micro-manipulationtechniques. In experiment I the zona pellucida of hamster oocyteswas partially opened with a 32 gauge steel needle (macro-manipulation)and these oocytes were then co-incubated with human spermatozoaat 36.5°C in 5% CO₂ in air for 3.5 h. Zona-free and zona-intactoocytes similarly inseminated served as controls. Of 113 oocytes,30 (26%) lysed following zona-opening with the macro technique.The sperm penetration rates of zona-opened and zona-free oocyteswere 37% (31/83) and 60% (49/81) respectively (P<0.01), with12 and 28% oocytes respectively showing polyspermia (P<0.05).No sperm penetration occurred in zona-intact oocytes. In experimentII the zona pellucida of hamster oocytes was partially openedby macro-or micro-manipulation techniques before the oocyteswere coincubated with human spermatozoa as in experiment I.Nine of 156 (6%) macro-manipulated and 11 of 133 (8%) micro-manipulatedoocytes lysed following zona-opening. Of 147 remaining macro-manipulatedand 122 remaining micro-manipulated oocytes, 80 (55%) and 79(64%) respectively were penetrated (P>0.05), with 30 and37% respectively showing polyspermia (P>0.05). These resultsdemonstrate that macro-manipulation results in similar penetrationand polyspermia rates as compared to conventional micro-manipulation.     

Immunology: Diversity of the blocking effects of antisperm antibodies on fertilization in human and mouse

The blocking effects of complement-dependent sperm immobilizingantibodies in the sera of infertile women and monoclonal antispermantibodies against humans and mice on fertilization were investigated.The hemizona assay (HZA) and sperm penetration assay (SPA) wereused to study the inhibitory effects of sera from 22 infertilepatients positive for sperm immobilizing antibodies. Use ofthese tests allowed us to differentiate whether the antibodyblocked sperm—zona pellucida tight binding and/or spermpenetration into the ooplasm. The zona pellucida penetrationassay (ZPA) was also used to study the effects of four monoclonalantibodies (mAbs) on human sperm penetration into the zona pellucida.Seven mAbs against murine spermatozoa were tested for theirinhibitory effects on in-vitro fertilization (IVF) and HZA inmice. Of 22 patient sera with sperm immobilizing antibodies,21 (95.5%) inhibited HZA attachment and penetration, whereasthis did not occur in any of 13 patient sera without these antibodies.However, 19 of 22 (86.4%) patient sera with sperm immobilizingantibodies and eight of 13 (61.5%) patient sera without theseantibodies inhibited the SPA. Two (2C6, 1G12) of four mAbs againsthuman spermatozoa showed strong inhibitory effects in all theassays (HZA, ZPA and SPA). One mAb (3B10) did not inhibit HZAbut blocked ZPA and SPA. Another mAb (H6-3C4) seemed to haveno inhibitory effects on fertilization. Two (Vx 5 and Vx 8)of seven mAbs against murine spermatozoa inhibited IVF in micebut did not block mouse HZA. These findings suggest that antispermantibodies block fertilization at specific stages. Some of themmay inhibit sperm capacitation and thus prevent all processesof fertilization that follow. Some other antibodies may notaffect capacitation and sperm binding to zona pellucida butinhibit the acrosome reaction, followed by the blocking of spermpenetration through zona pellucida and ooplasm.     

Participation of the sperm proteasome in human fertilization

BACKGROUND: Fertilization in mammals comprises the sequential interactions of the sperm with the cumulus oophorus, zona pellucida, and oocyte plasma membrane. Here we investigate proteasome activity in human sperm and its possible involvement during the fertilization process. METHODS: Proteasome activity was measured in intact sperm and in sperm extracts using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC, in the presence or absence of the specific proteasome inhibitor, clasto-lactacystin beta-lactone. The participation of the proteasome was evaluated during (i) sperm-zona binding using the hemizona assay; (ii) zona pellucida-induced acrosome reactions with a pulse and chase design; (iii) progesterone-induced acrosome reactions incubating overnight capacitated sperm with progesterone; and (iv) progesterone-induced Ca(2+) influx using fura-2AM. RESULTS: Intact sperm and sperm extracts possessed proteasome activity, which was susceptible to inhibition by clasto-lactacystin beta-lactone. Sperm-zona binding was not inhibited by clasto-lactacystin beta-lactone. However, both zona pellucida- and progesterone-induced acrosome reactions were inhibited by clasto-lactacystin beta-lactone. The proteasome inhibitor also blocked the sustained phase of the Ca(2+) influx provoked by progesterone but not the peak. CONCLUSION: The human sperm proteasome is involved in the exocytosis of the acrosome, perhaps in events upstream of the plateau phase of the Ca(2+) influx.     

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.     

Analysis of the participation of N-acetylglucosamine in the different steps of sperm-zona pellucida interaction in hamster

Glycoproteins and lectin-like proteins mediate sperm-zona pellucida interaction. The present study analysed the participation of carbohydrates in the different stages of sperm interaction with the zona pellucida in hamster, by determining the effects of different monosaccharides. N-acetylglucosamine (GlcNAc, 1 mM) reduced sperm ability to bind to the zona pellucida. Surprisingly, spontaneous acrosome reaction (AR) was also inhibited by this sugar. In order to analyse the effect of GlcNAc on sperm-zona pellucida binding, independent of its effect on the AR, strontium (Sr) was used as a calcium (Ca) replacement in the sperm capacitation and co-incubation medium. Sr seemed to be able to replace Ca for sperm capacitation, at least when measured as the ability to bind to the zona pellucida, and undergo AR when Ca is provided. Moreover, sperm-zona pellucida binding could also take place in a Sr-modified medium. When binding assays were carried out in the Sr medium, GlcNAc also produced an inhibitory effect. This could be reproduced when sperm, but not oocytes, were pre-incubated with the monosaccharide. IVF assays were also carried out to analyse the participation of GlcNAc in the different steps of sperm-oocyte interaction. Taken together, the results support the involvement of the GlcNAc residues of the zona pellucida in the early steps of the interaction with sperm.     

Appropriate timing of the acrosome reaction is a major requirement for the fertilizing spermatozoon

This study was undertaken to determine the site of the acrosomereaction of spermatozoa penetrating into freshly inseminatedhuman oocytes. The inseminated oocytes were treated with ananti-acrosin monoclonal antibody and the bound antibody wasvisualized at the ultrastructural level with the use of a secondperoxidase-conjugated antibody. Quantitative analysis of serialthin sections cut throughout the specimens showed that the numberof spermatozoa within the zona pellucida (potentially fertilizingones) corresponded to the number of acrosin deposits associatedwith acrosomal ghosts on the zona pellucida surface. As it isknown that a large acrosin bundle is liberated from a spermatozoonat a well-defined point of the acrosome reaction, these findingsindicate that the acrosome reaction of the fertilizing spermatozoonmust be exactly synchronized with its penetration through theegg vestments, apparently by the action of specific acrosomereaction-promoting substances in the oocyte/cumulus complex.These results represent a theoretical basis for evaluation ofdirect and indirect laboratory tests for human sperm acrosomereaction. ‘Good’ sperm samples should display elevatedlevels of acrosome-reacted spermatozoa after the administrationof an appropriate stimulus and low levels of spontaneous acrosomereactions.     

Zona pellucida-induced acrosome reaction in human sperm: dependency on activation of pertussis toxin-sensitive G(i) protein and extracellular calcium,and priming effect of progesterone and follicular fluid

In these studies, we aimed to characterize the effects of the physiological, homologous agonists of the acrosome reaction, i.e. the zona pellucida (ZP) and progesterone/follicular fluid, on human sperm. The specific aims of our studies were: (i) to examine the dependency of the solubilized ZP-induced acrosome reaction on G(i) protein activation and presence of extracellular calcium; and (ii) to determine whether progesterone/follicular fluid exert a priming or synergist effect on the solubilized ZP-induced acrosome reaction. Highly motile sperm from fertile donors were exposed to the agonists in a microassay and the acrosomal status of live sperm was determined by indirect immunofluorescence using PSA-FITC/Hoechst double-staining. Pretreatment with pertussis-toxin (100 ng/ml) and EGTA (2.5 mmol/l) significantly inhibited the ZP-induced acrosome reaction without affecting the spontaneous rate of exocytosis. Progesterone (1.25 microg/ml) and human follicular fluid (10%) exerted a priming, time-dependent effect on the ZP-induced acrosome reaction. These studies demonstrated that: (i) acrosomal exocytosis of capacitated human sperm triggered by the homologous ZP is dependent on the activation of G(i) proteins (pertussis toxin-sensitive) and the presence of extracellular calcium; and (ii) progesterone and follicular fluid exert a priming effect on the ZP-induced acrosome reaction.     

Fertilization and early embryology: Timing of pronuclear development and first cleavages in human embryos after subzonal insemination: influence of sperm phenotype

The sequential transformations of human sperm nuclei in humaneggs after subzonal insemination (SUZI; n = 104) and the influenceof sperm defects on this timing were studied. This chronologywas compared to that of two control series of zygotes obtainedafter SUZI with normal spermatozoa (n = 35) and after in-vitrofertilization (IVF) with normal donor spermatozoa (D-IVF; n= 220). Pronuclear formation took place between 4.5 and 10.5h post-SUZI for 92.8% of the zygotes. They remained visiblefor 13 h and began to disappear 18.5 h post-SUZI. The time-spanbetween pronuclear disappearance and cleavage was 3 h. Zygotesobtained after D-IVF had a similar rate of pronuclear disappearancebut 4 h later. The second cell cycle was more rapid for zygotesobtained by D-IVF than by SUZI, but the developmental rate ofzygotes obtained by SUZI varied according to sperm phenotypes.For patients with previous unexplained IVF failures (controlgroup with normal spermatozoa), the developmental rate was lower,suggesting the influence of oocyte quality. In conclusion, theend of the first cell cycle of zygotes obtained by inseminationunder the zona pellucida appears 4 h earlier compared to zygotesobtained after insemination outside the zona pellucida.     

Species specificity of human and murine anti-ZP3 synthetic peptide antisera and use of the antibodies for localization and identification of ZP3 or ZPC domains of functional significance

The mammalian zona pellucida has an important function in the fertilization process. The zona pellucida protein 3 (ZP3 or ZPC) is the ligand for primary sperm binding and induces the acrosome reaction. In various species, ZP3 primary structures are highly conserved as revealed by cDNA cloning. The objective of these studies was to localize ZP3 protein using antisera generated against defined synthetic peptides that are specific for mouse or for human ZP3. Immunohistochemistry and transmission electron microscopy were applied to murine and human ovary sections. Immunochemical studies were performed in hemizonae pellucidae from microbisected human oocytes. Using the competitive hemizona assay and various anti-ZP3 antibodies, we further intended to identify human ZP3 epitopes of functional significance. Our results showed that antiserum AS ZP3-9 (mouse specific) detected mouse ZP3 protein in mouse oocytes and in immunoblots, whereas AS ZP3-14 (human specific) detected human ZP3 protein in human ovary sections, native hemizonae pellucidae and in immunoblots. ZP3 material was also detected in cumulus cells by immunohistochemistry. Ultrastructural studies showed an equal distribution of ZP3 throughout the zona pellucida. The human competitive hemizona assay revealed that none of the anti-ZP3 synthetic peptide antisera affected sperm binding suggesting that those epitopes are not involved in primary sperm binding. Anti-porcine ZP3 beta protein antibodies (polyclonal) blocked human sperm-zona pellucida binding. In summary, these anti-ZP3 synthetic peptide antibodies specifically reacted with intact ZP3 protein (murine and human) but did not inhibit human sperm-zona pellucida binding; anti-ZP3 antibodies can therefore be used as biomarkers for ZP3 localization and function.     

Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C

To facilitate our understanding of the role of zona pellucida glycoproteins during fertilization in humans, recombinant human zona pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of approximately 110, approximately 70-75 and approximately 65 kDa, respectively, as compared to approximately 80, approximately 65 and approximately 50 kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose alpha 1-3 or mannose alpha 1-6 residues) and Jacalin (alpha-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P=0.0005) and hZPC (P=0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase (P>0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway.     

Fertilization and early embrology: A sequential analysis of the effect of progesterone on specific sperm functions crucial to fertilization in vitro in infertile patients

The objective of these studies was to evaluate the modulatoryeffect(s) of progesterone on sperm functions crucial to fertilizationin infertile men with abnormal sperm parameters. A prospective,controlled study applying a sequential diagnostic analysis capableof identifying specific dysfunctions of the male gamete wasperformed. Patients (n = 14) were allocated to the study groupif they had a history of infertility of >1 year durationand after semen evaluation showed teratozoospermia (< 14%normal sperm forms as diagnosed by strict criteria) or terato-asthenozoospermia(< 50% progressive motility). After swim-up separation ofthe motile sperm fraction, the following functions were assessedwith and without previous exposure to progesterone (1.0 µg/ml):acrosome reaction (using Pisum sativum agglutinin), hyperactivatedmotility (using a computerized semen analyser), sperm-zona pellucidabinding (in the hemizona assay), sperm-zona pellucida penetration(in a sperm-zona penetration assay), and sperm-oocyte penetration(using the hamster zona-free oocyte/sperm penetration assay).Progesterone did not affect the percentage of acrosome-reactedspermatozoa after 1 or 3 h of incubation. Hyperactivated motilitywas significantly enhanced by progesterone after 1 h (12 ±4 versus 6 ± 2% in controls; P < 0.02). Although progesteronedid not affect sperm-zona binding, it significantly enhancedboth sperm-zona pellucida penetration (27 versus 12% in controls;P = 0.03) and sperm-oocyte penetration (15 versus 8% in controls;P < 0.05). Because those sperm functions enhanced by progesteroneare crucial to fertilization, the steroid may have value inthe treatment of some male-factor patients undergoing assistedreproductive therapy.     

Studies on the membrane integrity of human sperm treated with a new injectable male contraceptive

BACKGROUND: The aim of this study was to evaluate the integrityof sperm surface characteristics in the presence of a new malecontraceptive, RISUG [1 mg styrene maleic anhydride (SMA)/100µl dimethylsulphoxide (DMSO) in 1 ml sperm solution].METHODS: Progressively motile human sperm were treated in vitrowith RISUG. The cells were analysed for the release of 5'-nucleotidase(5'-NT) (a plasma membrane marker) using 3 mmol/l 5'-AMP and3 mmol/l -glycerophosphate as substrates. Hyaluronidase (anacrosomal membrane marker) was analysed using hyaluronic acidas a substrate. The contents of free and total acrosin, and% proacrosin (all acrosome markers) were assayed using 0.5 mmol/l-N-benzoyl-L-arginine ethylester (BAEE). RESULTS: RISUG causedalmost complete disintegration of the plasma membrane leadingto significant (P<0.0001) release of 5'-NT into the surroundingmedia. Complete dissolution of the acrosome with concomitantvesiculation of the membrane system, as judged from the lossof hyaluronidase, was observed. Total acrosin content in thesperm was also reduced to almost 10%, and proacrosin droppedto 13.2% in the presence of RISUG in comparison to 90.2% incontrol (P<0.0001), indicating dispersion of acrosomal contents.CONCLUSION: Under in vitro conditions, RISUG, at a concentrationof 1 mg SMA dissolved in 100 µl of DMSO, caused significantdamage to the acrosome and its contents, indicating loss offunctional ability of sperm.