Gonadotrophin-releasing hormone antagonists inhibit sperm binding to the human zona pellucida.

Previous work from our laboratory indicated that gonadotrophin-releasing hormone (GnRH) increases human sperm-zona pellucida binding. Here we present evidence that GnRH antagonists inhibit sperm-zona pellucida binding in humans. Motile spermatozoa (10(7) cells/ml) were incubated in modified Tyrode's medium at 37 degrees C, in 5% CO(2) in air. After 4.5 h, aliquots of spermatozoa were treated with saline (control) or with different concentrations of GnRH antagonists (test). Each sperm aliquot was then tested in the hemizona binding assay. In this assay, the control aliquot was incubated with half a human zona pellucida (hemizona) and the test aliquot was incubated with the matching half. After 20 min, the hemizonae were withdrawn and the number of zona-bound spermatozoa counted using phase-contrast microscopy. In addition, the effect of GnRH antagonists upon the pattern of sperm movement, frequency of sperm-zona pellucida collisions, and percentage of living and acrosome-reacted spermatozoa was determined. The results indicated that treatment with GnRH antagonists decreased the number of zona-bound spermatozoa and did not change the pattern of sperm movement, frequency of sperm-zona collisions, and percentage of acrosome-reacted spermatozoa. We suggest that this action of GnRH antagonists may be due to an effect on zona receptors on the sperm plasma membrane.     

Hyper-osmotic condition enhances protein tyrosine phosphorylation and zona pellucida binding capacity of human sperm

BACKGROUND: The aim of this study was to determine the effect of culture medium osmolality, in the range known to occur in the male and female reproductive tracts, on human sperm tyrosine phosphorylation and sperm-zona pellucida (ZP) interaction in vitro. METHODS: Motile sperm (2x10(6)), selected by swim-up from semen of normozoospermic men with normal sperm-ZP binding, were incubated with or without four oocytes in 1 ml human tubal fluid (HTF) medium with different osmolalities (150, 200, 280, 350, 400 mOsm/kg) adjusted by variation of the NaCl concentration. After 2 h incubation, the number of sperm bound to the four ZP was examined, sperm motility and velocities were assessed by Hamilton-Thorn Motility Analyzer (IVOS 10) and sperm tyrosine phosphorylation was assessed by both western immunoblotting and immunofluorescence with an anti-phosphotyrosine monoclonal antibody (PY20). The effect of hyper-osmolality (400 mOsm/kg) on the ZP-induced acrosome reaction (AR) was also determined. RESULTS: Incubation of human sperm in hyper-osmotic medium significantly increased tyrosine phosphorylation and the number of sperm bound to the ZP. In contrast, hypo-osmotic medium significantly decreased both tyrosine phosphorylation and sperm-ZP binding. Medium with high osmolality (400 mOsm/kg) significantly reduced the ZP-induced AR. Both hypo- and hyper-osmotic media significantly decreased average sperm percentage progressive motility and velocities. CONCLUSION: Incubation of human sperm in hyper-osmotic media was associated with significantly increased tyrosine phosphorylation and ZP-binding ability but severely reduced the ZP-induced AR.     

Case report: pregnancy outcome following ICSI of oocytes with abnormal cytoplasm and zona pellucida

A couple with unexplained infertility was referred for routine IVF and ICSI treatment. Ovulation was induced by the GnRH analogue protocol combined with HMG and HCG. Preparation of denuded oocytes revealed a major disorder of the zona pellucida and abnormal oocytes. During preparation of ova for ICSI, 15 retrieved oocytes were denuded, 14 of which underwent ICSI treatment. Four of the oocytes collapsed and the remaining 10 appeared to have irregular, fragile zona pellucida. Nevertheless, following ICSI, seven low-quality embryos developed, three of which were transferred into the uterus. Two implantations were achieved, but only one embryo resulted in an uneventful pregnancy and delivery by Caesarean section of a normal female neonate with an Apgar score of 10. It is hypothesized that infertility was due to the abnormal oocyte structure and abnormal zona pellucida, which prevented natural conception. This condition was successfully resolved by the ICSI procedure.     

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.     

Four zona pellucida glycoproteins are expressed in the human

BACKGROUND: The zona pellucida (ZP) is an extracellular glycoprotein matrix which surrounds all mammalian oocytes. Recent data have shown the presence of four human zona genes (ZP1, ZP2, ZP3 and ZPB). The aim of the study was to determine if all four ZP proteins are expressed and present in the human. METHODS: cDNA derived from human oocytes were used to amplify by PCR the four ZP genes. In addition, isolated native human ZP were heat-solubilized, trypsin-digested and subjected to tandem mass spectrometry (MS/MS). RESULTS: All four genes were expressed and the respective proteins present in the human ZP. Moreover, a bioinformatics approach showed that the mouse ZPB gene, although present, is likely to encode a non-functional protein. CONCLUSIONS: Four ZP genes are expressed in human oocytes (ZP1, ZP2, ZP3 and ZPB) and preliminary data show that the four corresponding ZP proteins are present in the human ZP. Therefore, this is a fundamental difference with the mouse model     

Defective sperm-zona pellucida interaction: a major cause of failure of fertilization in clinical in-vitro fertilization

Sperm-zona pellucida binding and penetration were assessed on the oocytes that failed to fertilize from couples with >/=3 oocytes treated by standard in-vitro fertilization (IVF). There were four groups: fertilization rate 0% (n = 369), 1-25% (n = 194), 26-50% (n = 81) and 51-95% (n = 100). Of the couples with zero fertilization rate 70% had 相似文献   

Detection of zona pellucida proteins during human folliculogenesis

BACKGROUND: The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage. METHODS: Follicles (from n = 18 patients, 14-40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined. RESULTS: All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. CONCLUSIONS: ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.     

High frequency of defective sperm-zona pellucida interaction in oligozoospermic infertile men

BACKGROUND: The ability of sperm to interact with the zona pellucida (ZP) plays a critical role during the process of human fertilization. The aim of this study is to determine frequency of defective sperm-ZP interaction in oligozoospermic infertile men. METHODS: Sperm-ZP binding assays and the ZP-induced acrosome reaction (AR) were performed in 72 infertile men with a sperm concentration <20 x 10(6)/ml. Oocytes that had previously failed to fertilize in a clinical IVF programme were used for the tests. Motile sperm (2 x 10(6)/ml) selected by swim-up from each semen sample were incubated with four oocytes for 2 h. The number of sperm bound per ZP and the ZP-induced AR were assessed. Under these conditions, an average of < or =40 sperm bound/ZP was defined as low sperm-ZP binding and a ZP-induced AR < or =16% was defined as low ZP-induced AR. RESULTS: In the 72 oligozoospermic men, 28% (20/72) had low sperm-ZP binding. Of those with normal sperm-ZP binding, 69% (36/52) had low ZP-induced AR. Overall, 78% (56/72) had either low ZP-binding or normal ZP binding but low ZP-induced AR. This means that only 22% (16/72) had both normal sperm-ZP binding and normal ZP-induced AR. CONCLUSION: Oligozoospermic men have a very high frequency of defective sperm-ZP interaction, consistent with their low natural fertility or low fertilization rate in conventional IVF. Infertile couples with oligozoospermic semen should be treated by ICSI rather than by conventional IVF.     

A simple method for the morphological analysis of human ova: zona penetration in oocytes failing to fertilize or fertilizing abnormally in vitro

A simple and rapid technique is described whereby several unfertilized oocytes and embryos can be processed simultaneously for embedding and sectioning for histological analysis. Oocytes are encased in a serum matrix and thereafter can be handled with ease. A total of 112 unfertilized oocytes and abnormal embryos following insemination in vitro have been examined. Only 31% of oocytes showed pyknotic nuclei after a mean of 4.0 +/- 1.24 days in culture and 37% had recognizable metaphase II chromosomes. Twenty per cent showed evidence of fertilization, but spermatozoa had failed entirely to penetrate the zonae pellucidae of 34% of oocytes. The mean percentages of penetrating sperm were not significantly different between oocytes which did and did not fertilize. Under these in-vitro conditions, approximately 1-2% of spermatozoa reaching the oocyte penetrated into the zona pellucida and a lesser number to the perivitelline space. Polar bodies were also observed and the incidence of divisions, retention and parthenogenesis estimated. This technique may advance our understanding of the reasons for the success or failure of fertilization in vitro.     

Scanning electron microscopy analysis of the human zona pellucida: influence of maturity and fertilization on morphology and sperm binding pattern

Human oocytes from the same as well as from different patients have an extremely heterogeneous morphology of the zona pellucida surface as shown by scanning electron microscopy. For years it has been believed that this heterogeneous morphology plays an important part in the sperm-oocyte interaction. It was the aim of this investigation to analyse the morphology and the sperm binding patterns of the human zona pellucida. Oocytes were divided into four categories: mature, immature, fertilized and unfertilized. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between zona type and oocyte maturity or zona type and achieved fertilization. However, fertilized (polyploid) oocytes had a more compact and smooth zona surface than unfertilized ones. The analysis of the number and distribution patterns of bound spermatozoa on the zona pellucida revealed extremely variable patterns regardless of the zona morphology. Significant differences between mature and immature oocytes did not appear. In both groups there were oocytes with either no or numerous bound spermatozoa on the zona pellucida. Oocytes overloaded with spermatozoa could only be found in the mature group. Unfertilized oocytes had fewer bound spermatozoa on average than polyploid zygotes.     

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     

Carbohydrate analysis of the zona pellucida and cortical granules of human oocytes by means of ultrastructural cytochemistry

BACKGROUND: The zona pellucida (ZP), the mammalian oocyte coat, consists of a restricted number of highly glycosylated proteins. In vitro sperm binding studies suggest a higher binding affinity for the outer region of the ZP compared to its inner region in different species. However, the reason for this difference in binding distribution remains unresolved. Many studies suggest that the carbohydrate sequences linked to ZP glycoproteins act as ligands for sperm binding to this matrix. METHODS: Lectins and antibodies that recognize different carbohydrates were employed to perform an ultrastructural analysis of human ZP and cortical granule glycosylation. RESULTS: This study reveals variable glycosylation of the human ZP throughout its thickness, with pronounced differences between the most external and internal regions of this matrix. The binding studies also indicate that ZP glycoproteins express some carbohydrate sequences not previously detected in other species. Finally, cytochemical analysis of human cortical granules suggests similarities in glycosylation to ZP glycoproteins but not to cortical granules from other mammalian species. CONCLUSION: A heterogeneous carbohydrate composition was observed in the thickness of the human ZP that could be responsible for the different sperm binding affinity detected between the outer and inner regions of the ZP.     

Regulators of sperm function: Composition of the human zona pellucida and modifications following fertilization

The composition of individual human zonae pellucidae and modificationsto this extracellular coat both before and after fertilizationwere analysed using a rapid, sensitive, non-radioactive biotinylation-or lectin-based detection system; these assays use commerciallyavailable reagents and can be performed on fragments of individualzonae pellucidae. The zona pellucida from unfertilized eggsis composed of three glycoprotein species designated as huZP1,huZP2 and huZP3. Under non-reducing conditions, the molecularweights of these proteins are 150 kDa, 100 kDa, and 55–65kDa respectively. Following fertilization, huZP1 was not detectedunder either non-reducing or reducing conditions. In contrast,after fertilization huZP2 was detected under non-reducing conditions,but not under reducing conditions. The ability to detect pre-and postfertilization changes in a single human zona pellucidais discussed in relation to its value in assessing deficienciesin clinical and laboratory protocols used for in-vitro fertilization.     

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.     

Fertilization and early embryology: Scanning electron microscopy of the zona pellucida of human oocytes during intracytoplasmic sperm injection (ICSI)

During intracytoplasmic sperm injection (ICSI) approximately10% of all injected oocytes degenerate. The reason for thisprocess is unknown. It has been speculated that the mechanicalprocedure of the insertion of the ICSI needle induces injuriesto the zona pellucida which lead to the death of the cell. Byscanning electron microscope (SEM), it could be shown that thesurface structure of mature oocytes is extremely elastic sothat the injection needle penetrates the zona pellucida withoutdestroying the mesh-like or more compact surface. No tissuepieces or zona fragments were detectable. After a culture timeof 15 min the penetration site on the zona was no longer easilyvisible. We believe that oocyte degeneration is not caused bythe penetration of a glass needle into the ooplasm but by aninjury to the meiotic spindle or by an excessive dose of fluid[polyvinylpyrrolidone (PVP) or medium] during sperm injection.     

The impact of cigarette smoking on zona pellucida thickness of oocytes and embryos prior to transfer into the uterine cavity

BACKGROUND: Smoking has been reported to promote infertility. The zona pellucida plays an important role in fertilization and implantation. We report, for the first time, the effect of cigarette smoking on zona pellucida thickness of oocytes and embryos as one of the factors that may interfere with fertility. METHODS: This study comprised 169 women, grouped according to their smoking habits: 31 active smokers, whose husbands do not smoke; 44 active smokers, whose husbands smoke; 65 passive smokers, because of smoking husbands and 29 non-smokers (women and husbands). Zona pellucida thickness was measured prospectively on printed photos of 903 oocytes and 456 embryos. RESULTS: The zona pellucida thickness of oocytes and embryos of non-smoking women was significantly thinner than those of active and passive smokers. However, no significant differences were observed in the natural ability of the zona pellucida to become thinner after 48 h in culture. CONCLUSIONS: Our study demonstrates that active and passive cigarette smoking increases the zona pellucida thickness of oocytes and embryos. Our findings also show that active and passive smoking has no significant effect on the thinning mechanism of the zona pellucida, which implies that it is independent of the initial zona pellucida thickness.     

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.     

High magnitude of light retardation by the zona pellucida is associated with conception cycles

BACKGROUND: Failures in expression of zona proteins correlate to subfertility in animals. Low expression of the zona proteins by the growing human oocyte may indicate reduced developmental potential. Therefore, we non-invasively analysed the thickness and the structure of the zona pellucida (ZP) of human oocytes with respect to embryo fate after ICSI. METHODS: Retardance magnitude and thickness of the inner, middle and outer layers of the ZP were quantitatively analysed by a Polscope in 166 oocytes selected for transfer after ICSI (63 patients; 32.8 +/- 4.4 years) on the basis of pronuclear score at day 1. Blastomere number was determined at day 2. Data were compared between conception cycles (CC; 65 oocytes/23 patients) and non-conception cycles (NCC; 101 oocytes/40 patients) and with respect to maternal age. RESULTS: The thickness was slightly elevated (P < 0.001), and the mean magnitude of light retardance was nearly 30% higher (P < 0.001) in the inner layer of the zona pellucida of oocytes contributing to CC compared to NCC. Embryos in the CC group tended to develop faster. CONCLUSIONS: The magnitude of light retardance by the zona pellucida inner layer appears to present a unique non-invasive marker for oocyte developmental potential.     

Exposure of actin on the surface of the human sperm head during in vitro culture relates to sperm morphology, capacitation and zona binding

BACKGROUND: The aim of this study was to determine the relationship between the proportion of motile sperm with actin exposed on the surface of the head and sperm function. METHODS: Semen samples were obtained from normozoospermic men and sperm function tests were performed. Motile sperm selected by swim-up were incubated with actin monoclonal antibody (A-mAb, 1:100) for 2 h, then anti-mouse IgG Dynabeads were used to detect sperm-bound A-mAb. Sperm capacitation was increased by phorbol myristate acetate (PMA) and decreased by bicarbonate-free medium. RESULTS: The proportion of sperm with exposed actin increased with time for up to 2 h incubation. Bicarbonate-free medium significantly decreased the proportion of sperm with exposed actin. PMA significantly enhanced this phenomenon. Sperm bound to zona pellucida (ZP) had a significantly higher proportion with exposed actin than did sperm remaining in medium. Of the 79 samples studied, an average of 9.4% (range 1-27%) of motile sperm had exposed actin after 2 h incubation and this was significantly correlated with sperm normal morphology and ZP binding. CONCLUSION: Exposure of actin on the surface of the sperm head during in vitro culture may be related to membrane modification during sperm capacitation and hence may be a useful marker for this subpopulation of sperm.     

Factors associated with accidental fractures of the zona pellucida and multipronuclear human oocytes following in-vitro fertilization

A total of 101 multipronuclear oocytes (7.4% of fertilizations)were retrospectively identified in this in-vitro fertilizationprogramme. The use of a manual syringe suction system, insteadof an electric pump, to aspirate follicles, was associated witha significant increase in the proportion of oocytes with fracturedzonae pellucidae (P < 0.001), a lower normal fertilizationrate (P < 0.05) and a higher proportion of multipronuclearfertilizations (P < 0.001). Irrespective of the mode of follicularaspiration, significantly more multipronuclear fertilizationsoccurred following stimulation with a combination of clomipheneand human menopausal gonado-trophin, than after clomiphene alone(P < 0.05). It was concluded that the aspiration pressures,created by syringe suction, were more likely to rupture thezona pellucida of some oocytes, while in others it predisposedto an increased multipronuclear fertilization rate.