Isolated Zonae Pellucidae Research Articles

Expression of Tas1 taste receptors in mammalian spermatozoa: functional role of Tas1r1 in regulating basal Ca²⁺ and cAMP concentrations in spermatozoa.

BackgroundDuring their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined.Methodology/Principal FindingsThe present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca2+ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca2+ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes.Conclusions/SignificanceSince Ca2+ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract.

The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein

Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 (+/- 120 Kd) and ZP3 (+/- 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.

Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.

Binding of recombinant human proacrosin/acrosin to zona pellucida (ZP) glycoproteins. I. Studies with recombinant human ZPA, ZPB, and ZPC

To characterize proacrosin/acrosin interaction with isolated zona pellucida (ZP) components. Prospective study. Basic research laboratory. Recombinant proteins derived from human proacrosin (Rec-40, Rec-30, Rec-20, Rec-10, and Rec-6) and from human ZP glycoproteins (rec-hZPA, ZPB, and ZPC). In vitro binding assay developed to assess proacrosin/acrosin-ZP interaction. Zona pellucida glycoprotein binding to proacrosin/acrosin; estimation of binding affinity. Of all ZP proteins, rec-hZPA demonstrated the highest binding activity toward acrosin (Rec-30) (rec-hZPB: 42% of rec-hZPA; rec-hZPC: 39% of rec-hZPA; P<.0005). Rec-hZPA interaction was disturbed by dextran sulphate (75% inhibition with 10 microM), fucose (67% inhibition with 1.5 microM), and mannose (69% inhibition with 333 mM). Comparing binding activity of proacrosin with other N-terminal acrosin fragments, Rec-40 showed 2.6-3 times higher levels. Moreover, saturable high affinity binding of Rec-40 to ZP components was observed (Kd: 34 nM for rec-hZPA, 38 nM for rec-hZPB, 63 nM for rec-hZPC). The rec-hZPA is the major ZP ligand for human proacrosin/acrosin. The interaction involves mannosyl, fucosyl, and sulfated glycans. Binding sites for rec-hZP would be located both at the N- and C-terminus of proacrosin, revealing a key role of the proenzyme in the interaction.

Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida.

The recognition and binding of sperm cells to the zona pellucida (the extracellular matrix of the oocyte) are essential for fertilization and are believed to be species specific. Freshly ejaculated sperm cells do not bind to the zona pellucida. Physiologically this interaction is initiated after sperm activation in the female genital tract (capacitation) via a yet unknown mechanism, resulting in the binding of a receptor in the apical sperm plasma membrane to the zona pellucida. In order to mimic this biochemically, we isolated zona pellucida fragments from gilt ovaries to prepare an affinity column with the intact zona pellucida structure and loaded this column with solubilized apical plasma membranes of boar sperm cells before and after in vitro capacitation. With this technique we demonstrated that two plasma membrane proteins of capacitated boar sperm cells showed high affinity for zona pellucida fragments. Further analysis showed that these proteins were tyrosine phosphorylated. Plasma membrane proteins from freshly ejaculated sperm cells did not exhibit any zona pellucida binding proteins, likely because these proteins were not tyrosine phosphorylated.

Immunoreactivity with native zona pellucida of antibodies against a 19 amino acid synthetic peptide corresponding to human ZP3

Immunization of female Syrian hamsters with a 19mer-synthetic peptide corresponding to amino acid sequence 323–341 of human ZP3 and coupled to diphtheria toxoid (DT) led to generation of antibodies against both the peptide as well as DT. Antisera showed positive reaction in ELISA with solubilized isolated zona pellucida (SIZP) from human oocytes. In an indirect immunofluorescence assay, the anti-peptide antibodies bound to zona pellucida of human and bonnet monkey but failed to recognize that of mouse, rabbit, hamster and dog. These studies will help in designing a synthetic peptide based ZP immunocontraceptive vaccine for human application.

Structural and functional relationships between mouse and hamster zona pellucida glycoproteins

The hamster egg's extracellular coat, or zona pellucida, consists of three glycoproteins, designated hZP1, hZP2, and hZP3, that exhibit extensive heterogeneity on SDS-PAGE. hZP1 is a relatively minor component of hamster zonae pellucidae, as compared with hZP2 and hZP3. In the presence of reducing agents, hZP1, 200,000 apparent M r, migrates on SDS-PAGE with an apparent M r of 103,000. This suggests that hZP1, like mouse ZP1, is composed of two polypeptides held together by intermolecular disulfides. When purified hamster ZP glycoproteins were tested at relatively low concentrations in an in vitro competition assay, employing either hamster or mouse gametes, only hZP3 (56,000 apparent M r) exhibited sperm receptor activity (i.e., inhibited binding of sperm to eggs). Thus, apparently hZP3 is the hamster counterpart of mouse ZP3, the mouse egg receptor for sperm. Furthermore, at relatively high concentrations, solubilized hamster egg ZP preparations induced both hamster and mouse sperm to undergo the acrosome reaction in vitro. hZP3 is encoded by a relatively abundant ovarian mRNA that is detected by a mouse ZP3 cDNA probe and is the same size, about 1.5 kb, as mRNA encoding the mouse sperm receptor, ZP3 (83,000 apparent M r). Like mouse ZP2, hZP2 undergoes limited proteolysis following artificial activation of hamster eggs in vitro. Results of in vitro assays employing intact eggs and isolated zonae pellucidae demonstrate that hamster eggs possess a ZP2-proteinase which has a substrate specificity similar to that of the mouse enzyme. These observations are discussed in terms of structural and functional relationships that may exist between hamster and mouse zona pellucida glycoproteins.

Electro-rotation of mouse oocytes: single-cell measurements of zona-intact and zona-free cells and of the isolated zona pellucida

Passive electrical properties of oocytes and of zonae pellucidae, and the mechanical coupling between them, can be elucidated by means of rotating-field-induced rotation. In low-conductivity media (25–100 μS/cm) rotation of mouse oocytes (with or without their zonae) requires fields in the 1–100 kHz frequency range. However, an isolated zona shows weak rotation in the opposite direction to that of a cell, and in response to much higher field frequencies (approx. 1 MHz). In zona-intact mouse oocytes, the rotation of cell and zona are not rigidly coupled: thus rotation of the cell can still be induced when the zona is held stationary. However, rotation of freely suspended zona-intact cells is much slower than that of zona-free cells and requires an optimum field frequency that is approximately 1.5 kHz higher. These observations show that the electrical properties of the oocyte that are measured by rotation are altered by the presence of the zona pellucida, even though no such influence has been detected using micro-electrodes. The data are consistent with the zona acting as a porous shell with a conductivity of 40 μS/cm (preliminary estimate made a a single medium conductivity of 26 μS/cm). Measurements on cells from which the zonae had been removed gave values for the membrane capacity and resistivity of 1.2–1.3 μF/cm 2 and 400 Ω · cm 2, respectively. These values may reflect the presence of plasmalemma microvilli. The results strongly suggest that the technique may be useful for studies of cell maturation and for in vitro fertilization, because the cells may be further cultured after measurement.

Bicarbonate is essential for fertilization of mouse eggs: mouse sperm require it to undergo the acrosome reaction.

Fertilization of mouse eggs by capacitated mouse sperm occurs readily in vitro in a defined culture medium designated medium CM, derived from Krebs-Ringer bicarbonate medium by supplementation with glucose, lactate, pyruvate, and albumin. Medium CM contains 25 mM HCO-3 equilibrated with CO2 to pH 7.4. Replacement of the CO2/HCO-3 buffer with 25 mM N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES) while otherwise maintaining pH, ionic strength, and composition identical to that of medium CM, yielded medium HM in which the percentage of in vitro fertilization observed was consistently zero. Addition of 10 mM HCO-3 to medium HM yielded medium HMB, in which the percentage of eggs fertilized was 70%, equal to that observed in medium CM. This percentage applies to cumulus-free, zona-intact eggs. With zona-free eggs, the percentage fertilization increased to 90% in media CM and HMB, but remained zero in medium HM. This result shows that HEPES does not inhibit fertilization while omission of CO2/HCO-3 blocks it completely. Capacitation of mouse sperm, as determined by chlortetracycline fluorescence assay, proceeded at the same rate in media CM and HMB, but was retarded in medium HM. Sufficient sperm became capacitated, however, that this could not be the block of fertilization. The acrosome reaction in sperm bound to isolated zonae pellucidae, as also determined by chlortetracycline fluorescence assay, occurred with the same time course in media CM and HMB, but did not occur in medium HM.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoelectrophoretic analysis of the porcine zona pellucida.

Antigens of the porcine zona pellucida were evaluated by 2-dimensional and line immunoelectrophoretic techniques. Zona antigen preparations studied included heat-solubilized isolated zonae pellucidae (SIZP), a purified 60 000 Mr glycoprotein (PPZA, purified pig zona antigen), and two fractions of this 60 000 Mr zona component which had been exposed to SDS (ZP3-E1C and ZP3-E2C). Antisera were raised to intact zonae pellucidae (IZP), SIZP and PPZA. Collectively, electrophoretic data revealed that the porcine zona system is antigenically complex with each zona antiserum tested detecting numerous antigens in the various zona preparations. These antigens, however, all had similar electrophoretic mobilities, and this limited the resolution of these techniques. The 60 000 Mr pig zona macromolecule (ZP3) appeared to be the most immunogenic of the three major pig zona glycoproteins since antisera prepared against IZP or SIZP reacted primarily with this component. However, the 60 000 Mr component does share antigenic determinants with the other major zona glycoproteins as revealed by cross-reactions of the antisera with the various zona preparations. Electrophoretic studies also suggested that the various zona antisera could distinguish, with different degrees of sensitivity, multiple antigenic determinants on the individual zona macromolecules. These studies also indicated that SDS treatment of zona glycoproteins does alter the antigenicity of the macromolecule, both with respect to the total number and individual identity of antigens detected.

Electrophoretic and Chromatographic Properties of Boar Sperm Plasma Membranes: Antigens and Polypeptides with Affinity for Isolated Zonae Pellucidae

Two methods were used to assess the ability of plasma membrane (PM) polypeptides to bind to isolated zonae pellucidae (ZP). Crossed immunoelectrophoresis patterns of detergent solubilized PM were compared in the presence and absence of ZP. Two antigens which represented less than 20% of the total antigen response were significantly changed in electrophoretic mobility and precipitin peak height. In the second experiment, detergent solubilized PMS, labeled with iodine‐125 (1125), were absorbed to a column containing purified ZP. Washed columns eluted with SDS‐urea were found to be enriched in several polypeptides which migrate in PAGE‐SDS gels below 22 Kd. Comparison of PAGE patterns of PM originating primarily from the sperm head with those enriched in PM from other regions of the sperm surface suggest that these low molecular weight polypeptides with affinity for isolated ZP are concentrated in the sperm head. These experiments provide a base for determining which PM components are involved in spermegg interactions in vivo.

Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte's zona pellucida

The zona pellucida is an acellular coat which surrounds the plasma membrane of fully grown mammalian oocytes and which performs a variety of important functions during oogenesis, fertilization, and preimplantation development. In this investigation the proteins of the mouse oocyte's zona pellucida have been identified and characterized by using zonae pellucidae isolated individually from fully grown oocytes with mouth-operated micropipets. Various morphological and biochemical criteria were employed to assess the purity of the isolated zonae pellucidae and, in most cases, they were found to be virtually free of contamination by other oocyte proteins. It was determined that each zona pellucida contains 4.8 ng of protein, which represents 80% or more of the dry weight of the zona pellucida and about 17% of the oocyte's total protein. Electrophoretic analyses of as few as five isolated zonae pellucidae treated with diazotized [ 125I]iodosulfanilic acid revealed the presence of only three radiolabeled proteins, designated ZP1, ZP2, and ZP3. The same three proteins were identified by Coomassie blue staining when large numbers of isolated zonae pellucidae (approximately 750) were subjected to SDS-polyacrylamide gel electrophoresis. These three proteins migrate as broad bands on SDS-polyacrylamide gels, consistent with their being glycoproteins, with apparent molecular weights of 200,000 (ZP1), 120,000 (ZP2), and 83,000 (ZP3). The same proteins were radiolabeled when intact oocytes were treated with diazotized [ 125I]iodosulfanilic acid, a reagent which does not penetrate the oocyte's plasma membrane, or when isolated zonae pellucidae were treated with 3H-labeled 1-dimethylaminonaphthalene-5-sulfonyl chloride. Results of amino acid analysis and high-resolution two-dimensional electrophoresis of the individual proteins suggest that each protein represents a unique polypeptide chain. The proteins ZP1, ZP2, and ZP3 represent about 36, 47, and 17%, respectively, of the total protein of the zona pellucida. In the presence of reducing agents which cause dissolution of the zona pellucida, ZP1 is converted into a species which migrates with an apparent molecular weight of 130,000, suggesting that it exists as an oligomer, stabilized by disulfide bonds, in the unreduced state. The results of these experiments are discussed in terms of the properties of the zona pellucida before and after fertilization and are compared with results obtained using vitelline envelopes of eggs from nonmammalian animal species.

Inhibition of fertility in mice by passive immunization with antibodies to isolated zonae pellucidae.

A chromatographically purified preparation of gamma globulin produced against isolated zonae pellucidae of mice was used to immunize mice. A single 2.5 mg dose totally inhibited fertility for a minimum of 11 days.

Effect of antisera against eggs and zonae pellucidae on fertilization and development of mouse eggs in vivo and in culture.

A single intraperitoneal injection of female mice with rabbit antisera against denuded mouse eggs and isolated zonae pellucidae inhibited fertilization for at least 25--30 days. Intraperitoneal injection appeared to be more effective and lasted longer than subcutaneous or intramuscular injection but oral administration had no effect. Such a passive immunization of females given after fertilization had no effect on embryonic development or the sex ratio of fetuses. The fertilizing capacity of mouse spermatozoa was suppressed by treatment of the sperm suspension with anti-egg but not with anti-zona serum. Treatment of mouse eggs with these antisera did not affect their development to blastocysts in culture but did inhibit the shedding of the zona pellucida and attachment to the dish.

Surface interactions between mammalian sperm and egg: variation of spermatozoa concentration as a probe for the study of binding in vitro.

The pre-penetration binding interactions between gametes of the golden hamster were investigated in vitro. Binding between capacitated spermatozoa and the surface of eggs, that is the zonae pellucidae with intact vitelli, as a function of the concentration of spermatozoa, followed a sigmoidal curve. This was in sharp contrast to the linear binding obtained with mechanically isolated zonae pellucidae (zonae lacking vitelli). Penetration of eggs as a function of the concentration of spermatozoa paralleled the binding curve that occurred between gametes. The binding curve obtained with uncapacitated spermatozoa and eggs was not sigmoidal but was linear after a slight lag and parallel to the curve obtained with uncapacitated spermatozoa and isolated zonae pellucidae. Taken together these results support previous work which implicated a vitelline factor in the binding reaction between the surfaces of eggs and capacitated spermatozoa. By scoring binding at one minute intervals it was possible to relate the rapid uninterrupted binding that occurs between capacitated spermatozoa and isolated zonae pellucidae with the equally rapid but transient and vitellus-influenced binding that occurs between gametes. It was concluded that the vitelline factor acts by preventing most of the early type of binding that occurs between spermatozoa and isolated zonae pellucidae and not by terminating the early, rapid, initial binding as previously postulated. Thus, this early binding never occurs between most of the gametes that finally bind 30 to 40 minutes later and, therefore, does not play a role in the establishment of the late binding step which leads to penetration.

Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs.

Receptors for Ricinus communis agglutinin I (RCAI), concanavalin A (Con A), and wheat germ agglutinin (WGA) were localized on the zonae pellucidae and plasma membranes of hamster, mouse, and rat eggs with ferritin-lectin conjugates. Intact eggs labeled with the ferritin conjugates showed dense concentrations of RCAI and WGA receptors in the outermost regions of their zonae pellucidae and sparse distributions of Con A receptors throughout the zonae. Ferritin-lectin labeling was specific, since inhibitory saccharides effectively blocked labeling. The asymmetric density of RCAI receptors across the zona was confirmed by ferritin-RCAI and fluorescein-RCAI labeling of mechanically isolated zonae pellucidae, indicating that the RCAI-binding sites are more densely distributed in the exterior zona regions. Plasma membranes of rodent eggs contained RCAI, WGA, and Con A receptors. These receptors were found to be more or less randomly distributed on surfaces of aldehyde-fixed eggs or on eggs labeled near 0 degrees C. However, eggs incubated at 25 degrees C showed aggregated WGA- and Con A-binding site distributions on their plasma membranes. This indicates that lectin-induced receptor redistribution occurs at this temperature. The possibility that plasma membrane receptor mobility is a requirement for sperm-egg fusion is discussed.

Contact between hamster spermatozoa and the zona pellucida releases a factor which influences early binding stages.

Summary. The studies reported here demonstrate that isolated hamster zonae pellucidae affect the binding between capacitated hamster spermatozoa and eggs included in the same drop. Mouse zonae pellucidae fail to affect binding between hamster gametes. These results suggest that binding, which occurs only between homologous spermatozoa and isolated zonae pellucidae causes a factor (S1 factor) to be released. The SI factor is diffusible and influences the two successive binding steps (B1 and B2) previously shown to occur between capacitated spermatozoa and eggs. The B2 step is delayed by the action of the SI factor during the early part of the interval between B1 and B2 suggesting that the effect may be mediated through Bl. It is proposed that the SI factor is also released during the first binding interaction (B1) between the spermatozoa and the egg which appears to be an analogous but terminated form of the binding to the isolated zona pellucida. The data are consistent with the possibility that the B1 step is terminated by the product of the interaction between the S1 factor and a factor from the vitellus or perivitelline space.

Nature of the pre-penetration contact interactions between hamster gametes in vitro.

The biphasic species-specific binding of hamster spermatozoa to eggs or isolated zonae pellucidae was manipulated in vitro in microdroplets in experiments with the trypsin inhibitor p-aminobenzamidine (pABA). Epidiymal spermatozoa capacitated by incubation for 5 hours in modified Medium 199 mixed with contents of an oviduct collected 17 hours after an injection of human chorionic gonadotropin were observed in 20 mcl drops with 15-20 fresh eggs or isolated zonae pellucidae. Sperm binding was scored after 3 washes in fresh medium. Normally 25-40% of the ova bound 1 or 2 sperm in 3 minutes then released them a process designated B1. 30 minutes later the ova bound 5-10 spermatozoa a process called B2 until penetration by 1 spermatozoon takes place 40-45 minutes after mixing. B1 B2 and binding to isolated zonae pellucidae were species specific since mouse ova included in the droplets did not bind hamster sperm. Further mouse eggs could not precondition hamster eggs for the B2 binding as shown by exposing sperm which had been incubated with mouse eggs for 25 minutes to hamster eggs to accelerate the B2 binding. Incubating sperm with pABA 5/10000 M for 30 minutes delayed B1 10-20 minutes and abolished B2 but did not inhibit binding to isolated zonae pellucidae. When both gametes were treated with pABA 50% and 80% of the ova bound sperm after 10 and 20 minutes. At 20 minutes binding had gradually declined to zero; no B2 phase took place. With zonae pellucidae pretreated with pABA B1 binding was not terminated. These and previous experiments were taken to imply that the ovum releases a substance that preconditions sperm for B2 and that a protease is involved in releasing sperm bound in B1.

Early contact interactions between mammalian gametes in vitro: evidence that the vitellus influences adherence between sperm and zona pellucida.

A study in vitro of interactions between gametes of the golden hamster showed that the spermatozoon associates with the zona pellucida, or outer coat of the ovum, by two successive steps termed attachment and binding. Attachment, apparently the first step in fertilization, is not species-specific; it is insensitive to temperature (2 degrees ) and is reversible. Binding, on the other hand, is species-specific, temperature-sensitive, and irreversible. Experiments with isolated zonae pellucidae indicated that the vitellus (cellular portion of the ovum) influences the interaction between the sperm and the zona pellucida by increasing the time required for the sperm to bind. This effect is exerted on the sperm while they are attached to the zona pellucida.