Zona Penetration Research Articles

Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro.

The minimum and maximum extracellular Ca2+ concentrations required to promote capacitation, the acrosome reaction, hyperactivated motility, zona penetration and gamete fusion in the mouse have been established. The traces of free calcium in Ca2+-deficient medium were shown not to enhance capacitation since the inclusion of EGTA to chelate free ions during a 120 min preincubation failed to alter the kinetics of capacitation from those observed in the absence of EGTA; 1 h after addition of 1.80 mM-Ca2+, both suspensions were highly fertile. Complete capacitation, when suspensions were immediately functional upon the addition of 1.80 mM-Ca2+, required the presence of greater than or equal to 90 microM-Ca2. Considerably higher concentrations were required to initiate optimal sperm responses: acrosome reaction, 900 microM; gamete fusion, 900 microM; hyperactivated motility, 1.80 mM; zona penetration, 1.80 mM. None of these changes was effected when Ca2+ was less than 450 microM. The responses to elevated Ca2+ were dependent on the length of incubation, being initially positive and then negative. A short (30 min) exposure to 3.40 mM-Ca2+ (x 2 the standard) accelerated capacitation, as evidenced by significantly increased acrosome loss, precocious expression of hyperactivated motility and enhanced fertilizing ability when Ca2+ was reduced to 1.80 mM. However, extended (120 min) preincubation irreversibly damaged sperm function. In the presence of 7.20 mM-Ca2+ (x 4), fertilizing ability was inhibited at both 30 and 120 min, despite a high incidence of acrosome loss. The primary deleterious effect appeared to be on motility which was judged to be more erratic than in 1.80 mM-Ca2+, possibly due to elevated intracellular Ca2+. Because of the considerable difference in threshold Ca2+ concentrations, it is now possible to dissociate the Ca2+-dependent events of capacitation from those of the acrosome reaction and motility changes.

Mouse sperm antigens that participate in fertilization: IV. A monoclonal antibody prevents zona penetration by inhibition of the acrosome reaction

To investigate the molecular basis of gamete interaction in mammals, monoclonal antibodies (mAbs) have been generated by syngeneic immunization with mouse testis. Previous work has described two particular mAbs, M41 and M42, which localize indistinguishably to the plasma membrane overlying a restricted portion of the acrosome, but recognize different antigens. One of the mAbs, M42, inhibits mouse fertilization in vitro significantly, but only in the presence of the zona pellucida, whereas M41 has no apparent effect upon any assayable event in the fertilization process. The experiments described here were performed to identify the precise event of sperm-zona interaction (sperm-zona binding, induction of the acrosome reaction, or penetration through the zona) that is affected by M42 mAb. Capacitated mouse sperm binding to the zona pellucida was undiminished following pretreatment with M42 mAb, when compared to levels achieved using either no mAb- or to M41 mAb-treated control sperm. When the effect of mAbs on the zona-induced AR was examined, the percentage of acrosome reacted (AR) sperm at the zona surface increased with time, plateauing at approximately 90 min post-insemination, with 78% of the bound cells AR in the control and the M41 mAb-treated groups. M42-treated sperm never achieved greater than 23% AR cells over the 120-min interval assayed. To quantitate this effect, capacitated sperm were exposed to increasing concentrations of acid-solubilized zonae. Increased proportions of AR sperm were found in the control and M41 mAb-treated groups, up to a maximum of 70–76% AR cells with 8 or 12 zonae/μl. In contrast, M42-treated sperm displayed only 21–28% AR cells over the entire range of zonae concentrations tested. An entirely different result emerged when acrosome reactions were induced with A23187: M42 was no longer able to prevent the AR. This ability of A23187 to override M42 mAb's inhibitory effect on the AR permitted specific examination of the possible effect of M42 mAb on sperm penetration through the zona pellucida. In the presence of A23187, zona penetration levels for M42 mAb-treated sperm were equivalent, both qualitatively and quantitatively, to control and to M41 mAb-treated sperm under the same conditions. It appears, therefore, that M42 mAb identifies a high molecular weight doublet (220–240 kDa) of mouse sperm that participates specifically in the induction of the sperm's acrosome reaction as it occurs under physiological conditions.

GROUP DISCUSSION

GROUP DISCUSSION

The evolution of hamster sperm motility during capacitation and interaction with the ovum vestments in vitro

AbstractMovement characteristics of golden hamster spermatozoa were studied upon collection from the cauda epididymis, during an incubation which capacitates the spermatozoa in vitro, during penetration of the cumulus, and during attachment to and penetration of the zona pellucida. High‐speed videomicrography was employed to quantitate flagellar beat frequency and shape. The status of the acrosome was also assessed. During capacitation, hamster spermatozoa become increasingly invigorated before the onset of hyperactivated motility. Within the cumulus, beat frequency and curvature are reduced, apparently in response to the physical resistive properties of the matrix material. These properties appear to vary within the cumulus. Initial attachment to the zona precedes completion of the acrosome reaction, is non‐rigid, and is accompanied by increased beat frequency and curvature. Subsequently, the onset of rigid binding to the zona, completion of the acrosome reaction, and increased flagellar beat frequency are very closely associated in time. The latter produces an increase in thrust against the zona. Preliminary results indicate that ensuing zona penetration requires not more than five minutes, is at oblique angles, and is associated with a continuation of vigorous flagellar beating.

An electron microscopic study of sperm penetration into the human egg investments

The ultrastructure of human spermatozoa located in the cumulus cells and the zona pellucida of a pro-nuclear egg, and in the zona pellucida of a two-cell egg, both fertilized in-vivo, has been analysed in order to understand how the human spermatozoon penetrates the investing coats of the oocyte. Among the 36 spermatozoa found in the cumulus cells, 31 were phagocytosed by cumulus cells and 5 were wedged in the matrix between the cells. These spermatozoa were acrosome-reacted and their equatorial segment was intact. Six of the seven spermatozoa found in the zona pellucida (four spermatozoa in the pronuclear egg and three in the two-cell egg) had lost the equatorial segment, while the other one was partially reacted. The sperm heads were located in slits with sharp edges. From these findings it was concluded that in the human (1) only few and normal spermatozoa seem to reach the cumulus cells after natural insemination, (2) the acrosome reaction probably occurs sometime before the spermatozoa reach the vicinity of the corona cells, (3) the reaction of the equatorial segment seems to occur during or before the initial phase of zona penetration, since the spermatozoa located in the matrix of the zona pellucida had no equatorial segment. No evidence of the presence of spermatozoa with an intact acrosome in the matrix of cumulus cells or with an intact equatorial segment in the zona pellucida were found.

Blocking of human fertilization in vitro by sera with sperm-immobilizing antibodies

Serum samples with sperm-immobilizing antibody activity from six women were examined for ability to block sperm-egg interaction by a zona penetration test where human follicular ova matured in vitro were used. Exposure of spermatozoa from a fertile healthy donor to the sera impaired binding to and penetration through the zona pellucida of the spermatozoa completely in five cases and incompletely in one case. Successful fertilization in vitro was achieved by using fetal cord serum instead of autoserum of the patient included in the in vitro fertilization and embryo transfer program. These results suggest that interference with sperm-egg interaction may be an additional mechanism of infertility that is caused by antisperm antibodies.

Fertility control in the bitch by active immunization with porcine zonae pellucidae: use of different adjuvants and patterns of estradiol and progesterone levels in estrous cycles.

To determine the changes in patterns of 17 beta-estradiol and progesterone levels underlying abnormal cycles in bitches immunized with solubilized crude porcine zonae pellucidae (cPZP), to attempt to circumvent these problems by immunizing with a purified zona fraction (pPZP), and to test the effectiveness of different adjuvants, bitches were immunized with cPZP or pPZP 2-6 times with no adjuvant, Freund's adjuvant, alum adjuvant, or the adjuvant CP-20,961. The bitch immunized without adjuvant had a low titer with a normal cycle and fertility. Immunization with cPZP and adjuvant produced moderate to high titers of antizona antibodies and infertility. Bitches with high titers experienced abnormal estrous cycles. Estradiol rose during proestrus, but instead of falling sharply in early estrus as in controls, it remained elevated. Progesterone did not rise. The moderate-titered bitches had normal cycles and steroid patterns. Bitches immunized with pPZP had moderate titers. Cycles were normal after 3 injections, but after 6 injections one bitch had an abnormal cycle. One pPZP-immunized bitch remained fertile but the others were infertile. Alum was the mildest adjuvant, causing no injection site lesions, but the highest titers occurred with Freund's and CP-20,961 adjuvants. All three adjuvants induced titers sufficient to inhibit fertility. Infertility in bitches immunized with PZP may be due to prevention of zona penetration, because their antisera inhibited zona penetration of oocytes by spermatozoa in vitro. However, alterations in ovarian function preventing ovulation and luteinization could be involved in high-titered bitches.

The kinetics of human sperm binding to the human zona pellucida and zona‐free hamster oocyte in vitro

AbstractIn these experiments the mean number of sperm bound to the zonae pellucidae of immature human oocytes and to the vitelli of zona‐free hamster oocytes were counted after centrifuging the oocytes through a discontinuous dextran gradient into a fixative. Sperm suspensions (107 sperm/ml) were preincubated in BWW medium containing 35 mg/ml of human serum albumin for 0 to 20 hr and coincubated with oocytes for 4 hr.The kinetics of sperm binding and sperm penetration were clearly different for the two types of oocytes in this system. Sperm binding to the zona pellucida appeared to be associated with zona penetration; when the zona was penetrated, many sperm were tightly bound and vice versa. A similar association between human sperm binding to the zonafree hamster oocyte and hamster oocyte penetration was not so apparent. Transmission electron microscopy revealed that all sperm which were “bound” to the hamster vitellus were acrosome reacted. On the surface of the human zona pellucida acrosome intact and acrosome reacted sperm were observed.These results suggest that tight binding of spermatozoa to the zona pellucida may be an important preliminary step in human fertilization. If this is true the activity of the sperm's “receptor for zona” may not be detected by the assay with zona‐free hamster oocytes.

Monoclonal antibodies to rabbit sperm autoantigens. I. Inhibition of in vitro fertilization and localization on the egg

Biochemical and antigenic similarities exist among members of what can be considered a family of low molecular weight rabbit sperm autoantigens. These autoantigens are intrinsic plasma membrane glycoproteins specific to spermatogenic cells and spermatozoa. The amino acid and carbohydrate compositions of rabbit sperm autoantigen-1 (RSA-1) and RSA-2 were compared and monoclonal antibodies (mAb) were raised in mice against rabbit sperm autoantigens. The epitopes recognized by the antibodies were present on RSA-1, 2 and 3. A monoclonal anti-RSA-1, 2 and 3 (designated A.F. 1) was used to localize the antigen on spermatozoa and testis cells and investigate the epitope's tissue specificity. This mAb inhibited in vitro fertilization but did not block the sperm from dispersing the cumulus cells surrounding the egg. The mAb also demonstrated the presence of RSA-1, 2 and 3 on the plasma membrane of the egg after fertilization. It is concluded that the RSA family plays a central role in zona penetration.

Relationship between the length of sperm preincubation and zona penetration in the golden hamster: A scanning electron microscopy study

AbstractHamster spermatozoa are able to fertilize a high percentage of zona‐intact hamster oocytes when they are preincubated for 2 hr in a chemically defined medium. From this time on, the longer the preincubation time the lower the percentage penetration. Spermatozoa preincubated for 6 or more hr are unable to cross the zona pellucida, retaining however their ability to fuse with zona‐free hamster oocytes.Zona‐intact hamster oocytes, as described above, were observed with the scanning electron microscope. When the oocytes were inseminated with spermatozoa preincubated for 1 to 5 hr the outer surface of the zona showed the penetrating spermatozoa and the sperm tracks made by those that failed to cross it. With longer preincubation times no penetrating spermatozoa were observed, and very few sperm tracks were present on the outer surface of the zona.Control experiments showed that neither eggs, spermatozoa, nor fertilization were affected by the medium recovered after long preincubations.These results show that care should be taken regarding the preincubation time when using the in‐vitro fertilization technique.

Ultrastructural localization of proacrosin and acrosin in ram spermatozoa

AbstractProacrosin and acrosin were localized immunocytochemically at the electron microscope level in ram spermatozoa undergoing an ionophore‐induced acrosome reaction. Antigenicity was preserved after fixation with 0.5% w/v ethyl‐(dimethylaminopropyl)‐carbodimide, and an antibody preparation was used that reacted with all major forms of ram acrosin. All stages of the acrosome reaction could be observed in a single preparation. At the earliest stage, labeling was observed throughout the acrosomal contents, which were just beginning to disperse. As dispersal proceeded, labeling diminished, being associated only with visible remnants of the acrosomal matrix. By the time the acrosome had emptied, almost no labeling could be detected on the inner acrosomal membrane.The relationship between matrix dispersal and proacrosin activation was studied in isolated ram sperm heads. While proacrosin was prevented from activating, the acrosomal matrix remained compact; but as activation proceeded, the matrix decondensed and dispersed in close parallel. By the time proacrosin activation was complete, the acrosomal contents had almost entirely disappeared.We conclude that proacrosin is distributed throughout the acrosomal contents as an intrinsic constituent of the acrosomal matrix. During the acrosome reaction, proacrosin activation occurs, resulting directly in decondensation of the matrix. All the contents of the acrosome including acrosin disperse and, by the time the acrosome is empty and the acrosomal cap is lost, only occasional traces of acrosin remain on the inner acrosomal membrane. Since the acrosomal cap is normally lost during the earliest stages of zona penetration, acrosin's role in fertilization is unclear: it does not appear to be a zona lysin bound to the inner acrosomal membrane.

Monoclonal antibody against mouse sperm blocks a specific event in the fertilization process.

Hybridoma cell lines were produced by PEG (polyethylene glycol)-induced fusion of myeloma cells (NS-1/X63 Ag 8) with dispersed spleen cells from a Balb/c male mouse immunized syngeneically with homogenized Balb/c testis. Indirect immunofluorescence on mature (cauda epididymal) mouse sperm was employed as the primary screening assay. This procedure allowed the immediate identification of hybridomas which produce monoclonal antibodies of interest with respect to the fertilization process. One hybridoma cell line, M29, secretes an antibody of the IgM class that localizes to the equatorial segment of the mouse sperm head. This antibody is not species specific, but is restricted to the homologous functional area, the equatorial segment, in all other sperm examined (hamster, rabbit, human). When tested for a possible effect on the fertilization process, ascites fluid containing M29 antibody was very effective in blocking the fertilization of mouse gametes in vitro. The same inhibiting activity was observed regardless of whether the M29-treated sperm first encountered the cumulus cell layer, the zona pellucida, or the egg plasma membrane. A large number of M29-treated sperm were capable of penetrating zonae pellucidae when this layer was present. It appears, therefore, that M29 monoclonal antibody prevents fertilization specifically at the level of interaction between the sperm and egg plasma membranes; the other prefertilization events (hyperactivated motility, zona binding, acrosome reaction, zona penetration) seem little affected. Using the M29 antibody and related probes that are specific for particular biological events, it should be possible to map, in molecular terms, the functional domains of the sperm cell's membrane.

What functions of the sperm cell are measured by in vitro fertilization of zona-free hamster eggs?

Human spermatozoa were capacitated in media containing either high concentrations (3.5%) of human serum albumin (HSA) or low concentrations (0.3%) of bovine serum albumin (BSA). The effects of both capacitation media were assessed immediately and after overnight preincubation (18 to 24 hours) by adding a mixture of nonliving human oocytes and living zona-free hamster eggs to the sperm suspension. Overnight preincubation of sperm in media containing 3.5% HSA enhanced sperm fusion with hamster vitelli, but the capacity for zona penetration was lost. These effects could be attributed to the concentration of HSA rather than a general effect of albumin concentration, because overnight preincubation in 3.5% BSA did not interfere with zona penetration. During overnight incubation in 3.5% HSA, the percentage of acrosome reactions increased, as did alterations in the equatorial segment of the acrosome-reacted sperm. The percentage of motile sperm remained high after overnight incubation in 3.5% HSA, but both the mean swimming speed and flagellar activity on the zona surface declined. The sperm also lost their ability for strong zona binding after overnight incubation in 3.5% HSA.

Immunocytochemical localization of acrosin on both acrosomal membranes and in the acrosomal matrix of porcine spermatozoa.

Immunocytochemical techniques were employed to determine, at the ultrastructural level, the location of acrosin in porcine spermatozoa. Antisera to highly purified porcine acrosin was produced in rabbits. The (Fab')2 fragments of the immunoglobulins were purified and conjugated with horseradish peroxidase (HRP). Washed, formaldehyde-fixed spermatozoa were reacted with the labeled antiacrosin immunoglobulins, utilizing a direct staining technique. Electron microscopy revealed that the peroxidase reaction product of HRP-antiporcine acrosin was distributed evenly over the outer acrosomal membrane of spermatozoa with intact acrosomes. The labeled antibody was also distributed evenly over the inner acrosomal membrane of cells when the overlying acrosomal structures were absent. In some spermatozoa, labeling was noted throughout the acrosomal matrix. No significant labeling was observed in control specimens when spermatozoa were exposed to HRP-antiporcine acrosin immunoglobulins that had been adsorbed previously with excess purified acrosin or exposed to HRP-conjugated rabbit antiporcine immunoglobulins. This pattern of labeling is consistent with the hypothesis that acrosin may function as a zona lysin. The observation that the outer acrosomal membrane and acrosomal matrix are labeled suggests that acrosin is not exclusively located on the inner acrosomal membrane and, thus, could participate in physiologic events other than zona penetration.

Ultrastructural Observations on the Penetration of Human Sperm into the Zona Pellucida of the Human Egg In Vitro

Sperm penetration into the zona of 46 human eggs was examined by electron microscopy. Preovulatory oocytes were aspirated at laparoscopy and inseminated by methods which produced normal pregnancies. These were routinely fixed 3–72 hours after insemination and examined for zona penetration.The mechanism of zona penetration was similar in the non‐activated oocytes, fertilized ova, and embryos obtained, and resembled that which was reported in most mammals. Sperm were tightly bound to the zona by their plasma membranes. Both acrosome‐reacted and unreacted sperm were seen in the cumulus and entering the zona. The acrosome reaction involved multiple fusions and vesiculation of the plasma and outer acrosome membranes. Fine and coarse vesiculation was observed, and the sperm digested a clear pathway through the zona. Sperm were rarely seen in the inner zona and never seen in the perivitelline space of monospermic ova, but freely penetrated these regions in polyspermie ova. A block to polyspermy seemed to operate at the level of the inner zona. Morphologic evidence for the involvement of the inner acrosome membrane in the acrosome reaction and a possible sequence of events in zona penetration is presented. The findings confirm most of late Professor Pierre Soupart's work on zona penetration.

Anti‐Mouse Sperm Antiserum

Rabbit anti‐mouse sperm (αMS) Fab fragments have been prepared, which are capable of inhibiting spermzona interaction (both binding and penetration) and sperm‐egg fusion in vitro. In cumulusintact eggs, αMS Fab did not inhibit zona penetration, but did inhibit sperm‐egg fusion, implying that separate and distinguishable antigens are involved in these two fertilization events. Preliminary identification of the antigens recognized by αMS IgG, using a gel overlay technique, revealed four major antigens of 200 Kd, 55–63 Kd, 37 Kd, and 33 Kd, and three minor antigens of 110 Kd, 48 Kd, and 40 Kd.

Time Resolution of the Reactions Preceding Penetration of the Zona Pellucida

3‐Quinuclidinyl benzilate (QNB) is a potent inhibitor of fertilization of mouse eggs in vitro: it acts by blocking penetration of the zonae by sperm. The time course of this inhibition was examined by addition of 50 μM QNB to the in vitro system for fertilization of cumulusfree mouse eggs at increasing times post‐insemination. QNB, added prior to and at intervals up to 20 minutes post‐insemination, inhibited fertilization by more than 90%. When added at longer times, QNB inhibition decreased; at 60 minutes post‐insemination, added QNB had no effect on fertilization. The time course of onset of insensitivity to QNB inhibition was compared to the time courses of other reactions identified as precursors to fertilization in the mouse system. The most rapid reaction is Ca2+‐mediated binding of sperm to zonae, which is reversible by EGTA; it has no time lag and is complete by 15 minutes post‐insemination. The second reaction is onset of insensitivity of this binding to reversal by EGTA; it has a lag time of 5 minutes post‐insemination and all bound sperm become insensitive 20 minutes post‐insemination. The acrosome reaction occurring in sperm bound to zonae has a lag time of 25 minutes, and is not completed in all bound sperm until 240 minutes post‐insemination. The time course of loss of QNB inhibition lies between the second reaction and the acrosome reaction. These studies allow the time resolution of four sequential reactions, which lead to fertilization of mouse eggs in vitro and occur prior to penetration of the zona pellucida. These are: EGTA‐reversible, Ca2+‐mediated binding of sperm to zona; onset of insensitivity to EGTA reversal of binding; onset of insensitivity to QNB inhibition of zona penetration; and onset of the acrosome reaction in sperm bound to zonae.

Effect of albumin on fertilization of mouse ova in vitro

AbstractSerum albumin is an obligatory component of the incubation medium for the fertilization of mouse ova. Normal, untreated bovine serum albumin supports high rates of fertilization of cumulus‐free ova both with and without their zonae pellucidae. Heat‐treated or trichloroacetic acid‐extracted bovine serum albumin is unable to support the fertilization of a majority of zona‐intact ova but fertilization of zona‐free ova is unimpaired. Spermatozoa incubated in medium containing heated bovine serum albumin fertilize zona‐intact ova when 2 mM caffeine is present but the progress of sperm head decondensation is delayed when compared to normal controls. Trichloroacetic acid extracted BSA preferentially and irreversibly inhibits zona penetration by spermatozoa, but this effect is not mediated by an inhibition of spermatozoal motility or zona‐binding ability. This effect occurs after only a 10‐min preincubation of the spermatozoa in the extracted BSA or when the medium contains only a 10% (v/v) proportion of this albumin. It is estimated that mouse spermatozoa under the conditions used take 2 hr to penetrate the zonae pellucidae of 50% of ova and effect fertilization.

Only acrosome-reacted spermatozoa can bind to and penetrate zona pellucida: a study using the guinea pig.

We have examined the behavior of guinea pig spermatozoa towards the guinea pig zona pellucida in vitro. Experiments were designed to determine whether the state of capacitation or the acrosome reaction determined the ability of the spermatozoa to attach to the zona. Freshly collected cauda epididymal spermatozoa are acrosome-intact (AI) and, by definition, uncapacitated. These cells could not attach (at least persistently) to the zona. We then sampled spermatozoa from the progressively capacitating population at various time intervals. At no point did AI spermatozoa from such samples have the ability to attach to the zona. On the other hand, acrosome-reacted (AR) spermatozoa that appeared in increasing number during this capacitation period rapidly and firmly adhered to the zona. Adhesion by AR spermatozoa prepared in several ways culminated in successful zona penetration and fertilization of the ovum. The data provide unequivocal evidence that AR guinea pig spermatozoa attach to zonae and fertilize the ova. We find no evidence that AI spermatozoa firmly attach or bind to the zona, regardless of their state of capacitation.

Antisperm antibody inhibition of sperm penetration of zona-free hamster eggs

Serum samples with sperm-immobilizing antibody activity from six women were examined for ability to block sperm-egg interaction by a zona penetration test where human follicular ova matured in vitro were used. Exposure of spermatozoa from a fertile healthy donor to the sera impaired binding to and penetration through the zona pellucida of the spermatozoa completely in five cases and incompletely in one case. Successful fertilization in vitro was achieved by using fetal cord serum instead of autoserum of the patient included in the in vitro fertilization and embryo transfer program. These results suggest that interference with sperm-egg interaction may be an additional mechanism of infertility that is caused by antisperm antibodies.