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The block to sperm penetration in zona-free mouse eggs
DEVELOPMENTAL
BIOLOGY
64,
l-10
(1978)
The Block to Sperm Penetration
in Zona-Free
Mouse Eggs
DON P. WOLF
with an Appendix by KEITH SOPER’ Division of Reproductive Biology, Department of Obstetrics and Gynecology, and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received September 10,1976; accepted in revised form December IS,1977 The rate of sperm penetration and the number of sperm penetrating zona-free mouse eggs were found to he dependent on sperm concentration. At the lowest sperm concentrations examined (IO’ cells/ml, sperm-egg ratios of approximately l:l), most eggs were penetrated (75%), and polyspermy was low (19%) following 3 hr of incubation. The number of sperm penetrating the egg was logarithmically related to sperm concentration. All eggs showed a delay of at least 20 min between insemination and penetration, and penetration was complete in approximately 2 hr at lo4 sperm/ml; this penetration block was attributed to egg-related changes. The existence and timing of the egg plasma membrane block to polyspermy were evaluated by reinsemination experiments. In this approach, the block was triggered in zona-free eggs with a low concentration of capacitated epididymal sperm at time 0, and the eggs were subsequently challenged with high sperm concentrations. The presence or absence of a block was inferred from the degree of polyspermy observed in these eggs after 3 hr of incubation. Adjusting for sperm concentration-dependent delays between insemination and sperm penetration, a blocking time of approximately 40 min was obtained. INTRODUCTION
block in mammalian eggs is limited. Barros and Yanagimachi (1972), studying the susceptibility of zona-free hamster eggs to refertilization, concluded that formation of the egg plasma membrane block required 2 to 3.5 hr. Baaed on temporal requirements, the zona reaction precedes the plasma membrane block in the hamster, challenging the significance of the latter event. In the mouse (Pavlok and McLaren, 1972) and the rat (Toyoda and Chang, 1968; Niwa and Chang, 1975), evidence for the existence of an egg plasma membrane block derives from the observation that zona-free eggs display polyspermy levels comparable to those of zona-intact controls. However, proteolytic enzymes were employed during zonae removal in these studies, raising the possibility that the egg’s receptivity to sperm was abnormal (Wolf et al., 1976). The development of a procedure for the mechanical removal of zonae (Wolf et al., 1976) provided the basis for the present
Normal development of mammalian embryos is dependent upon monospermic fertilization and the exclusion of supernumerary spermatozoa. While evidence for the existence of a zona reaction, a block to sperm penetration of the zona pellucida following penetration of the fertilizing sperm, has been presented (Braden et al. 1954;Barros and Yanagimachi, 1971; Gwatkin et al., 1973; Wolf and Hamada, 1977), monospermic embryos recovered from natural matings or from in vitro inseminations occasionally contain sperm in the pervitelline space, i.e., supplemental sperm (Austin, 1968). In the rabbit, large numbers of supplemental sperm are always seen. These observations necessitate an egg plasma membrane block to sperm penetration. Direct experimental evidence defining the ’ Epidemiology Unit, Department Medicine, University of Pennsylvania, Pennsylvania.
of Research
Philadelphia,
1 0012-1606/78/0641-ooO1$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
DEVELOPMENTAL BIOLOGY VOLUME64,1978
2
tion, as measured following 3 hr of incubation, was defined over a 105-fold range in sperm concentration (Fig. 1). Included in the figure are the unadjusted values for the mean number of sperm per egg at each MATERIALS AND METHODS sperm concentration, representing data Unfertilized tubal ova were recovered from six experiments each employing a diffrom superovulated Swiss mice (6-12 weeks ferent epididymal sperm preparation. Since old) and freed from surrounding cumulus sperm-egg interaction is, in part, dependent cells by exposure to hyaluronidase (O.l%, on the particular sperm preparation emSigma Type I) in a modified Krebs-Ringer ployed, an analysis of covariance was apbicarbonate medium previously described plied to give values adjusted for differences (Inoue and Wolf, 1974; Wolf and Inoue, among sperm preparations (see Appendix). 1976). Such eggs are regarded as normal, The higher concentrations studied here since they produce viable young at the con- were similar to those routinely employed trol frequency when transferred to foster for in vitro fertilization of intact eggs with females (Gates, 1971). Zonae pellucidae sperm-egg ratios of 5 or 10 X lo5 to 1. For were then removed by aspiration through comparison, penetration levels for intact micropipets (Wolf et al., 1976), washed eggs, calculated from Wolf and Inoue three times and transferred to 200~~1drops (1976), have been included in Fig. 1. At the of culture medium under silicone oil (Dow- lowest concentrations, sperm-egg ratios apCorning 200 Fluid; 50 cs). Unless otherwise proached the presumed physiological range indicated, eggs were inseminated with ap- of 1 to 1. Virtually all zona-free eggs were proximately 2 x lo5 capacitated sperm/ml penetrated at concentrations above lo2 (Wolf and Inoue, 1976). Experiments were cells/ml (75% at 102; 96-100% at ~10~). terminated by washing eggs free of adher- Polyspermy levels increased from 19% at ent sperm, followed by transfer to a slide lo2 cells/ml to r60% at the higher sperm for mounting and staining with acetolac- concentrations, and the mean number of moid (Toyoda and Chang, 1974). Ova were sperm per inseminated egg was logarithconsidered penetrated when a sperm tail(s) mically related to sperm concentration. and head remnant(s) or a male pronucleus were observed within the cytoplasm. References for the statistical treatment of the data are included in the Appendix; for statistical purposes, batches of eggs were considered homogeneous, as they were always derived from lo-animal pools and were all potentially penetrable. For an analysis of pseudo-first-order rate processes, see Segel (1968). investigation in which the occurrence and kinetics of a block to polyspermy were defined in zone-free mouse eggs inseminated in vitro.
RESULTS
Sperm Concentration Dependency in Penetration In setting out to examine the zona-free egg’s ability to modulate its own penetration, the influence of sperm concentration on the kinetics and maximum levels of penetration was studied. Maximum penetra-
LOG SPERM CONCENTRATION
FIG. 1. Sperm concentration
dependency in the penetration of mouse eggs. The mean number of sperm recovered at each inseminating concentration was calculated for zona-free eggs (closed circles) and corrected for differences in sperm preparations by an analysis of covariance (open circles). Comparable uncorrected values are included for zona-intact eggs (closed squares) from Wolf and Inoue (1976).
DON P. WOLF
Block to Sperm Penetration
The kinetics of sperm penetration (lo4 cells/ml) of zona-free eggs are presented in Fig. 2. Routinely, a delay was seen between insemination and penetration, as shown in Fig. 2 by the percentage of unfertilized eggs remaining at 100% for a time following the addition of sperm. This period, observed with both washed and unwashed preincubated epididymal sperm, may reflect the time requirements for sperm binding and for the occurrence of an acrosome reaction. However, it was not subject to reduction by sperm preincubation (30 min) in the presence of zona-free eggs. It should also be noted that the method employed here to assess penetration (microscopic examination of acetolacmoid-stained whole mounts) does not allow detection of the earliest stages of sperm penetration. Following this delay, the percentage of unfertilized eggs decreased as eggs became monospermic, and subsequently polyspermic, with penetration ceasing at approximately 2 hr. These relationships can be described by equation E+S-
kl
ES+S-
kz
ES2 + S ~
ks
loo-~:
0 --Y 180 TIME (min.) FIG. 2. The kinetics of penetration of zona-free mouse eggs inseminated at lo4 sperm/ml. The distribution of unfertilized (crosses), monospermic (open circles), and polyspermic (solid circles) eggs are presented as a function of insemination time. Data points represent means of four individual experiments.
...,
where E = egg, S = sperm, ES = monospermic egg, and ES2 = dispermic egg. As anticipated, the penetration rate for the first sperm (kd is seen in Fig. 2 to be greater than that for subsequent sperm (kz). The rate constants (k1 and kz) were estimated assuming that penetration behaved as a pseudo-first-order rate process; and therefore, log E = -kt/2.303. Figure 3 is a plot of log percentage of unfertilized or monospermic eggs versus time, where the slope equals -k/2.303. Based on these plots, the treatment of penetration as a pseudo-first-order rate process appears valid, although data collected at the longer times were unreliable. The rate of penetration of the first sperm was 0.036 min-’ and for subsequent sperm, 0.012 min-‘. By extrapolating the data in Fig. 3 to 100% un-
I
I
I
0
60
I
120
180
TIME (Min.)
FIG. 3. Time-dependent decreases in the log percentage of unfertilized and monospermic xona-free eggs in the presence of lo4 sperm/ml. Log E versus t for unfertilized (open circles) and monospermic (solid circles) eggs where the slope = -k/2.303 and t,,* =
0693/k.
fertilized (log E = 2), the time required for the sperm to acquire its penetrating capacity can be estimated (the delay time). This value was 26 min for the first sperm and, although the data are less precise, of the same magnitude for subsequent sperm. The ~I/Z values for penetration by the first and
4
DEVELOPMENTALBIOLOGY
subsequent sperm were 20 and 57 min, respectively. Sperm concentration-dependent variations in the kinetic parameters defining initial penetration can be seen in Table 1. With increasing sperm concentration, the rate of penetration and the number of penetrated sperm increased, while the delay between insemination and penetration decreased. At the lowest sperm concentration, interexperimental variability was large, an observation that is critical to the interpretation of the reinsemination experiments described below. The recorded k1 value of 0.089 was derived from only two experiments; in three others, penetration rates at 60 min were too low to allow accurate quantitation. These results illustrate the variability attributable to different sperm preparations. Experiments conducted with different concentrations of a single sperm preparation corroborate the findings in Table 1 (data not included).
The Block to Sperm Penetration Direct experimental evidence for an egg plasma membrane block to sperm penetration is provided by the demonstration that maximal penetration is reached and maintained in zona-free eggs following 2 hr of insemination at lo4 sperm/ml (Fig. 4). In the absence of an egg plasma membrane block, penetration should increase as a function of time until all sperm have exTABLE
1
SPERMCONCENTRATIONDEPENDENCYINTHE KINETICSOFPENETRATIONOFZONA-FREEEGG@ Sperm concentration
(cells/ml) 16-163 lo4 106
Penetration rate kl (min-‘)
Delay time (min)
t, (min)
Mean number of sperm
per egg 0.001-0.089 0.036 0.091
30-45 26 22
20 8
1.20 2.34 3.55
“Least squares regression lines were determined from data collected from four or five experiments at each sperm concentration. Kinetic parameters were then calculated as described in the text: tl = 0.693/k. The k, value for lb-l@ cells/ml represents the maximum rate seen in two of five experiments, the other penetration rates being less than 0.001 min-‘.
VOLUME 6‘i,1978 3r
1
60
I
120 TIME (Min.)
I
180
FIG. 4. Establishment of an egg plasma membrane block to sperm penetration in zona-free eggs. Eggs were inseminated with lo4 capacitated epididymal sperm/ml at time zero. At the indicated times, samples were removed and processed for the quantitation of penetrated sperm, as described in Materials and Methods. Both uncorrected (open circles) and corrected values (closed circles; analysis of covariance accounting for sperm preparation differences) for the mean number of sperm per egg are presented. The fitted curve was generated by an equation with a quadratic term in time.
pired. A quadratic term in time (see Appendix) indicates that the mean number of sperm per egg levels off to. a maximum value by 140 min (P
DON P. WOLF
5
Block to Sperm Penetration
original eggs, original eggs plus fresh sperm, and fresh eggs plus 2-hr sperm, respectively.
Time Requirements of the Block to Polyarmy In order to investigate the existence and timing of the presumed physiological block which maintains a monospermic embryo, reinsemination experiments were undertaken. In this approach, eggs were inseminated initially with low sperm concentrations designed to trigger an egg response while maintaining the monospermic condition, i.e., low levels of polyspermy (Rothschild and Swann, 1952; Paul, 1975). At timed intervals thereafter, eggs were challenged with high sperm concentrations, and subsequent determinations of polyspermy levels were used to evaluate the presence or absence of a block. The results of two such experients, each conducted with a single sperm preparation and pooled eggs from 10 animals, were averaged for presentation in Fig. 5. At early insemination times (15, 30, and 45 min), polyspermy levels and the
100 c
mean number of sperm/penetrated egg were similar to the values obtained for the challenge concentration alone (80 and 2.18%, respectively), indicating that a block had not been established at the time of challenge. In contrast, reinsemination at 60, 75, or 90 min was without effect on sperm incorporation, consistent with the presence of a block. The midpoints of these curves at about 50 min represent unadjusted block times. However, as noted above, a sperm concentration-dependent delay is always observed between insemination and penetration. Since we assume that sperm-egg fusion represents the triggering event in block formation, a refined block estimate can be obtained by first subtracting the delay required for the initiating sperm concentration to trigger the block and then adding the delay required for the challenging sperm to acquire penetrating capacity. In this manner, an adjusted block time of approximately 40 min was calculated with 95% confidence limits of 8.8 and 72.4 min (see Appendix). It should be noted that in this adjustment delay times were derived from the experiments summarized in Table 1 which represent different sperm preparations. DISCUSSION
I
I 20
I 40
1 60
I 80
TIME (Min.) FIG. 5. Polyspermy and the mean number of sperm per egg in reinseminated zona-free mouse eggs. Eggs were inseminated with lb sperm/ml at time = 0 and reinseminated with 105 cells/ml at the indicated times. Polyspermy levels (open circles) and the mean number of sperm per egg (closed circles) were determined following 3 hr of incubation. Control values for polyspermy and mean number of sperm per egg for the low and high sperm concentrations were 147k1.12 and 30%2.18, respectively.
The present results demonstrate that sperm penetration of zona-free eggs is proportional to concentration and that high penetration levels can be obtained at low sperm-egg ratios. This finding contrasts with that obtained for intact eggs from the mouse and other mammals (summarized in Wolf and Inoue, 1976) where high sperm concentrations (105-lo6 cells/ml) are required to effect maximal penetration levels in vitro. The finding that zona-free eggs are more readily penetrated is consistent with the observation that supplemental sperm are seldom found in penetrated mouse eggs (Braden et al., 1954), i.e., that zona penetration is followed rapidly by sperm-egg fusion. A rate-limiting step in sperm penetration must, therefore, precede or involve
6
DEVELOPMENTAL BIOLOGY
zona passage. The zona as a glycoprotein layer undoubtedly contains a set of sperm binding sites different from those in the egg plasma membrane, and the concentration and affinity of these sites would be important in determining penetration rates. The condition of the sperm must also be considered in this regard. Penetration of the egg plasma membrane of intact eggs clearly involves an acrosome-reacted sperm, and available evidence indicates a similar situation obtains with zona-free eggs in vitro (Yanagimachi and Noda, 1970a; Yanagimachi, 1972; Wolf et al., 1976). In contrast, the status of the acrosome is not as clearly defined when capacitated sperm contact with the zona is considered. The series of events through which the sperm progresses prior to contacting the egg is generally conceded to include, in sequence: capacitation, induction of an acrosome reaction, penetration of the cumulus matrix, and zona penetration (McRorie and Williams, 1974; Chang and Hunter, 1975). Interestingly, a delay in zona penetration by acrosome-reacted sperm has been noted in the hamster by Yanagimachi (1969). This delay may represent the time required for sperm to insert their anterior tips into the zona material (Yanagimachi and Noda, 1970b) or for the completion of a series of complex prepenetration interactions between sperm and egg (Hartmann and Hutchison, 1974a,b). While the condition of acrosomes was not evaluated directly in the present study, evidence is available supporting a sequence in which the induction of an acrosome reaction immediately precedes zona penetration and, as such, may represent a rate-limiting event (Wolf et al., 1977). Thus many sperm observed at the outer margin of the zona following in vitro inseminations retain intact acrosomes (Thompson et al., 1974; Anderson et al., 1975; Nicosia and Wolf, unpublished results), and sperm penetration of zona-intact and -free mouse eggs is enhanced and occurs sooner in the presence of the ionophore A23187 (Wolf, manuscript in preparation), which is known to
VOLUME 64,1978
promote the induction of acrosome reactions in sea urchin and mammalian sperm (Talbot et al., 1976). Unfortunately, little definitive evidence exists concerning the mechanisms involved in the induction of the mammalian acrosome reaction. Cumulus cells and follicular and tubal fluids have been implicated in sperm capacitation and, in some cases, in inducing acrosomal vesiculation (for review, see Bedford, 1973, or Yanagimachi, 1977). It is clear, however, that the reaction can occur in a simple synthetic medium alone in the hamster (Yanagimachi, 1977) and in the presence only of zona-free eggs in the mouse. Accordingly, the egg itself should be reconsidered as a possible source of an acrosomeinducing activity (Bedford, 1973) and, in this regard, the premature release of cortical granules gains added significance (Nicosia et al., 1977). The egg plasma membrane block to polyspermy in zona-free mouse eggs, as defined in the present study, occurred within approximately 40 min of penetration. Further refinements in this blocking time estimate ideally would involve elimination of the delay time between insemination and initial penetration. Alternatively, experiments must be conducted with a single sperm preparation throughout, i.e., in the determination of the kinetics of penetration at the low initiating and at the high challenging sperm concentrations, as well as in the reinsemination experiment. Further refinements of these block times are, however, of limited value in view of other considerations. Thus, while we presume that sperm-egg fusion represents the triggering event in block formation, we can’t precisely define the initiating event nor, for that matter, that event in sperm-egg interaction which is susceptible to blockage (see Paul, 1975, and Paul and Gould-Somero, 1976, for additional discussion). Blocking times of several seconds have been reported for the “fast” block to polyspermy in invertebrate eggs (Rothschild and Swann, 1952; Paul, 1975), while Barros
DON P. WOLF
and Yanagimachi (1972) concluded that an egg plasma membrane block does not develop in hamster eggs until several hours after initial penetration. The latter authors recovered penetrated eggs at various times after artificial insemination, the earliest of which corresponded to approximately 2 hr postpenetration. Zonae were removed, and the susceptibility of these naked eggs to a second penetration was subsequently scored. However, early times postpenetration were not evaluated, and the possibility remains that the hamster undergoes a relatively rapid but transient block comparable to the mouse. Mechanistically, the egg plasma membrane block in marine invertebrates is thought to involve electrical depolarization of the membrane upon penetration by the fertilizing sperm (Jaffe, 1976). In mammals, it has been suggested that, like the zona reaction, the egg plasma membrane block involves alteration of sperm binding sites by the extruded contents of the egg’s cortical granules (Austin, 1968). A cortical granule trypsin-like protease has been implicated in the zona reaction in the hamster (Gwatkin et al., 1973) and mouse (Wolf and Hamada, 1977) and in the analogous event in sea urchins, the elevation of a fertilization membrane (Vacquier et al., 1972a,b, 1973; Schuel et al., 1973). An examination of the temporal sequence in cortical granule release in zona-intact mouse ova does not rule out this possibility, as a rapid release of granules is associated with early sperm-egg fusion events (Nicosia et al., 1977). Clearly, however, modification by cortical granule exudate is not the only mechanism to be considered, in view of the preceding discussion and the observations TABLE Source Experiment Log concentration Residual Total
-
7
Block to Sperm Penetration
that cortical events can be separated temporally from the establishment of an egg plasma membrane block in eggs of Urechis caupo (Paul, 1975), Mytilus (Humphries, 1967), and Spisula (Rebhun, 1962). APPENDIX
Analysis of Covariance for Fig. 1 The mean number of sperm per egg in Fig. 1 varies at different concentrations and in different experiments. Using the technique of analysis of covariance (for an introduction, see Snedecor and Co&ran, 1967), the effects due to sperm concentration or to experiment may be separated out. From the analysis of variance (Table 2), we see that the log concentration explains more of the total variance (9.48) than does the experiment (6.50). The difference among experiments is significant (P < O.Ol), and the effect of log concentration is significant (P < 0.001). The estimated differences among experiments can be removed, giving the open circles in Fig. 1.
Establishing the Existence of a Block to Sperm Penetration (Fig. 4) If there were no block to sperm penetration, the mean number of sperm per egg would increase at a steady rate in Fig. 4, so that there would be a linear term in time. If, on the other hand, there is a block established, then the mean number of sperm per egg should eventually increase at a slower and slower rate until a maximum is reached. This “leveling off’ can be approximated by a quadratic term in time. Differences among experiments may also be taken into account using an analysis of covariance (Table 3). Even after adjusting 2
ANALYSIS OF COVARIANCE FOR FIG. 1 ___. -.-______ Sum of squares Degrees of Mean square freedom 6.5022 9.4776 2.1001
5 1 10
18.0799
16
1.3004 9.4776 0.2100
~
F
6.19 45.13
~__-.
~~
P
8
DEVELOPMENTAL BIOLOGY TABLE
VOLUME 64,1978 3
ANALYSIS OF COVARIANCE FOR FIG. 4 Source
Sum of squares
Degrees of freedom
Mean square
F
P
Experiment Linear in time Quadratic in time Residual
2.3334 9.0037 1.1861 1.4570
3 1 1 19
0.7778 9.0037 1.1861 0.0767
10.14 117.41 15.47
13.9802
24
Total
~
-~~__
for differences among experiments and a cause all eggs are blocked before refertililinear trend in time, the quadratic trend in zation can occur, or time is significant (F = 15.47, P < 0.001). tB + tL + CL 2 tR + tH. Calculations for the estimated linear and quadratic coefficients show that a maxi- At the time tR2 when the percentage of mum is reached at about 140 min. polyspermy levels reach a maximum, we have Estimation of Block Time tR2 + tH = tB + tL + CL. (2) The egg plasma membrane blocking time Let tip be the intervention time for which may be derived from analysis of reinsemination experiments. From Table 1, it can the percent polyspermy is halfway between the levels for very small or very large interbe seen that we must ahow for different vention times. If the decrease in polydelay times at low and high sperm concenspermy seen between 45 and 60 min fails at trations. Let tL be the time before the first an approximately constant rate, than tip egg is penetrated by the triggering sperm and let tL + CL be the time until the last = %( tR1 + tR2), or substituting into formulas egg is penetrated. tH and CH are the corre- (1) and (2), we have sponding times for sperm at the high contB = t1,2 + tH - tL + (CH CL). centration, and tR is the time the chahenging sperm is given. Let tB be the time re- Based on the maximum k1 values in Table 1, the times CH and CL are similar and can quired for the block after penetration. Then the first egg achieves a block at time tL + be neglected, leaving tg, and all eggs are blocked by time tL + CL tB = tl/2 + tH - tL (3) + tB. When tR is less than 45 min (before any decrease is seen in the polyspermy level as the formula for estimating the block time tB. If the delay times tL and tH were not in Fig. 5), all eggs are exposed to refertilithen we would zation before any eggs are blocked, and the concentration-dependent, resulting percentage of polyspermy is de- have tL = tH, and the above formula for estimating block time would be just tip, the termined by the challenging concentration. time most often reported in the literature. That is, Our block time is an adjustment of the tR + tH + CH s tL + tB. conventional time to allow for different delay times for the triggering and challenging At the time tR1 when the percent polyspermy first starts to decrease in Fig. 5, we concentrations. The variability in this estimate of tB can be calculated from the varihave ability in tl,2, h,, and tH. tR1 + tH + CH = tL + tB. (1) When tR is very large (over 60 min), the challenging concentration has no effect be-
Variability in the Estimates for tL, tw t1/2 tli2. Chi-square analyses of the differ-
DON P. WOLF
Block to Sperm Penetration
ences in polyspermy levels seen with the high sperm concentration alone or at reinsemination times of 15, 30, and 45 min indicate that no significant decreases occur by 45 min (0.75 > P > 0.5). Similarly, no significant differences (0.9 > P > 0.75) are observed in polyspermy levels between the low sperm concentration alone and reinsemination times of 60, 75, and 90 min. These tests provide evidence that tR1 > 45 min and that tR2 < 60 min, so that tlj2 is between 45 and 60 min. The best estimate for tip is then tl/2 = %(45 + 60) = 52.5 min. tL and tH. Since penetration can be regarded as a pseudo-first-order rate process (see Results), sperm delay times can be estimated from the regression of time on the logarithm of the percentage of unfertilized eggs for a specific sperm concentration. For this purpose, the high sperm concentration employed was lo6 cells/ml and the low was lo’-lo3 cells/ml (see Table 1). Only the two “best” preparations at the low concentration were used, partly by necessity and partly based on the rationale that Fig. 5 represented reinsemination experiments in which relatively rapid drops in polyspermy levels were seen. The delay time at the low sperm concentration was 33.4 min with a variance of 17.9 and at the high concentration 21.5 min with a variance of 12.6. Therefore, tL - tH is 11.9 min with a 95% confidence interval of -12.4 and 36.2 min. Summarizing, tB = 52.5 + 21.5 - 33.4 = 41 min, with a 95% confidence region given by 45 - 36.2, 60 - (-12.4) = 8.8 and 72.4 min. This large confidence interval is due partly to the fact that tl,2 cannot be more closely determined than the range 45 to 60 min. The interval given is quite conservative. The experimental design for estimating block time does not allow adjustments for differences due to different sperm preparations and has further contributed to the large interval. The author expresses appreciation to Ms. Patricia Park for secretarial assistance and to Dr. Jerry L. Hedrick for his critical reading of the manuscript and his many helpful suggestions. Supported by USPHS
9
HD-07635. A portion of this work was conducted at the Marine Biological Laboratories, Woods Hole, Massachusetts. REFERENCES ANDERSON, E., HOPPE, P. C., WHITTEN, W. K., and LEE, G. S. (1975). In vitro fertilization and early embryogenesis: A cytological analysis. J. Ultrastrut. Res. 50, 231-252. AUSTIN, C. R. (1968). “Ultrastructure of Fertilization.” Holt, Rinehart and Winston, New York. BARROS, C., and YANAGIMACHI, R. (1971). Induction of the zona reaction in golden hamster eggs by cortical granule material. Nature (London) 233,
268-269. BARROS, C., and YANACIMACHI, R. (1972). Polyspermy-preventing mechanisms in the golden hamster egg. J. Exp. Zool. 180,251-266. BEDFORD, J. M. (1973) Mechanisms involved in penetration of spermatozoa through the vestments of the mammalian egg. In “Physiology and Genetics of Reproduction, Part B” (E. M. Coutinho and F. Fuchs, eds.), Basic Life Sciences, Vol 4. Plenum Press, New York. BRADEN, A. W. H., AUSTIN, C. R., and DAVID, H. A. (1954). The reaction of the zona pellucida to sperm penetration. Aust. J. Biol. Sci. ‘7, 391-409. CHANG, M. C., and HUNTER, R. H. F. (1975). Capacitation of mammalian sperm: biological and experimental aspects. In “Handbook of Physiology” (R. 0. Greep and E. B. Astwood, eds.), Sect. 7, Vol. 5. American Physiological Society, Washington, D.C. GATES, A. H. (1971). Maximizing yield and developmental uniformity of eggs. In “Methods in Mammalian Embryology (J. C. Daniel, Jr., ed.), pp. 64-75. W. H. Freeman and Co., San Francisco. GWATKIN, R. B. L., WILLIAMS, D. T., HARTMANN, J. F., and KNIAZUK, M. (1973). The zona reaction of hamster and mouse eggs: Production in vitro by a trypsin-like protease from cortical granules. J. Re-
prod. Fert. 32.259-265. HARTMANN, J. F., and HUTCHISON, C. F. (1974a). Nature of the pre-penetration contact interactions between hamster gametes in vitro. J. Reprod. Fert. 36,49-57. HARTMANN, J. F., and HUTCHISON, C. F. (1974b). Contact between hamster spermatozoa and the zona pellucida releases a factor which influences early binding stages. J. Reprod. Fert. 37, 61-66. HUMPHRIES, W. J. (1967). Fine structure of cortical granules in eggs and gastrulae of Mytilus edulis. J. Ultrastruct. Res. 17, 314-326. INOUE, M., and WOLF, D. P. (1974). Comparative solubility properties of the zonae pellucidae of unfertilized and fertilized mouse ova. Bill. Reprod. 11, 558-565. JAFFE, L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature (London) 261,68-71.
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DEVELOPMENTALBIOLOGY
MCRORIE, R. A., and WILLIAMS, W. L. (1974). Biochemistry of mammalian fertilization. Annu. Rev. B&hem. 43,777-803. NICOSIA, S. V., WOLF, D. P., and INOUE, M. (1977). Cortical granule distribution and cell surface characteristics of mouse ova. Develop. Biol. 57,208-226. NIWA, K., and CHANG, M. C. (1975). Requirement of capacitation for sperm penetration of zona-free rat eggs. J. Reprod. Fert. 44,305-308. PAUL, M. (1975). The polyspermy block in eggs of Urechis caupo. Exp. Cell Res. 90, 137-142. PAUL, M., and GOULD-SOMERO,M. (1976). Evidence for a polyspermy block at the level of sperm-egg plasma membrane fusion in Urechis caupo. J. Exp. 2001. 196, 105-112. PAVLOK, A., and MCLAREN, A. (1972). The role of cumulus cells and the zona pellucida in fertilization of mouse eggs in vitro. J. Reprod. Fert. 29, 91-97. REBHUN, L. I. (1962). Electron microscopic studies on the vitelline membrane of the surf clam, Spisulu solidissima. J. Ultrastruct. Res. 6, 107-122. ROTHSCHILD, L., and SWANN, M. M. (1952). The fertilization reaction in the sea-urchins; the block to polyspermy. J. Exp. Biol. 29,496-483. SCHUEL, H., WII,SON, W. L., CHEN, K., and LORAND, L. (1973). A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization. Develop. Biol. 34, 175-186. SEGEL, I. H. (1968). “Biochemical Calculations,” pp. 390-392. Wiley, New York. SNEDECOR,G. W., and COCHRAN,W. G. (1967). “Statistical Methods,” 6th ed., pp. 419-443. Iowa State University Press, Ames, Iowa. TALBOT, P., SUMMERS, R. G., HYLANDER, B. L., KEOUGH, E. M., and FRANKLIN, L. E. (1976). The role of calcium in the acrosome reaction: An analysis using ionophore A23187. J. Exp. 2001. 198.383-392. THOMPSON,R. S., MOORE-SMITH, D., and ZAMBONI, L. (1974). Fertilization of mouse ova in vitro: An electron microscopy study. Fert. Steril. 25,222-249. TOYODA, Y., and CHANG, M. C. (1968). Sperm penetration of rat eggs in vitro after dissolution of zona pellucida by chymotrypsin. Nature (London) 220, 589-591. TOYODA,Y., and CHANG,M. C. (1974). Fertilization of
VOLUME 64. 1978
rat eggs in vitro by epididymal spermatozoa and the development of eggs following transfer. J. Reprod. Fert. 36.9-22. VACQUIER, V. D., EPEL, D., and DOIJGLAS, L. A. (1972a). Sea urchin eggs release protease activity at fertilization. Nature (London) 237.34-36. VACQUIER, V. D., TEGNER, M. J., and EPEL, D. (197213).Protease activity establishes the block against poiyspermy in sea urchin eggs. Nature @ondon) 240, 352-353. VACQUIER,V. D., TEGNER,M. J., and EPEL, D. (1973). Protease released from sea urchin eggs at fertilization alters the vitelline layer and aids in preventing polyspermy. Exp. Cell Res. 80,111-119. WOLF, D. P., and HAMADA, M. (1977). Induction of zonal and egg plasma membrane blocks to sperm penetration in mouse eggs with cortical granule exudate. Bial. Reprod. 17,350-354. WOLF, D. P., HAMADA, M., and INOUE, M. (1977). The kinetics of sperm penetration into and the zona reaction of mouse ova insemination in vitro. J. Exp. Zool. 201.29-36. WOLF, D. P., and INOUE, M. (1976). Sperm concentration dependency in the fertilization and zonae sperm binding properties of mouse eggs inseminated in vitro. J. Exp. Zool. 196, 27-38. WOLF, D. P., INOUE, M., and STARK, R. A. (1976). Penetration of zona-free mouse eggs. Biol. Reprod. 15, 213-221. YANAGIMACHI, R. (1969).In vitro capacitation of hamster spermatozoa by follicular fluid. J. Reprod. Fert. 18, 275-286. YANAGIMACHI, R. (1972). Fertilization of guinea pig eggs in vitro. Anat. Rec. 174.9-20. YAMAGIMACHI, R. (1977). Specificity of sperm-egg
interaction. In “Immunobiology of Gametes” (M. Edidin and M. H. Johnson, eds.), pp. 255-295. Cambridge University Press, Cambridge. YANAGIMACHI, R., and NODA, Y. D. (1970a). Ultrastructural changes in the hamster sperm head during fertilization. J. Ultrastruct. Res. 31, 465-485. YANAGIMACHI, R., and NODA, Y. D. (1970b). Physiological changes in the post-nuclear cap region of mammalian spermatozoa: A necessary preliminary to the membrane fusion between sperm and egg cells. J. Ultrastruct. Res. 31,486-493.
BIOLOGY
64,
l-10
(1978)
The Block to Sperm Penetration
in Zona-Free
Mouse Eggs
DON P. WOLF
with an Appendix by KEITH SOPER’ Division of Reproductive Biology, Department of Obstetrics and Gynecology, and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received September 10,1976; accepted in revised form December IS,1977 The rate of sperm penetration and the number of sperm penetrating zona-free mouse eggs were found to he dependent on sperm concentration. At the lowest sperm concentrations examined (IO’ cells/ml, sperm-egg ratios of approximately l:l), most eggs were penetrated (75%), and polyspermy was low (19%) following 3 hr of incubation. The number of sperm penetrating the egg was logarithmically related to sperm concentration. All eggs showed a delay of at least 20 min between insemination and penetration, and penetration was complete in approximately 2 hr at lo4 sperm/ml; this penetration block was attributed to egg-related changes. The existence and timing of the egg plasma membrane block to polyspermy were evaluated by reinsemination experiments. In this approach, the block was triggered in zona-free eggs with a low concentration of capacitated epididymal sperm at time 0, and the eggs were subsequently challenged with high sperm concentrations. The presence or absence of a block was inferred from the degree of polyspermy observed in these eggs after 3 hr of incubation. Adjusting for sperm concentration-dependent delays between insemination and sperm penetration, a blocking time of approximately 40 min was obtained. INTRODUCTION
block in mammalian eggs is limited. Barros and Yanagimachi (1972), studying the susceptibility of zona-free hamster eggs to refertilization, concluded that formation of the egg plasma membrane block required 2 to 3.5 hr. Baaed on temporal requirements, the zona reaction precedes the plasma membrane block in the hamster, challenging the significance of the latter event. In the mouse (Pavlok and McLaren, 1972) and the rat (Toyoda and Chang, 1968; Niwa and Chang, 1975), evidence for the existence of an egg plasma membrane block derives from the observation that zona-free eggs display polyspermy levels comparable to those of zona-intact controls. However, proteolytic enzymes were employed during zonae removal in these studies, raising the possibility that the egg’s receptivity to sperm was abnormal (Wolf et al., 1976). The development of a procedure for the mechanical removal of zonae (Wolf et al., 1976) provided the basis for the present
Normal development of mammalian embryos is dependent upon monospermic fertilization and the exclusion of supernumerary spermatozoa. While evidence for the existence of a zona reaction, a block to sperm penetration of the zona pellucida following penetration of the fertilizing sperm, has been presented (Braden et al. 1954;Barros and Yanagimachi, 1971; Gwatkin et al., 1973; Wolf and Hamada, 1977), monospermic embryos recovered from natural matings or from in vitro inseminations occasionally contain sperm in the pervitelline space, i.e., supplemental sperm (Austin, 1968). In the rabbit, large numbers of supplemental sperm are always seen. These observations necessitate an egg plasma membrane block to sperm penetration. Direct experimental evidence defining the ’ Epidemiology Unit, Department Medicine, University of Pennsylvania, Pennsylvania.
of Research
Philadelphia,
1 0012-1606/78/0641-ooO1$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
DEVELOPMENTAL BIOLOGY VOLUME64,1978
2
tion, as measured following 3 hr of incubation, was defined over a 105-fold range in sperm concentration (Fig. 1). Included in the figure are the unadjusted values for the mean number of sperm per egg at each MATERIALS AND METHODS sperm concentration, representing data Unfertilized tubal ova were recovered from six experiments each employing a diffrom superovulated Swiss mice (6-12 weeks ferent epididymal sperm preparation. Since old) and freed from surrounding cumulus sperm-egg interaction is, in part, dependent cells by exposure to hyaluronidase (O.l%, on the particular sperm preparation emSigma Type I) in a modified Krebs-Ringer ployed, an analysis of covariance was apbicarbonate medium previously described plied to give values adjusted for differences (Inoue and Wolf, 1974; Wolf and Inoue, among sperm preparations (see Appendix). 1976). Such eggs are regarded as normal, The higher concentrations studied here since they produce viable young at the con- were similar to those routinely employed trol frequency when transferred to foster for in vitro fertilization of intact eggs with females (Gates, 1971). Zonae pellucidae sperm-egg ratios of 5 or 10 X lo5 to 1. For were then removed by aspiration through comparison, penetration levels for intact micropipets (Wolf et al., 1976), washed eggs, calculated from Wolf and Inoue three times and transferred to 200~~1drops (1976), have been included in Fig. 1. At the of culture medium under silicone oil (Dow- lowest concentrations, sperm-egg ratios apCorning 200 Fluid; 50 cs). Unless otherwise proached the presumed physiological range indicated, eggs were inseminated with ap- of 1 to 1. Virtually all zona-free eggs were proximately 2 x lo5 capacitated sperm/ml penetrated at concentrations above lo2 (Wolf and Inoue, 1976). Experiments were cells/ml (75% at 102; 96-100% at ~10~). terminated by washing eggs free of adher- Polyspermy levels increased from 19% at ent sperm, followed by transfer to a slide lo2 cells/ml to r60% at the higher sperm for mounting and staining with acetolac- concentrations, and the mean number of moid (Toyoda and Chang, 1974). Ova were sperm per inseminated egg was logarithconsidered penetrated when a sperm tail(s) mically related to sperm concentration. and head remnant(s) or a male pronucleus were observed within the cytoplasm. References for the statistical treatment of the data are included in the Appendix; for statistical purposes, batches of eggs were considered homogeneous, as they were always derived from lo-animal pools and were all potentially penetrable. For an analysis of pseudo-first-order rate processes, see Segel (1968). investigation in which the occurrence and kinetics of a block to polyspermy were defined in zone-free mouse eggs inseminated in vitro.
RESULTS
Sperm Concentration Dependency in Penetration In setting out to examine the zona-free egg’s ability to modulate its own penetration, the influence of sperm concentration on the kinetics and maximum levels of penetration was studied. Maximum penetra-
LOG SPERM CONCENTRATION
FIG. 1. Sperm concentration
dependency in the penetration of mouse eggs. The mean number of sperm recovered at each inseminating concentration was calculated for zona-free eggs (closed circles) and corrected for differences in sperm preparations by an analysis of covariance (open circles). Comparable uncorrected values are included for zona-intact eggs (closed squares) from Wolf and Inoue (1976).
DON P. WOLF
Block to Sperm Penetration
The kinetics of sperm penetration (lo4 cells/ml) of zona-free eggs are presented in Fig. 2. Routinely, a delay was seen between insemination and penetration, as shown in Fig. 2 by the percentage of unfertilized eggs remaining at 100% for a time following the addition of sperm. This period, observed with both washed and unwashed preincubated epididymal sperm, may reflect the time requirements for sperm binding and for the occurrence of an acrosome reaction. However, it was not subject to reduction by sperm preincubation (30 min) in the presence of zona-free eggs. It should also be noted that the method employed here to assess penetration (microscopic examination of acetolacmoid-stained whole mounts) does not allow detection of the earliest stages of sperm penetration. Following this delay, the percentage of unfertilized eggs decreased as eggs became monospermic, and subsequently polyspermic, with penetration ceasing at approximately 2 hr. These relationships can be described by equation E+S-
kl
ES+S-
kz
ES2 + S ~
ks
loo-~:
0 --Y 180 TIME (min.) FIG. 2. The kinetics of penetration of zona-free mouse eggs inseminated at lo4 sperm/ml. The distribution of unfertilized (crosses), monospermic (open circles), and polyspermic (solid circles) eggs are presented as a function of insemination time. Data points represent means of four individual experiments.
...,
where E = egg, S = sperm, ES = monospermic egg, and ES2 = dispermic egg. As anticipated, the penetration rate for the first sperm (kd is seen in Fig. 2 to be greater than that for subsequent sperm (kz). The rate constants (k1 and kz) were estimated assuming that penetration behaved as a pseudo-first-order rate process; and therefore, log E = -kt/2.303. Figure 3 is a plot of log percentage of unfertilized or monospermic eggs versus time, where the slope equals -k/2.303. Based on these plots, the treatment of penetration as a pseudo-first-order rate process appears valid, although data collected at the longer times were unreliable. The rate of penetration of the first sperm was 0.036 min-’ and for subsequent sperm, 0.012 min-‘. By extrapolating the data in Fig. 3 to 100% un-
I
I
I
0
60
I
120
180
TIME (Min.)
FIG. 3. Time-dependent decreases in the log percentage of unfertilized and monospermic xona-free eggs in the presence of lo4 sperm/ml. Log E versus t for unfertilized (open circles) and monospermic (solid circles) eggs where the slope = -k/2.303 and t,,* =
0693/k.
fertilized (log E = 2), the time required for the sperm to acquire its penetrating capacity can be estimated (the delay time). This value was 26 min for the first sperm and, although the data are less precise, of the same magnitude for subsequent sperm. The ~I/Z values for penetration by the first and
4
DEVELOPMENTALBIOLOGY
subsequent sperm were 20 and 57 min, respectively. Sperm concentration-dependent variations in the kinetic parameters defining initial penetration can be seen in Table 1. With increasing sperm concentration, the rate of penetration and the number of penetrated sperm increased, while the delay between insemination and penetration decreased. At the lowest sperm concentration, interexperimental variability was large, an observation that is critical to the interpretation of the reinsemination experiments described below. The recorded k1 value of 0.089 was derived from only two experiments; in three others, penetration rates at 60 min were too low to allow accurate quantitation. These results illustrate the variability attributable to different sperm preparations. Experiments conducted with different concentrations of a single sperm preparation corroborate the findings in Table 1 (data not included).
The Block to Sperm Penetration Direct experimental evidence for an egg plasma membrane block to sperm penetration is provided by the demonstration that maximal penetration is reached and maintained in zona-free eggs following 2 hr of insemination at lo4 sperm/ml (Fig. 4). In the absence of an egg plasma membrane block, penetration should increase as a function of time until all sperm have exTABLE
1
SPERMCONCENTRATIONDEPENDENCYINTHE KINETICSOFPENETRATIONOFZONA-FREEEGG@ Sperm concentration
(cells/ml) 16-163 lo4 106
Penetration rate kl (min-‘)
Delay time (min)
t, (min)
Mean number of sperm
per egg 0.001-0.089 0.036 0.091
30-45 26 22
20 8
1.20 2.34 3.55
“Least squares regression lines were determined from data collected from four or five experiments at each sperm concentration. Kinetic parameters were then calculated as described in the text: tl = 0.693/k. The k, value for lb-l@ cells/ml represents the maximum rate seen in two of five experiments, the other penetration rates being less than 0.001 min-‘.
VOLUME 6‘i,1978 3r
1
60
I
120 TIME (Min.)
I
180
FIG. 4. Establishment of an egg plasma membrane block to sperm penetration in zona-free eggs. Eggs were inseminated with lo4 capacitated epididymal sperm/ml at time zero. At the indicated times, samples were removed and processed for the quantitation of penetrated sperm, as described in Materials and Methods. Both uncorrected (open circles) and corrected values (closed circles; analysis of covariance accounting for sperm preparation differences) for the mean number of sperm per egg are presented. The fitted curve was generated by an equation with a quadratic term in time.
pired. A quadratic term in time (see Appendix) indicates that the mean number of sperm per egg levels off to. a maximum value by 140 min (P
DON P. WOLF
5
Block to Sperm Penetration
original eggs, original eggs plus fresh sperm, and fresh eggs plus 2-hr sperm, respectively.
Time Requirements of the Block to Polyarmy In order to investigate the existence and timing of the presumed physiological block which maintains a monospermic embryo, reinsemination experiments were undertaken. In this approach, eggs were inseminated initially with low sperm concentrations designed to trigger an egg response while maintaining the monospermic condition, i.e., low levels of polyspermy (Rothschild and Swann, 1952; Paul, 1975). At timed intervals thereafter, eggs were challenged with high sperm concentrations, and subsequent determinations of polyspermy levels were used to evaluate the presence or absence of a block. The results of two such experients, each conducted with a single sperm preparation and pooled eggs from 10 animals, were averaged for presentation in Fig. 5. At early insemination times (15, 30, and 45 min), polyspermy levels and the
100 c
mean number of sperm/penetrated egg were similar to the values obtained for the challenge concentration alone (80 and 2.18%, respectively), indicating that a block had not been established at the time of challenge. In contrast, reinsemination at 60, 75, or 90 min was without effect on sperm incorporation, consistent with the presence of a block. The midpoints of these curves at about 50 min represent unadjusted block times. However, as noted above, a sperm concentration-dependent delay is always observed between insemination and penetration. Since we assume that sperm-egg fusion represents the triggering event in block formation, a refined block estimate can be obtained by first subtracting the delay required for the initiating sperm concentration to trigger the block and then adding the delay required for the challenging sperm to acquire penetrating capacity. In this manner, an adjusted block time of approximately 40 min was calculated with 95% confidence limits of 8.8 and 72.4 min (see Appendix). It should be noted that in this adjustment delay times were derived from the experiments summarized in Table 1 which represent different sperm preparations. DISCUSSION
I
I 20
I 40
1 60
I 80
TIME (Min.) FIG. 5. Polyspermy and the mean number of sperm per egg in reinseminated zona-free mouse eggs. Eggs were inseminated with lb sperm/ml at time = 0 and reinseminated with 105 cells/ml at the indicated times. Polyspermy levels (open circles) and the mean number of sperm per egg (closed circles) were determined following 3 hr of incubation. Control values for polyspermy and mean number of sperm per egg for the low and high sperm concentrations were 147k1.12 and 30%2.18, respectively.
The present results demonstrate that sperm penetration of zona-free eggs is proportional to concentration and that high penetration levels can be obtained at low sperm-egg ratios. This finding contrasts with that obtained for intact eggs from the mouse and other mammals (summarized in Wolf and Inoue, 1976) where high sperm concentrations (105-lo6 cells/ml) are required to effect maximal penetration levels in vitro. The finding that zona-free eggs are more readily penetrated is consistent with the observation that supplemental sperm are seldom found in penetrated mouse eggs (Braden et al., 1954), i.e., that zona penetration is followed rapidly by sperm-egg fusion. A rate-limiting step in sperm penetration must, therefore, precede or involve
6
DEVELOPMENTAL BIOLOGY
zona passage. The zona as a glycoprotein layer undoubtedly contains a set of sperm binding sites different from those in the egg plasma membrane, and the concentration and affinity of these sites would be important in determining penetration rates. The condition of the sperm must also be considered in this regard. Penetration of the egg plasma membrane of intact eggs clearly involves an acrosome-reacted sperm, and available evidence indicates a similar situation obtains with zona-free eggs in vitro (Yanagimachi and Noda, 1970a; Yanagimachi, 1972; Wolf et al., 1976). In contrast, the status of the acrosome is not as clearly defined when capacitated sperm contact with the zona is considered. The series of events through which the sperm progresses prior to contacting the egg is generally conceded to include, in sequence: capacitation, induction of an acrosome reaction, penetration of the cumulus matrix, and zona penetration (McRorie and Williams, 1974; Chang and Hunter, 1975). Interestingly, a delay in zona penetration by acrosome-reacted sperm has been noted in the hamster by Yanagimachi (1969). This delay may represent the time required for sperm to insert their anterior tips into the zona material (Yanagimachi and Noda, 1970b) or for the completion of a series of complex prepenetration interactions between sperm and egg (Hartmann and Hutchison, 1974a,b). While the condition of acrosomes was not evaluated directly in the present study, evidence is available supporting a sequence in which the induction of an acrosome reaction immediately precedes zona penetration and, as such, may represent a rate-limiting event (Wolf et al., 1977). Thus many sperm observed at the outer margin of the zona following in vitro inseminations retain intact acrosomes (Thompson et al., 1974; Anderson et al., 1975; Nicosia and Wolf, unpublished results), and sperm penetration of zona-intact and -free mouse eggs is enhanced and occurs sooner in the presence of the ionophore A23187 (Wolf, manuscript in preparation), which is known to
VOLUME 64,1978
promote the induction of acrosome reactions in sea urchin and mammalian sperm (Talbot et al., 1976). Unfortunately, little definitive evidence exists concerning the mechanisms involved in the induction of the mammalian acrosome reaction. Cumulus cells and follicular and tubal fluids have been implicated in sperm capacitation and, in some cases, in inducing acrosomal vesiculation (for review, see Bedford, 1973, or Yanagimachi, 1977). It is clear, however, that the reaction can occur in a simple synthetic medium alone in the hamster (Yanagimachi, 1977) and in the presence only of zona-free eggs in the mouse. Accordingly, the egg itself should be reconsidered as a possible source of an acrosomeinducing activity (Bedford, 1973) and, in this regard, the premature release of cortical granules gains added significance (Nicosia et al., 1977). The egg plasma membrane block to polyspermy in zona-free mouse eggs, as defined in the present study, occurred within approximately 40 min of penetration. Further refinements in this blocking time estimate ideally would involve elimination of the delay time between insemination and initial penetration. Alternatively, experiments must be conducted with a single sperm preparation throughout, i.e., in the determination of the kinetics of penetration at the low initiating and at the high challenging sperm concentrations, as well as in the reinsemination experiment. Further refinements of these block times are, however, of limited value in view of other considerations. Thus, while we presume that sperm-egg fusion represents the triggering event in block formation, we can’t precisely define the initiating event nor, for that matter, that event in sperm-egg interaction which is susceptible to blockage (see Paul, 1975, and Paul and Gould-Somero, 1976, for additional discussion). Blocking times of several seconds have been reported for the “fast” block to polyspermy in invertebrate eggs (Rothschild and Swann, 1952; Paul, 1975), while Barros
DON P. WOLF
and Yanagimachi (1972) concluded that an egg plasma membrane block does not develop in hamster eggs until several hours after initial penetration. The latter authors recovered penetrated eggs at various times after artificial insemination, the earliest of which corresponded to approximately 2 hr postpenetration. Zonae were removed, and the susceptibility of these naked eggs to a second penetration was subsequently scored. However, early times postpenetration were not evaluated, and the possibility remains that the hamster undergoes a relatively rapid but transient block comparable to the mouse. Mechanistically, the egg plasma membrane block in marine invertebrates is thought to involve electrical depolarization of the membrane upon penetration by the fertilizing sperm (Jaffe, 1976). In mammals, it has been suggested that, like the zona reaction, the egg plasma membrane block involves alteration of sperm binding sites by the extruded contents of the egg’s cortical granules (Austin, 1968). A cortical granule trypsin-like protease has been implicated in the zona reaction in the hamster (Gwatkin et al., 1973) and mouse (Wolf and Hamada, 1977) and in the analogous event in sea urchins, the elevation of a fertilization membrane (Vacquier et al., 1972a,b, 1973; Schuel et al., 1973). An examination of the temporal sequence in cortical granule release in zona-intact mouse ova does not rule out this possibility, as a rapid release of granules is associated with early sperm-egg fusion events (Nicosia et al., 1977). Clearly, however, modification by cortical granule exudate is not the only mechanism to be considered, in view of the preceding discussion and the observations TABLE Source Experiment Log concentration Residual Total
-
7
Block to Sperm Penetration
that cortical events can be separated temporally from the establishment of an egg plasma membrane block in eggs of Urechis caupo (Paul, 1975), Mytilus (Humphries, 1967), and Spisula (Rebhun, 1962). APPENDIX
Analysis of Covariance for Fig. 1 The mean number of sperm per egg in Fig. 1 varies at different concentrations and in different experiments. Using the technique of analysis of covariance (for an introduction, see Snedecor and Co&ran, 1967), the effects due to sperm concentration or to experiment may be separated out. From the analysis of variance (Table 2), we see that the log concentration explains more of the total variance (9.48) than does the experiment (6.50). The difference among experiments is significant (P < O.Ol), and the effect of log concentration is significant (P < 0.001). The estimated differences among experiments can be removed, giving the open circles in Fig. 1.
Establishing the Existence of a Block to Sperm Penetration (Fig. 4) If there were no block to sperm penetration, the mean number of sperm per egg would increase at a steady rate in Fig. 4, so that there would be a linear term in time. If, on the other hand, there is a block established, then the mean number of sperm per egg should eventually increase at a slower and slower rate until a maximum is reached. This “leveling off’ can be approximated by a quadratic term in time. Differences among experiments may also be taken into account using an analysis of covariance (Table 3). Even after adjusting 2
ANALYSIS OF COVARIANCE FOR FIG. 1 ___. -.-______ Sum of squares Degrees of Mean square freedom 6.5022 9.4776 2.1001
5 1 10
18.0799
16
1.3004 9.4776 0.2100
~
F
6.19 45.13
~__-.
~~
P
8
DEVELOPMENTAL BIOLOGY TABLE
VOLUME 64,1978 3
ANALYSIS OF COVARIANCE FOR FIG. 4 Source
Sum of squares
Degrees of freedom
Mean square
F
P
Experiment Linear in time Quadratic in time Residual
2.3334 9.0037 1.1861 1.4570
3 1 1 19
0.7778 9.0037 1.1861 0.0767
10.14 117.41 15.47
13.9802
24
Total
~
-~~__
for differences among experiments and a cause all eggs are blocked before refertililinear trend in time, the quadratic trend in zation can occur, or time is significant (F = 15.47, P < 0.001). tB + tL + CL 2 tR + tH. Calculations for the estimated linear and quadratic coefficients show that a maxi- At the time tR2 when the percentage of mum is reached at about 140 min. polyspermy levels reach a maximum, we have Estimation of Block Time tR2 + tH = tB + tL + CL. (2) The egg plasma membrane blocking time Let tip be the intervention time for which may be derived from analysis of reinsemination experiments. From Table 1, it can the percent polyspermy is halfway between the levels for very small or very large interbe seen that we must ahow for different vention times. If the decrease in polydelay times at low and high sperm concenspermy seen between 45 and 60 min fails at trations. Let tL be the time before the first an approximately constant rate, than tip egg is penetrated by the triggering sperm and let tL + CL be the time until the last = %( tR1 + tR2), or substituting into formulas egg is penetrated. tH and CH are the corre- (1) and (2), we have sponding times for sperm at the high contB = t1,2 + tH - tL + (CH CL). centration, and tR is the time the chahenging sperm is given. Let tB be the time re- Based on the maximum k1 values in Table 1, the times CH and CL are similar and can quired for the block after penetration. Then the first egg achieves a block at time tL + be neglected, leaving tg, and all eggs are blocked by time tL + CL tB = tl/2 + tH - tL (3) + tB. When tR is less than 45 min (before any decrease is seen in the polyspermy level as the formula for estimating the block time tB. If the delay times tL and tH were not in Fig. 5), all eggs are exposed to refertilithen we would zation before any eggs are blocked, and the concentration-dependent, resulting percentage of polyspermy is de- have tL = tH, and the above formula for estimating block time would be just tip, the termined by the challenging concentration. time most often reported in the literature. That is, Our block time is an adjustment of the tR + tH + CH s tL + tB. conventional time to allow for different delay times for the triggering and challenging At the time tR1 when the percent polyspermy first starts to decrease in Fig. 5, we concentrations. The variability in this estimate of tB can be calculated from the varihave ability in tl,2, h,, and tH. tR1 + tH + CH = tL + tB. (1) When tR is very large (over 60 min), the challenging concentration has no effect be-
Variability in the Estimates for tL, tw t1/2 tli2. Chi-square analyses of the differ-
DON P. WOLF
Block to Sperm Penetration
ences in polyspermy levels seen with the high sperm concentration alone or at reinsemination times of 15, 30, and 45 min indicate that no significant decreases occur by 45 min (0.75 > P > 0.5). Similarly, no significant differences (0.9 > P > 0.75) are observed in polyspermy levels between the low sperm concentration alone and reinsemination times of 60, 75, and 90 min. These tests provide evidence that tR1 > 45 min and that tR2 < 60 min, so that tlj2 is between 45 and 60 min. The best estimate for tip is then tl/2 = %(45 + 60) = 52.5 min. tL and tH. Since penetration can be regarded as a pseudo-first-order rate process (see Results), sperm delay times can be estimated from the regression of time on the logarithm of the percentage of unfertilized eggs for a specific sperm concentration. For this purpose, the high sperm concentration employed was lo6 cells/ml and the low was lo’-lo3 cells/ml (see Table 1). Only the two “best” preparations at the low concentration were used, partly by necessity and partly based on the rationale that Fig. 5 represented reinsemination experiments in which relatively rapid drops in polyspermy levels were seen. The delay time at the low sperm concentration was 33.4 min with a variance of 17.9 and at the high concentration 21.5 min with a variance of 12.6. Therefore, tL - tH is 11.9 min with a 95% confidence interval of -12.4 and 36.2 min. Summarizing, tB = 52.5 + 21.5 - 33.4 = 41 min, with a 95% confidence region given by 45 - 36.2, 60 - (-12.4) = 8.8 and 72.4 min. This large confidence interval is due partly to the fact that tl,2 cannot be more closely determined than the range 45 to 60 min. The interval given is quite conservative. The experimental design for estimating block time does not allow adjustments for differences due to different sperm preparations and has further contributed to the large interval. The author expresses appreciation to Ms. Patricia Park for secretarial assistance and to Dr. Jerry L. Hedrick for his critical reading of the manuscript and his many helpful suggestions. Supported by USPHS
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