Release of a Factor During Early Stages of Contact Between Hamster Sperm and Eggs In Vitro JOHN F. HARTMANN AND CAMERON F. HUTCHISON Merck Institute for Therapeutic Research, Rahway, New Jersey 07065
ABSTRACT A sensitive bioassay was devised which detected the release of a factor resulting from contact between the surfaces of sperm and zona pellucida of the golden hamster in vitro. This assay is based upon the ability of the factor to induce premature binding between the gametes. Release of the factor occurred in a dose-dependent manner as a function of increasing concentration of sperm, but only after they were capacitated, i.e., subjecting sperm to those conditions which endow them with the ability to penetrate the egg. The factor was released, in what appeared to be pulses of activity, throughout the 40-minutes prepenetration period, and this release culminated in a large pulse which was rapidly terminated soon after penetration began. The factor was also detected following contact with homologous zonae pellucidae from which the vitellus had been mechanically removed. Thus, factor release and cessation of its release occurred independently of the vitellus. When hamster eggs or isolated zonae were replaced by those of the mouse, the factor was not detected even though hamster sperm attached to them; nor was it recovered when isolated zonae or eggs of the hamster were treated with trypsin before exposure to sperm. Factor release and penetration of eggs were inhibited in a similar manner as a function of increasing concentrations of trypsin. This finding and the observation that the factor was not detected when the sperm were not capacitated, and therefore incapable of penetration, suggests that a relationship may exist between factor release and penetration. In order for fertilization to occur in the mammal, a spermatozoon must penetrate through the zona pellucida, a 5 to 10-p-thick envelope which invests the cellular portion of the ovum or vitellus (Austin, '61). Although a wide range of studies have been published, progress in the understanding of the mechanism of the early stages of mammalian fertilization has been hampered by the limited quantity of material available. For example, biochemical investigators have tended to focus on the more abundant gamete, the sperm (McRorie and Williams, '74). Data obtained from such studies are, however, difficult to relate to the process of fertilization. A large number of ultrastructural studies of mammalian fertilization have appeared (Bedford, '68; Yanagimachi and Noda, '70; Bedford, '72). Although useful for showing morphological relationships, the data are J. CELL. PHYSIOL.,93: 41-48.
difficult to interpret in terms of the dynamics of the process of fertilization. Other studies have focused on those changes in the sperm induced by residence in the female reproductive tract which endows them with the ability to penetrate the egg, a process termed capacitation (Austin, '52). A number of in vitro methods are available for capacitating sperm; these include incubation of sperm with complex biological fluids (Yanagimachi, '69, '701, crude commercial enzymes (Gwatkin and Hutchison, '71), hypertonic salt solutions (Oliphant and Brackett, '73b) and cells (Gwatkin, Andersen and Hutchison, '72). The biochemical nature of the change that capacitation produces is unknown, but appears to involve the surface of the sperm (Ericsson, '67; Hartmann and Gwatkin, '71; Oliphant and Brackett, '73a). Received Dee. 23, '76. Accepted May 2, '77.
41
42
JOHN F. HARTMANN AND CAMERON F. HUTCHISON
Our laboratory has been studying mammalian fertilization using an in vitro system with hamster gametes. We have concentrated on the interaction between the gametes after surface contact is made but before the zona is penetrated. By simultaneously studying both gametes a t the earliest contact stages, it was felt that a more direct approach to the mechanism of zona penetration could be made. In addition, penetration of the zona pellucida is a species-specific phenomenon (Barros, '68; Yanagimachi, '72) and i t seemed a reasonable expectation that the pre-penetration contact stages would also be specific. Superficially, this appears not to be the case. For example, hamster sperm will associate rapidly (within 15-30 seconds) by their heads with the zona surface of either homologous or heterologous eggs (Hartmann et al., '721, but only hamster sperm will penetrate hamster eggs. However, through the use of a simple assay, which combines a washing procedure and observation with a dissecting microscope, it has been possible to define and quantitate surface associations between the gametes. We have found that penetration of the zona by the sperm is preceded by two distinct contact stages. The first, termed attachment, is relatively loose and occurs during the initial 35 minutes of contact. The second, termed binding, follows and is tighter than attachment, is speciesspecific and immediately precedes penetration (Hartmann et al., '72; Hartmann and Hutchison, '74a). We also observed that binding between hamster gametes was delayed when hamster, but not mouse, isolated zonae pellucidae (zona lacking a vitellus) were included in the same drop (Hartmann and Hutchison, '74b). Based on the data it was tentatively concluded that a soluble factor, which we termed S1, was generated by contact between hamster sperm and the surface of the isolated zonae which for some unknown reason affected binding between gametes. Further study of the factor was prevented by the limitations of the assay. To overcome this, a new assay for the factor has been devised which offers considerable versatility and which has enabled us to answer a number of important questions. The assay and the results obtained with it are reported here, and indicate that a factor can be detected in the supernatant soon after hamster sperm make contact with either homologous isolated zonae or homologous eggs suggesting that it is released by a specific
sperm-zona contact mechanism. Factor recovery and penetration responded in an identical manner to experimental manipulations, indicating that a relationship between these events may exist. MATERIALS AND METHODS
Epididymal spermatozoa of the golden hamster Mesocricetus auratus were collected in Medium 199M2 (Gwatkin e t al., '72). They were capacitated a t a concentration of 107/ml according to the method of Gwatkin and Williams ('70). Capacitation is used here to designate the sum total of all changes which endow epididymal sperm with the ability to penetrate the egg. Essentially, this method consists of incubating the spermatozoa for five hours a t 37°C in 40-pl drops of 199M2 containing an unwashed cumulus cell mass obtained from an oviduct 17 hours after injection of human chorionic gonadotropin (HCG). Those eggs which had been introduced with the cumulus masses were removed before the binding assays were conducted. Unfertilized eggs for use in the binding assay were removed from oviducts 21 hours after HCG injection. They were pooled in Dulbecco's phosphate buffered-saline containing 1% polyvinylpyrolidone and hyaluronidase (450 units/ml) to remove the cumulus cells. The eggs were washed by transferring them through several changes of 199M2. The binding assay was conducted according to the general procedure described earlier (Hartmann and Gwatkin, '71). This consisted of transferring eggs with associated spermatozoa through three changes of Medium 199M2 with a micropipette of diameter of approximately 140 p , and the number of spermatozoa bound to the eggs were recorded. A Wild M5 dissecting microscope set a t x 37.5 was used to monitor the transfers. Bound sperm were counted a t a magnification of x 75. One micropipette was used. to manipulate gametes in all drops on a given day. Penetration of sperm into the vitelli was observed with a phase-contrast microscope as previously described (Hartmann and Hutchison, '76). The viability of the gametes was assessed every day by scoring penetration of ten eggs by capacitated sperm (1X 107/ml). Only those results were considered which were obtained on those days when all of the eggs were pentrated. Zonae pellucidae were isolated by forcing eggs through a micropipette with an inner di-
FACTOR RELEASE BY HAMSTER GAMETES
43
ameter of about 50 pm. The separated vitelli were discarded and the isolated zonae pellucidae washed by passing them through three changes of Medium 199M2. Most isolated zonae had a single “crack” of varying length. The assay for the soluble factor was conducted using two separate 4O-pl drops. The contents of the first drop (experimental drop) varied depending on the experiment; it contained sperm, cumulus cells if capacitated sperm were used, isolated zonae pellucidae or eggs. The contents of the second drop (assay drop) was invariant; it always contained capacitated hamster sperm (1 X 107/ml), cumulus cells and ten hamster eggs. Factor was assayed by transferring 1p1 of the supernatant from the experimental drop to the assay drop after the sperm and eggs in the assay drop had been mixed for 20 minutes. Binding between the gametes in the assay drop was scored ten minutes later as detailed above. RESULTS
The S1 factor was originally detected by adding isolated zonae (zona with its vitellus removed) to sperm and eggs (zona with vitellus intact) a t the time that the gametes were mixed (Hartmann and Hutchison, ’74b). This resulted in a delay in the binding between gametes. Although the delay was reproducible, an assay had to be devised which was less subject to nonspecific artifacts. In addition, combining the isolated zonae, eggs and sperm in the same drop severely limited the assay. In an effort to overcome these disadvantages a new method was devised which incorporated two major changes. The first change involved the time of addition of the zonae to the sperm and eggs. We found that by introducing the zonae into the drop 20 minutes after the sperm and eggs were combined, instead of a delay in binding, premature binding between the gametes occurred. Figure 1 shows the results of such an experiment in which premature binding was induced between hamster sperm and eggs by the presence of homologous isolated zonae pellucidae. Using half-maximal binding, as a reference point, it can be seen that premature binding occurred about eight minutes earlier than normal binding. Mouse isolated zonae pellucidae failed to induce premature binding between hamster sperm and eggs. Pre-washing of the zonae zero to ten times in Medium 199M2 failed to affect their ability to induce premature binding between the gametes
Time (min.) Since Sperm and Eggs Mixed
Fig. 1 Induction of premature binding between capacitated sperm and eggs in the presence of isolated zonae. Ten isolated zonae in 2-3p1 of 199M2 were added to each drop 20 minutes after gametes were mixed. Each point with the range marker is the mean of a duplicate experiment.
(data not shown). Thus, induction of premature binding is not caused by passive diffusion of material from the isolated zonae. Figure 2 shows that the degree of premature binding, that is the number of sperm bound to eggs 30 minutes after mixing the gametes, is a function of the time that the isolated zonae were added. When isolated zonae were added a t the same time that the gametes were combined no sperm were bound prematurely. However, premature binding between gametes did occur when addition of the isolated zonae was delayed; the later the addition the greater the premature binding. Maximum premature binding occurred when the hamster isolated zonae were added 20 minutes after the gametes were mixed. The induction of premature binding was incorporated into the second major change in the assay, which was the use of two drops instead of one (MATERIALS AND METHODS). From the experimental drop one microliter of the supernatant was transferred to a second drop, the assay drop. The induction of premature binding of gametes in the assay drop was scored ten minutes later. Figure 3 shows
44
JOHN F. HARTMANN AND CAMERON F. HUTCHISON B
,o 4 0 1
T Hamster lsoloted Zonoe
Mouse lsoloted Zonae 10 Time (min.) Zanoe Added After Mixing Gametes
Fig. 2 Induction of premature binding as a function of the time of addition of isolated zonae pellucidae. Ten isolated zonae in 2-3 pl of 1991112 were added to each drop a t the indicated times after the gametes bad been mixed. Premature binding between the gametes was scored 30 minutes after combining them. Each point is the mean of a duplicate experiment with the ranger marker.
the results of the 2-drop assay; i t compares the induction of premature binding by the supernatant of isolated zonae and eggs combined with various concentrations of sperm in the experimental drop. The supernatant of the drops containing either the isolated zonae or eggs increased premature binding directly as a function of the sperm concentration. In contrast, figure 3 also shows that premature binding was not induced by the supernatant when hamster sperm were combined, in the experimental drop, with either isolated zonae or eggs of the mouse instead of those of the hamster. In addition, the supernatant of hamster isolated zonae or eggs combined with or lacking cumulus cells also failed to induce premature binding when added to the assay drop. These results demonstrate the following: (1) That a factor from the supernatant, capable of inducing premature binding between gametes in a second drop, is present only when either homologous gametes or homologous sperm and isolated zonae are mixed; (2) That the factor induces premature binding when added to the assay drop in a dose-dependent manner as a function of the concentration of spermatozoa in the experimental drop.
5
10
Spermotozoa/mt ( x lo-’)
Fig. 3 Induction of premature binding by a supernatant of drops containing capacitated sperm and isolated zonae (A) or capacitated sperm and eggs (B) as a function of the concentration of sperm. Isolated zonae or eggs were mixed with sperm in the experimental drop, and after 30 minutes 1 p1 of the supernatant was transferred to an assay drop and the induction of premature binding scored as detailed in MATERIALS AND METHODS. Two duplicate determinations were made for each point on separate days. The range marker is the standard error of the mean.
The effect of dilution on the ability of the factor to induce premature binding was explored. Figure 4 shows that the supernatant of drops containing capacitated hamster sperm combined with either homologous isolated zonae or eggs was affected in a similar manner by dilution. At dilution levels of 60: 1 and 8O:l there was a sharp drop in the ability of the supernatants to induce premature binding, but a t greater dilutions the decline became less pronounced. An experiment was conducted to determine if factor activity could be detected after mixing epididymal sperm, which were not capacitated, with isolated zonae for various periods of time. Such sperm, although incapable of penetration, do bind to eggs and isolated zonae but by a mechanism which is different from that of capacitated sperm (Hartmann and Gwatkin, ’71; Hartmann and Hutchison, ’77). The results in figure 5 compare the recovery of factor from drops containing uncapacitated with those containing capaci-
FACTOR RELEASE BY HAMSTER GAMETES '
'I
45
Final Dilution of Factor
Fig. 4 Effect of dilution on ability of supernatant of capacitated hamster sperm and isolated zonae (A) and capacitated hamster sperm and eggs (B) to induce premature binding between gametes. The dilution of 41:l represents the standard experiment in which 1pi of the supernatant from the experimental drop is transferred to a 40-p1 assay drop containing capacitated sperm and egg. All of the other dilutions were performed by removing 2 p l from the assay drop and mixing i t with an appropriate volume of 199M2. One microliter of this diluted solution was then diluted 41: 1by transfer to the assay drop. Each point in figures 4A and 4B is the mean of four and two replicates, respectively.
I
11
Uncapacitated Sperm
10
20
I
30
a
4(
Minutes After Addition of Sperm
Fig. 5 Induction of premature binding by the supernat a n t of drops containing isolated zonae pellucidae combined with uncapacitated or capacitated sperm for various periods of time. Spermatozoa were used a t a concentration of 1 X lO'/ml. Each point is the mean of a duplicate experiment with the range.
tated sperm in the presence of isolated zonae. Factor is not recovered when uncapacitated sperm are used, even after sperm and zonae have been combined for 40 minutes. In contrast to this, a significant quantity of factor is
recovered from drops containing capacitated sperm when assayed a t 2 and 40 minutes but not a t 15 minutes (see below). The failure to detect the factor with uncapacitated sperm suggested that a relationship between factor recovery and penetration might exist. The possibility of such a relationship was explored. Previously, it was shown that pretreatment of eggs with trypsin blocked penetration of sperm (Hartmann and Gwatkin, '71; Oikawa et al., '75). We, therefore, treated isolated zonae and eggs with trypsin and scored factor release and penetration, respectively. The results in table 1 show that trypsin treatment, in addition to blocking penetration of eggs, prevented factor recovery from drops containing either eggs or isolated zonae. Neuraminidase and a mixture of other glycosidases failed to block factor recovery and penetration; neuraminidase, in fact, appeared to increase the amount of factor recovered. To explore more thoroughly the correlation between recovery of the factor and penetration, we compared these parameters as a function of increasing concentration of trypsin from 0.02-0.2 pg/ml in blind experiments. The sensitivity of factor recovery to trypsin was assessed after sperm and isolated zonae were combined for two minutes (fig. 6A) and 30 minutes (fig. 6B). The results show that factor recovery and penetration re-
46
JOHN F. HARTMANN AND CAMERON F. HUTCHISON
... 0
;I
10-
2
I i 9 - ; 2
Oo
a02
om
Tiypsin Conmentrotion W g / m l )
Fig. 6 Comparison of the effect of trypsin treatment on factor release and penetration. Isolated zonae pellucidae and eggs were treated with trypsin (Worthington, 3 X cryst., 200 unitdmg) for 60 minutes in Dulbecco's phosphate buffered saline (PBS)at 37°C and washed three times by transfer through three changes of PBS. Factor activity in the supernatant was assayed after the sperm and trypsin isolated zonae had been combined for 2 minutes (A) and 30 minutes (B). In separate drops penetration of trypsin treated eggs by sper.n was scored 90 minutes after mixing the gametes.
sponded similarly to increasing concentrations of trypsin. At a level of 0.02 p g trypsinf ml there was little or no reduction of either factor recovery or penetration. However, a t 0.05 p g trypsinfml penetration was virtually abolished. An effect on factor recovery was also apparent a t this level of enzyme; after two minutes recovery was reduced 77% a t a level of 0.05 p g trypsinfml; whereas after 30 minutes factor recovery was reduced only
about 20%.At a level of 0.1 p g trypsidml, the factor was no longer detected a t either time. In view of the observation that the quantity of factor recovered was affected by time (fig. 51, we assessed more thoroughly the recovery as a function of the period of time that sperm were combined with isolated zonae or eggs. Figure 7A shows that with the isolated zonae, activity of the factor peaked at two minutes and dropped sharply a t three minutes. The activity remained low until a second peak appeared a t 20 minutes. A third peak of activity appeared a t 31 minutes followed by a sharp drop a t 34 minutes. At 40-50 minutes, a final broad peak of activity occurred which was rapidly terminated. Activity of the soluble factor was not detected a t any time when mouse isolated zonae were combined with hamster sperm. Figure 7B shows the activity of the factor when isolated zonae were replaced by eggs. In general, the pattern of activity is similar to that obtained with isolated zonae; there are, however, differences: (1) throughout the first 40-minute period the peaks of factor activity are larger in drops containing isolated zonae, (2) the timing of the second peak fails to coincide in the isolated zona and egg preparations, appearing a t 20 and 25 minutes, respectively and (3) the final peak was sharper in the drops which contained egg than its counterpart in the drops which contained the isolated zonae. Both sets of results reveal a pattern of peaks which might be described as pulses of discrete activity culminating in a final large peak a t the time that binding is completed and penetration begins (arrow: fig. 7B). DISCUSSION
In a previous study it was observed that
TABLE 1
Factor recouery and penetration after treatment of isolated zonae and eggs with enzymes Factor recovery (% of control) Enzyme
Trypsin Neuraminidase Glycosidase mixture
Concentration ((rgiml)
Isolated zonae
0.1 10.0 10.0
0 200 91
Eggs
0
-
Penetration of eggs (% of control)
0 100 100
All enzymes were dissolved in PBS and exposed to isolated zonae and eggs for 60 minutes at 37°C in COzincubator. The isolated zonae and eggs were washed and then added to sperm in the experimental drop for 31 minutes. Factor was assayed as detailed in MATERIALS AND METHODS. Trypsin (bovine pancreatic, three times crystallized, 200 unitslmg) and neuraminidase fClostridiumperfringen, 0.80 unitslmg) were obtained from Worthington Biochemicals. The glycosidase mixture fcharonia lampus) was obtained from Miles Laboratory.All enzymes in the glycosidase mixture except P-xylosidase were assayed and contained the activity claimed. Lima bean inhibitor (1&g/ml)was added to the neuraminidase and mixed glycosidase preparations to inactivate contaminating trypsin.
FACTOR RELEASE BY HAMSTER GAMETES
47
Minutes After Addition to Sperm
Fig. 7 Induction of premature binding by one microliter of the supernatant of drops containing isolated zonae (A) and eggs (B) as a function of the time combined with sperm at a concentration of 1 X lO'/ml. Each point with the range marker is the mean of four determinations (experimentin duplicate on two separate days). Each range marker is the standard error of the mean. Points lacking the range marker are the means of two determinations fa single duplicate experiment).The arrow in figure 7B indicates the time when binding is completed and penetration begins.
binding between hamster eggs and capacitated sperm was delayed when homologous but not heterologous isolated zonae pellucidae were introduced into drops a t the same time that the gametes were combined (Hartmann and Hutchison, '74b). We tentatively concluded that a factor termed S1 was generated by contact between isolated zonae and sperm which, for an unknown reason, delayed binding between the gametes. The technical restrictions imposed by combining all of the components into a single drop prevented us from further investigating this perplexing but potentially interesting phenomenon. The 2-drop assay described in this report has overcome many of the limitations of the one drop method. The additional refinement of inducing premature binding proved to be a more reliable indicator of factor activity than did the delay of binding. Because the new assay differs from the previous one (Hartmann and Hutchison, '74b) and because the nature of the factors is still unknown we cannot be certain that the material detected by the two assays are identical. However, with this reservation in mind and to avoid possible unwarranted confusion, iden-
tity will be assumed and the term S1 will be applied to the factor described in this paper. The results of the new method shows that the S1 factor can be recovered from the supernatant of capacitated hamster sperm mixed with either homologous eggs or homologous isolated zonae throughout the pre-penetration period. Prior to mixing, the S1 factor was not present in the supernatants of either eggs or capacitated sperm. Like binding (Hartmann and Hutchison, '76) the quantity of factor recovered was found to be sperm dose-dependent. The release of the S1 factor is very specific, requiring more than just contact between the surfaces of sperm and zona pellucida. It fails to appear (1)when uncapacitated sperm bind to isolated zonae, (2) following treatment of isolated zonae trypsin and (3) when isolated zonae or eggs of the mouse are mixed with hamster sperm. Taken together these results point to the conclusion that the S 1 factor is released from the sperm and/or the zonae by a specific contact mechanism. Our studies with isolated zonae clearly show that release of the factor operates independently of the vitellus. Whether the differences in the patterns of peaks observed
48
JOHN F. HARTMANN AND CAMERON F. HUTCHISON
between eggs and isolated zonae reflect a vitelline influence or perhaps a non-specific effect related to the isolation procedure remains to be determined. The activity of the factor throughout the pre-penetration period is evident as peaks and culminates in a very large peak a t the time that penetration begins (Hartmann and Hutchison, '76). The multiphasic pattern suggests that the S1 factor may be unstable. I t is striking that the factor is not detected after penetration is initiated; this suggests that its release may reflect some preparative activity for penetration. A relationship between the appearance of the factor in the supernatant and penetration is also suggested by the sensitivity of both of these parameters to trypsin (fig. 16). Such a relationship is further supported by the finding that the S1 factor is not detected when uncapacitated sperm, which are incapable of penetration, bind to the surface of the zona pellucida (fig. 5 ) . I t is interesting to note that the sperm acrosome reaction, an essential prelude to penetration, occurs only after the sperm are capacitated (Bedford, '72). In addition, like factor release, the acrosome reaction has been shown to be dependent upon the concentration of spermatozoa (Talbot et al., '74). Thus release of the S1 factor may be related to the acrosome reaction. LITERATURE CITED Austin, C. R. 1952 The capacitation of mammalian sperm. Nature, 170: 326-327. 1961 The Mammalian Egg. Charles C Thomas, Springfield, Illinois. Barros, C. 1968 In vitro capacitation of golden hamster spermatozoa with fallopian tube fluid of the mouse and rat. J. Reprod. Fert., 17: 203-206. Bedford, J. M. 1968 Ultrastructural changes in the sperm head during fertilization in the rabbit. Am. J. Anat., 123: 329-358. 1972 Sperm capacitation in mammals. Biol. Reprod. (Suppl.), 2: 123-158. Ericsson, R. J. 1967 A fluorimetric method for measuring sperm capacitation. Proc. Soc. Exp. Biol. Med., 125: 1115-1118. Gwatkin, R. B. L., 0. F. Andersen and C. F. Hutchison 1972
-
Capacitation of hamster spermatozoa in uitro:The role of cumulus components. J. Reprod. Fertil., 30: 389-394. Gwatkin, R. B. L., and C. F. Hutchison 1971 Capacitation of hamster spermatozoa by P-glucuronidase. Nature, 229: 343-344. Gwatkin, R. B. L., and D. L. Williams 1970 Inhibition of sperm capacitation in uitro by contraceptive steroids. Nature, 227: 182-183. Hartmann, J. F., and R. B. L. Gwatkin 1971 Alteration of sites on the mammalian sperm surface following capacitation. Nature, 234: 479-481. Hartmann, J. F., R. B. L. Gwatkin and C. F. Hutchison 1972 Early contact interactions between mammalian gametes in uitro: Evidence that the vitellus influences adherence between sperm and zona pellucida. Proc. Natl. Aead. Sci. (U.S.A.), 64: 191-195. Hartmann, J. F., and C. F. Hutchison 1974a Nature of the pre-penetration contact interactions between hamster gametes in uitro. J. Reprod. Fertil., 36: 49-57. - 197413 Contact between hamster spermatozoa and the zona pellucida releases a factor which influences early binding stages. J. Reprod. Fertil., 37: 61-66. 1976 Surface interactions between mammalian sperm and egg: Variation of spermatozoa concentration as a probe for the study of binding in vitro. J. Cell. Physiol., 88: 219-226. 1977 Involvement of two carbohydrate-containing components in the binding of uncapacitated spermatozoa to eggs of the golden hamster in vitro. J. Exp. Zool., 201: 383-390. McRorie, R. A., and W. L. Williams 1974 Biochemistry of mammalian fertilization. Ann. Rev. Biochem., 43: 777-803. Oikawa, T., G . L. Nicolson and R.Yanagimachi 1975 Trypsin-mediated modification of the zona pellucida glycopeptide structure of hamster eggs. J. Reprod. Fertil., 43: 133-136. Oliphant, G., and B. G. Brackett 1973a Immunological assessment of surface charges of rabbit sperm undergoing capacitation. Biol. Reprod., 9: 404-414. 1973b Capacitation of mouse spermatozoa in media with elevated ionic strength and reversible decapacitation with epididymal extracts. Fertil. Steril., 24: 948-955. Talbot, P., L. E. Franklin and E. N. Fussell 1974 The effect of the concentration of golden hamster spermatozoa on the acrosome reaction and egg penetration in uitro. J. Reprod. Fert., 36: 429-432. Yanagimachi, R. 1969 In uitro capacitation of hamster spermatozoa by follicular fluid. J. Reprod. Fert., 18: 275-286. 1970 In uitro capacitation of golden hamster spermatozoa by homologous and heterologous b l d sera. Biol. Reprod., 3: 147-153. 1972 Penetration of guinea pig spermatozoa into hamster eggs In uitro. J. Reprod. Fert., 28: 477-480. Yanagamachi, R., and R. D. Noda 1970 Ulstrastructural changes in the hamster head during fertilization. J. U1tras6uct. Res., 31: 365-485.
-
-
ABSTRACT A sensitive bioassay was devised which detected the release of a factor resulting from contact between the surfaces of sperm and zona pellucida of the golden hamster in vitro. This assay is based upon the ability of the factor to induce premature binding between the gametes. Release of the factor occurred in a dose-dependent manner as a function of increasing concentration of sperm, but only after they were capacitated, i.e., subjecting sperm to those conditions which endow them with the ability to penetrate the egg. The factor was released, in what appeared to be pulses of activity, throughout the 40-minutes prepenetration period, and this release culminated in a large pulse which was rapidly terminated soon after penetration began. The factor was also detected following contact with homologous zonae pellucidae from which the vitellus had been mechanically removed. Thus, factor release and cessation of its release occurred independently of the vitellus. When hamster eggs or isolated zonae were replaced by those of the mouse, the factor was not detected even though hamster sperm attached to them; nor was it recovered when isolated zonae or eggs of the hamster were treated with trypsin before exposure to sperm. Factor release and penetration of eggs were inhibited in a similar manner as a function of increasing concentrations of trypsin. This finding and the observation that the factor was not detected when the sperm were not capacitated, and therefore incapable of penetration, suggests that a relationship may exist between factor release and penetration. In order for fertilization to occur in the mammal, a spermatozoon must penetrate through the zona pellucida, a 5 to 10-p-thick envelope which invests the cellular portion of the ovum or vitellus (Austin, '61). Although a wide range of studies have been published, progress in the understanding of the mechanism of the early stages of mammalian fertilization has been hampered by the limited quantity of material available. For example, biochemical investigators have tended to focus on the more abundant gamete, the sperm (McRorie and Williams, '74). Data obtained from such studies are, however, difficult to relate to the process of fertilization. A large number of ultrastructural studies of mammalian fertilization have appeared (Bedford, '68; Yanagimachi and Noda, '70; Bedford, '72). Although useful for showing morphological relationships, the data are J. CELL. PHYSIOL.,93: 41-48.
difficult to interpret in terms of the dynamics of the process of fertilization. Other studies have focused on those changes in the sperm induced by residence in the female reproductive tract which endows them with the ability to penetrate the egg, a process termed capacitation (Austin, '52). A number of in vitro methods are available for capacitating sperm; these include incubation of sperm with complex biological fluids (Yanagimachi, '69, '701, crude commercial enzymes (Gwatkin and Hutchison, '71), hypertonic salt solutions (Oliphant and Brackett, '73b) and cells (Gwatkin, Andersen and Hutchison, '72). The biochemical nature of the change that capacitation produces is unknown, but appears to involve the surface of the sperm (Ericsson, '67; Hartmann and Gwatkin, '71; Oliphant and Brackett, '73a). Received Dee. 23, '76. Accepted May 2, '77.
41
42
JOHN F. HARTMANN AND CAMERON F. HUTCHISON
Our laboratory has been studying mammalian fertilization using an in vitro system with hamster gametes. We have concentrated on the interaction between the gametes after surface contact is made but before the zona is penetrated. By simultaneously studying both gametes a t the earliest contact stages, it was felt that a more direct approach to the mechanism of zona penetration could be made. In addition, penetration of the zona pellucida is a species-specific phenomenon (Barros, '68; Yanagimachi, '72) and i t seemed a reasonable expectation that the pre-penetration contact stages would also be specific. Superficially, this appears not to be the case. For example, hamster sperm will associate rapidly (within 15-30 seconds) by their heads with the zona surface of either homologous or heterologous eggs (Hartmann et al., '721, but only hamster sperm will penetrate hamster eggs. However, through the use of a simple assay, which combines a washing procedure and observation with a dissecting microscope, it has been possible to define and quantitate surface associations between the gametes. We have found that penetration of the zona by the sperm is preceded by two distinct contact stages. The first, termed attachment, is relatively loose and occurs during the initial 35 minutes of contact. The second, termed binding, follows and is tighter than attachment, is speciesspecific and immediately precedes penetration (Hartmann et al., '72; Hartmann and Hutchison, '74a). We also observed that binding between hamster gametes was delayed when hamster, but not mouse, isolated zonae pellucidae (zona lacking a vitellus) were included in the same drop (Hartmann and Hutchison, '74b). Based on the data it was tentatively concluded that a soluble factor, which we termed S1, was generated by contact between hamster sperm and the surface of the isolated zonae which for some unknown reason affected binding between gametes. Further study of the factor was prevented by the limitations of the assay. To overcome this, a new assay for the factor has been devised which offers considerable versatility and which has enabled us to answer a number of important questions. The assay and the results obtained with it are reported here, and indicate that a factor can be detected in the supernatant soon after hamster sperm make contact with either homologous isolated zonae or homologous eggs suggesting that it is released by a specific
sperm-zona contact mechanism. Factor recovery and penetration responded in an identical manner to experimental manipulations, indicating that a relationship between these events may exist. MATERIALS AND METHODS
Epididymal spermatozoa of the golden hamster Mesocricetus auratus were collected in Medium 199M2 (Gwatkin e t al., '72). They were capacitated a t a concentration of 107/ml according to the method of Gwatkin and Williams ('70). Capacitation is used here to designate the sum total of all changes which endow epididymal sperm with the ability to penetrate the egg. Essentially, this method consists of incubating the spermatozoa for five hours a t 37°C in 40-pl drops of 199M2 containing an unwashed cumulus cell mass obtained from an oviduct 17 hours after injection of human chorionic gonadotropin (HCG). Those eggs which had been introduced with the cumulus masses were removed before the binding assays were conducted. Unfertilized eggs for use in the binding assay were removed from oviducts 21 hours after HCG injection. They were pooled in Dulbecco's phosphate buffered-saline containing 1% polyvinylpyrolidone and hyaluronidase (450 units/ml) to remove the cumulus cells. The eggs were washed by transferring them through several changes of 199M2. The binding assay was conducted according to the general procedure described earlier (Hartmann and Gwatkin, '71). This consisted of transferring eggs with associated spermatozoa through three changes of Medium 199M2 with a micropipette of diameter of approximately 140 p , and the number of spermatozoa bound to the eggs were recorded. A Wild M5 dissecting microscope set a t x 37.5 was used to monitor the transfers. Bound sperm were counted a t a magnification of x 75. One micropipette was used. to manipulate gametes in all drops on a given day. Penetration of sperm into the vitelli was observed with a phase-contrast microscope as previously described (Hartmann and Hutchison, '76). The viability of the gametes was assessed every day by scoring penetration of ten eggs by capacitated sperm (1X 107/ml). Only those results were considered which were obtained on those days when all of the eggs were pentrated. Zonae pellucidae were isolated by forcing eggs through a micropipette with an inner di-
FACTOR RELEASE BY HAMSTER GAMETES
43
ameter of about 50 pm. The separated vitelli were discarded and the isolated zonae pellucidae washed by passing them through three changes of Medium 199M2. Most isolated zonae had a single “crack” of varying length. The assay for the soluble factor was conducted using two separate 4O-pl drops. The contents of the first drop (experimental drop) varied depending on the experiment; it contained sperm, cumulus cells if capacitated sperm were used, isolated zonae pellucidae or eggs. The contents of the second drop (assay drop) was invariant; it always contained capacitated hamster sperm (1 X 107/ml), cumulus cells and ten hamster eggs. Factor was assayed by transferring 1p1 of the supernatant from the experimental drop to the assay drop after the sperm and eggs in the assay drop had been mixed for 20 minutes. Binding between the gametes in the assay drop was scored ten minutes later as detailed above. RESULTS
The S1 factor was originally detected by adding isolated zonae (zona with its vitellus removed) to sperm and eggs (zona with vitellus intact) a t the time that the gametes were mixed (Hartmann and Hutchison, ’74b). This resulted in a delay in the binding between gametes. Although the delay was reproducible, an assay had to be devised which was less subject to nonspecific artifacts. In addition, combining the isolated zonae, eggs and sperm in the same drop severely limited the assay. In an effort to overcome these disadvantages a new method was devised which incorporated two major changes. The first change involved the time of addition of the zonae to the sperm and eggs. We found that by introducing the zonae into the drop 20 minutes after the sperm and eggs were combined, instead of a delay in binding, premature binding between the gametes occurred. Figure 1 shows the results of such an experiment in which premature binding was induced between hamster sperm and eggs by the presence of homologous isolated zonae pellucidae. Using half-maximal binding, as a reference point, it can be seen that premature binding occurred about eight minutes earlier than normal binding. Mouse isolated zonae pellucidae failed to induce premature binding between hamster sperm and eggs. Pre-washing of the zonae zero to ten times in Medium 199M2 failed to affect their ability to induce premature binding between the gametes
Time (min.) Since Sperm and Eggs Mixed
Fig. 1 Induction of premature binding between capacitated sperm and eggs in the presence of isolated zonae. Ten isolated zonae in 2-3p1 of 199M2 were added to each drop 20 minutes after gametes were mixed. Each point with the range marker is the mean of a duplicate experiment.
(data not shown). Thus, induction of premature binding is not caused by passive diffusion of material from the isolated zonae. Figure 2 shows that the degree of premature binding, that is the number of sperm bound to eggs 30 minutes after mixing the gametes, is a function of the time that the isolated zonae were added. When isolated zonae were added a t the same time that the gametes were combined no sperm were bound prematurely. However, premature binding between gametes did occur when addition of the isolated zonae was delayed; the later the addition the greater the premature binding. Maximum premature binding occurred when the hamster isolated zonae were added 20 minutes after the gametes were mixed. The induction of premature binding was incorporated into the second major change in the assay, which was the use of two drops instead of one (MATERIALS AND METHODS). From the experimental drop one microliter of the supernatant was transferred to a second drop, the assay drop. The induction of premature binding of gametes in the assay drop was scored ten minutes later. Figure 3 shows
44
JOHN F. HARTMANN AND CAMERON F. HUTCHISON B
,o 4 0 1
T Hamster lsoloted Zonoe
Mouse lsoloted Zonae 10 Time (min.) Zanoe Added After Mixing Gametes
Fig. 2 Induction of premature binding as a function of the time of addition of isolated zonae pellucidae. Ten isolated zonae in 2-3 pl of 1991112 were added to each drop a t the indicated times after the gametes bad been mixed. Premature binding between the gametes was scored 30 minutes after combining them. Each point is the mean of a duplicate experiment with the ranger marker.
the results of the 2-drop assay; i t compares the induction of premature binding by the supernatant of isolated zonae and eggs combined with various concentrations of sperm in the experimental drop. The supernatant of the drops containing either the isolated zonae or eggs increased premature binding directly as a function of the sperm concentration. In contrast, figure 3 also shows that premature binding was not induced by the supernatant when hamster sperm were combined, in the experimental drop, with either isolated zonae or eggs of the mouse instead of those of the hamster. In addition, the supernatant of hamster isolated zonae or eggs combined with or lacking cumulus cells also failed to induce premature binding when added to the assay drop. These results demonstrate the following: (1) That a factor from the supernatant, capable of inducing premature binding between gametes in a second drop, is present only when either homologous gametes or homologous sperm and isolated zonae are mixed; (2) That the factor induces premature binding when added to the assay drop in a dose-dependent manner as a function of the concentration of spermatozoa in the experimental drop.
5
10
Spermotozoa/mt ( x lo-’)
Fig. 3 Induction of premature binding by a supernatant of drops containing capacitated sperm and isolated zonae (A) or capacitated sperm and eggs (B) as a function of the concentration of sperm. Isolated zonae or eggs were mixed with sperm in the experimental drop, and after 30 minutes 1 p1 of the supernatant was transferred to an assay drop and the induction of premature binding scored as detailed in MATERIALS AND METHODS. Two duplicate determinations were made for each point on separate days. The range marker is the standard error of the mean.
The effect of dilution on the ability of the factor to induce premature binding was explored. Figure 4 shows that the supernatant of drops containing capacitated hamster sperm combined with either homologous isolated zonae or eggs was affected in a similar manner by dilution. At dilution levels of 60: 1 and 8O:l there was a sharp drop in the ability of the supernatants to induce premature binding, but a t greater dilutions the decline became less pronounced. An experiment was conducted to determine if factor activity could be detected after mixing epididymal sperm, which were not capacitated, with isolated zonae for various periods of time. Such sperm, although incapable of penetration, do bind to eggs and isolated zonae but by a mechanism which is different from that of capacitated sperm (Hartmann and Gwatkin, ’71; Hartmann and Hutchison, ’77). The results in figure 5 compare the recovery of factor from drops containing uncapacitated with those containing capaci-
FACTOR RELEASE BY HAMSTER GAMETES '
'I
45
Final Dilution of Factor
Fig. 4 Effect of dilution on ability of supernatant of capacitated hamster sperm and isolated zonae (A) and capacitated hamster sperm and eggs (B) to induce premature binding between gametes. The dilution of 41:l represents the standard experiment in which 1pi of the supernatant from the experimental drop is transferred to a 40-p1 assay drop containing capacitated sperm and egg. All of the other dilutions were performed by removing 2 p l from the assay drop and mixing i t with an appropriate volume of 199M2. One microliter of this diluted solution was then diluted 41: 1by transfer to the assay drop. Each point in figures 4A and 4B is the mean of four and two replicates, respectively.
I
11
Uncapacitated Sperm
10
20
I
30
a
4(
Minutes After Addition of Sperm
Fig. 5 Induction of premature binding by the supernat a n t of drops containing isolated zonae pellucidae combined with uncapacitated or capacitated sperm for various periods of time. Spermatozoa were used a t a concentration of 1 X lO'/ml. Each point is the mean of a duplicate experiment with the range.
tated sperm in the presence of isolated zonae. Factor is not recovered when uncapacitated sperm are used, even after sperm and zonae have been combined for 40 minutes. In contrast to this, a significant quantity of factor is
recovered from drops containing capacitated sperm when assayed a t 2 and 40 minutes but not a t 15 minutes (see below). The failure to detect the factor with uncapacitated sperm suggested that a relationship between factor recovery and penetration might exist. The possibility of such a relationship was explored. Previously, it was shown that pretreatment of eggs with trypsin blocked penetration of sperm (Hartmann and Gwatkin, '71; Oikawa et al., '75). We, therefore, treated isolated zonae and eggs with trypsin and scored factor release and penetration, respectively. The results in table 1 show that trypsin treatment, in addition to blocking penetration of eggs, prevented factor recovery from drops containing either eggs or isolated zonae. Neuraminidase and a mixture of other glycosidases failed to block factor recovery and penetration; neuraminidase, in fact, appeared to increase the amount of factor recovered. To explore more thoroughly the correlation between recovery of the factor and penetration, we compared these parameters as a function of increasing concentration of trypsin from 0.02-0.2 pg/ml in blind experiments. The sensitivity of factor recovery to trypsin was assessed after sperm and isolated zonae were combined for two minutes (fig. 6A) and 30 minutes (fig. 6B). The results show that factor recovery and penetration re-
46
JOHN F. HARTMANN AND CAMERON F. HUTCHISON
... 0
;I
10-
2
I i 9 - ; 2
Oo
a02
om
Tiypsin Conmentrotion W g / m l )
Fig. 6 Comparison of the effect of trypsin treatment on factor release and penetration. Isolated zonae pellucidae and eggs were treated with trypsin (Worthington, 3 X cryst., 200 unitdmg) for 60 minutes in Dulbecco's phosphate buffered saline (PBS)at 37°C and washed three times by transfer through three changes of PBS. Factor activity in the supernatant was assayed after the sperm and trypsin isolated zonae had been combined for 2 minutes (A) and 30 minutes (B). In separate drops penetration of trypsin treated eggs by sper.n was scored 90 minutes after mixing the gametes.
sponded similarly to increasing concentrations of trypsin. At a level of 0.02 p g trypsinf ml there was little or no reduction of either factor recovery or penetration. However, a t 0.05 p g trypsinfml penetration was virtually abolished. An effect on factor recovery was also apparent a t this level of enzyme; after two minutes recovery was reduced 77% a t a level of 0.05 p g trypsinfml; whereas after 30 minutes factor recovery was reduced only
about 20%.At a level of 0.1 p g trypsidml, the factor was no longer detected a t either time. In view of the observation that the quantity of factor recovered was affected by time (fig. 51, we assessed more thoroughly the recovery as a function of the period of time that sperm were combined with isolated zonae or eggs. Figure 7A shows that with the isolated zonae, activity of the factor peaked at two minutes and dropped sharply a t three minutes. The activity remained low until a second peak appeared a t 20 minutes. A third peak of activity appeared a t 31 minutes followed by a sharp drop a t 34 minutes. At 40-50 minutes, a final broad peak of activity occurred which was rapidly terminated. Activity of the soluble factor was not detected a t any time when mouse isolated zonae were combined with hamster sperm. Figure 7B shows the activity of the factor when isolated zonae were replaced by eggs. In general, the pattern of activity is similar to that obtained with isolated zonae; there are, however, differences: (1) throughout the first 40-minute period the peaks of factor activity are larger in drops containing isolated zonae, (2) the timing of the second peak fails to coincide in the isolated zona and egg preparations, appearing a t 20 and 25 minutes, respectively and (3) the final peak was sharper in the drops which contained egg than its counterpart in the drops which contained the isolated zonae. Both sets of results reveal a pattern of peaks which might be described as pulses of discrete activity culminating in a final large peak a t the time that binding is completed and penetration begins (arrow: fig. 7B). DISCUSSION
In a previous study it was observed that
TABLE 1
Factor recouery and penetration after treatment of isolated zonae and eggs with enzymes Factor recovery (% of control) Enzyme
Trypsin Neuraminidase Glycosidase mixture
Concentration ((rgiml)
Isolated zonae
0.1 10.0 10.0
0 200 91
Eggs
0
-
Penetration of eggs (% of control)
0 100 100
All enzymes were dissolved in PBS and exposed to isolated zonae and eggs for 60 minutes at 37°C in COzincubator. The isolated zonae and eggs were washed and then added to sperm in the experimental drop for 31 minutes. Factor was assayed as detailed in MATERIALS AND METHODS. Trypsin (bovine pancreatic, three times crystallized, 200 unitslmg) and neuraminidase fClostridiumperfringen, 0.80 unitslmg) were obtained from Worthington Biochemicals. The glycosidase mixture fcharonia lampus) was obtained from Miles Laboratory.All enzymes in the glycosidase mixture except P-xylosidase were assayed and contained the activity claimed. Lima bean inhibitor (1&g/ml)was added to the neuraminidase and mixed glycosidase preparations to inactivate contaminating trypsin.
FACTOR RELEASE BY HAMSTER GAMETES
47
Minutes After Addition to Sperm
Fig. 7 Induction of premature binding by one microliter of the supernatant of drops containing isolated zonae (A) and eggs (B) as a function of the time combined with sperm at a concentration of 1 X lO'/ml. Each point with the range marker is the mean of four determinations (experimentin duplicate on two separate days). Each range marker is the standard error of the mean. Points lacking the range marker are the means of two determinations fa single duplicate experiment).The arrow in figure 7B indicates the time when binding is completed and penetration begins.
binding between hamster eggs and capacitated sperm was delayed when homologous but not heterologous isolated zonae pellucidae were introduced into drops a t the same time that the gametes were combined (Hartmann and Hutchison, '74b). We tentatively concluded that a factor termed S1 was generated by contact between isolated zonae and sperm which, for an unknown reason, delayed binding between the gametes. The technical restrictions imposed by combining all of the components into a single drop prevented us from further investigating this perplexing but potentially interesting phenomenon. The 2-drop assay described in this report has overcome many of the limitations of the one drop method. The additional refinement of inducing premature binding proved to be a more reliable indicator of factor activity than did the delay of binding. Because the new assay differs from the previous one (Hartmann and Hutchison, '74b) and because the nature of the factors is still unknown we cannot be certain that the material detected by the two assays are identical. However, with this reservation in mind and to avoid possible unwarranted confusion, iden-
tity will be assumed and the term S1 will be applied to the factor described in this paper. The results of the new method shows that the S1 factor can be recovered from the supernatant of capacitated hamster sperm mixed with either homologous eggs or homologous isolated zonae throughout the pre-penetration period. Prior to mixing, the S1 factor was not present in the supernatants of either eggs or capacitated sperm. Like binding (Hartmann and Hutchison, '76) the quantity of factor recovered was found to be sperm dose-dependent. The release of the S1 factor is very specific, requiring more than just contact between the surfaces of sperm and zona pellucida. It fails to appear (1)when uncapacitated sperm bind to isolated zonae, (2) following treatment of isolated zonae trypsin and (3) when isolated zonae or eggs of the mouse are mixed with hamster sperm. Taken together these results point to the conclusion that the S 1 factor is released from the sperm and/or the zonae by a specific contact mechanism. Our studies with isolated zonae clearly show that release of the factor operates independently of the vitellus. Whether the differences in the patterns of peaks observed
48
JOHN F. HARTMANN AND CAMERON F. HUTCHISON
between eggs and isolated zonae reflect a vitelline influence or perhaps a non-specific effect related to the isolation procedure remains to be determined. The activity of the factor throughout the pre-penetration period is evident as peaks and culminates in a very large peak a t the time that penetration begins (Hartmann and Hutchison, '76). The multiphasic pattern suggests that the S1 factor may be unstable. I t is striking that the factor is not detected after penetration is initiated; this suggests that its release may reflect some preparative activity for penetration. A relationship between the appearance of the factor in the supernatant and penetration is also suggested by the sensitivity of both of these parameters to trypsin (fig. 16). Such a relationship is further supported by the finding that the S1 factor is not detected when uncapacitated sperm, which are incapable of penetration, bind to the surface of the zona pellucida (fig. 5 ) . I t is interesting to note that the sperm acrosome reaction, an essential prelude to penetration, occurs only after the sperm are capacitated (Bedford, '72). In addition, like factor release, the acrosome reaction has been shown to be dependent upon the concentration of spermatozoa (Talbot et al., '74). Thus release of the S1 factor may be related to the acrosome reaction. LITERATURE CITED Austin, C. R. 1952 The capacitation of mammalian sperm. Nature, 170: 326-327. 1961 The Mammalian Egg. Charles C Thomas, Springfield, Illinois. Barros, C. 1968 In vitro capacitation of golden hamster spermatozoa with fallopian tube fluid of the mouse and rat. J. Reprod. Fert., 17: 203-206. Bedford, J. M. 1968 Ultrastructural changes in the sperm head during fertilization in the rabbit. Am. J. Anat., 123: 329-358. 1972 Sperm capacitation in mammals. Biol. Reprod. (Suppl.), 2: 123-158. Ericsson, R. J. 1967 A fluorimetric method for measuring sperm capacitation. Proc. Soc. Exp. Biol. Med., 125: 1115-1118. Gwatkin, R. B. L., 0. F. Andersen and C. F. Hutchison 1972
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Capacitation of hamster spermatozoa in uitro:The role of cumulus components. J. Reprod. Fertil., 30: 389-394. Gwatkin, R. B. L., and C. F. Hutchison 1971 Capacitation of hamster spermatozoa by P-glucuronidase. Nature, 229: 343-344. Gwatkin, R. B. L., and D. L. Williams 1970 Inhibition of sperm capacitation in uitro by contraceptive steroids. Nature, 227: 182-183. Hartmann, J. F., and R. B. L. Gwatkin 1971 Alteration of sites on the mammalian sperm surface following capacitation. Nature, 234: 479-481. Hartmann, J. F., R. B. L. Gwatkin and C. F. Hutchison 1972 Early contact interactions between mammalian gametes in uitro: Evidence that the vitellus influences adherence between sperm and zona pellucida. Proc. Natl. Aead. Sci. (U.S.A.), 64: 191-195. Hartmann, J. F., and C. F. Hutchison 1974a Nature of the pre-penetration contact interactions between hamster gametes in uitro. J. Reprod. Fertil., 36: 49-57. - 197413 Contact between hamster spermatozoa and the zona pellucida releases a factor which influences early binding stages. J. Reprod. Fertil., 37: 61-66. 1976 Surface interactions between mammalian sperm and egg: Variation of spermatozoa concentration as a probe for the study of binding in vitro. J. Cell. Physiol., 88: 219-226. 1977 Involvement of two carbohydrate-containing components in the binding of uncapacitated spermatozoa to eggs of the golden hamster in vitro. J. Exp. Zool., 201: 383-390. McRorie, R. A., and W. L. Williams 1974 Biochemistry of mammalian fertilization. Ann. Rev. Biochem., 43: 777-803. Oikawa, T., G . L. Nicolson and R.Yanagimachi 1975 Trypsin-mediated modification of the zona pellucida glycopeptide structure of hamster eggs. J. Reprod. Fertil., 43: 133-136. Oliphant, G., and B. G. Brackett 1973a Immunological assessment of surface charges of rabbit sperm undergoing capacitation. Biol. Reprod., 9: 404-414. 1973b Capacitation of mouse spermatozoa in media with elevated ionic strength and reversible decapacitation with epididymal extracts. Fertil. Steril., 24: 948-955. Talbot, P., L. E. Franklin and E. N. Fussell 1974 The effect of the concentration of golden hamster spermatozoa on the acrosome reaction and egg penetration in uitro. J. Reprod. Fert., 36: 429-432. Yanagimachi, R. 1969 In uitro capacitation of hamster spermatozoa by follicular fluid. J. Reprod. Fert., 18: 275-286. 1970 In uitro capacitation of golden hamster spermatozoa by homologous and heterologous b l d sera. Biol. Reprod., 3: 147-153. 1972 Penetration of guinea pig spermatozoa into hamster eggs In uitro. J. Reprod. Fert., 28: 477-480. Yanagamachi, R., and R. D. Noda 1970 Ulstrastructural changes in the hamster head during fertilization. J. U1tras6uct. Res., 31: 365-485.
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