Gamete Interactions In Vitro*†

Vol. 28, No.5, May 1977 Printed in U.S.A.

FERTILITY AND STERILITY Copyright" 1977 The American Fertility Society

GAMETE INTERACTIONS IN VITRO*t

RICHARD BRONSON, M.DJ YASUO HAMADA, M.D.§ Department of Obstetrics and Gynecology, Pennsylvania Hospital, and the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19107

A method was devised that allowed observation of individual sperm in vitro in the presence or absence of unfertilized eggs. Sperm traversing microcapillary tubes in the absence of eggs moved in linear tranjectories. Sperm in the environs of unfertilized eggs moved in erratic paths, owing to repeated adherence ofthe sperm head to the wall of the microcapillary tube. However, translational velocity was unaltered. Increased adhesiveness was not noted when sperm were exposed to unfertilized eggs following removal of the cumulus oophorus with hyaluronidase. Gamete interactions in vitro were also studied at varying sperm to egg ratios. When gametes were mixed at high sperm to egg ratios (approximately 1 ()6:1) the cumulus oophorus was rapidly dissolved, and sperm became adherent to the zona pellucida. At low ratios (approximately 1(J2:1), the cumulus oophorus remained intact. Binding of sperm to individual cumulus cells was noted. A hypothesis is presented that cumulus oophorus plays a role in fertilization by selectively trapping mature sperm within the perimeter of the egg and, in so doing, extending the duration of time in which gametes remain within close proximity, thus increasing the likelihood of egg contact by capacitated sperm.

In mammals, the passage of spermatozoa through the oviduct has not been accessible to direct observation. Although the early events of fertilization have been studied extensively in vitro, extrapolation of these findings to gamete interactions occurring in vivo may not be completely valid. Recent observations by ZambonP indicate that the ratio of sperm to eggs within the ampulla of the mouse oviduct (approximately 1:1) is much lower than that which has been used for in vitro fertilization. The close confinement of unphysiologic numbers of sperm in culture may then increase the likelihood of gamete contact and, in so doing, bypass sperm-trapping mechanisms Accepted January 10, 1977. *Supported by General Research Support Grant S501 RR05590-11 (to R. B.). tPresented at the Thirty-Second Annual Meeting of The American Fertility Society, April 5 to 9,1976, Las Vegas, Nev. tPresent address: Department of Obstetrics and Gynecology, TripIer Army Medical Center, APO San Francisco, Calif. 96438. To whom reprint requests should be addressed. §Ford Foundation Fellow in Reproductive Biology.

that may be operative within the ampulla, in the environs of eggs. Circumstantial evidence exists which suggests that eggs may alter the pattern of sperm motility in vivo. Harper2 has found that, in the rabbit, sperm transport through the oviduct is accelerated by the presence of eggs within the ampulla. Dickmann3 has studied binding of rabbit sperm to the zona pellucida of rat and rabbit eggs transferred to the rabbit oviduct following coitus. Selective binding of rabbit sperm to rabbit eggs was noted in vivo but not in vitro. To study these mammalian gamete interactions in vitro, a technique was devised which allowed observation of sperm motility in the presence or absence of unfertilized eggs. Eggs and sperm were placed in separate droplets in a tissue culture dish. The sperm droplet was connected by microcapillary tubes to the egg droplet and an empty droplet. Sperm leaving a uniform suspension within one droplet entered the microcapillary tubes leading toward either the egg-containing droplet or the empty droplet. Differences in the pattern 570

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GAMETE INTERACTIONS IN VITRO

of motility of these otherwise similar sperm would then be accounted for by differences in the local environments within the two microcapillary tubes. In this manner, those alterations in sperm motion which might occur within the proximity of eggs could be directly observed.

from previously siliconized Pasteur pipettes (Siliclad; Clay Adams, Parsippany, N. J.) by using an alcohol lamp. Lengths of micro capillary tube were inspected at x 48 magnification, and those segments of uniform 50- to 100-/-tm inner diameter were then broken into l-cm lengths with jeweler's forceps. Care was taken not to handle the microcapillary tubes, since skin secretions adhering to the outer glass surface made later observations within the tissue culture dish difficult. The microcapillary tubes were then filled by touching their tips to a droplet of medium. By using bright back-illumination, the interface between medium and air could be seen to rise quickly within the microcapillary tube and reach the opposite end. DUring this filling it was also important to prevent wetting of the mid portion of the microcapillary tube which was to rest within the oil phase of the tissue culture dish. Medium invariably extended along the outer wall of the micro-

MATERIALS AND METHODS

Tissue culture dishes were prepared as follows: three 25-/-t1 droplets of Toyoda's medium which had been equilibrated against 5% CO 2 in air were placed under light paraffin oil (Fisher white, light, paraffin oil, Saybolt viscosity 125/135), at equidistant points in a 35 x 10 mm polystyrene tissue culture dish (Falcon Plastics, San Francisco, Calif.). This medium was chosen for its known ability to allow capacitation of mouse sperm in vitro.4 Glass microcapillary tubes were pulled by hand

40mm Culture

571

TOP

VI EW

SIDE

VIEW

Tissue Dish

DROPLETS

OF

MEDIUM

1-

Epididymal segment

2-

Empty

droplet

or

eggs

with

3-

Empty

droplet

or

eggs

without

added

to create

sperm

suspension.

cumulus.

cumulus.

FIG. 1. Eggs and sperm were placed in separate droplets of medium which had been connected by microcapillary tubes. One of the distal droplets remained empty. The motilities of sperm traversing the microcapillary tubes exposed to and in the absence of eggs within the distal droplets were compared.

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BRONSON AND HAMADA

capillary tube when this step was not adhered to, forming a channel through the oil between droplets. Two filled microcapillary tubes were then placed in each dish, joining the three droplets in a V-shaped configuration (Fig. 1). At least two dishes were prepared for each experiment. All dishes were kept in an incubator at 31' C and gassed continuously with 5% CO 2 in air. Prior to the addition of gametes, completed culture dishes were equilibrated for at least 1 hour in the incubator. This time was chosen by preliminary observations of red blood cells which had been added to the droplets of medium in the test dish. These cells, which possess no inherent motility, were used as detectors of currents within the microcapillary tubes. Vnfertilizedeggs were obtained from randomly bred, mature albino mice which had been superovulated by intraperitoneal administration of gonadotropins. Mice were housed under artificial lighting, with a 10-hour period of darkness centered at midnight. Pregnant mare's serum gonadotropin (Gestyl; Organon Inc., West Orange, N. J.), 10 IV, was administered at 4 P.M., followed 45 hours later by human chorionic gonadotropin (Pregnyl; Organon Inc.), 10 IV. Animals were killed the morning after administration of human chorionic gonadotropin. The cumulus mass was freed from the ampulla of the oviduct and either ' washed once with Toyoda's medium and pipetted into the test dish or placed in a 0.5% hyaluronidase solution (Sigma type 1, 350 NF units/mg; Sigma Chemical Co., St. Louis, Mo.), to dissolve the cumulus oophorus. This solution consisted of 10 NF units/ml, made up in phosphate-buffered saline containing 1 mg/ml of polyvinyl pyrrolidone. Following dispersion of the cumulus, the naked eggs were washed three times in Toy-

May 1977

oda's medium, under paraffin oil, and transferred to the test dish. The cauda epididymidis of mature male mice was incised in an avascular location. The tip of a 30-gauge needle was touched to the fluid, which exuded onto the surface of the epididymis. The needle was passed once through Toyoda's medium to remove blood contamination and then placed within the proximal droplet of the test dish. Motile sperm swam out of the epididymal fluid at the tip ofthe needle toward the edge of the droplet of medium and then entered the microcapillary tube (Fig. 2). An ideal concentration was reached within seconds, and the needle was removed. This concentration during the development of the test system was 10 to 20 million sperm/ml. At lower concentrations, few sperm entered the microcapillary tubes. At higher concentrations, crowding of sperm within the microcapillary tubes made tracking of individual sperm difficult. Observations were made in room air at x 48 and x 160 magnification. The microscope stage was heated with incident infrared light (Sears Heatlamp) to maintain temperature at 31' C. By using bright, oblique back-illumination, sperm could be seen traversing the microcapillary lumen. To determine sperm velocity, this movement across a calibrated eyepiece reticule was timed with a stopwatch. Dishes were consecutively examined each 15 minutes during the 1st hour, and at least at halfhour intervals thereafter. Sperm motility was observed over a 2-minute period, and the test dish was returned to the incubator. The duration of exposure to room air was always less than 5 minutes. Phenol red, which had been added to the medium, was used to monitor pH during each

Droplet #-2

~,------+--- Medium

Microcapilla ry Tubes

~--+---

30 gauge neadle

' - - - - Segment of Cauda Epididymldi. Droplet #3

FIG. 2. A droplet of fluid from the cauda epididymidis was washed once in Toyoda's medium and held within the proximal droplet (Droplet #1) of the test dish by using a 30-gauge needle. Sperm swimming out of the epididymal fluid traversed the droplet and entered the microcapillary tubes.

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GAMETE INTERACTIONS IN VITRO

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experiment. In addition, direct preliminary measurements of the medium revealed that the pH remained stable during the short periods of exposure to room air. A separate series of observations was also carried out in the later experiments to compare the interaction of gametes in cases when the ratio of sperm to eggs was either high or low. Those sperm which initially traversed the microcapillary tube were observed as they entered the cumulus mass within the distal droplet. In addition, several small aliquots of sperm from the proximal droplet were transferred directly to the eggcontaining droplet to increase serially the sperm to egg ratio from approximately 1:1 to 100:!. At the conclusion of the later sperm velocity measurements, eggs were also transferred from the distal droplet into the sperm suspension within the proximal droplet, and the cumulus oophorus was observed. To aid the observation of gamete interaction at the low sperm concentration, single eggs were teased from the cumulus mass with 30-gauge needles, and the cumulus cells surrounding the egg were spread out along the bottom of the tissue culture dish, within the distal droplet of medium, prior to the addition of sperm (see Fig. 1).

RESULTS

The mean velocity of sperm traversing the microcapillary tube toward empty droplets of Toyoda's medium remained stable over a 5-hour period of observation (Table 1). The pattern of motility and velocity of sperm within the microcapillary tube was similar to that of sperm which had traversed the entire microcapillary and entered the empty distal droplet. These observations confirmed the supposition that no alteration in sperm motion had occurred within the confines of the microcapillary tube as a result of the configuration of the test system. When intact cumulus masses containing unfertilized eggs were added to one of the distal droplets, an alteration in sperm motility was observed. Shortly after the cauda epididymidis was placed in the proximal droplet, sperm entered the microcapillary lumen at a mean velocity no different from that noted previously. However, as the duration of in vitro culture lengthened, progression within the distal portion of the microcapillary became poor. Thereafter, sperm adhered to the inner wall of the microcapillary and progression stopped, although flagellar

573

TABLE 1. Mean Sperm Velocity a versus Time in Culture Time (I)

Velocity ± SO

hr

J1.m/sec

t= 0 t< 1 2< t < 3 4< t< 5

118 146 125 116

± 46.0 (5)b ± 21.9 (5) ± 60.0 (5) ± 26.8 (3)

a All experiments. The velocity of sperm traversing microcapillary tubes toward empty droplets of Toyoda's medium was measured over a 3-hour period of observation in five separate runs and over an additional 2 hours on three occasions. bNumbers in parentheses are numbers of runs.

beating continued. On the other hand, there was no alteration in velocity of sperm traversing the microcapillary toward the empty droplet (Table 2). The interval from initiation of in vitro culture to the onset of increased adhesiveness was found to depend upon the number of eggs in culture (Table 3). When 10 to 20 eggs were placed within the distal droplet, altered sperm motility occurred only after 4 hours of incubation. As the number of eggs in culture was increased, the time interval to the initiation of increased adhesiveness diminished. In certain instances after the addition of gametes, cumulus cells could be seen slowly drifting within the microcapillary lumen toward the sperm-containing droplet. In these cases, the initial period of incubation had been insufficient to ensure a complete absence of currents between droplets. Data from these cases were compiled separately until it was later determined that the mean velocity of sperm was not significantly different from that in which completely static conditions prevailed. In order to determine the possible role played by the cumulus oophorus in mediating the increased adhesiveness of sperm, unfertilized eggs were pretreated with hyaluronidase to

Velocity ± SO Time Eggs' with cumulus

Empty droplet

hr, min

3,35 3,45 4, 10 4, 15 4,30

pm/sec

96 ± 23.7 125 ± 25.1 145 ± 59.5 163 ± 53.3

136 ± 33.8 87.0 ± 40.3 65.3 ± 43.5 59.5 ± 30.3

aThe velocities of sperm traversing microcapillary tubes toward empty droplets and droplets containing unfertilized eggs were compared. The data from a single experiment are presented (see Table 3). bSeventeen eggs in droplet.

TABLE 3. Incubation Time in Vitro to Onset of Increased Sperm AdMsiveness a Ni,;;

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BRONSON AND HAMADA

574

~ft~~WS

Time interval

1hr

10.;; 20 20.;; 40 40.;; 80

*** **

2hr

3hr

4hr

5hr

*** * *

**

*

* *

*

*

*

aSpenn traversing microcapillary tubes toward unfertilized eggs were observed until the initiation of increased adhesiveness. The hour at which altered adhesiveness was first noted (asterisks) has been plotted against the number of eggs in culture for each of 18 separate runs. This incubation time correlated inversely with the number of eggs in culture (correlation coefficient - 0.81; N = 18; P < 0.01). bEggs were added to test droplet at time zero.

remove the cumulus before they were placed in the test dish. In the absence of cumulus there was no alteration in the pattern of sperm motility over a 5-hour period of observation. The diminished velocity of sperm in the presence of cumulus oophorus was found by observation at x 160 magnification to be due to intermittent adherence of the sperm head to the inner wall of the microcapillary. Sperm velocity decreased as the number of contacts with the microcapillary increased. An erratic pattern of motion was created, in contrast to the linear trajectories taken by sperm in the absence of cumulus (Fig. 3). However, when the velocity of such "sticky" sperm between points of adherence was compared with that of sperm in the absence of eggs, no significant difference was found. In order to eliminate the influence of prolonged in vitro culture upon sperm, unfertilized eggs were added to the distal droplets of the test dish and preincubated before the addition of sperm.

Toward Eggs Without Cumulus

Following varying intervals, sperm were added to the proximal droplet of the test dish as previously described and immediately observed. In these experiments, eggs with cumulus oophorus were added to one of the distal droplets and eggs without cumulus oophorus were added to the other (Table 4). An alteration in sperm motility was again noticed in the presence of cumulus. Following 4 to 5 hours' preincubation of eggs, the number of contacts of sperm to the inner wall of the microcapillary increased markedly, and sperm progression diminished. There was no change in velocity in the absence of cumulus. In the later series of observations, when cumulus masses were added directly to the spermcontaining proximal droplet, the cumulus oophorus was soon dissolved and many sperm adhered to the zona pellucida. On the other hand, when small numbers of sperm were added to the eggcontaining droplet so that the gamete ratio was close to 1:1, the majority of sperm rapidly adhered to the cumulus cells. Although flagellar beating continued, no sperm were seen to penetrate through the cumulus oophorus to the zona pellucida during a 1-hour period of observation. DISCUSSION

These observations suggest that, in the mouse, intact, unfertilized eggs have no direct effect upon the velocity of sperm. Alterations in sperm motility were demonstrated, however, which appear to be the result of changes in adhesiveness of the sperm head. Such changes seem to be secondary to diffusion of a substance into the microcapillary tube from the distal egg-containing droplet.

Toward Eggs With CumulUS

FIG. 3. As represented in this schematic drawing, spenn observed at x 160 magnification moved in linear trajectories toward eggs without cumulus oophorus and in erratic paths in the presence of cumulus.

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GAMETE INTERACTIONS IN VITRO

TABLE 4. Mean Sperm Velocity versus Preincubation Time a Preincubation time (t)

Velocity ± SD Eggs without cumulus

hr

t",;; 1

2< t < 3 4< t < 5

Eggs with cumulus p.mlsec

146 ± 21.0 (4)b 176 ± 38.0 (4) 168 ± 13.4 (3)

129 ± 18.0 (4) 137 ± 12.7 (2) 67.9 ± 21.6 (3)

aEggs either with cumulus oophorus present or following cumulus dispersion with hyaluronidase were pre incubated in distal test droplets. Velocity was measured immediately after addition of sperm. Four separate experiments were performed. bNumbers in parentheses are numbers of instances sperm entered both microcapillary tubes.

The correlation seen between the number of eggs added to the test droplet and the time of onset of increased adhesiveness supports this thesis. The failure to observe an alteration in adhesiveness following removal of the cumulus oophorus with hyaluronidase implicates the cumulus as mediating this effect. Metabolic products of the cumulus cells, or secretions of these cells (e.g., steroids, prostaglandins, or enzymes), might also be possible mediators of this effect. Alternatively, solubilization of the hyaluronic acid matrix of the cumulus mass within the tissue culture medium might alter the surface properties ofthe microcapillary tube. The consistently observed adherence of the sperm head to the microcapillary wall with continued free motion of the tail tends to rule against the likelihood of such a generalized, nonspecific increase in adhesiveness of the microcapillary wall . Evidence exists in several species which supports the contention that cumulus oophorus plays a role in fertilization. Although the exact mode of action of cumulus cells has yet to be defined, this body of information increases the likelihood that the altered sperm adhesiveness noted in these experiments in the presence of cumulus oophorus is of physiologic consequence. In 1962, Chang and Bedford 5 showed a decreased frequency of fertilization in the rabbit when eggs were transferred to the oviduct following removal of the cumulus oophorus and corona radiata. It was unclear from these data, however, whether cumulus was playing a role in maintaining the viability of the egg, and hence its fertilizability, or acting at the level of gamete interaction. Dickmann6 subsequently found that prior removal of the cumulus oophorus lowered the number of sperm in contact with the zona pellucida following transfer of such treated eggs to the oviducts of rabbits postcoitus. These observations

575

suggested that cumulus influences gamete contact within the oviduct. More recently, it has been found that cumulus oophorus may playa role in capacitation of sperm in the mouse, where the proportion of eggs penetrated and undergoing fertilization in vitro is higher in the presence of cumulus than without it.7. 8 Oliphant9 has also provided evidence in the rabbit that removal of seminal plasma components bound to sperm is a prerequisite to the induction of the acrosome reaction. Defined media which allow capacitation of sperm in vitro have similarly been shown in the mouse to remove epididymally derived protein coats from the sperm head. 10 The alteration in sperm adhesiveness noted in the present experiments may thus be a reflection of the removal of such sperm surface coats. In prior studies of in vitro fertilization, gametes have been mixed at high sperm to egg ratios (approximately 106 :1). Observations by Zambonj1 indicate that the ratio of sperm to eggs within the ampulla of the mouse oviduct is close to 1:1. Indeed, the experimental conditions utilized in current studies of fertilization in vitro are probably not similar to those in vivo. As illustrated in these experiments, cumulus oophorus rapidly dissolves when cumulus masses are placed within a droplet containing sperm at high concentration. Following dissolution ofthe cumulus, many sperm adhere to the zona pellucida. It is well known that the release of hyaluronidase from the high numbers of dead sperm present in vitro is responsible for this phenomenon. On the other hand, when fertilized eggs (as confirmed by the presence of a second polar body) are obtained from the oviductal ampulla on the morning following coitus, these eggs are found to be within the undispersed cumulus mass. Rat l1 and rabbit12 eggs are also penetrated by sperm before any dispersion of the cumulus is visible. Following the addition of small numbers of motile sperm to the egg-containing droplet, adhesion of sperm to individual cumulus cells occurred frequently. Attachment occurred at the sperm head and was never noted at mid piece or tail. These observations are quite similar to those noted by Gwatkin et al. 13 in their recent study of fertilization in the hamster. In that species, as well, sperm were bound to the cumulus cells which surrounded the egg. The sperm cell surface appeared to be altered by this interaction,14 perhaps through the mediation of a glucosidase,15 and, during the period of contact with the cumulus

576

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BRONSON AND HAMADA

cells, capacitation occurred. A dialyzable factor from the cumulus matrix was also required for capacitation. If all sperm were permanently immobilized within the periphery of the cumulus mass by binding to cumulus cells, fertilization could not occur. Binding must hence either be reversible or selective. The observations by Gwatkin et al. 13 in the hamster indicate that sperm in this species are released within 3 to 4 hours after initial binding to cumulus cells. Although sperm release was not noted in the present work, sperm-cumulus interactions were observed for only 1 hour following initial adhesion. It is difficult to state whether selective binding of sperm to cumulus cells took place. In each of the individual experiments performed, some sperm were seen to move freely within the distal droplet without adhering to cumulus. It remains unclear whether this represents a failure to contact cumulus cells owing to the chance distribution of these cells within the droplet or to a failure to bind despite cell contact. Selective binding could theoretically take place if the surface properties of sperm were not uniform. Alterations in the cell surface of sperm have been noted during their passage through the epididymis. 16 These changes might not occur uniformly within the population of sperm that reach the cauda epididymidis. The acquisition of the ability to bind to cumulus cells and undergo capacitation may be dependent upon such cell surface alteration. Cohen 17 has provided circumstantial evidence that all ejaculated sperm do not, indeed, possess an equal capacity to fertilize. Although these observations must be viewed with caution, both the alteration in adhesiveness of sperm within the microcapillary lumen in the presence of cumulus oophorus and the binding of sperm to individual cumulus cells may be reflections of a selection mechanism operative within the egg environs. Cumulus oophorus may act as a cellular net, by selectively binding a subpopulation of sperm that is capable of subsequently undergoing capacitation. This could increase the likelihood of fertilization by trapping sperm and maintaining the continuous release of capacitated sperm within the perimeter of the egg.

Acknowledgments. The authors wish to thank Patrice Harris and Thomas Henry for their technical assistance.

REFERENCES 1. Zamboni L: Fertilization in the mouse. In Biology of Mammalian Fertilization and Implantation, Edited by KS Moghissi, ESE Hafez. Springfield Ill, Charles C Thomas, 1972, p 213 2. Harper MJK: Stimulation of sperm movement from the isthmus to the site of fertilization in the rabbit oviduct. BioI Reprod 8:369, 1973 3. Dickmann Z: Chemotaxis of rabbit spermatozoa. J Exp BioI 40:1, 1963 4. Toyoda Y, Yokoyama M, Hosi T: Studies on the fertilization of mouse eggs in vitro. In vitro fertilization of eggs by fresh epididymal sperm. Jap J Anim Reprod 16:147, 1971 5. Chang MC, Bedford JM: Fertilizability of rabbit ova after removal of the corona radiata. Fertil Steril 13:421, 1962 6. Dickmann Z: Fertilization and development of rabbit eggs following the removal of the cumulus oophorus. J Anat 98:397, 1964 7. Cross PC, Brinster RL: In vitro development of mouse oocytes. BioI Reprod 3:298, 1970 8. Miyamoto H, Chang MC: Fertilization in vitro of mouse and hamster eggs after the removal offollicular cells. J Reprod Fertil 30:309, 1972 9. Oliphant G: Removal of sperm-bound seminal plasma components as a prerequisite to induction of the rabbit acrosome reaction. Fertil Steril 27:28, 1976 10. Oliphant G, Brackett BG: Capacitation of mouse spermatozoa in media with elevated ionic strength and reversible decapacitation with epididymal extracts. Fertil Steril 24:948, 1973 11. Austin CR: Function of hyaluronidase in fertilization. Nature 162:63, 1948 12. Chang MC: Fertility and sterility as revealed in the study of fertilization and development of rabbit eggs. Fertil Steril 2:205, 1951 13. Gwatkin RBL, Andersen OF, Hutchinson CF: Capacitation of hamster spermatozoa in vitro: the role of cumulus components. J Reprod Fertil 30:389, 1972 14. Gwatkin RBL, Carter HW: Cumulus oophorus. In Scanning Electron Microscopic Atlas of Mammalian Reproduction, Edited by ESE Hafez. Tokyo, Igaku Shoin Ltd, 1975, P 270 15. Gwatkin RBL, Andersen OF: Effect of glycosidase inhibitors on the capacitation of hamster spermatozoa by cumulus cells in vitro. J Reprod Fertil 35:565, 1973 16. Bedford JM: Components of sperm maturation in the human epididymis. Adv Biosci 10:145, 1973 17. Cohen J: Gametic diversity within an ejaculate. In The Functional Anatomy of the Spermatozoon. Proceedings of the Second International Symposium, Stockholm, August 1973. New York, Pergamon Press, p 329