Capacitation of Large Numbers of Hamster Sperm in Vitro

Transcription

1 BIOLOGY OF REPRODUCTION 9, (1973) Capacitation of Large Numbers of Hamster Sperm in Vitro BRUCE MORTON, B. J. ROGERS, AND T. S. K. CHANG Department of Biochemistry and Biophysics, University of Hawaii, Honolulu, Hawaii Accepted April 13, 1973 A test tube system that capacitates large numbers of hamster sperm is described in detail. The 1-mi capacitating media contained heat-detoxified human serum or a dialysate of human serum to which human serum albumin had been added. Up to 2.5 >< 10 sperm were incubated therein for 4 h at 37#{176}C in the absence of mineral oil and ova. When aliquots of these sperm were combined with ova and the ova were examined 1-2 h later, up to 100% contained swelling sperm heads within them. Fertilization was often polyspermic. After the 4 h capacitation incubation, up to 60% of the motile sperm were without acrosomes, suggesting a high capacitation elficiency. This macrocapacitation system has proved useful in the clarification of certain biochemical events in sperm capacitation. A key event in mammalian conception is the maturation of sperm within the female through a little understood process called capacitation (Austin, 1951; Chang, 1951; Bedford, 1970). Without this step, sperm are unable to fertilize the ovum. Capacitation of hamster sperm has been achieved in vitro by use of media containing: fallopian tube fluid from the mouse and rat (Barros, C., 1968), bovine follicule fluid (Yanagimachi, 1969a; Cwatkin and Anderson, 1969), serum from several species (Yanagimachi, 1969; Barros and Garavagno, 1970), crude fl-glucuronidase (Gwatkin and Hutchinson, 1971), Sendai virus (Ericson et al., 1971), and components from the cumulous oophorus of hamster ova (Gwatkin et a!., 1972). The small number of sperm present in these in vitro hamster sperm capacitation systems precludes the use of most biochemical assays. This has impeded the investigation of the molecular events of capacitation. Here we report that large numbers of sperm can be capacitated in vitro in a reproducible manner. This has made it possible to begin detailed investigations of the biochemistry of sperm capacitation (Morton and Albagli, 1973; Rogers and Morton, 1973a; Morton and Chang, 1973; Rogers and Morton, 1973b). METHODS AND MATERIALS Epididymides removed from 60- to 90-day-old golden hamsters were washed with saline (room temperature) to remove blood, which is toxic to the spermatozoa. Each epididymis was held with a hemostat so that the caudal portion was isolated on one side and placed under pressure. A stainless-steel needle (No. 22) was passed through this portion of the organ at about ten different points. The sperm exudate was placed in a small petri dish containing 2 ml Tyrode s solution (Tyrode, 1910; Paul, 1970) at room temperature. This was covered and placed in a 37#{176}Cincubator for about 5 mm to induce sperm dispersal. The suspension was then rinsed into a 12-ml calibrated conical centrifuged tube to a volume of 5 ml with Tyrode s solution. This stock sperm suspension was kept at 37#{176}Cand contained (1- S) x 10 cells/ml as determined with a hemocytometer. To capacitate the spermatozoa, 0.5-ml aliquots of the sperm stock were placed in 15-mi Corex test tubes at 37#{176}Cwith 0.5 ml heat detoxified human serum or 0.5 ml human serum dialysate and 10 mg crystalline human albumin (Nutritional Biochemicals). The tubes were tightly stoppered 356 Copyright 1973 by The Society for the Study of Reproduction. All rights of reproduction in any form reserved.

2 In vitro SPERM CAPACITATION 357 and incubated for 4 h at 37#{176}C, at which time the sperm were assayed for capacitation. Sperm motility was evaluated with a phase contrast microscope at loox magnification without cover slip for percentage of motile sperm, rate of sperm motility (0-10), and percentage head to head agglutination. The percentage of motile sperm without acrosomes was determined by observations at 400x with a cover slip in place. After 4 h of incubation the sperm were assayed for capacitation by the following modification of a method of Yanagimachi (1969a). At least 45- day-old female hamsters which had been previously grouped according to their day in the fourday estrous cycle (Orsini, 1961) were injected ip with 30 IU of PMS (Equinex brand of serum gonadotropin, Ayerst Laboratories, New York) in 0.3 ml at day 1, 9-12 AM, and with 30 IU of HCG (A.P.L. Brand of chorionic gonadotropin, Ayerst Laboratories) in 0.3 ml at day 3, 9-10 Pat. Ovulation occurs on day 4, h after the HCG injection. At h after the HCC injection the oviducts were excised and washed in Tyrode s solution to remove blood. Each oviduct was placed in a 5-cm watch glass containing 1 ml Tyrode s solution. Using a stereo zoom dissecting microscope and bent needle, the oviduct was punctured at the point showing the most internal streaming and transparency. The cumulus mass either extruded spontaneously or was pulled out using the bent needle. Several of these masses were then transferred to another watch glass containing Tyrode s solution, divided, and placed on 2-cmdiam watch glasses containing 0.1 ml Tyrode s solution and covered with mineral oil. Each oviduct contained ova. A 50-tl aliquot of the sperm to be evaluated for capacitation was next placed with these cumulus masses. After 1 or 2 h of incubation at 37#{176}C, the ova, by then free from cumulus masses, were gathered in the center of the watch glass by use of the bent needle. A frosted glass microscope light source increased the visibility of the eggs. Using the capillary action of a Pasteur pipette, the ova were transferred into a second 2-cm watch glass containing 0.1 ml Tyrode s solution in order to wash off excess adhering sperm. After gathering the eggs together a second time, they were transferred to a microscope slide and again grouped together. Four 1-mm-diam beads, composed of 20% paraffin and 80% vaseline, were placed on the slide around the ova in order to accomodate a cover slip. This was carefully lowered over the eggs while they were still under the dissecting scope. The cover slip was gently pressed down until the eggs were compressed to about one-half their normal diameter. This provided improved optics when the eggs were next examined under phase contrast at 400x for sperm with swollen heads within the vitellus (Fig. 1). Even if the Fic. 1. Swelling sperm heads within ovum 1 h after gamete combination. The line beside the ovum represents 10 m.

3 358 MORTON, ROGERS, AND CHANC penetrating head became invisible, the midpiece and tail could still unmistakably be seen within the ova as the microscope was focused through the egg. To obtain the human serum fractions, 0.5 liter blood was drawn without anticoagulants. After clotting 1-2 h at 0#{176}C,it was centrifuged at 15,000 g for 20 mm. Aliquots of 40 ml of the serum were then frozen. When needed, such an aliquot was heated at 56#{176}Cfor 30 mm to remove toxicity, adjusted with 0.2 N HCI to ph 7.5, passed through a Millipore filter of 0.22-tm pore size, further subdivided into 20-mi samples, and frozen. These were either used directly to capacitate sperm or dialyzed for h against an equal volume of Tyrode s solution to produce the serum dialysate. Because capacitated sperm cannot be unequivocally identified visually, each serum batch was tested for ability to capacitate sperm by the fertilization assay. We have fcund that some sera had lower capacitating potency. Stored sera was also tested for deterioration on a monthly basis. Some retained activity for over 4 mo. RESULTS As indicated in Table 1, when sperm were incubated 4 h in the absence of capacitating components, they did not agglutinate, undergo the acrosome reaction, or fertilize ova in vitro. Their rate of motility and percentage motile decreased in a way that could be prevented by the addition of 10 m caffeine, an agent elevating sperm camp levels and motility (Garbers et a!,, 1971) in a manner similar to that observed in capacitating hamster sperm (Morton and Albagli, 1973; Rogers and Morton, 1973). However, elevation of sperm camp levels alone was not sufficient to confer on these sperm the ability to fertilize (Morton and Chang, 1973). In contrast, sperm incubated in the presence of serum-derived capacitating components retained their original vigorous motility while passing through a head-to-head agglutination stage lasting at least 2.5 h. When the sperm were added to ova at 4 h, up to 100% of the eggs were fertilized within the subsequent 1-2 h. Figure 1 shows the presence of swelling heads within one of these fertilized ova. Polyspermy was common. The average percent of ova fertilized in the dialysate-albumin system, 74± (a range of) 20%, was somewhat higher than that observed for the serum system, 64 ± 35%. Conversely, the percent of all sperm present that were motile and had lost their acrosome cap at 4 h was greater in the serum system, 20 ± 10%, than in the dialysate-albumin system, 3 ± 2%. If sperm in the latter system were incubated 7 instead of 4 h, the percentage undergoing the acrosome reaction rose toward that TABLE 1 EFFECT OF CAPACITATING CONDITIONS ON SPERM PERFORMANCE Motility#{176} Head_headb.&crosome Fertilized / Percent rate (%) aggregates reaction total ova fertilized Tyrode s, 0 h % 0% - - Tyrode s, 4 h % 0% 0/33 0% Serum, 0 h % 0% - - Serum, 4 h ± 20% 40 ± 20% 145/ ± 35% 1)ial-Alb, 0 h % 0 Dial-AIb, 4 h ± 20% 8 ± 5% 92/ ± 20% #{176}Motility is described as the rate of sperm movement (0-10) and percentage of sperm moving. Head-to-head aggregates refers to the rosettes formed by sperm temporarily agglutinated by intercellular binding at the acrosome region. #{176}Acrosome reaction refers to the percentage of the moving sperm that have no visible acrosome. To obtain the percentage of the total sperm population that are both motile and without acrosomes, multiply this figure times the percentage of the total sperm that are moving. d Dial-Alb is an abbreviation for the incubation medium containing human serum dialysate and albumin described in the text. The dispersions indicated refer to the range of experimental variation.

4 In vitro SPERM CAPAC1TATION 359 seen in the serum system at 4 h. Only a slight increase was observed in the serum system as a result of this additional 3 h of incubation. DISCUSSION In previously reported hamster sperm capacitation systems the number of sperm present ranged from 2 X 10 through 1.25 X 10#{176} per container (Barros, 1968; Yanagimachi, 1969a, b; Gwatkin and Anderson, 1969; Hartmann and Gwatkin, 1971). We have fertilized ova after up to 2.5 X 10 sperm per tube were incubated in capacitation media. Capacitation of larger numbers was not attempted because 10 cells have been a sufficient quantity to examine biochemically. There are two additional advantages to the system reported here: Mineral oil is not used to cover the suspension of capacitating sperm. This greatly simplffies the removal of uncontaminated samples during the incubation. More important, in this method the sperm are capacitated before they are placed with ova. This simplifies the capacitating environment substantially and avoids uncertainty about the contribution of the ovum to sperm maturation. However, the exposure of the ova to sperm for only 1-2 h instead of throughout the incubation may account for the less than 100% of ova fertilized on the average in the present work. The use of the same time sequence by Yanagimachi resulted in a similar percentage of hamster ova fertilized in a follicular fluid-based system (Yanagimachi, 1969a). Although Gwatkin et a!. (1972) found that cumulous ooophorous components could capacitate hamster spermatozoa in vitro, these components clearly are not required for in vitro hamster sperm capacitation: Yanagimachi and Noda (1970) observed that hamster sperm, incubated in a follicular fluid medium in the absence of eggs, fertilized 100% of ova which had been previously denuded and washed free of cumulous oophorous. At present no method exists to determine the percentage of sperm capacitated by in vitro methods. The loss of acrosomes from motile sperm might be expected to be such an indicator, because capacitation appears to be a prerequisite for this true acrosome reaction (Bedford, 1970). However, our observation that sperm capacitated in dialysate-albumin fertilize better while manifesting much less acrosome reaction than those capacitated in serum suggests otherwise (Rogers and Morton, 1973a); unless one postulates that the heat-treated serum, but not its dialysate, partially inhibits fertilization. Thus failure to penetrate 100% of ova may not be a sign of incomplete sperm capacitation. Enhanced, sperm_ova interaction may be promoted by rocking of ova in the presence of sperm (Gwatkin and Anderson, 1969). Although serum albumin appears to be the only macromolecule required for hamster capacitation (Yanagimachi, 1970a), its function is not well understood. Not only does it promote a temporary head-to-head agglutination (Yanagimachi, 1969b), but also appears to be involved in the release of sperm acrosomal hyaluronidase (Rogers and Morton, 1973a). Even less is known about the identity and possibly multiple function of the small molecules required for capacitation. The have been found to activate the motility of hamster sperm (Yanagimachi, 1970b; Morton and Chang, 1973) and to stimulate oxygen consumption (Iritani, 1969) of rabbit and ram sperm. Using the capacitation system described here, we have verified these observations (Morton and Chang, 1973), and in addition have found that the small molecules of capacitating media markedly lower sperm ATP levels (Rogers and Morton, 1973b) while elevating camp synthetic rates by sperm adenyl cyclase (Albagli and Morton, 1973). A hypothesis regarding the molecular basis of the changes in energy metabolism that occur in capacitated sperm has been proposed (Rogers and Morton, 1973b).

5 6O MORTON, ROGERS, ANfl CHANC ACKNOWLEDGMENTS We thank R. Yanagimachi for his interest and for demonstrating his capacitation assay. T. S. K. Chang was supported by a Ford Foundation Predoctoral Fellowship ( A) from the Department of Anatomy and Reproductive Biology, University of Hawaii. This research was supported in part by a research grant from the NIH (HD 04738). REFERENCES AUSTIN, C. R. (1951). Observations on the penetration of the sperm into the mammalian egg. Aus. J. Sci. Res. B 4, BARmios, C. (1968). In vitro capacitation of golden hamster spermatozoa with fallopian tube fluid of the mouse and rat. I. Reprod. Fert. 17, BARROS, C., AND GARAVAGNO, A. (1970). Capacitation of hamster sperm with blood sera. 1. Reprod. Fert. 22, BEDFORD, J. M. (1971). Sperm capacitation and fertilization in mammals. Biol. Reprod. Sup pl. 2, CHANG, M. C. (1951). Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168, 697. ER1CSON, R. J. BUTHALA, D. A., AND NORLAND, J. F. (1971). Fertilization of rabbit ova in vitro by sperm with absorbed sendal virus. Science 173, GARnERS, D. L., LUST, W. D., FIRST, N. L., AND LARDY, H. A. (1971). Effects of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility. Biochemistry 10, GWATKIN, R. B. L., AND ANDERSON, 0. F. (1969). Capacitation of hamster spermatozoa by bovine follicular fluid. Nature 224, GWATKIN, H. B. L., ANDERSON, 0. F., AND HUTCHINSON, C. F. (1972). Capacitation of hamster spermatozoa in vitro: The role of cumulus components. 1. Revrod. Fert. 30, GWATK1N, R. B. L., AND HUTCHINSON, C. F. (1971). Capacitation of hamster spermatozoa by beta-glucuronidase. Nature 229, HARTMAN, J. G., AND GWATKIN, R. B. L. (1971). Alteration of sites on the mammalian sperm surface following capacitation. Nature 234, IRITANI, A., COMES, W. R., AND VANDEMARK, N. L. (1971). The effect of whole, dialyzed, and heated female genital tract fluids on respiration of rabbit and ram spermatozoa. Biol. Reprod. 1, MORTON, B., AND ALI3AGLI, L. (1973). Modification of sperm adenyl cyclase by capacitation in vitro. Biochem. Biophys. Res. Comm. 50, MORTON, B., AND CHANG, T. S. K. (1973). The effects of fluid from the cauda epididymidis, serum components and caffeine upon the survival of deluted epididymal hamster Spermatozoa. J. Reprod. Fert. 35, 203. ORSINI, M. W. (1961). External vaginal phenomena characterizing the stages of the estrous cycle, pregnancy, pseudopregnancy, lactation, and the anestrous hamster, mesocricatus auratus waterhouse. Proc. Amin. Care Panel 11, PAUL, J. (1970). Cell and Tissue Culture, 4th ed., p. 91. Williams and Wilkin, Baltimore. Rocans, B. J., AND MORTON, B. (1973a). The release of hyaluronidase from capacitating hamster spermatozoa. 1. Reprod. Fert. In press. ROGERS, B. J., ANn MORTON, B. (1973b). ATP levels in hamster spermatozoa during capacitation in vitro. Biol. Reprod. 9, in press. TYRODE, M. V. (1910). The mode of action of some purgative salts. Arch. mt. Pharmacodyn. 20, YANAGIMACHI, R. (1969a). In vitro capacitation of hamster spermatozoa by follicular fluid. I. Reprod. Fert. 18, YANAGIMACHI, R. (1969b). In vitro acrosome reaction and capacitation of golden hamster spermatozoa by bovine follicular fluid and its fractions. I. Exp. Zool. 170, YANAGIMACIII, R., AND NODA, Y. D. (1970). Physiological changes in the post nuclear cap region of mammalian spermatozoa: A necessary preliminary to the membrane fusion between sperm and egg cells. I. Ultrastruct. Res. 31, YANAGIMACHI, R. (1970a). In vitro capacitation of golden hamster spermatozoa by homologous blood sera. Biol. Reprod. 3, YANACIMACHI, R. (1970b). The movement of golden hamster spermatozoa before and after capacitation. J. Reprod. Fert. 23,