The regulation of acrosomal exocytosis

DEVELOPMENTAL BIOLOGY 128,453-463

(1988)

The Regulation of Acrosomal Exocytosis I. Sperm

Capacitation Is Required for the Induction of Acrosome Reactions by the Bovine Zona Pellucida in Vitro HARVEY M. FLORMANAND NEAL L. FIRST

Department ofMeat and Animal Science, University of Wisconsin, Mad&n, Accepted April

Wisconsin 58706

18, 1988

The regulation of acrosomal exocytosis in capacitated bovine spermatozoa by soluble extracts of zonae pellucidae was examined. Kinetic studies demonstrated that zonae pellucidae stimulated synchronous acrosome reactions. The .& of this process was 5-10 min and response was maximal at 20 min. The apparent initial rate of exocytosis in sperm populations was dependent upon the concentration of zona pellucida protein, with an EDSo and a maximally effective dosage of 20 and 50 ng protein/PI, respectively. Zonae pellucidae caused up to a 4%fold increase in the apparent initial rate and a 3- to I-fold stimulation in the net occurrence of exocytosis. In contrast, solubilized zonae pellucidae did not induce acrosome reactions in uncapacitated sperm. The development of a capacitated state, as assayed by the ability of sperm to fertilize eggs in vitro, was compared to the expression of zona pellucida-regulated acrosome reactions in a series of kinetic experiments. Both activities were manifest with similar kinetics and displayed identical dependencies toward stimulatory and inhibitory agents in vitro. It is concluded that capacitation is an essential prerequisite for the induction of acrosomal exocytosis in bovine sperm by the zona pellucida. o 1988Academic PRESS,1,~.

INTRODUCTION

Mammalian sperm undergo a terminal structural modification, or acrosome reaction, during fertilization. This is an exocytotic reaction, consisting of fusion and extensive vesiculation between the plasma membrane and the subjacent membrane of the acrosomal vesicle. It results in the display of a new cell surface domain in the apical region of the sperm head and in the externalization of vesicular contents (reviewed by Meizel, 1985; Wassarman, 1987). The acrosome reaction is an important control mechanism of the cellular interactions during fertilization. In mammals, it determines the ability of sperm to adhere to the egg’s extracellular coat, or zona pellucida (Saling and Storey, 1979; Huang et aZ., 1981; Florman and Storey, 1982; Bleil and Wassarman, 1983,1986; Cherr et aL, 1986; Myles et al. 1987); to penetrate the zona pellucida; and to fuse with the egg’s plasma membrane (Yanagimachi and Noda, 1970; Yanagimachi, 1981). Yet, premature exocytosis is associated with decreased ability to fertilize eggs in viva and in vitro (Gwatkin, 1976; Fleming and Yanagimachi, 1982). Clearly, the occurrence of acrosome reactions must be coordinated with the availability of eggs. Exocytosis is controlled at two sites. First, acrosome reactions can be regulated indirectly through the state of cellular activation. Mammalian sperm are released from the male reproductive tract in a functionally immature state (Austin, 1951,1952; Chang, 1951). During subsequent maturation, or capacitation, sperm acquire

the ability to fertilize eggs. One consequence of capacitation is an increase in the intrinsic rate of exocytosis. Such constitutive acrosome reactions are regulated autonomously by sperm and do not require direct extrinsic stimuli (Bedford, 1983; Austin, 1985). Second, exocytosis is a receptor-mediated process regulated directly by agonists in the zona pellucida. Agonist activity is retained in soluble extracts of the zona pellucida from a number of mammalian species (Florman and Storey, 1982; Florman et aL, 1982; Bleil and Wassarman, 1983; Florman et al, 1984; Wassarman et al, 1985; Cherr et aL, 1986; O’Rand and Fisher, 1987; Wassarman, 1987; Cross et aL, 1988). ZP3, a 83-kDa glycoprotein, accounts for agonist activity in the mouse (Bleil and Wassarman, 1983; Florman et al, 1984). These agents may initiate exocytosis by activating a GTP-binding regulatory protein-mediated signal transduction system (Kopf et ah, 1986; Endo et aL, 1987). The relationship between these control mechanisms is uncertain. Prior capacitation may modulate induction of exocytosis directly (Ward and Storey, 19841, by regulating receptor access or the function of a signal transduction system. Alternatively, capacitation may control the zona pellucida induced acrosome reaction indirectly, by supporting the preliminary step of sperm adhesion to the zona pellucida (Inoue and Wolf, 1975; Saling et aL, 1978; Heffner and Storey, 1982) that is essential for agonist-receptor interaction. To determine which is the valid model, we have examined the effects of soluble extracts of bovine zonae pellucidae on 453

001%1606/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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DEVELOPMENTALBIOLOGY

sperm in vitro. This permits the agonist-regulated acrosome reaction to be dissected from other modes of sperm interaction with the zona pellucida. These studies demonstrate that the acrosome reaction of bovine sperm is triggered by the zona pellucida and that induction is regulated directly by capacitation. MATERIALS AND METHODS

Gamete culture. Sperm were cultured in a modified Tyrode’s medium, designated mTALP,’ containing (mM): NaCl (lOO), KC1 (3.1), MgClz (1.5), CaClz (2.1), KHzPOl (0.29), NaHCOs (25), Na-Hepes (lo), pyruvic acid (l), Na-lactate (21.6), BSA (6 mg/ml essentially fatty acid free BSA, Sigma Chemical Co., St. Louis), gentamycin (50 rg/ml), and phenol red (10 pg/ml; pH 7.4 with 1 N NaOH/HCl). Media stock was prepared fresh and sterilized by Millipore filtration (0.22 pm pore). Bovine sperm (American Breeder Service; DeForest, WI) from three ejaculates were pooled, washed twice (2OOg;10 min) with 10 vol of mTALP, adjusted to a concentration of 5 X lO’/ml, and capacitated during a 4-hr preincubation at 39°C in an environment of 5% COzin air in mTALP, supplemented with 10 pg/ml heparin (First and Parrish, 1987;Parrish et aL, 1988).Samples with less than 70% motile sperm at the onset were discarded. All of the experiments reported here utilized neat semen, although the same results were obtained when extended semen was used. Bovine ovaries were obtained from a local abattoir. Fully grown, germinal vesicle-intact oocytes were aspirated from small (l-5 mm in diameter), antral follicles. Oocyte-cumulus complexes were selected based upon the presence of a complete, unexpanded cumulus investment and a dark, uniformly pigmented cytoplasm. Oocyte-cumulus complexes were cultured for 22 hr at 39°C in tissue culture medium 199 supplemented with 10% heat-treated (56°C; 30 min) fetal bovine serum, 0.25 mM pyruvic acid, 0.5 kg/ml FSH, 5 pg/ml LH, 1 pg/ml estradiol-1’7& and 50 pg/ml gentamycin. During this time oocytes undergo meiotic maturation and the cumulus layers expand (Leibfried-Rutledge et al, 1987). Fertilization was carried out in vitro in 50-~1 drops containing 8-10 cumulus-intact, in vitro matured oocytes and lo6 sperm/ml, in a medium containing (mM): NaCl (114), KC1 (3.2), MgClz (0.5), CaClz (2), NaHCOs (25), NaH2P04 (0.34), Na-lactate (lo), pyruvic acid 1Abbreviations used: BSA, bovine serum albumin; EDm, dose producing half-maximal effect; EDTA, ethylenediamine tetraacetic acid; mTALP, modified Tyrode’s buffer; NP-40, Nonidet P-40; PMSF, phenylmethylsulfonyl fluoride; PVP, Polyvinylpyrrolidone; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; tlIz, half time.

VOLUME B&1988

(0.25), BSA (6 mg/ml essentially fatty acid free BSA; Sigma), gentamycin (50 pg/ml), and phenol red (10 rg/ml; pH 7.4) (Parrish et aL, 1985,1988).Gametes were cultured for 4 hr at 39OC,at which time oocytes were stripped of cumulus cells, fixed in 1% gluteraldehyde, stained with acetoorcein, and examined under phasecontrast optics for signs of fertilization (decondensed sperm head in oocyte cytoplasm). Zona pellucidu preparations. Zonae pellucidae were obtained from abattoir-derived ovaries that had been maintained in 0.154 M NaCl (39°C) during transit and used within 4 hr of collection. Ovaries (200-300) were rinsed with a harvesting medium (medium HM) consisting of (mM): NaCl (137); KC1 (2.7); NazHP04 (8.1); KHzP04 (1.47);Naz-EDTA (0.59);PVP-40 (4 mg/ml; MW average, 40,000Da; Sigma); and D-glucose (l.l), pH 7.2. In some experiments oocytes were aspirated, cultured in vitro (as described above) to permit meiotic maturation, and cleaned of adherent cumulus cells, and zonae pellucidae were removed manually with a mouth-operated micropipet. A portion of these oocytes were cultured in vitro with sperm, in order to demonstrate that zonae pellucidae were obtained from fertilizable oocytes. Alternatively, follicles were ruptured and oocytes were collected by a modification of the procedure of Dunbar et al. (1980). Briefly, ovaries were rinsed, submerged in medium HM, and sliced with a bank of ganged razor blades. Ovarian debris was removed during three cycles of filtration using screens of decreasing mesh sizes (230-2000 pm) and oocytes in various stages of growth were isolated on 36-pm screens. Zonae pellucidae were collected in large quantities by a procedure essentially identical to that of Bleil and Wassarman (1986). Briefly, oocytes were recovered from 36 pm screens, collected by centrifugation (2OOg;5 min), and resuspended in 4 ml of homogenization buffer (25 mM triethanolamine, pH 8.5; 150 mlK NaCl; 1 mlK MgClz; 1 mM CaClz; and 0.1 m&f PMSF) supplemented with 1 mg/ml each of DNase and hyaluronidase (Sigma; Types I and IV, respectively). Preparations were chilled to 4”C, homogenized with a Polytron (2 bursts of 5 set at 4.75; 1 burst of 5 set at 5.25), brought to 1% NP-40, and rehomogenized as above. Homogenates were collected by centrifugation (11,OOOg; 5 min), resuspended in homogenization buffer, brought to a final concentration of 50% Percoll (Pharmacia; Piscataway, NJ), and centrifuged at 25,000~for 45 min at 4°C in a 70Ti rotor (Beckman Instruments; Palo Alto, CA). Fractions (1 ml) containing zonae pellucidae were extracted with 10 ml of a medium containing 10 mM Hepes (pH 7.5), 1 M NaCl, and 1% NP-40 for 15 min at 4°C; washed three times with 10 ml HBS-PVP (10 mM Hepes, 140 mM NaCl, 0.4% PVP-40, pH 7.4; 3000~ 10 min); resuspended at a

FLORMANANDFIRST

RephAm

of Exoqtosti

455

in Sperm

concentration of 1.25-2.5 mg protein/ml in HBS-PVP containing 50% glycerol; and stored at -20°C. To prepare soluble extracts, zonae pellucidae were centrifuged (11,OOOg;5 min), resuspended at a concentration of 1.25 mg protein/ml in dilute CH&OOH (pH 2.5) and incubated at 80°C for 20 min. This treatment solubilizes in excess of 85% of zona pellucida protein, with >95% of the solubilized protein migrating in the included volume of Sephadex G-150 columns (Florman and First, unpublished). Given the apparent molecular weight values for bovine zona pellucida constitutents (Fig. l), this solubilization scheme must eliminate protein-protein interactions that exist in the native zona pellucida. Samples were lyophilized and reconstituted with mTALP. Zonae pellucidae were radioiodinated with chloramine-T. Protein determinations. Protein was determined by bicinchoninic acid assay (Pierce Chemical Co.; Rockford, IL). The concentration of standard solutions was determined by Am, using El’ l-cm values of 14.06 for

human transferrin (Kirschenbaum, 19’77a) and 8.14 for human al-acid glycoprotein (Kirschenbaum, 1977b). Electrophoresis. SDS-PAGE (10% acrylamide) was carried out as described by Laemmli (1970). Isoelectric focusing was carried out in polyacrylamide gels (5% T, 4% C), using 2% pH 5-7 and 0.5% pH 3-10 ampholytes (Biolyte; BioRad). Gels were prefocused (200 V x I5 min; 300 V X 30 min; 400 V X 30 min), loaded with

samples, and focused at 400 V for 12.5 hr, followed by 1 FIG. 1. Autoradiographic analysis of the bovine zona pellucida by hr at 800 V. Acetylated cytochrome c pl markers (Cal- two dimensional polyacrylamide gel electrophoresis.Zonaepellucidae Biochem; La Jolla, CA) were used to determine pH were isolated manually from fertilizable oocytesthat had undergone gradients.

Acrosome reaction assaps. Capacitated sperm were diluted stepwise with mTALP. The final sperm concentration was set at 1,3.125,6.25,12.5, or 25 X 106/ml, the concentration of soluble zona pellucida protein was set as desired, and the final volume was 25 ~1. Sperm were cultured for up to 1 hr at 39°C. Parallel sperm samples

meiotic maturation in vitro (A) or were obtained following bulk isolation from follicular oocytes(B). lzj-I-zonae pellucidae were solubilized by heating (8O"C, 10 min) in 2 ~11% SDS,cooledto room temperature, and mixed with 18 ~1of an isoelectric focusing samplesbuffer containing 2% NP-40.Molecular weight standards are indicated (220, 94,60,43, and 27 kDa). (A) Gel loadedwith one zonepellucida (50,000 dpm); one zona pellucida = 27.1 rt 1.1 ng protein/zona pellucida. (B) Gel loadedwith 500 ng zona pellucida protein (65,000 dpm).

received either buffer without added protein or unrelated glycoproteins and served as negative controls. Each experiment included as a positive control capacitated sperm which were incubated up to 60 min with 10 PLMionomycin, conditions that will result in a maximal response in terms of acrosome reactions (data not shown). Aliquots were taken to determine viability, as assayed by sperm motility, and to assess the occurrence

of acrosome reactions. The presence of intact acrosomal membranes was determined histochemically, by the method of Bryan and Akruk (1977). We have previously confirmed this assay by correlation with transmission electron microscopy (Lenz et al, 1983). Data an&s&. The statistical significance of the data presented here was analyzed by a Student t test on paired observations.

RESULTS

Behavior of Bovine Sperm in Culture Ejaculated bovine sperm, washed from seminal plasma, maintain a relatively uncapacitated state during a culture

of 5-6 hr in mTALP.

Under these condi-

tions sperm are inefficient in fertilizing eggs in vitro, as is expected of poorly capacitated preparations (Table 1). Cell viability, as assayed by sperm motility, did not decline significantly during this period (~10%). These preparations displayed a low basal rate of acrosomal exocytosis: 5-10% of the sperm had undergone the acrosome reaction

at the beginning

of this culture

pe-

riod and this fraction did not increase significantly over this time frame.

456

DEVELOPMENTALBIOLOGY

TABLE 1 CHARACTERISTICS OF EJACULATEDBOVINE SPERMATOZOA IN

VITRO

Time in culture (hr) Parameter 36 Motileb

Culture conditions”

mTALP mTALP + heparin % Acrosome mTALP reacted” mTALP + heparin mTALP Eggs fertilizedd mTALP + heparin

0

4

6

81 + 3 73 + 4 66 + 5 34k4 71 f 3 61+ 3 8fl 10 + 2 12 + 2 9+2 11 + 2 15 + 3 O/60 (0) l/60 (2) 3/60 (5) O/60 (0) 52/60 (37) 50/60 (33)

’ Sperm were washed, adjusted to a concentration of 5 X 107/ml in mTALP or in mTALP supplemented with 10 rg heparin/ml, and incubated at 39°C for O-6 hr. bAliquots (1 ~1) were added to 9 ~1 mTALP on a warmed (39°C) microscope slide, coverslipped, and examined under phase-contrast optics. Fifty sperm were evaluated in each of the four randomly chosen fields. Sperm displaying any motion were scored as motile. The results are the means and standard errors of three independent experiments, where triplicate samples of 200 sperm were evaluated per experimental point. ” Aliquots (2.5 ~1) were air-dried onto a warmed microscope slide, stained with erythrosin B, and naphthol yellow S as described under Materials and Methods, and evaluated for the presence of an acrosome. Two hundred sperm were scored in each aliquot. The results represent the means and standard errors of three independent experiments, with each experiment consisting of triplicate samples. ‘Sperm were incubated for the indicated times in mTALP or in mTALP supplemented with 10 pg heparin/ml. Aliquots (1 ~1) were added to 49-pl drops of fertilization medium containing 8-10 in vitro matured bovine oocytes (see Materials and Methods). Gametes were cocultured for an additional 4 hr and eggs were assayed for signs of fertilization (sperm head in oocyte cytoplasm). Data represent the total number of eggs fertilized as a fraction of the total number examined during three separate experiments. Figures in parentheses indicate the percentage of total eggs that were fertilized.

Sperm capacitate during a 4-hr incubation in the presence of 10 pg/ml heparin (Parrish et aL, 1985,1988; First and Parrish, 198’7), as indicated by an enhanced ability to fertilize eggs in vitro. Cell viability is not compromised by these conditions for the first 5-6 hr; beyond 6 hr there is a more pronounced loss of motility. The basal rate of acrosomal exocytosis was not enhanced significantly during capacitation (Table 1). Thus, stable cultures of uncapacitated and capacitated bovine sperm are available for 5-6 hr. We have therefore restricted our observations to this time frame.

Characterization of Zfma Pellucida Preparations Two different preparations of zonae pellucidae were used in this study. First, material was harvested manually from fully grown (ea. 150 pm in diameter) bovine oocytes that had completed meiotic maturation in vitro, yielding approximately 4 pg of zona pellucida protein/ preparation. These zonae pellucidae were considered

VOLUME 123.1933

functionally competent, as the oocytes from which they were derived were fertilized efficiently by capacitated sperm (37145 = 82% fertilized; n = 3). This material was radioiodinated and examined by two-dimensional gel electrophoresis (Fig. 1A). Three components were resolved, each displaying a pronounced microheterogeneity: apparent M, and pI ranges were 94-110 kDa and pI = 7.0~8.3,82-94 kDa and pI = 4.3-4.8, and 70-76 kDa and pl = 5.8-7.3 A second preparation was used to obtain greater quantities of material. Ovarian follicles were ruptured and oocytes were collected by sieving, as described under Materials and Methods. Zonae pellucidae were isolated from oocytes greater than 36 I.crnin diameter by homogenization and density gradient centrifugation, yielding 100-200 pg zona pellucida protein/preparation. The eleetrophoretic profile of zonae pellucidae isolated by density gradient centrifugation was similar to the pattern observed for material from in vitro matured oocytes (Fig. 1B). In addition, both preparations affected bovine sperm identically. Therefore experiments were carried out using bulk prepared material. All results were subsequently confirmed with preparations from fertilizable oocytes. As no significant differences were found in the biological effects of these two preparations, these data have been pooled for presentation.

Zonae PeUucidae Induce the AcrosonzeReaction Soluble extracts of zonae pellucidae were prepared by heating in an acidified medium of low ionic strength (80°C; 20 min; dilute CH&OOH; pH 2.5), as described under Materials and Methods, and added to sperm suspensions. In these initial experiments sperm were capacitated at a high cell density (5 X 107/ml) and incubated with zona pellucida protein at a lower cell density (106/ml). Sperm preincubated for 4 hr under conditions that support capacitation underwent acrosomal exocytosis following addition of solubilized zonae pellucidae. Only 6-8% of the sperm population had undergone the acrosome reaction at the end of the capacitation interval and during an additional 1 hr incubation this increased to only 8-12% in untreated control preparations (Fig. 2). Low concentrations of zona pellucida proteins (6.25 ng/pl; one bovine zona pellucida = 27.1 f 1.1 ng protein) had no effect on this background rate of acrosome reaction. In contrast, soluble zona pellucida protein ranging in concentrations from 12.5-50 ng/pl stimulated the occurrence of the acrosome reaction. The EDso for this process was 18-20 ng/pl. Maximal stimulation was observed when sperm were incubated with 50 ng zona pellucida protein/pi; under these conditions 30-35% of the population completed the acrosome reaction, representing a three- to fourfold net enhancement of exocytosis.

FLORMANAND FIRST

0 20

0

40

457

Regulation of Exocytosis in Sperm

60

Time (Min) FIG. 2. Kinetic analysis of the effects of soluble extracts of zonae pellucida on acrosomal exocytosis. Capacitated sperm (106/ml) were incubated in mTALP containing either control glycoproteins (- - -, 50 and 500 ng/pl al-acid glycoprotein or transferrin; data were pooled and the standard error of all data points was fl.l%) or solubilized zona pellucida protein (0,6.25 ng/pl; l ,12.5 ng/rl; Cl, 18.75 ng/pl; K, 25 ng/rl; A, 50 ng/nl; A, 100 ng/&. Aliquots were assayed for the occurrence of acrosomal exocytosis. Data represent the means and standard errors of five separate experiments, with each experiment consisting of triplicate samples and with 200 sperm assayed/sample.

While soluble extracts of zonae pellucidae stimulated acrosomal exocytosis in a concentration-dependent fashion, there was no effect on the time course of this process (Fig. 2). The acrosome reaction induced by 12.5-100 ng zona pellucida protein/p1 occurred without an apparent lag, had a t1j2 of 5-10 min, and attained maximal levels by 20 min. Yet the apparent initial rate of this reaction, and hence the maximal response, was determined by the concentration of zona pellucida protein (Fig. 3). The initial rate of acrosome reaction in untreated control preparations of capacitated bovine

spermatozoa was estimated at 0.05% /min. This was increased to 2.4% /min during incubation with 50 ng zona pellucida protein/p& a 48-fold stimulation. Acrosome reaction inducing activity was a specific feature of solubilized zonae pellucidae, since control glycoproteins (50-500 ng1p.l transferrin and cY1-acid glycoprotein) and proteins (all experiments, including controls, were carried out in the presence of 6 pg/pl BSA) failed to trigger acrosome reactions above the levels observed in untreated, control cultures. In addition, the stimulatory activity was not a consequence of decreased cell viability, with its associated generalized demembranation. Sperm motility was not affected by incubation with zona pellucida proteins for up to 1 hr (Fig. 4). During more prolonged incubations (2-3 hr) we noted a secondary increase in acrosome reactions, but this was associated with a loss of motility from sperm suspensions and likely represents cell death (data not shown). The kinetics of the zona pellucida-induced acrosome reaction were not altered when sperm concentrations were increased over the range l-25 X 106/ml. In all cases the time courses of the acrosome reaction induced by soluble extracts of zonae pellucidae were indistinguishable from that shown in Fig. 2. Yet, we have observed that the sperm motility was more stable when higher sperm concentrations were used (data not shown). In addition, we have found that the magnitude of response to solubilized zona pellucida protein was de60 L

600

20

40

60

60

100

Protein Added @g/pi)

-0

20

40

60

60

100

Zona Pellucida Protein (ng/pl) FIG. 3. The effects of solubilized zona pellucida protein on the apparent initial rates of acrosomal exocytosis by bovine spermatozoa. The initial rates of exocytoeis were estimated from the kinetic data presented in Fig. 2. Data represent the means and standard errors of initial rate estimates on five separate experiments.

FIG. 4. The effects of solubilized zona pellucida proteins on the motility of bovine sperm. Capacitated sperm(lO’/ml) were incubated with solubilized zona pellucida proteins (m)or with control glycoproteins (Cl, cY,-acidglycoprotein, traneferrin) for 60 min. Aliquots were observed under coverslips and the fraction of motile sperm was determined. Each data point represents observations on four randomly chosen fields of 50 sperm each and the means of five separate experiments are displayed. Standard errors were less than 10% of the meansat all concentrations and the motility of the two treatment groups did not differ significantly (P > 0.1).

458

DEVELOPMENTALBIOLOGY TABLE 2 INFLUENCEOF SPERMCULTURECONDITIONSON ZONA PELLUCIDA-INDUCEDACROSOMALEXOCYTOSIS % Acrosome reacted sperm*

Culture conditions” [HCOa]

BSA Fraction V Fatty acid free Fatty acid free Fatty acid free Fatty acid free

WW 25 25 10 0 25

Temperature (“C) 39 39 39 39 37

Control 12 + 1 12 + 2 11 f2 91tl 11+2

+50 ng zona pellucida 21 + 30 + 30 + 12 * 16 f

2 2 3 1 2

‘Sperm were washed, adjusted to a concentration of 5 x 107/ml, and incubated for 4 hr at the indicated temperature in mTALP containing 10 ag heparin/ml and supplemented as indicated. NaHCCs was replaced with Hepes in order to maintain osmolarity and to stabilize pH. ‘Aliquots (12.5 al) were diluted 1:l with an identical culture medium containing either &i-acid glycoprotein (control) or soluble extracts of bovine zonae pellucidae (+ zona pellucida), such that the final concentration of protein other than BSA was 50 ng/pl. Samples were incubated for 1 hr under the conditions indicated and assayed for the occurrence of acrosomal exocytosis. The results represent the means and standard errors of three independent experiments, with triplicate samples of 200 sperm evaluated per experimental point.

pendent on culture conditions. A maximal response was observed (1) when essentially fatty acid free BSA was used. Preparations of Cohn’s Fraction V BSA also supported zona pellucida-induced acrosome reactions, albeit at lower levels (Table 2). (2) HCO; anion must be present (Table 2). Zona pellucida-induced exocytosis did not occur in HCOi-free medium, even under conditions where extracellular pH was maintained. In contrast, 10 mMHC0; supported an identical exocytotic response to soluble extracts of zona pellucida as did mTALP (25 mM HCOi). (3) Temperature must be maintained at 39°C (bovine core body temperature). This is likely an indirect effect, since at lower temperatures (37°C) bovine sperm capacitate less efficiently (Lenz et al, 1983).

Eflects of Sperm Capacitation on the Zona PellucidaInduced Acrosome Reaction We sought to determine whether the zona pellucidadependent acrosome reaction of bovine sperm required prior capacitation. Previously, it was reported that mTALP supplemented with 10 pg heparin/ml supports capacitation; this process requires about 4 hr (First and Parrish, 1987; Parrish et al, 1988). We therefore incubated sperm for 5 hr, either in the presence or the absence of added heparin, and at intervals assayed the ability to undergo acrosome reactions in response to 50 ng zona pellucida protein/g1 (Fig. 5). In these experiments, 6-8s of the sperm had completed the acrosome

VOLUME128,1988

reaction at the earliest time point (10 min). This value did not increase significantly in preparations incubated under noncapacitating conditions (no heparin), even when challenged with soluble zona pellucida protein after 4 hr of incubation. In contrast, sperm acquired the ability to undergo a zona pellucida-regulated acrosome reaction during incubation in the presence of heparin. A rapid initiation of acrosome reactions was observed when zonae pellucidae were added to sperm treated for 4 hr with heparin, as noted earlier (Figs. 2 and 5). The expression of zona pellucida-regulated acrosome reactions required 3-4 hr to develop, as sperm treated with heparin for shorter periods of time (10 min to 3 hr) did not undergo acrosomal exocytosis in response to 50 ng/pl soluble zona pellucida protein. This was the case even when the concentration of zona pellucida protein was increased to 250 ng/pl (data not shown). While sperm were nonresponsive to zona pellucida protein during this initial phase, acrosome reactions could nevertheless be triggered with the divalent cationophore ionomycin. This suggests that the exocytotic pathways in the sperm cytoplasm were functional. Rather, sperm incubated under capacitating conditions for up to 3 hr specifically failed to initiate acrosome reactions in response to zonae pellucidae.

60 0 al ij 3 cc

40

2 % ti 4

20

c

&

G-+@ 0,i

0

L 100

200

300

Time (Min)

FIG. 5. Kinetic analysis of the expression of zona pellucida regulated acrosome reactions in bovine sperm. Sperm were incubated for 5 hr under noncapacitating (0, mTALP) or capacitating conditions (a 10 pg heparin/ml mTALP). Soluble extracts of zona pellucida protein (50 ng/pl) were added to aliquots of sperm at the indicated times (arrows) and the occurrence of the acrosome reaction was monitored for 20-60 min. The induction of acrosomal exocytosis by 10 pM ionomycin (A) and by control glycoproteins (A; 50-250 ng/pl transferrin and al-acid glycoprotein) was also assayed. Data represent the means and standard errors of five independent experiments, with each experiment consisting of triplicate samples and with 200 sperm assayed/sample. Standard errors were less than 10% of the means at all times and in all treatments and error bars were omitted from lines that were not different (+ heparin vs - heparin, 10 min to 3.5 hr; control protein vs - heparin, 4-5 hr; P > 0.1).

FLORMANANDFIRST

Zona pellucida-dependent acrosomal exocytosis is both first observed and reaches its maximal expression between 3 and 4 hr of incubation under capacitation conditions (Fig. 5). It is unlikely that this is a secondary consequence of compromised sperm viability, since there was no selective decrease in sperm motility associated with incubation under capacitating conditions. Bovine sperm will aggregate to a greater extent in medium containing heparin (capacitating conditions); yet, when aggregates were dispersed by gentle pipetting no heparin-dependent differences in motility were noted (Fig. 6). In addition, a second phase of acrosome reactions was observed during prolonged culture (6-8 hr). This reflects an increase in the zona pellucida-independent rate of exocytosis. This is likely due to cell death, since it is associated with a decline in motility (data not shown). A series of experiments were carried out in order to explore the relationship between the expression of zona pellucida-regulated acrosome reactions and the development of a capacitated state. Since only capacitated sperm can penetrate eggs, the progress of capacitation can be monitored by observing gamete interaction (Austin, 1951,1952; Chang, 1951). Sperm were preincubated in the presence of heparin (10 pg/ml; capacitating conditions) for 10 min to 4 hr, diluted 50-fold into heparin-free medium containing in vitro matured oocytes, and incubated an additional 4 hr. The final conditions of gamete coculture do not support capacitation and fertil-

80

60om 100

200

300

459

&q&&on of Exocytosis in Sperm

400

Time (Min)

FIG. 6. The effects of solubilized zona pellucida proteins on the motility of bovine sperm cultured under noncapacitating and capacitating conditions. Sperm (5 X 107/ml) were incubated for 5 hr under noncapacitating (Cl, mTALP) or capacitating conditions (W,10 pg heparin/ml mTALP). To assay motility, the fraction of motile cells was observed at intervals in aliquots under coverslips. Four randomly chosen fields of 50 sperm were assayed for each experimental point and the data presented are the means of five separate experiments. At all times the standard errors were less than 10% of the means (not shown) and these data sets are not different (P z 0.1).

100 I D 2 ” .-

z 2

Eii

80 -

60

40 -

G g?

20100

Time (Min)

FIG. 7. Kinetic analysis of the expression of fertilizing ability in bovine sperm incubated under noncapacitating and capacitating conditions. Sperm (5 X lO’/ml) were incubated under noncapacitating (0, mTALP) and capacitating conditions (0,lO pg heparin/ml mTALP). Aliquots (1 ~1) were taken at the times indicated and added to 49-$ droplets of fertilization medium containing 8-10 oocytes. Gametes were cocultured for 4 hr at 39’C at which time oocytes were fixed and assayed for fertilization. Data represent the means and standard errors of three separate experiments, with each experiment utilizing 20 oocytes/point.

ization will not occur without prior treatment of sperm (Table 1). Sperm failed to fertilize eggs following incubation in the presence of heparin for as long as 2.5 hr (Fig. 7). The fertilizing ability of sperm is expressed at some time during the interval of 2.5-4 hr of culture and is manifested fully by 4 hr. Thus, the acquisition of a capacitated state occurs with approximately the same kinetics as the development of zona pellucida-regulated acrosome reaction (Figs. 5 and 7). In a second series of experiments we examined the effects of an inhibitor of capacitation on the development of zona pellucida-regulated acrosome reactions in bovine sperm. D-Glucose (5 mM) inhibits the heparininduced capacitation of sperm under the experimental conditions that we have utilized (First and Parrish, 1987). Sperm were preincubated in mTALP (noncapacitating conditions) or in mTALP supplemented with heparin (10 pg/ml; capacitating conditions) for 4 hr. Aliquots were assayed for the onset of capacitation, as demonstrated by the ability to fertilize eggs under the noncapacitating conditions described previously, and for the expression of zona pellucida-dependent acrosome reactions (Table 3). Sperm cultured under noncapacitating conditions failed either to display zona pellucida-dependent acrosome reactions or to fertilize eggs efficiently (Table 3, Line 1). These low, basal rates were not suppressed by the presence of D-glucose during preincubation (Line 3). In contrast, a 4-hr incubation in a capacitating environ-

460

DEVELOPMENTALBIOLOGY

TABLE 3 EFFECTSOFD-GLUCOSEONTHE DEVELOPMENTOF ZONA PELLUCIDAREGULATEDACROSOMEREACTIONSIN BOVINE SPERMATOZOA

O-4 hr 1. mTALP 2. mTALP 3. mTALP + glucose 4. mTALP + heparin 5. mTALP + heparin + glucose 6. mTALP 7. mTALP + heparin

>4 hr

pacitated state, subsequent addition without effect (Line 7).

of glucose was

DISCUSSION

% Acrosome reacted spermb Culture conditionsa

VOLUME 128,1988

+ Control protein

+ Zona pellucida protein

Fertilization” p % Total eggs

mTALP 10 df ionomycin

12 + 3

14 + 3

a+4

52 + 4+

56 + 3”

-

mTALP

12 + 2

13 f 1

5+3

mTALP

11+3

30 f 2*

85 + 5*

mTALP mTALP + glucose mTALP + glucose

13 k 3

16 f 2

13 + 3

10 k 3

13 + 1

8k2

13 + 3

32 + 2’

83 + 5*

‘Sperm were incubated at a high cell density (5 X lO’/ml) in mTALP, or in mTALP supplemented as indicated (O-4 hr), for 4 hr at 39°C. Aliquots were then cultured at a lower cell density (see below) in mTALP or in mTALP supplemented as indicated (>4 hr) and assayed for the occurrence of basal and zona pellucida-stimulated acrosome reactions and for the ability to fertilize eggs. bAliquots (12.5 ~1) of sperm were diluted 1:l with the culture medium containing either control glycoproteins or soluble extracts of zona pellucida protein (50 ng/Nl). Sperm were incubated 1 hr at 39°C and then assayed for the occurrence of acrosome reactions. Each experiment consisted of triplicate samples and 200 sperm were scored per sample. The results represent the means and standard errors of three separate experiments. Superscripted data points (*) were significantly different (p < 0.001) from untreated control samples incubated the entire period in mTALP (Line 1). ’ Aliquots (1 ~1) of sperm were added to 49-pl drops of fertilization medium containing 10 in vitro matured bovine oocytes. Gametes were cocultured an additional 4 hr at 39°C and assayed for indications of fertilization (see Materials and Methods). Data are expressed as the percentages of total eggs that were fertilized. Each experiment consisted of duplicate samples and the results represent the means and standard errors of three independent experiments. Superscripted data points (s) are significantly different (P < 0.001) from untreated control samples incubated the entire period in mTALP (Line 1).

ment resulted in the full expression of both the induced acrosome reaction and the fertilizing ability (Line 4). Both of these biological activities were completely inhibited by the presence of D-glUCOSe during preincubation (Line 5). In addition, neither zona pellucida-regulated acrosome reactions nor the ability to fertilize eggs developed during incubation in another medium containing D-glucose, tissue culture medium 199 (data not shown). This inhibitory activity was only evident if the monosaccharide were present during the preincubation phase: when sperm were first permitted to attain a ca-

Our central finding is that soluble extracts of the bovine zona pellucida can initiate acrosomal exocytosis in spermatozoa through a capacitation-regulated pathway. Three lines of evidence support this conclusion. First, these two processes display an identical stimulation by heparin. When sperm are cultured in the absence of heparin they do not attain a capacitated state, as indicated by an inability to fertilize eggs in vitro, and soluble extracts of zonae pellucidae are unable to initiate acrosomal exocytosis. Addition of heparin supports both capacitation and the expression of zona pellucidaregulated acrosome reactions. It should be noted that the level of acrosome reactions in sperm preparations incubated with heparin, but in the absence of zona pellucida proteins were indistinguishable from the levels in untreated control cultures (Fig. 5). This is consistent with heparin acting specifically as a capacitating agent and not as a direct inducer of exocytosis (First and Parrish, 1987; Parrish et aL, 1988). Second, the stimulatory effects of heparin on the expression of both fertilizing ability and zona pellucidaregulated acrosome reactions can be inhibited by 5 mM D-glucose (Table 3) (First and Parrish, 1987). Glucose does not function as a direct antagonist of zona pellucida agonist activity, since full acrosome reaction inducing activity was observed when glucose was added to previously capacitated sperm; rather, its action was exerted specifically during heparin preincubation (Table 3). Metabolizable hexoses, including glucose, have previously been reported to delay the capacitation of sperm that occurs in vitro in defined media and in oviductal fluid in certain mammalian species, such as bovine and guinea pig (Rogers and Yanagimachi, 1975; Hyne and Edwards, 1985; Murphy et aL, 1986; First and Parrish, 1987). It is well established that these compounds can act as metabolic regulators, modulating a wide range of sperm functions (Lardy and Phillips, 1941; Mann, 1964; Rogers et aL, 1979; Inskeep et CN!,1985). Glucose utilization can alter a number of intracellular and extracellular parameters which have previously been associated with capacitation, including sperm ATP and respiratory levels, as well as external pH (Mann, 1964, Hyne and Garbers, 1981; Hammerstedt and Lardy, 1983; Murphy and Yanagimachi, 1984; Inskeep and Hammerstedt, 1985; Hyne and Edwards, 1985; Inskeep et al., 1985; Fraser and Lane, 1987; see reviews by Harrison, 1977; Rogers, 1978; Garbers and Kopf, 1980). Additional experimentation is required to define the mechanism of glucose inhibition of heparin-induced capacitation of bovine sperm.

FLORMANAND FIRST

461

Regulation of Exocytosis in Sperm

Third, kinetic experiments demonstrate that sperm develop a capacitated state and the ability to undergo zona pellucida-dependent acrosomal exocytosis during preincubation with heparin with similar time courses. We have again monitored capacitation indirectly, by observing the time-dependent manifestation of the ability of spermatozoa to fertilize eggs during a subsequent incubation in a noncapacitating milieu. Following 2.5-3 hr of incubation with heparin, sperm have not completed capacitation and also are unable to undergo acrosome reactions upon addition of soluble extracts of zonae pellucidae. Both of these activities develop completely during an additional l-l.5 hr or preincubation in heparin-supplemented medium (Figs. 5 and 7). After a 4-hr preincubation, sperm can initiate acrosomal exocytosis without apparent time lag following the addition of soluble extracts of zonae pellucidae, indicating that all preparatory events have been completed. Sperm in these preparations have either attained a capacitated state or can complete any final steps in this process in a noncapacitating medium. Taken together, these data argue that ejaculated, bovine sperm must be capacitated in order to manifest zona pellucida regulated acrosomal exocytosis. Ward and Storey (1984) have reached a similar conclusion in the case of cauda epididymal mouse sperm. A functional activation, or capacitation, of spermatozoa following release from the male reproductive tract is apparently required in all eutherian mammals (Bedford, 1983). The biochemical basis of reprogramming is unclear, although this process is associated with diverse alterations in metabolism, as indicated above, as well as changes in motility patterns, membrane function, and cell surface features (see reviews by Yanagimachi, 1981; O’Rand, 1982; Clegg, 1983). As a consequence of these events sperm first display the ability to fertilize eggs (Austin, 1951,1952; Chang, 1951). One function of capacitation is to regulate gamete interactions during fertilization. Uncapacitated sperm fail to adhere to the zona pellucida (Overstreet and Bedford, 1974; Inoue and Wolf, 19’75;Saling et aL, 19’78;Heffner and Storey, 1982) or to undergo zona pellucida-triggered acrosome reactions (Ward and Storey, 1984; present study). While adhesion capability is developed early in the process of capacitation (Inoue and Wolf, 1975; Heffner and Storey, 1982), the kinetic data presented here suggest that the expression of zona pellucida-regulated exocytosis is a late, possibly final, event. Delayed expression of agonist sensitivity has apparent advantages if capacitation is to function as a spatial and temporal integrator of sperm and egg availability (Bedford, 1983). At present, the basis of suppression in uncapacitated sperm and of the release from inhibition during capacitation are unknown. This may occur by a capacitation-regulated oc-

elusion of sperm surface receptors for zona pellucida agonists, as has been suggested for gamete adhesion (Shur and Hall, 1982), or by modulation of a signal transduction system (Endo et al, 1987). Previously, we (Florman and Storey, 1982; Florman et uL, 1984) and others (Bleil and Wassarman, 1983, Cherr et u,!, 1986; O’Rand and Fisher, 1987; Cross et al, 1988) have observed that acrosome reaction inducing activity is retained in soluble extracts of the zona pellucida of mouse, hamster, human, and rabbit. The present study extends these observations to the bovine. A maximum of 30-35s of bovine sperm initiate acrosomal exocytosis during incubation with solubilized zona pellucida proteins. The level of zona pellucida-induced exocytosis in the bovine is approximately 25%, similar to the range noted in other systems (25-40s). While a higher level of acrosomal exocytosis has been reported in the mouse (60-70%), this is a consequence of an elevated rate of background (zona pellucida-independent) acrosome reactions (20-30s) (Florman and Storey, 1982; Bleil and Wassarman, 1983; Florman et al, 1984). The response in the bovine is not limited by the cellular exocytotic machinery, since 50-60s of these sperm undergo acrosome reactions following administration of a Ca’+/Mp ionophore. Rather, this may reflect the relative efficiencies of in vitro capacitation systems available in these species. In the mouse, where capacitation of individual sperm can be monitored, there is a correlation between the fraction of sperm capacitated and that undergoing zona pellucida-induced acrosome reactions (Ward and Storey, 1984). It is likely that a similar relationship applies to the bovine and that the culture conditions utilized in this study permit complete capacitation of only 25-33s of the sperm population. The mechanism of agonist-induced acrosomal exocytosis and its regulation during capacitation requires further examination. We are grateful to Drs. Robert Bremel, Henry Lardy, and Linda Schuler for generously providing access to their laboratories. In addition, we found discussions with Drs. John Parrish and Bayard Storey valuable during the course of this research. Patient secretarial assistance was provided by Julie Busby. This work was supported by the Department of Meat and Animal Science, College of Agricultural and Life Sciences, and a grant from the W. R. Grace and Company. REFERENCES AUSTIN, C. R. (1951). Observations in the penetration of sperm into the mammalian egg. Au&al J Ski Res. (B) 4,581-592. AUSTIN, C. R. (1952). The “capacitation” of the mammalian sperm. Nature (Lundon) 170,326. AUSTIN, C. R. (1935). Sperm maturation in the male and female genital tracts. In “Biology of Fertilization” (C. B. Metz and A. Monroy, Eds.), Vol. 2, pp. 121-155. Academic Press, New York. BEDFORD,J. M. (1983). Significance of the need for sperm capacitation before fertilization in eutherian mammals. Biol. Reprod. 28, 108-120.

462

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in Spm

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