Proc. Natl. Acad. Sci. USA Vol. 75, No. 10, pp. 4954-4958, October 1978 Cell Biology Stimulation of in vitro activation and the acrosome reaction of hamster spermatozoa by catecholamines (capacitation/adrenergic receptors/epinephrine/membranes) LAWRENCE E. CORNETT AND STANLEY MEIZEL* Department of Human Anatomy, School of Medicine, University of California, Davis, California 95616 Communicated by Edwin C. Krebs, June 29,1978

ABSTRACT Capacitation and the acrosome reaction of point for identification of the motility factor because the large mammalian spermatozoa are essential for fertilization. In vitro size of the bovine adrenal gland enabled us to separate the results are presented that demonstrate that catecholamines stimulate activation (a whiplash flagellar movement charac- adrenal cortex and medulla (two very different functional teristic of capacitated hamster spermatozoa) and the acrosome tissues) for testing as sources of the factor(s). The results pre- reaction. Protein-free ultrafiltrates of bovine adrenal cortex and sented in this paper demonstrate that catecholamines are one medulla preparations stimulated motility, activation, and of the components of the adrenal gland motility factor and that acrosome reactions of hamster spermatozoa in the presence of catecholamines can stimulate activation and the acrosome re- bovine serum albumin. The medulla preparation was more ef- action of hamster spermatozoa in vitro. This work has been fective than the cortex preparation in the stimulation of acti- vation and acrosome reactions. Epinephrine (0.5-50 paM) and reported in preliminary form (13). norepinephrine (50.0 pM) in the presence of bovine serum al- bumin and a partially purified protein-free cortex preparation MATERIALS AND METHODS also stimulated activation and the acrosome reactions. Both Preparation of Motility Factor from Bovine Adrenal activation and acrosome reactions in the presence of epineph- rine were inhibited by the adrenergic antagonists phentolamine Glands. The medulla and cortex of several steer adrenal glands, and propranolol, suggesting the involvement of a- and a-ad- trimmed of fat (Pel-Freez Biologicals), were separated by dis- renergic receptors in the stimulation of capacitation and the section and homogenized in 0.9% NaCl (0.3 ml of saline per 30 acrosome reaction. In addition, phenylephrine, an a-adrenergic mg wet weight) in a VirTis homogenizer (15,000 rpm for 3 min agonist, was as potent as epinephrine in the stimulation of at 40). The homogenate was centrifuged at 27,000 X g for 30 acrosome reactions, but activation was reduced. Isoproterenol, min, and the supernatant was heated to 56° for 60 min to de- a P-adrenergic agonist, was as potent as epinephrine in the stimulation of activation, but acrosome reactions were reduced. stroy complement and then centrifuged once again at 27,000 High percentages of both activation and acrosome reactions X g for 30 min. The supernatant was then passed through a were observed only in the presence of epinephrine, norepi- Millipore PSAC ultrafiltration membrane (cutoff 1000 mo- nephrine, or phenylephrine and isoproterenol together. lecular weight at 30 pounds/inch2 (200 kPa) with N2 gas in an Sperm capacitation (subtle cellular changes occurring within Amicon cell. The protein-free ultrafiltrate was treated with the female reproductive tract or in vitro) and the resulting Florisil, a steroid adsorbent, that had previously been washed acrosome reaction (a fusion and vesiculation of the outer with twice-distilled H20 to remove fine particles (14). Florisil acrosomal membrane and its overlying sperm head plasma (60 mg/ml) was added to the ultrafiltrate, and the suspension membrane) are essential for mammalian fertilization (1, 2). The was vortex mixed for 60 sec. The supernatant was removed and molecular events of capacitation and of the acrosome reaction, passed through a Millipore filter (0.45 gm pore diameter) into including its initiation, are only partially understood (3). sterile tubes and stored at -20°. Capacitation and the acrosome reaction can be induced in For those experiments testing the effect of catecholamines hamster spermatozoa in vitro by detoxified follicular fluid (4, and adrenergic agonists and antagonists, the protein-free ul- 5) or by blood sera (6). A nondialyzable serum or follicular fluid trafiltrate derived from the cortex was not extracted with factor (labile at 900) was required for the acrosome reaction to Florisil, but was treated by exposure to alumina as described occur and a dialyzable heat-stable factor derived from serum for adsorption of plasma catecholamines (15) and then was or follicular fluid was necessary for sperm motility, activation extracted twice with 4 vol of chloroform for 60 min with con- (the whiplash flagellar movement characteristic of capacitated stant shaking. The aqueous fraction resulting from this ex- hamster spermatozoa), and the occurrence of the optimum traction was then lyophilized overnight, redissolved to the number of acrosome reactions (4-6). It has now been shown that original volume with twice-distilled H20, passed through a certain serum albumins can replace the nondialyzable factor Millipore filter (0.45 ,um) into sterile tubes, and stored at (6-9) and that the bovine follicular fluid factor is serum albumin -20°. (9). Sperm Washing Procedure. Spermatozoa recovered from Recently, it has been reported that the dialyzable factor can the cauda epididymides of 3- to 4-month-old golden hamsters be replaced by a "motility factor" of less than 1000 molecular weighing 130-150 g were washed by the method of Bavister weight obtained from extracts of hamster adrenal gland (10) and Yanagimachi (11) except for the following modifications: or hamster, guinea pig, or human spermatozoa (11, 12). The the distal tubule of one epididymis was punctured, the contents motility factor derived from spermatozoa also seems to maintain were carefully layered on 10 ml of washing medium (equal hamster sperm viability upon dilution in vitro (12). In the volumes of Dulbecco's phosphate-buffered saline and isotonic present paper the bovine adrenal gland was chosen as a starting sucrose) containing penicillin at 100 units/ml in a conical centrifuge tube, and the spermatozoa were allowed to disperse The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviation: AC cortex preparation, alumina-treated and chloro- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate form-extracted cortex preparation. this fact. * To whom reprint requests should be addressed. 4954 Downloaded by guest on September 27, 2021 Cell Biology: Cornett and Meizel Proc. Nati. Acad. Sci. USA 75 (1978) 4955 until homogeneous. After the second centrifugation step, the more vigorous in the presence of the medulla preparation (data sperm pellet was gently resuspended in 0.5 ml of washing not shown). The protein-free ultrafiltrates of the bovine adrenal medium, but the suspension was not centrifuged again. gland vary in their ability to induce activation (Fig. 1A). The An aliquot of the washed sperm suspension was diluted 1:14 cortex preparation was ineffective in inducing activation; only with modified Tyrode's buffer (containing 25.0 mM bicar- a low percentage of activation (never more than 20%) was ob- bonate, glucose at 0.9 mg/ml, 0.2 mM pyruvate, 10.0 mM served during 4.5 hr of incubation. However, the medulla lactate, and penicillin at 200 units/ml and adjusted to pH 7.4 preparation induced a high degree of activation, reaching a with CO2 prior to sperm addition) to a concentration of 5.3-6.8 plateau at 50% after only 3.5 hr. When the cortex and medulla X 106 sperm per ml. Aliquots of this sperm suspension were used preparations were present together, a higher percentage of in all incubations. activation (40%) was seen early in the incubation and the pla- Sperm Incubation Procedure. Incubations were carried out teau (60%), also reached at 3.5 hr, was higher. at 370 in a humidified 95% air/5% CO2 atmosphere in sterilized The protein-free ultrafiltrates of the bovine adrenal gland 1.5-ml polypropylene Eppendorf tubes (Brinkmann) in the also differ in their ability to stimulate the acrosome reaction absence of oil (16). In all cases, the pH of the incubation mix- (Fig. 1B). The Florisil-treated cortex preparation never stim- tures was maintained at 7.5-7.6 throughout the incubation. In ulated more than 20% acrosome reactions up to 4 hr (although experiments testing the medulla and cortex preparations, in- as many as 40% were detected at 4.5 hr). However, in the cubation tubes contained 50 AI of sperm suspension, 40 ,.l of presence of Florisil-treated medulla preparation, acrosome bovine serum albumin dissolved in Tyrode's buffer (pH 7.4, 12 reactions were observed as early as 2.5 hr and the percentage mg/ml), 5 Ml of either cortex preparation or medulla prepara- increased to greater than 70% by 4.5 hr. High numbers of tion (Florisil treated), and 5 Ml of phosphate-buffered saline. acrosome reactions (80% at 4 hr) also were seen in spermatozoa When the two preparations were tested together, 5 Ml of each incubated in the presence of both the medulla and cortex was used and the phosphate-buffered saline was omitted. preparations. In experiments testing epinephrine, norepinephrine, and Epinephrine and Norepinephrine. Because epinephrine and adrenergic agonists, the incubation tubes contained 50 JAl of norepinephrine are present in the adrenal medulla in high sperm suspension, 40 Ml of bovine serum albumin, 5 Ml of the amounts, each was tested in place of the medulla preparation. alumina-treated and chloroform-extracted cortex preparation However, spermatozoa did not survive in the presence of bovine (AC cortex preparation), and 5 Ml of a stock solution of the serum albumin and catecholamine alone but required the compound being tested. The compounds were dissolved in presence of the cortex preparation (data not shown). The AC phosphate-buffered saline and passed through Nucleopore cortex preparation was ineffective in inducing activation (Figs. filters (0.45 Mm), immediately before addition to the incubation 2A and 3A). Only 10-20% of motile incubated spermatozoa tubes. were activated during a 4.5-hr incubation. Those spermatozoa In experiments testing the adrenergic antagonists, the incu- that were motile were less vigorous than spermatozoa incubated bation tubes contained 50 Ml of sperm suspension, 40 Ml of bo- vine serum albumin, 5 Ml of the AC cortex preparation, 5 Al of 100 a stock solution of epinephrine, and 5 Ml of a stock solution of the antagonist being tested. The antagonists were dissolved in twice-distilled H20 and passed through Nucleopore filters (0.45 80 Mm) immediately before addition to the incubation tubes. c Determination of Sperm Motility, Activation, and Acro- r. 60 some Reactions. At various times during the incubation, in- dividual tubes were removed from the incubator and the per- C 40 centage of motile spermatozoa and the percentage of motile spermatozoa showing activation at 370 by phase-contrast mi- 20 croscopy were estimated (16). Activated spermatozoa were identified by a vigorous whiplash flagellar movement charac- teristic of capacitation (17). The percentage of motile sper- 5.0 matozoa that had undergone an acrosome reaction was deter- mined by phase-contrast microscopy, using the morphological criteria established by Yanagimachi (4) and observing at least 100 motile spermatozoa. Chemicals Used. Pyruvic acid sodium salt, L-(+)-lactic acid 0 (30%), D-(+)-glucose, penicillin G sodium salt, (-)-epinephrine, u (-)-norepinephrine, (-)-phenylephrine-HCI, Florisil, and CD E alumina (grade I, neutral) were purchased from SIGMA 0 Chemical Co. Sucrose was purchased from J. T. Baker. Phen- 0 u tolamine-HCl, (-)-propranolol-HCl, and (-)-isoproterenol- CU (+)-bitartrate were gifts from Ciba Pharmaceutical Co., Ayerst Laboratories, and Sterling-Winthrop Research Institute, re- spectively. Pentex fraction V bovine serum albumin was ob- tained from Miles Laboratories. Incubation time, hr FIG. 1. Effect of bovine adrenal gland preparations (Florisil- RESULTS treated) on activation and the acrosome reaction of hamster sperm. Florisil-Treated Bovine Adrenal Cortex and Medulla 0, Cortex preparation; 0, medulla preparation; *, cortex preparation + medulla preparation; (A) Each point is the mean (vertical line is Preparations. While the percentage of spermatozoa alive range) of estimated percent sperm showing activation (n = 4). (B) during an incubation (% motility) was constant irrespective of Each point is the mean ± SEM of percent sperm with acrosome re- which source of "motility factor" was used, sperm motility was actions (n = 4). Downloaded by guest on September 27, 2021 4956 Cell Biology: Cornett and Meizel Proc. Nati. Acad. Sci. USA 75 (1978)

100r A A 80 F 80 c C jr- 60 0Ago, 60

m4-0 4u 40 mu40

20 I,e 20 0O,0jj.4 0 2.5 3.0 3.5 4.0 4.5 5.0 2.5 3.0 3.5 4.0 4.5 5.0

B B

in c c 0 r._

CD E E 0 0 (n 0.4 0

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5.0 3.0 3.5 4.0 Incubation time, hr Incubation time, hr

FIG. 2. Effect of (-)-epinephrine on activation and the acrosome FIG. 3. Effect of (-)-norepinephrine on activation and the reaction of hamster sperm. 0, 5 ,d of AC cortex preparation; *, 50.0 acrosome reaction of hamster sperm. 0, 5 MAl ofAC cortex preparation; *, 50.0 M (-)-norepinephrine; *, 5.0 AM (-);norepinephrine; E, 0.5 MAM (-)-epinephrine; 0, 5 MAM (-)-epinephrine; *, 0.5 EM (-)-epi- nephrine). (A) Each point is the mean (vertical line is range) of esti- MM (-)-norepinephrine. (A) Each point is the mean (vertical line is mated percent sperm showing activation (n = 4). (B) Each point is range) of estimated percent sperm showing activation (n = 4). (B) Each point is the mean + SEM of percent sperm with acrosome re- the mean ± SEM of percent sperm with acrosome reactions (n = 4). actions (n = 4). in the presence of the AC cortex preparation in the presence of added epinephrine or norepinephrine (data (Table 1). With not shown). The results in Fig. 2A also show that epinephrine, epinephrine, which is known to interact with both a- and fl- adrenergic receptors (18), the percentage of acrosome reactions in the presence of AC cortex.preparation, stimulated activation. and activation is greatly increased over the control values (AC The highest concentration tested, 50.0 ,gM, gave a high per- cortex preparation only) at the three concentrations tested. The centage of activation (50%) early in the incubation and reached a-adrenergic agonist at 50.0 MAM and 5.0 MM was as effective a plateau at 60%. In the presence of lower concentrations, 5.0 as epinephrine in the induction of the acrosome reaction, but ,MM and 0.5 MM, the same plateau was reached by 4.5 hr, but the percentage of activated spermatozoa was reduced. Sper- at earlier times the percentage of activation was lower than that matozoa incubated in the presence of isoproterenol showed a seen with 50.0 ,M epinephrine. high degree of activation equal to that seen with spermatozoa Epinephrine in the presence of the AC cortex preparation incubated in the presence of epinephrine at the three concen- also stimulated acrosome reactions (Fig. 2B). With 50.0 ,M trations tested. However, isoproterenol only increased the epinephrine, the percentage of acrosome reactions increased percentage of acrosome reactions over control values at the from 10% at 3 hr to 80% at 4.5 hr. In the absence of added highest concentration tested, 50.0 MiM. When phenylephrine epinephrine, 20% acrosome reactions were seen at 4.0 hr and and isoproterenol were present together, the percentage of the percentage increased to over 40% at 4.5 hr. In the presence activated spermatozoa was equal to that with epinephrine at of 5.0 MAM and 0.5 ,M epinephrine, there were 70% acrosome all three concentrations tested and the percentage of acrosome reactions at 4.5 hr. reactions was equal to that seen in tubes with epinephrine Norepinephrine also stimulated activation in the presence presented except at the highest concentration, 50.0,MM. of the AC cortex preparation (Fig. 3A). With 50.0MuM norepi- Adrenergic Antagonists. The a-adrenergic antagonist were nephrine, activation was high (30%) early in the incubation and phentolamine and the (3-adrenergic antagonist propranolol tested for their ability to inhibit activation and acrosome re- reached a plateau (60%) at 4 hours. The lower concentrations actions in the presence of 10.0 MM of norepinephrine, while better than the AC cortex preparation epinephrine (Table 2). Phentolamine, 25.0 ,M and 10.0 MAM, reduced acrosome reac- were effective at activation than alone, less inducing equal tions and activation a 4.5-hr incubation. with 2A during However, concentrations of epinephrine (Figs. and 3A). 1.0 ,M phentolamine, there was no effect on either acrosome Norepinephrine also stimulated acrosome reactions (Fig. 3B). reactions or activation. Propranolol, 25.0 MM, reduced acrosome With 50.0 MM norepinephrine, the percentage of acrosome reactions and completely inhibited activation. With 10.0 ,M reactions increased from 5% at 3 hr to nearly 80% at 4.5 hr. The and 1.0 ,M propanolol, activation was reduced, but there was lower concentrations tested, 5.0MM and 0.5 ,M, did not stim- no effect on acrosome reactions. The percentages of acrosome ulate acrosome reactions above the control values. reactions obtained in the presence of either 25.0 MAM phento- Adrenergic Agonists. The a-adrenergic agonist phenyl- lamine or 25.0 MAM propranolol (Table 2) were also lower than ephrine and the f3-adrenergic agonist isoproterenol were tested the percentage of acrosome reactions in the presence of bovine for their ability to induce the acrosome reaction and activation serum albumin and the AC cortex preparation (Fig. 2B). Downloaded by guest on September 27, 2021 Cell Biology: Cornett and Meizel Proc. Natl. Acad. Sci. USA 75 (1978) 4957 Table 1. Effect of adrenergic agonists on the hamster sperm DISCUSSION acrosome reaction* We have shown in this paper that the "motility factor" previ- % motile,t % activatedt % AR,§ ously found in hamster adrenal glands (10) is also present in Treatment(n) x (range) x (range) x ± SEM bovine adrenal glands. The results of the experiments indicated that the Control (13) 50 (40-60) 0 (0-20) 27 ± 4 adrenal medulla contained factors that sustained nor- (-)-Epinephrine mal motility and also stimulated both activation and acrosome 50.0MM (12) 60 (40-70) 70 (50-90) 67 4 reactions. The adrenal cortex preparation did not stimulate 5.0 AM (5) 50 (50) 50 (40-60) 48 + 7 acrosome reactions or activation as well as the medulla prepa- 0.5 MM (4) 50 (50) 50 (40-60) 41 4 ration, but did support motility and seemed to facilitate the (-)-Phenylephrine activity of the medulla preparation. The cortex preparation was 50.0 AM (4) 50 (50) 40 (20-40) 68 + 4 treated with Florisil or extracted with chloroform to remove 5.0AM (4) 50 (50) 20 (10-40) 46 ± 6 several steroids (cortisol, progesterone, testosterone, and es- 0.5 AM (4) 50 (50) 20 (10-30) 26 + 7 trogen) that in high concentrations (10-100 ,uM) inhibit in wtro (-)-Isoproterenol acrosome reactions of hamster spermatozoa (unpublished data). 50.0 AM (4) 60 (60) 80 (80-90) 53 + 4 The adrenal gland motility factor is probably more than one 5.0MM (4) 50 (50-60) 50 (30-70) 25 2 molecule, because spermatozoa did not survive in the presence 0.5MuM (4) 50 (50-60) 30 (30-40) 23 ± 3 of bovine serum albumin and catecholamine alone but required (-)-Phenylephrine + the presence of the cortex preparation. (-)-Isoproterenol Although activation has only been observed in hamster and 25.0 + 25.0AM (5) 60 (60-70) 80 (70-80) 53 ± 8 guinea pig spermatozoa, the occurrence of acrosome reactions 2.5 + 2.5 AM (5) 60 (50-70) 50 (40-70) 47 + 7 in motile hamster and guinea pig spermatozoa together with 0.25 + 0.25 MM (5) 60 (50-70) 40 (40-50) 44 ± 3 a high percentage of activation is generally considered to be an * Incubations carried out in the presence ofthe AC cortex preparation indication that capacitation and subsequent physiological for 4 hr. acrosome reactions have occurred (17, 19, 20). In the present t Approximate percentage of motile sperm at 37°. results, the acrosome reactions stimulated by catecholamines Approximate percentage of motile sperm at 370 with whiplash fla- in the presence of bovine serum albumin and the cortex prep- gellar movement characteristic of capacitated sperm. aration do appear to be physiological. Unpublished results (D. § Percentage of motile sperm (at least 100 observed) that had un- Friend and S. dergone an acrosome reaction, observed by phase contrast micros- Meizel) indicate that the ultrastructural mor- copy. phology of the hamster sperm acrosome reaction induced in the presence of bovine serum albumin and a protein-free prepa- ration of the bovine adrenal gland is typical of the physiological It should be noted that, in the presence of 25.0 ,uM pro- acrosome reaction, by previously established criteria (1). In pranolol, abnormalities of the sperm head occurred in ap- addition, spermatozoa incubated in the presence of bovine proximately 30% of the motile spermatozoa. In these sperma- serum albumin, the AC cortex preparation, and 20.0 1AM epi- tozoa the tip of the acrosomal cap assumed a swollen blunt nephrine fertilize a high percentage of hamster eggs, while shape, making it impossible to determine if an acrosome reac- spermatozoa incubated in the presence of albumin and tion had occurred. These spermatozoa were not included in the the AC acrosome reaction counts. As yet we do not know whether the cortex preparation only did not fertilize eggs (L. E. Cornett, B. abnormalities of the sperm head represent a stage of inhibited Bavister, and S. Meizel, unpublished data). membrane fusion or nonspecific damage of the membranes of Because phentolamine and propranolol in the presence of some spermatozoa. added epinephrine reduced the percentage of acrosome reac- tions and activation below that obtained in the presence of only bovine serum albumin and the cortex preparations, it is likely Table 2. Effect of adrenergic antagonists on the hamster sperm that low levels of catecholamines remaining in the AC cortex acrosome reaction in the presence of (-)-epinephrine* preparation or bound to albumin were responsible for the in- % AR,§ duction of acrosome reactions and low activation in the absence % motile,t %.activated, i of added catecholamines. The ultrastructural morphology of Treatment (n) x (range) x (range) SEM these acrosome reactions observed in the absence of activation will have to be studied before we can determine whether or not Control 10.0MM 50(50-70) 70 (50-80) 67 ± 3 (-)-epinephrine (11) such acrosome reactions are physiological. Phentolamine + 10.0 iM Ahlquist (21) divided adrenergic responses into two types on epinephrine the basis of relative potencies of six catecholamines on different 25.0,MM (4) 50 (30-50) 10 (0-20) 11 ± 4 physiological preparations. Moran and Perkins (22) were able 10.0MAM (4) 40(30-50) 40 (10-60) 34 ± 4 to define two types of adrenergic antagonists that supported 1.0MtM (4) 50(50) 60(50-80) 63 ±8 Ahlquist's findings. The nature of the behavior of the agonists (-)-Propranolol + 10.0 gM and antagonists led to a definition of an a-adrenergic receptor epinephrine and aj3-adrenergic receptor to explain the many different re- 25.0M4M (4) 40 (30-50) 0 (0) 20 ± 3 sponses to catecholamines. 10.0MM (4) 50 (50-60) 40 (30-50) 64 ± 4 Our results with adrenergic agonists and antagonists suggest 1.0MAM (4) 50 (50-60) 50 (30-80) 69 ± 8 the involvement of both a- and f3-adrenergic receptors in the * Incubations carried out in the presence of the AC cortex preparation stimulation of activation and the acrosome reaction. Both ac- for 4.5 hr. tivation and acrosome reactions were inhibited by either an- t Approximate percentage of motile sperm at 37°. tagonist. The a-adrenergic agonist was as potent as Approximate percentage of motile sperm at 370 with whiplash fla- epinephrine gellar movement characteristic of capacitated sperm. in the stimulation of acrosome reactions, but activation was § Percentage of motile sperm (at least 100 observed) that had un- reduced. The,B-adrenergic agonist was as potent as epinephrine dergone an acrosome reaction, observed by phase contrast micros- in the stimulation of activation but had a reduced ability to copy. stimulate acrosome reactions. A high percentage of activation Downloaded by guest on September 27, 2021 4958 Cell Biology: Cornett and Meizel Proc. Nati. Acad. Sci. USA 75 (1978) and acrosome reactions was seen only when epinephrine or Davis, in partial fulfillment of the requirements for a Ph.D. degree in norepinephrine was used or when the a- and f3-adrenergic Physiology and was supported by National Institutes of Health Grant agonists were used together. Epinephrine is known to have both HD 06698. a- and f3-adrenergic effects, and norepinephrine acts pre- dominantly on a-adrenergic receptors but has some effect on 1. Bedford, J. M. (1970) Biol. Reprod. Suppl. 2, 128-158. fl-adrenergic receptors (18). 2. Yanagimachi, R. (1977) in Immunobiology of Gametes, eds. Obviously an increased number of capacitated spermatozoa Edidin, M. & Johnson, M. H. (Cambridge Univ. Press, New York), pp. 225-291. could lead to an increased number of acrosome-reacted sper- 3. Meizel, S. (1978) in Development in Mammals, ed. Johnson, M. matozoa (provided an acrosome reaction initiator was also (Elsevier, Amsterdam, Netherlands), Vol. 3, pp. 1-64. present). Further experiments will be necessary in order to 4. Yanagimachi, R. (1969) J. Exp. Zool. 170,269-280. determine in a definitive manner whether catecholamines 5. Yanagimachi, R. (1969) J. Reprod. Fertil. 18,275-280. stimulate the acrosome reaction primarily through some direct 6. Yanagimachi, R. (1970) Biol. Reprod. 3, 147-153. effect, indirectly through stimulation of capacitation, or 7. Bavister, B. D. & Morton, D. B. (1974) J. Reprod. Fertil. 40, through both mechanisms. 495-498. The expression of fl-adrenergic receptors is usually mediated 8. Lui, C. W. & Meizel, S. (1977) Differentiation 9,59-66. through increased cyclic AMP levels (23). There is evidence that 9. Lui, C. W., Cornett, L. E. & Meizel, S. (1977) Biol. Reprod. 17, cyclic AMP may be involved in the capacitation process (24). 34-41. 10. Bavister, B. D., Yanagimachi, R. & Teichman, R. J. (1976) Biol. The expression of a-adrenergic receptors is not as well under- Reprod. 14, 219-221. stood, but there is evidence that a-adrenergic activation leads 11. Bavister, B. D. & Yanagimachi, R. (1977) Biol. Reprod. 16, to a calcium influx in hepatocytes (25) and in parotid gland (26). 228-237. Calcium has been shown to be essential for activation and the 12. Bavister, B. D. (1974) J. Reprod. Fertil. 38,431-440. acrosome reaction of mammalian spermatozoa (20, 27). The 13. Cornett, L. & Meizel, S. (1977) J. Cell Biol. 75, A164 (abst.). manner in which putative increased levels of sperm cyclic AMP 14. Srott, C. A. (1975) Methods Enzymol. 36,34-48. or calcium might then stimulate activation or acrosome reac- 15. Anton, A. H. & Sayre, D. F. (1962) J. Pharmacol. Exp. Ther. 138, tions is not yet known. Several such mechanisms for the acro- 360-375. some reaction have been suggested, involving cyclic AMP and 16. Meizel, S. & Lui, C. W. (1976) J. Exp. Zool. 195, 137-144. 17. Yanagimachi, R. (1970) J. Reprod. Fertil. 23, 193-196. calcium stimulation of sperm hydrolytic enzymes (3). 18. Innes, I. R. & Nickerson, M. (1975) in The Pharmacological Basis It having been established that epinephrine and norepi- of Therapeutics, eds. Goodman, L. S. & Gilman, A. (Macmillan, nephrine interact with hamster spermatozoa in vitro to stim- New York), pp. 477-513. ulate activation and the acrosome reaction, it will be important 19. Austin, C. R. (1975) J. Reprod. Fertil. 44, 155-166. to determine whether or not catecholamines stimulate capac- 20. Yanagimachi, R. & Usui, N. (1974) Exp. Cell Res. 89, 161- itation and/or the acrosome reaction of spermatozoa of other 174. species and in vivo. Although the catecholamine content of 21. Ahlquist, R. P. (1948) Am. J. Physiol. 153,586-600. whole oviduct and uterus (the sites of in vivo capacitation) have 22. Moran, N. C. & Perkins, M. E. (1958) J. Pharmacol. Exp. Ther. been determined in several mammalian species (28, 29), no such 124,223-237. assay has been made of a female reproductive tract fluid. In 23. Wolf, B. B., Harden, T. K. & Molinoff, P. B. (1977) Annu. Rev. Pharmacol. Toxicol. 17,575-604. addition, catecholamines have been demonstrated to be present 24. Hoskins, D. D. & Casillas, E. R. (1975) in Molecular Mechanisms in unfertilized rat ova (30). It will be of interest to determine of Gonadal Hormone Action, eds. Senhal, R. & Thomas, J. A. if the motility factor derived from mammalian spermatozoa (University Park, Baltimore, MD), pp. 293-324. (11) contains catecholamines. 25. Assimacopoulos-Jeannet, F. D., Blackmore, P. F. & Exton, J. H. In conclusion, these results demonstrate a positive effect of (1977) J. Biol. Chem. 252,2662-2669. a hormone on activation (an indication of hamster sperm 26. Peterson, 0. H., Veda, N., Hall, R. A. & Gray, T. A. (1977) Eur. capacitation) and the acrosome reaction of mammalian sper- J. Physiol. 372,231-237. matozoa. Further experiments may add to our understanding 27. Talbot, P., Summers, R. G., Hylander, B. L., Keough, E. M. & of the molecular events essential for mammalian fertilization Franklin, L. E. (1976) J. Exp. Zool. 198,685-688. extension of these results to other lead 28. Dujovne, A. R., deLaborde, N. P., Carrel, L. M., Cheviakoff, S., and future species may Pedroza, E. & Rosner, J. M. (1976) Am. J. Obstet. Gynecol. 124, to new approaches to contraception. 229-233. 29. Bodkhe, R. R. & Harper, M. J. K. (1972) Biol. Reprod. 6,288- We thank Ms. Sarah Goodlin for her assistance in preliminary ex- 299. periments. This work is based on part of the dissertation to be submitted 30. Burden, H. W. & Lawrence, I. E., Jr. (1973) Am. J. Anat. 136, by L.E.C. to the Graduate Division of the University of California, 251-257. Downloaded by guest on September 27, 2021