MOLECULAR REPRODUCTION AND DEVELOPMENT 26143-149 (1990)

Effects of Cold Shock and Phospholipase A2 on Intact Boar Spermatozoa and Sperm Head Plasma Membranes L. ROBERTSON,' J.L. BAILEY: AND M.M. BUHR2 'Department of Physiology, Royal Veterinary College, London, England; 'Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada

ABSTRACT Head plasma membranes (HPM) isolated from cryopreserved boar sperma-

permeability. Secondary to this change, cellular components such as phospholipids, proteins, and ions tozoa show an excessive fluidization (Buhr et al., are released, and Ca2 and Na' gain intracellular Gamete Res 23:441-449, 1989), which might be access. Spermatozoa are rendered immotile and cell involved in the loss of fertility. The current study metabolism is disrupted (reviewed Watson, 1981). assessed the ability of cold shock (5°C) and phosAlthough the phenomenon of cold shock has been pholipase A, (PA,) to duplicate these effects on extensively investigated, the nature of the changes membrane structure and to affect 45Ca2+uptake that underlie the loss of membrane selective permeand gross morphological characteristics of whole, ability is unclear. Commercial cryopreservation of fresh boar sperm. The HPM from cold-shocked boar sperm has been shown to cause a fluidization of sperm showed a significantly greater rate of fluidthe head plasma membranes (HPM) relative to HPM ization over time than did HPM from control sperm. from fresh sperm (Buhr et al., 1989). The change in Addition of PA, (bee or snake venom, 0.1 or 10.0 fluidity reflects a change in membrane molecular ng/ml) to HPM from control sperm caused fluidizaorganization (Sklar et al., 19791, which could impact on the functional ability of such membrane-bound tion similar to cold shocking, but to a lesser degree enzymes as Ca2+-adenosine triphosphate (ATPase) ( P < 0.05). Cold-shocked intact sperm exhibited (Breitbart et al., 1983). Disruption of membrane severe acrosomal disruption, loss of motility, and phospholipids could cause this fluidity shift, and increased 45Ca2+uptake relative to control sperm. Watson and Morris (1987) theorized that an initial Addition of PA, (bee or snake venom, 0.1, 1.0., action of cold shock was the induction of uncontrolled 10.0, and 1,000 ng/ml) to control sperm had no lateral phase changes within the membrane. effect on gross morphology or motility while maintaining or increasing sperm extrusion of 45c~2+. To test this theory and simultaneously investigate a possible causative mechanism, the effects of phosphoTherefore, although PA, can, to some extent, dulipase A2 (PA,) were compared to the effects of cold plicate the effects of cold shock on HPM molecular shock on boar sperm and isolated membranes. PA, is organization, its lipid hydrolytic action is insufficient an acyl hydrolase that specifically cleaves the acyl esto cause all the gross disruptions of severe thermal ter at position 2 of the glycerol moiety of phospholipids. shock. Both PA, and cold shock disrupted HPM Phospholipid hydrolyis releases lysophospholipids and structure, but only cold shock increased 45Ca2+ free fatty acids, which disrupt membrane structure uptake, suggesting that cold shock may be in(Poole et al., 1970; Lucy, 1978). Lysophosphatidylchocreasing 45Ca2+ uptake in areas other than the line is known to promote plasma membrane and achead. Cold shock disrupts sperm on three levels; rosomal membrane disruption in mammalian spermamembrane molecular organization, intracellular tozoa (Jones, 1976; Fleming and Yanagimachi, 1981). Ca2+ regulation, and gross morphology/motility. PA2 from two sources was utilized in this study, PA, Key Words: Fertility, Ca2+ uptake, Head plasma +

membrane

INTRODUCTION Boar spermatozoa are particularly sensitive to rapid cooling (cold shock) (Lasley and Bogart, 1944; Polge, 1956; Hood et al., 1970; Purse1 et al., 1972; Robertson et al., 1988). It is generally agreed that the initial injury of cold shock is loss of membrane selective

0 1990 WILEY-LISS, INC.

Received September 15, 1989; accepted January 2,1990. L. Robertson's present address is Dept. SurgeryIReproduction, Glasgow University Veterinary School, Glasgow, Scotland G61/1QH. J.L. Bailey's and M.M. Buhr's present address is Dept. of Animal and Poultry Science, Ontario Agricultural College, University of Guelph, Guelph, Ontario, Canada N1G 2W1.Address reprint requests to M.M. Buhr there.

144

L. ROBERTSON ET AL.

derived from bee venom (Apis mellifer) and PA2 derived from snake venom (Crotalus adamanteus), since they have been shown to have differing substrate preferences (Reed, 1981). The impacts of cold shock and PA, on several levels of sperm organization were assessed. Whole spermatozoa were examined for motility, membrane integrity as judged by vital staining, and acrosomal damage. As a measure of membrane function, the ability of intact cells to regulate intracellular calcium was measured using a 45Ca2+ radioassay (Robertson and Watson, 1986). Molecular organization of the HPM was measured by fluorescence polarization (Canvin and Buhr, 1989). Comparison of PA, effects with those of cold shock could elucidate the mechanism of chillinginduced damage to sperm.

MATERIALS AND METHODS Semen Collection Semen was collected from Landrace-cross boars by the gloved hand method into a vacuum flask, maintaining the temperature at 30°C. The gel-free, spermrich fraction of the ejaculates was used for the experiments. Only semen of good initial motility was used. Sperm concentrations were estimated using a previously calibrated colorimeter. Light Microscopy Semen from five boars was diluted tenfold into HBGS (20 mM HEPES-NaOH, 40 mM glucose, 125 mM NaCl, pH 7.0, 310 mOsm/kg) containing 0, 0.1, 1.0, 10.0, or 1,000 ng/ml PA, from bee or snake venom (Sigma Chemical Co., St. Louis, MO) or was subjected to cold shock by rapid dilution a t 5°C; motility, percentage motile, percentage unstained cells, and degree of acrosomal damage were monitored over a 4 hr period following dilution. Spermatozoa were scored for motility by visual examination of duplicate samples in random order using a heated microscope stage to maintain samples a t 37°C. The vigor of motility was scored on a scale of 0-4 (Emmens, 1947). The percentage motile sperm was estimated (Martin, 1963) to the nearest 5%. Eosin-nigrosin differential staining was used to estimate the percentage of unstained (live) sperm (Mortimer, 1985). Acrosomal damage was scored on a scale of 1-4 using Giemsa-stained smears (Watson, 1975). &Ca2' Radioassay Semen from each of five boars was extended tenfold in HBGS containing 45Ca2+as CaCI, with a specific activity of 0.1 pCi/ml at a final calcium concentration of 300 pM. Semen was incubated in the presence of 0, 0.1, 1.0, 10.0, or 1,000 ng/ml PA, from bee or snake venom a t 30°C or with no PA, at 5°C (cold shock). Aliquots were removed immediately upon dilution (time 01, and a t 30 min intervals for 240 min after dilution. Calcium movement was terminated and the spermatozoa were prepared for scintillation counting as previ-

ously described (Robertson and Watson, 1986). Data were converted to nmol Ca2+/109spermatozoa.

Membrane Isolation Whole sperm were subjected to cold shock by rapid cooling to 5"C, held for 30 min, then rewarmed to 30°C prior to processing. Parallel samples were held a t 30°C for this 30 min period. During subsequent processing the temperature of all treatments was maintained at 30°C. Membrane isolation was carried out by the method of Canvin and Buhr (1989). Briefly, semen was filtered twice through miracloth (Calbiochem, La Jolla, CAI, diluted 1:l with Tris buffer (5 mM Tris HCl, 0.25 M sucrose, pH 7.4; Gillis et al., 1978) and centrifuged (1,50Og, 10 min) through an oil mixture (D.C. 550 and D.C. 1150 mixed 1:1, (v/v); Dow Corning Oil Co.) t o remove gel particles. The sperm pellet was washed twice in Tris buffer (pH 7.4) by centrifugation (2,50Og, 10 min), resuspended in buffer, subjected to nitrogen cavitation in a Parr bomb at 116 kg/cm2N, for 10 min, and extruded into Tris buffer (pH 5.0). The membrane fraction was collected by harvesting the supernate from three centrifugation washes (l,OOOg, 10 min) in Tris buffer (pH 5.0). The combined supernates were centrifuged (6,00Og, 10 min) to remove remaining intact cells. The membrane suspension was concentrated by centrifugation (365,00Og, 70 min), then washed (365,00Og, 40 min) in Tris NaCl buffer (10 mM Tris, 0.9% NaC1, pH 7.4). The pellet was resuspended in Tris NaCl and used immediately for fluorescence determination. Final protein concentrations were determined (Bradford, 1976) using bovine gamma-globulin as the standard. Plasma membrane enrichment was assessed using an alkaline phosphatase assay (Linhardt and Walter, 1963). Fluorescence Polarization Measurements Fluorescence measurements were made on a model LS5 Perkin Elmer spectrofluorometer with a polarizing accessory using excitation and emission slit widths of 10 and 5 nm, respectively, utilizing the fluorescent probe trans-parinaric acid (tPNA) (Molecular Probes, Inc., Eugene, OR). Probe solutions were freshly prepared and stirred in the dark prior to use. Membrane fractions were mixed with Tris NaCl (pH 7.4) to give 50 pg proteidml, 10 mM CaCl,, and 1 pM tPNA in 3 ml volume in the cuvette. PA, solutions were made immediately prior to use and added to the cuvette in 20 ~1 volume to achieve final PA, concentrations of either 0.1 or 10 ng/ml. Temperature was maintained at 30°C using a circulating water bath and monitored with a thermocouple placed directly in the sample solution. Fluorescence was assessed continuously for 120 min a t 30°C using excitation and emission wavelength maxima of 324 and 420 nm, respectively. Fluorescence intensities were transformed into polarization values using the Perrin equation (Shinitzky and Barenholz, 1978).

COLD SHOCK AND PHOSPHOLIPASE AFFECT BOAR SPERM

145

TABLE 1. Sperm Characteristics in Response to Cold Shock or PA,?

Incubation (min) 0 60 120 180 240

Motility" Cond P A P CSd MSd 3.5 3.6 3.3 3.4 3.1

3.5 3.5 3.4 3.2 3.0

O* O* 0" O*

O*

0.22 0.23 0.18 0.20 0.19

Con 69 72

70 67 60

Percent motile PA, CS MS 69 70 70 66 60

O* O* O* O* O*

89 92 47 33 72

Con 69 66 74 67 66

Unstainedb PA, CS MS

Con

Acrosomec PA, CS

1.2 1.1 1.2 1.3 1.3

1.1 1.1 1.3 1.2 1.2

MS

~~

72 69 70 74 71

16* 2*

O* 5* 2*

193 119 112 141 114

2.1* 1.9" 2.6* 2.4* 2.4"

0.04 0.03 0.10 0.04 0.05

I.N = 5 for all treatments. *Different from control (P < 0.05). "Scored 0-4. 'Vital staining. 'Acrosomal damage scored 1-4. dCon, control; PA,, phospholipase A,; CS, cold shock; MS, error mean square. "Pooled responses to 0.0001,0.001, 0.01,or 1.0 pg PA,/ml from snake and bee venom.

Statistical Analysis 45Ca2 + radioassay and morphology data were subjected to analysis of variance; percentage data were transformed to angles before analysis. Initial polarization values were analyzed for differences using least square means and their predicted differences. Polarization values over time were adjusted within each trial by subtracting the value at time zero from all subsequent values to reduce variation among trials due to differences in initial values. Data were analyzed by stepwise regression on a polynomial equation, with time and powers of time as the dependent variables. The slopes of linear data were compared using a t test for parallelism.

motility and percent motile scores than did 10.0 ng/ml snake venom PA,. 4sCaa+Radioassay

Intracellular Ca2+ increased slightly but significantly (P < 0.01) in control sperm over time (Fig. 1). Calcium accumulation in response to cold shock (Fig. 1) was approximately 50-fold greater than that of control (P < 0.001). Incubation in the presence of 1,000 ng/ml PA, from bee venom (Fig. 2b) resulted in no increase in intracellular 45Ca2+above that of the starting point (time 0). This calcium uptake was significantly lower than that of the control from 120 min onwards (P < 0.05).In the presence of 10 ng/ml PA, from bee venom, calcium RESULTS uptake was significantly lower than control from 180 Light Microscopy Cold shock resulted in a severe drop in motility, per- min onwards ( P < 0.05). No consistent effect of PA, centage motility, and percentage unstained as well as from snake venom was detected at any concentration. an increase in acrosomal damage (P < 0.001; Table 1). A preliminary experiment (not shown) established that Data for all PA, treatments are pooled for clarity of samples treated with 10 or 100 pg/ml PA, from bee or presentation, since few statistically significant differ- snake venom had no further significant effect. ences were detected by pairwise comparisons. No PA, treatment differed from control in any morphological Fluorescence Polarization characteristic within any one time period, with the sinAlkaline phosphatase activity of the head plasma gle exception of 1,000 ng/ml bee venom exceeding (P < 0.05) the percent unstained of control a t 180 min only. membrane preparation was 257% k 16% (mean k Bee venom PA, caused some significant morphological s.e.m.) for control preparations and 269% k 51% for differences compared with that of snake venom PA,. preparations subjected to cold shock compared with Treatment with 1,000 ng/ml bee venom PA, increased 100% for intact spermatozoa. Fluorescence measurements were made for 120 rnin the percent unstained over some doses of the snake venom PA, a t 60,180, and 240 rnin and also increased at 30°C; extending the measurement period to 240 min the giemsa score a t 240 min over that of 0.1 ng/ml bee had no effect on results. Plasma membrane preparavenom and the three highest doses of snake venom. tions that received no treatment other than incubation Motility characteristics were rather more sensitive to at 30°C (Fig. 3, control) showed a significant decrease PA2 than was morphology. Over time, motility de- in fluidity over the 120 min period (P < 0.05). Memcreased (P < 0.05) for five of the eight PA, treatments brane preparations from spermatozoa that were subbut not for the control. The percent motile decreased jected to cold shock (Fig. 3, cold shock) prior to processover time (P < 0.05) for control, 1,000ng/ml bee venom ing showed a significant increase in fluidity over time PA, and 1,000 and 10.0 ng/ml snake venom PA,. The (P < 0.05). Membrane preparations incubated in the 1,000 nglml dose of bee venom PA, also gave higher presence of PA, (bee or snake venom, 0.1 or 10 nglml;

146

L. ROBERTSON ET AL.

b

a

60

120

180

240

Time(mi nu tes)

Fig. 1. Accumulation of Ca2+ (nmol Ca"' /lo9 sperm; t 5.e.) over 240 min (N = 5). Spermatozoa at 30°C (a) accumulated less Ca2+ ( P < 0.001) than spermatozoa cold shocked to 5°C (b).

Fig. 4) maintained starting fluidity, with no change in fluidity over the 2 hr period (Table 2, slopes). The unadjusted initial polarization values, indicating the fluidity of control membranes at the start of the fluorescence incubation, did not differ significantly among treatments (IPV, Table 2). There was a slight tendency for membranes from cold-shocked sperm to be more fluid initially than membranes from control sperm.

DISCUSSION Cold shock caused substantial disruption t o all three levels of sperm organization examined. Cold-shocked spermatozoa displayed gross disruption of morphology and motility at a light microscope level. Substantial Ca2' accumulation was detected by radioassay, reflecting the inability of the sperm membrane to regulate intracellular Ca2+ (Robertson and Watson, 1986). Using fluorescence polarization, it was found that head plasma membranes isolated from normal spermatozoa showed a consistent decrease in fluidity with time, whereas membranes from cold-shocked spermatozoa underwent a significant increase in fluidity (slopes, Table 2). Since tPNA preferentially inserts into the most ordered areas of the lipid bilayer (Sklar et al., 1979), the decrease in fluidity shown by normal membranes suggests that HPM from normal spermatozoa either increase their amount of gel phase lipid and/or

increase their overall membrane order. Thus the HPM from normal spermatozoa naturally undergo molecular reorganization, which is detectable as a fluidity change. Slow cooling and rewarming of isolated membranes have been shown to disrupt the steady decline in fluidity over time (Canvin and Buhr, 1989). Membranes isolated from commercially cryopreserved boar sperm displayed a significantly retarded ability to decrease fluidity over time (Buhr et al., 19891, and the current results demonstrate that cold shocking intact spermatozoa causes the head plasma membranes actually to increase in fluidity. Since spermatozoa subjected to cold shock were rewarmed to 30°C prior to processing, it is apparent that the membrane changes induced by cold shock were not reversible upon rewarming. Recent evidence suggests that boar spermatozoa are susceptible to damage due to rapid warming as well as rapid cooling (Bamba and Cran, 1985, 1988). "Warm shock" was manifest as acrosomal damage detectable by light and elecron microscopy, but with little effect on motility, glutamicoxaloacetic transaminase release, or respiration. In this study, spermatozoa that were rapidly cooled to 5°C and rapidly rewarmed to 30°C showed the loss of plasma membrane integrity, acrosomal disruption, and loss of motility classically defined as cold shock. Therefore, cold shock has been demonstrated to cause major

COLD SHOCK AND PHOSPHOLIPASE AFFECT BOAR SPERM

147

-t

Time(minutes)

Fig. 2. Control spermatozoa (a)accumulated more Ca2+than spermatozoaincubated with 1,000 ng/ml (b)or 10.0 ng/ml (e) PA, from bee venom from 120 or 180 min onward, respectively. Neither bee venom PA, at 0.1 or 1.0 ng/ml nor any dose of PA, from snake venom affected Ca” accumulation (results not shown).

and membrane organization. Acrosomal integrity, sperm vitality, motility, and percent motility showed 3 0.09{ ------ Cold Shock little or no difference with exposure to either source of PA,. Thus, PA, had minimal effect on microscopically visible sperm characteristics, which directly contrasted with the dramatic effects of cold shock. PA, did affect Ca2+ uptake, although again in a manner dissimilar to that of cold shock. Where cold shock caused a massive Ca2 influx, and even control sperm experienced some J I increase in intracellullar Ca2+,the highest doses of bee $ -0.061 4 venom PA, maintained the internal Ca2+ levels at -0.09! I 1 I I I I ( I I I I I starting values. This implies that there was increased 0 10 20 30 40 50 60 70 80 90 100110120 extrusion of Ca2+ by the cell exposed to bee venom Time (Minutes) PA,, which may be due to stimulation of a Ca2+transport pump as a consequence of increased fluidity of the Fig. 3. Changes in fluidity at 30°C of membranes isolated from membrane. Stimulation of Ca2+-ATPasein ram sperm spermatozoa incubated for 30 min prior to processing at 30°C (control) or 5°C (cold shock). Increasing polarization value indicates decreasing membrane preparations by bee venom PA, has been fluidity. For each line, N = 5. Slopes differ significantly ( P < 0.05). reported previously (Holt and North, 19861, and there is much evidence to suggest that integral membrane enzymes of this type are kinetically modified by damage to motility, morphology, Ca2+ regulation, and changes in their annular lipid environment (Kimelmembrane molecular organization. berg, 1977). The phenomenon of increased Ca2+extruPA, caused much less dramatic alterations in sperm sion was not observed during incubation in the presmorphology than did cold shock, and exhibited some ence of snake venom PA2. This may reflect the relative unexpected and interesting effects on sperm function preference of the two types of PA, for different phos0.121

-Control

+

L.ROBERTSON ET AL.

148

TABLE 2. Effect of Cold Shock or PA, on Sperm Membrane Fluidity Treatment Control 10.0ng SVb/ml 0.1 ng SV/ml 10.0 ng BVc/ml 0.1 ng BV/ml Cold shock

Slope k S.E. (PV/min x 10'

37.2

k

5.0d

- 7.6 f 5.0"" - 15.0 2 5.3e*f

- 2.5 f 5.1f

- 3.9 2 7.6e*f - 17.2 f 6.7"

IPV" (X f s.e.) 0.3425 f 0.0170d 0.3307 k 0.023gd 0.3484 2 0.023gd 0.3412 f 0.0208d 0.3067 f 0.0157d 0.3013 f 0.0231d

"Initial polarization value.

bPA, from snake venom. "PA, from bee venom. d-fValues within a column with different superscripts differ (P < 0.05).

0.121

Bee Venom PA2

P,

2 -0.03

:

,4j

-0.09

0

10 20 30 40 50 60 70 80 90 100110120 Time (Minutes)

Fig. 4. Changes in fluidity a t 30°C of membranes isolated from spermatozoa a t 30°C. PA, from bee venom or snake venom was added at time 0. For each line, N = 5.

pholipid substrates (Reed, 1981) and may also imply that the Ca2+ pump is located in an environment containing the preferred lipid substrate of bee venom PA,. PA, caused a significant increase in the rate of fluidization (slopes, Table 2 ) of the head plasma membrane in comparison with control membranes. This fluidization was of a nature similar to that caused by cold shock, but less extensive. PA, from bee venom prevented the decrease in fluidity exhibited by mem-

branes from control sperm, whereas snake venom PA2 caused a slight time-dependent increase in fluidity. PA, causes phospholipid hydrolysis; a product of this hydrolysis is lysophosphatidylcholine (lysolecithin), known to cause membrane disruption (Poole et al., 1970; Lucy, 1978). Upon incorporation into a lipid bilayer, lysophosphatidylcholine is thought to produce, as a consequence of its wedge shape, a transition from bimolecular leaflet to radially oriented molecules, inducing micellar formation. Micelle formation has been suggested to be part of the sequence of events associated with increased membrane fluidity and membrane fusion (Lucy, 1978; Howell et al., 1973). This behavior could explain the increased membrane fluidity observed on incubation of head membranes or whole spermatozoa with PA,. Apparently, the membrane disruption caused by PA2 was similar to that caused by cold shock a t a molecular level but not at a microscopic level. This study has shown that cold shock disrupts boar spermatozoa on three levels of organization: gross morphology, membrane function assessed as the ability to regulate intracellular calcium, and membrane molecular organization measured as rate of fluidity change. PA, added to intact spermatozoa could not duplicate cold shock's effect on morphology and function, but when added directly to head membranes produced a cold shock-like disruption of molecular organization. The disruption of the normal dynamics of membrane molecular interactions demonstrated here provides concrete evidence to support the theory that the initial action of cold shock is the induction of abnormal lateral phase changes within the membrane.

ACKNOWLEDGMENTS Thanks to Dr. P.F. Watson for his critical review of the manuscript. L.R. was supported by an award from the Prof. J.G. Wright Memorial Fund, awarded by the Royal College of Veterinary Surgeons Trust Fund. J.L.B. was a recipient of a University of Manitoba Graduate Scholarship. This research was generously supported by the Manitoba Hog Producers' Marketing Board of Canada. REFERENCES Bamba K, Cran DG (1985):Effect of rapid warming of boar semen on sperm morphology and physiology. J Reprod Fertil75:133-138. Bamba K, Cran DG (1988): Further studies on rapid dilution and warming of boar semen. J Reprod Fertil82:509-518. Bradford MM (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72248-254. Breitbart H, Stern B, Rubinstein S (1983): Calcium transport and Ca2+-ATPase activity in ram spermatozoa1plasma membrane vesicles. Biochim Biophys Acta 728:349-355. Buhr MM, Canvin AT, Bailey J L (1989): Effects of semen preservation on boar spermatozoa plasma membranes. Gamete Res 23:441449. Canvin AT, Buhr MM (1989):Effect of temperature on the fluidity of boar sperm membranes. J Reprod Fertil-85533-540.

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