CRYOBIOLOGY

29, 106-117 (1992)

Differential Effects of Butylated Hydroxytoluene Analogs on Bull Sperm Subjected to Cold-Induced Membrane Stress’ Biochemistry

JAMES

K. GRAHAM

Program,

The Pennsylvania

AND ROY H. HAMMERSTEDT2 State

University,

University

Park,

Pennsylvania

16802

Previous reports established that butylated hydroxy toluene (BHT) minimized cold-induced membrane rupture in sperm from several species. No data regarding the specificity of its effect is available. In this study 25 BHT analogs were tested for their effect on bovine sperm membrane stability. Fourteen were membrane lytic at 2s”C and 6 were neither membrane lytic nor membrane stabilizing. The remaining 5 compounds, a family of 2,6-tert-butyl phenols with substitutions at position 4 of hydrogen, methyl (BHT), ethyl, butyl, hexyl, or octyl, afforded effective membrane protection to cold shock. Since membrane protection is a function of both the ability of a compound to partition into the membrane and a molecule’s effectiveness once there, an analysis of each analog’s membrane partitioning, assessed by measuring the cellular analog/cholesterol ratio, showed the following extents of transfer for the analogs: ethyl = butyl > methyl = hydrogen > hexyl > octyl. Thus, an optimum chain length exists for partitioning from micellar donors into cells. A separate experiment established that all analogs, when incorporated in equivalent amounts, protect equally plasma and mitochondrial membranes from cold shock. No effect on acrosomal membrane stability was noted. BHT, but not the other analogs, reduced sperm motility. Addition of egg yolk to extender containing BHT analog protected sperm motility from cold shock but had little effect on membrane stabilization. Analysis of sperm membrane compartments revealed that little to no analog was partitioned into the outer acrosomal membrane or the plasma membrane overlying the acrosome, but rather was localized in other portions of the sperm. We conclude that (a) the effective BHT analogs, if partitioned into the membrane, are indistinguishable with regard to their capacity to eliminate cold-induced membrane lysis; (b) membrane-linked events (e.g., motility) are uniquely disrupted by a subset of this analog family; and (c) when concentrations of egg yolk and BHT analogs are carefully controlled, unique synergistic effects are noted. o 1992Academic P~.XS, IX

Cooling sperm from body temperature to 0-W can result in irreversible cellular damage, often referred to as cold shock (30). Reports of release of phospholipids into the surrounding medium (7), altered cell metabolism (4), and cation distribution (25) are consistent with the concept that membrane damage is induced by cold shock (30). The discovery that egg yolk (EY) (22) and in particular the egg yolk phospholipids (16, 24, 29) minimized sperm damage dur-

ing cooling led to the widespread use of egg yolk-based semen diluents. Recently, purified phospholipid liposomes have been shown to be effective in protecting bull (11, 21) and boar (5) sperm from cold shock damage. The mechanism by which liposomes prevent sperm damage has yet to be established. Hammerstedt et al. (13, 14) reported that butylated hydroxytoluene (BHT) protected bull and ram sperm from membrane damage during cold shock. Others reported that the addition of BHT to sperm suspensions increased the percentage of motile cells and sperm with intact acrosomes after cooling, compared with untreated sperm, for the boar (23) and the ram (3 1). Combinations of 20% EY and 2 mM BHT did not appear to be any more effective than the individual

Received November 16, 1990; accepted March 20, 1991. ’ Supported by USDA 85-CRCR-1-1878. * To whom correspondence should be addressed at 406 Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802. 106

OOll-2240/92 $3.00 Copyright Q 1952 by Academic Press. Inc. All rights of reproduction in any form reserved.

EFFECTS

components (23). However, the mechanism by which BHT benefits sperm during cooling remains unknown. In an effort to understand the effects of BHT at the cellular level, experiments were designed to (a) screen BHT analogs for their capacity to protect the sperm functions of membrane integrity, motility, and metabolism from damage during cooling; (b) determine membrane compartments of the sperm into which the analogs partition; (c) evaluate the ability of these compounds, when present in the membrane(s) at equivalent concentrations, to eliminate cold shock damage; (d) establish the extent of interaction between BHT and egg yolk in preventing cold shock and freeze damage; and (e) use flow cytometric analyses to test for selective damage (or protection) of the sperm subcompartments of plasma membrane, mitochondria, and acrosomal membranes during cooling. MATERIALS

AND

METHODS

Experiment 1. Evaluation of Protection from Cold Shock Afforded Sperm and the Toxicity of BHT-Like Compounds 1 .I. Semen collection and analog addition. Bull ejaculates were collected at the

Penn State University Dairy Breeding Research Center using an artificial vagina and transported to the laboratory within 30 min. Semen was diluted 1:3 (v:v) with Hanks’ buffered saline (HBS) (15) and washed twice by centrifugation at 300g for 10 min and resuspended in HBS at a concentration of 2 x lo8 cells/ml. BHT analogs were dissolved in ethanol at 100 times the desired final concentration. A volume of 20 ul of stock analog solution was added to 2 ml washed spermatozoa at 2 x lo8 cells/ml, the solution was immediately mixed by hand swirling and then incubated for 30 min at 25°C. 1.2. Electron paramagnetic resonance spectroscopy (EPR) analysis of spermato-

OF

107

BHT

zoa. BHT analog-treated samples were prepared for EPR analysis as described by Hammerstedt et al. (14). Briefly, 180 ~1 of the sperm/analog mixtures was placed into glass tubes and 10 p.1 of tempone (1 mM in water) and IO t.~l of EDTA-MnO (1 M in water) were added immediately prior to assay of the sperm suspension. Aliquots (50 ~1) were analyzed using a Varian E9 spectrometer (Varian Associates, Instruments Division, Palo Alto, CA) with settings as follows: scan range, 10 G; field setting, 3196.2 G; modulation frequency, 100 Hz; receiver gain, 4 x 104, and microwave frequency, 8.992 GHz. A basal spectrum for each sample was obtained prior to cold shock, after which each sample, within a 5O-tJ capillary tube, was cold shocked by plunging into an ice-water bath and incubating for 30 min. A second spectrum was obtained on each cold-shocked sample immediately after rewarming the sample to 25°C. The peak height of the high field spectral peak in each spectrum was determined as described by Hammerstedt et al. (13). Peak height is directly proportional to cellular water volume sequestered behind membranes and inaccessible to the broadening agent (EDTA-MnO). The ratio (peak height after cold shock/peak height before cold shock) indicates the percentage of cells retaining intact membranes after cold shock treatment. Analogs were defined as “toxic” if sperm spectra were completely broadened by EDTA-MnO (indicating membrane rupture) when analyzed for a basal spectrum prior to plunging into ice-water. Experiment

2. Extent of BHT Analog into Sperm and Evaluation of Their Effects on Membrane Stability

Incorporation

2.1.

EPR

analysis

of spermatozoa.

Sperm were collected and prepared as described in experiment 1 and then tested for the effect BHT and five analogs that differ

108

GRAHAM

AND HAMMERSTEDT

only in the substitution at ring position 4 have on protecting sperm from cold shock (Fig. 1). EPR analysis was conducted as described in experiment 1.

and the solutions were stored at 5°C until analyzed. The analog/cholesterol ratio was determined using HPLC analysis. Samples (l& 2.2. Analysis of cholesterol and analog(s) 25 ~1) were injected on a 5-cm LC Si colwithin sperm. Extracellular analog was re- umn (Supelco, Inc., Bellefonte, PA) and moved using a modified procedure of Hameluted with hexane:isopropanol(200: 1; v:v) merstedt et al. (13), each sperm/analog mixeluent at a flow rate of 1 ml/min; total analture was diluted to 5 ml with HBS and ysis time was ~15 min. A Waters 490 dual washed four times by centrifugation at SOOg wavelength detector (Milford, MA) was for 10 min, using new tubes after each used to assay cholesterol (209 nm) and the wash. The sperm pellet was resuspended to analogs (277 nm) simultaneously. Amounts 0.5 ml with HBS, after which 5 ml methanol of cholesterol and analogs were determined was added and the suspension irradiated by comparison to a standard curve of peak using an ultrasonic bath cleaner (Model areas to known masses of each compound. 8855; Cole-Parmer Instrument Co., Chi- A small amount of material, absorbing at cago, IL) for 10 min. A volume of 10 ml of 277 nm, was present in control samples that chloroform then was added; the sample was eluted at the same time as BHT and its anstoppered and stored overnight at 5°C. alogs (~2 min). This blank value was subSamples then were filtered through a tracted from experimental samples to proglass wool column into 30 ml glass centrivide a true estimate of the analog./cholesfuge tubes and lipids extracted for analysis terol ratio. as described by Folch et al. (9). The aqueous phase was discarded, the lower layer Experiment 3. Protective Ability of Equal Amounts of BHT or Its Analogs (chloroform) was transferred into conical teflon tubes, and the chloroform was evapSperm were prepared as described in exorated under N, atmosphere until the samperiment 1, except that the concentrations ple volume was 0.1 ml. A volume of 0.8 ml of each analog (calculated from results of hexane:isopropanol (200: 1) was added to experiment 2) used in this experiment were each tube, the mixture was vortexed 30 s, An-O, 225 PM; An-l, 275 FM; An-2, 125 FM; An-4, 125 PM; and An-6, 450 FM. An-8 was omitted from this experiment due OH to its minimal incorporation into sperm membranes in experiment 2. The protection CH3 from cold shock damage offered by the anA-CH, alogs and the analog/cholesterol ratio of treated sperm were determined as dehH3 scribed in experiments 1 and 2, respectively.

FIG. 1. Basic structure of BHT analogs tested in this study. The BHT analogs studied in detail differed in the nature of the substituent at position 4, where R = hydrogen (An-O); R = methyl (An-l; BHT); R = ethyl (An-2); R = butyl (An-4); R = hexyl (An-6); R = octyl (An-S).

Experiment 4. Preservation of Sperm Membrane Integrity and Motility after Cold Shock in the Presence of Mixtures of BHT Analogs and Egg Yolk 4.1. Treatment of spermatozoa with BHT analogs and egg yolk. Sperm were col-

lected and prepared as described in exper-

EFFECTS

iment 1. Concentrations of analogs were the same as those used in experiment 3. After incubation of sperm with analogs for 30 min, samples were diluted to 80 x lo6 cells/ml with Tris-Citrate (trizma base, 200 mM; citric acid, 65 rniW; glucose, 55 mM) medium (EYT) containing 0, 0.25, 0.5, or 1.0% egg yolk, prepared as described by Parks et al. (21). These low EY concentrations were chosen to avoid masking any analog effect on the sperm cell, while retaining maximal EY protection from cold shock damage at the highest (1%) EY concentration (3). 4.2. Analysis of plasma membrane integrity using EPR. Since the spermatozoa con-

centration was lower in this study than in experiment I the amounts of nitroxide (tempone) and broadening agents were increased by treating 180 ~1 of spermatozoa with 10 111of tempone (5 mM in water) and 20 p,l of EDTA-MnO (1 M In water). These increased concentrations of tempone and EDTA-MnO had no effect on the percentage of motile sperm, estimated visually, or the integrity of the sperm plasma membrane, measured using EPR (data not presented). Samples (50 t~.l) were analyzed as described in experiment 1. 4.3. Analysis of the percentage of motile sperm. Spermatozoa prepared in 4.1 were

cold shocked by plunging them into a water bath at 5°C. After 30 min equilibration an equal volume of EYT containing 14% glycerol was added to each sample, and the sperm were packaged into 0.5-ml straws (IMV, Minneapolis, MN) and frozen 4.5 cm over static liquid nitrogen (11). Sperm were stored under liquid nitrogen until thawed at 39°C for 30 s. The percentage of motile sperm in each treatment was determined (a) immediately after dilution in EYT, prior to cooling; (b) after dilution with glycerol containing extender at 5°C and rewarming to 39°C; and (c) after freezing and thawing (warmed to 39°C). The percentage of motile sperm in a sample was estimated by a single observer on a 6-tJ subsample placed on a

OF

109

BHT

slide at 35°C and examined with a phase microscope equipped with a 10X objective. A television monitor connected to the microscope permitted visualization of the sperm at 625X. Experiment 5. Flow Cytometric Analysis of Cold-Shocked Spermatozoa Treated with BHT Analogs 5.1. Analysis of spermatozoa treated with An-l and An-2 analogs after cold shock. Spermatozoa were prepared as de-

scribed in experiment 1. In this experiment, only An- 1 and An-2 were used at initial concentrations of 275 and 125 @4, respectively, because both compounds protect the plasma membrane from cold shock damage but differed in their effect on spermatozoal motility. A volume of 12 p,l rhodamine 123 (R123; detects “energized” mitochondria) at 0.01 mg/ml was added to 1 ml of sperm at the same time the analog was added. After 30 min of incubation, sperm were diluted to 20 x lo6 cells/ml and 20 ~1 of propidium iodide (PI; detects sperm with damaged plasma membranes making the DNA accessible to PI staining from the suspending medium and is used to evaluate cell viability) at 1 mg/ml and 24 p,l of phycoerythrinlabeled lectin pisum sativum (PSA; detects sperm with exposed acrosomal compartment) at 0.1 mg/ml was added to 800 p,I of sperm. Samples were split and one half was cold shocked by plunging into ice water for 30 min and rewarming to 25°C. Samples were assayed for cell viability, acrosomal integrity, and mitochondrial function using flow cytometry as described by Graham et al. (12). Experiment 6. Determination of the Distribution of BHT Analogs into Membrane Fractions of Spermatozoa 6.1 Membrane

isolation

and analysis.

Spermatozoa were collected and treated with An-l and An-2 (275 and 125 PM, respectively) as described in experiment 1.

110

GRAHAM

AND

Three membrane fractions were isolated [plasma membrane over the acrosome (PMA), outer acrosomal membrane (OAM), and the remainder of the sperm membranes after removal of PMA and OAM (RSM)] using an isolation procedure of membrane fractions modified from that of Parks et al. (20). After N, cavitation, the fractionation scheme was modified to isolate both the PMA and the OAM in a single step. A sucrose step gradient with densities of 1.05, 1.16, and 1.24 g/ml was prepared and the entire cavitate preparation layered onto it. After centrifugation at 100,OOOg for 3.5 h at 5°C PMA was recovered at the 1.050.16 g/ml interface, OAM was recovered at the 1,16/1.24 g/ml interface, and the RSM was collected as the pellet at the bottom of the tube. Individual membrane fractions were prepared for analog and cholesterol analysis as described in experiment 2. The phospholipid content of each membrane fraction was determined using the procedure of Chen et al. (6). This ratio (analog/cholesterol/phospholipid) was used to compare analog incorporation as the choIesteroUphospholipid ratio’s across these several membrane fractions were nearly equivalent (20). Statistical

Analysis

Analyses were conducted on arcsine transformations of all data. The analog/ cholesterol ratios, the percentage of motile sperm, acrosome intact sperm, and sperm with energized mitochondria were analyzed by analysis of variance and the significance of mean differences was tested using the Student-Newman-Keuls multiple range test (27). RESULTS

Experiment 1. An initial screening of 25 BHT-like compounds for protection against cold shock damage, by EPR analysis of membrane integrity, is shown in Table 1. The compounds which protected sperm

HAMMERSTEDT

from cold shock without toxic side effects all have a similar structure (Fig. l), differing only in the length of the carbon chain at position 4 of the aromatic ring. Conversion from beneficial to toxic effects occurs by removal of one of the butyl residues (compare compounds (a) with (p), and (b) with (q) in Table 1); replacing one butyl residue with a methyl residue (compare compound (a) with (r) in Table 1); or rearrangement of the two butyl residues (compare compound (a) with (u) or(h) in Table 1). Changes in the substituent at carbon 4 also can change the property of the analog, but only from being protective to having no effect. Analogs containing aldehyde and carboxyl residues offer no protection, while BHT, containing a methyl residue, is protective (compare compounds (b) with (m) or (n)). Extending the carbon chain at position 4 resulted in failure to protect sperm from cold shock (compare compounds a-e with j-l). The five compounds that were nontoxic and protected sperm from cold shock were selected for further studies. Experiment 2. Further examination of six analogs which protected sperm from cold shock damage indicated that increasing the initial concentration of every analog resulted in greater protection from cold shock damage and greater incorporation of each analog into the sperm (Fig. 2). However, differences were detected between analogs, both in their abilities to protect sperm from cold shock damage and their partitioning into the sperm. Experiment 3. Based upon the results of experiment 2, initial analog concentration was adjusted to provide equivalent molar incorporation into spermatozoa. When the analog/cholesterol ratios were the same (ratio for An-6 was higher), all analogs afforded equal protection to sperm from cold shock damage (Table 2). Experiment 4. The capability of analogs to protect both the sperm plasma membrane from cold shock, membrane integrity evaluated by EPR analysis, and sperm mo-

111

EFFECTS OF BHT TABLE 1 The Capacity of 25 BHT Analogs to Protect Sperm from Cold Shock Damage

Compound tested

% Protection from cold shock

Protect sperm from cold shock without toxic effects (An-O) 61-11 2,6-di-terr-butylphenol 2,6-di-rert-butyWmethylpheno1 (An-l) 74-85 62-97 2,6-di-tert-butyl-4-ethylphenol (An-2) (An-4) 49-59 2,6-di-tert-butyl-4-butylphenol 21-34 2,6-di-rert-butyl-4-hexylphenol (An-6) Protect sperm from cold shock but are toxic (f) 4-tert-butylphenol 35-57 35-48 (g) 3-tert-butylphenol (h) 3,5-di-tert-butylphenol 40-46 (i) 2,4-di-tert-butylphenol 31-44 No protection from cold shock and are not toxic (i) 2,6-di-tert-butyl-4-octylphenol (An-8) (k) 2,6-di-tert-butyl-4-decylphenol (1) 2,6-di-tert-butyl-4-dodecylphenol (m) 3,5-di-tert-butyl-4hydroxybenzoic acid (n) 3,5-di-tert-butyl-4-hydroxybenzaldehyde (0) 2,4-di-(err-butylbenzene No protection from cold shock and are toxic (p) 2-(err-butylphenol (q) 2-tert-butyl-4-methylphenol (r) 2-tert-butyl&methylphenol (s) 4-tert-butyl methylphenol (t) 4-terr-butylcatachol (u) 2,5-di-tert-butylphenol (v) 2,5-di-rert-butylhydroquinone (w) 4,6-di-terr-butylresorcinol (x) 3,5-di-tert-2,6-dihydroxybenzoic acid (y) 2,6-di-tert-butylbenzoquinone

Toxic at (mM)

(a) (b) (c) (d) (e)

5 5 5 5 1 5 5 1 1 1

Note. Analogs (at 0.5, 1, 5, and 100 mM final concentration; N = 2) were tested for capacity to protect against membrane lysis induced by cold shock (assessed via EPR analysis) and general toxicity. Percentage protection from cold shock is presented as the range observed after combining results for all nontoxic concentrations tested. The concentrations at which toxic effects were observed are indicated.

from both cold shock and freeze-thaw damage was evaluated in this experiment. The initial percentage of motile sperm, after treatment with most analogs or with egg yolk, did not differ from that of untreated sperm (Table 3). The exception was An-l (BHT), where a lower percentage of motile sperm was observed. After cooling to 5°C the percentage of motile sperm increased (from 18% in the absence of EY) with each increasing concentration of EY (to 68% in tility

the presence of 1% EY). The addition of BHT analogs to the medium, however, did not affect the percentage of motile sperm compared to control samples except in the case of AN-l which was detrimental. These BHT analogs did, however, protect the plasma membrane from cold-induced membrane rupture (Table 3B). The addition of BHT analogs (except An-l) did not affect the percentage of motile sperm post-thaw, whereas the addition of even 0.25% EY in-

112

GRAHAM

AND HAMMERSTEDT

TABLE 2 The Percentage of Whole Cell Volume Remaining after Cold Shock and the Analog/Cholesterol Ratio of Sperm after Addition of BHT Analogs at Concentrations which Would Equalize Incorporation

Analog

Concentration added (PM)

None An-O An-l An-2 An-4 An-6

250 275 12.5 125 450

SEM

3

% cell volume after cold shock

Analog/ cholesterol

40” 646 686 61’ 65’ 69’

0.03” 0.36’ 0.3Y 0.436,’ 0.42’,’ 0.49’ 0.03

Note. Mean values from six replicates are presented. Means within columns having different superscripts (a, b, c) denote differences at P < 0.05.

creased the percentage of motile sperm post-thaw. Additional increases in the EY concentration up to 1% (v/v) resulted in additional increases in sperm motility after freezing and thawing (Table 3A3). Experiment 5. In this experiment, flow cytometry was used to assess the effects that An-l and An-2 have on sperm cell viability (PI staining), acrosomal integrity (PSA binding) and mitochondrial function (R123 staining) prior to and after cold shock (Table 4). Cell viability of analog-treated sperm was higher than that of untreated sperm after cold shock, a result similar to the evaluation of sperm plasma membrane integrity via EPR. Analogs did not affect the percentage of intact acrosomes on live sperm initially or after cold shock, but analog-treated cells did retain a higher percentage of sperm with normal mitochondrial function following cold shock. Experiment 6. The extent of incorporation of two analogs (AN-l and AN-2) into each of several sperm membrane fractions, isolated by sucrose gradients, are presented in Table 5. Cholesterol/phospholipid ratios of 0.26, 0.42, 0.32, and 0.32 were measured for

whole sperm, PMA, OAM, and RSM sperm fractions, respectively. For purposes of this discussion, results are expressed in terms of moles analog/moles P-lipid. Small amounts of analog were found in the PMA and OAM fractions, but these were not statistically different from those of control preparations. DISCUSSION

Small changes in the structure of BHT result in very different cellular effects, ranging from protecting sperm from cold shock damage to lysing the sperm. The two butyl residues at carbons 2 and 6 appear to be extremely important to membrane protective effects of BHT (Table 1). Removal, modification or rearrangement of one butyl residue location eliminates membrane protection and results in toxicity to sperm. Changes at carbon 4 are less drastic in that several analogs differing in carbon 4 residues protect sperm from cold shock damage while others had no noticeable effect. The primary effect here might be on the capacity of the molecule to transfer into the cell (long carbon chains may prevent transfer of monomers from the analog micelles through the aqueous medium into the sperm) to affect membrane function. Analogs with very short chains at carbon 4 (hydrogen and methyl groups) transferred to a lesser extent than did analogs having ethyl or butyl groups at carbon 4, and increasing the carbon 4 groups to 6 or 8 carbons decreased analog incorporation (Fig. 2B). These differences are probably due to the manner in which the analogs interact within the analog micelles and at the aqueous/ micelle interface (2). Movement of analogs having chains of >4 carbons at position 4 may be precluded by their hydrophobicity and inability to transfer though the aqueous medium into sperm membranes. The capability of analogs to prevent sperm membrane damage from cold shock, determined using EPR techniques, corre-

EFFECTS

OF

113

BHT

TABLE 3 Effect of BHT Analog and of Egg Yolk on the Percentage of Motile Sperm and on Sperm Cell Volume (% Protection) Initially, after Cooling, and after Freezing and Thawing Percentage egg yolk Experiment condition

Analog

Al. Motility (after dilution and before cooling)

None AN-O AN-l AN-2 AN-4 AN-6

A2. Motility (after cooling)

A3. Motility (after freezing and thawing)

B. % Protection (after cold shock)

0

0.25

0.5

1

Mean

69 72 54 74 71 67

74 74 67 15 75 75

17 75 13 71 77 15

78 76 15 76 76 76

Mean

68*

73

76

76

None AN-O AN-l AN-2 AN-4 AN-6

18 20 6 25 20 18

55 49 20 48 44 55

61 55 26 65 50 58

68 62 40 68 62 66

Mean

18***

45**

52*

61

3

None AN-O AN-l AN-2 AN-4 AN-6

12

10 0 11 7

31 23 13 26 23 30

35 28 20 30 26 30

25 21

4

22 24 6 19 22 23

Mean

I**

19*

24

28

2

74 14 67+ 15 75 13

1 SEM 50 46 23+ 51

44 49

SEM

lo+ 21 19 22

None AN-O AN-l AN-2 AN-4 AN-6

42 51 61 65 59 63

41 53 56 51 55 54

45 52 56 55 54 53

51 52 56 56 53 53

47+ 54 51 51 55 56

Mean

58

53

53

53

2

SEM

SEM

Note. Mean values from five replicates. Differences in the main effect of egg yolk concentration are denoted by different numbers of asterisks ( *, **, ***) in rows, P < 0.05. Differences in the main effect of analogs are denoted by an (+) in columns, P < 0.05.

sponded with analog capacity to incorporate into the sperm (Fig. 2A). An additional study, to determine if analogs differ in their capacity to protect sperm membranes present at equivalent molar concentrations, established that all analogs affect membrane properties equivalently once inserted into the cell membrane (Table 2). Therefore, differences in ability of an analog to protect sperm from cold shock are due en-

tirely to differences in their ability to partition into the membrane, and not to differences in the way individual analogs interact with the membrane once incorporated. The ability of BHT to readily incorporate into membranes and prevent membrane damage after exposure to cold has been demonstrated previously for sperm (13, 14, 23, 31) as well as mammalian fibroblast cells in culture (18, 26).

114

GRAHAM

AND

TABLE 4 The Percentage of Live Cells, Percentage of the Live Cells which Are Acrosome Intact, and the Percentage of Live Cells with Functioning Mitochondria of Sperm Treated with BHT Analogs prior to and after Cold Shock Cell function analyzed Viability (PI --I

LiveiAcrosome intact (PI -, PSA -) Live/functional mitochondria (PI -, R123 +)

Fresh

Cold shocked

None AN-l AN-2

86 86 85

21 79* 70*

SEM

2

4

None AN-l AN-2

84 80 85

87 91

SEM

4

4

None AN-l AN-2

61 64 56

26 61* 49*

SEM

8

9

90

Note. Mean values of five replicates are presented. Differences between treatment means in columns, within a cell function, are denoted by an (*), P < 0.05.

The addition of analogs to sperm in the presence of egg yolk revealed differences in several of the parameters tested: First, addition of An-l (BHT) reduced the percentage of motile sperm immediately after dilution, after cooling to YC, and post-thaw; compared to control samples or TABLE 5 Analysis of BHT Analogs (Treated Samples) and Background Material (Control Samples) in Several Membrane Compartments of Bull Spermatozoa Treatment

Whole sperm

PMA

OAM

RSM

Control An-l An-2 SEM

0.00 0.14* 0.13* 0.03

0.01 0.01 0.01 0.00

0.18 0.05 0.55 0.31

0.00 0.13* 0.14* 0.03

Note. Ratios have been adjusted for membrane phospholipid content as described in the text. Mean values of four replicates are presented. Differences between ratios of analog detected in each membrane preparation, columns, are denoted by an (*), P < 0.05.

HAMMERSTEDT

samples treated with other analogs (Table 3). A similar reduction in sperm motility has been reported for human sperm treated with BHT (1). The mechanism by which BHT reduces motility is not known. Second, increasing concentrations of egg yolk in BHT-treated sperm improved motility. This is probably due to the presence of the lipid vesicles in the egg yolk and their ability to act as an alternative (competitive) site for BHT transfer. This reduces the amount of BHT available to interact with and become incorporated into the sperm, thus lowering the effective BHT concentration and reducing the effect on sperm motility. Other lipid sources (milkfat) produce a similar phenomenon; Killian et al. (17) reported higher percentages of motile sperm 4 h post-thaw in samples treated with 0.5 mM BHT when diluted in whole milk (48%) than skim milk (6%). Third, addition of increasing concentrations of egg yolk increased the percentage of cells surviving cold shock, although the addition of analogs protected sperm more efficiently. The combination of analog and egg yolk was better than egg yolk treatment alone, but inferior to treatment with analog alone. These data suggest that, at the concentrations tested, analogs are more effective than egg yolk in protecting sperm against cold shock. As seen previously, the addition of egg yolk to the medium effectively reduced the amount of analog available to interact with sperm and therefore resulted in less protection than samples not treated with egg yolk. The motility of BHT-treated spermatozoa, in the absence of egg yolk, was not different from control spermatozoa after cooling to 5°C but was lower than control samples after freezing and thawing. Law et al. (19) reported BHT increased membrane fluidity in the nonpolar regions of the membrane, but also increased the sensitivity of mammalian tibroblast cells to freezing damage. They hypothesized that this increased

EFFECTS

TA

OF

115

BHT

AAn-

jAn-4

/

.cj 80 ij 2 % 70 E z

& 60 a

n -..----------m&r8

50 I

0

I, I

I

100

I I

I I

I I

200 I.IM

I I

I I

300

I, I

400

I

I I

I I

500

Analog

0

I I 0

100

-~--------~An-8 ) I I : I I 200

PM

300

400

I

~ ,

500

Analog

FIG. 2. Effect of increasing concentration of BHT analog on protection from damage after cold shock and incorporation into sperm. (A) The percentage of cells which survived membrane rupture due to cold shock. The X axis values indicate the concentration of analog added to sperm and are not adjusted for differences in analog incorporation into spermatozoa. (B) The amount of analog partitioning into sperm, determined as described in the text. Mean values of six replicates are presented. Different types of lines (-, . . ., - - -, - . . -) denote differences between analogs in rates of analog incorporation and protection at P < 0.05.

sensitivity was due to the formation of hexagonal phase changes in membrane induced by the BHT. Similar results were reported for fibroblast cells frozen at slow rates (0.3”Umin) using a combination of BHT and dimethyl sulfoxide for cryoprotection, although a fast cooling rate (3.0”C/min) improved freeze-thaw cell survival (10). Flow cytometric analysis of analogtreated sperm, using PI uptake as a measure of membrane damage, confirmed the results obtained using EPR analysis. Although BHT reduced the percentage of motile cells after addition, it was very effective in protecting the sperm mitochondria from cold shock damage. This indicates that incorporation of BHT (An-l), but not An-2, alters some aspect of the sperm motility apparatus unrelated to mitochondrial metabolism. Perhaps this could be an interaction of the various tail fibril components with themselves or their surrounding membrane (8).

Flow cytometric analysis also revealed no beneficial effect of the analog on the percentage of intact acrosomes in the live cell population after cold shock. Watson and Anderson (31) reported sperm samples had a higher percentage of cells with intact acrosomes after cold shock when treated with 2 mM BHT than in untreated samples. Purse1 (23) reported similar results with BHT concentrations ranging from OS-2 mit4, however, BHT at 0.1 miU failed to protect boar sperm acrosomes from cold shock damage. If BHT affects boar sperm and bull sperm similarly, the absence of acrosomal protection, after BHT addition, in this study is likely to be due the low concentration of BHT analog used (0.275 and 0.125 mM of AN-l and AN-2, respectively) . Values for the cholesterol/phospholipid ratios of the different sperm membrane compartments (whole sperm, PMA, OAM) presented here are similar to those reported

116

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previously (20). Analysis of the incorporation of analog into the several membrane compartments confirmed that the amount of An- 1 and An-2 (added at 275 and 125 pM, respectively) partitioning into the membranes were the same (Table 5). These analyses also indicate that very little analog (An-l or An-2) partitions into either the OAM or the PMA, but rather resides in the other portions of the sperm. The difference in partitioning most likely is due to differences in composition of the various sperm membranes, yielding a situation where analogs are selectively accommodated. The assumption is made that selective partitioning occurred before membrane isolation. Furthermore, these data establish that the protective effect at the plasma metnbrane is achieved with accumulation of very little analog into that specific membrane. Such an hypothesis is not unreasonable as significant membrane alterations can be observed in liposomes with the addition of anesthetics at molar ratios as low as 0.05 (28). CONCLUSION

BHT analogs differ in their ability to partition into the membranes of sperm, with partitioning depending upon the substituent at carbon 4. Analogs selectively partition into the different membrane compartments of individual sperm with minimal residence in the PMA or the OAM but extensive partitioning into the other sperm compartments. In addition, analogs, once incorporated into sperm, can act in distinct manners. If present in equivalent amounts, all protect the plasma membrane from rupture due to cold shock. Both An-l and An-2 offered equal protection to cold shock damage for mitochondrial membranes but have no effect on the stability of acrosomal membranes. BHT, but not the other analogs, reduces sperm motility. Reasons for either the differential incorporation into membrane compartments or specific alterations of cellular motility are not known but are likely to be due to membrane unique com-

position(s) and/or dependence on membrane-linked events. Finally, the studies demonstrate the need for special attention to interactions between additives when designing cryoprotective mixtures. This study demonstrates that EY can mask the properties of other additives with desirable features unless the concentrations of each are carefully balanced. The need for measuring more than one sperm parameter (plasma membrane integrity, motility, and acrosoma1 status) was established as additives can affect each of these sperm parameters in a different manner. REFERENCES 1. Aitken, R. J., and Clarkson, J. S. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J. Androl. 9, 367-376 (1988). 2. Alauddin, M., and Verrall, R. E. Apparent molal volume studies of 2,6-Di-tert-butyl-4methylphenol, 2-tert-Butyl-4-methoxyphenol, and 2,6-Di-tert-butyl-4-(hydroxymethyl)phenol in aqueous micelle solutions of sodium dodecanoate as a function of micelle concentration and temperature. J. Phys. C/tern. 88,5725-5730 (1984). 3. Blackshaw, A. W. The prevention of temperature shock of bull and ram spermatozoa. Amt. J. Biol. Sci. 7, 573-582 (1954). 4. Blackshaw, A. W., and Salisbury, G. W. Factors influencing metabolic activity of bull spermatozoa. II. Cold-shock and its prevention. J. Dairy Sci. 40, 1099-l 106 (1957). 5. Butler, W. J., and Roberts, T. K. Effects of some phosphatidyl compounds on boar spermatozoa following cold shock or slow cooling. J. Reprod. Fe&. 43, 183-187 (1975). 6. Chen, P. S., Jr., Toribara, T. Y., and Warner, H. Microdetermination of phosphorus. Anal. Chem. 28, 1756-1758 (1956). 7. Darin-Bennett, A., Poulos, A., and White, I. G. The effects of cold shock and freeze-thawing on the release of phospholipids by ram, bull and boar spermatozoa. Amt. J. Biol. Sci. 26, 140% 1420 (1973). 8. Dentler, W. L. Linkages between microtubules and membranes in cilia and flagella. In “Ciliary and Flageller Membranes” (R. A. Bloodgood, Ed.), pp. 31-64. Plenum, New York, 1990. 9. Folch, J., Lees, M., and Sloane-Stanley, G. H. A simple method for the isolation and purification

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