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BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008607
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BIOLOGY OF REPRODUCTION 68, 1308–1317 (2003)
DOI: 10.1095/biolreprod.102.008607
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Structural Features of Sterols Required to Inhibit Human Sperm Capacitation1

Matthew R. Nimmoa, and Nicholas L. Cross2,a

a Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of cholesterol is an essential step in the capacitation of human sperm. Experimentally maintaining a high level of cholesterol inhibits capacitation, but the mechanism is unknown. The present study investigated the structural features that are required for cholesterol's inhibitory activity. Human sperm also contain much desmosterol, which is lost from sperm during capacitation. Preventing the loss of desmosterol inhibited capacitation (as assessed by acrosomal responsiveness), with an effectiveness approximately equal to cholesterol's inhibitory activity. Other structural analogs were added to the incubation medium to replace sperm cholesterol and desmosterol. Most inhibited capacitation, including those that lacked cholesterol's 3ß-OH group (cholesteryl methyl ether and epicholesterol) and those with modified C17 groups (ergosterol and diosgenin). Two steroids did not inhibit capacitation well. Coprostanol, which has a nonplanar steroid nucleus, had low inhibitory activity that could be explained by an elevated endogenous cholesterol concentration. Epicoprostanol, which has a nonplanar ring structure and a 3{alpha}-OH group, promoted rather than inhibited capacitation. The inhibitory activity of the analogs was correlated with their ability to promote order of egg phosphatidylcholine as measured by fluorescence anisotropy. In summary, a planar ring structure is required for sterol inhibitory activity, but a 3ß-OH group and a saturated cholesterol-like aliphatic tail on C17 are not required. The present results support the hypothesis that sperm sterols block capacitation by increasing order of phospholipids.

acrosome reaction, fertilization, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian sperm must undergo the acrosome reaction (exocytosis of the acrosome) before they can fertilize an egg. Freshly ejaculated sperm do not acrosome-react. However, when they are incubated in a suitable medium, a few sperm spontaneously acrosome-react, and others acquire the ability to react in response to agents such as the egg's zona pellucida, progesterone (P4), and Ca2+/H+-exchanging ionophores (reviewed in [1]). Acquisition of acrosomal responsiveness is believed to model capacitation, the time-dependent acquisition of fertilizing ability that normally occurs in the female reproductive tract [2, 3].

The mechanisms that render sperm acrosomally responsive are not well understood. Events that have been implicated in this process include loss of sperm sterols (reviewed in [4]), loss of molecules from the cell surface (reviewed in [5]), membrane hyperpolarization [6], production of reactive oxygen species [7], elevated concentrations of calcium and cAMP [7–9], protein phosphorylation [10–15], and increased intracellular pH [16–20].

Of the many events that accompany capacitation, loss of sterols is one of the few that are known to be obligatory. The most abundant sterol in human sperm is cholesterol, and most work has concentrated on it. However, desmosterol and a small amount of cholesteryl-3-sulfate are also present [21, 22]. All three inhibit capacitation when added to the medium in which sperm are suspended [23]. They are generally assumed to act after uptake by sperm, although to our knowledge, a close correlation between sperm content and inhibition of capacitation has only been shown for cholesterol [24].

How might sterols control capacitation? Cholesterol is abundant in the sperm plasma membrane [25, 26], and most models propose that it acts there. The critical event is often suggested to be an increase in phospholipid fluidity or bilayer permeability resulting from loss of cholesterol [27]. This model follows from well-studied interactions of cholesterol with phospholipids. Cholesterol orients perpendicular to the plane of a bilayer and, above the phospholipid transition temperature, increases the order of phospholipid acyl chains, with effects on related properties: It reduces the average molecular surface area, increases the bilayer thickness, and reduces the bilayer permeability (reviewed in [28]). Changes in phospholipid order can directly affect the activities of some membrane proteins (see [29] and references therein). Cholesterol's ability to order saturated phospholipids may also be important in the formation of lipid rafts, sterol-rich regions in membranes that have characteristic protein compositions and that may modify signaling pathways [30, 31].

In spite of the common suggestion that capacitation follows a cholesterol loss-dependent increase in lipid "fluidity," little supporting evidence is available. A preliminary report found no difference in the lipid order (as determined with steady-state fluorescence anisotropy) of fresh and capacitated human sperm [32]. With the spin label, 16-doxyl stearate, Purohit et al. [33] reported increased rotational function during capacitation of human sperm but no change in membrane packaging or lateral diffusion of molecules. Likewise, lateral diffusion of lipids measured by recovery of fluorescence after photobleaching, although of questionable relationship to sterol content [34], does not change during capacitation of hamster or mouse sperm [35, 36]. Capacitating boar sperm exhibit increased labeling with merocyanine 540 (thought to detect increased membrane lipid structural disorder), but this has been correlated with phospholipid "scrambling" and its relationship to sterol content is not yet clear [37, 38].

The present study tested the hypothesis that capacitation requires a decrease in phospholipid order following loss of cholesterol. The molecular features required for sterols to affect phospholipid order have been well studied. In most model systems and cellular membranes, these effects require a ß-oriented OH on C3, a cholesterol-like tail on C17, and a planar ring structure (reviewed in [39]). The ability of structural analogs to inhibit capacitation was tested and compared with their ability to influence phospholipid order.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

The following chemicals were used: phosphatidylcholine (PC; Avanti Polar Lipids, Inc., Alabaster, AL), methanol and chloroform (EM Science, Gibbstown, NJ), hexane and ethanol (Pharma Products, Brookfield, CT), BSA (Pentex Bovine Albumin, Fraction V, Reagent Grade; catalog no. 81-066-7, lot 46; Miles, Inc., Kankakee, IL), Pisum sativum agglutinin (Vector Laboratories, Burlingame, CA), and 1,6-diphenyl-1,3,5-hexatriene (DPH; Molecular Probes, Inc., Eugene, OR). Most steroids were obtained from Steraloid (Newport, RI). Cholesterol, stigmastadienone, {alpha}-cholestane, and all other chemicals were obtained from Sigma Chemical (St. Louis, MO). The purity of steroids was confirmed by gas chromatography. The steroids are depicted in Figure 1.



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  		FIG. 1. Steroids used in the present study. 1) 16, (5{alpha}) Androsten-3{alpha}-ol. 2) 16, (5{alpha}) Androsten-3ß-ol. 3) 5{alpha}-Androstan-3-one. 4) 5{alpha}-Androstan-3ß-ol. 5) 3ß-Chloro-5-cholestene. 6) 4-Cholesten-3-one. 7) 5{alpha}-Cholestane. 8) 5-Cholestene. 9) 5-Cholestene-3ß,19-diol. 10) 5-Cholestene-7-one. 11) Cholesterol. 12) Cholesteryl-3-formate. 13) Cholesteryl methyl ether. 14) Coprostanol. 15) Desmosterol. 16) Diosgenin. 17) Epicholesterol. 18) Epicoprostanol. 19) Ergosterol. 20) Fucosterol. 21) Stigmasterol

Sperm Preparation

Except where noted otherwise, human sperm were washed, incubated in vitro, and exposed to P4 as previously described [40, 41]. Informed consent was obtained from the semen donors, and an institutional review board approved this investigation. Semen were obtained by masturbation, and motile sperm were selected by centrifugation through a Percoll gradient, washed, and suspended in a medium modified from that described by Suarez et al. [42]: 117.6 mM NaCl, 0.36 mM NaH2PO4, 8.6 mM KCl, 2.4 mM CaCl2, 0.49 mM MgSO4·7H20, 25 mM NaHCO3, 2 mM glucose, 0.25 mM Na-pyruvate, 19 mM Na-lactate, 0.05 mg/ml of streptomycin sulfate, 0.075 mg/ml of penicillin, and 26 mg/ml of BSA.

Sperm suspensions (1.0–2.5 ml, 2 x 106 sperm/ml) were incubated in triplicate with or without steroid for 24 h in 15-ml polystyrene conical centrifuge tubes (Sarstedt, Inc., Newton, NC). The tubes were flooded with a humidified mixture of 5% CO2/20% oxygen/75% nitrogen (v/v/v), capped loosely, and reclined at an angle of approximately 5° above horizontal so that the sperm would not collect at the point of the tube. The tubes were incubated at 37°C in a chamber containing a humidified atmosphere of 5% CO2/95% air (v/v).

Steroids were administered either by direct addition to the medium or contained in PC liposomes. To prepare liposomes, 1 mg of sterol and 2–6 mg of PC were combined, and solvent was evaporated under flowing N2. The lipids were twice dissolved in diethyl ether and dried in a thin film, and residual solvent was removed by vacuum at less than 100 µm Hg for approximately 15 h. To the dried lipids were added 2 ml of medium lacking BSA, through which nitrogen had been bubbled to remove oxygen. The suspension was covered with nitrogen, vortexed vigorously for 15 min, and then sonicated in a bath sonicator (Laboratory Supply Co., Inc., Hicksville, NY) at 30–40°C for 15–30 min. Aggregated lipids were removed by centrifugation (10 000 x g, 1 h), and the liposomes were stored at -20°C. After dilution into incubation medium on the day of use, the mixture was passed through a filter (pore size, 22 µm) to remove particulate material. Liposomes containing no steroid were prepared as a control. The liposome steroid content was assayed by extraction and gas chromatography as described below, and the PC concentration was determined by assaying phosphate [43]. Liposomes containing cholesterol or desmosterol were used at less than 1 µM PC. Although PC liposomes can increase the loss of sperm cholesterol and increase acrosomal responsiveness [24, 44], 1 µM is less than the effective concentration (data not shown). Some other steroids required the use of higher concentrations of liposomes, as discussed in Results.

Alternatively, a steroid dissolved in dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethanol, or isopropanol was diluted 1000-fold in incubation medium during vortexing. The solution was agitated gently at room temperature for 15–60 min and then passed through a filter (pore size, 0.22 µm). In these experiments, steroid-treated sperm received 0.05% (v/v) solvent, and control sperm received only solvent.

After 24 h, sperm were tested for acrosomal responsiveness and steroid content. To induce acrosome reactions, 75-µl aliquots of sperm suspension were exposed for 10 min to 0.5 µg/ml of Hoechst 33258 (to label dead sperm) and either 1 µg/ml of P4 (to induce acrosome reactions in capacitated sperm) or solvent control (0.1% [v/v] DMSO). The acrosomal status of living (Hoechst 33258-negative) sperm was determined using P. sativum agglutinin [41]. Sperm that were acrosome-reacted in the solvent control sample are termed spontaneously reacted. The incidence of capacitated sperm is the percentage of sperm that acrosome-reacted following exposure to P4 corrected for the number of spontaneously reacted sperm. During 24 h of incubation, the incidence of spontaneously reacted sperm is usually less than 5%; the incidence of capacitated sperm is initially 0% and then rises during incubation [24]. When sperm were treated with exogenous steroids, inhibition of capacitation was calculated by comparing the incidence of capacitated sperm in the experimental group to the incidence in the control group. Data from 14 experiments (of 175 total) were excluded, because in these experiments, the percentage of control capacitated sperm was too low (<=15%) to make this calculation with confidence.

Whether the steroid was delivered in the form of liposomes or from a solution, sperm were not washed free of the steroid before the response to P4 was tested. Additional experiments, in which sperm that had been capacitated in control medium for 24 h were simultaneously exposed to the steroid and to P4, tested whether steroid in the medium affected the response of sperm to P4.

Steroid Assays

The initial sperm sterol content was determined immediately after motile sperm were prepared from semen. An aliquot of suspension containing 5 x 106 sperm was added to 5 ml of PBS (138 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, 1.5 mM KH2PO4, 1.0 mM CaCl2, and 0.5 mM MgS04), centrifuged (800 x g, 10 min), and suspended in PBS. The concentration of sperm was determined in a hemocytometer, and a measured aliquot was frozen (-20°C). Sterol assays were completed within 3 days. The 24-h incubated sperm were centrifuged, washed in 5 ml of PBS, and suspended in PBS. Because of the large number of samples, the sperm concentration after washing was determined with CyQuant, a fluorescent DNA-binding dye (Molecular Probes). Triplicate 20-µl aliquots were combined in the wells of a 96-well plate with 200 µl of the CyQuant reagent according to the manufacturer's directions. To convert fluorescence units to sperm/ml, the sperm concentrations in two tubes were determined in a hemocytometer, and the ratio of fluorescence to sperm concentration was calculated.

To assay steroid content, {alpha}-cholestane was added to each tube of washed sperm as an internal standard, and lipids were extracted with chloroform and methanol as previously described [45–47]. When the interaction of {alpha}-cholestane with sperm was analyzed, stigmastadienone was used as an internal standard. The extracted material was dissolved in hexane and analyzed by gas chromatography using a Perkin-Elmer Autosystem XL with Turbochrom 4.1 for control and analysis and a DB-17 column (inner diameter, 0.53 mm; length, 30 m; J&W Scientific, Folsom, CA). The carrier was helium (18 ml/min), and the flame ionization detector was supplied with hydrogen (45 ml/min) and air (450 ml/min). Preliminary experiments determined that the ratios of steroids to {alpha}-cholestane were not altered by the extraction procedure. To assure that the washing procedure removed soluble steroid from the sperm suspension, blank samples were prepared with steroid in incubation medium but lacking sperm. This protocol assays free, unesterified steroids; in the present study, the term sterol or steroid means the unesterified form.

Fluorescence Anisotropy

Liposomes were prepared on the day of use with egg PC containing 1 mol % butylated hydroxytoluene as described above, with the following differences: The PC and the steroid (0–30 mol %) were sonicated at a total lipid concentration of 1 mM in nitrogen-saturated Hepes-buffered saline (HBS; 150 mM NaCl and 20 mM Hepes, pH 7.1). The DPH was diluted to 2 µM in HBS from a stock solution of 1 mM in acetone. To remove the acetone, nitrogen was passed through the solution for 7 min at 65°C. All work with DPH was performed in low light to reduce photobleaching. The DPH and liposomes were combined in HBS to make 100 µM lipid and 1 µM DPH and were maintained in the dark for at least 30 min. The phosphate and steroid content of the liposomes was determined as described above.

Steady-state anisotropy was measured using a PTI Quantamaster spectrofluorimeter (Lawrenceville, NJ) in L-configuration (excitation wavelength, 358 nm; emission wavelength, 427 nm; slit widths, 3 nm). A 400-nm, long-pass filter in the emission path blocked scattered light, and a 12% transmission neutral-density filter in the exciting path reduced photobleaching. The solution was magnetically stirred and maintained at 23–24°C. Fluorescence was recorded for 4 sec with an integration time of 0.25 sec. Anisotropy (r) is given by

where IVV and IVH are the fluorescence emission intensities measured with the polarizer polarization axis vertical in both cases and the analyzer polarization axis vertical or horizontal, respectively. The G factor corrects for unequal response of the instrument to vertical and horizontal polarized light and is given by

where IHV and IHH are the fluorescence emission intensities measured with the polarizer polarization axis horizontal in both cases and the analyzer polarization axis vertical or horizontal, respectively. Before calculation, fluorescence intensities were corrected for the minimal scatter caused by liposomes by subtracting a blank sample that did not contain DPH.

Statistics

Means were compared by analysis of variance with Bonferroni posttests using InStat (GraphPad, Inc., San Diego, CA). Percentage data were transformed before analysis (arcsin [%/100]1/2;). The value of 1 x 10-7 was substituted for values of zero. When appropriate, repeated-measures tests were used to accommodate variations among ejaculates. The slope of the linear midranges of dose-response curves (inhibition of capacitation vs. sperm sterol content) were calculated by linear regression; median effective concentrations (EC50) were calculated from the resulting curve using InPlot (GraphPad).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Freshly prepared human sperm contain two major sterols, cholesterol and desmosterol [21, 22]. The content of both sterols decreases during capacitation in vitro, and although their relative effectiveness has not been reported, both inhibit capacitation when they are added to the incubation medium [23, 24, 47]. Effectiveness was assessed by incubating sperm 24 h in medium supplemented with either cholesterol or desmosterol in a range of concentrations (0–2.5 µM), then measuring the sperm sterol content and acrosomal responsiveness to P4. The results obtained using sterol-containing liposomes and by dilution from a DMSO stock solution were not significantly different and were combined for analysis.

Cholesterol

Sperm incubated 24 h in cholesterol-supplemented medium had an elevated cholesterol content, and their response to P4 was inhibited compared to sperm incubated without cholesterol (Fig. 2). The cholesterol content was increased from 1.13 ± 0.12 nmole/107 sperm in control sperm to 3.42 ± 0.24 nmole/107 sperm in the highest treatment group (mean ± SEM, n = 9). Desmosterol content was not affected, except in highest treatment group, in which it was slightly increased compared to control sperm (0.320 ± 0.030 vs. 0.202 ± 0.037 nmole/107 sperm, mean ± SEM, n = 9, P < 0.01).



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  		FIG. 2. Effect of cholesterol on capacitation as assessed by P4-induced acrosome reactions. Results of a representative experiment are shown. Sperm in five treatment groups were incubated with (from left to right) 0.0, 0.312, 0.625, 1.25, or 2.5 µM cholesterol (delivered in PC liposomes). After 24 h, sperm were assessed for capacitation and incorporation of sterol. A) Inhibition of capacitation. Data are mean ± SEM (n = 3). B) Composition of the sperm sterol fraction in each treatment group: desmosterol (solid bars); cholesterol (open bars). For comparison, horizontal dotted lines indicate the sterol composition of freshly prepared (T0) sperm: cholesterol (C); desmosterol (D)

Curves such as in Figure 2 were used to calculate the sperm total sterol content (cholesterol + desmosterol) that inhibited the response to P4 by 50% (i.e., EC50). The EC50 for cholesterol treatment was 2.10 ± 0.18 nmol sterol/107 sperm (mean ± SEM, n = 9); this accounted for 51% of the total sterol content of uncapacitated sperm. The effect of sterol on acrosomal responsiveness reached a plateau at the highest doses. The maximum inhibition observed was 90% ± 3% at a total sperm sterol content of 2.99 ± 0.32 nmole/107 sperm (mean ± SEM, n = 9). This accounted for 73% of the total sterol content of uncapacitated sperm. The slope of the approximately linear midrange of the dose-response plots indicated that each added nmol total sterol/107 sperm inhibited capacitation by 82.4%.

Desmosterol

Sperm were incubated 24 h in desmosterol-supplemented medium. Desmosterol was 2.63 ± 0.527 nmole/107 sperm in the highest treatment group, compared to only 0.252 ± 0.022 nmole/107 sperm in control sperm (mean ± SEM, n = 11) (Fig. 3). Cholesterol was 1.22 ± 0.11 nmole/107 sperm in the highest treatment group, compared to 1.04 ± 0.07 in control sperm, but the difference was not statistically significant. Incubating sperm with desmosterol inhibited capacitation (Fig. 3). The EC50 for desmosterol treatment was 2.07 ± 0.17 nmol total sterol/107 sperm. The slope was 77 ± 14% inhibition per nmol added total sterol/107 sperm (mean ± SEM, n = 10). In most experiments, inhibition did not reach a plateau; when it did, inhibition was near 100%.



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  		FIG. 3. Effect of desmosterol on capacitation as assessed by P4-induced acrosome reactions. Results of a representative experiment are shown. Sperm in five treatment groups were incubated with 0.0, 0.312, 0.625, 1.25, or 2.5 µM desmosterol. After 24 h, sperm were assessed for capacitation and incorporation of sterol. A) Inhibition of capacitation. Data are mean ± SEM (n = 3). B) Composition of the sperm sterol fraction in each treatment group: desmosterol (solid bars); cholesterol (open bars). Horizontal dotted lines indicate the sterol composition of freshly prepared (T0) sperm: cholesterol (C); desmosterol (D)

To compare the effectiveness of desmosterol versus cholesterol in inhibiting capacitation, we first compared the EC50 for each treatment. The EC50 for cholesterol treatment was 2.10 ± 0.18 nmole/107 sperm (n = 9), and the EC50 for desmosterol treatment was 2.07 ± 0.17 nmol total sterol/107 sperm (n = 10). These EC50 values were not significantly different. Considering the number of samples and experimental scatter in the data, we had a 95% probability of detecting a difference of 1.0 nmole/107 sperm in the EC50 as significant at P < 0.05.

The slopes of the two dose-response curves were also similar. The slope was 82.5% ± 10.9% per nmole/107 sperm (mean ± SEM, n = 9) for cholesterol treatment and 77.0% ± 14.0% per nmole/107 sperm (mean ± SEM, n = 10) for desmosterol treatment. These SEMs are fairly large, however, producing an 80% probability of detecting a difference of 40 units as significant at P < 0.05.

Structural Analogs

Steroid analogs were delivered to sperm in two forms. A few steroids were used in the form of PC liposomes. To achieve significant transfer of steroid into sperm, liposomes were used at 30 µM PC. This high concentration increases the loss of sperm sterols and elevates the response of sperm to P4 [24]. The appropriate control is sperm treated with steroid-free liposomes, but data are also presented for sperm that were not exposed to liposomes. Unless it is stated that the steroid was incorporated into liposomes, it was diluted into medium from a stock solution in an organic solvent. Ethanol was used as a solvent most often, but for some steroids, it was necessary to use isopropanol, DMSO, or DMF. The solvent concentration did not exceed 0.05% (v/v) and did not affect sperm viability, number of spontaneous acrosome reactions, or response to progesterone (data not shown). When more than one solvent was used for a steroid, the results were pooled.

One group of steroids did not efficiently transfer into sperm and had no effect on capacitation or spontaneous acrosome reactions. This group include 5{alpha}-cholestane and 5-cholestene (2 µM in liposomes), 3ß-chloro-5-cholestene (5 µM), and stigmasterol (15 µM). These steroids were 6% or less of the sperm steroid content after 24 h.

A second group of steroids comprised 4-cholesten-3-one and the androstane-derivatives 16, 5{alpha}-androstan-3ß-ol; 16, (5{alpha}) androsten-3{alpha}-ol; 16, (5{alpha}) androsten-3ß-ol; and 5{alpha}-androstan-3-one. These steroids had varying abilities to transfer to sperm. All of them significantly inhibited capacitation, but all of them also induced significant numbers of sperm to acrosome react without exposure to P4. Two of these steroids were studied in detail. 4-Cholesten-3-one transferred to sperm efficiently from liposomes (15 µM steroid), representing 62% of the sperm steroid content and elevating the total steroid content by 1.28 nmole/107 sperm compared to sperm incubated with steroid-free liposomes (Fig. 4). Capacitation was inhibited 85% compared to sperm incubated with steroid-free liposomes and 68% compared to untreated control sperm. The unexpected finding was that the incidence of spontaneously reacted sperm was significantly elevated compared to control sperm (Fig. 4). When 4-cholesten-3-one was added to 24-h sperm (5–15 µM) either alone or with P4, it did not induce acrosome reactions or alter the percentage of sperm that reacted in response to P4 (Table 1). Therefore, induction of acrosome reactions by 4-cholesten-3-one differs from induction by P4. Although 4-cholesten-3-one inhibited capacitation effectively, the fact that it induced acrosome reactions suggests that it acts on sperm differently from the endogenous sterols, cholesterol and desmosterol.



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  		FIG. 4. Effect of 4-cholesten-3-one. Sperm were incubated 24 h with steroid in liposomes (4C3), liposomes alone (PC), or with no addition (Con). A) Sterol content was determined in freshly prepared control sperm (Con-T0) and in 24-h sperm (T24) of each treatment group: cholesterol (open bars); desmosterol (striped bars); 4-cholesten-3-one (solid bar; n = 3). B) Effect of 4-cholesten-3-one on capacitation was assessed at 24 h: spontaneously acrosome-reacted sperm (S); sperm that acrosome-reacted in response to P4 (P). Incorporation of 4-cholesten-3-one triggered acrosome reactions and reduced the number of sperm that can respond to P4. Error bars indicate the SEM (n = 4)


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  		TABLE 1. Percentage of acrosome-reacted sperm occurring when 24-h capacitated sperm were exposed to steroids either alone or simultaneously with P4.a

Sperm incubated with liposomes containing 5{alpha}-androstan-3ß-ol incorporated a small amount of the steroid (Fig. 5), which increased the total steroid content relative to sperm incubated with steroid-free liposomes but not relative to untreated control sperm. Nevertheless, capacitation was inhibited approximately 65% relative to both groups of sperm (Fig. 5). The incidence of acrosome-reacted sperm was elevated in the 5{alpha}-androstan-3ß-ol-treated group (Fig. 5). Similar results were obtained for sperm incubated with 15 µM 5{alpha}-androstan-3ß-ol diluted from stock solutions in DMSO or DMF (data not shown). The elevated incidence of reacted sperm suggested that this steroid was behaving anomalously, and this was confirmed when the steroid was applied to 24-h capacitated control sperm (Table 1). A small but significant number of sperm acrosome-reacted. When 5{alpha}-androstan-3ß-ol was added together with P4, the percentage of responding sperm was reduced by almost half.



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  		FIG. 5. Effect of 5{alpha}-androstan-3ß-ol on capacitation. Sperm were incubated 24 h with steroid in liposomes (And), liposomes alone (PC), or with no addition (Con). A) Sterol content was determined in freshly prepared control sperm (Con-T0) and in 24-h sperm (T24) of each treatment group: cholesterol (open bars); desmosterol (striped bars); 5{alpha}-androstan-3ß-ol (solid bar; n = 3). B) Effect of 5{alpha}-androstan-3ß-ol on capacitation was assessed at 24 h: spontaneously acrosome-reacted sperm (S); sperm that acrosome-reacted in response to P4 (P). Incorporation of 5{alpha}-androstan-3ß-ol triggered acrosome reactions and reduced the number of sperm that could respond to P4. Error bars indicate the SEM (n = 6)

A third group of sterols comprised epicholesterol, cholesteryl methyl ether, ergosterol, and diosgenin. These transferred to sperm efficiently and inhibited capacitation without affecting the amount of endogenous sterols or triggering acrosome reactions. Furthermore, when these sterols were added to control 24-h capacitated sperm, they did not induce acrosome reactions or alter the response of the sperm to P4 (Table 1). Each was inspected over a range of concentrations. Although quantitative measures like the EC50 were quite variable among replicates, both uptake of steroid and inhibition of capacitation were dependent on dose. Lower concentrations caused less uptake and less inhibition of capacitation, so the data presented below, obtained at the highest doses, are not in a plateau region of maximal effects.

Sperm incubated with 20 µM cholesteryl methyl ether incorporated 0.554 ± 0.011 nmole/107 sperm (n = 4) (Fig. 6). This accounted for 27% ± 3% of the total sperm sterol. The response of the sperm to P4 was inhibited (P < 0.05) by 43% ± 13% (n = 5).



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  		FIG. 6. Effect of cholesteryl methyl ether on capacitation. A) Sterols of freshly prepared sperm (Con-T0) and 24-h sperm (T24) that had been incubated with cholesteryl methyl ether (CME) or without steroid (Con): cholesterol (open bars); desmosterol (striped bars); cholesteryl methyl ether (solid bar; n = 4). B) Effect of cholesteryl methyl ether on capacitation was assessed at 24 h: spontaneously acrosome-reacted sperm (S); sperm that acrosome-reacted in response to P4 (P). Error bars indicate the SEM (n = 5)

Sperm incubated with 12.6 µM ergosterol incorporated 0.259 ± 0.051 nmole/107 sperm (n = 4) (Fig. 7). This represented 14% ± 2% of the total sperm sterol. The response of the sperm to P4 was inhibited (P < 0.05) by 51% ± 11% (n = 4) compared to control sperm.



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  		FIG. 7. Effect of ergosterol on capacitation. A) Sterols of freshly prepared sperm (Con-T0) and of 24-h sperm (T24) that had been incubated with ergosterol (Erg) or without steroid (Con): cholesterol (open bars); desmosterol (striped bars); ergosterol (solid bar; n = 4). B) Effect of ergosterol on capacitation was assessed at 24 h: spontaneously acrosome-reacted sperm (S); sperm that acrosome-reacted in response to P4 (P). Error bars indicate the SEM (n = 4)

Sperm incubated with 12 µM diosgenin incorporated 1.34 ± 0.14 nmole/107 sperm (n = 3) (Fig. 8). This represented 47% of the total sperm sterol. The response of the acrosome to P4 was inhibited (P < 0.01) by 96% ± 2% compared to control sperm.



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  		FIG. 8. Effect of diosgenin on capacitation. A) Sterols of freshly prepared sperm (Con-T0) and of 24-h sperm (T24) that had been incubated with diosgenin (Dio) or without steroid (Con): cholesterol (open bars); desmosterol (striped bars); diosgenin (solid bar; n = 3). B) Effect of diosgenin on capacitation was assessed at 24 h: spontaneously acrosome-reacted sperm (S); sperm that acrosome-reacted in response to P4 (P). Error bars indicate the SEM (n = 4)

Sperm incubated with 6–13 µM epicholesterol incorporated 0.961 ± 0.149 nmole/107 sperm (n = 3) (Fig. 9). This represented 46% ± 3% of the total sperm sterol. The response of the sperm to P4 was inhibited 85% ± 7% (n = 6).



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  		FIG. 9. Effect of epicholesterol on capacitation. A) Sterol content in freshly prepared sperm (T0), and in 24-h sperm that had been incubated with epicholesterol (ECh) or without steroid (Con): cholesterol (open bars); desmosterol (striped bars); epicholesterol (solid bar; n = 3). B) Effect of epicholesterol on capacitation was assessed at 24 h: spontaneously acrosome-reacted sperm (S); sperm that acrosome-reacted in response to P4 (P). Error bars indicate the SEM (n = 6)

Four additional steroids may belong to this group: cholesteryl-3-formate, fucosterol, 5-cholesten-7-one, and 5-cholesten-3ß,19-diol. Each of these steroids inhibited capacitation effectively (Table 2), but the amount of steroid in the sperm and whether the concentrations of endogenous sterols were affected have not been determined.


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  		TABLE 2. Steroids for which the sperm steroid content was not measured

The final group of steroids comprised coprostanol and epicoprostanol, two steroids with nonplanar ring structures. When sperm were incubated 24 h with 5–18 µM coprostanol, they incorporated 1.58 ± 0.31 nmole/107 sperm (n = 9) (Fig. 10). This represented 43% ± 5% of the total sperm steroid content. Unexpectedly, cholesterol of coprostanol-treated sperm was 0.50 nmole/107 sperm greater than in control sperm. The response to P4 was significantly (P < 0.01) reduced by 33% ± 7% (n = 12) in coprostanol-loaded sperm compared to control sperm, but this inhibition was small considering the amount of steroids in the treated sperm (see Discussion). Sperm incubated 24 h with 6 µM epicoprostanol incorporated 0.452 ± 0.052 nmole/107 sperm (n = 4) (Fig. 11). This accounted for 27% ± 2% of the total sperm steroid content. Epicoprostanol did not induce acrosome reactions alone, but the number of epicoprostanol-treated sperm that responded to P4 was 70% ± 27% (n = 4) (P < 0.05) greater than the number of control sperm that responded. Adding epicoprostanol to 24 h-incubated control sperm (alone or simultaneously with P4) had no effect (Table 1).



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  		FIG. 10. Effect of coprostanol on capacitation. A) Sterol content in freshly prepared sperm (T0) and in 24-h sperm that had been incubated with coprostanol (Cop) or without steroid (Con): cholesterol (open bars); desmosterol (striped bars); coprostanol (solid bar; n = 9). B) Effect of coprostanol on capacitation was assessed at 24 h: spontaneously acrosome-reacted sperm (S); sperm that acrosome-reacted in response to P4 (P). Error bars indicate the SEM (n = 12). Coprostanol inhibited capacitation, but the amount of sperm cholesterol was greater than in control 24-h sperm



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  		FIG. 11. Effect of epicoprostanol on capacitation. A) Sterol content in freshly prepared sperm (T0) and in 24-h sperm that had been incubated with epicoprostanol (ECp) or without steroid (Con): cholesterol (open bars); desmosterol (striped bars); epicoprostanol (solid bar; n = 4). B) Effect of epicoprostanol on capacitation was assessed at 24 h: spontaneously acrosome-reacted sperm (S); sperm that acrosome-reacted in response to P4 (P). Error bars indicate the SEM (n = 4). Epicoprostanol increased the number of capacitated sperm

None of the structural analogs affected sperm viability (data not shown). Gas chromatography of the extracts of sperm that had been treated with structural analogs did not reveal significant conversion to other molecules, because the only appreciable steroid peaks other that of than the analog being tested were cholesterol and desmosterol (data not shown).

The ability of steroids to promote a tightly packed and ordered state of phospholipids was assessed by fluorescence anisotropy of DPH in egg PC liposomes. Most steroids were easily incorporated into liposomes by sonication. No results were obtained for 5-cholesten-7-one, which incorporated less than 5 mol % into liposomes, and for 5-cholesten-3ß,19-diol, which could not be reliably quantified by our gas chromatographic assay. For the remainder of the steroids, the anisotropy at 20 mol % steroid was interpolated from plots of r versus mol % steroid. Desmosterol was approximately as effective as cholesterol at increasing the anisotropy (creating order). The other steroids presented a range of abilities to order egg PC, with epicoprostanol, 4-cholesten-3-one, coprostanol, and 5{alpha}-androsten-3ß-ol being the least effective (Table 3).


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  		TABLE 3. DPH fluorescence anisotropy in egg PC with or without 20 mol % steroid.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study investigated the structural features of the cholesterol molecule that are required for controlling sperm capacitation. Although cholesterol inhibits fertilization of rabbit and rodent eggs (reviewed in [27]), capacitation of human sperm cannot be assessed by fertilization, so the ability of sperm to acrosome-react when exposed to P4 was employed.

One goal was to test the hypothesis that cholesterol's role is to control the orderly packing of phospholipids. The approach was to use structural analogs of cholesterol that should vary in their ability to create phospholipid order and determine whether they inhibited sperm capacitation. The structural features that most affect a steroid's ability to create order are the ß-oriented OH on C3, cholesterol-like tail on C17, and planar ring structure (reviewed in [39]).

First, the inhibitory activities of the two main endogenous sterols were examined. Cholesterol and desmosterol differ in their degree of saturation of the C17 tail. Although it was not possible experimentally to replace all the endogenous sterol with a single sterol, the amount and composition of the endogenous sterol content could be manipulated. Adding desmosterol or cholesterol to the medium maintained a high level of the added sterol in sperm, probably by reducing its net efflux [48], whereas the other sterol decreased normally. The results show that cholesterol and desmosterol have similar inhibitory activities. First, when inhibition of capacitation was plotted against the total sterol concentration, the EC50 values were the same for desmosterol and cholesterol treatments. At the EC50, the sterol content of sperm treated with cholesterol was almost entirely cholesterol, whereas desmosterol-treated sperm contained a mix of approximately equal amounts of cholesterol and desmosterol. Second, the slopes of the linear midranges of the dose-response curves were not significantly different for desmosterol and cholesterol treatments, demonstrating that regardless of the sterol, each 1 nmol added inhibited a similar amount.

Because of the abundance of desmosterol in sperm and its robust inhibitory activity, it should be considered as important as cholesterol in the control of capacitation. Although sperm start with less desmosterol, the molar losses of the two sterols are approximately the same, because a greater fraction of desmosterol is lost [47]. Perhaps, as in other cells, desmosterol efflux is faster than cholesterol efflux [49].

Next, the activities of 19 other steroids were tested. The only steroid that was incorporated into the sperm and completely failed to inhibit capacitation was epicoprostanol. In fact, it had the unexpected effect of increasing the number of capacitated sperm. Its structure differs from that of cholesterol in that it has a nonplanar ring structure and its 3-OH group is in {alpha} orientation. Coprostanol, having a nonplanar ring structure but a cholesterol-like ß-OH group, weakly inhibited capacitation. The cholesterol content of coprostanol-treated sperm was elevated by 0.5 nmole/107 sperm. Considering the efficacy of cholesterol to inhibit capacitation as determined in the first part of the present study, the additional cholesterol is more than sufficient to explain the modest inhibition of capacitation observed during treatment with coprostanol. For this reason, coprostanol itself likely has little or no inhibitory activity, corroborating the importance of a planar ring structure. Whether the capacitation-promoting effect of epicoprostanol is unique is unclear at this point. The {alpha}-OH group cannot be the sole cause, because epicholesterol effectively inhibited capacitation. The effect must result either from the nonplanar rings or from the combination of nonplanar rings and the {alpha}-OH orientation. Possibly, coprostanol would also show this effect if it had been incorporated into sperm with less cholesterol.

Other modifications of the ring structure were tolerated. Ergosterol is 7-unsaturated and was an effective inhibitor. The oxysterol 5-cholesten-3ß,19-diol also inhibited capacitation. The addition of the 19-OH group to the region of the molecule that is normally deep in the hydrophobic core of the bilayer should markedly alter the orientation of the molecule in the membrane, as has been shown for other oxysterols [50]. The present results with this sterol should be considered provisional, because its effect on sperm sterol content has not been determined.

Two sterols with modified 3-OH groups effectively inhibited capacitation. Cholesteryl methyl ether has an oxygen atom in ether linkage on C3, and is incapable of contributing a hydroxyl hydrogen atom for hydrogen bonding with adjacent molecules. Epicholesterol has a hydroxyl group in {alpha}-linkage, in contrast to the ß-OH group in cholesterol. The orientation of the 3-OH group is evidently not critical for inhibitory activity. The definitive test regarding the importance of the 3-OH group is whether 5-cholestene, which completely lacks this group, has inhibitory activity, but it has not been possible to transfer it into sperm. The ketosteroid 5-cholesten-7-one lacks a C3 oxygen yet strongly inhibited capacitation, as did cholesteryl-3-formate and cholesteryl-3-sulfate [23, 51]. (Again, one must be cautious about interpreting the results with 5-cholesten-7-one, cholesteryl-3-formate, and cholesteryl-3-sulfate, because the effect of these steroids on the amounts of endogenous sterols is not known.) Because steroids that lack a 3-OH group inhibit capacitation, we can rule out a model in which cholesterol's 3-OH group is covalently modified (e.g., as in cholesterol's formation of a lipid adduct to the hedgehog family of signaling molecules [52]).

The 3-ketosteroid 4-cholesten-3-one was incorporated by sperm and caused approximately 20% of the sperm to acrosome-react during 24-h incubation. Although this steroid shares a 3-keto group with P4, this does not account for its ability to induce acrosome reactions. The 3-keto group is not required for the activity of P4 [53], and 24-h capacitated control sperm did not acrosome-react when exposed to 4-cholesten-3-one. The effect may be related to the ability of 3-ketosteroids to increase the permeability of phospholipid bilayers [54, 55].

To examine the role of the C17 aliphatic tail, several androstane derivatives lacking C17 groups were tested. They proved to be potent inhibitors of capacitation. However, these steroids also elevated the number of spontaneous acrosome reactions (i.e., in the absence of P4). Moreover, when 5{alpha}-androstan-3ß-ol was added to control sperm at 24 h, it induced some sperm to acrosome-react and reduced the response of the remaining sperm to P4. This antagonist-weak agonist behavior is similar to the effects of testosterone and related molecules on the sperm intracellular calcium concentration [53]. Possibly, the androstane derivatives interact with the putative sperm P4 receptor to activate pathways that interfere with the response to P4. These results suggest that the mechanism by which androstane derivatives inhibit capacitation is different from the endogenous sterols, so these steroids do not presently help us to understand the effects of cholesterol and desmosterol on sperm.

The similar efficacy of desmosterol and cholesterol as inhibitors suggests that some modification of the C17 tail can be tolerated without loss of inhibitory activity, and this was confirmed with two additional steroids. Diosgenin is a spirosten derivative having two oxygen-containing rings in place of the iso-octane chain of cholesterol, one of them fused to the cyclopentane ring of the steroid nucleus. This produces a much bulkier and more rigid group than the iso-octane chain of cholesterol. Nevertheless, diosgenin effectively inhibited capacitation. Compared to cholesterol, ergosterol has an additional methyl group at C24 as well as a rigidifying double bond at C22, and it inhibited capacitation. Fucosterol, with a bulkier tail than ergosterol, also inhibited capacitation. Similarly, sitosterol, which has a C24 ethyl group, has been reported to render capacitated sperm unresponsive to P4 [56].

The ability of steroids to inhibit capacitation correlated with their ability to order egg PC, as assessed by anisotropy of DPH. All steroids for which r >= 0.120 (i.e., those that were best at promoting PC order) inhibited capacitation, and the two steroids with the least ability to inhibit capacitation (coprostanol and epicoprostanol) were also poor at ordering egg PC. Desmosterol and cholesterol had similar abilities to produce order and equal abilities to inhibit capacitation. It is surprising, though, that the large difference between the ordering effects of cholesterol and of the weaker steroids, epicholesterol and cholesteryl methyl ether, was not reflected in their ability to inhibit capacitation. The ordering effects of the steroids reported here are consistent with previous reports [29, 57–60].

One might also consider whether these data fit a model in which the loss of sperm sterols alters sterol-rich lipid rafts, in turn triggering capacitation. The emerging view derived from model systems is that the ability of a sterol to promote raft formation depends on its ability to create order among saturated phospholipids [30, 31], so a plausible model for the downstream effect of sterol loss could include modification of raft structure. Such comments are highly speculative at this point, however.

In summary, steroids require a planar ring structure to be inhibitory, but the C17 tail and C3-OH can be extensively modified without loss of activity. The ability of steroids to inhibit capacitation correlates with their ability to create order in egg PC. The results rule out a model in which sterols inhibit capacitation via reaction of the 3-OH group and support the idea that a bulk effect on phospholipid order is important in the control of capacitation.


   ACKNOWLEDGMENTS
 
We appreciate the excellent technical assistance of Angela Sitterly.


   FOOTNOTES
 
1 Supported by NIH grant HD30763. Back

2 Correspondence: Nicholas L. Cross, Department of Physiological Sciences, 264 McElroy Hall, Oklahoma State University, Stillwater, OK 74078. FAX: 405 744 8263; ncross{at}okstate.edu Back

Received: 18 June 2002.

First decision: 11 July 2002.

Accepted: 23 October 2002.


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