Role of Cholesterol in Sperm Capacitation¹

^a Department of Anatomy, Pathology, and Pharmacology, Oklahoma State University, Stillwater, Oklahoma 74078

	INTRODUCTION

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Forty years ago, M.C. Chang found that exposing capacitated rabbit sperm to seminal plasma made them incapable of fertilizing eggs in vivo [1]. The results of this rather unphysiological experiment had a strong impact on the study of sperm capacitation, which Chang [2] and Austin [3] had discovered six years earlier. It was widely believed that understanding how seminal plasma reverses capacitation would shed light on how sperm become capacitated, and considerable effort was devoted to identifying the inhibitory agents in seminal plasma. The candidates included proteins, peptides, and lipids [4, 5].

Beginning with his work on the inhibitory activity of seminal plasma, Brian Davis and his coworkers built a strong case for lipids playing an important role in capacitation. Davis showed that sperm could be "decapacitated" by a vesicle fraction prepared from seminal plasma (reviewed in [6]). The activity of the fraction was diminished by partially extracting the lipids and could be mimicked by synthetic phospholipid vesicles containing cholesterol. Davis suggested that the vesicles changed the lipid composition of the sperm plasma membrane and, secondly, that the changes were the reverse of alterations that normally occur during capacitation.

In Davis's model the cholesterol/phospholipid (C/PL) ratio of the sperm plasma membrane determines the capacitation state of the sperm. A freshly ejaculated sperm has a high C/PL ratio; and during capacitation, cholesterol moves from the sperm membrane to soluble protein acceptors, and/or phospholipid moves into the sperm membrane. At the time this model was formulated, it was generally believed that capacitation caused sperm to acrosome-react spontaneously (that is, without exposure to a specific inducer). In Davis's model, the falling C/PL ratio triggered an acrosome reaction. Explanations for how this might happen were based on information obtained in other systems; and it was suggested that a lower C/PL ratio decreased the membrane microviscosity, relaxed the packing of phospholipids in the membrane, and perhaps permitted greater calcium influx, all leading through unspecified intermediate steps to fusion of the plasma and outer acrosomal membranes.

The early work that supported a role for cholesterol in the control of sperm function has been extensively reviewed [6–9]. The present review will emphasize more recent work and will summarize current information regarding how cholesterol might act. To conform to the format of a minireview, not all of the available literature is cited, and preference is given to citing recent papers; their bibliographies will lead to earlier work.

	CONTENT AND DISTRIBUTION OF STEROLS IN MAMMALIAN SPERM

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In most species that have been studied, cholesterol is the major sterol of ejaculated sperm. Other sterols are often present, including desmosterol (the immediate precursor of cholesterol), cholesta-7,24-dien-3ß-ol, desmosterol sulfate, cholesterol sulfate, and cholesterol esters [10–14]. Some of these molecules have been suggested to be important regulators of sperm function [15], but this review will focus on cholesterol because of its abundance. The extent to which sperm can interconvert these forms needs to be better understood. Most of the work that will be discussed below assumes that cholesterol is the active agent, but in fact a molecule whose concentration mirrors the concentration of cholesterol could be just as important or even more so.

In somatic cells, most unesterified cholesterol is in membranes, with the plasma membrane containing the highest concentration [16]. Consistent with this distribution, the C/PL ratio of isolated sperm plasma membrane fraction is higher than that of the acrosomal membrane fraction [17]. Studies employing filipin, a polyene antibiotic that binds to 3ß-hydroxysterols to form complexes visible by electron microscopy, confirm this distribution and also suggest that sterols are not uniformly distributed in the plane of the plasma membrane. Filipin-sterol complexes are concentrated in the plasma membrane overlying the acrosome, with less in the posterior region of the head and the flagellum [18]. These observations should be interpreted with caution, however, because filipin-sterol complexes may redistribute in the plane of the membrane [19].

The concentration of cholesterol in the sperm plasma membrane varies considerably among species. The C/PL molar ratios for isolated plasma membrane fractions are about 0.20 in boar sperm [20], 0.36 in stallion sperm [20], about 0.40 in bovine sperm [17], 0.43 in ram sperm [21], and 0.83 in human sperm [22]. Sperm plasma membrane fractions usually originate principally from the anterior head, so these ratios may be more typical of that region than of the entire plasma membrane. It is important to note that these C/PL ratios are not unusual for plasma membranes. The red blood cell membrane has a C/PL molar ratio of about 0.9, and the ratios in plasma membranes of nucleated cells range from 0.4 to 0.8 [23]. These data argue against the view that a recently ejaculated sperm is incapable of acrosomal exocytosis because its plasma membrane is somehow "frozen" by an extremely high concentration of cholesterol.

What is the source of sperm cholesterol? Human sperm lysates supplemented with NADPH can synthesize cholesterol from acetate [24], but the activity is probably too low to contribute significantly to the free cholesterol content of 2.9 x 10^-16 mol/sperm [25]. It is generally believed that sperm obtain most of their cholesterol from their environment. Little is known about sterol dynamics of testicular sperm membranes. In some species the plasma membrane cholesterol concentration changes as the sperm passes through the epididymis. The C/PL ratio of ram and goat sperm plasma membrane fractions increases during epididymal transit [21, 26], as does the content of filipin-reactive sterols in the anterior head plasma membrane of hamster sperm [18]. An increase in sterol concentration of the plasma membrane is not universal, however. The sterol content of rat and equine sperm plasma membranes decreases as sperm move through the epididymis [27, 28]. The C/PL ratio of the plasma membrane fraction of boar sperm does not change [12], although filipin detects an increase of about 20% in the sterol concentration of the anterior head plasma membrane [29]. How these changes come about is not clear, especially in those instances in which the concentration of cholesterol in the plasma membrane varies in the opposite direction from the total cell cholesterol.

Ejaculated sperm may obtain additional cholesterol from seminal plasma. Human seminal plasma contains about 250 µg cholesterol per milliliter at a C/PL molar ratio of 5.9, much higher than the ratio of 1.0 present in sperm [30, 31]. The cholesterol resides in lipoproteins and in vesicles [32–34].

	CHOLESTEROL AND SPERM FUNCTION IN VITRO

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When sperm are incubated in a capacitating medium in vitro, the sperm cholesterol content gradually decreases [25, 35]. The loss of cholesterol from human sperm is initially linear, but acrosomal responsiveness appears only after a delay of several hours, suggesting that cholesterol loss precedes the development of responsiveness [25]. Incubation reduces the density of filipin-sterol complexes in the plasma membrane of the anterior sperm head [36], or in some cases, causes clearing of filipin-sterol complexes from small areas of the membrane [37].

Where does the sperm cholesterol go? It must either be converted to another molecule or exit the cell. Davis et al. [38] reported that sperm can convert a substantial portion of radiolabeled cholesterol to another form, but the material was not identified, and little is known about the ability of sperm to carry out such reactions. Current thinking is that most cholesterol loss is due to slow diffusion from the cell, and a net transfer of cholesterol from rat and bovine sperm to the medium has been demonstrated [38, 39]. Most incubation media contain serum albumin, which binds cholesterol efficiently, reducing cholesterol influx and therefore increasing the net loss of cholesterol. Albumin preparations often contain lipid transfer protein-I, which further accelerates the movement of lipids [40].

The amount of sperm cholesterol lost during incubation in vitro can be manipulated by adjusting the cholesterol concentration or the cholesterol-accepting ability of the medium, allowing one to test the dependence of a sperm function on the sperm cholesterol content. Adding cholesterol to the medium prevents sperm from becoming able to undergo an acrosome reaction [25] or fertilize eggs [35]. When the loss of cholesterol was progressively diminished through addition of increasing amounts of cholesterol to the medium, the number of sperm that became acrosomally responsive was reduced in an amount that matched the inhibition of cholesterol loss [25]. On the other hand, increasing cholesterol loss by augmenting the cholesterol-binding capacity of the medium with phosphatidylcholine liposomes caused a parallel increase in the number of responsive sperm [25]. Thus there is strong evidence that the sperm concentration of free cholesterol controls sperm acrosomal responsiveness.

	HOW DOES CHOLESTEROL CONTROL SPERM FUNCTION?

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In the years since Davis's work, many ideas have been offered to explain how cholesterol controls sperm function, but few of the hypotheses have been tested experimentally. It is often suggested that loss of cholesterol directly affects the sperm plasma membrane lipid bilayer to make it "fusogenic." This idea is consistent with the observations that the acrosomal and plasma membranes of cholesterol-inhibited sperm do not fuse upon treatment of the sperm with inducers of the acrosome reaction [41], and that membranes isolated from bovine sperm can fuse in vitro only if the membranes are prepared from previously capacitated sperm [42]. There is no evidence, however, that these effects are under direct control of cholesterol. The early reports that cholesterol inhibited membrane fusion in somatic cell systems have largely been reevaluated, and it is now appreciated that cholesterol generally promotes membrane fusion rather than inhibiting it [43]. So, while this model has not been disproven, there is little evidence to support it.

One model that is currently being investigated is that cholesterol loss leads to exposure on the sperm surface of a receptor for mannose [9]. It is proposed that the receptor functions in sperm-zona pellucida interaction because 1) mannose and mannosylated proteins interfere with sperm binding to the zona pellucida, 2) mannose and mannosylated proteins induce the acrosome reaction, and 3) the zona pellucida contains mannose (see [44]). This model would not explain how cholesterol inhibits the development of responsiveness to progesterone and Ca²⁺/H⁺-exchanging ionophores [41].

The mannose receptor has been detected by labeling sperm with fluoresceinated, mannosylated BSA (FM-BSA) in buffers containing 20 mM calcium. Human sperm incubated 18 h in vitro exhibit increased labeling with FM-BSA, increased spontaneous acrosome reactions, and loss of sperm cholesterol. Sperm of a small group of males did not lose cholesterol, had few spontaneous acrosome reactions, and bound little FM-BSA, suggesting that in sperm of normal males, loss of sterol leads to expression of the mannose receptor [45]. Binding of FM-BSA to the anterior head of uncapacitated sperm increased when the sperm membrane was disrupted, so it was suggested that the mannose receptor initially resides beneath the plasma membrane and that cholesterol efflux causes the mannose receptor to move into the phospholipid bilayer [45]. Support for the idea that cholesterol loss triggers expression of the mannose receptor came from evidence that cholesterol-enriched medium prevented the expression of mannose receptors [46]. This result is questionable, however, because the experimental treatment increased the sperm cholesterol content well above its normal concentration.

The subcellular location and identity of the mannose receptor have also been questioned. Simultaneous assessment of the FM-BSA labeling and the viability of individual sperm showed that FM-BSA preferentially labels dead sperm [44, 47]. The similarity of the FM-BSA-labeling pattern to the distribution of acrosomal contents [48], the increased labeling seen when sperm membranes were experimentally disrupted, and the preferential labeling of dead sperm with leaky membranes led Chen et al. [44] to suggest that the receptor might be the acrosomal protein, proacrosin. More work is required to identify the mannose receptor and to locate it unequivocally.

Another model under investigation is that cholesterol loss leads to a rise in intracellular pH (pH_i) that is required in order for sperm to become acrosomally responsive. The development of acrosomal responsiveness in hamster, bovine, and mouse sperm is associated with elevations of pH_i [49–51], and treatments that prevent pH_i from increasing also prevent sperm from becoming acrosomally responsive [51, 52].

During 24-h incubation of human sperm in vitro, the fluorescent pH-sensitive probe 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein showed that the average pH_i increased 0.14 units, from 6.94 to 7.08 [53]. The pH_i did not increase when cholesterol loss was prevented by incubating sperm in cholesterol-enriched medium, demonstrating the cholesterol dependence of this event. To determine whether the rise in pH_i is required for the development of acrosomal responsiveness in human sperm, we exploited the observation of Hamamah et al. [54] that human sperm pH_i varies with the extracellular pH. Incubating sperm in media of low pH prevented the rise of sperm pH_i, and under those conditions, fewer sperm became acrosomally responsive (unpublished results).

These results make a case for cholesterol loss triggering a rise in pH_i that in turn promotes the development of acrosomal responsiveness. We have been unable, however, to overcome cholesterol inhibition by experimentally increasing pH_i [53]. Therefore it appears that the rise of pH_i is only one of several consequences of cholesterol loss that are required in order for a sperm to become acrosomally responsive.

Effects of cholesterol on pH_i have been noted before, using platelets and fibroblasts [55, 56]. How could membrane cholesterol modify pH_i? The control of pH_i is complex and depends on intracellular buffers, production of metabolic acids, and acid and/or base transport activities. Cholesterol alters the activity of the Na⁺-H⁺ antiporter [55–57] and the erythrocyte Cl^-/HCO₃^- exchanger. In the latter case, however, the direction of the effect is such that a loss of cholesterol would probably lower pH_i rather than raise it [58]. The pH_i is not determined solely by the activity of the plasma membrane exchangers, and it appears that during capacitation of bovine sperm, pH_i rises because sperm lose the ability to regulate pH_i, not because a new set point has been established [50]. In this regard it may be relevant that the passive proton permeability of mitochondrial membranes is inversely related to their cholesterol content [59].

	DOES CHOLESTEROL CONTROL SPERM FUNCTION IN VIVO?

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One would like to recover capacitated sperm from the site of fertilization and determine their cholesterol content, but the small number of sperm available for such observations makes this experiment difficult. Most studies so far have examined whether incubating sperm in vitro with fluids of the female reproductive tract causes cholesterol loss, with positive results. For example, incubating bovine sperm in vitro with oviductal fluid caused a net transfer of about 25% of the sperm cholesterol to high-density lipoproteins [60]. In other studies, radiolabeled cholesterol transferred from sperm to lipoproteins or albumin in uterine fluid and follicular fluid [61, 62].

In one study, the sterol concentration of boar sperm was determined before and after incubation in ligated uteri under capacitating conditions, and no change was found [63]. On the other hand, when the density of filipin-sterol complexes was compared on ejaculated boar sperm and sperm recovered from uteri 2 h after mating, the density on the capacitated sperm was reduced 13–21%, depending on the region of the head [29]. As noted by Parks and Ehrenwald [8], the cholesterol concentration of the boar sperm plasma membrane is so low that cholesterol loss may not be as important during capacitation in this species.

	RELATIONSHIP TO MALE FERTILITY

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In humans, the amount of cholesterol in sperm varies considerably among ejaculates, and although the number of men who have been studied is small, there is some reason to believe that elevated sperm cholesterol content might contribute to infertility. The C/PL ratio in sperm of patients with unexplained infertility is about twice that of fertile donors [64]. Interestingly, the difference is due solely to a lower phospholipid content of the patient sperm. Sperm of normospermic men who failed to fertilize eggs in vitro were found to be characterized by abnormally high cholesterol content, or by a slow loss or even an increase in cholesterol during in vitro incubation [65]. These sperm also failed to undergo spontaneous acrosome reactions. A possible explanation for these observations was offered by Hoshi et al. [66], who reported that sperm with a high C/PL ratio are slower to capacitate (assayed by penetration of zona-free hamster oocytes) than sperm with a lower C/PL ratio. Why the lipid composition of sperm varies among men is unclear. There appears to be no correlation between the amount of cholesterol or phospholipid in blood serum and that in sperm or seminal plasma, suggesting that sperm cholesterol content is regulated locally within the male reproductive tract [31].

	SUMMARY

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There is now substantial evidence that the cholesterol content of mammalian sperm affects the development of acrosomal responsiveness and fertilizing ability in vitro. There may be some species that do not fit this model, and this needs to be clarified. The major challenges in the near future are to better understand the mechanism of cholesterol action in susceptible species and to determine whether control of sperm function by cholesterol is important in vivo.

	FOOTNOTES

 
¹ Supported by NIH grant HD30763 and the Oklahoma Center for the Advancement of Science and Technology.

² Correspondence: FAX: (405) 744-8263; ncross{at}vm1.ucc.okstate.edu

Accepted: February 22, 1998.

Received: November 20, 1997.

	REFERENCES

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