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From the Program for the Topical Prevention of Conception and Disease
(TOPCAD) and * Departments of Obstetrics and
Gynecology, Rush University Medical Center, Chicago, Illinois; and the
Biopharmaceutical Sciences, University of
Illinois at Chicago, Chicago, Illinois.
| Correspondence to: Robert A. Anderson Jr, Ob/Gyn Research, Rush Medical Center, Chicago, IL 60612 (e-mail: randerso{at}rush.edu). |
| Received for publication August 29, 2005; accepted for publication February 10, 2006. |
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key words: Topical contraceptive microbicide, signal transduction, spermatozoa, mechanism, calcium channels
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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More than 1 in 5 individuals over the age of 12 in the United States are infected with the herpes simplex virus (HSV-2) (Lafferty, 2002). HSV and other genital tract infections enhance HIV transmission (Cohen, 1998; Mbopi-Keou et al, 2000). Gonorrhea rates among adolescents are high in several countries, including the United States (Barberis et al, 1998; Panchaud et al, 2000). Chlamydial infections are higher among female teenagers than among males of the same age (Panchaud et al, 2000).
Unplanned pregnancies present additional risk to women's reproductive health. Nearly half of these in the United States are terminated (Henshaw, 1998). Safe contraceptive microbicides will improve the reproductive health of women and the population at large.
Sulfuric acid modified mandelic acid (SAMMA) is a carboxylated oligomeric (average molecular weight of less than 2 kd) compound from the Topical Prevention of Conception and Disease (TOPCAD) Program, under development as a contraceptive microbicide. A license has been secured (Yaso Biotechnology Inc, Coppell, Tx) for its commercialization under US patent no. 5932619 (Zaneveld et al, 1999). It is active against HIV, HSV, and Chlamydia trachomatis, among other sexually transmitted pathogens (Zaneveld et al, 2002). SAMMA is active against spermatozoa; it inhibits hyaluronidase and acrosin (required for fertilization), causes premature acrosomal loss (AL), and is contraceptive in the rabbit. However, sperm motility is unaffected, suggesting that SAMMA is not acting as a spermicide (Zaneveld et al, 2002).
SAMMA's relatively simple method of synthesis and its low molecular weight lends itself to chemical modifications that could improve efficacy and selectivity of action. Prerequisite to more active SAMMA derivatives is the knowledge of its mechanism(s) of action. SAMMA disrupts acrosomes in noncapacitated spermatozoa.
The acrosome overlies the sperm head and contains enzymes required for fertilization (eg, Yanagimachi, 1988). Normally, acrosomes of capacitated spermatozoa are disrupted (acrosome reaction) upon contacting the zona pellucida, that surrounds the oocyte. Premature AL during the residence of spermatozoa in the vagina would hinder or prevent fertilization, and be contraceptive. The acrosome reaction requires entry of extracellular calcium ions (Ca2+) (Breitbart and Naor, 1999; Guraya, 2000). This work was carried out to evaluate pathways of Ca2+ entry in SAMMA-induced acrosomal loss (SAL).
The results show that SAL, similar to the acrosome reaction, occurs by a Ca2+-dependent mechanism. However, unlike acrosome reactions, entry of Ca2+ in response to SAMMA does not involve capacitative entry through store-operated channels, but rather may only be through T-type voltage-dependent channels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human Subjects— All subjects gave informed consent to participate in this study, which was carried out in compliance with and approved by the institutional review board. In each experiment, fresh semen was collected from 2 to 3 individuals out of a total pool of 9 healthy donors (mean age = 32 + 3.5 [SEM] years). All subjects were free from illness requiring medical intervention for at least 1 year before their participation in this study; they were not taking prescription, over-the-counter, or recreational drugs at the time of the study. Alcohol consumption was low to moderate, with no subject consuming more than 4 to 7 drinks per week. No subject reported urinary infection, prostatic inflammation, or contraction of sexually transmitted infection within 3 years of the study. Inclusion criteria for participation included maximum allowable abnormal sperm forms of 30% (World Health Organization, 1992), minimum proportion of spermatozoa responding to hypoosmotic medium (HOS test; Jeyendran et al, 1984) of 60% and minimum of 30% of capacitated spermatozoa that undergo acrosome reaction in response to the calcium ionophore A23187 (De Jonge et al, 1989). Semen quality was consistently high, with average volume, sperm count and motility within 1 hour of collection of 3.7 ± 0.43 mL, 70 (90% confidence limits = 57.5–84.2) x 106/mL and 71% (68.4–74.4), respectively (n = 30).
Methods
In this context, acrosomal loss refers to the disruption of the sperm
acrosome in response to exposure to a treatment or chemical entity. No
inference is made as to whether this response is identical to the
physiological acrosome reaction, during which the acrosome is also lost.
Preparation of Spermatozoa and Induction of Acrosomal Loss— Semen was allowed to liquify for at least 30 minutes after collection. Within 90 minutes, semen was layered over buffered 11% Ficoll, and spermatozoa were sedimented and separated from seminal plasma by centrifugation. Spermatozoa were resuspended (5 x 106 cells/mL) in a modified BWW medium (Biggers et al, 1971), less bovine serum albumin (Anderson et al, 1994). Composition of the medium is as follows: 94.6 mM NaCl, 4.8 mM KCl, 1.28 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.1 mM NaHCO3, 0.25 mM sodium pyruvate, 23.3 mM sodium lactate, 5.6 mM glucose, and antibiotics (100 IU penicillin, 50 µg/mL streptomycin). The sperm suspension was divided into 0.5-mL portions. A small portion (approximately 10 µL) was reserved for sperm motility assessment by light microscopy.
After 5 minutes of equilibration at 37°C, modulators were added as indicated. Ten minutes thereafter, acrosomal loss was induced by SAMMA at different concentrations as indicated. Fifteen minutes after addition of SAMMA, 10 µL of the suspension were removed for motility assessment. Spermatozoa were fixed with buffered glutaraldehyde (Anderson et al, 1992) and stained for acrosome visualization with Bismarck Brown and Rose Bengal (De Jonge et al, 1989). Results are expressed as percent of maximal acrosomal loss induced by a maximally stimulating concentration of the calcium ionophore, A23817 (Anderson et al, 1992).
Data Analysis— All frequency (%) data were subjected to arcsine transformation before further analysis (Sokal and Rohlf, 1981). Values are presented as average percentage of maximal acrosomal loss, with the 90% confidence limits. Analysis of variance and the Newman-Keuls multiple range test were used to identify differences among treatment groups within individual experiments. Dose–response data were best fit to transitional-type curves (eg, Sigmoidal) with TableCurve 2D curve-fitting software (SPSS Statistical Software, Chicago, Ill), from which appropriate constants (eg, ED50 values) were derived. Differences among treatment groups were considered significant at P < .05. Differences were not considered significant at P > .10.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SAL is Ca2+-dependent. When SAMMA is present at 2 µg/mL, acrosomal loss increases with increasing concentration of added extracellular Ca2+. The concentration of extracellular Ca2+ required for half-maximal effect is 85 µM. This is similar to the concentration of extracellular Ca2+ required for half-maximal response to a maximally stimulating concentration of the calcium ionophore, A23187 (Figure 1). Nearly maximal (99.9%) response to SAMMA is observed at 0.55 mM Ca2+.
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SAL is not blocked by 10 µM nifedipine, an L-type voltage-dependent Ca2+ channel blocker (Figure 4). However, nifedipine inhibits acrosomal loss induced by 1 µM thapsigargin by 40% (t = 11.06; P < .001; df = 6). Thapsigargin is a Ca2+-ATPase inhibitor, and is thought to act by releasing intracellular Ca2+ stores (Meizel and Turner, 1993; Dragileva et al, 1999; O'Toole et al, 2000).
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2-Aminoethoxydiphenylborate (2-APB), an inositol-1,4,5-trisphosphate (InsP3) receptor antagonist and store-operated Ca2+ channel blocker (Bilmen and Michelangeli, 2002), is without effect on SAL. This is in contrast to nearly 91% inhibition by 2-APB (t = 13.51, df = 7; P < .001) of AL induced by 1 µM thapsigargin (Figure 6).
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SAL is inhibited by diphenylhydantoin, a relatively selective blocker of T-type Ca2+ channels (Suzuki et al, 1990; Perez-Reyes, 2003). Inhibition by this channel blocker is dose-dependent (Figure 7). The approximate IC50 is 210 µM, with nearly complete (99.9%) inhibition at 2.1 mM. Similarly, SAL is inhibited by the T-type channel inhibitor, NiCl2 (Lee et al, 1999; Perez-Reyes, 2003), with 50% and 99.9% inhibition observed at 75 µM and 236 µM, respectively (Figure 7).
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= .098; n = 7; P
> .1). Because motility is unaffected, changes in acrosomal loss are not
likely secondary to decreased cell viability. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SAL requires extracellular Ca2+ (Figure 1). The Ca2+ requirement of SAL agrees well with that for stimulus-induced acrosome reactions of capacitated spermatozoa (eg, Thomas and Meizel, 1989). SAL inhibition by verapamil (Figure 2) suggests that it may be mediated through voltage-dependent Ca2+ channels.
Voltage-dependent Ca2+ channels consist of four or five
subunits. Electrophysiological and pharmacological properties are determined
primarily by the 
1 subunit
(Catterall et al, 2003).
Evidence exists for 1A (P/Q-type channels),
1E (R-type channels)
(Lievano et al, 1996),
1G, 1H (T-type channels),
C (L-type channels)
(Espinosa et al, 1999), and
1B (N-type channels)
(Wennemuth et al, 2000) in
mouse, 1C in rat
(Goodwin et al, 1998), and
1B, 1E, 1G,
1H, and 1I (T-type channels) in human
(Goodwin et al, 1997;
Park et al, 2003)
spermatogenic cells and spermatozoa. Any or all of these channels may allow
stimulus-induced Ca2+ influx into the cell.
Ca2+ influx in response to the zona pellucida has been attributed to activation of T-type Ca2+ channels and subsequent activation of store-operated channels, possibly through transient receptor potential protein 2 (Trp2) (Darszon et al, 2001; Primakoff and Myles, 2002; Felix, 2005). These conclusions are based primarily on studies carried out with mouse spermatozoa and spermatogenic cells (Arnoult et al, 1999; O'Toole et al, 2000; Jungnickel et al, 2001). Ca2+ transients, measured over a period of approximately 20–200 milleseconds, and inhibition by 1 µM pimozide and 50 µM Ni2+, suggest involvement of T-type channels (Arnoult et al, 1999). However, peak Ca2+ levels are inhibited by 0.1 µM PN200-110 (a dihydropyridine [DHP]). Although T-type channels are not completely insensitive to DHPs (Bean, 1989; Perez-Reyes, 2003), this concentration of PN200-110 is generally considered selective for L-type channels (Lacinova et al, 2000; Grumann et al, 2003). Further, although not definitive, work by Kobori et al (Kobori et al, 2000) suggests that inhibition of Ca2+ influx into mouse spermatozoa by pimozide may be secondary to L-type channel blockade.
Inhibition of sustained Ca2+ increase by PN200-110, nifedipine,
and verapamil is attributed to their initial effects on T-type currents
(O'Toole et al, 2000).
However, nifedipine is also generally regarded as selective for L-type
channels and has little effect on T-type channels
(Morita et al, 2002). Some
T-type currents are inhibited by low concentrations of nifedipine
(Kd = 5 µM) (Akaike et al, 1989). However, this current is
largely insensitive to Ni2+ (Kd = 0.6 mM), suggesting
that they are formed by either 1G or 1I
(Perez-Reyes, 2003). Recent
evidence favors expression of primarily 1H channels in mouse
spermatocytes (Stamboulian et al,
2004), which are about 20 times more sensitive to Ni2+
(Perez-Reyes, 2003). Our data
demonstrate that processes inhibited by the T-type channel blockers and by
nifedipine appear to be different, because nifedipine inhibits
thapsigargin-induced AL, but not SAL
(Figure 3). Thus, the role of
T-type currents in DHP-sensitive spermatozoal processes remains open to
question, and participation of high voltage activated (HVA) channels can not
be excluded. Splice variants of DHP-sensitive channels in spermatozoa may have
electrophysiological properties of T-type channels
(Benoff, 1998).
Inhibition of SAL by verapamil suggests that SAL occurs through activation of voltage-dependent Ca2+ channels. However, this antagonist is relatively non-selective. For example, verapamil has similar effects on L- and T-type channels (Bean, 1989; De Paoli et al, 2002a). Inhibition of L-type channels by phenylalkylamines such as verapamil is use-dependent. Upon repeated stimulation of L-type channels, their sensitivity to inhibition by channel blockers increases (Reuter, 1983; Godfraind, 1984; Emanuel et al, 1998). Absence of use-dependent inhibition of SAL by verapamil (see Results; Figure 3), failure of nifedipine to inhibit SAL (Figure 4), and SAL sensitivity to Ni2+ (Zamponi et al, 1996) make it unlikely that L-type channels are involved.
Although there are no selective T-type channel antagonists (Catterall et al, 2003), the pharmacological profile of SAL is more consistent with involvement of T-type channels than other sperm-derived channels. SAL inhibition by diphenylhydantoin and Ni2+ is similar to inhibition by these agents of Ca2+ current in T-type channels in other cell types (McCarthy et al, 1990; Suzuki et al, 1990; Viana et al, 1997). Other channels are considered below.
Verapamil inhibits R-type (1E or Cav2.3)
channels, although apparently much less than either L- or T-type channels. The
IC50 against R-type channels is much higher (170 µM
(Benquet et al, 1999) than
against SAL (0.4 µM). The IC50 for Ni2+ against SAL
(75 µM; Figure 7) is higher
than generally required for R-type currents
(Tottene et al, 2000); it is
in the same range as concentrations used in other tissues to inhibit T-type
channel activity (Wolfart and Roeper,
2002; Watanabe et al,
2004). Our data argue against a role of R-type channels in
SAL.
The 1A and 1B subunits form part of the
P/Q-type (Cav2.1) and N-type (Cav2.2) channels,
respectively. The relative insensitivity to verapamil (<1 µM) of
1A (Hirasawa et al,
2001; Miranda et al,
2001) and 1B
(Molderings et al, 2000;
Ichida et al, 2005) and to
Ni2+ (,100 µM) of 1A
(Zamponi et al, 1996;
Hirasawa et al, 2001) and
1B (Zamponi et al,
1996; Hirasawa et al,
2001) argue against their involvement in SAL.
Sustained entry of Ca2+ is required for AL. For zona- and progesterone-induced AL, this sustained entry is thought to be mediated by the activation of store-operated channels (Breitbart et al, 2005; Felix, 2005). However, the present data do not support involvement of store-operated channels in SAL (see below).
T-type channels, under appropriate circumstances, may allow sustained Ca2+ entry. T-channel inactivation can be slow and incomplete, leading to sustained Ca2+ influx (Bean, 1989). Window currents produced in T-type channels by potentials from -50 to -20 mV can lead to sustained entry of Ca2+ (Jensen et al, 2004). Noncapacitated spermatozoa have resting potential of around -26 mV (Arnoult et al, 1999), making these cells candidates for T-type window currents, which may account for Ca2+ influx responsible for SAL.
Findings in this study differentiate AL in response to SAMMA from that caused by either physiological stimuli or to thapsigargin. Unlike AL induced by physiological stimuli (Luconi et al, 1996; O'Toole et al, 2000), SAL is unaffected by agents that modulate intracellular Ca2+ stores or store-operated channels. BAPTA (introduced as BAPTA-AM) is an intracellular Ca2+ chelator that depletes Ca2+ released from intracellular stores and inhibits Ca2+ influx occurring by capacitative entry (Ng et al, 1990; Cloutier et al, 1993). Intracellular BAPTA reverses increases in Ca2+ secondary to release of intracellular Ca2+ stores (O'Toole et al, 2000; Yeromin et al, 2004). Further, intracellular BAPTA completely inhibits the Ca2+ response to progesterone in human spermatozoa (Luconi et al, 1996), but has no effect on SAL. In contrast, BAPTA inhibits thapsigargin-induced AL (releases Ca2+ stores). BAPTA has a smaller effect on ionophore A23187-induced AL (Figure 5). This is consistent with release of Ca2+ stores by A23187 (Pittman et al, 1994; Roy and Lee, 1995), which represents a minor contribution to increased intracellular Ca2+. SAL is unaffected by 2-APB (Figure 7). 2-APB blocks InsP3 receptors (responsible for release of intracellular Ca2+ stores) and store-operated channels. This provides additional evidence against involvement of store-operated channels or release of Ca2+ stores by InsP3 in the mechanism of SAL. The concentration of 2-APB is within the range of effective concentrations of 2-APB used in other cell types (Chen et al, 2002; Yeromin et al, 2004). 2-APB inhibits thapsigargin-induced AL; this agrees with the ability of 2-APB to inhibit store-operated channels and thapsigargin-induced capacitative Ca2+ entry (Enfissi et al, 2004; Dedos et al, 2005). Our findings are inconsistent with Ca2+ influx through store-operated channels. Further, SAL is clearly different from AL induced by thapsigargin.
Thapsigargin and ZP3 activate the same Ca2+ influx pathway in spermatozoa (O'Toole et al, 2000). The similarity of the Ca2+ pathway promoted by ZP3 and thapsigargin is of particular importance regarding the present study. Thapsigargin-induced AL differs from SAL (Figures 3, 5, and 6). By inference, the Ca2+-dependent pathway leading to SAL differs from that leading to ZP3-induced AL. Further, involvement of Trp2 in zona pellucida-induced AL (Jungnickel et al, 2001) suggests that SAL is not likely mediated by Trp2. Jungnickel and coworkers suggest that Trp2 may be identified as or closely related to store-operated channels. Store-operated channels do not mediate SAL (Figures 5 and 6).
Activities of SAMMA as a broad-spectrum contraceptive microbicide suggest common underlying mechanisms for these effects. As a microbicide, SAMMA is classified as an entry inhibitor (Herold et al, 2002; Cheshenko et al, 2004). Signal transduction perturbations may be partly responsible for the multiple activities of this contraceptive microbicide. Infection of target cells by HSV and HIV are associated with Ca2+ signaling in the target cells (Haughey et al, 1999; Dellis et al, 2002; Cheshenko et al, 2003). SAMMA may exert some of its microbicidal properties through altered Ca2+ signaling.
Voltage-activated Ca2+ ions are typically identified by their
biophysical and pharmacological properties and by their transcripts. However,
assigning a channel to a biological process is complicated by the presence of
several channel types, by association of the 1 subunit with
different combinations of auxiliary subunits, and by the occurrence of splice
variants. These variables contribute to blurring the distinction of channel
types based on pharmacological and physical properties; they give rise to
channel variants that may have atypical properties
(Bean, 1989;
Benoff, 1998;
Publicover and Barratt, 1999;
Morita et al, 2002;
Jensen et al, 2004). Further,
T-type channels, although usually responsible for transitory currents, can
also support sustained Ca2+ entry
(Bean, 1989;
Jensen et al, 2004). This
problem is further complicated in spermatozoa, cells that are not amenable to
characterization by conventional electrophysiological techniques and that do
not uniformly respond to stimuli. The molecular identity of Ca2+
channel(s) participating in zona pellucida-induced AL has not been established
(Darszon et al, 2001). In
spite of these limitations, pharmacological characterization of relevant
responses such as AL remains useful. Results from the present provide evidence
that the pharmacological sensitivity of SAL, although supporting involvement
of T-type channels, is not the same as that of zona pellucida-induced AL; SAL
and zona pellucida-induced AL likely occur through different
Ca2+-mediated pathways. We propose that SAMMA's contraceptive
activity lies in its ability to induce premature AL, caused by dysregulation
of Ca2+ signaling.
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Footnotes |
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