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From the * Unit of Reproductive Biology, Faculty
of Health Sciences, and the Chemistry
Department, Faculty of Basic Sciences, University of Antofagasta, Antofagasta,
Chile.
| Correspondence to: Dr Patricio Morales, Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, PO Box 170, Antofagasta, Chile (e-mail: pmorales{at}uantof.cl). |
| Received for publication November 19, 2002; accepted for publication January 16, 2003. |
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Abstract |
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-hydroxy-7-oxoazorellano (azorellanone), a cyclic
diterpene extracted from Azorella yareta Hauman, on in vitro human sperm
physiology. Sperm aliquots, capacitated for 4.5 or 20 hours, were incubated
for 15 minutes with different concentrations of azorellanone. Then, the
effects of azorellanone on sperm motility, viability, binding to the human
zona pellucida, progesterone-induced acrosome reactions and increase in
intracellular Ca2+ concentration, and trypsin and
chymotrypsin-like protease activities were evaluated. Sperm motility was
evaluated according to World Health Organization (WHO) guidelines; sperm
viability with the supravital dye Hoescht 33258; sperm–zona binding with
the hemizona assay; progesterone-induced acrosome reaction with fluorescent
lectin; intracellular Ca2+ level with fura 2; and
protease activity with the synthetic substrates
N-t-Boc-Gln-Ala-Arg-Amido-4-methylcoumaryn and
Succinyl-Leu-Leu-Val-Tyr-Amido-4-methylcoumaryn. The results obtained indicate
that azorellanone inhibited sperm motility in a concentration-dependent manner
at 0.15, 1.5, and 3 mM, while sperm viability was only inhibited at 3 mM.
Treatment with azorellanone significantly inhibited sperm–zona binding,
progesterone-induced acrosome reactions, and intracellular
Ca2+ concentration. Treatment of free-swimming sperm
with azorellanone did not affect protease activity; however, the incubation of
sperm extracts with azorellanone significantly inhibited both trypsin-like and
chymotrypsin-like protease activities. In conclusion, azorellanone has a
significant effect on the different parameters that characterize human sperm
physiology.
Key words: Sperm–zona binding, cyclic terpenes, intracellular Ca2+, protease activity
In the high Andes in the north of Chile grows Azorella yareta
Hauman, a shrub species that belongs to the Umbeliferae family. It is known as
"yareta," and it has been used as an herb medicine. Historical
records indicate that yareta has been used to treat the common cold and ache,
to reduce sugar in the blood, and possibly as an ointment to treat
dermatological disorders. This species constitutes a valuable source of
mulinane and azorellane diterpenoids (Loyola et al,
1997,
1998a,b).
In a previous communication, we reported the structures of the mulinane
derivatives mulinolic acid and mulin-11,13-dien-20-oic acid and those of the
azorellane derivatives azorellanol, 13--hydrozyazorellane, and
13-ß-hydrozyazorellane, which were obtained from a petroleum ether
(petrol) extract of A yareta Hauman
(Loyola et al, 2001).
In this report, we studied the more polar chromatographic fractions of the
same extract and described the isolation of a new diterpenoid with an
azorellane skeleton, the structure of which was established by spectroscopic
analysis and by oxidation of 7-desacetylazorellanol 1, to our compound
13--hydroxy-7-oxoazorellano (azorellanone). Its molecular formula is
C20H32O2, and it corresponds to a tricyclic
diterpene. Previous studies have in dicated that cyclic terpene compounds such
as triptolide decreased sperm motility and concentration in the rat
(Lue et al, 1998;
Sinha Hikim et al, 2000). In
this work, we present the result of the isolation of the diterpenoid
azorellanone from A yareta and its effect on several parameters that
characterize human sperm function (ie, sperm motility and viability, sperm
binding to the human zona pellucida, progesterone-induced acrosome reaction,
and increase in intracellular Ca2+
concentration—[Ca2+]i]) and protease
activity.
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Materials and Methods |
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Extraction and Isolation of the Azorellanone
A yareta Hauman plants were collected in January 1999 in
"Quebrada de las Llaretas" in Vallenar, Chile
(Loyola et al, 2001). A
voucher specimen was deposited in the Herbarium of the Universidad de
Concepción. The dried and finely powdered whole plant of A
yareta Hauman (3.0 kg) was extracted with petrol at room temperature to
give a gum (220 g). This extract was chromatographed by flash chromatography
on Si gel (Loyola et al,
2001). Fraction 5, eluted with petrol-ethyl acetate (12: 8) (23
g), was further purified on an Si gel column and eluted with petrol-ethyl
acetate (14:6) to yield azorellanone (3.2 g). To test the effect of
azorellanone on sperm, it was diluted in 0.5% ethyl acetate (vol/vol) at
0.015, 0.15, 1.5, and 3 mM.
Semen Samples
Semen samples were obtained after 2–3 days of sexual abstinence. All
donors signed a form consenting to the use of their sperm cells for research
purposes. All samples used were normal, according to World Health Organization
(WHO) guidelines (1999).
Motile sperm were separated using a double Percoll gradient, as described
previously (Morales et al,
2000). Briefly, aliquots of semen were deposited on the upper
Percoll layer and centrifuged at 300 x g for 20 minutes. The
pellet was resuspended in 10 mL of modified Tyrode medium consisting of 117.5
mM NaCl, 0.3 mM NaH2PO4, 8.6 mM KCl, 25 mM
NaHCO3, 2.5 mM CaCl2, 0.5 mM MgCl2, 2 mM
glucose, 0.25 mM sodium pyruvate, 19 mM sodium lactate, 70 µg/mL penicillin
and streptomycin, phenol red, and 0.3% BSA
(Suarez et al, 1986); was
centrifuged again at 300 x g for 10 minutes; and finally, was
resuspended in the same medium but supplemented with 2.6% BSA. The sperm
concentration was adjusted to 10 x 106 cells/mL, and the
suspension was incubated at 37°C with 5% CO2 in air for 4.5 or
20 hours to promote capacitation.
Zona Pellucida Collection
Human oocytes were dissected from ovarian tissue obtained from cadavers and
stored at -80°C as previously described
(Overstreet et al, 1980; Cross et al, 1988). After
thawing, the oocytes were freed of remaining cumulus cells by passing them
through a narrow-bore pipette. As a result of freezing and thawing, these
oocytes were not viable.
Evaluation of Sperm Motility and Viability
Sperm aliquots (400 µL) were incubated with different concentrations of
azorellanone for 30 and 60 minutes at 37°C with 5% CO2 in air.
Control sperm were incubated with 0.5% ethyl acetate (vol/vol). The motility
of the sperm was evaluated according to WHO guidelines
(1999). Sperm viability was
assessed by labeling the sperm with the supravital dye Hoescht 33258 and
examining them with a fluorescence microscope. Results were expressed as the
percentage of motile and alive sperm.
Sperm–Zona Pellucida Binding Assay
The sperm capacity to bind to the zona pellucida was evaluated using the
hemizona assay (Burkman et al,
1988; Franken et al,
1989). Briefly, 49-µL droplets of 4.5-hour capacitated sperm
were treated by adding 1 µL of test (azorellanone) or control solution
(0.5% ethyl acetate [vol/vol]) under oil in a plastic petri dish. Then, 1
hemizona was added to the control sperm droplet, and the matching hemizona was
added to the test sperm droplet. Control and test sperm droplets containing
hemizonae were incubated for 10 minutes at 37°C with 5% CO2 in
air. After incubation, each hemizona was removed and gently washed with a
wide-bore pipette. The tightly bound spermatozoa on the outer surface of each
hemizona were counted under a phase-contrast microscope. These procedures have
been described in detail elsewhere (Morales et al,
1994b,
1999;
Morales, 1998).
Progesterone-Induced Acrosome Reactions
After the spermatozoa were incubated for 20 hours, some aliquots were
incubated for 15 minutes with different concentrations of azorellanone.
Control sperm were incubated with 0.5% ethyl acetate (vol/vol). Then, the
sperm were treated for 15 minutes with 0.69 µM progesterone or its solvent
(0.1% DMSO). Control sperm received 0.1% DMSO. Acrosomal status was evaluated
using FITC-labeled PSA, as described (Cross
et al, 1986; Morales et al,
1992).
Measurement of Intracellular Ca2+
Concentration
The progesterone-induced increase in
[Ca2+]i was evaluated as described
(Garcia and Meizel, 1999;
Morales et al, 2000). Briefly,
4.5-hour capacitated sperm were incubated with 3 µM of fura 2 for 30
minutes at 37°C with 5% CO2 in air. The sperm were then washed
in Tyrode medium with 2.6% BSA without phenol red and centrifuged twice at 300
x g for 5 minutes. Sperm aliquots were incubated with different
concentrations of azorellanone for 15 minutes at 37°C with 5%
CO2 in air at a final concentration of 7–8 x
106 cells/mL. Control sperm were incubated with 0.5% ethyl acetate
(vol/vol). Then, 1-mL sperm aliquots were used for spectrofluorometry,
resuspending them directly into stirred fluorescence cuvettes. All these
procedures were carried out in the dark to prevent sample photobleaching.
Fluorescence caused by [Ca2+]i under various
experimental conditions was monitored using a Shimadzu model 1501 (Kyoto,
Japan) spectrofluorometer at an excitation wavelength pair of 340/380 nm and
an emission wavelength of 510 nm. After equilibration for 2 minutes,
measurements of [Ca2+]i were started.
Approximately 50 seconds after the beginning of each sample run, progesterone
(0.69 µM) was added to the sperm suspension. Sequential additions of 20
µM digitonin and 10 mM Tris-EGTA were made near the end of each experiment
to facilitate determination of [Ca2+]i, as
previously described. To further analyze the results, the highest value of
[Ca2+]i (peak) for each treatment was
obtained, and the area under the curve during the first 300 seconds of
treatment was measured using a planimeter GTCO Quickruler (Columbia, Md)
(Morales et al, 2002).
Preparation of Sperm Extracts
Crude sperm extracts to evaluate protease activity were obtained as
described previously (Morales et al,
1994a,
2002). Briefly, sperm were
separated from seminal plasma, other cell types, and cellular debris by
centrifugation through a column of Percoll as described above except that the
Percoll was prepared in 50 mM HEPES and 191 mM NaCl, pH 7.4
(Morales et al, 1994a). The
resulting sperm pellet was washed 2 times by centrifugation at 300 x
g for 10 minutes and then resuspended in homogenization buffer (50 mM
HEPES and 10% glycerol, pH 7.4) at a concentration of 25 x
107 sperm/mL. The sperm suspension was then sonicated (Virsonic,
Gardiner, NY) with seven 60-W bursts of 30 seconds each and centrifuged for 30
seconds at 5000 x g in a Beckman microfuge to remove nuclear
and flagellar material. The supernatant was used as the enzyme stock
preparation. All these procedures were performed at 4°C. The protein
concentration in each sperm extract preparation, obtained using the Bradford
method (Bradford, 1976), ranged
between 0.3 and 0.8 mg/mL. The trypsin-like and chymotrypsin-like activities
of the sperm extracts were assayed using the fluorogenic substrates
Suc-LLVY-AMC and Boc-GAA-AMC. Aliquots of 100 µL of enzyme extract were
incubated in a final volume of 2 mL containing 10 mM CaCl2, 50 mM
HEPES, pH 7.4, and 10 µM substrate. The assay was run at 37°C, and the
fluorescence was monitored with excitation at 380 nm and emission at 460 in a
Shimadzu model 1501 spectrofluorometer.
To test the effect of the azorellanone on the sperm proteolytic activity, 100-µL aliquots of the extract were preincubated with different concentrations of azorellanone for 15 minutes at 37°C before adding the substrates. Control sperm were incubated with 0.5% ethyl acetate (vol/vol). In some experiments, free-swimming sperm were incubated with azorellanone prior to preparation of the extracts.
Statistical Analysis
The data were analyzed by analysis of variance for repeated measures using
the StatView program (SAS Institute, Cary, NC) on an Apple Power Macintosh
6500/225. Differences between individual groups were examined with Fisher's
protected least significant difference test. Paired comparisons were conducted
using a paired t test, and all data are presented as mean values plus
or minus standard errors of the mean. Differences were considered significant
at the .05 level of confidence.
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Results |
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-hydroxy-7-oxoazorellano. The proposed structure was
confirmed by the transformation of known 7-desacetylazorellanol 1 into
13--hydroxy-7-oxoazorellano (azorellanone) by means of oxidation with
pyridium chlorochromate. Table
1 shows the data for the NMR 1H and NMR 13C
spectra of azorellanone.
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Biological Effect of Azorellanone
Regarding sperm motility, treatment with azorellanone significantly reduced
the number of free-swimming sperm. This effect was observed starting at 0.15
mM and was concentration-dependent (Table
2). There was no further decline in motility when the cells were
incubated for 60 minutes instead of 30 minutes. This effect of azorellanone on
sperm motility, however, was not strictly associated with a decrease in sperm
viability. In effect, the concentration of azorellanone necessary to decrease
the number of living sperm was higher than that required to reduce sperm
motility (Table 2).
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With regard to the sperm's ability to bind to the human zona pellucida, the results indicated that azorellanone significantly inhibited sperm–zona pellucida binding at all concentrations tested (Figure 2), including the concentration of 0.015 mM, which did not affect sperm movement. In addition, treatment with 1.5 mM azorellanone significantly inhibited the occurrence of the acrosome reaction stimulated by progesterone (Figure 3). Thus, while the percentage of acrosome-reacted sperm rose from 18.6% plus or minus 4.4% (control) to 41.4% plus or minus 6.1% after only progesterone treatment (P < .001), in the sperm previously treated with 1.5 mM azorellanone, the percentage of acrosome-reacted sperm after progesterone treatment was only 16.2% plus or minus 2.2%.
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With regard to the progesterone-induced increase in [Ca2+]i, this was about 3 times the basal value (Figure 4A). The basal Ca2+ levels were the same whether the sperm were treated with 0.1 DMSO or 1.5 mM azorellanone (data not shown). Thus, progesterone treatment alone increased the intracellular Ca2+ concentration from 317 plus or minus 25 nM to 993 plus or minus 63 nM. However, previous treatment of the sperm with azorellanone significantly inhibited the maximum value reached in the intracellular Ca2+ concentration stimulated by progesterone, where azorellanone concentrations were 0.15 and 1.5 mM. The [Ca2+] levels were 503 ± 48 nM and 683 ± 58 nM, respectively. This was also evident when the area under the Ca2+ peak was analyzed (Figure 4B).
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Finally, with regard to the effect of azorellanone on the protease activity of human sperm extracts, incubation with azorellanone significantly inhibited both the trypsin-like (Figure 5A) and the chymotrypsin-like activity of the extracts (Figure 5B). The specific activity toward both trypsin and chymotrypsin substrates was significantly inhibited in a concentration-dependent manner (Table 3). The inhibition of protease activities was observed only when the sperm extracts were incubated with azorellanone; incubation of free-swimming sperm with azorellanone did not inhibit the protease activity of the extracts (data not shown).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-hydroxy-7-ox oazorellano (azorellanone), extracted from the high
Andes shrub A yareta inhibited several parameters that characterize
human sperm function. In effect, treatment of human sperm with azorellanone
inhibited 1) sperm motility without significantly affecting sperm viability;
2) sperm–zona pellucida binding; progesterone-induced acrosomal
exocytosis and an increase in [Ca2+]i; and 3)
trypsin-like and chymotrypsin-like sperm protease activities. With regard to sperm motility and viability, the number of motile sperm was significantly reduced with azorellanone in a concentration-dependent manner, while the latter was affected only at the highest concentration tested (3 mM). The mechanism by which azorellanone decreased flagellar beat is not clear. However, the work of Huynh et al (2000) with triptolide demonstrated that this diterpene extracted from the Chinese medicinal plant T wilfordii inhibited the motility of rat epididymal sperm. In addition, electron microscopy studies suggested that triptolide provoked severe alterations in the sperm ultrastructure, the most conspicuous being the absence of a sperm plasma membrane over the middle and principal piece of the flagellum and premature decondensation of the nuclei (Sinha Hikim et al, 2000). The loss of the plasma membrane from these sperm cells explained their loss of motility. Therefore, the viability of the sperm was also affected in the same manner as motility. According to this, azorellanone could be a potential contraceptive agent, since mammalian sperm must use their flagellar apparatus to move along the female genital tract, from their site of deposition in the vagina to the site of fertilization in the ampulla of the oviduct (Yanagimachi, 1994). In addition, it is highly unlikely that immotile mammalian sperm could traverse the zona pellucida (van Kooij et al, 1985; Mellinger and Goldstein, 1987; Bedford, 1998).
We also showed in this study that azorellanone inhibited the sperm's ability to bind to the human zona pellucida. This inhibition by azorellanone cannot be attributed only to a decrease in sperm motility after treatment, because the effect was also caused by an azorellanone dose of 0.015 mM, which did not affect sperm motility. Perhaps more subtle alterations of the sperm plasma membrane could explain this result.
Azorellanone inhibited the progesterone-induced acrosomal exocytosis and increase in [Ca2+]i. It has been shown that plasma membrane molecules in mammalian sperm are important for the sperm's migration to the site of fertilization and that the functional integrity of the plasma membrane is an important factor in sperm motility, sperm acrosome reaction, capacitation, metabolism, and binding to the zona pellucida (Fraser and Ahuja, 1988). Therefore, it is possible that azorellanone may be altering a surface molecule(s) responsible for the effects mentioned above. Similar changes in the plasma membrane could be involved in altering its ionic conductance, in this case to Ca2+. The latter could also be related to changes in the flagellar movement (Porat, 1990).
With regard to the inhibition of the trypsin-like and chymotrypsin-like protease activities of the sperm, azorellanone inhibited both activities in a concentration-dependent manner. This was the case when sperm extracts were incubated with azorellanone. Thus, this could be an additional mechanism by which azorellanone impaired sperm function. Other plant extracts such as gossypol also inhibited the activity of certain enzymes that are involved in the metabolic regulation of the sperm (Maugh, 1981; Nakamura et al, 1988; Rajpurohit and Giridharan, 1988). Moreover, gossypol inhibition of the serine protease acrosin (EC 3.4.21.10, an enzyme of acrosomal origin with trypsin-like activity) in pigs has also been suggested as a mechanism of sperm function impairment (Sadykov et al, 1985). The scanty or nil effect of azorellanone on the enzyme activity when added to whole sperm did not necessarily imply that this compound did not have an effect on the enzyme activity. Damage to the plasma membrane could be a first event in the interaction between azorellanone and the sperm, and then azorellanone would enter the cytoplasm, inhibiting its protease activities. Experiments with longer incubation times than the ones used in this study may confirm this hypothesis.
In summary, the plant origin compound azorellanone significantly inhibited several functions that are essential for fertilization, such as sperm motility, sperm–zona binding, sperm acrosome reaction and Ca2+ influx, and sperm protease activities. Azorellanone had a spermicidal effect only at the highest dose tested (3 mM). All these results allow us to think that azorellanone may be a potential candidate as a contraceptive agent to be used in the manufacture of vaginal jellies or creams. Experiments are under way to determine the in vivo effect of azorellanone.
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Footnotes |
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