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176 and rhuZP3b177348 and Their Mechanism
,¶
From the * Department of Reproductive Physiology,
Zhejiang Academy of Medical Sciences, Hangzhou, China;
Department of Laboratory Medicine, Wenzhou
Medical College, Wenzhou, China; Shanghai
Institute of Planned Parenthood Research, Shanghai, China;
Zhejiang Provincial Institute of Planned
Parenthood Research, Hangzhou, China; and the ||
Women's Hospital, School of Medicine, Zhejiang
University, Hangzhou, China.
| Correspondence to: Qi-xian Shi, Department of Reproductive Physiology, Zhejiang Academy of Medical Sciences, 182 Tian Mu Shan Road, Hangzhou, Zhejiang 310013, China (e-mail: qxshi{at}mail.hz.zj.cn). |
| Received for publication July 24, 2006; accepted for publication September 11, 2006. |
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Abstract |
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176 and
rhuZP3b177348, we examined whether
rhuZP3a22176 or rhuZP3b177348 trigger the
acrosome reaction (AR) of human spermatozoa and we investigated the underlying
mechanism. The assessment of AR was performed using chlortetracycline
staining. The intracellular free calcium concentration ([Ca2+]i) in
Fura-2/AM-loaded human sperm was monitored with a spectrofluorophotometer. We
found that rhuZP3a22176 and rhuZP3b177348 were
capable of eliciting AR at different concentrations. With the addition of
either peptide, the [Ca2+]i level was raised to a peak with or
without a plateau. The AR could be inhibited by pertussis toxin (PTX), EGTA,
and pimozide (a T-type calcium channel blocker), whereas verapamil was less
effective in this regard. The results of the present study suggest that
peptides rhuZP3a22176 and rhuZP3b177348 have a
role similar to human ZP3, and that the mechanism of the response to the
peptides involves influx of calcium, the G protein pathway, and a T-type
calcium channel.
Key words: Human sperm, pertussis toxin, intracellular calcium, G protein, calcium channel, chlortetracycline
ZP3 binds to the ZP3 receptor and induces AR via a pertussis toxin (PTX)-sensitive GTP-binding protein (Bastiaan et al, 1999; Bray et al, 2002; Schuffner et al, 2002). With the activation of G proteins, intracellular Ca2+ increases due to influx through voltage-operated calcium channels (VOCC) (Baldi et al, 1996). The increase in intracellular Ca2+ concentration ([Ca2+]i) is required for ZP-initiated acrosomal exocytosis (Babcock and Pfeiffer, 1987; Florman et al, 1992, 1994; Arnoult et al, 1996b). Although the exact nature of the VOCC has not been identified, a T-type VOCC (VOCCT) or the low voltage-activated calcium channel family may be involved (Arnoult et al, 1996a).
Due to the limited availability of native human ZP, recombinant human ZP3s
(rhuZP3s) are being developed as alternative sources. Various expression
systems have been used to prepare rhuZP3, including Chinese Hamster ovary
(CHO) cells (van Duin et al,
1994; Bray et al,
2002), Escherichia coli (E coli)
(Chapman et al, 1998), human
ovarian teratocarcinoma (PA-1) cells (Dong
et al, 2001), human embryonic kidney 293T cells
(Martic et al, 2004),
baculovirus (Chakravarty et al,
2005), and Spodoptera frugiperda sf9 insect ovary cells
(Caballero-Campo et al, 2006).
We have developed a high-yield E coli thermoinducible system for the
preparation of rhuZP3, and we have obtained two rhuZP3 polypeptides,
rhuZP3a22176 and rhuZP3b177348, the molecular
weights of which are about 22 kDa and 19 kDa, respectively
(He et al, 2005). The yields
of rhuZP3a and rhuZP3b products, as assessed on SDS-PAGE gel, were
approximately 10% of the total cell proteins
(He et al, 2005).
To explore the biological characteristics of rhuZP3a22176
and rhuZP3b177348, we investigated the effects of the two
polypeptides on the [Ca2+]i of capacitated human sperm and the
induction of AR, and explored the possible mechanism using PTX, EGTA, and
pimozide (a T-type calcium channel blocker).
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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176 and rhuZP3b177348 were
prepared using a previously described method
(He et al, 2005). Human ZP was
obtained from IVF with patient consent for donation and approval by the local
ethics committee of the Women's Hospital at Zhejiang University.
Media
Human tubule fluid (HTF), with some modifications, was constituted as
follows: 90 mM NaCl, 5.06 mM KCl, 25.3 mM NaHCO3, 1.8 mM
CaCl2, 1.17 mM KH2PO4, 1.01 mM
MgSO4·7H2O, 0.27 mM sodium pyruvate, 21.6 mM
sodium lactate, 5.56 mM glucose, 20 mM Hepes, 60 mg/L penicillin and 4 g/L
BSA. This medium was maintained in equilibrium with 5% CO2 in air,
had pH of 7.5 at 25°C and an osmolality of 300 mOsmol/kg.
Renaturation of rhuZP3a22176 and rhuZP3b177348
RhuZP3a22176 and rhuZP3b177348 were
separated and purified using an improved method of preparative gel
polyacrylamide gel electrophoresis. Each of the peptides was dissolved for
renaturation in a buffer that contained 8 mM urea, 20 mM Tris-HCl (pH 8.0), 1
mM EDTA, 100 mM NaCl, 2 mM GSH, 1 mM GSSG, 10 mM DTT, and 0.1 mM PMSF. After
adjustment to pH 10.7 with 10 mM KOH, the pH was readjusted to 8.0 with HCl.
The solutions were placed at room temperature for 30 minutes before
centrifuging at 12 000 x g for 15 minutes. The supernatants
were placed in dialyzable bags to dialyze for 5 hours in PBS (pH 7.4) that
contained 2 mM GSH and 1 mM GSSG. After the PBS was replaced with a larger
volume of PBS (pH 7.4), the solutions were dialyzed overnight and centrifuged
at 12 000 x g for 15 min. The supernatants were identified by
15% SDS-PAGE electrophoresis and frozen to dryness.
Human Sperm Preparation
Human semen samples were obtained by masturbation from 24 healthy donors
through the Zhejiang Provincial Institute of Planned Parenthood Research. All
samples were examined according to World Health Organization
(WHO, 1999) criteria. Only
samples with motility equal to or greater than 70%, viability equal to or
greater than 85%, and sperm concentration equal to or greater than 20 x
106 cells/mL were used. The sperm were washed with an equal volume
of HTF by centrifugation at 500 x g for 15 min. The pellets
were resuspended in 1 mL fresh HTF before centrifugation at 600 x
g for 15 minutes through a discontinuous Percoll gradient, which
contained 90% and 45% Percoll. The pellets were resuspended in approximately
10 volumes of HTF and centrifuged at 300 x g for 5 min. The
supernatants were discarded and resuspended in fresh HTF. The sperm
concentration was adjusted to 23 x 107 sperm/mL. At this
stage, 95% of the sperm were viable. Then sperm were incubated at 37°C for
5 hours for capacitation.
Preparation of Solubilized ZP
ZP was obtained from IVF following appropriate patient consent for donation
and approval by the local ethics committee of the Women's Hospital at Zhejiang
University. The ZP were stored in PBS buffer (pH 7.4) at –20°C. On
the day of the experiment, the ZP were thawed and solubilized by incubation at
60°C for 1 hour. The preparation was centrifuged at 13 000 x
g for 8 minutes at 4°C to remove particulate debris, and the
supernatant was used for the experiment.
Assessment of AR
To estimate the AR of human sperm in response to peptides
rhuZP3a22176 and rhuZP3b177348, spermatozoa
were stained with the supervital stain Hoechst 33258, which does not stain
cells with intact plasma membranes, and with the CTC stain, as previously
described (Ward and Storey,
1984; DasGupta et al,
1993; Shi et al,
1997). Briefly, a stock solution of 100 mg/mL Hoechst 33258 was
prepared by dissolving the dye in triple-distilled water. The stock solution
was stored in a foil-wrapped bottle at 4°C. The final concentration of dye
when added to the sperm suspension was 1 µg/mL. Spermatozoa were stained
for 10 minutes and then washed through 45% and 90% Percoll by centrifugation
at 600 x g for 10 minutes to remove free dye. The pellet was
resuspended in HTF and the spermatozoa were then stained with CTC. In a 0.5-mL
Eppendorf tube, 200 µL of CTC solution (500–700 µM CTC, 130 mM
NaCl, 5 mM cysteine, 20 mM Tris-Cl, [pH 7.8]) was added to 200 µL of sperm
suspension and mixed well by gently aspiration with a cut pipette tip. The
mixture was immediately added to 70 µL of the relevant fixative (10%
formalin in 2.5 M Tris Base [1:1]), mixed for a while, and stored at 4°C
in the dark until observation. Each sample was scored for more than 250
spermatozoa under a fluorescence microscope (Nikon Eclipse 80i; Nikon Inc,
Tokyo, Japan). Three patterns can be observed with CTC staining
(Lee et al, 1987;
Shi et al, 1997): pattern F
(also referred to as pattern A), which consists of fluorescence over the
entire head and equatorial region, is characteristic of uncapacitated
acrosome-intact sperm; pattern B, which consists of a fluorescence-free band
in the postacrosomal region, represents capacitated acrosome-intact sperm; and
pattern AR, which consists of very low fluorescence over the head, corresponds
to sperm that have undergone acrosomal exocytosis. The number of
Hoechst-positive (dead) spermatozoa corresponded closely to the number of
immotile cells (5%), which indicates that rhuZP3 peptide-induced AR is
not due to dead sperm that have lost their acrosome after the addition of the
peptides and other reagents.
Measurement of [Ca2+]i
After incubation at 37°C for 4 hours, the sperm suspension was loaded
with 2 µM Fura-2/AM for 30 minutes at 37°Cin the dark, and the sperm
suspension was centrifuged in HTF culture media 3 times at 300 x
g for 5 minutes, in order to wash away the extracellular free
Fura-2/AM. Sperm were finally resuspended to a concentration of 10 x
106 cells/mL in HTF medium. After loading with Fura-2/AM, the sperm
were subjected to alternating determinations at the excitation wavelengths of
340 nm and 380 nm (emission 510 nm), at a speed of once every 40 seconds,
using the Shimadzu RF-540 spectroflurometer (Shimadzu Co, Kyoto, Japan). In
these processes, the cells were treated with either
rhuZP3a22176 or rhuZP3b177348. The
[Ca2+]i is expressed as the ratio of the fluorescence intensities
(F340/F380) at the 2 excitation wave-lengths
(Domínguez et al, 1992).
Statistical Analysis
Statistical analyses were carried out using the SPSS 13.0 software. All the
AR percentages are transformed into [arcsin [root] (% acrosome
reactions/100)]. The results are presented as the mean ± SE and were
analyzed by 1-way ANOVA. When the test of homogeneity of variances was
significant (P < .05), the data were analyzed by the Duncett C
test. Otherwise, the LSD test was used. P values less than .05 were
considered to be significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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176 and rhuZP3b177348 Peptides Induce Sperm AR176 or rhuZP3b177348 on human sperm
AR, sperm were incubated with HTF for 5 hours and then stimulated with
550 µg/mL rhuZP3a22176 or
rhuZP3b177348 for 15 minutes before assaying for AR. In a
preliminary study, spermatozoa were preincubated for 5 hours and then
challenged with the peptides at different times. Some spermatozoa became
responsive after exposure to peptides for 5 minutes, with a considerable
proportion of the spermatozoa becoming responsive after 10 minutes. The
proportion of spermatozoa that was capable of undergoing AR in response to
peptides increased as the exposure time was extended, reaching maximum values
after exposure for 15–20 minutes. For this reason, the subsequent
experiments in the present study were carried out with exposure of the
spermatozoa for 15 minutes after preincubation for 5 hours. PBS or the vehicle
for rhuZP3a22176 and rhuZP3b177348 served as
the negative control. Soluble native human ZP (1 ZP/µL), in agreement with
the results of Chapman et al
(1998), was used as a positive
control. The amount of ZP3 resident in a human ZP has been estimated at 5
ng/zona (Van Duin et al 1994).
As shown in Figure 1A,
rhuZP3a22176 induced human sperm AR in a
concentration-dependent manner. RhuZP3b177348 also induced
sperm AR to some extent (Figure
1B). These data suggest that rhuZP3a22176 and
rhuZP3b177348 stimulate sperm AR in a manner similar to
natural human ZP3.
|
|
176 or rhuZP3b177348. After
incubation with rhuZP3 for 15 minutes, CTC staining was performed. PTX
completely abolished AR induced by rhuZP3a22176 and
rhuZP3b177348 (Figure
2). The results indicate that both rhuZP3a22176
and rhuZP3b177348 act via a G protein.
RhuZP3 Requires Extracellular Ca2+ for the Induction of AR
To assess whether the effects of these peptides on the induction of AR were
related to Ca2+ influx via Ca2+ channels, the
capacitated sperm were incubated with either 2 mM EGTA (a Ca2+
chelator) or 100 µM pimozide (a T-type calcium channel blocker) or 10 µM
verapamil (an L-type calcium channel blocker) for 15 minutes, before the
addition of 50 µg/mL rhuZP3a22176 or
rhuZP3b177348. At the same time, capacitated sperm without
EGTA, pimozide or verapamil were used as controls. EGTA significantly reduced
rhuZP3-induced AR (Figure 3A).
Pimozide also abolished AR triggered by these two peptides
(Figure 3B). Verapamil was less
effective, whereas verapamil inhibited the AR induced by
rhuZP3a22176(23.5 ± 2.8%) and
rhuZP3b177348(25.5 ± 2.8%). These results suggest that
the rhuZP3s induce AR via a major T-type calcium channel.
|
Rhuzp3a22176or Rhuzp3b177348 Induces a Rise in Intracellular Calcium
The addition of rhuZP3a22176 to capacitated sperm loaded
with Fura-2/AM caused a rapid rise in the intracellular Ca2+ levels
(peak) but almost no sustained elevation of intracellular Ca2+. The
addition of pimozide or EGTA before the addition of
rhuZP3a22176 significantly reduced the peak values
(Figure 4A). The addition of
ZP3b177348 to capacitated sperm loaded with Fura-2/AM caused a
sudden rise in the intracellular Ca2+ levels (peak) and sustained
elevation of intracellular Ca2+ (plateau). Pimozide or EGTA
significantly reduced both the peak and the sustained values
(Figure 4B). These results
suggest that rhuZP3 induces sperm AR via increases in intracellular calcium
levels.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The construction, expression, and purification of
rhuZP3a22176 and rhuZP3b177348 have been
described in our previous study (He et al,
2005). Both peptides are able to generate higher antibody titers
in rabbits. Each antiserum specifically recognizes or binds to each target
peptide expressed in E coli and the native human ZP in vitro. The
antisera also inhibit sperm-egg binding in the competitive hemizona assay
(HZA) (Song et al, 2005). To
study further whether rhuZP3a22176 and
rhuZP3b177348 are capable of triggering the AR, the effects of
different concentrations of these 2 peptides have been analyzed in the present
study. Our results show that 5 µg/mL rhuZP3a22176 incubated
with capacitated human sperm for 15 minutes induces the AR. The concentrations
of rhuZP3a22176 and rhuZP3b177348 that induced
AR ranged from 5 to 50 µg/mL (Figure 1A
and B). These results show that the abilities of
rhuZP3a22176 and rhuZP3b177348 to initiate the
AR are not identical. This is probably due to the different lengths of the
amino acid sequences of the two peptides, since rhuZP3b177348
has a longer amino acid chain than rhuZP3a22176. Compared to
rhuZP3 expressed in CHO cells (Bray et al,
2002), rhuZP3b177348 appears to be more potent.
The rhuZP3 from CHO cells induced about 30% of AR at 100 µg/mL, whereas
this rate could be achieved by rhuZP3b22176 at 50
µg/mL.
E coli-expressed, intact ZP3-induced AR in human spermatozoa, as
reported by Chapman et al
(1998), requires 18 hours,
whereas rhuZP3a22176 and rhuZP3b177348
induced-AR required 15 minutes. These different results raise four major
possibilities. First, the purified recombinant human ZP3 used by Chapman et al
(1998) was expressed in E
coli as a C-terminal fusion to the dimeric GST from S japonicum.
The peptide fragments and lengths are different from those used in the present
work. Alternatively, some differences exist in the domains of the peptide
fragment. Second, the method of isolation of motile spermatozoa is also
different. Chapman et al
(1998) used a direct swim-up
technique, which ensures a longer time for sperm capacitation than washing the
spermatozoa by centrifugation through a 3-step Percoll gradient (Ni et al,
unpublished data). Third, in a previous study, we demonstrated that the
antisera against rhuZP322176 and rhuZP3176384
blocked human sperm-egg binding using a competitive hemizona assay (HZA),
which indicates that these peptides are similar to the domain of the native
ZP3 (Song et al, 2005).
Finally, the AR depends on the sperm capacitation status. Due to a lack of
agreement on how female-derived factors affect the process of capacitation, it
is not clear whether the time required for completion of the capacitation
process is different in vitro. The time required for sperm capacitation in the
human is a topic for debate (Jaiswal and
Eisenbach, 2002).
It is generally accepted that the carbohydrate domain displayed by the ZP3 glycoprotein plays a central role in sperm-egg binding (Wassasman, 1990). However, although both native human ZP and purified, glycosylated rhuZP3 secreted by CHO cells have been shown to stimulate AR in human spermatozoa, it has yet to be firmly established whether the ability to initiate AR resides in the carbohydrate moieties of the ZP3 glycoprotein alone, the polypeptide backbone of ZP3 or elements of both (Chapman et al, 1998). Although glycosylation of ZP3 is required for the induction of acrosomal exocytosis (Gahlay and Gupta, 2003), the significance of the polypeptide backbone should still be considered. Different recombinant ZP3s expressed from E coli are free of carbohydrate modifications but provide diverse results (Chapman et al, 1998; Gahlay and Gupta, 2003). This phenomenon may be caused by the different polypeptide backbones of recombinant ZP3, since one is effective (Chapman et al, 1998) and another is ineffective (Gahlay and Gupta, 2003). Furthermore, our results from the present study are distinct from the results of these previous studies. The different polypeptide backbones may explain why these results are discordant.
In the present study, PTX was shown to inhibit the AR induced by both
rhuZP3a22176 and rhuZP3b177348
(Figure 2), suggesting that a
pertussis toxin-sensitive G protein is involved in the signal pathways. Our
results are consistent with the observations obtained using other rhuZP3s in
different laboratories (Dong et al,
2001; Bray et al,
2002). To test the effects of PTX on peptide-initiated AR, sperm
were preincubated without PTX under capacitation conditions for 5 hours and
then exposed to PTX at various concentrations for 10 minutes before the
addition of rhuZP. After incubation, the sperm were challenged with rhuZP3 for
15 minutes, stained, and fixed. According to the inhibition test of
concentration response, 0.52.0 µg/mL PTX caused a significant
dose-dependent inhibition of rhuZP3-stimulated AR, with maximal effects seen
with 1.02.0 µg/mL of PTX. There were no effects on sperm motility or
integrity. It is generally believed that the peptides are sensitive to
pertussis toxin. Although the magnitude of the PTX concentration (0.52.0
µg/mL) used in the present study are slightly higher than those used by
Bray et al (2002) and Dong et
al (2001), who reported that
PTX inhibited the AR induced by recombinant human ZP3 at a concentration of
100 ng/mL. They pretreated the spermatozoa with PTX (100 ng/mL) for 3 hours
before stimulating with rhuZP3. This is a longer time period than we used. In
other words, this difference is probably the result of spermatozoa exposure
time to PTX. Therefore, we consider that peptide-induced AR is mediated by a
transmembrane signaling pathway that involves the activation of PTX-sensitive
G proteins.
The removal of extracellular Ca2+ from the medium by the
addition of EGTA significantly inhibited the AR induced by
rhuZP3a22176 or rhuZP3b177348, which suggests
that extracellular Ca2+ is required for the signal pathway.
Pretreatment with EGTA did not inhibit the basal or spontaneous acrosomal
exocytosis, which suggests that intracellular sources of calcium may be
sufficient for sustaining basal levels of AR
(Schuffner et al, 2002).
The AR stimulated by rhuZP3a22176 or
rhuZP3b177348 was possibly mediated by a VOCCT. It
has been demonstrated that T-type current, but not L-type current, is involved
in mouse sperm AR induced by ZP (Arnoult et
al, 1996a). We demonstrated that the AR induced by these 2
peptides was blocked by the VOCCT inhibitor pimozide but less
effectively by verapamil. However, this does not rule out the potential
influence of phospholipase A2
(Thakkar et al, 1984). It is
well known that verapamil is an L-type calcium channel blocker that inhibits
calcium influx, resulting in the inhibition of sperm AR. However, it is still
not known how verapamil inhibits phospholipase A2 activity.
[Ca2+]i elevation was initiated by rhuZP3a22176
and rhuZP3b177348(Figure 4A
and 4B). The elevation of [Ca2+]i induced by ZP3 has
been demonstrated to proceed via two signaling pathways in sperm
(Felix, 2005). ZP3 activates a
heterotrimeric GTP-binding protein and PLC, thus elevating [Ca2+]i.
After binding to the same receptor(s), ZP3 can also stimulate a transient
influx of calcium through T-type channels. The influx of Ca2+
through T-type Ca2+ channels is transient (500 ms)
(Darszon et al, 2001;
Jagannathan et al, 2002).
Furthermore, the initial response to ZP is a large, transient
[Ca2+]i spike with kinetics comparable to those of T currents
(Arnoult et al, 1999). This
initial Ca2+entry induces a second, sustained Ca2+
influx. Recent evidence indicates that the sustained component of
Ca2+influx is mediated primarily by store-operated channels (SOCs)
that are activated after the depletion of a small Ca2+store
(O'Toole et al, 2000;
Jungnickel et al, 2001),
probably in the acrosome. When sperm were treated with
rhuZP3a22176 or rhuZP3b177348, the ratio of
fluorescence intensity (F340/F380) rose, indicating the
elevation of [Ca2+]i. Both rhuZP3a22176 and
rhuZP3b177348 enable the capacitated sperm to increase the
[Ca2+]i, which indicates that they have biological activities
similar to those of the native human ZP3. To our knowledge, this is the first
report that rhuZP3 peptides generated from a prokaryotic cell induce changes
in the intracellular calcium levels of human spermatozoa. It has been reported
that native ZP3 causes a spike followed by activation of a sustained influx of
[Ca2+]i (Baldi et al,
1996). We found that rhuZP3a22176 caused a spike
without plateau elevation of [Ca2+]i, while
rhuZP3b177348 caused a spike followed by activation of a
sustained influx of [Ca2+]i. This suggests that
ZP3b177348 is similar to natural ZP3 and can act as an
alternative for native human ZP3.
In conclusion, rhuZP3a22176 and
rhuZP3b177348 obtained from E coli were capable of
inducing AR at different concentrations. With the addition of each of these 2
peptides, the [Ca2+]i was raised, although the patterns differed.
The rhuZP3-induced AR was inhibited by PTX, EGTA, and pimozide. The present
study demonstrates that rhuZP3a22176 and
rhuZP3b177348 have the same efficiency as natural human ZP3 to
induce human AR, and that they induce AR via the activation of a G protein and
the induction of extracellular calcium influx through a T-type calcium
channel.
|
Footnotes |
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¶ Y.N. and K.L. contributed equally to this work. 
|
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6, except for ZP, where n = 2). For each graph, the letters above
the bars indicate statistically significant differences. (A) a compared
to the negative control, P < .05; c compared to b and d,
P > .05; d compared to b, P < .05. (B) a
compared to the negative control, P < .05; c compared to d,
P > .05; d and c compared to b, P < .05.
