letters to nature
Received 23 April; accepted 21 October 1999.
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Univ. Press, New York, 1992).
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reanalysis of 1987 survey data. Conserv. Biol. 10, 1142±1153 (1996).
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Supplementary information is available on Nature's World-Wide Web site
(http://www.nature.com) or as paper copy from the London editorial of®ces of Nature.
Acknowledgements
We thank A. Baynes, J. H. Brown, N. Czaplewski, B. Danielson, T. Franklin, M. Kaspari,
B. Maurer, K. Pandora, D. Perault, K. Perez, S. Pimm, G. A. Smith and C. Vaughn for
advice and comments on this paper, and J. M. Scott, D. Steadman and L. Carbyn for
information on the distribution of several species. R.C. was supported by the Department
of Zoology, University of Oklahoma, while conducting this research, and M.V.L. was
supported by grants from the US National Science Foundation.
Correspondence and requests for materials should be addressed to R.C.
(e-mail: [email protected]).
86
.................................................................
Reduced vas deferens contraction
and male infertility
in mice lacking P2X1 receptors
K. Mulryan*, D. P. Gitterman*, C. J. Lewis*, C. Vial*, B. J. Leckie²,
A. L. Cobb³, J. E. Brown³, E. C. Conley§, G. Buellk#, C. A. Pritchard¶
& R. J. Evans*
* Department of Cell Physiology & Pharmacology, Medical Sciences Building,
² Department of Medicine, ³ Transgenic Unit, Biomedical Services,
§ Department of Pathology and Centre for Mechanisms of Human Toxicity &
¶ Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK
k Glaxo-Wellcome Geneva Biomedical Research Institute, 14 chemin des Aulx,
Plan-les-Ouates, 1228 Geneva, Switzerland
# Present address: Serono Pharmaceuticals, 14 chemin des Aulx, Plan-les-Ouates,
1228 Geneva, Switzerland
..............................................................................................................................................
P2X1 receptors for ATP are ligand-gated cation channels, present
on many excitable cells including vas deferens smooth muscle
cells1±5. A substantial component of the contractile response of the
vas deferens to sympathetic nerve stimulation, which propels
sperm into the ejaculate, is mediated through P2X receptors1.
Here we show that male fertility is reduced by ,90% in mice with
a targeted deletion of the P2X1 receptor gene. Male mice copulate
normallyÐreduced fertility results from a reduction of sperm in
the ejaculate and not from sperm dysfunction. Female mice and
heterozygote mice are unaffected. In P2X1-receptor-de®cient
mice, contraction of the vas deferens to sympathetic nerve
stimulation is reduced by up to 60% and responses to P2X
a
exon 1
ATG
Probe
The index of centrality (C) ranged from 0, where the extant range fell completely outside
the central portion of the historical range, to 1, where the extant range fell completely
within the central portion of the historical range. We designated species with C values
greater than 0.5 as `central species', and those species with C values less than 0.5 as
`peripheral species'. We then used a binomial test to determine whether the ratio of central
to peripheral species differed signi®cantly from 1 : 1.
We used maps for species with multiple patches in their historical range to test whether
persistence was higher for populations inhabiting larger patches. We ®rst assigned patches
to one of two size categories (`large' or `small'), based on their area relative to the median
patch size. If a species had an odd number of patches in its historical range, the mediansized patch was excluded from the analysis. For each species, we counted the number
of large and small patches maintaining persistent populations (P1 and Ps, respectively).
We counted the number of species (S1) for which P1 was greater than Ps and the number
of species (Ss) where Ps was greater than P1. Species with ties (P1 ˆ Ps ) were excluded
from analysis. We used a binomial test to determine whether the ratio of S1 to Ss
differed signi®cantly from 1 : 1. This analysis was done for 124 continental and 44 insular
species24.
To compare the relative persistence of mainland and island patches, we ®rst calculated
the total area of all of the historical patches (ATH) and the area of the historical mainland
patches (AMH) for 44 species. We multiplied AMH/ATH by the total number of persisting
patches (PTP) to generate the expected number of patches persisting on the mainland.
If the number of patches persisting on the mainland (PMP) was greater than expected,
we classi®ed the species as a mainland species, otherwise it was classi®ed as an island
species. There were no ties (PMP ˆ expected number of patches). We tested whether the
ratio of mainland species and island species differed signi®cantly from 1 : 1 using a
binomial test.
Wild-type gene
1Kb
CD
X
HSV-TK
b
X
A B
TK-neo
lacZ
TK-neo Mutated gene
c
+/+
Targeting
vector
lacZ
d
–/– +/+
+/+ +/– –/–
+/–
WT
neo
P2X1
KO
P2X1
actin
Figure 1 Generation of P2X1-receptor-de®cient mice. a, Genomic maps of the wild-type
gene, targeting vector and mutated gene. BamHI sites (indicated by arrows) and the probe
used for detection of the homologous recombination events by Southern analysis are
shown. Polymerase chain reaction (PCR) primers used for genotyping of mouse-tail DNA
are indicated (A±D). b, Southern blot analysis of tail genomic DNA from +/+ and -/animals. Genomic DNA was digested with BamHI and hybridized with the probe indicated
in a which detects a 4.8-kb band in +/+ DNA and a 3.7-kb band in -/- DNA. WT, wildtype; KO, knock-out. c, PCR genotyping of mouse-tail DNA. Primers A, B, C and D were
used in one PCR reaction to genotype mouse-tail genomic DNA. Primers A and B amplify a
519-bp product from the neoR gene, whereas primers C and D amplify a 317-bp product
from the deleted region of the P2X1 receptor gene. c, RT-PCR analysis. A PCR product of
442 bp from the P2X1-receptor gene was ampli®ed from bladder complementary DNA
from a +/+ animal but not from bladder cDNA of a -/- animal. As a control, ampli®cation
of 199-bp product from the actin gene was detected in both samples.
© 2000 Macmillan Magazines Ltd
NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com
letters to nature
receptor agonists are abolished. These results show that P2X1
receptors are essential for normal male reproductive function and
suggest that the development of selective P2X1 receptor antagonists
may provide an effective non-hormonal male contraceptive pill.
Also, agents that potentiate the actions of ATP at P2X1 receptors
may be useful in the treatment of male infertility.
ATP is co-released with noradrenaline from sympathetic nerves
and acts through P2X receptors on smooth muscle to mediate
membrane depolarization and contraction1. Seven P2X receptors
have been identi®ed at the molecular level1. P2X receptors were
originally isolated from the rat vas deferens and can account for the
rapidly inactivating a,b-methylene ATP (a,b-meATP)-sensitive
native vas deferens P2X receptor phenotype6. In smooth muscle
the P2X1 receptor is the dominant form expressed5. Owing to the
lack of potent and subtype-selective P2X-receptor antagonists the
physiological role of P2X1 receptors has been dif®cult to determine.
To overcome these problems we have generated a P2X1-receptorde®cient mouse.
The P2X1-receptor-targeting vector was designed to remove the
®rst 45 amino acids of the P2X1 receptor (exon 1) and results in the
functional inactivation of the P2X1 receptor gene (Fig. 1). Male and
female mice heterozygous for the P2X1 receptor de®ciency (+/-) are
phenotypically normal and when inter-crossed produced progeny
with a mendelian genotype distribution of 1+/+ : 2.1+/- : 1-/- (sex
ratio 1.05 male : 1 female, n ˆ 320) indicating that there is no
selective fertilization or mortality in utero. Con®rmation of the
de®ciency of the P2X1 receptor in homozygous mutant mice was
obtained at the messenger RNA and protein levels. Polymerase
chain reaction after reverse transcription of RNA (RT-PCR), using
primers speci®c for a region 39 to that deleted by the targeting
vector, ampli®ed a 442-base pair (bp) product from +/+ mice but
not from -/- mice, indicating that mRNA for a truncated form of
the P2X1 receptor is not produced by -/- mice (Fig. 1d). A P2X1
receptor antibody raised against the carboxy terminus of the protein
produced high levels of immunoreactivity in the smooth muscle
layer of +/+ vas deferens as reported previously7. However, no P2X1
receptor immunoreactivity was detected in the vas deferens of -/mice (Fig. 2a and b).
Interbreeding of mutant -/- mice did not result in pregnancy
(n ˆ 8) even though coitus had taken place. Mutant -/- female
mice produced litters when mated with +/+ (n ˆ 5) or +/- males
(n ˆ 3). When -/- males were mated with +/+ females only 13.7%
of matings resulted in pregnancy (n ˆ 51 matings of 22 -/- males)
compared to a 100% pregnancy rate for +/- males (n ˆ 12). These
results indicate that the reduced fertility was speci®c to the male
P2X1-receptor-de®cient mice. The mean litter size was also
reduced for -/- males by 30% (mean litter size -/- 6:6 6 1:0,
n ˆ 6; +/- 9:5 6 0:46, n ˆ 12; P , 0:05). Taking together the
reduction in pregnancy rate and litter size, the fecundity of -/males is reduced by 90.4%.
A vaginal coagulum/plug is formed following copulation. This
results from the action of enzymes in the seminal plasma secreted
from the male sex accessory glands. Coagulum weight was essentially the same following mating with +/+ or +/- mice
(47:2 6 2:7 mg, n ˆ 5) or -/- males (57:3 6 3:7 mg, n ˆ 7) indicating that similar ejaculate volumes were produced. Given that the
¯uids secreted from accessory sexual glands account for ,80% of
total semen volume these ®ndings suggest that the contraction and
function of these glands is unaffected in the P2X1-receptor-de®cient
mouse. The reduction in male fertility could result from problems
with spermatogenesis or sperm quality and/or the reduction of
sperm numbers in the ejaculate. P2X1 receptor immunoreactivity
was detected in blood vessels in the +/+ testis; however, no
immunoreactivity was detected in the seminiferous tubules
(Fig. 2c), indicating that P2X1 receptors are not involved in
spermatogenesis. In addition, haematoxylin- and eosin-stained
sections of testis from -/- mice appeared normal. Vas deferens
from +/+, +/- and -/- mice appeared equally full of sperm.
Motile sperm were recovered from +/+, +/- and -/- epididymis and vas deferens in similar numbers (epididymal count
…3:6 6 0:8† 3 106 ml 2 1 for +/+ and +/-, …5:1 6 1:6† 3 106 ml 2 1
for -/-, n ˆ 5±6) and were equally effective at fertilizing ova in
vitro (Fig. 2b and d, % success at 4 days 70:0% 6 2:6% for +/+ and
65:5% 6 5:9% for -/-, n ˆ 3). Thus infertility does not result from
a
+/+
b
+/–
–/–
–/–
c
125µm
Figure 2 Immunohistochemical detection of P2X1 receptors and in vitro fertilization
studies. a, b, Confocal images of transverse sections of vas deferens show
immunohistochemical detection of the P2X1 receptor protein in the smooth-muscle layers
of the wild-type vas deferens (a); no immunoreactivity was present in the -/- mutant
animal (b). c, Confocal image of a transverse section of testis shows P2X1-receptor
immunoreactivity associated with blood vessels, but no reactivity with the seminiferous
tubules. d, Fertilization of ova in vitro by sperm extracted from -/- epididymis,
photomicrograph taken at 4 days.
NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com
1 nA
2s
500 ms
e
Contraction (g)
1.5
1.0
0.5
0
d
150µm
Contraction (g)
150µm
150µm
–/–
ATP
α,ββ,γmeATP meATP
d
b
+/+
ATP
20 mV
ATP
a
c
1
0.1
10 100
α,β-meATP (µM)
1.5
1.0
0.5
0
1
0.1
10 100
Noradrenaline (µM)
Figure 3 Response to purinergic agonists, nerve stimulation and noradrenaline of wildtype and P2X1-receptor-de®cient mouse vas deferens. a, ATP (100 mM), a,b-meATP and
1-b,g-meATP (both 10 mM) evoked transient contractions of +/+ vas deferens but had
no effect on the tone of -/- vas deferens. b, Intracellular membrane potential recordings
from vas deferens smooth-muscle cells. Sympathetic nerve stimulation (10 pulses at
10 Hz, 0.5 ms pulse width) evoked excitatory junction potentials in +/- but not -/- vas
deferens (resting membrane potential 2 87:7 6 1 mV and 87:6 6 1:7 mV for +/- and
-/- mice respectively, n ˆ 16 for each). c, Whole-cell patch-clamp recordings from
acutely dissociated vas deferens smooth muscle cells. ATP evoked a rapidly inactivating
inward current in +/+ but had no effect on -/- acutely dissociated vas deferens smooth
muscle cells (holding potential -60 mV). (a, c, Agonist applications indicated by bar, 60s
for a). d, e, Concentration±effect relationship for a,b-meATP (d) and noradrenaline (e)
for +/+ (open circles), +/- (shaded circles) and -/- (solid circles) vas deferens segments.
© 2000 Macmillan Magazines Ltd
87
letters to nature
a sperm dysfunction. Similarly reduced fertility is unlikely to have
been caused by blockage of the vas deferens as this results in
degeneration of sperm in the epididymis8. Analysis of lavage from
the uterus, taken at the junction with the coagulum, showed that
sperm were abundant in mice mated with +/+ (n ˆ 3) or +/- mice
(n ˆ 2), but no sperm could be detected in the lavage from matings
with -/- males (n ˆ 7). These results show that the reduction in -/male fertility results from a reduced sperm count.
Firing of sympathetic nerves associated with ejaculation results in
contraction of the vas deferens and emission of sperm into the
semen. A substantial component of this sympathetic-nerve-evoked
contraction is mediated through the activation of P2X receptors9.
P2X1-receptor-de®cient mice may therefore be compromised in vas
deferens function and this could explain the absence of sperm in the
ejaculate. In the +/+ vas deferens the purinergic agonists ATP, a,bmeATP and 1-b,g-meATP evoked transient contractions through
the activation of P2X receptors, which declined to baseline during
the continued presence of the agonist as described previously10,11
(Fig. 3a). The pEC50 values of a,b-meATP for evoking contraction
of the vas deferens for +/+ and +/- mice are 5:83 6 0:05 and
5:72 6 0:04, respectively (Fig. 3d). P2X receptors are thought to
underlie the excitatory junction potentials (EJPs) recorded from
smooth muscle12. Spontaneous EJPs and EJPs evoked by sympathetic nerve stimulation were recorded from all impalements of
the +/- vas deferens (n ˆ 16 impalements from four vasa deferentia) (Fig. 3b). In patch clamp studies on acutely dissociated +/+
vas deferens smooth muscle cells a,b-meATP and ATP evoked
transient inward currents (amplitude 2861 6 410 pA, n ˆ 8 and
2096 6 175 pA, n ˆ 3, respectively) with latency ,10 ms and with
10%±90% rise times of 22:7 6 7:7 ms (n ˆ 4), demonstrating the
direct activation of a ligand-gated P2X-receptor channel3,11 (Fig. 3c).
In contrast, the purinergic agonists a,b-meATP and ATP failed to
evoke contractions (Fig. 3a, d) or inward currents (Fig. 3c) and
spontaneous or evoked EJPs were not detected (Fig. 3b) (n ˆ 16
impalements from four vasa deferentia) from the vas deferens of the
-/- P2X1-receptor-de®cient mouse. It has been suggested, on the
b –/–
a +/+
control
0.2g
0.2g
control
d
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Contraction (g)
Contraction (g)
c
control
α,βα,βmeATP meATP
+ praz
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
α,βα,βmeATP meATP
+ praz
60p
20Hz
10p
20Hz
5p
10Hz
Figure 4 Sympathetic nerve-mediated contraction of +/+ and -/- P2X1-receptorde®cient vas deferens. a, b, Nerve stimulation, 60 pulses at 20 Hz (period indicated by
bar) evoked contractions of +/+ (a) and -/- (b) vas deferens. Following desensitization of
P2X-receptor-mediated responses with a,b-meATP (10 mM), the residual contraction
was abolished by the a1-adrenoceptor antagonist prazosin (0.1 mM). c, Summary of the
effects of P2X-receptor-desensitization with a,b-meATP and the a1-adrenoceptor
antagonist prazosin on neurogenic contractions of the mouse vas deferens; +/+ (white
columns), +/- (grey columns) and -/- (black columns). d, Effects of stimulus frequency
and duration on neurogenic contractions of the mouse vas deferens. Histogram shows
peak amplitude of contraction to stimulation with 60 pulses at 20 Hz, 10 pulses at 20 Hz
and 5 pulses at 10 Hz for +/+ (open columns) and -/- (black columns) mice.
88
basis of contraction studies, that there may be multiple P2X receptor
subtypes in the vas deferens (see references in ref. 13). In the present
study vas deferens P2X receptors showed the hallmark properties of
homomeric P2X1 receptors (transient a,b-meATP and 1-b, gmeATP sensitive responses). The lack of functional P2X receptors
in the -/- vas deferens shows that if subtypes do exist the expression
of P2X1 receptors is essential for the production of functional
receptors.
The lack of contraction of the -/- vas deferens to purinergic
agonists does not result from an impairment of the contractile
function, as responses evoked by 100 mM potassium chloride were
the same for +/+ and -/- P2X1-receptor-de®cient mice
(1:36 6 0:12 g and 1:17 6 0:08 g, respectively; n ˆ 14±17). In addition, +/+, +/- and -/- vas deferens contracted in response to
noradrenaline (Fig. 3e). The vas deferens of the -/- mice were more
sensitive to noradrenaline than the +/+ (pEC50 values of 5:65 6 0:15
and 4:79 6 0:09, respectively, P ˆ 0:005, n ˆ 4±6) and the +/- mice
had an intermediate sensitivity with a pEC50 of 5:1 6 0:27 (n ˆ 5).
The increase in sensitivity to noradrenaline in -/- animals suggests
a compensatory change in -/- animals which could be accounted
for by an increase in a1-adrenoceptor numbers14.
The lack of P2X receptor-mediated contractions in -/- vas
deferens suggests that contractions in response to sympathetic
nerve stimulation may also be affected. In rats the duration of
copulation from the ®rst pelvic thrust to dismount is 1±3 s (ref. 15).
To determine the response of the vas deferens to nerve stimulation
the vas deferens was stimulated with a train of 60 pulses at 20 Hz
(train duration 3 s) (Fig. 4). Electrically evoked responses were
mediated through the activation of sympathetic nerves, as they were
abolished by tetrodotoxin (0.3 mM) or the adrenergic neuron
blocker guanethidine (3 mM) (n ˆ 4). The peak amplitude of
contraction of +/+ mouse vasa deferentia was 1:16 6 0:08 g
(n ˆ 21). Desensitization of P2X receptors with a,b-meATP
revealed that the P2X-receptor-mediated contraction accounted
for 79 6 5:5% of the response (n ˆ 7). The residual response was
abolished by the a1-adrenoceptor antagonist prazosin (Fig. 4a and
c). Similar responses were recorded from +/- males (Fig. 4c). In
contrast, the peak amplitude of contractions of -/- vas deferens was
only ,60% (0:7 6 0:04 g, n ˆ 36) of the +/+ response (n ˆ 16),
was reduced by ,10% by a,b-meATP treatment and was abolished
by prazosin (n ˆ 4) (Fig. 4b and d). This increase in magnitude of
the a1-adrenoceptor-mediated component in -/- mice, like the
response to exogenously applied noradrenaline, can be accounted
for by an increase in a1-adrenoceptor number14. Long trains of
high-frequency stimulation are known to favour the noradrenergicmediated component of smooth-muscle contraction; however, they
probably do not re¯ect the in vivo nerve traf®c.
The physiological sympathetic nerve ®ring pattern resulting in
ejaculation remains to be determined. It is likely that the burst of
sympathetic nerve activity to the vas deferens associated with
ejaculation is considerably shorter than the duration of copulation.
We therefore determined the effects of 0.5-s trains of stimuli at
different frequencies. The magnitude of the contractile response was
frequency dependent (Fig. 4d) and in response to 10-Hz stimulation
the resonse of -/- vas deferens was only 40% of the +/+ response. A
similar decrease in ejaculate sperm count results in infertility16.
Thus, this reduction in the neurogenic vas deferens contraction can
account for the decrease in male fertility rate and suggests that in the
majority of cases, in vivo contraction of the -/- vas deferens is below
the threshold required to eject the sperm into the semen.
In addition to the vas deferens, P2X1 receptors are also present on
a variety of other smooth-muscle preparations, including the
urinary bladder and arteries, as well as parts of the nervous
system. There was no obvious effect on the behaviour of P2X1receptor -/- mice, and heart rate (572:2 6 20:3 beats per minute for
-/- and 556 6 19 beats per minute for +/+, n ˆ 8 for each), and
bladder function all appeared normal. There was, however, a small
© 2000 Macmillan Magazines Ltd
NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com
letters to nature
increase in systolic blood pressure at rest (115:8 6 2 mm Hg for -/and 108 6 1:8 mm Hg for +/+, as measured by the tail cuff method;
P ˆ 0:016, n ˆ 8 for each).
We have shown that P2X1 receptors are involved in the contraction of the vas deferens and that P2X1 receptor de®ciency results in a
90% decrease in male fertility through a reduction in sperm in the
ejaculate associated with a decrease in neurogenic vas deferens
contraction. In mice it appears that the residual a1-adrenoceptormediated neurogenic vas deferens contraction is insuf®cient for
normal ejaculatory function. Selective a1-adrenoceptor antagonists
do not cause azoospermia and infertility in man17 and contractile
studies have indicated the presence of a substantial non-adrenergic
component of contraction of prostatic portions of the human vas
deferens18. This suggests that P2X1-receptor antagonists may provide a target for the development of a non-hormonal male contraceptive pill. In addition, agents that potentiate the actions of ATP at
P2X1 receptors may be useful in the treatment of male infertility19. M
Methods
12. Burnstock, G. & Holman, M. E. The transmission of excitation from autonomic nerve to smooth
muscle. J. Physiol. 155, 115±133 (1961).
13. Ralevic, V. & Burnstock, G. Receptors for purines and pyrimidines. Pharmacol. Rev. 50, 413±492
(1998).
14. Kenakin, T. in Pharmacologic Analysis of Drug-Receptor Interaction Vol. 3, 63±67 (Lippincott-Raven,
Philadelphia, 1997).
15. Yang, L. & Clemens, L. G. In¯uence of male-related stimuli on female postejaculatory refractory
period in rats. Physiol. Behav. 63, 675±682 (1998).
16. Anderson, R. A., Oswald, C., Willis, B. R. & Zaneveld, L. J. D. Relationship between semen
characteristics and fertility in electroejaculated mice. J. Reprod. Fertil. 68, 1±7 (1983).
17. Hoffman, B. B. & Lefkowitz, R. J. in The Pharmacological Basis of Therapeutics (eds Hardman, J. G.,
Limbird, L. E., Molinoff, P. B., Ruddon, R. W. & Gilman, A. G.) Vol. 9, 199±248 (McGraw-Hill, New
York, 1996).
18. Hedlund, H., Andersson, K. & Larsson, B. Effect of drugs interacting with adrenoceptors and
muscarinic recetors in the epididymal and prostatic parts of the human isolated vas deferens. J.
Autonom. Pharmacol. 5, 261±270 (1985).
19. Jacobson, K. A. et al. A pyridoxine cyclic phosphate and its 6-azoaryl derivative selectively potentiate
and antagonize activation of P2X1 receptors. J. Med. Chem. 41, 2201±2206 (1998).
20. Hogan, B., Constantini, F. & Lacy, E. Manipulating the Mouse Embryo: A Laboratory Manual (Cold
Spring Harbour, New York, 1986).
21. Lewis, C., Surprenant, A. & Evans, R. J. 29,39-O-(2,4,6-trinitrophenyl) adenosine 59-triphosphate
(TNP-ATP)Ða nanomolar af®nity antagonist at rat mesenteric artery P2X receptor ion channels. Br.
J. Pharmacol. 1463±1466 (1998).
Generation of P2X1 receptor-de®cient mice
Acknowledgements
The targeting vector includes 5.8 kilobases (kb) of P2X1-receptor genomic DNA, the lacZ
gene, the neoR gene driven by the TK promoter and the HSV-tk gene. Homologous
recombination of this vector with the wild-type gene results in deletion of 350 bp of DNA
which includes exon 1 and the initiating ATG. The deleted 350 bp are replaced by the lacZ
gene and the neoR gene of the targeting vector. The targeting vector was electroporated into
E14.1a embryonic stem (ES) cells derived from the 129Ola mouse strain, and colonies were
selected with G418 and gancyclovir. Positive colonies were identi®ed by the presence of the
3.7 kb band in BamHI-digested genomic DNA. Four ES colonies containing the targeting
event were microinjected into F1 (CBA 3 C57BL=6) blastocysts and chimaeras were
derived. Germline transmission of the targeted allele was obtained from all four ES
colonies upon mating to MF-1 animals. The mice analysed here have 129Ola-MF-1 genetic
backgrounds.
We thank L. Vulchanova and R. Elde for the P2X1 receptor antibody, B. Grubb and
S. Giblett for help with the immunohistochemistry and C. d'Lacey for help with the
confocal images. We also thank J. Luckett and S. Monkley for help at the bench and S. R.
Nahorski for comments on the manuscript. This work was supported by the Medical
Research Council and the Wellcome Trust.
Immunohistochemistry
Detection of the distribution of the P2X1 receptor using an antibody raised against the C
terminus of the receptor was as described7. In vitro fertilization was as described20.
Physiological studies
Mice were produced by crossing +/- mice. Littermates were genotyped by PCR, animals
were sexually mature (4±6 months old), weight was 37:5 6 0:8, 39:6 6 1:3 and 37:36
1:0 g for wild type, +/- and -/-, respectively (n ˆ 15±19). Mouse vasa deferentia were
mounted in Ringers solution at 36 8C in 15-ml organ baths under an initial load of 1 g, and
tension was monitored isometrically. Agonists were applied to the bath at 30-min intervals
and removed by washing; this solution was also used for intracellular recordings from the
vas deferens using standard methods. Trains of electrical stimuli were delivered through
silver chloride electrodes, 20±40 V 0.5 ms pulse width. Vas deferens smooth-muscle cells
were enzymatically dissociated and responses to purinergic agonists applied rapidly by a
U-tube perfusion system were determined in voltage-clamp recordings as described21.
Received 22 July; accepted 25 October 1999.
1. Burnstock, G. The past, present and future of purine nucleotides as signalling molecules. Neuropharmacology 36, 1127±1139 (1997).
2. Surprenant, A., Buell, G. & North, R. A. P2X receptors bring new structure to ligand-gated ion
channels. Trends Neurosci. 18, 224±229 (1995).
3. Evans, R. J. & Surprenant, A. P2X receptors in autonomic and sensory neurons. Semin. Neurosci. 8,
217±223 (1996).
4. Brading, A. F. & Williams, J. H. Contractile responses of smooth muscle strips from rat and guinea-pig
urinary bladder to transmural stimulation: effects of atropine and a,b-methyleneATP. Br. J.
Pharmacol. 99, 493±498 (1990).
5. Collo, G. et al. Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended
family of ATP-gated ion channels. J. Neurosci. 16, 2495±2507 (1996).
6. Valera, S. et al. A new class of ligand-gated ion channel de®ned by P2X receptor for extracellular ATP.
Nature 371, 516±519 (1994).
7. Vulchanova, L. et al. Differential distribution of two ATP-gated ion channels (P2X receptors)
determined by immunohistochemistry. Proc. Natl Acad. Sci. USA 93, 8063±8067 (1996).
8. Costa, S. L., Boekelheide, K., Vanderhyden, B. C., Seth, R. & McBurney, M. W. Male infertility caused
by epididymal dysfunction in transgenic mice expressing a dominant negative mutation of retinoic
acid receptors alpha 1. Biol. Reprod. 56, 985±990 (1997).
9. Allcorn, R. J., Cunnane, T. C. & Kirkpatrick, K. Actions of a,b-methylene ATP and 6-hydroxydopamine on sympathetic neurotransmission in the vas deferens of the guinea-pig, rat and mouse:
support for co-transmission. Br. J. Pharmacol. 89, 647±659 (1986).
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mouse vas deferens: suramin-sensitive and suramin-insensitive components in the contractile effect
of ATP. Naunyn Schmiedeberg's Arch. Pharmacol. 342, 198±205 (1990).
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NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com
Correspondence and requests for materials should be addressed to R.J.E.
(e-mail: [email protected]).
.................................................................
Turning of nerve growth cones
induced by localized increases
in intracellular calcium ions
James Q. Zheng
Department of Neuroscience and Cell Biology, University of Medicine and
Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane,
Piscataway, New Jersey 08854, USA
..............................................................................................................................................
Guidance of developing axons involves turning of the motile tip,
the growth cone, in response to a variety of extracellular cues1,2.
Little is known about the intracellular mechanism by which the
directional signal is transduced. Ca2+ is a key second messenger in
growth cone extension3,4 and has been implicated in growth-cone
turning5,6. Here I report that a direct, spatially restricted elevation
of intracellular Ca2+ concentration ([Ca2+]i) on one side of the
growth cone by focal laser-induced photolysis (FLIP) of caged
Ca2+ consistently induced turning of the growth cone to the side
with elevated [Ca2+]i (attraction). Furthermore, when the resting
[Ca2+]i at the growth cone was decreased by the removal of
extracellular Ca2+, the same focal elevation of [Ca2+]i by FLIP
induced repulsion. These results provide direct evidence that a
localized Ca2+ signal in the growth cone can provide the intracellular directional cue for extension and is suf®cient to initiate
both attraction and repulsion. By integrating local and global Ca2+
signals, a growth cone could thus generate different turning
responses under different environmental conditions during guidance.
To examine the response of Xenopus growth cones to spatially
restricted cytosolic Ca2+ signals, FLIP of caged Ca2+ was used to
elevate [Ca2+]i directly in a spot ,2 mm in diameter. The ef®cacy
and spatial restriction of FLIP was ®rst examined in vitro using
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