BACKGROUND
for approximately three decades, the xenopus oocyte has been a useful cell model to determine the underlying mechanisms responsible for the increase of the cytoplasmic ca2+ concentration through its release from intracellular reservoirs  <cit>  and by calcium influx either through ca2+-dependent voltage-dependent channels or via store-operated ca2+  channels  <cit> .
the latter results from the activation of the phenomenon known as store-operated ca2+ entry , which allows the replenishment of emptied reservoirs  <cit>  after the stimulation of ca2+ release through ip3/diacylglycerol synthesis by phospholipase c .
release of ca2+ from intracellular reservoirs and soce activation are common responses in the xenopus oocytes since they endogenously express the machinery that activates plc by stimulating endogenous g protein-coupled receptors ; cytoplasmic ca2+-increase, through either release or influx, opens ca2+-dependent cl− channels in the oocyte membrane generating conspicuous current responses  <cit> .
soce activation in the membrane of the xenopus oocyte was first detected by measuring the transient inward  current response  <cit>  after ca2+ release in the oocyte.
the tin response is generated by hyperpolarizing steps, and is mainly due to the ca2+-influx that subsequently opens ca2+-dependent cl− channels; this membrane response has been used as a reliable monitor of soc channel activation  <cit> .
the soce current is most likely driven through ca2+-permeable channels formed by orai, a channel activated by association with the stromal interaction molecule   <cit> , a protein that is localized mainly in the endoplasmic reticulum  membrane and that senses the ca2+ concentration in its lumen  <cit> .
although transcripts for endogenous orai and stim molecules have been reported in the oocyte  <cit> , the role for the different types and isoforms of these proteins and their relation with endogenous responses in the oocyte have not been thoroughly studied; these issues are of interest given that the roles for the different soc molecular elements are also incompletely understood, and their study in a well-known model such as the xenopus oocyte might reveal important information.
two stim proteins, stim <dig> and stim <dig>  are expressed in eukaryotic cells  <cit> .
a different role for each of them has been proposed; for example, the er ca2+ content must be greatly reduced in order to activate stim <dig> protein, while the more ca2+-sensitive stim <dig> seems to require only a slight reduction in er ca2+ concentration  <cit> .
it has been proposed that stim <dig> participates in maintaining the cytoplasmic ca2+ concentration  <cit> .
although the fundamental role of stim <dig> in activating soce has been demonstrated in several cell types  <cit> , other information indicates that stim <dig> is the main protein involved in soce generation in neurons, dendritic cells, and mammary epithelial cells  <cit> .
thus, it is plausible that the specific functions of stim <dig> and stim <dig> depend on the cell type, their relative rates of expression, and other factors such as interactions among them or with regulatory proteins.
it has also been shown that during maturation, the ca2+-signaling pathway in the oocyte is significantly reconfigured, probably as part of the mechanism that prepares the gamete for fertilization and subsequent embryonic development.
this reconfiguration includes orai <dig> channels and stim <dig>  which are regulated during maturation thus eliminating the soce response  <cit> .
due to the importance of this phenomenon for cell cycle control in general, it is also of interest to explore the effects on oocyte maturation of altered stim expression  <cit> .
in the present study, we specifically knocked down stim <dig> or stim <dig> in the xenopus oocyte to analyze the effect on two endogenous phenomena, the generation of the tin current response  and the maturation process.
we found that stim <dig> expression was essential in both phenomena, while stim <dig> expression was not.
methods
cell preparation
xenopus laevis frogs were obtained from xenopus i .
ovary lobules  <cit>  were surgically removed under sterile conditions from frogs that had been anaesthetized using  <dig> % aminobenzoic acid ethyl ester and rendered hypothermic.
after surgery, frogs were sutured and allowed to recover from anesthesia.
frogs were maintained for 3– <dig> days in individual tanks until healing was complete; they were then housed in larger groups, and no further oocytes were taken from them for at least 2 months.
procedures were approved by the institutional animal committees .
the lobules were placed in sterile barth’s solution containing :  <dig> nacl,  <dig> kcl,  <dig>  nahco <dig>   <dig>  ca <dig>   <dig>  cacl <dig>   <dig>  mgso <dig>  and  <dig> hepes, with 75 μg/ml gentamicin and adjusted to ph  <dig> .
studies were carried out using oocytes at stage vi  <cit>  dissected from the ovaries and defolliculated by collagenase  treatment at room temperature for 30 min in normal frog ringer’s solution .
after washing, the oocytes were stored at 18°c in sterile barth’s solution, and electrical recordings were performed over a period of 2– <dig> days in either uninjected oocytes or in those injected with crna for specific receptors and/or with antisense oligonucleotide to knock down specific proteins.
reverse transcription polymerase chain reactions
total rna from the oocytes was purified using trizol reagent .
first-strand cdna was synthesized using 2 μg of dnase-treated rna as template and 1 μg of oligo ,  <dig>  μg random hexamers, and reverse transcriptase.
the cdna was used as template in a polymerase chain reaction to amplify cdna fragments for stim <dig> and stim <dig>  and the ribosomal protein s <dig>  was used as a control.
all the pcr programs started at 95°c for 2 min.
the amplification in the 35 cycles consisted in 45 s at 95°c, 40 s at 55°c, and 35 s at 72°c, and a final extension at 72°c for 5 min.
the sequences of oligonucleotides used were: stim <dig>  forward, 5'-cgacgagtttctcagggaag-3' and reverse, 5'-cttcatgtggtcctcggagt-3'; stim <dig>  forward, 5'-ccagccttgaggcaatatgt-3' and reverse, 5'-gcaacctccaactccgatta-3'; rps <dig>  forward, 5'-tggtaacaggggaggtttccgc-3' and reverse, 5'-ataccagccatcatgagcagc-3'.
the amplified products were isolated, purified , and subcloned into the pjet  <dig>  vector .
finally, their nucleotide sequences were confirmed by sanger sequencing .
western blot
protein expression was assessed by western blot in either control oocytes or in those injected with as-stim <dig> or as-stim <dig>  for each group,  <dig> oocytes were homogenized 72 h post-injection in a buffer containing :  <dig> tris–hcl ph  <dig> ,  <dig> edta ph  <dig>   <dig> sucrose, and 1x complete mini protease inhibitor .
then samples were centrifuged at 4°c and 500 rpm for 5 min, at 3500 rpm for 10 min, and at  <dig>  rpm for 20 min.
subsequently, the final pellets were resuspended in 50 μl of buffer containing :  <dig> tris–hcl ph  <dig> ,  <dig> edta ph  <dig>   <dig> nacl,  <dig> mgcl <dig>  and 1x complete mini protease inhibitor.
total membrane protein concentration was quantified with a bradford assay.
for electrophoresis, samples  were fractionated in a 10% sds-polyacrylamide gel and transferred to a nitrocellulose membrane .
membranes were blocked for 1 h at room temperature in tbs-t solution :  <dig> nacl,  <dig> tris, ph  <dig> , and  <dig> % tween  <dig>  containing 5% nonfat dry milk and then incubated overnight at 4°c with a 1: <dig> dilution of rabbit primary antibody.
the antibody denoted nh-stim <dig>  was directed against a region of the amino-terminus of the stim <dig> protein, and the antibodies denoted nh-stim <dig>  and cooh-stim <dig>  were against the amino and carboxy termini, respectively, of stim <dig>  western blot analysis was also used to detect serca <dig> expression, used as a loading control .
after incubation, the membranes were washed with tbs-t and incubated for 45 min at room temperature with hrp-conjugated goat anti-rabbit antibody  in tbs-t. the immunoreactive proteins were detected by chemiluminescence, and analyzed with imagej software ; the results were normalized against the control condition and expressed in optical density units.
to analyze loading controls such as serca <dig>  the same membranes used to detect stim proteins were incubated for 30 min in striping solution :  <dig> tris ph  <dig> ,  <dig> β-mercaptoethanol, and 2% sds at 55°c and then washed twice with tbs-t. then the membranes were treated with a primary antibody against the serca <dig> protein and finally with an hrp-conjugated goat anti-rabbit antibody  in tbs-t and quantified as above.
expression of purinergic and muscarinic receptors and transcript knockdown using antisense oligonucleotides in xenopus laevis oocytes
in order to express the desired membrane receptors, cdna coding for p2y <dig>  p2y <dig>  or m <dig> receptors were cloned into the plasmid pexenex <dig> and linearized with sali or hindiii, then purified and transcribed to capped rna with t <dig> polymerase using the mmessage mmachine kit .
oocytes were injected with 25–50 ng of the respective crna .
for purinergic receptors the p2y <dig> xenopus laevis , and the p2y <dig> xenopus tropicalis  subtypes were used, and for muscarinic receptors, the m <dig> subtype .
another group was injected with 25–50 nl of h2o for control experiments.
the antisense sequences were designed to target the initiation translation region, a strategy that has been successfully used in several experimental protocols; antisense oligonucleotide strongly inhibits mrna expression via an rnase-h-dependent mechanism  <cit> .
expression of endogenous stim <dig> or stim <dig> was knocked down by the injection of 25–50 ng of antisense oligonucleotides with the following sequences: for antisense oligonucleotide stim <dig> , 5´-atagcagagtccgacaccaaagcattccgc-3´, and for antisense oligonucleotide stim <dig> , 5´-tcctcttcttctttctcccgttcatggctg-3´.
control experiments for antisense oligonucleotides were performed injecting  scrambled sequences for both as-stim, and a second control for antisense oligonuleotide injection  was made knocking down the expression of connexin  <dig>  which was monitored measuring the ic current in ca2+-free ringer solution  <cit> , the sequence for as-cx <dig> was: 5´-gctttagtaattcccatcctgccatgtttc-3´.
in general, after injection, oocytes were incubated at 18°c in barth’s solution, and the effects of these procedures on protein expression and current responses were examined by biochemical and electrophysiological methods.
unless otherwise stated, groups of injected oocytes that were induced to express purinergic receptors were incubated in barth’s solution containing  <dig> u/ml apyrase to hydrolyze the atp that is released from the oocyte into the medium, thus avoiding stimulation of purinergic receptors during the incubation period  <cit> .
electrophysiology
oocyte membrane currents were monitored using the two-electrode voltage-clamp technique.
the cells were continuously superfused  with nr solution and held at −10 mv.
voltage steps to −100 mv with a duration of 4 s were applied every 40 s to activate the tin current response, and the oocytes were stimulated for 120 s  with one of the agonists  added to the bath solution.
for long-lasting stimulation, gpcr-expressing oocytes were incubated for 1–4 h with 1 μm agonist, and p2y8- or p2y2-expressing oocytes were incubated in medium devoid of apyrase; in this condition, endogenously released atp activated the receptors in most cases.
intra-oocyte injection of antibodies during electrophysiological recording was achieved by pneumatic pressure ejection from a third micropipette  <cit> .
the injection micropipette was loaded with antibody dissolved in 5 mm hepes, adjusted to ph  <dig>  with koh.
oocyte maturation assays
maturation studies were carried out on batches of 15– <dig> defolliculated oocytes, stage vi, incubated in 2 ml of barth’s solution plus 10 μm progesterone.
gvbd was scored by white-spot formation and confirmed by cutting the oocytes through the equator after incubating them in hot nr for 1 min  <cit> .
maturation was analyzed in groups of oocytes that had been injected 72 h earlier with as-stim <dig> or as-stim <dig>  and they were compared with uninjected oocytes or those injected with h2o.
electrical properties of oocytes from the different groups were analyzed after 9–12 h in the presence of progesterone.
reagents
atp, ach, apyrase, collagenase type i, progesterone, fbs, and all salts were from sigma chemical co. .
statistical analysis
all data are expressed as mean ± sem of at least 10– <dig> oocytes from three different frogs for each condition.
statistical analysis was performed using the igor pro wavemetrics, inc. software through analysis of variance .
the means of two different experimental groups were compared using a student’s t-test.
differences were considered to be significant at p < <dig> .
RESULTS
expression of endogenous stim <dig> and stim <dig> in xenopus oocytes
expression of rna transcripts stim <dig> and stim <dig> was determined in oocytes using rt-pcr.
in rna samples from control  oocytes  the use of oligonucleotide primers for stim <dig> resulted in an amplicon of 463 bp, while stim <dig> primers amplified a fragment of 494 bp.
both had the expected size for the corresponding transcript, and the amplified fragments were then cloned into the pjet  <dig>  vector, sequenced, and analyzed using blast.
the sequences obtained were highly homologous to those reported for stim <dig>  from xenopus laevis  and for stim <dig>  from xenopus tropicalis  .
control amplifications without rt or without a cdna template did not produce any pcr products .
groups of oocytes that had been injected 48–72 h earlier with either the as-stim <dig> or the as-stim <dig> oligonucleotide sequences showed a dramatic decrease in the corresponding transcripts.figure  <dig>
stim expression in the
xenopus
oocyte and its downregulation by as-stim injection.
a) shows the rt-pcr amplification of products that corresponded to the size expected for either stim <dig> or stim <dig> in native oocytes ; the corresponding amplicons were absent in oocytes from the same batch that had been injected with either as-stim <dig> or as-stim <dig> 48 h before the assay.
the rps <dig> amplicon indicates the reaction efficiency, and -rt and h2o lanes correspond to negative controls, either rna without rt, or to the reaction mix without a cdna template, respectively.
b) stim <dig> and stim <dig> were identified by western blot analysis in protein extracts from oocytes  or mouse brain  using either nh-stim <dig>  or cooh-stim <dig>  as antibody.
c) a similar analysis as in b was made for batches of oocytes injected with h2o as control , or with as-stim <dig> or as-stim <dig> 48 h before the protein extraction, in which cases proteins were eliminated.
.
to determine whether the injection of antisense oligonucleotides induced a parallel reduction in level of stim <dig> and stim <dig> proteins, these were evaluated using western blot analysis with specific antibodies .
as expected, nh-stim <dig> detected a band above  <dig> kda in total membrane fractions from control oocytes, and from the mouse brain   <cit> .
then, a group of oocytes injected with as-stim <dig> was tested; as illustrated in figure 1c, antisense-injected oocytes showed a significant stim <dig> decrease compared to the control group.figure  <dig>
knockdown of stim expression in oocytes co-injected with gpcr mrna.
a) rt-pcr amplification of stim <dig>  stim <dig>  or rps <dig> in batches of oocytes injected with h2o  or with crna  coding for either p2y <dig> or m <dig> gpcr.
in oocytes co-injected with as-stim <dig> or as-stim <dig>  together with p2y <dig> or m <dig> crna, the corresponding stim amplicon was downregulated.
control reactions illustrate specificity; rps <dig> amplicons are positive controls, and -rt and h2o lanes show negative controls.
b) similar groups of oocytes as in a) were assayed using the western blot technique; in this case oocytes from the same donor injected with one gpcr mrna  alone, or co-injected with as-stim <dig>  were tested with nh-stim <dig>  while as-stim2-injected oocytes were probed with cooh-stim <dig>  in both as-stim groups serca was used as gel-loading control.
c) the graph shows the densitometric analysis of bands, summarizing the results obtained in different preparations of  <dig> oocytes per group and repeated in 3– <dig> frogs.
both pcr products and bands detected by western blot  were analyzed for batches of oocytes injected with h2o  or with either 50 ng as-stim <dig> or as-stim <dig> alone .
similar analysis was made for batches of control oocytes injected with p2y <dig> or m <dig> crna alone, and oocytes from the same frogs co-injected with either as-stim or as-stim together with the gpcr crna.
optical density units  for each band were normalized against the value obtained in the corresponding cnt conditions .
similarly, stim <dig> was detected using two distinct antibodies, cooh-stim <dig>  and nh-stim <dig>  which revealed stim <dig> as a band about  <dig> kda, in both the total membrane preparation of control oocytes and in total protein from mouse brain, in agreement with previous reports  <cit> .
western blot analysis in oocytes injected with as-stim <dig> indicated that antisense produced a large decrease in the amount of stim <dig> as compared to control oocytes .
these results showed that the antisense sequences used specifically decreased the endogenous transcripts for stim <dig> or stim <dig>  with a concomitant depletion of stim <dig> and stim <dig> proteins.
stim <dig> and stim <dig> levels were decreased by injection of antisense-stim sequences in oocytes co-expressing gpcr
most of the following experiments were made using oocytes exogenously expressing muscarinic or purinergic receptors due to injection of the respective crna in order to get a robust and consistent response; therefore, it was determined if gpcr expression affected either the endogenous expression of stim or its decrease due to as-stim injection.
seventy-two hours after injection with crna coding for gpcr, oocytes exhibited strong current responses in the presence of their respective agonists ; this gpcr expression did not affect the level of stim <dig> or stim <dig> transcripts, as illustrated in figure 2a.
moreover, the decrease of stim <dig> or stim <dig> expression due to antisense injection was also not affected in oocytes that were co-injected with either p2y <dig> or m <dig> receptor crna .
consistent with this result, knockdown of neither stim <dig> nor stim <dig> protein was altered by co-injecting oocytes with crna to express gpcr, as shown in figure 2b.
in all cases, serca <dig>  did not show significant changes in response to the various experimental conditions .
the results obtained for all conditions described above are summarized in figure 2c.
taken together, they confirm that antisense knockdown of endogenous stim proteins in the oocyte was specific and effective and that this was not affected by simultaneous exogenous gpcr expression.
stim protein knockdown did not affect the generation of the oscillatory cl− current induced by agonist
to evaluate the role of stim <dig> and stim <dig> in soc current activation, we monitored the tin response generation.
prior to this analysis we tested whether or not as-stim injection affected the ca2+-dependent cl− oscillatory current  generated by the first application of one of the agonists .
for this, groups of oocytes were exposed to either fbs )  <cit> , ach, or atp in order to monitor the iosc amplitude, as a measure of the oocyte capacity to release ca2+ from intracellular reservoirs  <cit> .
iosc elicited by fbs were recorded from native oocytes, and those generated by ach or atp were recorded from oocytes expressing m <dig> or p2y receptors.
the iosc amplitude was then compared among control oocytes and oocytes co-injected with as-stim <dig> or as-stim <dig>  the results showed no significant difference among the various groups of oocytes tested for a particular agonist , although average current amplitude was consistently smaller for lpar stimulation, and larger for m1-stimulated responses.
together, these results showed that the injection of antisense oligonucleotides did not affect iosc activation, strongly suggesting that the ca2+-release mechanism remained intact and showing that its strength was dependent on the receptor type stimulated.figure  <dig>
i
osc
and
t
in
responses activated by agonist stimulation.
a) strength of i
osc elicited by first agonist application did not change by knockdown of stim <dig> or stim <dig>  compared with that obtained in cnt oocytes; top traces are typical responses elicited by ach, similar responses were obtained by fbs or atp applications, and the graph shows the average i
osc responses obtained in oocytes held at −60 mv.
b) record illustrating the activation of t
in current obtained in an oocyte expressing the m <dig> receptor by a single ach  application for 40 s .
oocytes were held at −10 mv while being superfused with nr solution and stepped to −100 mv for 4 s every 40 s; sudden hyperpolarization generated t
in current responses that follow consistent kinetics with a peak amplitude response at 280–360 s ; after that the response was washed out with a similar time course.
c) shows the t
in current during the steps from − <dig> to −100 mv indicated with letters in panel b).
d) a similar t
in current response elicited in an oocyte from the same frog that was pre-incubated with 1 μm ach for 4 h , then monitored with the same electrical recording parameters and stimulated with 100 μm ach.
e) shows the t
in responses indicated with the same letters as in d).
in this protocol t
in current was consistently activated from the beginning of the record, and a transient inhibition of the response was noted during application of the agonist ; after that, t
in recovered and remained fully activated for a long period of time.
similar responses were obtained using oocytes expressing p2y receptors and stimulating with atp.
participation of stim <dig> in tin generation
to analyze the role of stim <dig> and stim <dig>  tin current was monitored by applying hyperpolarizing voltage steps of −90 mv every 40–60 s, from a holding potential of −10 mv .
as illustrated in figure 3b, the tin current amplitude generally increased after acute agonist application , reached a peak after 400–480 s, and then slowly returned to basal levels after 680–800 s .
consistent with previous studies  <cit> , tin was a cl− current that was dependent on extracellular ca2+, and it was blocked by lanthanides with an ic <dig> for la3+ of 41 ±  <dig>  nm and for gd3+ of 7 ±  <dig>  μm, potencies similar to those shown to block soc channels in other studies  <cit> .
then the effect of as-stim <dig> injection on tin current generation  was assessed.
in control oocytes, application of fbs  elicited tin current responses of  <dig>  ±  <dig>  μa .
however, in as-stim1-injected oocytes, the average tin generated was  <dig>  ±  <dig>  μa, which represented a decrease of 60 ±  <dig> %.
similarly, oocytes exposed to 100 μm ach  showed a 70 ±  <dig> % decrease in tin in the as-stim1-injected group.
however, oocytes expressing p2y <dig> and exposed to 100 μm atp exhibited a 20 ±  <dig> % decrease in tin and oocytes expressing p2y <dig> exhibited a reduction of only 15 ±  <dig> % .
the results clearly indicated that elimination of stim <dig> did not cause a complete loss of the tin response elicited by any of the agonists used.figure  <dig>
specific stim knockdown by oocyte injection of as-stim differentially decreased the
t
in
current.
a) oocytes induced to express m <dig>  p2y <dig>  or p2y <dig> receptors were stimulated with either ach or atp , and lpar in native oocytes were stimulated by fbs ; the resulting t
in currents  were compared with the t
in obtained in oocytes from the corresponding group that were also injected with 50 ng as-stim <dig> ; all responses were monitored 48–72 h after oocyte injection.
b) the graph shows the results obtained using the different experimental conditions illustrated in a).
c) in a set of experiments similar to those shown in a), t
in currents were monitored, and the peak amplitudes of non-injected cnt oocytes were compared with those of oocytes injected  with 50 ng as-stim <dig> and stimulated with the agonists.
d) the graph shows the results obtained using the different experimental conditions illustrated in c).
bars correspond to the mean  of the t
in peak amplitude of 10– <dig> oocytes from 5– <dig> frogs .
stim <dig> knockdown potently inhibited tin generation regardless of the receptor stimulated
we next tested whether stim <dig> knockdown affected tin currents activated either by p2y or m <dig> receptors with experiments similar to those described above.
in contrast to what happened with stim <dig> knockdown, as-stim <dig> injection drastically reduced the tin response elicited by the acute stimulation of any of the receptors tested .
tin current amplitude was reduced by 96 ±  <dig> % in oocytes stimulated with fbs, by 96 ±  <dig> % with ach, by 93 ±  <dig> % with atp for oocytes expressing p2y <dig> receptors, and by 94 ± 8% for those expressing p2y <dig>  in this case, the amount of decrease observed did not differ among the receptors studied .
as shown above, it was evident that the decrease in the tin response was not due to uncoupling of the ip3/ca2+-release system since the oscillatory responses in all the oocyte groups remained unchanged.
control experiments were also made using scrambled oligonucleotide sequences as well as as-cx <dig> a different antisense oligonucleotide sequence to rule out the possibility that injection per se yielded nonspecific results, in these cases no effects were observed on tin current amplitude.
for example, as-cx <dig> was used to knockdown connexons formed by cx <dig>  whose opening by superfusion of ca2+-free ringer’s solution  <cit>  results in a fast and reliable test for cx <dig> expression.
thus, in control oocytes the ic current response was elicited by superfusion of ca2+-free ringer’s solution  while in as-cx <dig> injected oocytes the current response was eliminated.
however, in the same oocytes from both groups, the tin current amplitude was similar, regardless the membrane receptor stimulated, either m <dig>  or p2y <dig>  .
all together, these results indicate that tin generation in the xenopus oocyte requires stim <dig> protein.
cooh-stim <dig> antibody enhances the tin current response
envisioning that specific binding of antibody to stim <dig> or stim <dig> might affect the function of these proteins and then serve as a specific tool to evaluate the involvement of stim in a particular response, we tested the same antibodies used in the western blot for their effect on tin generation.
for this purpose, antibodies were microinjected into the oocyte cytoplasm to reach a final dilution of 1: <dig>  figure  <dig> shows that ach application onto oocytes pre-loaded with cooh-stim <dig> resulted in a robust potentiation of the tin response, increasing the amplitude by 158 ± 25% .
cooh-stim <dig> injection also potentiated by 168 ± 30% the tin responses elicited by fbs, and a similar effect was observed in oocytes stimulated through the p2y <dig>  or the p2y <dig> receptor  .
however, in oocytes  from the same frogs that were injected with denatured cooh-stim <dig> , tin potentiation was completely abolished .
also, injection of nh-stim <dig> or nh-stim <dig> antibody did not produce any changes in the tin response, nor did the injection of a p2y <dig> antibody.figure  <dig>
oocyte injection with cooh-stim <dig> antibody produced a strong potentiation of
t
in
current response.
a)
t
in current responses were monitored in two conditions: non-loaded oocytes  and oocytes loaded with cooh-stim <dig> antibody .
t
in responses were elicited by ach, fbs, or atp application, depending on the receptor to be stimulated.
in all cases, a strong potentiation of the response was observed in ab-loaded oocytes.
b) oocytes stimulated by ach  loaded with denatured cooh-stim <dig> had control-like responses, while nh-stim <dig> or nh-stim <dig> loading did not produce t
in potentiation.
c) the graph shows the results obtained using the different experimental conditions illustrated in a and b; each bar corresponds to the mean  of the t
in peak amplitude normalized against the cnt current of 10– <dig> oocytes from 3– <dig> frogs .
all these results clearly indicated that the cooh-stim <dig> antibody specifically potentiated the tin current, regardless of the receptor stimulated.
role of stim <dig> and stim <dig> during long-lasting agonist stimulation
the following experiments were designed to explore the possibility that stim <dig> and stim <dig> have different effects, depending on the duration of the stimulus.
thus, oocytes injected with as-stim <dig> or with as-stim <dig> and expressing m <dig>  p2y <dig>  or p2y <dig> receptors were incubated for 1–4 h in the presence of their respective agonists at 1 μm .
extended agonist incubation generated strong tin currents that remained stable for more than the 60-min recording time, even under constant superfusion of the oocytes with nr solution, and it began to decrease after 120–180 min of wash; we assumed that in this condition the soc machinery was over-stimulated, and that the time spent in the activated state reflected the time necessary to refill the reservoirs.
in the oocytes knocked down for stim <dig>  tin currents activated by long-lasting stimulation with any of the agonists analyzed were no different from those observed in control oocytes.
in contrast, in oocytes injected with as-stim <dig> and expressing p2y or m <dig> receptors that had been stimulated for long intervals with their respective agonists, the tin current was no longer generated , strongly suggesting that stim <dig> in the oocyte was essential for responses generated through both the acute and long-lasting stimulation protocols.
stim proteins and the maturation process
during the maturation process, molecular elements that control the ca2+ dynamics in the xenopus oocyte undergo an important reconfiguration; this observation has been extended to different species, and similar changes are known to occur during mitosis  <cit> .
given the importance of these events for cell cycle control, we asked whether or not the knockdown of stim proteins affected the maturation process.
thus, batches of control oocytes, and those that were injected with as-stim <dig> or as-stim <dig> were assayed 48–72 h after injection with 10 μm progesterone in barth’s solution to induce maturation.
oocyte maturation entry was scored by the appearance of gvbd after 8–12 h in the presence of progesterone.
the gvbd score obtained was compared against progesterone-treated control oocytes.
the results are illustrated in figure 6a; as-stim1-injected oocytes did not show any effect on the efficiency of maturation, while stim <dig> knockdown produced a strong inhibition of the process .
lack of gvbd in as-stim2-injected oocytes seemed to indicate a failure to enter meiosis and a consequent incomplete maturation process; this interpretation was also supported by monitoring electrophysiological parameters in all of the groups tested.
as illustrated in figure 6b-c, electrical parameters of as-stim2-injected oocytes  were different from those displayed by as-stim1-injected oocytes and control oocytes maintained in progesterone for the same period of time.figure  <dig>
effect of as-stim <dig> on gvbd and oocyte membrane characteristics during maturation induced by progesterone.
a) the maturation process promoted by progesterone  was analyzed in uninjected oocytes, or in oocytes injected 72 h prior to the assay with either as-stim <dig> or as-stim <dig>  and compared with control oocytes in the absence of progesterone.
gvbd was quantified after 8–12 h in presence of progesterone  and is normalized against the value observed in uninjected oocytes.
b) resting membrane potential was monitored 8–12 h after addition of progesterone in the same groups of oocytes  as in a).
c) the input membrane resistance  was estimated over the range from − <dig> to −20 mv in the different oocyte groups treated in the same conditions.
control groups, without progesterone, included both uninjected and antisense-injected oocytes.
in all cases, values for as-stim2-injected groups were different from as-stim1-injected or uninjected groups .
taken together, these results clearly showed that in oocytes where stim <dig> was knocked down, the process of maturation was inhibited at some early point.
the first manifestation of this was the complete blockage of gvbd, i.e., of the signal for meiosis entry; this result was clearly different from that observed in stim1-deprived oocytes.
discussion
here, using biochemical strategies and electrophysiology, we studied what effects the knockdown of endogenous stim proteins had on two important xenopus oocyte responses: the activation of soce monitored by measuring tin current generation, and the maturation process induced by progesterone.
we found that: i) both stim <dig> and stim <dig> proteins were endogenously expressed in the xenopus oocyte; ii) injection of antisense oligonucleotide sequences of stim <dig> or stim <dig> potently knocked down the expression of both the corresponding mrna and the protein; iii) stim <dig> or stim <dig> knockdown did not seem to affect the ca2+-signaling machinery responsible for generating oscillatory ca2+-signals in the oocyte; iv) stim <dig>  but not stim <dig>  proved to be fundamental for tin current generation; this was observed both in acute stimulation protocols or after long-lasting stimulation periods, and it did not depend on the receptor type stimulated; v) stim <dig> protein knockdown blocked entry into the process of maturation induced by progesterone, while stim <dig> elimination did not affect this process; and vi) an antibody against the cooh terminus of stim <dig> potentiated tin current generation.
calcium release and influx are two phenomena well studied in the xenopus oocyte.
the main subject addressed here is the identity and role of stim proteins during calcium influx stimulated through endogenous responses.
it is known that after gpcr stimulation, both endogenous as well as exogenously expressed gpcr generate in the oocyte mainly two ca2+-dependent cl− ion currents, one due to intracellular ca2+ release that is normally followed by another current dependent on ca2+ influx; this pattern is generated through an enzymatic cascade involving ip <dig> synthesis, a common mechanism in most cell systems  <cit> .
following the original nomenclature, in the oocyte the first response is named iosc, while the second generates the tin current response  <cit> .
the ca2+-influx magnitude is directly related to the amplitude of ca2+-release; the main molecular element responsible for this linkage is the stim protein, since it is the ca2+-sensor within the ca2+ reservoir.
as in previous studies  <cit> , to monitor the i increase produced by both mechanisms in the oocyte, here we used the ca2+-dependent cl− current as an endogenous sensor whose amplitude accurately reflects the concentration of ca2+ beneath the plasmatic membrane.
this is especially true for ca2+ influx, since this occurs in the plasma membrane where the ca2+-dependent cl− channels are co-expressed with the soc channels responsible for the influx.
thus, monitoring an endogenous ca2+ sensor such as the cl− channel offers not only spatial and temporal advantages, but also amplifies the normally small ca2+ current through soc channels and avoids altering the ca2+ dynamics with further pharmacological manipulations.
stim protein expression and function was then studied in the xenopus oocyte using this tool.
it is well known that when ca2+ is released from the er, stim proteins are activated, rapidly translocated, and oligomerized into junctions formed between the er and the plasma membrane, where they bind to and activate highly selective ca2+ channels formed by orai proteins that allow ca2+ influx  <cit> .
the main trigger for this phenomenon is a decrease in er ca2+ content; however, evidence indicates that isoforms of stim <dig> protein might maintain a basal activation of orai channels without prior ca2+ release, thereby controlling the cytoplasmic ca2+ concentration  <cit> .
the ratio of stim <dig> to stim <dig> expression seems to depend on the cell type, and perhaps on the pathophysiological state; as shown here for the xenopus oocyte, other cells such as t cells, myoblasts, skeletal muscle, and liver cells also co-express both stim proteins  <cit> .
studies to distinguish the roles of stim <dig> and stim <dig> in various cells have employed diverse silencing strategies and overexpression.
the injection of an antisense oligonucleotide sequence for each stim protein was chosen here for its simplicity and because it excludes potential non-specific effects caused by protein over-expression.
the effect of antisense injection on stim expression was demonstrated by analyzing both its mrna and protein expression by rt-pcr and western blot, respectively.
this analysis demonstrated that expression of stim <dig> or stim <dig> was strongly downregulated in oocytes injected with the corresponding antisense oligonucleotide sequence.
this antisense effect was not affected by co-expression of gpcr proteins, used experimentally to stimulate ca2+-release.
also, it was shown that stim protein knockdown did not affect the ip <dig> increase and subsequent ca2+ release, as indicated by the iosc amplitude responses evoked at the beginning of each experiment by acute application of one of the agonists studied.
in addition, preliminary results using rt-pcr showed amplification of transcripts for orai <dig> and orai <dig> in the oocyte .
oocytes injected with either antisense stim <dig> or stim <dig> were then monitored to analyze their ability to generate tin current using two stimulation protocols.
in the first, acute application of the agonist produced the typical iosc response; in control conditions it was followed by tin generation that declined after 680–800 s. in the second protocol, long-lasting  stimulation of control oocytes with a low concentration of agonist  gave strong tin current responses that remained active for 60–180 min, even in constant superfusion with nr solution.
a possible explanation for this difference in response kinetics is that prolonged stimulation produced a stronger activation of the soce mechanism, probably due to a more marked decrease in er ca2+ concentration.
both protocols were applied in oocytes in which stim <dig> or stim <dig> expression had been eliminated.
the stim <dig> knockdown produced a severe decrease in tin current generation  in both stimulation protocols, indicating that stim <dig> was indispensable to induce the tin current response.
using acute receptor stimulation, elimination of stim <dig> caused a smaller but significant decrease in tin current generation thus, stim <dig> alone was unable to support full tin activation during acute stimulation, suggesting that an association of stim <dig> with stim <dig> was necessary in order to activate the endogenous response.
also it was observed that stim <dig> requirement seemed to be minor during p2y purinergic stimulation, this difference cannot be explained by the amplitudes of iosc generated by the agonists, given that ach and lpa were the more and less effective, respectively, in generating the response, and both agonists showed similar patterns of tin decrease by stim <dig> knockdown.
thus, this result might indicate some type of molecular specificity, perhaps intrinsic to the molecules involved or as a consequence of their differential expression and localization in the oocyte membrane.
differential insertion of several proteins expressed in the oocyte membrane has been demonstrated; thus, membrane domains with greater expression of stim <dig> together with p2y receptors are plausible.
a central role for stim <dig> protein in soce generation has been shown before in some cell types such as neurons  <cit>  and dendritic cells  <cit> ; however, there is no information indicating whether or not the expression of stim <dig> might affect the full endogenous response in these cases.
for example, it has been shown the essential role of stim <dig> in soce activation in dendritic spines  <cit> , which was not substituted by overexpression of stim <dig>  the authors concluded that this is due to differences in stim-ca2+ sensitivity and subcellular localization of the proteins.
in many other cell systems, co-participation or complementary roles for the two stim proteins have been postulated  <cit> .
finally, when the oocytes were stimulated using the long-lasting protocol, elimination of stim <dig> had no effect indicating that, in this case, the tin current was activated by stim <dig> alone.
the central role for stim <dig> is supported by the latter result as well as by the finding that injecting the antibody  against stim <dig> specifically increased the current amplitude by more than 100%, regardless of the agonist used to generate the tin response.
it is known that the cooh region, in both stim <dig> and stim <dig>  contains the domain necessary to interact and activate the soc channel formed by orai  <cit> .
thus, a potentiating effect of the cooh-stim <dig> antibody indicates that the strength of the stim2-orai interaction might be regulated, either positively or negatively, through a site that is affected by the antibody, indirectly confirming the central role of stim <dig> during tin generation.
as expected given the stim structure proposed, the two antibodies that recognized domains close to the amino-terminus had no effect on the tin response.
significant inhibition of the maturation process was observed in oocytes devoid of stim <dig> protein.
here, we provided clear-cut evidence of stim <dig> involvement during or in preparation for maturation, since its absence eliminated the process of gvbd.
once again, this result contrasted with the lack of effect in stim1-knockdown oocytes, whose maturation was similar to that of control oocytes.
indeed, it has been shown that the function of stim <dig> is downregulated during the maturation process, which contributes to elimination of the soc response in xenopus oocytes  <cit> ; a similar condition has been shown in the mammalian oocyte  <cit> , although in the latter this phenomenon remains controversial  <cit> .
there is no previous information regarding the effect produced by lack  of stim <dig> during maturation either in frog or mammalian oocytes, as most previous studies focused on the role of stim <dig>  however, mouse oocyte is known to express stim <dig> protein in the er; during maturation, stim <dig> re-localizes from a homogeneous distribution to one closer to the meiotic spindles, suggesting a role during this process  <cit> .
in frog, is possible that the inhibitory effect of stim <dig> knockdown was unrelated to its role in soce activation, since meiosis entry in xenopus does not require ca2+-influx  <cit> .
further studies will be necessary to characterize the level at which the lack of stim <dig> had such a dramatic effect on the maturation process, and to determine if it might have more general implications.
one possibility is that as-stim <dig> might cause downregulation of a stim <dig> isoform different from that involved in soce activation.
another possibility relates to its role regulating cytoplasmic , in which case as-stim <dig> might affect the activation of ca2+-dependent processes required prior to meiosis entry.
CONCLUSIONS
in this study, stim <dig> is fundamental for the endogenous soc response in the oocyte, although an association with stim <dig> seemed to be necessary for its full generation.
the mechanism responsible for the clear dependence of meiosis entry on stim <dig> expression is a fundamental question that remains open, and its elucidation might help to understand the function of stim proteins in the xenopus oocyte and in other cell types as well.
additional file
additional file 1:
blast analysis for the amplified sequences of stim <dig> and stim <dig>  alignment produced by blast for the amplified sequences of stim <dig>   and stim <dig>   with the sequences reported for stim1x <dig>  and stim2xt , respectively.
competing interests
the authors declare that they have no competing interests.
author’s contributions
b.s.-f. performed experiments, collected data, provided input in data analysis and was involved in drafting the manuscript.
f.g.v.-c. and e. g. performed experiments and provided substantial input in design and interpretation of data.
r.o.a.
designed research, performed experiments and drafted the manuscript.
all authors gave final approval to the version to be published.
the author’s thank to dr. dorothy d. pless for editing the manuscript.
we also thank mr. horacio ramírez leyva, m. en c. leonor casanova, ing.
ramón martínez olvera, and isc omar gonzález hernández, for their expert technical assistance.
b.s.-f. as a doctoral student from programa de doctorado en ciencias biomédicas at universidad nacional autónoma de méxico  is grateful that she received fellowship  <dig> from conacyt-méxico.
r.o.a.
received grants by conacyt-méxico  <dig> and papiit-unam-méxico in <dig>  lastly, f.g.v.-c. is grateful for the grants received by conacyt-méxico no.
<dig> and papiit-unam-méxico no.
in <dig>

 for approximately three decades, the xenopus oocyte has been a useful cell model to determine the underlying mechanisms responsible for the increase of the cytoplasmic ca2+ concentration through its release from intracellular reservoirs and by calcium influx either through ca2+-dependent voltage-dependent channels or via store-operated ca2+ channels. the latter results from the activation of the phenomenon known as store-operated ca2+ entry, which allows the replenishment of emptied reservoirs after the stimulation of ca2+ release through ip3/diacylglycerol synthesis by phospholipase c. release of ca2+ from intracellular reservoirs and soce activation are common responses in the xenopus oocytes since they endogenously express the machinery that activates plc by stimulating endogenous g protein-coupled receptors; cytoplasmic ca2+-increase, through either release or influx, opens ca2+-dependent cl− channels in the oocyte membrane generating conspicuous current responses. soce activation in the membrane of the xenopus oocyte was first detected by measuring the transient inward current response after ca2+ release in the oocyte. the tin response is generated by hyperpolarizing steps, and is mainly due to the ca2+-influx that subsequently opens ca2+-dependent cl− channels; this membrane response has been used as a reliable monitor of soc channel activation. the soce current is most likely driven through ca2+-permeable channels formed by orai, a channel activated by association with the stromal interaction molecule, a protein that is localized mainly in the endoplasmic reticulum membrane and that senses the ca2+ concentration in its lumen. although transcripts for endogenous orai and stim molecules have been reported in the oocyte, the role for the different types and isoforms of these proteins and their relation with endogenous responses in the oocyte have not been thoroughly studied; these issues are of interest given that the roles for the different soc molecular elements are also incompletely understood, and their study in a well-known model such as the xenopus oocyte might reveal important information. two stim proteins, stim and stim are expressed in eukaryotic cells. a different role for each of them has been proposed; for example, the er ca2+ content must be greatly reduced in order to activate stim protein, while the more ca2+-sensitive stim seems to require only a slight reduction in er ca2+ concentration. it has been proposed that stim participates in maintaining the cytoplasmic ca2+ concentration. although the fundamental role of stim in activating soce has been demonstrated in several cell types, other information indicates that stim is the main protein involved in soce generation in neurons, dendritic cells, and mammary epithelial cells. thus, it is plausible that the specific functions of stim and stim depend on the cell type, their relative rates of expression, and other factors such as interactions among them or with regulatory proteins. it has also been shown that during maturation, the ca2+-signaling pathway in the oocyte is significantly reconfigured, probably as part of the mechanism that prepares the gamete for fertilization and subsequent embryonic development. this reconfiguration includes orai channels and stim which are regulated during maturation thus eliminating the soce response. due to the importance of this phenomenon for cell cycle control in general, it is also of interest to explore the effects on oocyte maturation of altered stim expression. in the present study, we specifically knocked down stim or stim in the xenopus oocyte to analyze the effect on two endogenous phenomena, the generation of the tin current response and the maturation process. we found that stim expression was essential in both phenomena, while stim expression was not. methods cell preparation xenopus laevis frogs were obtained from xenopus i. ovary lobules were surgically removed under sterile conditions from frogs that had been anaesthetized using % aminobenzoic acid ethyl ester and rendered hypothermic. after surgery, frogs were sutured and allowed to recover from anesthesia. frogs were maintained for 3– days in individual tanks until healing was complete; they were then housed in larger groups, and no further oocytes were taken from them for at least 2 months. procedures were approved by the institutional animal committees. the lobules were placed in sterile barth’s solution containing: nacl, kcl, nahco ca cacl mgso and hepes, with 75 μg/ml gentamicin and adjusted to ph . studies were carried out using oocytes at stage vi dissected from the ovaries and defolliculated by collagenase treatment at room temperature for 30 min in normal frog ringer’s solution. after washing, the oocytes were stored at 18°c in sterile barth’s solution, and electrical recordings were performed over a period of 2– days in either uninjected oocytes or in those injected with crna for specific receptors and/or with antisense oligonucleotide to knock down specific proteins. reverse transcription polymerase chain reactions total rna from the oocytes was purified using trizol reagent. first-strand cdna was synthesized using 2 μg of dnase-treated rna as template and 1 μg of oligo,  μg random hexamers, and reverse transcriptase. the cdna was used as template in a polymerase chain reaction to amplify cdna fragments for stim and stim and the ribosomal protein s was used as a control. all the pcr programs started at 95°c for 2 min. the amplification in the 35 cycles consisted in 45 s at 95°c, 40 s at 55°c, and 35 s at 72°c, and a final extension at 72°c for 5 min. the sequences of oligonucleotides used were: stim forward, 5'-cgacgagtttctcagggaag-3' and reverse, 5'-cttcatgtggtcctcggagt-3'; stim forward, 5'-ccagccttgaggcaatatgt-3' and reverse, 5'-gcaacctccaactccgatta-3'; rps forward, 5'-tggtaacaggggaggtttccgc-3' and reverse, 5'-ataccagccatcatgagcagc-3'. the amplified products were isolated, purified, and subcloned into the pjet vector. finally, their nucleotide sequences were confirmed by sanger sequencing. western blot protein expression was assessed by western blot in either control oocytes or in those injected with as-stim or as-stim for each group, oocytes were homogenized 72 h post-injection in a buffer containing: tris–hcl ph , edta ph  sucrose, and 1x complete mini protease inhibitor. then samples were centrifuged at 4°c and 500 rpm for 5 min, at 3500 rpm for 10 min, and at  rpm for 20 min. subsequently, the final pellets were resuspended in 50 μl of buffer containing: tris–hcl ph , edta ph  nacl, mgcl and 1x complete mini protease inhibitor. total membrane protein concentration was quantified with a bradford assay. for electrophoresis, samples were fractionated in a 10% sds-polyacrylamide gel and transferred to a nitrocellulose membrane. membranes were blocked for 1 h at room temperature in tbs-t solution: nacl, tris, ph , and % tween containing 5% nonfat dry milk and then incubated overnight at 4°c with a 1: dilution of rabbit primary antibody. the antibody denoted nh-stim was directed against a region of the amino-terminus of the stim protein, and the antibodies denoted nh-stim and cooh-stim were against the amino and carboxy termini, respectively, of stim western blot analysis was also used to detect serca expression, used as a loading control. after incubation, the membranes were washed with tbs-t and incubated for 45 min at room temperature with hrp-conjugated goat anti-rabbit antibody in tbs-t. the immunoreactive proteins were detected by chemiluminescence, and analyzed with imagej software; the results were normalized against the control condition and expressed in optical density units. to analyze loading controls such as serca the same membranes used to detect stim proteins were incubated for 30 min in striping solution: tris ph , β-mercaptoethanol, and 2% sds at 55°c and then washed twice with tbs-t. then the membranes were treated with a primary antibody against the serca protein and finally with an hrp-conjugated goat anti-rabbit antibody in tbs-t and quantified as above. expression of purinergic and muscarinic receptors and transcript knockdown using antisense oligonucleotides in xenopus laevis oocytes in order to express the desired membrane receptors, cdna coding for p2y p2y or m receptors were cloned into the plasmid pexenex and linearized with sali or hindiii, then purified and transcribed to capped rna with t polymerase using the mmessage mmachine kit. oocytes were injected with 25–50 ng of the respective crna. for purinergic receptors the p2y xenopus laevis, and the p2y xenopus tropicalis subtypes were used, and for muscarinic receptors, the m subtype. another group was injected with 25–50 nl of h2o for control experiments. the antisense sequences were designed to target the initiation translation region, a strategy that has been successfully used in several experimental protocols; antisense oligonucleotide strongly inhibits mrna expression via an rnase-h-dependent mechanism. expression of endogenous stim or stim was knocked down by the injection of 25–50 ng of antisense oligonucleotides with the following sequences: for antisense oligonucleotide stim, 5´-atagcagagtccgacaccaaagcattccgc-3´, and for antisense oligonucleotide stim, 5´-tcctcttcttctttctcccgttcatggctg-3´. control experiments for antisense oligonucleotides were performed injecting scrambled sequences for both as-stim, and a second control for antisense oligonuleotide injection was made knocking down the expression of connexin which was monitored measuring the ic current in ca2+-free ringer solution, the sequence for as-cx was: 5´-gctttagtaattcccatcctgccatgtttc-3´. in general, after injection, oocytes were incubated at 18°c in barth’s solution, and the effects of these procedures on protein expression and current responses were examined by biochemical and electrophysiological methods. unless otherwise stated, groups of injected oocytes that were induced to express purinergic receptors were incubated in barth’s solution containing u/ml apyrase to hydrolyze the atp that is released from the oocyte into the medium, thus avoiding stimulation of purinergic receptors during the incubation period. electrophysiology oocyte membrane currents were monitored using the two-electrode voltage-clamp technique. the cells were continuously superfused with nr solution and held at −10 mv. voltage steps to −100 mv with a duration of 4 s were applied every 40 s to activate the tin current response, and the oocytes were stimulated for 120 s with one of the agonists added to the bath solution. for long-lasting stimulation, gpcr-expressing oocytes were incubated for 1–4 h with 1 μm agonist, and p2y8- or p2y2-expressing oocytes were incubated in medium devoid of apyrase; in this condition, endogenously released atp activated the receptors in most cases. intra-oocyte injection of antibodies during electrophysiological recording was achieved by pneumatic pressure ejection from a third micropipette. the injection micropipette was loaded with antibody dissolved in 5 mm hepes, adjusted to ph  with koh. oocyte maturation assays maturation studies were carried out on batches of 15– defolliculated oocytes, stage vi, incubated in 2 ml of barth’s solution plus 10 μm progesterone. gvbd was scored by white-spot formation and confirmed by cutting the oocytes through the equator after incubating them in hot nr for 1 min. maturation was analyzed in groups of oocytes that had been injected 72 h earlier with as-stim or as-stim and they were compared with uninjected oocytes or those injected with h2o. electrical properties of oocytes from the different groups were analyzed after 9–12 h in the presence of progesterone. reagents atp, ach, apyrase, collagenase type i, progesterone, fbs, and all salts were from sigma chemical co.. statistical analysis all data are expressed as mean ± sem of at least 10– oocytes from three different frogs for each condition. statistical analysis was performed using the igor pro wavemetrics, inc. software through analysis of variance. the means of two different experimental groups were compared using a student’s t-test. differences were considered to be significant at p <.  expression of endogenous stim and stim in xenopus oocytes expression of rna transcripts stim and stim was determined in oocytes using rt-pcr. in rna samples from control oocytes the use of oligonucleotide primers for stim resulted in an amplicon of 463 bp, while stim primers amplified a fragment of 494 bp. both had the expected size for the corresponding transcript, and the amplified fragments were then cloned into the pjet vector, sequenced, and analyzed using blast. the sequences obtained were highly homologous to those reported for stim from xenopus laevis and for stim from xenopus tropicalis. control amplifications without rt or without a cdna template did not produce any pcr products. groups of oocytes that had been injected 48–72 h earlier with either the as-stim or the as-stim oligonucleotide sequences showed a dramatic decrease in the corresponding transcripts.figure stim expression in the xenopus oocyte and its downregulation by as-stim injection. a) shows the rt-pcr amplification of products that corresponded to the size expected for either stim or stim in native oocytes; the corresponding amplicons were absent in oocytes from the same batch that had been injected with either as-stim or as-stim 48 h before the assay. the rps amplicon indicates the reaction efficiency, and -rt and h2o lanes correspond to negative controls, either rna without rt, or to the reaction mix without a cdna template, respectively. b) stim and stim were identified by western blot analysis in protein extracts from oocytes or mouse brain using either nh-stim or cooh-stim as antibody. c) a similar analysis as in b was made for batches of oocytes injected with h2o as control, or with as-stim or as-stim 48 h before the protein extraction, in which cases proteins were eliminated.. to determine whether the injection of antisense oligonucleotides induced a parallel reduction in level of stim and stim proteins, these were evaluated using western blot analysis with specific antibodies. as expected, nh-stim detected a band above kda in total membrane fractions from control oocytes, and from the mouse brain. then, a group of oocytes injected with as-stim was tested; as illustrated in figure 1c, antisense-injected oocytes showed a significant stim decrease compared to the control group.figure knockdown of stim expression in oocytes co-injected with gpcr mrna. a) rt-pcr amplification of stim stim or rps in batches of oocytes injected with h2o or with crna coding for either p2y or m gpcr. in oocytes co-injected with as-stim or as-stim together with p2y or m crna, the corresponding stim amplicon was downregulated. control reactions illustrate specificity; rps amplicons are positive controls, and -rt and h2o lanes show negative controls. b) similar groups of oocytes as in a) were assayed using the western blot technique; in this case oocytes from the same donor injected with one gpcr mrna alone, or co-injected with as-stim were tested with nh-stim while as-stim2-injected oocytes were probed with cooh-stim in both as-stim groups serca was used as gel-loading control. c) the graph shows the densitometric analysis of bands, summarizing the results obtained in different preparations of oocytes per group and repeated in 3– frogs. both pcr products and bands detected by western blot were analyzed for batches of oocytes injected with h2o or with either 50 ng as-stim or as-stim alone. similar analysis was made for batches of control oocytes injected with p2y or m crna alone, and oocytes from the same frogs co-injected with either as-stim or as-stim together with the gpcr crna. optical density units for each band were normalized against the value obtained in the corresponding cnt conditions. similarly, stim was detected using two distinct antibodies, cooh-stim and nh-stim which revealed stim as a band about kda, in both the total membrane preparation of control oocytes and in total protein from mouse brain, in agreement with previous reports. western blot analysis in oocytes injected with as-stim indicated that antisense produced a large decrease in the amount of stim as compared to control oocytes. these results showed that the antisense sequences used specifically decreased the endogenous transcripts for stim or stim with a concomitant depletion of stim and stim proteins. stim and stim levels were decreased by injection of antisense-stim sequences in oocytes co-expressing gpcr most of the following experiments were made using oocytes exogenously expressing muscarinic or purinergic receptors due to injection of the respective crna in order to get a robust and consistent response; therefore, it was determined if gpcr expression affected either the endogenous expression of stim or its decrease due to as-stim injection. seventy-two hours after injection with crna coding for gpcr, oocytes exhibited strong current responses in the presence of their respective agonists; this gpcr expression did not affect the level of stim or stim transcripts, as illustrated in figure 2a. moreover, the decrease of stim or stim expression due to antisense injection was also not affected in oocytes that were co-injected with either p2y or m receptor crna. consistent with this result, knockdown of neither stim nor stim protein was altered by co-injecting oocytes with crna to express gpcr, as shown in figure 2b. in all cases, serca did not show significant changes in response to the various experimental conditions. the results obtained for all conditions described above are summarized in figure 2c. taken together, they confirm that antisense knockdown of endogenous stim proteins in the oocyte was specific and effective and that this was not affected by simultaneous exogenous gpcr expression. stim protein knockdown did not affect the generation of the oscillatory cl− current induced by agonist to evaluate the role of stim and stim in soc current activation, we monitored the tin response generation. prior to this analysis we tested whether or not as-stim injection affected the ca2+-dependent cl− oscillatory current generated by the first application of one of the agonists. for this, groups of oocytes were exposed to either fbs ), ach, or atp in order to monitor the iosc amplitude, as a measure of the oocyte capacity to release ca2+ from intracellular reservoirs. iosc elicited by fbs were recorded from native oocytes, and those generated by ach or atp were recorded from oocytes expressing m or p2y receptors. the iosc amplitude was then compared among control oocytes and oocytes co-injected with as-stim or as-stim the results showed no significant difference among the various groups of oocytes tested for a particular agonist, although average current amplitude was consistently smaller for lpar stimulation, and larger for m1-stimulated responses. together, these results showed that the injection of antisense oligonucleotides did not affect iosc activation, strongly suggesting that the ca2+-release mechanism remained intact and showing that its strength was dependent on the receptor type stimulated.figure i osc and t in responses activated by agonist stimulation. a) strength of i osc elicited by first agonist application did not change by knockdown of stim or stim compared with that obtained in cnt oocytes; top traces are typical responses elicited by ach, similar responses were obtained by fbs or atp applications, and the graph shows the average i osc responses obtained in oocytes held at −60 mv. b) record illustrating the activation of t in current obtained in an oocyte expressing the m receptor by a single ach application for 40 s. oocytes were held at −10 mv while being superfused with nr solution and stepped to −100 mv for 4 s every 40 s; sudden hyperpolarization generated t in current responses that follow consistent kinetics with a peak amplitude response at 280–360 s; after that the response was washed out with a similar time course. c) shows the t in current during the steps from − to −100 mv indicated with letters in panel b). d) a similar t in current response elicited in an oocyte from the same frog that was pre-incubated with 1 μm ach for 4 h, then monitored with the same electrical recording parameters and stimulated with 100 μm ach. e) shows the t in responses indicated with the same letters as in d). in this protocol t in current was consistently activated from the beginning of the record, and a transient inhibition of the response was noted during application of the agonist; after that, t in recovered and remained fully activated for a long period of time. similar responses were obtained using oocytes expressing p2y receptors and stimulating with atp. participation of stim in tin generation to analyze the role of stim and stim tin current was monitored by applying hyperpolarizing voltage steps of −90 mv every 40–60 s, from a holding potential of −10 mv. as illustrated in figure 3b, the tin current amplitude generally increased after acute agonist application, reached a peak after 400–480 s, and then slowly returned to basal levels after 680–800 s. consistent with previous studies, tin was a cl− current that was dependent on extracellular ca2+, and it was blocked by lanthanides with an ic for la3+ of 41 ±  nm and for gd3+ of 7 ±   μm, potencies similar to those shown to block soc channels in other studies. then the effect of as-stim injection on tin current generation was assessed. in control oocytes, application of fbs elicited tin current responses of  ±   μa. however, in as-stim1-injected oocytes, the average tin generated was  ±   μa, which represented a decrease of 60 ±  %. similarly, oocytes exposed to 100 μm ach showed a 70 ±  % decrease in tin in the as-stim1-injected group. however, oocytes expressing p2y and exposed to 100 μm atp exhibited a 20 ±  % decrease in tin and oocytes expressing p2y exhibited a reduction of only 15 ±  %. the results clearly indicated that elimination of stim did not cause a complete loss of the tin response elicited by any of the agonists used.figure specific stim knockdown by oocyte injection of as-stim differentially decreased the t in current. a) oocytes induced to express m p2y or p2y receptors were stimulated with either ach or atp, and lpar in native oocytes were stimulated by fbs; the resulting t in currents were compared with the t in obtained in oocytes from the corresponding group that were also injected with 50 ng as-stim; all responses were monitored 48–72 h after oocyte injection. b) the graph shows the results obtained using the different experimental conditions illustrated in a). c) in a set of experiments similar to those shown in a), t in currents were monitored, and the peak amplitudes of non-injected cnt oocytes were compared with those of oocytes injected with 50 ng as-stim and stimulated with the agonists. d) the graph shows the results obtained using the different experimental conditions illustrated in c). bars correspond to the mean of the t in peak amplitude of 10– oocytes from 5– frogs. stim knockdown potently inhibited tin generation regardless of the receptor stimulated we next tested whether stim knockdown affected tin currents activated either by p2y or m receptors with experiments similar to those described above. in contrast to what happened with stim knockdown, as-stim injection drastically reduced the tin response elicited by the acute stimulation of any of the receptors tested. tin current amplitude was reduced by 96 ±  % in oocytes stimulated with fbs, by 96 ±  % with ach, by 93 ±  % with atp for oocytes expressing p2y receptors, and by 94 ± 8% for those expressing p2y in this case, the amount of decrease observed did not differ among the receptors studied. as shown above, it was evident that the decrease in the tin response was not due to uncoupling of the ip3/ca2+-release system since the oscillatory responses in all the oocyte groups remained unchanged. control experiments were also made using scrambled oligonucleotide sequences as well as as-cx a different antisense oligonucleotide sequence to rule out the possibility that injection per se yielded nonspecific results, in these cases no effects were observed on tin current amplitude. for example, as-cx was used to knockdown connexons formed by cx whose opening by superfusion of ca2+-free ringer’s solution results in a fast and reliable test for cx expression. thus, in control oocytes the ic current response was elicited by superfusion of ca2+-free ringer’s solution while in as-cx injected oocytes the current response was eliminated. however, in the same oocytes from both groups, the tin current amplitude was similar, regardless the membrane receptor stimulated, either m or p2y. all together, these results indicate that tin generation in the xenopus oocyte requires stim protein. cooh-stim antibody enhances the tin current response envisioning that specific binding of antibody to stim or stim might affect the function of these proteins and then serve as a specific tool to evaluate the involvement of stim in a particular response, we tested the same antibodies used in the western blot for their effect on tin generation. for this purpose, antibodies were microinjected into the oocyte cytoplasm to reach a final dilution of 1: figure  shows that ach application onto oocytes pre-loaded with cooh-stim resulted in a robust potentiation of the tin response, increasing the amplitude by 158 ± 25%. cooh-stim injection also potentiated by 168 ± 30% the tin responses elicited by fbs, and a similar effect was observed in oocytes stimulated through the p2y or the p2y receptor. however, in oocytes from the same frogs that were injected with denatured cooh-stim, tin potentiation was completely abolished. also, injection of nh-stim or nh-stim antibody did not produce any changes in the tin response, nor did the injection of a p2y antibody.figure oocyte injection with cooh-stim antibody produced a strong potentiation of t in current response. a) t in current responses were monitored in two conditions: non-loaded oocytes and oocytes loaded with cooh-stim antibody. t in responses were elicited by ach, fbs, or atp application, depending on the receptor to be stimulated. in all cases, a strong potentiation of the response was observed in ab-loaded oocytes. b) oocytes stimulated by ach loaded with denatured cooh-stim had control-like responses, while nh-stim or nh-stim loading did not produce t in potentiation. c) the graph shows the results obtained using the different experimental conditions illustrated in a and b; each bar corresponds to the mean of the t in peak amplitude normalized against the cnt current of 10– oocytes from 3– frogs. all these results clearly indicated that the cooh-stim antibody specifically potentiated the tin current, regardless of the receptor stimulated. role of stim and stim during long-lasting agonist stimulation the following experiments were designed to explore the possibility that stim and stim have different effects, depending on the duration of the stimulus. thus, oocytes injected with as-stim or with as-stim and expressing m p2y or p2y receptors were incubated for 1–4 h in the presence of their respective agonists at 1 μm. extended agonist incubation generated strong tin currents that remained stable for more than the 60-min recording time, even under constant superfusion of the oocytes with nr solution, and it began to decrease after 120–180 min of wash; we assumed that in this condition the soc machinery was over-stimulated, and that the time spent in the activated state reflected the time necessary to refill the reservoirs. in the oocytes knocked down for stim tin currents activated by long-lasting stimulation with any of the agonists analyzed were no different from those observed in control oocytes. in contrast, in oocytes injected with as-stim and expressing p2y or m receptors that had been stimulated for long intervals with their respective agonists, the tin current was no longer generated, strongly suggesting that stim in the oocyte was essential for responses generated through both the acute and long-lasting stimulation protocols. stim proteins and the maturation process during the maturation process, molecular elements that control the ca2+ dynamics in the xenopus oocyte undergo an important reconfiguration; this observation has been extended to different species, and similar changes are known to occur during mitosis. given the importance of these events for cell cycle control, we asked whether or not the knockdown of stim proteins affected the maturation process. thus, batches of control oocytes, and those that were injected with as-stim or as-stim were assayed 48–72 h after injection with 10 μm progesterone in barth’s solution to induce maturation. oocyte maturation entry was scored by the appearance of gvbd after 8–12 h in the presence of progesterone. the gvbd score obtained was compared against progesterone-treated control oocytes. the results are illustrated in figure 6a; as-stim1-injected oocytes did not show any effect on the efficiency of maturation, while stim knockdown produced a strong inhibition of the process. lack of gvbd in as-stim2-injected oocytes seemed to indicate a failure to enter meiosis and a consequent incomplete maturation process; this interpretation was also supported by monitoring electrophysiological parameters in all of the groups tested. as illustrated in figure 6b-c, electrical parameters of as-stim2-injected oocytes were different from those displayed by as-stim1-injected oocytes and control oocytes maintained in progesterone for the same period of time.figure effect of as-stim on gvbd and oocyte membrane characteristics during maturation induced by progesterone. a) the maturation process promoted by progesterone was analyzed in uninjected oocytes, or in oocytes injected 72 h prior to the assay with either as-stim or as-stim and compared with control oocytes in the absence of progesterone. gvbd was quantified after 8–12 h in presence of progesterone and is normalized against the value observed in uninjected oocytes. b) resting membrane potential was monitored 8–12 h after addition of progesterone in the same groups of oocytes as in a). c) the input membrane resistance was estimated over the range from − to −20 mv in the different oocyte groups treated in the same conditions. control groups, without progesterone, included both uninjected and antisense-injected oocytes. in all cases, values for as-stim2-injected groups were different from as-stim1-injected or uninjected groups. taken together, these results clearly showed that in oocytes where stim was knocked down, the process of maturation was inhibited at some early point. the first manifestation of this was the complete blockage of gvbd, i.e., of the signal for meiosis entry; this result was clearly different from that observed in stim1-deprived oocytes. discussion here, using biochemical strategies and electrophysiology, we studied what effects the knockdown of endogenous stim proteins had on two important xenopus oocyte responses: the activation of soce monitored by measuring tin current generation, and the maturation process induced by progesterone. we found that: i) both stim and stim proteins were endogenously expressed in the xenopus oocyte; ii) injection of antisense oligonucleotide sequences of stim or stim potently knocked down the expression of both the corresponding mrna and the protein; iii) stim or stim knockdown did not seem to affect the ca2+-signaling machinery responsible for generating oscillatory ca2+-signals in the oocyte; iv) stim but not stim proved to be fundamental for tin current generation; this was observed both in acute stimulation protocols or after long-lasting stimulation periods, and it did not depend on the receptor type stimulated; v) stim protein knockdown blocked entry into the process of maturation induced by progesterone, while stim elimination did not affect this process; and vi) an antibody against the cooh terminus of stim potentiated tin current generation. calcium release and influx are two phenomena well studied in the xenopus oocyte. the main subject addressed here is the identity and role of stim proteins during calcium influx stimulated through endogenous responses. it is known that after gpcr stimulation, both endogenous as well as exogenously expressed gpcr generate in the oocyte mainly two ca2+-dependent cl− ion currents, one due to intracellular ca2+ release that is normally followed by another current dependent on ca2+ influx; this pattern is generated through an enzymatic cascade involving ip synthesis, a common mechanism in most cell systems. following the original nomenclature, in the oocyte the first response is named iosc, while the second generates the tin current response. the ca2+-influx magnitude is directly related to the amplitude of ca2+-release; the main molecular element responsible for this linkage is the stim protein, since it is the ca2+-sensor within the ca2+ reservoir. as in previous studies, to monitor the i increase produced by both mechanisms in the oocyte, here we used the ca2+-dependent cl− current as an endogenous sensor whose amplitude accurately reflects the concentration of ca2+ beneath the plasmatic membrane. this is especially true for ca2+ influx, since this occurs in the plasma membrane where the ca2+-dependent cl− channels are co-expressed with the soc channels responsible for the influx. thus, monitoring an endogenous ca2+ sensor such as the cl− channel offers not only spatial and temporal advantages, but also amplifies the normally small ca2+ current through soc channels and avoids altering the ca2+ dynamics with further pharmacological manipulations. stim protein expression and function was then studied in the xenopus oocyte using this tool. it is well known that when ca2+ is released from the er, stim proteins are activated, rapidly translocated, and oligomerized into junctions formed between the er and the plasma membrane, where they bind to and activate highly selective ca2+ channels formed by orai proteins that allow ca2+ influx. the main trigger for this phenomenon is a decrease in er ca2+ content; however, evidence indicates that isoforms of stim protein might maintain a basal activation of orai channels without prior ca2+ release, thereby controlling the cytoplasmic ca2+ concentration. the ratio of stim to stim expression seems to depend on the cell type, and perhaps on the pathophysiological state; as shown here for the xenopus oocyte, other cells such as t cells, myoblasts, skeletal muscle, and liver cells also co-express both stim proteins. studies to distinguish the roles of stim and stim in various cells have employed diverse silencing strategies and overexpression. the injection of an antisense oligonucleotide sequence for each stim protein was chosen here for its simplicity and because it excludes potential non-specific effects caused by protein over-expression. the effect of antisense injection on stim expression was demonstrated by analyzing both its mrna and protein expression by rt-pcr and western blot, respectively. this analysis demonstrated that expression of stim or stim was strongly downregulated in oocytes injected with the corresponding antisense oligonucleotide sequence. this antisense effect was not affected by co-expression of gpcr proteins, used experimentally to stimulate ca2+-release. also, it was shown that stim protein knockdown did not affect the ip increase and subsequent ca2+ release, as indicated by the iosc amplitude responses evoked at the beginning of each experiment by acute application of one of the agonists studied. in addition, preliminary results using rt-pcr showed amplification of transcripts for orai and orai in the oocyte. oocytes injected with either antisense stim or stim were then monitored to analyze their ability to generate tin current using two stimulation protocols. in the first, acute application of the agonist produced the typical iosc response; in control conditions it was followed by tin generation that declined after 680–800 s. in the second protocol, long-lasting stimulation of control oocytes with a low concentration of agonist gave strong tin current responses that remained active for 60–180 min, even in constant superfusion with nr solution. a possible explanation for this difference in response kinetics is that prolonged stimulation produced a stronger activation of the soce mechanism, probably due to a more marked decrease in er ca2+ concentration. both protocols were applied in oocytes in which stim or stim expression had been eliminated. the stim knockdown produced a severe decrease in tin current generation in both stimulation protocols, indicating that stim was indispensable to induce the tin current response. using acute receptor stimulation, elimination of stim caused a smaller but significant decrease in tin current generation thus, stim alone was unable to support full tin activation during acute stimulation, suggesting that an association of stim with stim was necessary in order to activate the endogenous response. also it was observed that stim requirement seemed to be minor during p2y purinergic stimulation, this difference cannot be explained by the amplitudes of iosc generated by the agonists, given that ach and lpa were the more and less effective, respectively, in generating the response, and both agonists showed similar patterns of tin decrease by stim knockdown. thus, this result might indicate some type of molecular specificity, perhaps intrinsic to the molecules involved or as a consequence of their differential expression and localization in the oocyte membrane. differential insertion of several proteins expressed in the oocyte membrane has been demonstrated; thus, membrane domains with greater expression of stim together with p2y receptors are plausible. a central role for stim protein in soce generation has been shown before in some cell types such as neurons and dendritic cells; however, there is no information indicating whether or not the expression of stim might affect the full endogenous response in these cases. for example, it has been shown the essential role of stim in soce activation in dendritic spines, which was not substituted by overexpression of stim the authors concluded that this is due to differences in stim-ca2+ sensitivity and subcellular localization of the proteins. in many other cell systems, co-participation or complementary roles for the two stim proteins have been postulated. finally, when the oocytes were stimulated using the long-lasting protocol, elimination of stim had no effect indicating that, in this case, the tin current was activated by stim alone. the central role for stim is supported by the latter result as well as by the finding that injecting the antibody against stim specifically increased the current amplitude by more than 100%, regardless of the agonist used to generate the tin response. it is known that the cooh region, in both stim and stim contains the domain necessary to interact and activate the soc channel formed by orai. thus, a potentiating effect of the cooh-stim antibody indicates that the strength of the stim2-orai interaction might be regulated, either positively or negatively, through a site that is affected by the antibody, indirectly confirming the central role of stim during tin generation. as expected given the stim structure proposed, the two antibodies that recognized domains close to the amino-terminus had no effect on the tin response. significant inhibition of the maturation process was observed in oocytes devoid of stim protein. here, we provided clear-cut evidence of stim involvement during or in preparation for maturation, since its absence eliminated the process of gvbd. once again, this result contrasted with the lack of effect in stim1-knockdown oocytes, whose maturation was similar to that of control oocytes. indeed, it has been shown that the function of stim is downregulated during the maturation process, which contributes to elimination of the soc response in xenopus oocytes; a similar condition has been shown in the mammalian oocyte, although in the latter this phenomenon remains controversial. there is no previous information regarding the effect produced by lack of stim during maturation either in frog or mammalian oocytes, as most previous studies focused on the role of stim however, mouse oocyte is known to express stim protein in the er; during maturation, stim re-localizes from a homogeneous distribution to one closer to the meiotic spindles, suggesting a role during this process. in frog, is possible that the inhibitory effect of stim knockdown was unrelated to its role in soce activation, since meiosis entry in xenopus does not require ca2+-influx. further studies will be necessary to characterize the level at which the lack of stim had such a dramatic effect on the maturation process, and to determine if it might have more general implications. one possibility is that as-stim might cause downregulation of a stim isoform different from that involved in soce activation. another possibility relates to its role regulating cytoplasmic, in which case as-stim might affect the activation of ca2+-dependent processes required prior to meiosis entry.  in this study, stim is fundamental for the endogenous soc response in the oocyte, although an association with stim seemed to be necessary for its full generation. the mechanism responsible for the clear dependence of meiosis entry on stim expression is a fundamental question that remains open, and its elucidation might help to understand the function of stim proteins in the xenopus oocyte and in other cell types as well. additional file additional file 1: blast analysis for the amplified sequences of stim and stim alignment produced by blast for the amplified sequences of stim and stim with the sequences reported for stim1x and stim2xt, respectively. competing interests the authors declare that they have no competing interests. author’s contributions b.s.-f. performed experiments, collected data, provided input in data analysis and was involved in drafting the manuscript. f.g.v.-c. and e. g. performed experiments and provided substantial input in design and interpretation of data. r.o.a. designed research, performed experiments and drafted the manuscript. all authors gave final approval to the version to be published. the author’s thank to dr. dorothy d. pless for editing the manuscript. we also thank mr. horacio ramírez leyva, m. en c. leonor casanova, ing. ramón martínez olvera, and isc omar gonzález hernández, for their expert technical assistance. b.s.-f. as a doctoral student from programa de doctorado en ciencias biomédicas at universidad nacional autónoma de méxico is grateful that she received fellowship from conacyt-méxico. r.o.a. received grants by conacyt-méxico and papiit-unam-méxico in lastly, f.g.v.-c. is grateful for the grants received by conacyt-méxico no. and papiit-unam-méxico no. in 
thus, the endogenous soce response in xenopus oocytes depended mainly on stim and its expression was necessary for entry into meiosis induced by progesterone. selective elimination of their mrna and protein expression was confirmed by pcr and western blot analysis, and we then evaluated the effect of their absence on two endogenous responses: the opening of soc channels elicited by g protein-coupled receptor -activated ca2+ release, and the process of maturation stimulated by progesterone. it was found that stim but not stim was essential in both responses, and tin currents and gvbd were strongly reduced or eliminated in cells devoid of stim2; stim knockdown had no effect on the maturation process, but it reduced the tin response by to 70%. here, we used antisense oligonucleotides to investigate the role in the oocyte of stromal interaction molecule proteins that are fundamental elements of the store-operated calcium-entry phenomenon, as they are both sensors for ca2+ concentration in the intracellular reservoirs as well as activators of the membrane channels that allow ca2+ influx. activation of soc channels was monitored electrically by measuring the tin response, a ca2+-influx-dependent cl− current, while maturation was assessed by germinal vesicle breakdown scoring and electrophysiology. the xenopus oocyte is a useful cell model to study ca2+ homeostasis and cell cycle regulation, two highly interrelated processes.
 the xenopus oocyte is a useful cell model to study ca2+ homeostasis and cell cycle regulation, two highly interrelated processes. here, we used antisense oligonucleotides to investigate the role in the oocyte of stromal interaction molecule proteins that are fundamental elements of the store-operated calcium-entry phenomenon, as they are both sensors for ca2+ concentration in the intracellular reservoirs as well as activators of the membrane channels that allow ca2+ influx.  endogenous stim and stim expression was demonstrated, and their synthesis was knocked down 48–72 h after injecting oocytes with specific antisense sequences. selective elimination of their mrna and protein expression was confirmed by pcr and western blot analysis, and we then evaluated the effect of their absence on two endogenous responses: the opening of soc channels elicited by g protein-coupled receptor -activated ca2+ release, and the process of maturation stimulated by progesterone. activation of soc channels was monitored electrically by measuring the tin response, a ca2+-influx-dependent cl− current, while maturation was assessed by germinal vesicle breakdown scoring and electrophysiology.  it was found that stim but not stim was essential in both responses, and tin currents and gvbd were strongly reduced or eliminated in cells devoid of stim2; stim knockdown had no effect on the maturation process, but it reduced the tin response by to 70%. thus, the endogenous soce response in xenopus oocytes depended mainly on stim and its expression was necessary for entry into meiosis induced by progesterone. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords socestim1stim2xenopus oocyteca2+-entrymaturationissue-copyright-statement© the author 2014 