extfieldsofstudy: Medicine Biology

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The bacterial enhancer-binding protein NTRC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation.

NTRC is a prokaryotic enhancer-binding protein that activates transcription by sigma 54-holoenzyme. NTRC has an ATPase activity that is required for transcriptional activation, specifically for isomerization of closed complexes between sigma 54-holoenzyme and a promoter to open complexes. In the absence of ATP hydrolysis, there is known to be a kinetic barrier to open complex formation (i.e., the reaction proceeds so slowly that the polymerase synthesizes essentially no transcripts even from a supercoiled template). We show here that open complex formation is also thermodynamically unfavorable. In the absence of ATP hydrolysis the position of equilibrium between closed and open complexes favors the closed ones. Use of linear templates with a region of heteroduplex around the transcriptional start site--"preopened" templates--does not bypass the requirement for either NTRC or ATP hydrolysis, providing evidence that the rate-limiting step in open complex formation does not lie in DNA strand denaturation per se. These results are in contrast to recent findings regarding the ATP requirement for initiation of transcription by eukaryotic RNA polymerase II; in the latter case, the ATP requirement is circumvented by use of a supercoiled plasmid template or a preopened linear template.

crS4-Holoenzyme can bind to the glnA promoter in a closed recognition complex in the absence of other factors but requires the presence of NTRC and ATP to isomerize into the open complex, in which the DNA around the transcriptional start site is denatured locally 1Present address: Max-Planck-Institut fuer Biochemie, 82152 Martinsreid, Germany.
( Fig. 1; Kustu et al. 1991). Neither ATP~S nor ADP can substitute for ATP in allowing formation of open complexes although both inhibit the ATPase activity of NTRC, indicating that they bind to the protein . NTRC binds to an enhancer consisting of two tandem DNA-binding sites centered at -140 and -108 with respect to the transcriptional start site and contacts the polymerase by means of a DNA loop. To hydrolyze ATP and activate transcription, NTRC must be phosphorylated on an aspartic acid residue within its aminoterminal regulatory ("receiver")domain (Weiss and Magasanik 1988;Sanders et al. 1992). This phosphorylation, which is mediated by the NTRB protein, occurs under nitrogen-limiting conditions and is labile; the half-life of phosphorylated NTRC is ---4 min at 37°C.
A number of mutant forms of NTRC--so-called NTRC c°nstitutive proteinsmhave some ability to activate transcription without being phosphorylated (Popham et al. 1989;Weglenski et al. 1989;Dixon et al. 1991;Klose et al. 1993;Flashner et al. 1995). All of these forms also have the ability to hydrolyze ATP without being phosphorylated and depend on nucleotide hydrolysis to catalyze the formation of open complexes by ¢54-holoenzyme in vitro. The latter findings distinguish the nucleotide requirement for open complex formation from that  Kustu et al. 1991;Porter et al. 1995). crS4-Holoenzyme can bind to the glnA promoter in a closed recognition complex in the absence of other factors. Conserved promoter sequences lie in the -24 and -12 regions with respect to the start site of transcription. Dimers of NTRC bind to the enhancer, which is composed of two dyad-symmetrical sites centered at -140 and -108. NTRC contacts the polymerase by means of a DNA loop (not shown). When phosphorylated, NTRC catalyzes the isomerization of a closed complex between crS4-holoenzyme and the promoter to a transcriptionally competent open complex, in which the DNA around the transcriptional start site is denatured. To catalyze open complex formation, NTRC must hydrolyze ATP. In open complexes, the conformation of polymerase is changed such that its DNase I footprint is elongated and extends downstream of the transcriptional start site ).
for phosphorylation. Unlike phosphorylated wild-type NTRC, NTRC c°nstitutive proteins provide a homogenous population of active molecules.
There are two striking features that distinguish cr s4 from other cr factors. First, cx s4 bears no detectable sequence homology to other cr factors (Merrick 1993), all of which are homologous to one another (Hellman and Chamberlin 1988;Lonetto et al. 1992). Second, conserved dinucleotides in crsn-dependent promoters must be exactly one turn of the DNA helix apart (10 bp), whereas conserved promoter elements for other holoenzyme forms of polymerase are 2 to 2 '/2 turns apart. Moreover, cr s4 alone can bind to "strong" (high-affinity)promoters in the absence of core polymerase (Buck and Cannon 1992;Cannon et al. 1994).
In this paper we show that the barrier to open complex formation by orS4-holoenzyme is thermodynamic, as well as kinetic. Because NTRC acts catalytically in the for-mation of open complexes, this indicates that the energy of ATP hydrolysis must be coupled to open complex formation to drive the reaction and hence that NTRC is a simple molecular machine (Alberts and Miake-Lye 1992). We also provide evidence that the rate-limiting step in open complex formation is not DNA strand denaturation per se.

The barrier to open complex formation is both kinetic and thermodynamic
When an excess of crS4-holoenzyme, a supercoiled template bearing the glnA promoter, and an NTRC ¢°nstitutive protein (NTRC D54E' s16oF) were incubated together in the absence of ATP, no detectable open complexes accumulated even after 60 min ( Fig. 2A, lanes 1,2; assessed in a single-cycle transcription assay, see Materials and methods). However, when ATP was added at 60 rain, half of the promoters carried an open complex within 2 min ( Fig. 2A, lane 3 and legend). If NTRC was subsequently prevented from hydrolyzing ATP by addition of the inhibitor ADP at 62 min , the preformed open complexes slowly decayed ( Fig must be a kinetic barrier between the two forms. Therefore, the failure to detect formation of open complexes in the absence of ATP hydrolysis cannot be attributable to their lack of stability but must be attributable to their failure to form at all. Experiments in which open complexes were separated from ATP by sieving or were diluted to decrease the concentration of this nucleotide far below that required for open complex formation indicated that a slow rate of decay of open complexes did not depend on the presence of ATP or ADP and that the rate of decay was not affected by these nucleotides Wedel 1993 Even when an excess of cr -holoenzyme was incubated with the template in the presence of nucleotides to allow immediate synthesis of transcripts, essentially no transcripts were seen ( Fig. 2A, Popham 1989). Additional experiments indicated that crS4-holoenzyme could not transcribe even in the presence of NTRC and all four nucleotides if the activator was prevented from hydrolyzing the f~-~/bond of purine nucleotides Popham 1989;Wedel 1993). Hydrolysis of the ~-~/ bond was blocked by the inhibitors ADP or ATP~/S or, alternatively, by providing ~, ~/-methylene-ATP instead of ATP and using a low concentration of GTP.
As noted above ( Fig. 2A, lanes 3-5; Fig. 2B), after inhibition of the ATPase activity of NTRC, the number of  Klose et al. 1993) were incubated with a supercoiled template (pJES 463; 1 nM= 20 fmoles/20 ~xl) bearing the wild-type glnA promoter-regulatory region at 3 7°C in a final volume of 120 ~xl. At 10 and 60 min 20-~xl samples were taken (lanes 1,2) to which ADP and heparin were added to 4 mM and 100 gg/ml, respectively, to stop the subsequent formation of open complexes {see Results; Weiss et al. 1991); after 2 min at 37°C, an elongation mix was added containing ATP, GTP, CTP, and [o~-32p]CTP to allow any polymerase molecules in open complexes to synthesize single transcripts. Immediately after the second sample was taken at 60 min, ATP was added to the remainder of the reaction mixture to 0.4 raM. After incubation for 2 min at 37°C, formation of open complexes was stopped by addition of ADP to 4 mM and a third sample of 20 gl was taken to which the elongation mix was added (lane 3)(because the reaction mixture already contained ADP, only heparin was added before ribonucleotides). Under similar conditions, the formation of open complexes has been shown to cease within 2 rain after the addition of ATP (Popham 1989  Because closed complexes/active crS4-holoenzyme have a measurable activity~that is, the ability to form open complexes~the models described above can be differentiated by asking whether the product formed when open complexes decay can be made to reform open complexes. To determine this, we first formed open complexes and separated them from free crS4-holoenzyme by passage through a sieving column (diagram in Fig. 3A). The isolated open complexes were then allowed to decay, and the crS4-holoenzyme released from them was given an opportunity to reform open complexes either on the same template or on a second template that yielded a transcript of different length. As detailed below, open complexes reformed in both cases (Fig. 3A,(9)(10)(11)(12)quantified in Fig. 3B,C), indicating that decay was at least in part to active crS4-holoenzyme.
The template used for the initial formation of open complexes was a linear fragment carrying a variant of the glnA promoter with four A/T to G/C transitions in the melting region; the half-life of open complexes on this template is 5-10 min at 37°C and -~30 min at 4°C. (We sought a short half-life at 37°C to minimize polymerase inactivation during the course of the experiment.) After rapid purification away from free crS4-holoenzyme over a sieving column at 4°C, open complexes were split into three portions. ATP was added back to all three, whereas NTRC and NTRB were added back to portions 2 and 3 only (diagram in Fig. 3A). To aliquot 3 was also added a 17-fold excess of a second template that yields a shorter transcript. The three samples were then placed at 37°C to allow simultaneous decay and reformation of open complexes. In the absence of NTRC, open complexes decayed from the first template with a half-life of --~5 min ( Figure 3B to the zero time point shows that portion 2, to which NTRC was added, had no more open complexes than portion 1. Similarly, extrapolation of the line for the "Sum" in C to the zero time point and comparison with the extrapolation of either line in B indicates that there were no excess open complexes in portion 3, in spite of the fact that it contained an additional template with a very high apparent affinity for polymerase (see Materials and methods and below ;Wedel 1993). Results similar to those in Figure 3 were obtained in a second experiment in which template and/or NTRC and NTRB were added back after allowing different times for decay of open complexes from the first template (not shown). Because decay of open complexes was to reactantnactive closed complexes or free crSa-holoenzymenthe barrier to open complex formation is thermodynamic as well as kinetic {Fig. 2C, model I ).

Melting of the DNA around the transcriptional start site does not circumvent the requirement for either NTR C or A TP hydrolysis
Because the barrier to open complex formation lies between closed complexes and localized DNA strand denaturation around the transcriptional start site, it is possible that crSa-holoenzyme alone cannot carry out the melting reaction, and that NTRC catalyzes open complex formation by acting as a site-specific helicase. To ask whether strand denaturation was, in fact, the ratelimiting step in open complex formation, we constructed and tested several templates carrying glnA promoter sequences in which the DNA duplex around the transcriptional start site was destabilized by base mispairing {i.e., "preopened" templates} {Fig. 4A). The region that is denatured in natural open complexes at crSa-dependent promoters extends from approximately position -7 to position +3 or +4 with respect to the start site of transcription (Sasse-Dwight and Gralla 1988; Whitehall et al. 1992). The three templates that we tested had regions of heteroduplex from -10 to -2, -1 to +4, and -10 to + 4, respectively; note that the denatured region in the third heteroduplex is longer than that in natural open complexes.
In the presence of NTRC and ATP, all of the promoters carrying regions of heteroduplex were used with comparable efficiency to one another and to the wild-type glnA promoter {Fig. 4B, cf. lanes 3, 5, and 7 with lane 1), whereas in the absence of NTRC they were used poorly {lanes 4, 6, and 8). However, in contrast to the glnA promoter, each of the promoters carrying a region of heteroduplex did support a small amount of transcription in the absence of NTRC. For the promoters carrying heteroduplex from -1 to + 4 and -10 to + 4, transcription in the absence of NTRC was 1%-2% of that obtained with NTRC (not visible in Fig. 4B), whereas for the promoter carrying heteroduplex from -10 to -2, this value was 5%-10%. We have obtained three lines of evidence that transcription in the absence of NTRC results from the fact that crS4-holoenzyme can initiate transcription without going through the normal open complex intermediate when presented with the unusual DNA struc-  template labeled homoduplex carries the wild-type glnA promotet-regulatory region, whereas the templates labeled heteroduplex contain regions of heteroduplex between the promoter and the transcriptional start site (-10 to -2) or between the promoter and a position just downstream of the transcriptional start site {-1 to +4 and -10 to +41. Heteroduplexes were constructed by purifying individual single strands from phagemid particles, followed by renaturation and digestion with a restriction enzyme that releases the promoter-bearing fragment (see Materials and methods). Because the heteroduplexes were reconstituted from purified single strands, they are free of the corresponding alternative heteroduplexes. In all cases, transcripts lack U residues, which minimizes background transcription by core polymerase originating at the fragment ends. (B1 Transcription. Transcription was carried out using the above templates {1 riM) and 50 nM ~S4-holoenzyme (50 nM core polymerase and 200 nM ors4}. Reaction mixtures yielding transcripts shown in the odd lanes also contained wild-type NTRC and NTRB (100 and 20 riM, respectively}. Templates are indicated above the lanes. Shown is an autoradiograph of the gel on which transcripts were isolated. ( Fig. 4B, lanes 3, 5, and 7}. In contrast, transcriptionally competent complexes formed on the heteroduplex promoters in the absence of NTRC initiated primarily at + 3 (see especially Fig. 4B, lane 4), providing a first line of evidence that these complexes are not on the normal reaction pathway.
To confirm that the two sorts of complexes formed between crS4-holoenzyme and the heteroduplex promoters initiated differently, we labeled the 5' ends of such transcripts formed at the promoter carrying heterodu-plex between -1 0 and -2 with [~/-32P]ATP or [-¢-32p]GTP to identify transcripts initiating at + 1 or + 3, respectively; we also labeled with [a-a2P]CTP, as usual, to detect all transcripts. As at the wild-type glnA promoter, complexes between crS4-holoenzyrne and the heteroduplex promoter that were formed in the presence of NTRC initiated at both + 1 and + 3 {Fig. 5A, lanes 1-3}. However, complexes formed in the absence of NTRC initiated almost exclusively at + 3 {Fig. 5A, lanes 4-6}. Because the same high concentration of ATP was present in reaction mixtures with or without NTRC, the failure of complexes formed in the absence of NTRC to initiate at the + 1 A residue did not result from a lack of substrate. It is particularly interesting that the complexes formed without NTRC preferred to initiate at the + 3 rather than + 1 position because base rnispairing in the template extended only through -2 with respect to the start site of transcription. Nevertheless, crS4-holoenzyme alone had access to the template a few bases downstream of the end of the heteroduplex region. The same effect of NTRC on the start point of initiation was obtained with the templates carrying heteroduplex between -1 and + 4 and between -1 0 and + 4 (not shown}. To compare the stabilities of transcription complexes formed in the presence or absence of NTRC, we formed them on the promoter carrying heteroduplex from -10 to -2 at a subsaturating concentration of crS4-holoenzyme and in the presence of ATP. We then diluted them 50-fold into buffer containing heparin to minimize the formation of new complexes. After a further 30-rain incubation at 37°C, complexes formed in the presence of NTRC had decayed by only 1.6-fold relative to the undiluted control {i.e., 36% of the complexes had decayed; To provide further evidence that transcription complexes formed without NTRC on heteroduplex promoters are not on the natural reaction pathway, we constructed a "mutant" version of the -1 0 to -2 heteroduplex promoter lacking the conserved GG dinucleotide in the -2 4 region {deleted from -2 7 to -18; see Materials and methods}. In the absence of NTRC, transcription from this mutant promoter was the same as that from the parental heteroduplex {Fig. 5C, lanes 2,4}. However, whereas NTRC greatly stimulated transcription from the parental promoter {Fig. 4B, cL lane 3 with lane 4; Fig. 5C, cf. lane 1 with lane 21, it had no effect on transcription from the mutant promoter {Fig. 5C, cf. lane 3 with lane 4). This indicates that initiation of transcription from the parental heteroduplex promoter in the absence of NTRC and ATP results from a complex that is not dependent on the conserved promoter recognition sequence in the -24 region and therefore lies outside the normal reaction pathway.
Because the behavior of transcription complexes formed on heteroduplex promoters in the absence of GENES   reactions, each containing ATP at 400 ~M. Each reaction mixture was then split; one portion was immediately provided with elongation mix containing both ADP and heparin; the other was diluted 50-fold into a buffer lacking polyethylene glycol and containing ADP at 100 p~M and heparin at 100 ~g/ml and incubated for a further 30 min at 37°C before elongation mix was added. The initial formation of complexes between crS4-holoenzyme and the promoter was carried out with limiting core RNA polymerase (0.5 nM) SO that the forward association reaction of holoenzyme with the promoter would be minimized after dilution (to 0.01 nM), allowing any difference in the stabilities of complexes formed with or without NTRC to be detected. Elongation was carried out with the same nucleotide concentrations and the same specific activity of the labeled nucleotide in each case, and transcripts were precipitated from the entire reaction mixture and run on a denaturing gel; hence, all lanes are directly comparable. The presence or absence of NTRC and the use of dilution are indicated above the lanes. Shown are autoradiographs of the gels on which transcripts were isolated. There are four transcript bands rather than the two seen in A because the template was not digested with BstXI and hence there are two stops within the bank of T residues used for termination, as well as two starts. I C) Requirement for conserved promoter sequences.
Complexes were formed between 60 nM ~r54-holoenzyme (60 nM core polymerase and 180 nM cr 54) and template (1-kb PvuII fragments; 0.8 riM)with or without NTRC sl6°F and NTRB (each at 20 riM). Formation of complexes and their assessment by transcription were as described in B and Materials and methods, except that ADP was not added with heparin before elongation. Because heparin does not disrupt complexes between ~r54-holoenzyme and heteroduplex promoters, the prominent short transcript band in lane 1 is probably attributable to a second cycle of initiation. Templates were the -1 0 to -2 heteroduplex promoter (lanes 1,2J or the -1 0 to -2 heteroduplex promoter lacking conserved promoter sequences in the -24 region (lanes 3,4; see Materials and methods). The templates were not digested with BstXI (see legend to B). NTRC and NTRB were present for the samples in lanes 1 and 3, as indicated.  Aiyar et al. 1994). NTRC is a molecular machine that have been tested; these include promoters that are activator dependent in vivo and at which open complex formation is activator dependent under stringent conditions in vitro (e.g., the lac promoter; R. Ebright, per. comm.), promoters at which open complexes do not accumulate sufficiently to be detected physically (e.g., the trp promoter; Kierkegaard et al. 1983), and promoters at which physical detection of open complexes depends on the presence of initiating nucleotides (e.g., the rrnB Pl promoter; Newlands et al. 1991;Ohlsen and Gralla 1992).

There is a thermodynamic barrier to open complex formation by ~4-holoenzyme
In addition to the kinetic barrier to open complex formation by (rS4-holoenzyme, there is a thermodynamic barrier. That is, in the absence of ATP hydrolysis by NTRC, the position of equilibrium favors closed complexes. As indicated above, open complexes do not form in the absence of NTRC or ATP hydrolysis. Moreover, we have now demonstrated that open complexes formed in the presence of both of these factors and then separated from them decay back to closed complexes, as opposed to inactive polymerase (Fig. 3). Because NTRC does not remain associated with the polymerase in open complexes, a thermodynamic requirement indicates that there must be a mechanism for coupling ATP hydrolysis by NTRC to open complex formation by ~S4-holoenzyme. In this sense NTRC acts as a simple molecular machine (A1berts and Miake-Lye 1992). In contrast, nucleotide hydrolysis by G proteins plays a regulatory role (Bourne et al. 1991;Webb 1992). Binding of nucleoside triphosphate confers an "active conformation" on G proteins, whereas binding of nucleoside diphosphate yields an inactive conformation. In the case of NTRC, neither ATP~/S nor ADP yields an active conformation; rather, nucleotide hydrolysis is required for activity .

NTRC is not a site-specific helicase
We have considered two possible stages for the involvement of NTRC in open complex formation by orSa-holoenzyme. One is that NTRC acts as a site-specific helicase to perform the DNA strand-denaturation reaction per se, a mode of action that has been assumed by others (Laurent et al. 1993). A second possibility is that NTRC couples the energy of nucleotide hydrolysis to a change in conformation of (rS4-holoenzyme and that this conformational change enables the polymerase itself to carry out the strand-denaturation reaction. We have obtained evidence against the first possibility by showing that preopened promoter templates do not circumvent the requirement for either NTRC or ATP, although they are used very effectively if both are provided (Figs. 4 and 5). Hence, DNA strand separation does not appear to be the rate-limiting step in open complex formation by ~54-holoenzyme and NTRC is not a site-specific helicase. Therefore, we take as a working hypothesis that NTRC mediates a conformational change in polymerase. Such a conformational change is known to accompany (i.e., to occur before or concomitant with) open complex formation, as evidenced by a lengthening of the polymerase "footprint" to a position downstream of the transcriptional start site ( Fig. 1; Popham et al. 1989).
The inferred mode of action of NTRC is of interest with respect to the mechanism of open complex formation by crT°-holoenzyme and (r32-holoenzyme. Kinetic studies indicate that a conformational change in these polymerases is rate limiting for open complex formation, rather than denaturation of the DNA strands per se (Kadesch et al. 1982;Buc and McClure 1985;Roe et al. 1985;Duval-Valentin and Ehrlich 1987;for review, see Buc 1989;Leirmo and Record 1989). This conformational change characterizes the transition from the "first closed complex," an initial weak-binding recognition complex, to the "second closed complex," a tighter binding intermediate complex that is formed before DNA melting. The second closed complex has been characterized physically at temperatures too low to allow DNA strand separation [10-20°C for linear templates; Spassky et al. (1985) for lacUV5; Hofer et al. (1985) for fd PVIII; Duval-Valentin and Ehrlich (1986)for pSC101 tetR; Li and Mc-Clure (1988) for ~ PRM (up-l, A265); Schickor et al. (1990) for T7/A1; Cowing et al. (1989) and Mecsas et al. (1991) for groE]. In it, polyrnerase usually has a longer footprint than in the initial closed complex, and this footprint extends downstream of the transcriptional start site; lengthening of the footprint is inferred to precede DNA strand denaturation at higher temperatures.

The A TP requirement differs from that for initiation of transcription by eukaryotic RNA polymerase H
In contrast to the case for ~S4-holoenzyme, the requirement for ATP hydrolysis for initiation of transcription by eukaryotic RNA polymerase II is circumvented by use of a supercoiled plasmid template or a preopened linear template (Parvin and Sharp 1993;Tantin and Carey 1994;Timmers 1994). Use of a supercoiled plasmid template also obviates the need for the general transcription factor TFIIH, which has an ATP-dependent helicase activity (Schaeffer et al. 1993}. [Requirements for general transcription factors have not yet been determined with preopened linear templates (Tantin and Carey 1994).] At present it is not clear whether the helicase activity of TFIIH is required for open complex formation per se, promoter clearance, or both (Wang et al. 1992;Parvin and Sharp 1993;Goodrich and Tjian 1994;Timmers 1994).

Templates
Plasmid pIES750 is derived from pIES463, which carries the glnA promoter-regulatory region containing a number of synthetic restriction sites and yields a transcript containing 64 C residues (Porter et al. 1993;Wedel 1993). In pIES750, the sequence between the BclI site at position -96 of PIES463 and the NcoI site at -38 (containing the low-affinity NTRC binding sites III, IV, and V)has been replaced with the 403-bp BclI-NcoI fragment from pACYC184, positioning NTRC-binding sites I and II, which constitute the glnA enhancer, at -484 and -452 with respect to the start site of transcription (Porter et al. 1993). In addition, pJES750 contains four tandem oligonucleotides lacking T residues in the nontranscribed strand (ACCACA-GACCACACCACAGAC) that were inserted into the BstXI site of pJES463 at +28 (the site was regenerated). When UTP is omitted during the elongation phase of a single-cycle transcription assay, transcription from pJES750 terminates at about position + 113, the beginning of a bank of T residues in the nontranscribed strand, yielding a transcript of 112 bases containing 50 C residues. If pJES750 is cleaved with BstXI, the runoff transcript contains 108 bases and 49 C residues.
Plasmids pJES752, pJES754, and pJES755 are the same as pJES750 with the exception of the bases in the melting region downstream of the promoter. In pJES752, the sequence ATTTT from positions -6 to -2 is replaced with GCTCC; in pJES754, the sequence TTATATTTT from positions -10 to -2 is replaced with CCGCGCCCC; and in pJES755, the sequence TACGG from -1 to +4 is replaced with CGAAC. The base changes were made by PCR using the NcoI site at -38 and the HindIII site in the polylinker downstream of the transcript terminus, pJES760 is the same as pJES752, except that it carries six rather than four tandem oligonucleotides and hence yields a transcript of ---154 bases containing 70 C residues.
Plasmids pJES761 and pJES762 are the same as pJES750 and pJES754, respectively, with the exception of bases in the -24 region of the promoter. In both cases, PCR was used to delete bases -27 through -18.

Heteroduplex templates
Plasmid pJES750 was constructed in the vector pTZ19U (Mead et al. 1986), which produces and packages the template strand when replicated from the phage fl origin. The EcoRI-HindIII fragments bearing the promoter regions from pJES750, pJES754, pJES755, and pJES763 were also cloned into pTZ19R, which produces the opposite strand when replicated from the fl origin. Single-stranded plasmid carrying each of the three promoter regions was made from the constructs in both pTZ19U and pTZ19R (Ausubel et al. 1989) and purified using Qiagen tip-500 columns (Qiagen, cat. no. 12162). Two complementary single strands (10 }*g each)were heated to 95°C for 10 min in 800 }*1 of full-strength PvuII buffer (New England Biolabs buffer 2)and slowly cooled to room temperature. Then 100 units of PvuII was added, and digestion was allowed to proceed for 2 hr at 37°C. DNA was precipitated with ethanol, and the 1-kb PvuII fragment bearing the heteroduplex promoter region was purified on an agarose gel. After isolation from the gel, templates used for the experiments of Figures 3, 4, and 5A were digested with BstXI, which cleaves at the 3' end of the transcript-coding sequence just before the bank of T residues. When A, C, and G nucleotides are provided, the resulting 700-bp fragment yields a runoff transcript with a single 3' end. It was important that the concentration of single-stranded DNA in the annealing step be low to avoid formation of a large network of partially annealed DNA circles.
Core RNA polymerase from Escherichia coh was kindly provided by D. Hager and R. Burgess (University of Wisconsin, Madison).

Open complex formation
Formation of open complexes was detected in a single-cycle transcription assay, as detailed below. Studies from several laboratories have established the validity of this assay by showing that the requirements for the formation of open complexes are the same whether this assay is used to detect them or any one of four additional assays is used (e.g., Sasse-Dwight and Gralla 1988;Popham 1989;Popham et al. 1989;Weiss et al. 1991;Wedel 1993).
Core RNA polymerase and cr 54 were mixed together on ice, to which buffer [final concentration, 50 mM HEPES-acetate (pH 8.0), 100 mM potassium acetate, 8 mM magnesium acetate, 27 mM ammonium acetate, 1 mM dithiothreitol, and 3.5% (wt/vol) polyethylene glycol (6000-8000; Sigma), final pH 8.0], template and, if present, NTRC and NTRB, were added. The final concentrations of template and protein components for individual experiments are detailed in the figure legends.
Mixtures were then warmed to 37°C for 10 min, and reactions were initiated by adding ATP to a final concentration of 0.4 mM in a total volume of 24 }*1. After 10 min, formation of open complexes was terminated by addition of heparin to a final concentration of 0.1 mg/ml and ADP to a final concentration of 4 mM. After an additional 10 min, nucleotides were added to allow synthesis of transcripts [GTP to 0.4 mM and CTP to 0.1 raM; 5 mCi of [c~-'UP]CTP was also added (10 mCi/mmole, New England Nuclear)]. After 10 min, transcripts were precipitated and isolated by gel electrophoresis. Gels were dried and transcript bands were quantified using a Molecular Dynamics Phosphor-Imager. Any deviations from this protocol are detailed in the relevant figure legend.
The specific activity of CTP was determined by spotting 1 }*1 of a 1:1000 dilution of the heparin-nucleotide mix on the dried gel before visualization/quantitation on the PhosphorImager. The specific activity of transcripts was then determined by multiplying the specific activity of CTP by the number of C residues per transcript--70 for the transcript from pJES760 and 50 for transcripts from pJES750, pJES752, pJES754, pJES755, and each of the templates carrying a region of heteroduplex downstream of the promoter ("heteroduplex promoter templates"). Finally, radioactivity in transcript bands (pixel volume corrected for background} was converted to fmoles of transcript.

Decay and reformation of open complexes
Open complexes were formed on a PvuII fragment of ---1 kb derived from pJES760, as detailed below; this template yields a transcript of -~ 154 bases containing 70 C residues. Core RNA polymerase (50 nM), cr 54 (200 riM), NTRC (100 riM), NTRB (20 nM1, and template (10 nM) were mixed in a final volume of 140}.1 in the transcription buffer described above and incubated at 37°C for 10 min. Formation of open complexes was then initiated by addition of ATP to 1 mM. After a further incubation of 8 min, heparin was added to 100 }*g/ml and the mixture was chilled on ice. To assess the number of open complexes at this point, a 20-},1 sample was taken and incubated with elongation mix to allow synthesis of transcripts.
To separate open complexes from free proteins, 100 t*1 of the mixture described above was loaded on a 24-ml Superose-12 column (Pharmacia HR 10/30 prepacked) that had been equilibrated in transcription buffer without polyethylene glycol, and open complexes were eluted at 0.3 ml/min in the same buffer. The column fraction containing the bulk of the template and, hence, of open complexes, was identified by absorbance at 260 nm and contained DNA at a concentration of 0.3 nM. To monitor decay and reformation of open complexes, the column fraction identified above was split into three portions of 80 ~1 each on ice. Each portion was made 1 mM in ATP and 3.5% in polyethylene glycol, the second and third portions were also made 100 nM in NTRC and 20 nM in NTRB, and the third portion was made 5 nM {representing an excess of 17-fold over the first tem-plate} in the -10 to + 4 heteroduplex promoter template, which yields a transcript of ---113 bases containing 50 C residues (see Fig. 3). The heteroduplex template was used because ~54-holoenzyme binds to it with a very low apparent KD (Wedel 1993}. The final volume of all three portions was 100 ~1. The three mixtures were transferred to prewarmed glass tubes at 37°C to initiate the decay and reformation reactions. Beginning 2 min after transfer to 37°C, 20-~1 samples were removed at 5-rain intervals and added to an elongation mix containing heparin, GTP, and CTP plus oL32p]CTP to allow any open complexes to form transcripts. This procedure takes a "snapshot" of the number of open complexes at any given moment, and in portion 3, it shows the partitioning of holoenzyme between the two templates.
Before sieving on the Superose-12 column, 30% of the template molecules carried polymerase in an open complex, whereas after sieving that fraction was ---3%, meaning that ---10% of the open complexes survived the 45 min between addition of heparin before loading the column and collection of the template fraction off the column. The half-life of open complexes on this template is --30 min at 0°C in the absence of heparin and is shorter in the presence of heparin. Addition of heparin was found to be necessary to prevent free holoenzyme from remaining associated with the template during passage through the column, and the open complexes were therefore in contact with heparin until some time after they entered the column.
Control experiments indicated that incubation of polymerase with template at low concentrations of each (0.01 nM core polymerase, 0.01 nM cr 54 and 0.3 nM template in the presence of polyethylene glycol~ conditions of Fig. 3A, lanes 5-8) resulted in no loss of polymerase activity after 1 hr at 37°C. Hence, loss of activity in lanes 5-8 of Figure 3A cannot be accounted for on this basis.


extfieldsofstudy: Biology Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Mechanisms of Nucleobase Transport in Rabbit Choroid Plexus EVIDENCE FOR A Na (cid:49) -DEPENDENT NUCLEOBASE TRANSPORTER WITH BROAD SUBSTRATE SELECTIVITY*

The overall goal of this study was to determine the mechanisms by which nucleobases are transported in the choroid plexus. Choroid plexus tissue slices were obtained from the lateral ventricles of rabbit brains and depleted of ATP with 2,4-dinitrophenol. In the presence of an initial inwardly directed Na (cid:49) gradient, hypoxanthine accumulated in the tissue slices against a concen- tration gradient. Na (cid:49) -stimulated hypoxanthine uptake was saturable with a K m of 31.1 (cid:54) 9.71 (cid:109) M and a V max of 2.69 (cid:54) 0.941 nmol/g/s (mean (cid:54) S.E.). Na (cid:49) -stimulated hypoxanthine uptake was inhibited by (100) (cid:109) M naturally occurring purine and pyrimidine nucleobases (adenine, cytosine, guanine, hypoxanthine, thymine, uracil, and xanthine) as well as by the nucleoside analog, dideoxya- denosine. The stoichiometric coupling ratio between Na (cid:49) and hypoxanthine was 1.7:1. The data demonstrate the presence of a novel Na (cid:49) -dependent nucleobase transporter in the choroid plexus, which is distinct from the previously described Na (cid:49) -nucleoside transporter in choroid plexus and from Na (cid:49) -dependent nucleobase transporters in other tissues in terms of its kinetics, substrate selectivity, and Na (cid:49) -nucleobase stoichiometry. This transporter

Nucleobases and their structural analogs have become important drugs in the treatment of a number of viral infections of the central nervous system such as cytomegalovirus retinitis, herpes simplex encephalitis, and AIDS-related dementia complex. Therefore, nucleobase transport mechanisms at the major barriers of the central nervous system, namely the bloodbrain barrier and the blood-cerebrospinal fluid barrier (choroid plexus epithelium), may play a critical role in targeting nucleobase analogs to the affected tissues. Moreover, such transport systems may be important in the salvage of purines for nucleic acid synthesis in the brain.
Saturable, low affinity nucleobase transporters have been identified in the blood-brain barrier (1,2). However, it appears that these transporters may not be sufficient to mediate the flux of important quantities of hypoxanthine, the principal salvageable nucleobase, or other nucleobases into the brain. As a result, attention has focused on the choroid plexus as a possible route of entry for hypoxanthine and other nucleobases into the brain (3).
In mammalian cells, transcellular flux of nucleobases is mediated by specific transporters in the plasma membranes. Two major classes of nucleobase transporters have been characterized: equilibrative and concentrative. Equilibrative transporters are present in a number of cell types and are broadly selective for purine and pyrimidine nucleobases. Recent studies with acyclovir and ganciclovir, nucleobase/nucleoside analogs, demonstrate that these agents permeate the human erythrocyte by way of equilibrative mechanisms (14,15).
Recently, concentrative nucleobase transporters have been identified in LLC-PK 1 cells (4), guinea pig kidney (5,13), rat jejunal tissue (6), and guinea pig placenta (7). These transporters are secondary active and Na ϩ -dependent and mediate the influx of specific nucleobases. The substrate selectivity of the transporter in LLC-PK 1 cells has been studied in considerable detail and includes selected purine and pyrimidine nucleobases.
The goal of this study was to determine the mechanisms of nucleobase transport in the choroid plexus. Our data demonstrate the presence of a novel Na ϩ -dependent nucleobase transporter in the choroid plexus, which is distinct from previously described Na ϩ -driven nucleobase transporters in other tissues in terms of its substrate selectivity and kinetics. This transporter may play an important role in the delivery of both salvageable and therapeutic nucleobases to the central nervous system.

EXPERIMENTAL PROCEDURES
Preparation of ATP-depleted Choroid Plexus Tissue Slices-Choroid plexus tissue slices from rabbit were ATP-depleted by the method of Whittico (8). Choroid plexuses were obtained from the lateral ventricles of New Zealand White rabbits. The choroid plexus tissue was placed in KCl buffer (37°C) of the following composition: KCl (150 mM), mannitol (40 mM), and HEPES (25 mM), pH 7.4, with 1 M Tris. Choroid plexus tissue was cut into 2-3-mm pieces and ATP-depleted by incubating at 37°C for 20 min in 2,4-dinitrophenol (250 M) in KCl buffer. Under these conditions, the ATP concentration is reduced to less than 10% of control (9). Following this incubation, the choroid plexus slices in buffer were stored on ice until uptake experiments were performed.
Accumulation Studies-Uptake of [ 3 H]hypoxanthine was studied by methods published previously (10). Briefly, individual choroid plexus tissue slices were incubated with 140 l of reaction mixture containing whether metabolism or degradation of hypoxanthine had occurred. Procedures used were as described previously (10).
Data Analysis-The radioactive content from each choroid plexus tissue slice was expressed as a volume of distribution (V d ) as described previously (8,10). 3 H͔hypoxanthine/g choroid plexus dpm͓ 3 H͔hypoxanthine/ml media Ϫ dpm͓ 14 C͔mannitol/g choroid plexus dpm͓ 14 C͔mannitol/ml media Statistical analysis was carried out by a Student's unpaired t test. A probability, p, of less than 0.05 was considered significant. Data points were determined in triplicate for each experiment. Data, unless mentioned otherwise, are expressed as the mean Ϯ S.E. of data obtained from three experiments in choroid plexus tissue from separate animals. Standard methods were used to determine IC 50 values and Michaelis-Menten kinetics (10).
To determine the stoichiometric coupling between Na ϩ and hypoxanthine, a modified version of the Hill equation was used, in the reaction mixture. The data were transformed with a logarithm and linearly regressed to obtain a and n. This equation has been used previously in stoichiometry studies (12). Materials-[ 3 H]Hypoxanthine (11.6 Ci/mmol), [ 3 H]thymidine (65 Ci/ mmol), and [ 14 C]mannitol (56 mCi/mmol) were purchased from either Amersham Life Science, Arlington Heights, IL or Moravek Biochemicals, Inc., Brea, CA. Hypoxanthine, adenine, adenosine, dideoxyadenosine (ddA), guanine, xanthine, caffeine, cytosine, thymine, uracil, thymidine, and proline were purchased from either Sigma or Aldrich. All other chemicals were purchased from either Sigma, Fisher Scientific, or Aldrich. New Zealand White rabbits were purchased from Nitabell Rabbitry, Hayward, CA. Cytoscint ES scintillation fluid was purchased from ICN Biomedical Inc.

RESULTS
In the absence of a Na ϩ gradient, hypoxanthine accumulated in the tissue slices and reached an equilibrium (V d ϭ 2.57 Ϯ 0.29) in approximately 5 min (Fig. 1). This uptake was not reduced by any compound (including unlabeled hypoxanthine) in the concentration range used in these studies. These results suggest that in the absence of a Na ϩ gradient, the uptake of hypoxanthine represents a nonselective or low affinity binding process or transport process.
In contrast, in the presence of an initial inwardly directed Na ϩ gradient (150 mM), hypoxanthine accumulated temporarily ("overshoot phenomenon") in the tissue slices above the equilibrium value (V d ϭ 6.11 Ϯ 0.91 at 1 min) (Fig. 1). Thin layer chromatography (TLC) studies indicated that hypoxanthine was not significantly metabolized at 30 s or 5 min (data not shown).
Kinetic experiments (Fig. 2) were performed in which the rate of hypoxanthine uptake (at 30 s) as a function of concentration was determined in the presence of an inwardly directed Na ϩ gradient (150 mM). The data are consistent with a single saturable process. The data from each of the three experiments were fit to an appropriate Michaelis-Menten equation, which included a linear component (8). The K m and V max (mean Ϯ S.E.) for Na ϩ -stimulated hypoxanthine uptake were 31.1 Ϯ 9.71 M and 2.69 Ϯ 0.94 nmol/g/s, respectively.
To determine the stoichiometry (Fig. 3) of the Na ϩ -dependent nucleobase transport system, the Na ϩ -dependent uptake of hypoxanthine (0.24 M) was examined in the presence of increasing Na ϩ concentrations (0 -140 mM). The uptake of hypoxanthine (V d ) was sensitive to Na ϩ concentration. The data were fit to a Hill equation as described under "Experimental Procedures." The Hill coefficient was 1.7 Ϯ 0.4 (mean Ϯ S.E.) for Na ϩ :hypoxanthine and was significantly different from 1 but not from 2.
The effect of the nucleobases, hypoxanthine and thymine, on the Na ϩ -dependent uptake of the nucleoside, thymidine, was examined (Fig. 7). At concentrations of 100 M, hypoxanthine and thymine did not significantly inhibit Na ϩ -dependent thy- midine uptake at 30 s. These data suggest that nucleobases and nucleosides do not share the same Na ϩ -stimulated transporter in the choroid plexus. Further studies using NEM, an irreversible sulfhydryl modifying agent, significantly inhibited Na ϩthymidine uptake but not Na ϩ -hypoxanthine uptake (data not shown). This further demonstrates that these two compounds do not share the same transporter. DISCUSSION Previous studies demonstrated that hypoxanthine accumulates and is metabolized in rabbit choroid plexus tissue slices (3). However, the driving force of the transport mechanism(s) was not determined. By using ATP-depleted choroid plexus slices, we experimentally imposed an initial inwardly directed Na ϩ gradient and directly demonstrated that hypoxanthine transport is coupled to a Na ϩ gradient (Fig. 1). These data provide the first demonstration of a Na ϩ -nucleobase transporter in the choroid plexus.
The data in this study suggest that the Na ϩ -dependent hypoxanthine transporter in the choroid plexus is distinct from Na ϩ -dependent hypoxanthine transporters in other tissues. First, Na ϩ -dependent hypoxanthine uptake in the choroid plexus has a lower affinity (31.1 M) than Na ϩ -stimulated hypoxanthine uptake in guinea pig kidney (4.4 M) (5, 13) and LLC-PK 1 cells (0.79 M) (4) but a higher affinity than the saturable uptake system for hypoxanthine (400 M) identified in the blood brain barrier (2).
Second, the substrate selectivity of the Na ϩ -stimulated hypoxanthine transporter in the choroid plexus differs from that of Na ϩ -stimulated hypoxanthine transporters in other tissues. In the choroid plexus, the purine nucleobases, adenine, guanine, and xanthine, and the pyrimidine nucleobases, cytosine, thymine, and uracil, were potent inhibitors of Na ϩ -dependent hypoxanthine uptake (Figs. 4 and 5). In contrast, in LLC-PK 1 cells the nucleobases, adenine, cytosine, and xanthine, did not inhibit Na ϩ -stimulated hypoxanthine uptake (4). The transporter also appears to differ in its substrate selectivity from the Na ϩ -dependent nucleobase transporter in renal brush border membrane vesicles from guinea pig, which excludes adenine (5,13). A Na ϩ -dependent nucleobase transporter for pyrimidines in rat jejunal tissue has been characterized (6); however, the interactions of purines with the transporter were not examined. The interaction of ddA with the Na ϩ -dependent nucleobase transporter suggests that the choroid plexus may play a role in the transport of certain clinically relevant nucleoside analogs.
Our data are consistent with a 2:1 coupling ratio for Na ϩ - hypoxanthine transport. Na ϩ -hypoxanthine transport exhibits a 1:1 stoichiometry in LLC-PK 1 cells and a 2:1 stoichiometry in the guinea pig kidney (13). Similar data have been obtained for Na ϩ -nucleoside transport in choroid plexus. That is, a 2:1 Na ϩnucleoside stoichiometry has been obtained for the N-3, Na ϩnucleoside transporter in choroid plexus (10).
The Na ϩ -nucleobase transporter in the choroid plexus also appears to be distinct from the Na ϩ -nucleoside transporter in the same tissue. Neither cytidine (Fig. 5) nor thymidine (data not shown) inhibited Na ϩ -stimulated hypoxanthine uptake in the choroid plexus. Conversely, neither hypoxanthine (100 M) nor thymine (100 M) inhibited Na ϩ -stimulated thymidine uptake (Fig. 7). Moreover, previous studies in this laboratory have demonstrated that the sulfhydryl modifier, NEM, irreversibly inhibits Na ϩ -nucleoside transport in choroid plexus (11). However, NEM did not inhibit Na ϩ -hypoxanthine uptake in rabbit choroid plexus.
In conclusion, a Na ϩ -dependent nucleobase transporter in choroid plexus from rabbit has been characterized. This transporter is broadly selective for both purine and pyrimidine nucleobases and appears to differ from the recently characterized Na ϩ -nucleobase transporters in guinea pig kidney and LLC-PK 1 based on its kinetics and substrate selectivity. However, this cannot be confirmed until the transport has been cloned and sequenced. Further studies are also needed to determine the relative contribution of the choroid plexus (in comparison with the blood brain barrier) in transporting physiologically relevant quantities of nucleobases into the brain and the role of the transporter in targeting nucleobase and nucleoside analogs to the central nervous system.


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Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm

Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G alpha q/11 and the beta 1 isoform of phospholipase C (PLC beta 1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Kd = 26 nM, Bmax = 30 pmol/mg) and low affinity (Kd = 1.6 microM, Bmax = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome- reacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 microM IP3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca(2+)-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca2+ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of G alpha q/11, PLC beta 1 and a functional IP3R in the anterior acrosomal region of mammalian sperm, as well as thapsigargin's induction of the acrosome reaction, implicate IP3-gated calcium release in the mammalian acrosome reaction.

sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized Ge%n 1 and the [31 isoform of phospholipase C (PLCI31) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Ka = 26 nM, Bma x = 30 pmol/mg) and low affinity (K d = 1.6 IxM, Bma x = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosomereacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 IxM IP 3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca 2÷-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca 2÷ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of Getq/ll, PLC[M and a functional IP3R in the anterior acrosomal region of mammalian sperm, as well as thapsigargin's induction of the acrosome reaction, implicate IP3-gated calcium release in the mammalian acrosome reaction.
T HE acrosome is a specialized membrane-bound organelle located in the head of sperm cells that contains a rich store of hydrolytic enzymes. Binding of the sperm head to the zona pellucida (ZP) 1 of the egg triggers the "acrosome reaction," an exocytotic event that releases these enzymes from the acrosome to facilitate sperm penetration through the ZP, leading to sperm-egg fusion. The acrosome reaction is crucial for fertilization. Acrosomal exocytosis requires extracellular calcium, with zona binding initiating a signaling cascade that leads to calcium influx . The specific signal transduction mechanisms that trigger the acrosome re- 1. Abbreviations used in this paper. DAG, diacylglycerol; BHQ, 2,5-di-(t-butyl)-l,4-benzohydroquinone; IP3, inositol 1,4,5-trisphosphate; IP3R, inositol trisphosphate receptor; PLCIM, phospholipase CIM; ZP, zona pep lucida. action have not been fully clarified. ZP3, a protein of the mouse ZP, initiates the signal transduction process upon sperm binding (Bleil and Wassarman, 1983). Progesterone has also been shown to induce acrosomal exocytosis (Osman et al., 1989;Blackmore et al., 1990Blackmore et al., , 1991Roldan et al., 1994). Increases in intracellular calcium (Lee and Storey, 1989), pH (Florman et al., 1989), cAMP (Noland, T. D., D. L. Garbers, and G. S. Kopf. 1988. Biol. Reprod. 38:94a), IP 3 (Domino and Garbers, 1989; Thomas and Meizel, 1989), and diacylglycerol (DAG) (Roldan et al., 1994) have been associated with ligand-induced acrosomal exocytosis in sperm. Heterotrimeric G proteins of the Gi and Gz classes have been identified in mammalian sperm, while there is no evidence for the presence of Gs and Go proteins (Glassner et al., 1991). Recently, the ZP has been shown to activate Gil and Gi2 in mouse sperm preparations (Ward et al., 1994). The direct roles and relative importance of various signaling processes in promoting calcium influx and triggering acrosomal exocytosis remain unclear.
In the present study, we demonstrate the selective local-ization of signaling elements related to the phosphoinositide system in mammalian sperm. The heterotrimeric G proteins Gq and Gn selectively activate the [3 isoforms of the phosphatidylinositol-specific PLC family, and most effectively stimulate the [31 isotype (Lee et al., 1992). We have localized Gaq/n and PLC[31 to the anterior acrosomal region. We have also visualized IP3Rs in the acrosome cap of mammalian sperm and have enriched for IP3R protein in acrosomal subcellular fractions. We have demonstrated specific calcium release in digitonin permeabilized sperm by IP3 and observed stimulation of the acrosome reaction by thapsigargin, apparently by releasing calcium from an acrosomal IPB-sensitive calcium store. Antibodies. Peptide antibodies to Gctq/ll and PLCI31 and the corresponding immunizing peptides were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit anti-IP3R polyclonal antibody used in this study has been described previously (Sharp et al., 1993). FITC-conjugated goat anti-rabbit secondary antibodies and 5-nm colloidal goldconjugated anti-rabbit secondary antibodies were purchased from Boehringer Mannheim (Indianapolis, IN) and Amersham, respectively.

Materials
Animal Species. 325 g male Sprague-Dawley rats were purchased from Sasco, Inc. (Boston, MA) and CD1 retired breeder male mice from Charles River (Wilmington, MA).
[3H]IP3 binding, fractionation, and 45Ca2+ flux experiments were conducted using rat sperm in order to readily obtain sufficient sperm samples to perform the replicates required of each experiment. Acrosome reaction assays were performed using mouse sperm because this species is most widely used to study acrosomal exocytosis due to extensive characterization of the physiologic ZP3-induced mouse acrosome reaction Wassarman, 1980, 1983).

Sperm Isolation and Preparation
Adult rat, mouse, hamster, and dog cauda epididymides were removed and connective tissue carefully dissected away. The caudac were immersed in PBS and under a dissecting microscope, the tubules were pierced using a 27-gauge needle. Sperm were allowed to diffuse freely from the tubules at room temperature for 5-10 min, until the media became turbid. For preparation of Triton X-100 extracts, the cauda were removed and sperm collected and centrifuged at 600 g for 10 rain. Sperm were resuspended in PBS, centrifuged, and subsequently resuspended in buffer containing 50 mM Trizma, pH 7.4, 1 mM 13-mercaptoethanol, 1 mM EDTA, 1% Triton X-100, and protease inhibitors (4 ~zg/ml leupeptin, 2 p.g/ml antipain, 2 tzg/ml chymotrypsin, 2 i~g/ml pepstatin). After 30 min on ice with intermittent vortexing, samples were centrifuged for 20 min in a table top microfuge at maximal speed. Supernatants were collected and protein assayed. For [3H]IP3 binding, dispersed sperm were probe sonicared (Ultrasonics W-220F) on ice at 20% with one 5-s burst. After spinning in a table top centrifuge at maximal speed for 20 rain at 4°C, the pellet was resuspended by sonication in binding buffer (see below) and protein assayed. For the 45Ca 2+ loading assay, sperm were dispersed and washed in PBS containing 2 mM EGTA (to remove calcium) and 1 mM 13-mercaptoethanol. After pelleting at 600 g, sperm were resuspended in 20 mM Hepes (pH 7.4 with KOH) containing 1 mM 13-mercaptoethanol followed by addition of digitonin to a final concentration of 10 I~M. Sam-pies were incubated on ice for 10 min before addition to the 45Ca2+ loading assay.

Western Blotting
Protein samples (20-50 p.g) were subjected to electrophoresis using precast 6% or 4-12% gradient Tris-glycine gels. The separated proteins were semidry transferred to nitrocellulose and the blots blocked in 5% nonfat dry milk for 2 h, followed by overnight incubation at 4°C in antibody diluted with 3% BSA/PBS. The blots were developed using the ECL detection system according to the manufacturer's protocol. The polyclonal IP3R antibody dilution was 1:1,000 (0.5 i~g/ml). Controls were conducted by pretreating the primary antibody with purified and reconstituted IP3R (Ferris et al., 1989;Supattapone et al., 1988) (5 ixg/ml) overnight at 4°C. Gaq/11 and PLCIM antibodies were diluted to 1 ~g/ml, and preabsorbed antibody was prepared by adding the immunizing peptide at 10 p.g/ml followed by overnight incubation at 4°C.

[3H]IP3 Binding to Rat Sperm
Binding of [3H]IP3 to rat cauda sperm sonicates was measured using 12.5 txg of protein and 2.0 nM [3H]IP3 in 0.25 ml of 25 mM Trizma, pH 8.3, containing 1 mM EDTA. Binding equilibrium was achieved after incubation for 20 rain at 4°C. The equilibrated mixture was then centrifuged in a table top microfuge at maximal speed for 15 min at 4°C, the supernatant aspirated, and the pellet resuspended in 0.25 ml H20 by probe sonication. Bound radioactivity was measured by liquid scintillation in 5 ml of Formula 963. Specific binding represented <10% of added radioactivity. Binding in the presence of 25 ~M unlabeled IP3 was subtracted from total binding to yield specifically bound [3H]IP3. Competition experiments were performed with 0.001-25 ~zM unlabeled IP3.

Immunohistochemistry and Indirect Immunofluorescence
Adult Sprague-Dawley rats were perfused with PBS and 4% paraformaldehyde, and the testes and epididymides removed, postfixed and cryoprotected for frozen sectioning. Immunohistochemistry was conducted using the avidin-biotin-peroxidase system with diaminobenzidine as the chromogen, according to the manufacturer's protocol. The IP3R antibody dilution was 1:500 (1 p.g/ml). For immunostaining of sperm, cells were harvested from the cauda epididymis as described above and dispersed in PBS. Sperm were then mixed with an equal volume of 8% freshly depolymerized paraformaldehyde and incubated on ice for 30 min. Subsequently, fixed sperm were wet-mounted on slides and allowed to air dry. The slides were then washed in PBS for 3 x 5 min, 50 mM NH4CI for 3 x 15 rain, PBS for 3 x 5 min, permeabilized in 0.1% Triton X-100 for 15 rain, rinsed in PBS for 3 x 5 min, treated with 0.01% hydrogen peroxide for 10 rain, and then rinsed in PBS for 3 X 5 min. Slides were then blocked for 1 h at room temperature with 2% normal goat serum/l% BSA in PBS, suctioned dry, and incubated overnight at 4°C. For immunoperoxidase staining of mouse sperm with Gctq/n and PLCIM antibodies, slides were incubated with 1 tzg/ml antibody. Controls were conducted by preabsorbing the antibodies with 10 ixg/ml of the corresponding peptide. Slides were developed using the avidin-biotin-peroxidase system with diaminobenzidine as the chromogen. For indirect immunofluorescence staining, slides were incubated with IPaR antibody at a dilution of 1:500 (1 p~g/ml). Controls for IP3R staining were performed using antibody preabsorbed with purified protein at 5 p~g/ml. Slides were washed in PBS for 3 x 5 min, incubated for 1 h at room temperature with 30 izg/ml FITC-conjugated goat anti-rabbit IgG secondary antibody, washed again in PBS for 3 × 5 min, coverslipped with vectashield mounting medium, and examined by immunofluorescence and confocal microscopy.

Immunoelectron Microscopy
Rat cauda sperm pellets were fixed with 2% paraformaldehyde/0.5% glutaraldehyde for 30 rain on ice. Samples were centrifuged in a table top microfuge at maximal speed and washed with PBS for 3 × 5 rain, NH4C1 for 1 x 1 h, PBS for 2 x 5 min, followed by 0.1 M sodium cacodylate, pH 7.2, for i x 10 min. Pellets were then treated with 1% OsO4 in sodium cacodylate for i h at 4°C. After rinsing 2 x 5 min in distilled water, samples were dehydrated and embedded in LR White. Polymerized blocks were sectioned on a Reichert Ultracut E microtome with a diatome low angle diamond knife. 80-nm thin sections were picked up on fomvar-coated 200 mesh nickel grids. Grids were immunolabeled in droplet solutions. Sec-tions were treated with 1% sodium-meta-periodate for 15 min followed by a 15 min rinse in TBS, 15 min in 50 mM NI-~C1, and 10 min in 1% BSA/ TBS/0.05% Tween. An alternate procedure using tannic acid in place of OsO4 treatment was also used (Berryman et al., 1992). Grids were treated with IPaR antibody diluted 1:250 overnight at 4°C and then washed in TBS, followed by incubation with a 1:50 dilution of 5-nm colloidal goldconjugated anti-rabbit secondary antibody for 1 h at room temperature. The grids were then washed in TBS and distilled water, treated with 2% glutaraldehyde for 5 min, rinsed with distilled water, treated with 3 % uranyl acetate for 20 min, rinsed with distilled water and blotted dry. Sections were viewed on a Zeiss TEM 10A electron microscope at 60 kV. Control grids were incubated with primary antibody pretreated with reconstituted IP3R overnight at 4°C.

Sperm Fractionation
Rat cauda sperm were dispersed in PBS containing 2 mM EGTA and 1 mM 13-mercaptoethanol, and probe sonicated at 35% for four 10-s bursts. An equal volume of 1.8 M sucrose was added and the suspension layered over a discontinuous sucrose gradient containing equal volumes of 2.05 M and 2.2 M sucrose solutions. The sample was centrifuged at 100,000 g for i h at 4°C. The pellet and fractions at the 2.2 M/2.05 M and 2.05 M/0.9 M interfaces were isolated, resuspended in Trizma, pH 7.4, containing 1 mM EGTA, i mM 13-mercaptoethanol, and pelleted at 100,000 g for 45 rain at 4°C. Oil immersion phase microscopy of Coomassie stained fractions (1 g Coomassie brilliant blue per liter of methanol:acetic acid:water at 5:1:5) revealed acrosome-free heads in the pellet, intact and fragmented tails at the 2.2 M/2.05 M interface and acrosomes and acrosomal ghosts at the 2.05 M/0.9 M interface. Samples were protein assayed and equal protein loads subjected to Western analysis for IPaR.
In an alternate procedure, PBS-washed sperm were pelleted at 600 g, resuspended in 0.15 M NaCl/5 mM Hepes, pH 7.3, and homogenized in a glass-Teflon homogenizer with ten strokes at low speed. The sample was layered over a discontinuous sucrose gradient containing equal volumes of 1.75 M sucrose/0.9% NaC1 and 1.3 M sucrose/0.9% NaC1, and ultracentrifuged at 27,500 rpm for 2 h at 4°C. The pellet and 1.3 M/1.75 M interface were isolated, resuspended in PBS, and pelleted by ultracentrifugation at 33,000 rpm. The pelleted samples were subjected to Western analysis for IP3R.

Acrosome Reaction Assays
For isolation of membranes released by acrosomal exocytosis, rat cauda sperm were capacitated for i h at 37°C in M199 media supplemented with 25 mM Hepes (pH 7.4 with KOH), 30 p,g/ml sodium pyruvate and 4 mg/ml protease-free BSA. Only samples displaying more than 80% motility (as determined by cell counting) were used for further study. Sperm concentration was determined using a hemacytometer and set at 107/ml. A 1-ml aliquot of sperm was treated with 10 IxM A23187, while a control sample received vehicle (1% DMSO). After a 30-min incubation at 37°C, sperm were spun in a table top microfuge at 1,000 g. The supernatant was isolated and subsequently ultracentrifuged at 100,000 g for 1 h at 4°C. The pellets were resuspended by sonication in PBS and protein assayed. Equal protein loads were subjected to Western analysis for IP3R.
Mouse acrosome reaction assays were conducted essentially as previously described (Kinloch et al., 1991), using the supplemented M199 medium. Mouse sperm isolated from the cauda were capacitated for 1 h at 37°C. Only samples displaying vigorous motility with greater than 80% motile sperm were used for further evaluation. The sperm concentration was set at 106/ml. 50-1xl aliquots were treated with 10 ~M A23187,100 nM-10 ~M thapsigargin, and vehicle (1% DMSO). After a 1-h incubation at 37°C, the samples were fixed with 50 Ixl of 4% paraformaldehyde on ice for 15 min. Sperm were subsequently mounted on subbed slides, air-dried, and washed with PBS 3 × 5 rain, followed by Coomassie staining for 5 min. After washing with PBS 3 × 5 rain, the slides were coverslipped with 30% glycerol in PBS. Experiments were done in duplicate and 200 sperm per slide were evaluated for acrosomal status by oil immersion microscopy at 100× using Nomarski optics. Control and A23187-treated sperm were additionally subjected to anti-IP3R indirect immunofluorescence in order to document the presence or absence of acrosomal IP3Rs in acrosomereacted sperm.

Region of the Mouse Sperm Head
Western analysis using peptide antibodies to Gotq/11 and PLCI31 detects bands of ~42 kD and 115 kD, respectively, in 1% Triton X-100 extracts of cauda epididymal mouse sperm (Fig. 1). Immunohistochemical staining reveals a selective localization of Gaq/11 to the acrosomal cap region (arrowheads) and the midpiece (Fig. 1 A). No staining is evident in the principal piece or endpiece of the tail. The immunohistochemical staining and the 42-kD band on Western blot are blocked by preabsorbing the antibody with immunizing peptide (Fig. 1 B).
PLCIM is also selectively localized to the anterior acrosomal region of the head with somewhat lighter staining of the midpiece and no staining of the principal piece or endpiece of the tail (Fig. 1 C). The staining pattern and the 115-kD band on Western blot are blocked by preabsorbing the antibody with immunizing peptide (Fig. 1 D).

Localization of the IP3R to Acrosomes of Mammalian Sperm
Western blot analysis using a polyclonal antibody to purified brain IP3R, reveals a specific 260-kD band in 1% Triton X-100 extracts of rat, mouse, hamster, and dog sperm (Fig. 2). The sperm IP3 R band comigrates with the cerebellar IP3R. Immunoreactivity is blocked by preincubation of the antibody with purified and reconstituted IP3R.
Rat cauda sperm possess substantial IP3 binding capacity. Scatchard analysis of [3H]IP3 binding to sperm sonicates reveals a curvilinear plot containing discrete high and low affinity components with Ko values of 26 nM and 1.6 txM, respectively (Fig. 3). Bm~ values for the high and low affinity sites are 30 and 550 pmol/mg protein, respectively, reflecting an extraordinarily high receptor density. The Bmax value for the high affinity site in sperm is several times that reported for membranes of the rat cerebellum, the tissue generally regarded as having the highest IPaR density (Supattapone et al., 1988;Mourey et al., 1990  . The existence of two classes of [3H]IP3-binding sites may reflect different functional states of the IP3R or the presence of receptor subtypes with distinct binding specificities. Immunoperoxidase staining of the rat testis and epididyrnis demonstrates a selective localization of IP3R in maturing sperm cells. In rat testis, the developing acrosomes of round spermatids stain for IP3R (Fig. 4 A). These cells undergo morphologic differentiation and are ultimately re- Figure 2. Identification of IP3R in mammalian sperm by Western blotting. IP3R antibody (1:1,000, 0.5 ~g/ml) detects a specific band of ~260 kD in 1% Triton X-100 extracts of rat, hamster, mouse, and dog sperm. The sperm bands comigrate with IP3R protein detected in the cerebellum. Preincubation of the antibody with purified and reconstituted brain IP3 R blocks the immunoreactivity.  leased into the lumen of the seminiferous tubules. Sperm acquire the potential for motility and egg binding as they traverse the epididymis. Even at low magnification, a cross section through an epididymal tubule reveals intense immunoperoxidase staining of the acrosomal crescent of maturing sperm (Fig. 4 B).
Indirect immunofluorescence confocal microscopy reveals high densities of discretely localized IP3R in the acrosome cap of rat sperm (Fig. 5, A and B). A focus of staining is also present at the proximal midpiece of rat sperm (Fig. 5, A and B). A n IP3-gated calcium store at the proximal midpiece may participate in regulating motility in response to signals received by putative odorant receptors recently identified on the rat sperm midpiece (Walensky et al., 1995). IP3R staining is completely blocked by preabsorbtion with pure IP3R protein (Fig. 5 C).
IP3R is also localized to acrosomes in mouse, hamster, and dog sperm (Fig. 6, E-H). In each species, the shape of the anterior acrosome is distinct and can be visualized by Coomassie staining (Fig. 6, A-D). IP3R localization conforms to the shape of the acrosome in each case. In contrast to the rat, no IP3R staining is detected in the proximal midpiece of hamster, mouse, or dog sperm. At the electron microscopy level, we detect specific immunogold labeling of the acrosome (Fig. 7 A). The distribution of the gold particles conforms to the localization of the outer acrosomal membrane (Fig. 7 B). There is no labeling of the pefforatorium or the equatorial segment (Fig. 7 A). The nuclear envelope and the plasma membrane (in sections where it remained intact) similarly exhibit no immunolabeling (data not shown). Preincubation of IP3R antibody with pure IP3R protein abolishes acrosomal labeling.
The acrosome is bound by two discrete membranes, the outer and inner acrosomal membranes. During the acrosome reaction, the outer acrosomal membrane and the overlying plasma membrane fuse and vesiculate, resulting in release of the acrosomal contents and loss of the overlying membranes. The inner acrosomal membrane is subsequently exposed on the sperm head surface. To confirm the association of IP3R with the outer acrosomal membrane, we conducted indirect immunofluorescence staining of acrosome intact and A23187 acrosome-reacted mouse sperm. Acrosomal IP3R staining of intact sperm is absent in acrosome-reacted sperm (Fig. 8).
To further examine the association of IP3R with the acrosome and acrosomal membrane, we performed two sperm fractionation procedures (Purvis et al., 1982;Zahler and Doak, 1975). Rat sperm were probe sonicated, layered over a discontinuous sucrose gradient, and three fractions were isolated, Coomassie stained and visualized by phase microscopy. The 2.05 M/0.9 M interface contains acrosomes and acrosomal ghosts ( Fig. 9, a, A), the 2.2 M/2.05 M interface contains intact and fragmented tails, and the pellet is enriched in acrosome-free heads ( Fig. 9, a, B). Western blot analysis of the acrosome and acrosome-free head fractions reveals IP3R protein in the acrosome fraction ( Fig. 9, a, C). In a second procedure, rat sperm were homogenized using a glass-Teflon homogenizer, layered over a discontinuous sucrose gradient, and the 1.3 M/1.75 M interface containing acrosomal membranes isolated and analyzed by Western blotting. IP3R protein is also enriched in this acrosomal membrane fraction (data not shown). In a final experiment to link IP3R to the acrosome, we capacitated rat sperm and induced the acrosome reaction with 10 ~xM A23187. After centrifugation at 1,000 g, the medium containing membranes released during acrosomal exocytosis was centrifuged at 100,000 g for 1 h at 4°C. The 100,000 g pellet of sperm treated with A23187 is enriched with IP3R protein compared to the corresponding fraction of untreated capacitated sperm (Fig. 9 b). Because dying cells and a percentage of capacitated cells can autoacrosome react in a calcium-containing media, the presence of some IP3R protein is expected in the control fraction.

45Ca2+ Loading of Digitonin Permeabilized Rat Sperm
We used digitonin permeabilized rat sperm loaded with 45Ca2+ to examine IP3-mediated calcium release from intracellular stores. Sodium azide was included in the reaction buffer at a concentration known to inhibit mitochondrial 45Ca 2+ accumulation (Arab et al., 1990;Verma et al., 1992). 45Ca2+ accumulated in the presence of ATP, which activates CaZ+-ATPase loading of intracellular stores . No calcium accumulation occurs in the absence of ATP or in the presence of thapsigargin or Figure 8. Acrosomal anti-IP3R immunofluorescence is lost in acrosome-reacted sperm. Capacitated mouse sperm were treated with 10 I~M A23187 to induce acrosomal exocytosis. Control sperm treated with 1% DMSO are acrosome intact by Coomassie staining (A) and demonstrate the characteristic acrosomal anti-IP3R immunofluorescence (B). A23187-treated sperm undergo the acrosome reaction and lose the outer acrosomal membrane, the overlying plasma membrane, and acrosomal contents. A21387-treated sperm no longer exhibit Coomassie staining (D) or anti-IP3R staining (E) of the acrosome. Panels C and F are the corresponding phase images of B and E, respectively. Bars: (A and D) 14 ~m; (B, C, E, and F) 28 Ixm. BHQ, both of which specifically inhibit the Ca2÷-ATPase of nonmitochondrial intracellular membranes Llopis et al., 1991). The calcium ionophore, A23187, also prevents calcium loading. 10 ~M IP 3 dec r e a s e s 45Ca 2+ accumulation in sperm by 45% (Fig. 10), which is similar to the effect observed in cerebellar microsomes (Verma et al., 1992). Actions of IP3 on sperm calcium are blocked by heparin, an IP3R antagonist. In contrast, caffeine and ryanodine, which selectively release calcium from the calcium-induced-calcium-release (CICR) pool (Ehrlich et al., 1994), display no effect o n 45Ca2+ loading. The inability of IP 3 to release all of the accumulated calcium is consistent with data from the cerebellum, where IP3 and caffeine in combination can release no more than 65% of the accumulated calcium (Verma et al., 1992).

Induction of the Acrosome Reaction by Thapsigargin
Thapsigargin selectively inhibits the Ca2÷-ATPase of nonmitochondrial intracellular membranes and does not affect the plasma membrane pump (Lytton et al., 1991). The failure of caffeine or ryanodine to influence calcium accumulation suggests that thapsigargin releases calcium from IP3-sensitive stores. To determine whether an IP3-gated calcium store participates in acrosomal exocytosis, we examined the influence of thapsigargin on the acrosome reaction of mouse sperm. Thapsigargin triggers acrosomal exocytosis with a maximal effect equivalent to the maximal response elicited by the calcium ionophore, A23187 (Fig. 11). The potency of thapsigargin in stimulating the acrosome reaction (ECs0 --0.5 p~M) resembles its potency in elevating the intracellular calcium levels of pancreatic acinar cells (Metz et al., 1992). Thapsigargin also induces the acrosome reaction of human sperm (Meizel and Turner, 1993).

Discussion
The main findings of this study are the selective localization and functional association of IPaRs with the acrosomes of mammalian sperm,. The level of IP3 binding in sperm is among the highest observed in mammalian tissues. This is particularly striking since the IP3R is selectively localized to the acrosomal membrane, which constitutes only a small fraction of sperm membranes. Acrosome-reacted sperm lose their acrosomal IPaR immunostaining, and the medium in which the reaction takes place is correspondingly enriched with IP3R protein compared to control samples. These findings are consistent with the release of the outer acrosomal membrane and associated IPaRs from the sperm head into the medium during aerosomal exocytosis. Sperm IPaRs are functional, as evidenced by the IPa-mediated activation of calcium channels in a r e v e r s e 45Ca2+ flux assay using digitonin permeabilized sperm. Recently, type 1 and type 3 ryanodine receptors have been identified in Figure 9. (a) IP3R is associated with sperm acrosomes. Fractionating rat sperm sonicates over a discontinuous sucrose gradient produces a layer enriched in acrosomes and acrosomal ghosts at the 2.05 M/0.9 M interface (A) and a pellet containing acrosome-free heads (B). Western blotting of equal protein loads demonstrates IP3R in the acrosome-enriched fraction but not in the acrosome-free head fraction (C). (b) Capacitated rat sperm (107/ml) were treated with 10 p~M A23187 to induce the acrosome reaction, and control samples received vehicle (1% DMSO). Sperm were pelleted at 1,000 g and the supernatant containing membranes lost during acrosomal exocytosis was centrifuged at 100,000 g. The pellets were resuspended and subjected to Western analysis using anti-IP3R antibody. The sample derived from acrosome-reacted sperm is enriched with IP3R protein compared to untreated control samples. Bars: (A) 12 Ixm; (B) 6 Ixm. murine testis and in purified populations of spermatocytes and spermatids (Giannini et al., 1995). Although the presence or absence of ryanodine receptors in mature sperm awaits further study, we were unable to detect an effect of caffeine or ryanodine on sperm 45Ca2+ accumulation, suggesting that the phosphoinositide system is the major mechanism for intracellular calcium release in sperm. Accordingly, the finding that thapsigargin, which acts selec- In the absence of ATP or in the presence of the intracellular Ca2÷-ATPase inhibitors thapsigargin (10 ixM) and BHQ (10 IxM), no loading is observed. 10 p~M IP 3 d e c r e a s e s 45Ca2+ accumulation by 45%. The IP 3 effect is reversed by heparin (500 p~g/ml). In contrast, caffeine (5 mM) and ryanodine (5 ixM) have no effect on 45Ca2+ accumulation. The drug vehicles ethanol (1%) and DMSO (1%) similarly have no effect on 45Ca2+ accumulation. Specific loading represents ~<10% of added radioactivity. Experiments were performed in duplicate and repeated at least three times. Bars represent standard deviations of the mean.  Figure 11. Induction of the acrosome reaction by thapsigargin. Thapsigargin triggers the acrosome reaction of capacitated mouse sperm in a dose-dependent manner from 100 nM to 10 p,M. The maximal thapsigargin effect is equivalent to that elicited by the calcium ionophore, A23187 (10 I~M). Experiments were performed in duplicate and repeated twice. For each reaction sample, sperm were fixed, Coomassie stained, and 200 sperm per slide evaluated for acrosomal status under oil immersion microscopy at 100× using Nomarski optics. Bars represent standard deviations of the mean.
tively upon the intracellular calcium release system, triggers acrosomal exocytosis strongly implicates IP3-mediated calcium release in promoting the acrosome reaction. The acrosomal vesicle is similar to somatic secretory granules in that it packages proteins at an acidic pH and is exocytosed in response to specific ligand stimulation. The localization of IP3Rs to the acrosome suggests that this organelle additionally functions as a calcium store. Whereas the endoplasmic reticulum is the most widely recognized subcellular localization of IP3Rs, IP3-gated calcium channels have been identified in the plasma membrane , nuclear membrane (Malviya et al., 1990), and nerve terminals (Peng et al., 1991). The finding that IP3 directly triggers calcium release from bovine adrenal medullary secretory vesicles, indicates that IP3Rs are a component of chromaffin granules (Yoo et al., 1990). A recent study localized IP3R subtype 3 to the insulin and somatostatin secretory granules of pancreatic [3-cells and 8-cells, respectively (Blondel et al., 1994). These vesicles have been shown to release calcium upon cell stimulation (Abrahamsson et al., 1981). Calcium loading of secretory granules is a well known phenomenon, and may play a functional role in stimulus-secretion (Nicaise et al., 1992). High capacity calcium-binding proteins typically present in the core of secretory granules participate in calcium sequestration. Using video enhanced fluorescence microscopy of fura-2 loaded mouse sperm, (Herrick, S. B., and R. A. Cardullo. 1994. Mol. BioL Ceil 5:346a) recently lo-calized intracellular calcium to the acrosomal crescent. Interestingly, an endoplasmic reticulum protein, calreticulin, has been identified as a component of the rat sperm acrosome (Nakamura et al., 1993). Calreticulin possesses high and low affinity calcium-binding sites and may participate in acrosomal calcium sequestration. The identification of IP3Rs in [3-and 8-secretory granules has generated the hypothesis that secretagogue-induced IP3 production triggers calcium release from the granules that directly regulates exocytosis (Blondel et al., 1994). IP3-induced calcium mobilization has been directly shown to activate nuclear vesicle fusion in vitro (Sullivan et al., 1993). IP3-gated calcium release from the acrosome may likewise play an important role in promoting membrane fusion events and resultant acrosomal exocytosis.
What is the signal transduction mechanism leading to acrosomal exocytosis? The specific ligands known to trigger the acrosome reaction include ZP3 (Bleil and Wassarman, 1983), a glycoprotein of the mouse ZP, and progesterone (Osman et al., 1989;Blackmore et al., 1990Blackmore et al., , 1991Roldan et al., 1994), a steroid present in the cumulus oophorus which surrounds the egg. Several sperm ZP3binding proteins are candidate ZP3 receptors (Leyton et al., 1989(Leyton et al., , 1992Miller et al., 1992;Cheng et al., 1994). Indeed, ZP3 may have multivalent interactions with sperm surface proteins . A cell surface receptor has been hypothesized to transduce the progesterone signal (Blackmore et al., 1991;Mendoza and Tesarik, 1993). Progesterone may act through a sperm GABAA-Iike receptor (Wistrom and Meizel, 1993;Roldan et al., 1994) and perhaps additionally through direct activation of an unidentified calcium channel (Roldan et al., 1994). Although multiple signal transduction components have been identified and hypothesized to play important roles in acrosomal exocytosis, the specific nature of the ligand-receptor interaction, the relevant signaling pathways activated, and the sequence of transduction events remain unclear.
Like many ligand-activated secretory events, the physiologic acrosome reaction is calcium-dependent and has been long known to require extracellular calcium (Yanagimachi and Usui, 1974). In a wide range of somatic cell types, ligand-mediated generation of IP3 followed by calcium release from IP3-gated internal stores promotes extracellular calcium influx across the plasma membrane (Berridge and Irvine, 1989). This phenomenon of capacitative calcium entry is well documented but the mechanism is incompletely understood (von Tscharner et al., 1986;Putney, 1990;Bird et al., 1991;Schilling et al., 1992). In many secretory cells, the initial focal release of calcium from IP3-gated intracellular stores has a priming effect on exocytosis (Marty, 1991;Thorn, 1993;Blondel et al., 1994). In chromaffin cells and basophils, the release of intracellular calcium alone by ligand-activated generation of IP3 or by treatment with intracellular calcium release agents such as thapsigargin, is insufficient to promote secretion in the absence of extracellular calcium (Cheek and Barry, 1993;MacGlashan and Botana, 1993). The requirement of extracellular calcium to induce acrosomal exocytosis likewise suggests that IP3-gated acrosomal calcium release may be insufficient to trigger acrosomal exocytosis in the absence of external calcium. However, under physiological condi- Figure 12. Model of ligand-induced signaling events in the mammalian acrosome reaction leading to increased intracenular calcium in the sperm head. ZP3 (above) may have multivalent interactions with the sperm plasma membrane (below) and thereby trigger multiple signaling pathways. A pertussis-insensitive pathway may involve those signal transduction components identified in this study. Receptor activation of Gq stimulates PLCIM activity resulting in phosphatidylinositol 4,5-bisphosphate hydrolysis and the generation of IP 3 and DAG. Binding of IP3 to IP3Rs localized to the outer acrosomal membrane would trigger release of acrosomal calcium. Subsequent capacitative calcium entry through focal, voltage-insensitive channels (F) would further elevate intracellular calcium, trigger membrane depolarization, and activate L-like calcium channels (L). A pertussis-sensitive pathway involves ZP3 activation of G i which may directly activate L-like calcium channels, resulting in extracellular calcium influx. ZP3 binding to p95, a putative receptor tyrosine kinase (TK), may result in receptor phosphorylation and PLC~/stimulation. Activation of a Na+-H + exchanger would cause a Na ÷ influx, H ÷ efflux, a rise in intracellular pH, membrane depolarization, and resultant calcium influx through L-like channels. The increase in pH would enhance IP3-gated calcium release. Progesterone (PROG) can trigger calcium influx and initiate the acrosome reaction in a pertussisinsensitive manner. Progesterone may operate through a receptor-operated calcium channel, a GABAA-like receptor (GabaR), or a Gq-coupled receptor. The ligand-induced elevations of sperm intracellular calcium levels are required for membrane fusion events and resultant acrosomal exocytosis.
tions, IP3-gated calcium release may be an important mechanism for activating the extracellular calcium influx required for the acrosome reaction. Roldan and colleagues (1994) have demonstrated that progesterone and ZP activate sperm phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, which produces both IP3 and DAG. The generation of D A G is believed to be important in activating phospholipase A2, which generates fusogenic free fatty acids and lysophosphatides (Roldan and Harrison, 1993). Roldan and colleagues (1994) measure increases in whole cell D A G mass in response to ZP and progesterone, and indicate that the ligand-induced second messenger elevation is dependent upon extracellular calcium. Chelation of extracellular calcium, however, can deplete intracellular calcium and thereby preclude PLC activation. In addition, measuring whole cell D A G mass may be insufficient to detect ligand-induced generation of D A G in the absence of extracellular calcium due to the focal nature of second messenger elevations typical of polarized secretory processes (Marty, 1991;Thorn, 1993). Ultimately, it will be important to determine if the extracellular calcium-dependent increases in whole cell D A G mass correlate with acrosomal exocytosis. Our 4~Ca2+ ac-cumulation data indicate that sperm can load calcium in an ATP-dependent manner into an IP3-gated store and that thapsigargin can completely prevent this accumulation. The association of ATPase activity with the outer acrosomal membrane (Gordon et al., 1978;Gordon, 1973), coupled with the finding that thapsigargin can stimulate acrosomal exocytosis in human (Meizel and Turner, 1993) and mouse sperm, suggests that calcium release from the acrosome participates in triggering acrosomal exocytosis. Thus, the finding that early events of the acrosome reaction require micromolar Ca 2+ levels and later events require millimolar levels (Roldan and Harrison, 1993), is consistent with initial ligand-induced generation of IP3 and acrosomal calcium release, followed by extracellular calcium influx.
What agonist-induced calcium responses have been observed in mammalian sperm? Florman et al. (1992) (Florman, 1994 have characterized ZP-induced calcium channel activity in bovine sperm. Florman has identified two agonist-dependent calcium entry mechanisms. The first is a poorly selective, QNB-sensitive, and voltage-insensitive cation channel that is responsible for transient and focal calcium elevations in the sperm head. A transient, focal in-tracellular calcium elevation was similarly observed in mouse sperm heads in response to ZP (Storey et al., 1992). The second activity has several pharmacological characteristics of L-type voltage-sensitive calcium channels and is responsible for sustained calcium elevations throughout the entire sperm cell. This secondary massive influx of calcium is believed to be essential for acrosomal exocytosis. ZP3-activation of the first cation channel may produce membrane depolarization resulting in activation of sperm "L-like" channels. Whereas the focal calcium conductance is pertussis-insensitive, the L-like channel activity is pertussis-sensitive. ZP-induced acrosomal exocytosis is inhibited by QNB presumably by blocking focal channels. In the presence of pertussis toxin, the ZP-induced acrosome reaction is also inhibited, despite activation of focal channels. These findings suggest that sperm L-like channels are regulated by dual voltage-sensitive and voltage-insensitive mechanisms (Florman, 1994).
The inhibition of ZP3-induced acrosomal exocytosis with pertussis toxin (Endo et al., 1987), indicates that G proteins participate in regulating this secretory event. In mouse sperm preparations, Gil and Gi2 are directly activated by ZP3 in a pertussis-sensitive manner (Ward et al., 1994). The mechanism by which ZP3 activates L-like calcium channels and the manner in which pertussis-sensitive G proteins are involved in mediating this process is unknown. Another mechanism for triggering membrane depolarization, L-like channel activation, and calcium influx has been proposed by Fraser et al. (1993a,b) (Fraser, 1994) and involves early activation of a Na+-H + exchanger. Fraser et al. (1993a) have demonstrated that the Na+-ionophore monensin can trigger acrosomal exocytosis. Activation of Na + entry and H + extrusion would promote alkalinization, membrane depolarization, and subsequent activation of sperm L-like calcium channels. This sequence of events is supported by the ability of nifedipine to block the monensin-induced acrosome reaction (Fraser, 1993b). The mechanism by which the sperm Na+-H + exchanger is activated in response to ligand stimulation is not known. In somatic cells, agonist-induced activation of Na+-H + exchangers has been shown to occur through phosphorylation by protein kinase A, protein kinase C, and tyrosine kinase activities Turner, 1989, 1991;Borgese et al., 1992;Grinstein and Rothstein, 1986;Grinstein et al., 1989). A 95-kD candidate sperm receptor for ZP3 has tyrosine kinase activity (Leyton et al., 1992;Sating, 1991) and could potentially activate a sperm Na+-H+-exchanger (Fraser, 1994).
In this study, we have identified components of the phosphoinositide signaling pathway, including Gotq/ll , PLC[M, and an IP3-gated calcium store in the acrosomal region of mammalian sperm. Recently, Gq has independently been identified in the acrosomal region and midpiece of mouse sperm by P. E. Visconti and G. S. Kopf (personal communication). These pertussis-insensitive signaling components may promote acrosomal exocytosis by mobilizing intracellular calcium. In insulin secreting I~-TC3 cells, the muscarinic agonist carbachol stimulates the phosphoinositide pathway resulting in intracellular calcium release and the potentiation of glucose induced insulin secretion (Baffy et al., 1993). Cellular injection of anti-Gq antibodies inhibits the carbachol effect. Interestingly, a muscarinic antagonist, QNB, has been shown to attenuate ZP-induced focal, transient Ca 2+ elevations in the mouse sperm head (Storey et al., 1992) and inhibits focal channel activity in bovine sperm (Florman, 1994). Thus, ZP3 may initially activate a Gq-coupled receptor resulting in IP3-production, acrosomal calcium release and subsequent focal capacitative calcium entry through voltage-insensitive channels in the sperm head plasma membrane. This focal elevation of intraceUular calcium may participate in L-like channel activation by triggering membrane depolarization as proposed by Florman (1994). ZP3 binding to a Gi-linked receptor may directly activate L-like calcium channels, producing a global influx of extraceUular calcium. Phosphorylation activation of a Na+-H ÷ exchanger by PKC or a receptor tyrosine kinase may additionally promote L-like channel activation by increasing Na +, extruding H ÷, increasing pH, and causing membrane depolarization. It is noteworthy that the increase in pH during the acrosome reaction would potentiate calcium release from the acrosome, since binding of IP 3 to IPaRs and subsequent calcium release through IP3Rs are both favored by alkaline pH (Brass and Joseph, 1985;Worley et al., 1987). A model of ligand-induced signaling events leading to increased sperm intracellular calcium is presented in Fig. 12.
There are multiple signal transduction components in the acrosomal region of mammalian sperm poised to execute a single exocytotic event in response to the appropriate ligand. ZP3 may activate several signaling pathways through multivalent interactions with the sperm plasma membrane. The sequence of signaling events and the manner in which they are integrated to produce exocytosis is not definitively known. However, there appears to be alternate routes to exocytosis. Whereas ZP3 triggers the acrosome reaction in a pertussis-sensitive fashion, progesterone has been shown to initiate the acrosome reaction in a pertussis-insensitive manner . Multiple physiologic inducers or inhibitors may participate in the regulation of acrosomal exocytosis. Identification and characterization of the specific sperm receptors that trigger signaling pathways in response to physiologic ligands, will contribute enormously to our future understanding of this critical exocytotic event. It is clear, however, that elevated intracellular calcium is central to induction of the acrosome reaction. Whereas IP3-gated calcium release is well known to be essential for egg activation (Miyazaki et al., 1992;Xu et al., 1994), our data suggests that calcium flux through sperm IP3Rs may likewise play a pivotal role in the fertilization process.