Article |
Address correspondence to Kiyoko Fukami, Division of Biochemistry, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: 81-3-5449-5507. Fax: 81-3-5449-5417. E-mail: kfukami{at}ims.u-tokyo.ac.jp
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Abstract |
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Key Words: phospholipase C; acrosome reaction; zona pellucida; calcium; sperm
* Abbreviations used in this paper: IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; SBTI, soybean trypsin inhibitor; SOC, store-operated channel; ZP, zona pellucida.
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Introduction |
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The acrosome reaction, which entails exocytosis of the acrosomal vesicles, is an essential step for fertilization. In mammalian sperm, the acrosome reaction is thought to be initiated in vivo by binding to the zona pellucida (ZP), the extracellular matrix of the egg, and only sperm that have completed the acrosome reaction can penetrate the ZP and fuse with the egg plasma membrane (Wassarman, 1999; Wassarman et al., 2001; Primakoff and Myles, 2002). In general, it is thought that sperm binding to ZP3, one of the glycoprotein components of the ZP, induces a transient Ca2+ influx into the sperm through voltage-dependent nonselective cation channels, which in turn leads to activation of a pertussis toxinsensitive trimeric Gi/o proteincoupled PLC (Patrat et al., 2000; Darszon et al., 2001). A tyrosine kinaseregulated PLC may also be activated during ZP3 binding (Patrat et al., 2000). Activation of PLCs generates IP3, thereby mobilizing [Ca2+]i from the sperm's intracellular Ca2+ store, the acrosome, although the Ca2+ storing capacity of this organelle seems very limited (Rossato et al., 2001). Nonetheless, these early responses appear to promote a subsequent sustained Ca2+ influx signal via store-operated channels (SOCs) that results in the acrosome reaction (Florman, 1994; O'Toole et al., 2000; Breitbart, 2002). Recent studies have provided evidence for the expression in sperm of transient receptor potential protein channels 1, 3, and 6 (Trp1, Trp3, and Trp6), all putative Ca2+-permeant SOCs (Trevino et al., 2001), and Trp2 has been described to play a role in the ZP3-induced acrosome reaction in mouse sperm (Jungnickel et al., 2001). However, the precise molecular mechanism by which the acrosome reaction occurs has remained unclear.
In addition to ZP, thapsigargin, a specific blocker of the sarco/endoplasmic reticulum Ca2+-ATPase, which causes Ca2+ depletion from internal stores and leads to capacitative Ca2+ entry (Sabala et al., 1993), is also able to induce the acrosome reaction (Llanos, 1998). Furthermore, progesterone released from the cumulus cells, and thus one of the major components of follicular fluid, has also been shown to induce the acrosome reaction in a presumed physiological manner (Roldan et al., 1994; Kobori et al., 2000). Although the mechanism of action of progesterone on sperm is not yet fully understood, it is thought to induce Ca2+ influx by activating a GABAA-like progesterone receptor/Cl- channel (Meizel et al., 1997).
On the basis of the consensus that the acrosome reaction requires Ca2+ influx, global patterns of [Ca2+]i changes in single sperm have been observed. However, only a few studies have reported detailed spatio-temporal analysis of [Ca2+]i rises at the single-sperm level because of difficulties of the measurement in these cells. Here we report, using Ca2+ imaging of single sperm, that sperm show Ca2+ waves in response to ZP, progesterone, and thapsigargin, although the site of initiation of the rises appears to differ with the agonist, and that PLC4 is an important protein in the regulation of the Ca2+ responses that drive the acrosome reaction.
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Results |
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Treatment of PLC4+/+ sperm with thapsigargin or ionomycin induced continuous [Ca2+]i increases, and in most sperm, the [Ca2+]i rises were sustained for at least 240 s. Treatment of PLC
4-/- sperm with these reagents produced a similar pattern, but with less amplitude (Fig. 4 B). These results raise the possibility that PLC
4 may have an important role in the regulation of [Ca2+]i mobilization in sperm in response to numerous agonists.
Agonist-specific spatial distribution of [Ca2+]i rises in sperm
Typical [Ca2+]i increase patterns of sperm in response to ZP, progesterone, or thapsigargin over time are shown in Figs. 5 and 6 and Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200210057/DC1). When PLC4+/+ sperm preloaded with fluo-4 were treated with ZP, the [Ca2+]i increase was first detected in the acrosome area (2 s), then in the equatorial segment (4 s), and finally extended to the whole sperm head (around 14 s) (Fig. 6 A). Although there are a few reports that describe the global [Ca2+]i elevations in bovine or hamster sperm head during the ZP-induced acrosome reaction (Florman, 1994; Shirakawa and Miyazaki, 1999), this is the first observation that clearly notes the sequential [Ca2+]i mobilization induced by ZP. These data support the notion that the acrosome vesicle serves as the intracellular Ca2+ store and that ZP mobilizes [Ca2+]i before promoting Ca2+ influx during acrosome reaction. Interestingly, the acrosome reaction always occurred after peak [Ca2+]i values were attained, and the occurrence of the reaction was confirmed by simultaneous monitoring of fluo-4 and Alexa Fluor®594labeled SBTI (unpublished data). As expected, only a minor [Ca2+]i increase was detected in PLC
4-/- sperm after the addition of ZP (Fig. 5 B; Fig. 6 B).
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Monitoring of [Ca2+]i mobilization on sperm at the population level suggests a role of PLC4 on Ca2+ influx
To further understand the role of PLC4 in sperm [Ca2+]i mobilization and to confirm the validity of the Ca2+ responses obtained using single-sperm measurements, we examined at the population level the [Ca2+]i changes induced by the same agonists using a fluorescence microplate reader corresponding to a 96-well plate. We detected comparatively weak and slow increases in fluorescence in PLC
4+/+ sperm after addition of ZP (Fig. 7 A), reflecting, perhaps, the fact that the response to ZP may start over a course of several minutes within a population of sperm, as shown by others. In contrast, progesterone triggered a very rapid and large [Ca2+]i increase and so did ionomycin and thapsigargin (Fig. 7 B). This pattern of [Ca2+]i increase was also observed in PLC
4-/- sperm; however, the maximum intensity of [Ca2+]i uptake was about half of that observed in PLC
4+/+ sperm, which agrees with the results reported in Figs. 4 and 5. Because thapsigargin appears to promote SOC channel activity but not generation of IP3 (Sabala et al., 1993), and ionomycin induces Ca2+ influx through the formation of synthetic Ca2+ channels, these results raise the possibility that PLC
4 may play in sperm a novel functional role in the regulation of Ca2+ influx.
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Discussion |
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Using high resolution single-sperm Ca2+ imaging, we demonstrate that the agonists used in this study to induce the acrosome reaction trigger distinct Ca2+ responses, and that these responses are altered in intensity and duration in PLC4-/- sperm. First, we confirmed that in wild-type sperm, the addition of solubilized ZP induces the acrosome reaction by triggering Ca2+ release. We extended those observations by showing that the ZP-induced [Ca2+]i increase started in the acrosome region and spread to the whole sperm head (Fig. 6 A). As it has been reported that both the IP3 receptors (IP3Rs) and PLC
4 localize to the acrosome (Walensky and Snyder, 1995; Fukami et al., 2001), our results suggest that the ZP induces [Ca2+]i mobilization initially from the acrosome and that PLC
4 is responsible for the presumed generation of IP3. Evidence in support of this notion is provided by the findings that ZP almost failed to induce a [Ca2+]i increase in PLC
4-/- sperm. Therefore, we conclude that the abnormal acrosome reaction induced by ZP in PLC
4-/- sperm is most likely due to impaired intracellular [Ca2+]i mobilization in these sperm, and that this protein plays a crucial role in the acrosome reaction during natural fertilization.
Although the progesterone-induced [Ca2+]i increases in sperm exhibited different spatial and temporal patterns than those evoked by ZP, the intensity of these responses was still altered in PLC4-/- sperm. Our data show that most of the progesterone-induced Ca2+ response initiated in the post-acrosomal region, and that it was primarily due to Ca2+ influx, as it was not observed in Ca2+-free medium. Remarkably, in PLC
4-/- sperm, the amplitude and duration of the progesterone-induced Ca2+ response were greatly decreased, paralleled by a reduction in the number of sperm that underwent the acrosome reaction (Fig. 2 B), supporting the notion that a sustained [Ca2+]i increase is required for the completion of the acrosome reaction and that PLC
4 may regulate this aspect of the progesterone-induced Ca2+ signal.
The thapsigargin-induced Ca2+ responses were similar to those evoked by progesterone in that they depended on Ca2+ influx to elevate [Ca2+]i in sperm. In addition, direct assessment of SOC function using thapsigargin showed that capacitative Ca2+ entry through SOC channels is severely impaired in PLC4-/- sperm (Fig. 8 A), further supporting a role of PLC
4 in the regulation of Ca2+ influx through these channels. Moreover, in this manuscript, we determined for the first time that the site of Ca2+ influx after the addition of thapsigargin (and progesterone) appears to be the post-acrosomal region, near the connective piece. The question then arises as to why this site might mediate Ca2+ influx. It is possible that SOC channels may be localized to this region. Toward this end, it is worth noting that recent reports localize IP3Rs to an additional region on the base of the head and neck region in human and bovine sperm (Ho and Suarez, 2001; Naaby-Hansen et al., 2001). Whether there is a direct link between IP3R and SOC channels in sperm and whether the site of Ca2+ influx is conserved across species remain to be investigated.
It also remains unclear how PLC4 might regulate SOC channels. It has recently been shown that some Trp channels operate as SOC channels (Montell et al., 2002), and TrpC3 has been reported to associate directly with the IP3R (Boulay et al., 1999; Kiselyov et al., 1999). In addition, recent evidence has shown that TrpC2 is implicated in the acrosome reaction (Jungnickel et al., 2001) and the phosphoinositide pathway may regulate the activity of Trp channels (Montell et al., 2002; Runnels et al., 2002). Therefore it is possible that defective stimulation of the phosphoinositide pathway may be responsible for the abnormal Ca2+ responses detected in the mutant PLC
4 sperm. Lastly, we cannot discount the possibility that in PLC
4 mutant sperm, the number and/or function of SOC channels may be altered in a manner unrelated to the possibilities considered here. Nonetheless, our finding that PLC
4 is one of the important enzymes in the regulation of [Ca2+]i mobilization in sperm significantly contributes to the elucidation of the molecular pathways of mammalian fertilization. In addition, its possible role in the regulation of Ca2+ influx suggests novel areas of research. Regulation of Ca2+ influx is important for many vital cellular functions, such as cell growth, apoptosis, exocytosis, muscle contraction, and gene transcription (Berridge et al., 1999, 2000), and its abnormal function may lead to the development of disease (Peng and Hediger, 2002; Torbergsen, 2002). Further understanding of the relationship between phosphoinositide metabolism and Ca2+ channel regulation may contribute to elucidating the molecular basis of these diseases.
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Materials and methods |
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Measurements of [Ca2+]i mobilization in single sperm
Capacitated cauda epididymal sperm were loaded with 4 µM fluo4-AM (Molecular Probes) for 15 min at 37°C and immobilized on a laminin-coated glass-bottom dish just before calcium measurement. We monitored simultaneously in single sperm the fluorescence emission of two dyes, fluo-4 and Alexa Fluor®594labeled SBTI. Fluorescence Ca2+ images of fluo-4 and Alexa Fluor®594labeled SBTI images were collected alternately for 200 msec every 2 s with altering excitation filters (470490 nm for fluo-4 and 535550 nm for SBTI), a dual-band dichroic mirror (51016bs; Chroma Technology Corp.), and a dual-band emission filter (51016em; Chroma Technology Corp.) on an inverted microscope (IX-70; Olympus) through a 60x objective (n.a. 1.4; Olympus) with a digital CCD camera (ORCA-ERG; Hamamatsu Photonics). On-line control of the system, acquisition, and off-line analysis of the collected data were done with TI Workbench software (written by T. Inoue) running on a Macintosh computer. Levels of [Ca2+]i are calculated as F/F0 ratios after background subtraction in both images and time course plots. Five image frames just before drug application were averaged and used as an F0 image. The experiments were performed at 32°C with a heating chamber covering the stage and objective lens of the microscope. For measurements of SOC activity, immobilized sperm were washed three times in Ca2+-free HS buffer (containing 1 mM EGTA) and treated with thapsigargin, followed by the addition of 1 mM extracellular Ca2+.
Measurements of [Ca2+]i mobilization at the population level
Capacitated cauda epididymal sperm were loaded with 4 µM fluo4-AM for 15 min at 37°C. Approximately 1 x 106 sperm were placed on a 96-well plate and treated with ZP, progesterone, thapsigargin, or ionomycin at 32°C. The fluorescence output changes were monitored at 5-s intervals using a Fusion- fluorescence microplate analyzer (Packard BioScience) with a 495-nm excitation filter and a 515-nm emission filter. Increasing levels of [Ca2+]i were expressed as F/F0 ratios.
Expression of PLC4 isoforms, Western blot analysis, and RT-PCR analysis
COS-7 cells were maintained in DME supplemented with 10% FBS. PLC4, PLC
4/ALT-I, and PLC
4/ALT-II were subcloned into the expression vector pcDNA3 and transiently expressed in COS-7 cells by electroporation. At 48 h after transfection, cells were lysed with SDS sample buffer, and the proteins were separated by SDS-polyacrylamide electrophoresis, followed by transfer onto nitrocellulose membranes (Schleicher & Schuell). Western blot analysis was performed using a specific polyclonal antibody against PLC
4.
Sperm capacitated for the indicated period were collected by centrifugation, solubilized with SDS sample buffer, and subjected to electrophoresis. Tyrosine phosphorylation was assessed by immunoblot analysis using a 4G10 antibody (Upstate Biotechnology) that recognizes tyrosine-phosphorylated proteins.
Total RNA from testis and sperm was prepared using the QIAGEN RNeasy kit. 10 µg RNA was used for reverse transcription. PCR was performed for 40 cycles of 30 s at 95°C, 30 s at 54°C, and 60 s at 72°C with 5 µCi [32P]dCTP and PLC4-specific primers (forward primer, TGGCACACCATCTGATTGCG; reverse primer, TACACGGCATAGCTGTCTGG) to produce 491-bp (PLC
4), 587-bp (PLC
4/ALT-I), 533-bp (PLC
4/ALT-II), and 398-bp (PLC
4/ALT-III) fragments. PCR products were separated on a 5.0% acrylamide gel, followed by autoradiography.
Cholesterol assay
Cauda epididymal sperm were capacitated for the indicated times in HS medium at 37°C under CO2. Sperm were collected by centrifugation, and lipids were extracted by chloroform/methanol/HCl as previously described (Schacht, 1978). An aliquot of the extracted lipids was used for measuring cholesterol content using an Amplex Red Cholesterol Assay kit (Molecular Probes). The cholesterol content was normalized by protein content and expressed relative to the concentration before capacitation.
Online supplemental material
The supplemental material (Figs. S1 and S2 and Videos 13, available at http://www.jcb.org/cgi/content/full/jcb.200210057/DC1) shows [Ca2+]i mobilization in wild sperm treated with acrosome reaction inducers. Capacitated wild sperm were loaded with 4 µM fluo4-AM for 15 min, and Ca2+ images were monitored every 2 s after treatment with 3 Zp/µl solubilized mouse ZP (ZP/WT), 100 µM progesterone (PG/WT), or 5 µM thapsigargin (TG/WT).
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Acknowledgments |
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This work was supported by a special coordination fund for promoting science and technology from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by a grant from the United States Department of Agriculture to R.A. Fissore.
Submitted: 10 October 2002
Revised: 27 February 2003
Accepted: 3 March 2003
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