1 School of Biological Science, University of Birmingham, Birmingham, B15 2TT, and 2 Reproductive Biology and Genetics Research Group, Birmingham Women's Hospital,Birmingham B15 2TG, UK
Abstract
Evidence from pharmacological studies suggests that induction of the acrosome reaction of mammalian spermatozoa by solubilized zona pellucida, and possibly by progesterone, is dependent upon Ca2+ influx through voltage-operated Ca2+ channels. Studies on Ca2+ accumulation and membrane potential in ligand-stimulated or artificially depolarized spermatozoa support such a conclusion. Electrophysiological studies on rodent spermatogenic cells have revealed the presence of a `T' type voltage-operated Ca2+ current. This current has pharmacological attributes consistent with those of the putative channel responsible for Ca2+ influx mediating the acrosome reaction. However, use of molecular techniques to study human and rodent testis and spermatogenic cells has detected the presence of three different voltage-operated Ca2+ channel subunits. One of these (lE) may generate T-currents, though this is currently disputed. Voltage-operated Ca2+ channel structure and the relationship between channel subunit expression and the characteristics of consequent Ca2+ currents is briefly reviewed. The nature and function of T-channel-mediated Ca2+ influx is examined in the context of the time-course of ligand- and depolarization-induced elevation of [Ca2+]i in mammalian spermatozoa. It is likely that a secondary Ca2+ response (mobilization of stored Ca2+ or activation of a second Ca2+-influx pathway) is required for the acrosome reaction. Evidence for the existence and participation of various candidates is discussed (including voltage-operated Ca2+ channels, which may be functionally expressed only in mature spermatozoa), the available evidence favouring a secondary Ca2+-influx pathway. Immediate priorities for future research in this area are proposed.
Key words: acrosome reaction/calcium/ion channels/membrane potential/spermatozoa
The acrosome reaction and Ca2+ channels
The acrosome reaction (AR) of human spermatozoa, as in virtually all other forms of stimulus-activated exocytosis, is mediated by an elevation of [Ca2+]i. In various mammalian species, including man, this elevation of [Ca2+]i and the consequent AR (induced by solubilized zona or progesterone) is greatly attenuated by reduction of [Ca2+]o or by a non-specific blockade of Ca2+ channels by metal ions (Blackmore et al., 1990; Florman et al., 1992
; Florman, 1994
; Plant et al., 1995
; Aitken et al., 1996
). Furthermore, when mammalian spermatozoa are exposed to solubilized zona, both the [Ca2+]i response and the AR are blocked by 1,4-dihydropyridines (DHPs), a class of drugs that are specific to voltage-operated Ca2+ channels (VOCCs; Florman et al., 1992
; Florman, 1994
). DHPs can also block responses to progesterone and follicular fluid in both human and rodent spermatozoa (Blackmore et al., 1990
; McLaughlin and Ford, 1994
; Shi and Roldan, 1995
; O'Toole et al., 1996
), though the findings in this case are less consistent (see below). It has been reported that these drugs can cause reversible infertility of the human male in vivo (Hershlag et al., 1995
). The simplest interpretation of these data is that influx of Ca2+ through VOCCs, probably related to those of somatic cells, is necessary for elevation of [Ca2+]i and, therefore, for successful AR (Benoff, 1998
; Florman et al., 1998
).
Use of fluorescent dyes to monitor the membrane potential (Em) of spermatozoa has confirmed that the mean Em of bovine and rat spermatozoa depolarizes, from around 60mV to between 25 and 20 mV, upon exposure to solubilized zona (Arnoult et al., 1996b). This depolarization is sufficient to activate VOCCs. It also appears that hyperpolarization of the spermatozoon Em may be a necessary component of capacitation, releasing VOCCs from inactivation (Zeng et al., 1995
).
Thorough characterization of human sperm VOCCs is essential to a full understanding of the processes required for human fertilization and may also allow development of novel contraceptives. During the last few years, considerable progress has been made in elucidating the nature of the sperm VOCC(s). In particular, it has been shown that there is a T type channel (see below) present in the spermatogenic cells and probably in the spermatozoa of rodents (Florman et al., 1998). However, the findings from different techniques, from different laboratories and from studies on different species are difficult to harmonize. It is arguable that our understanding of this field, particularly in humans, is far from complete. The purpose of this manuscript is to summarize and briefly review the available evidence and to suggest potentially fruitful areas for future work.
Voltage operated Ca2+ channels
Using both electrophysiological and molecular techniques, VOCCs have been described and characterized in a wide variety of somatic cells. These currents can be classified into at least six types (T, L, N, P, Q, R), based upon their biophysical characteristics and sensitivity to a range of drugs and toxins (Birnbaumer et al., 1994; Dunlap et al., 1995
). All except one (T-type) channel require large depolarizations (to voltages
30 mV) to cause opening and are, therefore, referred to as high voltage activated (HVA) channels. The T-type typically activates at voltages
60 mV (Figure 1b
) and is referred to as low voltage activated (LVA). Molecular studies have shown that all these VOCC types are members of the same protein family and are structurally similar, the main component being an
1 (pore-forming) protein subunit. This protein contains four homologous domains (repeats IIV), each composed of six transmembrane alpha helical segments (S1S6) interspersed by linkers (Figure 1a
). Eight of these helices (S5, S6 and the 56 linker from each repeat) are believed to surround the channel pore (McClesky, 1994
). Helices IIIS5, IIIS6 and IVS6 form the primary DHP binding region of L-type, HVA channels (Grabner et al., 1996
; Sinnegger et al., 1997
; Striessnig et al., 1998
). Expression studies suggest that the
1 subunit may be able to form a functional channel on its own, but is usually associated with (and modulated by) ß and
2-
auxiliary subunits. Ten different homologous
1 gene products (
1A/B/C/D/E/F/G/H/I/S) and a range of splice variants have been described. This molecular diversity of the
1 subunit is believed to be the primary cause of the observed biophysical and pharmacological variation among voltage operated Ca2+ currents. However, an important role for the auxiliary subunits is also recognized, both in significantly modulating functional characteristics of the channels and also in regulating expression of
1 subunits in the plasmalemma (Birnbaumer et al., 1998
; Trimmer, 1998
).
|
Monitoring of [Ca2+]i and use of drugs
Various methods have been employed for detection and characterization of sperm VOCCs. Use of fluorescence techniques for monitoring [Ca2+]i in mammalian spermatozoa has shown that induction of AR by solubilized zona or by alkaline, depolarizing media is associated with elevation of [Ca2+]i and that activation of VOCCs is a necessary step in this response (Florman et al., 1992; Linares-Hernandez et al., 1998
). Imaging of individual, zona-stimulated cells shows that an initial focal influx through a non-specific cation channel precedes (and probably induces) a larger global elevation of [Ca2+]i (Florman, 1994
; Arnoult et al., 1996a
,b
). Blockade of VOCCs causes failure of the global (but not the focal) [Ca2+]i response to zona and consequent failure of the AR (Florman, 1994
; Arnoult et al., 1996a
,b
).
Similarly, activation of AR by progesterone may be VOCC dependent and it has been suggested that Cl efflux via a receptor/chloride channel, similar to the neuronal GABAA receptor, may provide the necessary depolarization (Turner and Meizel, 1995; Meizel et al., 1997
). However, the effects of DHPs and verapamil (a phenylalkylamine VOCC blocker) on responses to progesterone are not consistent. Different laboratories report that the [Ca2+]i transient and/or AR which occurs upon application of progesterone or follicular fluid to mouse and human spermatozoa is sensitive (Blackmore et al., 1990
; McLaughlin and Ford, 1994
; Shi and Roldan, 1995
; O'Toole et al., 1996
) or insensitive (Thomas and Meizel, 1989
; Foresta et al., 1993
; Aitken et al., 1996
) to blockade of VOCCs.
Linares-Hernandez et al. (1998) have recently undertaken a detailed study of the depolarization-induced elevation of [Ca2+]i in non-capacitated human spermatozoa. Valinomycin was used to increase membrane K+ permeability, thus `clamping' Em at the K+ equilibrium potential (EK), which was then controlled by adjusting medium K+ concentration. Differences in the responses of human spermatozoa, in comparison with rodent and bovine cells, included the ability of depolarization to induce Ca2+-influx without alkalinization (though alkalinization enhanced the effect) and an insensitivity to the DHP, nifedipine. The T channel blocker Ni2+ inhibited the depolarization-induced elevation of [Ca2+]i, but required very high (non-selective) concentrations to achieve a significant effect (see below).
Electrophysiological investigations
Ca2+-sensitive fluorescent dyes monitor the concentration of Ca2+ in the cytosol, not transmembrane Ca2+ fluxes, and thus provide only indirect information on the nature of Ca2+ channel(s). However, direct electrophysiological recording from spermatozoa is currently impracticable due to the combination of shape and minute size. Some brief, cell-attached patch (single channel) records have been reported (e.g. Espinosa et al., 1998), which have established that sperm membranes possess a range of anion and cation channels, similar to somatic cells, but these studies have provided little information on VOCCs of spermatozoa.
Reconstitution of purified sperm membrane proteins into artificial membranes has been used extensively and has occasionally revealed the presence of Ca2+ channels. A DHP-modulated channel has been described from boar spermatozoa, which shows very little voltage sensitivity of activation or inactivation (Tiwari-Woodruff and Cox, 1995). Purified membranes from mouse spermatozoa include a Ca2+ conducting channel of high, multistate conductance and low Ca2+ selectivity (Beltran et al., 1994
). These channels may play a role in the control of the AR but neither has the characteristics of the currents seen in spermatogenic cells (see below).
Electrophysiological studies of male germ cell VOCCs, employing the more informative `whole cell' technique, have been restricted to `spermatogenic cells'. Mature spermatozoa are not transcriptionally active so it is possible that spermatogenic cells express the same channels as mature spermatozoa (Arnoult et al., 1996a; Lievano et al., 1996
), though insertion into the sperm membrane of previously transcribed proteins may occur. Records obtained from spermatogenic cells of rat and mouse show a short-lived (fast inactivating), LVA current similar to the T-type VOCC of somatic cells (Hagiwara et al., 1984; Arnoult et al., 1996a
; Santi et al., 1996
). The channel shows a sensitivity to DHPs which is sufficient to account for the effect of these compounds on [Ca2+]i responses and on AR. Furthermore, it has a pharmacology (relative potency of various blockers in inhibiting currents) similar to that of the putative AR-inducing channel (Arnoult et al., 1996a
; Santi et al., 1996
; Florman et al., 1998
). Arnoult et al. (1997) showed that the current facilitates in response to depolarising prepulses, the facilitated current being enlarged but having similar kinetics to control currents. This effect is tyrosine phosphatase dependent (Arnoult et al., 1997
), so activation of tyrosine kinase associated with capacitation and/or activation of the spermatozoa (Ward and Kopf, 1993
; Visconti and Kopf, 1998
) may suppress facilitation. No other types of VOCC current have been seen in these cells.
Molecular investigations
Use of reverse transcriptionpolymerase chain reaction (RTPCR) to study expression of VOCC 1 subunits in male germ cells has produced evidence for the presence of three different VOCC
1 subunits. Studies by Darszon and colleagues on rat spermatogenic cells have detected primarily RNA coding for the
1E subunit (Lievano et al., 1996
).
1A was also detected, though at a lower level (Lievano et al., 1996
).
Benoff and colleagues report detection of message for 1C in rat and human testis (Goodwin et al., 1997
; Benoff, 1998
; Goodwin et al., 1998a
,b
,c
). In-situ RTPCR for
1C was carried out on frozen sections from rat testis and PCR product was present at all stages of germ cell differentiation (Goodwin et al., 1998b
). The laboratory has recently reported that they have successfully carried out RTPCR on RNA from motile, mature human spermatozoa and were able to detect mRNA for
1C (Goodwin et al., 1998a
). The testicular
1C is truncated at the 5' end and sequencing has revealed splice variants in regions IS6, IIIS2 and IVS3 of the molecule (Goodwin et al., 1997
; Benoff, 1998
; Goodwin et al., 1998a
,b
,c
; Figure 1a
), which may be functionally significant (Benoff, 1998
). Region IS6 is known to contribute significantly to determination of the kinetics of inactivation in channels expressed in Xenopus oocytes (Zhang et al., 1994
), though other features of the channel (particularly the type of ß subunit co-expressed) are very important in this respect (Birnbaumer et al., 1998
). Alternative splicing in segment IIIS2 of
1C may affect the voltage-dependence of channel block by DHPs. Human
1C has two splice variants in this region (Soldatov et al., 1995
). The variant which is expressed in the testis has been shown to have higher sensitivity to DHPs when cells are held at 90 mV, though at 40 mV the two splice variants showed similar sensitivity (Soldatov et al., 1995
). The main DHP-biding regions (IIIS5, IIIS6 and IVS6; Grabner et al., 1996
; Sinnegger et al., 1997
; Striessnig et al., 1998
) and the voltage sensors (S4 in each repeat; McClesky, 1994
) do not seem to be subject to alternative splicing in testicular
1C (Figure 1a
).
1G, a subunit believed to generate T-currents in neurons, is not expressed at detectable levels in rat testis (Perez-Reyes et al., 1998a
). Another possible T channel,
1H, has also been described and is expressed primarily in heart (Cribbs et al., 1998
; Perez-Reyes et al., 1998b
), but information on its presence/absence in male germ cells is not yet available.
Which 1 subunit generates the T-current of spermatogenic cells?
The data from electrophysiological and molecular studies on the VOCCs of male germ cells are not easily harmonized. It is generally accepted that 1A codes for the P/Q channel normally found in brain (Birnbaumer et al., 1994
), an HVA current. It is most unlikely that this subunit is responsible for the LVA current of germ cells. The situation with regard to
1E is more complex. This subunit was initially considered to be an LVA channel (Soong et al., 1993
), but subsequent investigations cast doubt upon this classification. The consensus is now that it probably codes for the R type HVA channel, but the identity of
1E is still open to question since different conclusions have been reached depending on the nature of the characteristics assessed (Williams et al., 1994
; Bourinet et al., 1996
; Randall and Tsien, 1997
). A feature of T currents which has been proposed as diagnostic is that current families (currents induced by a series of incremented voltage steps), when superimposed, cross over (Randall and Tsien; 1997; Figure 1b
). Currents carried by expressed recombinant
1E do not show crossing over (Randall and Tsien, 1997
) but currents seen in rodent spermatogenic cells do (Arnoult et al., 1996a
; Santi et al., 1996
).
Expression of 1C subunit normally produces HVA currents, of the L-type, which inactivate relatively slowly (Figure 1c
). Such currents were not detected in spermatogenic cells of rodents (see above). It has been argued that alternative splicing in the testis specific form of
1C could cause changes in both voltage dependence and kinetics of the channel such that, though the currents in spermatogenic cells are generated by testicular
1C, they have been wrongly identified as T type (Benoff, 1998
). There is no precedent for such a fundamental transformation of all biophysical characteristics in a splice variant. However, it is impossible to assess the impact of the alternative splicing in the testis-specific channel until it is expressed, with an appropriate ß subunit, in oocytes or a cell line, allowing the application of electrophysiological techniques.
A further complication in interpretation of the molecular data has been introduced by the recent findings of Meir and Dolphin (1998) that several 1 subunits (
1B,
1C and
1E) can generate T-like single channel currents upon expression in a VOCC-null (COS-7) cell line. The HVA channels observed in previous studies on expression of these subunits were also present, though when
1B was expressed without any auxiliary subunits only the T-like channels were observed (Meir and Dolphin, 1998
). There are, as yet, no reports of significant whole cell currents being generated in this way.
Thus, it is conceivable that the testicular 1E and/or
1C are responsible for the spermatocyte T type current. However, the weight of available evidence does not support such a conclusion and it appears probable that at least the testicular
1C (and possibly the
1E) is separate from the subunit responsible for the observed T currents. If so then (i) male germ cells also express
1 subunits for LVA channels, such as
1H or
1I (Cribbs et al., 1998
; Perez-Reyes et al., 1998b
) and (ii) currents carried by the subunits
1A, C and E are not detected by electrophysiological study of spermatogenic cells, possibly being available for activation only in mature spermatozoa (see below)
Is the T channel the primary Ca2+ influx pathway for AR?
Electrophysiological data (from rodent spermatogenic cells) and data from experiments on the effects of VOCC blockers on AR strongly suggest that spermatozoa posses a DHP-sensitive T channel that must be activated for successful induction of AR. Ca2+ flux through T channels can perform a second messenger function, such as in induction of steroidogenesis in adrenal cortex (Enyeart et al., 1993; Rossier et al., 1996
), but this does not appear to be typical. Rather than providing a major pathway for Ca2+ influx, T currents frequently act to modulate Em, supporting pacemaking, rhythmic or oscillatory activity (Carbonne and Swandulla, 1990; Huguenard, 1996
) and may provide a depolarizing current sufficient to recruit Ca2+ entry through HVA channels. Analysis of the currents in spermatogenic cells has shown some overlap between the voltage dependencies of activation and inactivation (i.e. some activation can occur at voltages at which steady-state inactivation is not complete; Santi et al., 1996
) and it is, therefore, theoretically possible for these channels to support sustained Ca2+ influx at suitable membrane potentials (65 to 45 mV). However, even at the centre of this range (55 mV) the current is likely to be only ~2.5% of the possible maximum. Furthermore, upon exposure to solubilized zona, Em is not held in this range but depolarizes to more positive values within a minute (Arnoult et al., 1996b
). T channel currents inactivate rapidly at voltages positive to 60 mV (Carbonne and Swandulla, 1990) and the currents carried by these channels, therefore, normally last <100 ms when induced by step depolarizations (Figure 1
). The ramp depolarization of Em that occurs in response to solubilized zona (Arnoult et al., 1996b
) may permit T channels to remain open for longer, but the consequent current would still be (probably) small and of no more than a few seconds in duration. In contrast, the AR-linked [Ca2+]i response in bovine spermatozoa persists for several minutes before AR (Florman, 1994
; Arnoult et al., 1996b
).
To support such a substantial and sustained [Ca2+]i response either repetitive activation of T currents (repetitive Ca2+ spiking) or a secondary event (mobilization of intracellular stores or activation of a second Ca2+ influx pathway), subsequent to T-current activation, must occur.
Repetitive Ca2+ spiking?
T currents of neurons can support Ca2+ action potentials, (known as low threshold spikes; LTSs) which are of lower amplitude but longer duration than normal action potentials (amplitude of 1525 mV and duration of 20150 ms). LTSs are normally induced by `rebound' following transient hyperpolarization of Em and consequent release of T channels from inactivation (Huguenard, 1996). When expressed in combination with other conductances which can cause a post-LTS hyperpolarization the cycle can repeat causing oscillation of Em and consequent repeated activation of T currents (Huguenard, 1996
). Though such activity could conceivably generate a prolonged (possibly oscillating) Ca2+ signal in spermatozoa induced to undergo AR, there is currently no evidence to this effect.
Is there a Ca2+ store in spermatozoa?
There is evidence that at least one Ca2+ store exists within mature rodent and bovine spermatozoa, probably in the acrosome (Walensky and Snyder, 1995; Spungin and Breitbart, 1996
). Whether this store is mobilized by inositol trisphosphate (IP3) is disputed (Walensky and Snyder, 1995
; Spungin and Breitbart, 1996
). Thapsigargin, which selectively releases Ca2+ from intracellular stores of somatic cells (Thastrup et al., 1990
), elevates [Ca2+]i of capacitated or uncapacitated human and non-human spermatozoa and initiates AR. However, these effects are observed only in the presence of extracellular Ca2+, responses being suppressed in `Ca2+-free media' (Blackmore, 1993
; Meizel and Turner, 1993
; Spungin and Breitbart, 1996
). Similar results are reported with 2,5-di(tert-butyl)hydroquinone (Perry et al., 1997
). The significance of a Ca2+ store in the events which follow zona binding and result in AR should, therefore, remain open to question.
Is there a second Ca2+ influx pathway?
Since three different 1 subunits have already been detected in spermatogenic cells (see above), it is likely that at least one HVA channel is present in these cells and could provide a route for a secondary, prolonged Ca2+ influx, or allow a major influx leading to mobilization of other sources of Ca2+. The novel splice variants of
1C detected by Benoff and colleagues (Goodwin et al., 1997
; Benoff, 1998
; Goodwin et al., 1998a
,b
,c
) are possible candidates, though it is important to know the activation and inactivation characteristics of expressed channels before their potential contribution to sustained Ca2+ influx can be assessed. The
1A detected at low levels by Lievano et al. (1996) may also be significant, particularly since the P/Q channels which are believed to reflect expression of this subunit do not show complete inactivation even at 0 mV and could, therefore, support sustained influx of Ca2+ over a wide voltage range (Regan, 1991
).
Since electrophysiological studies of spermatogenic cells have detected no HVA currents, it is likely that these channels, if present, are non-functional at this stage (see above). However, such channels could subsequently become inserted into the plasma membrane, or become functional through the activity of second messengers or addition of necessary auxiliary subunits. Plasma membrane vesicles from bovine spermatozoa possess a Ca2+ channel that is sensitive to DHPs and becomes functional only after stimulation of protein kinase C (Spungin and Breitbart, 1996). Dolphin and colleagues have recently demonstrated that auxiliary subunits may be able to reveal hidden currents. Undifferentiated NG10815 neuroblastoma/glioma hybrid cells express
1A, B, C, D and E proteins (
1 G and H were not tested) and low levels of auxiliary (
2-
, and ß) subunits, but electrophysiological recording detects almost exclusively LVA currents. However, after over-expression of auxiliary subunits by transfection of cDNAs an additional sustained, HVA component to the currents is seen (Wyatt et al., 1998
).
The data of Linares-Hernandez et al. (1998) on K+ (depolarization)-induced [Ca2+]i responses in non-capacitated human spermatozoa are consistent with the existence of both a T channel-like and a slowly-inactivating, voltage activated Ca2+ influx pathway in these cells. An early component of the response to depolarization was blocked by 50 µM Ni2+ but very high doses (600 µM) were required to achieve a lasting inhibitory effect. Doses for 50% block of T-channel currents, including those of mouse spermatogenic cells, are typically around 1050 µM, HVA currents being significantly blocked at 200300 µM Ni2+ (Fox et al., 1987; Narahashi et al., 1987
; Gu and Spitzer, 1993
; Arnoult et al., 1998
). The results of experiments on the persistence of the voltage-activated Ca2+ influx pathway are particularly striking. When cells were depolarized to 17 mV (mean for population) in a low Ca2+ medium (no added Ca2+, 0.5 mM EGTA) for up to 90 s, no elevation of [Ca2+]i occurred. However, subsequent addition of extracellular Ca2+ caused an immediate rise in [Ca2+]i, indicating that an influx pathway was available (Linares-Hernandez et al., 1998
). T channels should inactivate completely within 1 s at this value of Em and a separate voltage activated influx pathway must, therefore, exist in human spermatozoa. The [Ca2+]i response induced by depolarization of these cells was not inhibited by the DHP nifedipine.
Another possible (non-VOCC) Ca2+ influx route is capacitative Ca2+ entry. This influx pathway is not voltage activated, but is induced upon emptying of intracellular stores, possibly due to release from the depleted store of a `Ca2+ influx factor', which stimulates a Ca2+ release activated current (ICRAC; Fasolato et al., 1994). ICRAC is an important route for prolonged Ca2+ influx in somatic cells. Activation of such a pathway as a secondary response after initial VOCC activation is a possibility. It is also consistent with the ability of thapsigargin to elevate [Ca2+]i only in the presence of extracellular Ca2+ (see above), assuming that the Ca2+ store in spermatozoa is too small to cause significant elevation of [Ca2+]i. There is currently no other evidence regarding capacitative Ca2+ entry in spermatozoa.
Could the T channel have a function in spermatogenesis?
Since it has only been possible to obtain direct records of T currents from differentiating (spermatogenic) cells, it is pertinent to ask whether these currents could have a role in spermatogenesis (Santi et al., 1996). There is evidence that T currents are involved in generating periodic Ca2+ transients in immature neuronal cells, which are necessary for differentiation (Gu and Spitzer, 1993
; Gu et al., 1994
). In both excitable and non-excitable cells, T currents tend to be most strongly expressed early during the process of differentiation (HVA currents subsequently becoming prevalent) and their expression is enhanced upon artificial induction of differentiation (Gottmann et al., 1988
; Bickmeyer et al., 1993
; Gu and Spitzer, 1993
; Publicover et al., 1994
; Preston et al., 1996
). Both observations are consistent with a role for T currents in this process. The T-current of rodent spermatogenic cells shows voltage dependent facilitation, a process that is tyrosine phosphatase dependent (Arnoult et al., 1997
; see above). A similar voltage-induced enhancement of T currents has been observed in rat bone marrow stromal cells (Publicover et al., 1995
). This facilitation is greatly reduced in mature osteoblasts (M.R. Preston et al., unpublished data) again consistent with a function in differentiation. It is, therefore, conceivable that the T currents which have been observed in spermatogenic cells play a role in the differentiation of male germ cells, though this does not preclude a further role in signalling in the mature spermatozoon.
Conclusions
It is clear that, despite considerable progress, a number of questions regarding the VOCCs of human spermatozoa and their role in the AR are still to be answered. Though evidence for the necessary participation of a T current in activation of the AR is strong, it appears likely that at least one further Ca2+ influx mechanism is involved. Three different VOCC 1 subunits, which probably form non-T-type channels, have been detected in male germ cells and these must be candidates for secondary influx pathways. The characteristics of these VOCCs should be established. Intracellular Ca2+ stores and ICRAC are possible contributors to Ca2+ signalling during AR and merit further investigation. It is also crucial to establish whether the observations made on animal (particularly rodent) spermatozoa and the models based upon them hold good for the human. Studies that could be undertaken in the near future include: (i) recording of Ca2+ currents from human spermatogenic cells; (ii) cloning and expression of animal and human testis/sperm VOCCs. Expression of the human channel(s) in a system in which they can be analysed will allow both characterization and assessment of the importance of co-expression of auxiliary subunits; and (iii) use of preparations of permeabilized spermatozoa to determine the nature, capacity and regulation of Ca2+ stores.
Though not yet possible, the application of the whole cell patch technique to spermatozoa (allowing thorough assessment of ionic currents and their regulation in individual cells stimulated to undergo AR) may ultimately be required in order to answer many of the questions which have been raised here.
Notes
3 To whom correspondence should be addressed
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Submitted on October 6, 1998; accepted on December 21, 1998.