Molecular Human Reproduction, Vol. 5, No. 1, 38-45,
January 1999
© 1999 European Society of Human Reproduction and Embryology
Identification and localization of G protein subunits in human spermatozoa
1 Laboratoire de Fécondation In Vitro, Hôpital de Poissy, Poissy, France, 2 Metabolic Diseases Branch, NIDDKD, NIH, Bethesda, MD, 3 Laboratory of Cellular and Molecular Biophysics, NICHHD NIH, Bethesda, MD, 4 Center for Reproductive Research and Testing, Rockville, MD, USA and 5 Laboratoire de Biochimie et Biologie Moléculaire, Faculté de Médecine Paris-Ouest, Hôpital R.Poincaré, F92380, Garches, France
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Abstract |
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Antibodies to
and ß subunits of guanine nucleotide regulatory proteins (G proteins) were used to identify which G proteins are present in mature human spermatozoa and to determine their subcellular localization. Immunoblots of membranes from spermatozoa demonstrate the presence of Gi2, Gi3, Gq/11 and Gß35 and the absence of Gi1, Go, Gs, G12, G13, G16, Gz and Gß36. Indirect immunofluorescence demonstrates the presence of Gq/11 in the acrosome, with the highest proportion in the equatorial segment. Gi2 is present in the acrosome, midpiece and tailpiece and Gi3 in the postnuclear cap, midpiece and tailpiece. The Gß35 subunit is found mostly in the midpiece, with marginal labelling of the head, tailpiece and the equatorial segment of the acrosome. The distinct pattern of distribution of G proteins suggests that they may couple to receptors or effectors which also have discrete regions of localization in spermatozoa. These highly localized signal transduction pathways may regulate discrete functions, such as activation of the acrosome reaction, fusion with the oocyte and motility. acrosome/equatorial segment/guanine nucleotide regulatory protein/immunohistochemistry/spermatozoa
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Introduction |
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Heterotrimeric guanine nucleotide binding proteins (G proteins) couple the activation of membrane receptors to the modulation of intracellular effectors such as adenylyl cyclase, phospholipases C and A2, and ion channels (Spiegel et al., 1992
; Gilman, 1995; Bourne, 1997). G protein heterotrimers consist of three subunits: , ß and 
. Upon interaction of a G protein with an activated receptor, GTP is exchanged for GDP at the guanine nucleotide binding site on the subunit and the subunit dissociates from the tightly and non-covalently bound ß complex. The GTP-bound subunit and in some instances the dissociated ß complex may either activate or inhibit specific effectors (Clapham and Neer, 1993). An intrinsic GTPase activity present in the subunit hydrolyses GTP to GDP, resulting in reassociation of with ß and G protein inactivation.
The multiplicity of , ß and subunits may potentially result in a myriad of heterotrimers with specific functional receptor and effector interactions. G proteins are classically defined by their specific subunit, although it is now appreciated that ß and subunits are important in determining coupling specificity and in effector modulation (Birnbaumer, 1992). The functional roles of some G proteins are well established (e.g. Gs stimulates adenylyl cyclase; Gq and G11 stimulate phospholipase Cß). The pertussis toxin (PTX)-sensitive G proteins (Gi1, Gi2, Gi3, Go1 and Go2) have been implicated in the inhibition of adenylyl cyclase and the regulation of ion channels and membrane trafficking. However, in many cases it is unknown which Gi or Go is involved in specific signal transduction pathways. The functional roles of other G proteins such as Gz, G12 and G13 are poorly defined. Many G proteins are ubiquitously expressed while others such as the transducins (Gt's), gustducin (Ggust), G15 and G16 have a very limited tissue distribution.
Spermatozoa from various species contain both PTX-sensitive (Bentley et al., 1986; Kopf et al., 1986; Endo et al., 1987; Lee et al., 1992b*) and -insensitive (Glassner et al., 1991; Hinsch et al., 1992) G proteins. Functional experiments indicate that PTX-sensitive G proteins are involved in the zona pellucida-induced acrosome reaction (Ward et al., 1992; Tesarik et al., 1993*; Breitbart and Spungin, 1997*; Franken et al., 1996*; Brandelli, 1997*). Changes in intracellular Ca2+ and pH which induce acrosomal exocytosis may also occur as a result of signals which are transmitted by G proteins (Florman et al., 1989; Breitbart and Spungin, 1997).
In some polarized cells and in sperm cells, specific G proteins are distributed at discrete portions of the plasma membrane (Glassner et al., 1991; Hinsch et al., 1992). In mouse spermatozoa, Gi proteins were localized to the acrosomal region and Gz to the postacrosomal region of the head in mouse spermatozoa (Glassner et al., 1991). A poorly characterized G protein was identified in bovine and human tail membranes (Hinsch et al., 1992, 1995). The goal of this study was to identify which specific G protein subunits are present in human spermatozoa and to determine their subcellular localization. We have confirmed the presence of Gi2, Gi3, Gq/11 and Gß35 in sperm membranes and by indirect immunofluorescence have demonstrated that each subunit has a unique pattern of subcellular localization in mature spermatozoa.
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Materials and methods |
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Experimental subjects
Specimens were obtained from healthy adult males after informed consent was obtained. The volume, pH, cell number, percentage mobile and percentage abnormal forms were determined. Only samples meeting the World Health Organization criteria were utilized.
Preparation of human sperm membranes
Percoll (Pharmacia, Sweden) was prepared by dilution with Eagle's medium (Eurobio, Les Ulis, France) (18:2 vol). In order to obtain a discontinuous gradient for separation of motile sperm cells from seminal fluid and debris, the stock solution was diluted further with B2 Ménézo medium (Biomérieux, Marey l'etoile, France) and 1.0 ml of 90% Percoll was underlayed with 1.5 ml of 45% Percoll in a 15 ml conical tube. After liquefaction, 2 ml semen was placed onto the top of the Percoll gradient and centrifuged at 400 g for 20 min at room temperature. After centrifugation, the semen and the 45% fraction were discarded. The 90% Percoll fraction remaining in the tube and containing the motile sperm cells was washed once with 2 ml of B2 Ménézo medium by centrifugation at 600 g for 10 min at room temperature. These progressively motile spermatozoa were condensed into a pellet and were then either smeared for immunofluorescence studies or stored at –70°C prior to membrane preparation. Sperm membranes were prepared using a slight modification of a previously described method (Kopf et al., 1986). Sperm cells from 10 donors were thawed and diluted 10% (v:v) in a buffer containing Tris–HCl 10 mM, EGTA 1 mM, dithiothreitol 1 mM, glycerol 20%, phenylmethylsulphonyl fluoride (PMSF) 0.01 mM, soybean trypsin inhibitor 1 mg/ml, leupeptin 0.1 mg/ml, aprotinin 0.01 mg/ml, para-aminobenzamidine 1 mM, pH 8.0 at 4°C. The spermatozoa were sonicated (4 blasts of 10 s with VibraCell 72434, setting 40%) and centrifuged at 600 g for 10 min at 4°C to remove unbroken cells and nuclei. The supernatant was centrifuged for 1h at 48000 g at 4°C. The pellet was resuspended in Tris–HCl 25 mM, pH 6.8 containing PMSF 0.01 mM, soybean trypsin inhibitor 1 mg/ml, leupeptin 0.1 mg/ml, aprotinin 0.01 mg/ml and para-aminobenzamidine 1 mM and diluted with 1 volume of 2x Laemmli buffer (Laemmli, 1970) containing 10% ß-mercaptoethanol.
Miscellaneous membrane preparations
Membranes of human platelet, rat liver, bovine brain cholate extract, NIH-3T3 cells, Sabra rat kidney membranes, RINm5F cells (a rat insulinoma cell line) and rat pancreatic islets were prepared as described elsewhere (Denis-Henriot et al., 1998). NIH3T3 cells transfected with G12 cDNA were obtained from S.Gutkind (NiDR, NIH, Bethesda, MD, USA) and Escherichia coli recombinant G16 was obtained from M. Forte (Oregon Health Science Univ., Portland, OR, USA).
Immunoblotting
Membrane proteins were resolved on a sodium dodecyl sulphate (SDS)–polyacrylamide gel (0.1% SDS, 11% acrylamide, 0.08% bisacrylamide) and then transferred to PVDF membranes (Millipore, 150 mA, 20 h) in Tris–HCl 25 mM, glycine 192 mM buffer containing 20% methanol. PVDF membranes were blocked for 2 h at room temperature in TTBS (Tris–HCl 20 mM, NaCl 500 mM, Tween-20 0.05%) containing 2.5% gelatin (Biorad, Hercules, CA, USA) and incubated for 20 h at room temperature with antibodies (2–10 µg/ml affinity purified antibodies or antisera at 1:200 dilution) in TTBS–gelatin 2.5%. The PVDF membranes were washed with TTBS and then incubated with 125I-labelled protein A (Amersham, 0.2 µCi/ml) in TTBS–gelatin 2.5% for 1–2 h. Antigen–antibody complexes were detected by autoradiography.
Indirect immunofluorescence
Freshly prepared washed spermatozoa were smeared on a microscope slide at a density of 1.5–3x104 cells/cm2, air-dried and permeabilized in acetone for 10 min at room temperature. After rehydration of sperm cells in phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) for 15 min at room temperature, antibodies to specific G protein subunits (10 µg/ml in PBS containing 3% BSA) were added to the fixed, permeabilized spermatozoa. The slides were then incubated for 2 h at 37°C in a humidified chamber, washed three times for 5 min each in PBS, and incubated for 1 h at 37°C with fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit IgG (Kierkegaard and Perry, Gaithersburg, MD, USA) diluted 1:40 in PBS containing 3% BSA. The slides were then washed three times for 5 min in PBS and mounted with Citifluor (City University, London, UK). Exposure times during photography were kept uniform to allow for optimal comparison of fluorescence between test samples.
Antibodies
QL (anti-Gq, G11), AS (anti-Gt, Gi1, Gi2), RM (anti-Gs), EC (anti-Gi3), QN (anti-Gz), GO (anti-Go1), GC2 (anti-Go1, Go2) and SW (anti-Gß) antibodies were prepared from peptide-immunized rabbits as previously described (Goldsmith et al., 1988a,b; Simonds et al., 1989; Shenker et al., 1991; Murakami et al., 1992). Decapeptides corresponding to the carboxy termini of G12, G13 and G16 (Amatruda et al., 1991; Strathmann and Simon, 1991) were synthesized and used to immunize rabbits (Goldsmith et al., 1987), generating antibodies QE, HD and AR, respectively (Table I). Some antisera were affinity purified on Affi-Gel 15 columns (Bio-Rad) containing the corresponding immobilized peptide. Control IgG was obtained from Pel Freez Biologicals (Rogers, AR, USA).
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Indirect immunofluorescence with counterstaining for cell viability and acrosomal status
After liquefaction, a modified swim-up was done by dividing each sample into 1 ml aliquots in 15 ml Corning conical flasks and overlaying them with 3 ml of modified Biggers–Whitten–Whittingham medium (mBWW; Irvine Scientific, Irvine, CA, USA) with 0.3% BSA. After 60 min of incubation at 37°C the upper 2.9 ml of medium was aspirated from each tube, pooled, then divided into 2 equal parts in 15 ml conical tubes. These were centrifuged at 500 g for 10 min and suspended in the same volume of mBWW with 0.3% BSA. The wash was repeated and the final pellet resuspended in 4 ml of mBWW with 0.3% BSA.
The staining protocol was modified from a previously published method (Cross, 1993). Sperm suspension (75 µl) was added to three wells of a 96-well culture dish for each antibody tested. Then 50 µl of ethidium monoazide (EMA; Sigma, St Louis, MO, USA) in mBWW (1.25 µg/ml) was added to each well and incubated for 10 min under fluorescent light. Another 100 µl of mBWW was added to each well and mixed. The mixture from each well was overlaid on to 300 µl of 35% Nycodenz in a single well of an 8-well glass slide and centrifuged at 40 g for 5 min. The supernatant was removed and the glass slide dipped into ice-cold 100% ethanol. The ethanol was removed and the slide was dipped into fresh 100% ethanol and incubated for 10 min. The ethanol was removed and the slide was allowed to air dry. 100 µl of FITC–pisium sativum agglutinate (FITC-PSA, Vector Labs, lot D0310) in PBS (50 µg/ml) was added to each well, incubated for 10 min , and gently washed with water. After the slides were air-dried they were rehydrated in PBS with 3% BSA and stained for Gq/11 (QL antibody, 10 µg/ml) as described above, except that the secondary antibody was labelled with rhodamine. Cells were scored for the presence or absence of intact acrosomes (Cross et al., 1986) and correlated with antibody staining pattern using a Leitz Dialux fluorescence microscope with filter blocks to optimize the separation of FITC, EMA and rhodamine.
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Results |
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Immunoblotting was performed in order to determine which G proteins are present in human spermatozoa. In all immunoblots 400 µg of sperm membrane protein was loaded in each lane. QL, an antibody raised against the carboxy terminal decapeptide of G
q and G11, labelled a single 42 kDa band in spermatozoa which comigrated with a band in rat liver membranes previously identified as Gq and/or G11 (Figure 1) (Shenker et al., 1991). AS, an antibody raised against the carboxy terminal decapeptide of Gt and which recognizes Gi1 and Gi2 (Simonds et al., 1989), labelled a single 40 kDa band in human spermatozoa which comigrated with the lower band found in rat kidney membranes (Figure 1). This lower band has been previously characterized as Gi2 (Murakami et al., 1989). LD, an antibody directed to an internal sequence of Gi1 (Goldsmith et al., 1988b), did not detect a band in sperm membranes (Figure 1). Therefore the single band detected by AS in sperm membranes appears to represent Gi2 and not Gi1. EC, an antibody raised toward the carboxy terminal decapeptide of Gi3 (Simonds et al., 1989), labelled a single 41 kDa band in sperm membranes which comigrated with a band in RINm5F cells previously characterized as Gi3 (Figure 1) (Cormont et al., 1991). SW (anti-Gß) detected a single 35 kDa band comigrating with the lower band of the 35–36 kDa Gß doublet seen in pancreatic islet membranes (Figure 1). In other cell lines, the 35 kDa band is presumably ß2 and or ß3.
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HD, an antibody against G
13, detected no bands on immunoblots of sperm membranes. Prolonging of the film exposure up to 48h failed to reveal G13 in sperm membranes but did revealed a non-specific band in control membranes (Figure 1
). This higher molecular weight band is usually absent under standard film exposure (24 h). Antibodies RM (anti-Gs), QN (anti-Gz), QE (anti-G12), AR (anti-G16) and GO and GC (both anti-Go) detected no bands on immunoblots of sperm membranes (Figure 2). For each antibody an appropriate size band was detected in a positive control sample run in the same experiment.
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These antibodies were also used for indirect immunofluorescence studies of intact spermatozoa. Immunocytochemical analysis of human sperm cells stained with affinity-purified QL (anti-G
q/11) antibody revealed faint labelling of the apical acrosomal area with strong labelling of a band representing the equatorial segment of the sperm head (Figure 3A). No other component of the sperm cells appeared to be stained. FITC–PSA staining showed that the vast majority of cells had intact acrosomes and that all cells stained strongly for Gq/11 in the equatorial segment independently of the acrosomal status. EMA staining revealed that virtually all cells examined were viable (data not shown). Rabbit IgG (10 µg/ml) used as control exhibited no fluorescent staining (data not shown).
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Immunocytochemistry of sperm cells with affinity-purified AS revealed labelling in the acrosome and midpiece and marginal labelling of some of the tailpiece (Figure 3C
). In contrast to the experiments using QL, there was no staining of the equatorial segment. Based upon the immunoblot data, the immunocytochemical staining of spermatozoa by AS presumably represents Gi2. Affinity-purified EC (anti-Gi3) antibody labelled the posterior part of the head, known as the postnuclear cap, the initial portion of the midpiece and some of the tailpieces (Figure 3D).
Incubation of sperm cells with SW antibody revealed staining of the midpiece, less staining of the tailpiece and marginal staining of the equatorial segment (Figure 3B). There was little or no staining of the acrosome.
Incubation of sperm cells with affinity-purified QN, QE, RM, GO antibodies up to a concentration of 10 µg/ml did not reveal any significant staining (data not shown), consistent with the negative results obtained with these antibodies on immunoblots of sperm membranes.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Our findings represent the first determination of which G proteins are present in human spermatozoa and their subcellular distribution. Although it is known that spermatozoa contain G proteins (Kopf, 1988
, 1989; Hinsch et al., 1995), which G proteins are present in these cells and their subcellular localization have not been fully characterized. Based upon the results of immunoblotting and immunocytochemistry, Gi2, Gi3 and Gq (and/or G11) are present in sperm cells while Gs, Gi1, Go, Gz, G12, G13 and G16 appear to be absent. Moreover, each subunit present in spermatozoa has a discrete subcellular distribution, presumably related to specific functions of each G protein.
Based on our immunoblot experiments, the amount of G proteins in sperm membranes appears to be very low. For each antibody, 400 µg of sperm membranes gave a fainter band than 20–75 µg of controls (see legends to the figures). However, very low amounts of protein kinase C (PKC) are also present in sperm cells (Rotem et al., 1990), despite the fact that PKC probably plays an important role in sperm function (Rotem et al., 1990, 1992; Breitbart and Spungin, 1997).
It is likely that the staining of spermatozoa in our indirect immunofluorescence studies using these affinity-purified antibodies is specific. Spermatozoa were separated from other cells prior to membrane purification. Thus, the presence of G protein subunits on immunoblots does not reflect contamination by other cells which are found in human ejaculates (Hinsch et al., 1995). The antibodies which showed positive staining in immunofluorescence studies (QL, AS, EC and SW) only detected a single band of the appropriate size on immunoblots of sperm membranes. Seven other G protein-specific antibodies which did not detect a band on immunoblots did not stain sperm cells by immunofluorescence. The labelling of different specific regions of the sperm cells using different antibodies also suggests that the labelling is specific.
The G proteins found in human spermatozoa appear to be localized to discrete regions in the cell. Isoforms of another signalling protein, PKC, were also shown to have discrete subcellular localization in sperm cells (Rotem et al., 1992). The discrepancy between the subcellular localization of Gi2 and Gi3 suggests that these highly homologous proteins have different functions in spermatozoa. Such a discrepancy has already been described in LLC-PK1 renal epithelial cells (Ercolani et al., 1990). It is now clear that Gi proteins have variable subcellular localizations in many cell lines (Barr et al., 1991; Donaldson et al., 1991; Lewis et al., 1991; Stow et al., 1991a; Hermouet et al., 1992; Huang et al., 1992; Kumble et al., 1992). For example, Gi3 has been localized to both the Golgi and apical plasma membrane border in renal epithelial cells (Ercolani et al., 1990) but is almost exclusively restricted to the Golgi apparatus in LLC-PK1 cells (Stow et al., 1991a,b) or NIH3T3 fibroblasts (Hermouet et al., 1992).
The localization of Gi2 to the acrosome is consistent with prior studies which suggested the presence of one or more Gi in this region (Garty et al., 1988; Karnik et al., 1992). A protein isolated from the zona pellucida named ZP3 is considered to be the physiological inducer of the acrosome reaction. The induction of the acrosome reaction by ZP3 is inhibited by PTX (Lee et al., 1992b; Wilde et al., 1992; Brandelli, 1997; Breitbart and Spungin, 1997). The presence of Gi2 in this location together with the G protein-coupled receptor kinase GRK4 (Sallese et al., 1997) makes it the most likely candidate as the G protein which mediates this signalling pathway. Increased intracellular calcium concentrations, diacylglycerol and PKC have been implicated as mediators of the acrosome reaction (Roldan and Harrison, 1992; Rotem et al., 1992; Brandelli, 1997). It is therefore likely that stimulation of Gi2 in the acrosomal region induces the acrosome reaction by releasing ß subunits which stimulate phospholipase C (Camps et al., 1992; Katz et al., 1992; Carozzi et al., 1993). The role of Gi2 in the midpiece and Gi3 in the postnuclear region, midpiece and tailpiece is at present unknown. It might be predicted that these G proteins may play a role in flagellar motility. However PTX treatment does not appear to affect sperm motility (Lee et al., 1992b; Franken et al., 1996).
This is the first study to demonstrate the presence of members of the Gq/11 subfamily in human spermatozoa. Gq and G11 directly stimulate phospholipase C (primarily the ß1 isoform) (Taylor et al., 1991; Lee et al., 1992a). The localization of Gq/11 to the acrosome suggests that it may also play a role in the induction of the acrosome reaction as already reported in rodents (Walensky and Snyder, 1995). The highest concentration of Gq/11 is in the equatorial segment, a region which is the primary site of fusion with the oocyte. There is evidence that a G protein in the oocyte which is PTX-insensitive and raises intracellular calcium and inositol triphosphate concentrations is important in the initial steps of fertilization (Turner and Jaffe, 1989; Kline et al., 1991). This is likely to be a member of the Gq/11 subfamily. The localization of Gq/11 to the equatorial segment in spermatozoa suggests that Gq/11 present in the sperm cell membrane may also be important in the initial steps of fertilization. PKC and ßII also localize to equatorial segment (Rotem et al., 1992), making them likely candidates as molecules that are stimulated by the Gq/11 signalling pathway in this region.
Many of the G protein -subunits could not be detected in human sperm cells by either immunoblot or indirect immunofluorescence. Although Gs is ubiquitously expressed in virtually all somatic cells, it has previously been shown to be absent in sperm cells (Hildebrandt et al., 1985; Kopf et al., 1986; Karnik et al., 1992). Adenylyl cyclase, the classical effector coupled to Gs, is present in sperm cells. However, the regulation of this enzyme activity in sperm cells is not typical of that in most somatic cells and does not appear to be stimulated by Gs (Hildebrandt et al., 1985). Our inability to detect Go is consistent with previous studies which suggest that Go is absent in mouse spermatozoa (Karnik et al., 1992). Studies examining whether or not Go is present in bovine sperm cells are at present inconclusive (Garty et al., 1988; Hinsch et al., 1992). Although we did not identify Gi1 or Gz in human sperm cells, previous studies show evidence for the presence of Gi1 in mouse spermatids, spermatocytes and spermatozoa (Glassner et al., 1991; Karnik et al., 1992) and for Gz in the postacrosomal region in mouse spermatozoa (Glassner et al., 1991). Whether the contrasting results between the human and mouse studies are due to species-specific or technical differences is not known. Whether or not G12, G13 or G16 is present in sperm cells has not been previously studied. It is not surprising that G16 is not found in sperm cells, since its expression appears to be limited to haemopoietic cells (Amatruda et al., 1991). We observed only a 35 kDa band with SW antibody. This is consistent with a previous study which found only the 35 kDa ß2 subunit in tails of human and bovine spermatozoa (Hinsch et al., 1992, 1995). In the indirect immunofluorescence studies, there was little labelling of the acrosomal region with SW despite the fact that Gi2 and Gq/11 appear to be present in this region. There are several possible explanations for this discrepancy. It is possible that another ß subunit is present in the acrosome which this antibody does not recognize. It is also possible that SW does not detect ß subunits in the acrosome due to proteolysis of the protein during fixation, poor access of the antibody to the epitope or a modification of the ß subunit which alters its immunoreactivity. Such an alteration of immunoreactivity has already been described for Gi2 in adipocyte plasma membranes (Record et al., 1993) and might explain the discrepancy between our data and those obtained with different antibodies (Hinsch et al., 1995).
The identification and subcellular localization of G proteins in human spermatozoa is the first step towards our understanding of the role of these signalling molecules in the regulation of sperm functions such as the acrosome reaction, fertilization and motility. Examining the subcellular localization of other known signal transduction molecules, such as ZP3 receptors and phospholipase C isozymes, and determining what other receptors and effectors couple to G proteins in these cells will provide further clues to the role of G protein-coupled pathways in sperm physiology. A subfamily of the seven transmembrane domain receptors which are homologous to the odorant receptors found in olfactory epithelium has been cloned in germ cells (Meyerhof et al., 1991; Parmentier et al., 1992). Although some or all of these receptors are presumed to couple to G proteins in spermatozoa, their physiological ligands and G protein coupling specificity are at present unknown. Further studies will better define the role of G proteins in normal sperm physiology and will determine if G protein defects may explain some forms of male infertility.
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Acknowledgments |
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We wish to thank P.Cavelot and M.Dutay for their technical help, N.Parseghian and Dr M.Albert for their expert technical advice, Dr S.Gutkind for the gift of transfected NIH 3T3 membranes, M.Forte for the gift of recombinant G
16 and Dr A.M.Spiegel and Pr Y.Giudicelli for their support. This work was funded in part by Ipsen-Biotech and Organon. |
Notes |
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6 To whom correspondence should be addressed
* Studies performed on human spermatozoa.
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Submitted on May 27, 1998; accepted on October 2, 1998.
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