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Endocrinology Vol. 150, No. 1 473-484
Copyright © 2009 by The Endocrine Society

Progestin Signaling through an Olfactory G Protein and Membrane Progestin Receptor-{alpha} in Atlantic Croaker Sperm: Potential Role in Induction of Sperm Hypermotility

Christopher Tubbs and Peter Thomas

Marine Science Institute, The University of Texas at Austin, Port Aransas, Texas 78373

Address all correspondence and requests for reprints to: Christopher Tubbs, San Diego Zoo Conservation Research, 15600 San Pasqual Valley Road, Escondido, California 92027. E-mail: ctubbs{at}sandiegozoo.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progestin stimulation of sperm hypermotility remains poorly understood despite having been described in numerous vertebrate species. We show here that progestin stimulation of sperm hypermotility in a teleost, the Atlantic croaker (Micropogonias undulatus) is associated with activation of an olfactory G protein (Golf). Furthermore, we provide evidence that this progestin action is mediated by membrane progestin receptor-{alpha} (mPR{alpha}). Golf was identified in croaker sperm membranes and was specifically activated after treatment with the progestin 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S). Treatment of sperm membranes with 20β-S caused an increase in cAMP production, which was blocked by pretreatment with cholera toxin and two membrane adenylyl cyclase inhibitors: 2',5'-dideoxyadenosine and SQ22536. Moreover, preincubation of croaker sperm with 2',5'-dideoxyadenosine and SQ22536 resulted in a significant inhibition of 20β-S-stimulated hypermotility. Binding of [3H]20β-S to sperm membranes was decreased after pretreatment with GTP{gamma}S but not pertussis toxin, suggesting the receptor is coupled to a pertussis toxin-insensitive G protein. Golf and mPR{alpha} were coexpressed on the sperm midpiece and flagella and were coimmunoprecipitated from sperm membranes. Finally, expression of mPR{alpha} protein on sperm increased after in vivo treatment with LHRH and was associated with increased induction of sperm motility by 20β-S. These results suggest that 20β-S activates mPR{alpha} in croaker sperm, which in turn activates Golf and membrane adenylyl cyclase to stimulate sperm hypermotility. Taken together these findings provide a plausible mechanism by which progestins stimulate sperm hypermotility in croaker and provide the first evidence of hormonal activation of Golf in any species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rapid actions of progestins to induce sperm hyperactive motility have been described in mammalian and teleost species (1, 2, 3). Sperm hypermotility is characterized by increased flagellar movement resulting in changes in sperm swimming velocity and direction, which facilitate oocyte contact and subsequent fertilization (4, 5). However, the receptors and intracellular signaling pathways mediating progestin-induced sperm hypermotility are not currently known (2, 5, 6). Progestins can also initiate the acrosome reaction in mammalian sperm, which is marked by the fusion of the acrosome, an enzyme-filled vesicle, to the sperm plasma membrane. This ultimately results in the rupture of the acrosome and release of its contents allowing oocyte penetration and fusion (7, 8). Yet it is unclear whether the acrosome reaction and sperm hypermotility share a common progestin-mediated pathway (9, 10, 11). In the Atlantic croaker (Micropogonias undulatus) physiological levels of progestins have been shown to cause a concentration-dependent induction of sperm hypermotility (12). Treatment of croaker sperm with the endogenous progestin, 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S), results in a rapid induction of sperm hypermotility, which is correlated with increases in intracellular cAMP and Ca2+ concentrations (6, 12). These findings are similar to the observations in other vertebrate models (13, 14, 15) but are not complicated by potential progestin activation of signaling pathways associated with the acrosome reaction because croaker sperm lack an acrosome and do not undergo this process (16). Thus, croaker is an excellent model for examining progestin induction of sperm hypermotility.

A progestin receptor has been biochemically characterized on croaker sperm membranes and is believed to be the receptor that mediates progestin induction of sperm hypermotility in this species (17). Recent studies have identified the novel G protein-coupled receptor (GPCR)-like membrane progestin receptor-{alpha} (mPR{alpha}) as a candidate for this receptor because it is expressed in croaker sperm membranes and is localized to the sperm midpiece (17). Although it is not a member of the GPCR superfamily, mPR{alpha}, like GPCRs mediates intracellular changes through activation of G proteins. Both recombinant and native mPR{alpha}s have been shown to bind progestins and catalyze GDP/GTP exchange of inhibitory G proteins (Gi) to decrease cAMP concentrations in a variety of cell types in different vertebrate species (18, 19, 20). Based on the observation that both cAMP and Ca2+ concentrations increase in croaker sperm in response to progestins, it is unclear which G proteins might be activated by 20β-S. Candidates include the stimulatory family of G proteins (Gs), which has both a long and a short form of Gs, as well as the olfactory-type G protein (Golf) (21). Unlike Gs, Golf is expressed in high concentrations in the olfactory epithelium, but is also present in peripheral tissues such as the pancreas, testis and sperm (21, 22, 23, 24).

There are numerous reports of individual GPCRs activating multiple G proteins, although the mechanisms determining receptor/G protein selectivity are largely unclear (25). The type of G protein activated by a GPCR can be cell specific. For example, the neurotensin 1 receptor can activate Gq/11, Gi, or Gs proteins, depending on cell-type (26). Thus, the cellular environment of croaker sperm could potentially favor mPR{alpha} coupling and activation of a stimulatory G protein rather than Gi as has been described in all other cell types investigated to date.

Although Golf has been cloned only from a single nonmammalian species, Xenopus (27), immunoreactive proteins have been demonstrated using a Golf antibody in olfactory epithelia of three fish species (28, 29, 30). Golf expression has also recently been demonstrated in mammalian sperm, and therefore, it is possible that progestins could induce sperm hypermotility in croaker through activation of Golf, resulting in production of cAMP likely via activation of membrane adenylyl cyclases (mACs) and subsequent opening of cAMP-gated Ca2+ channels. Activation of mAC by GPCRs has been demonstrated in mammalian sperm (23, 31), but a physiological role for mAC or its association with Golf activation in sperm has not been established. In the present study, the hypothesis that 20β-S induction of croaker sperm hypermotility involves activation of a Golf pathway was tested. In addition, the potential involvement of the novel progestin receptor, mPR{alpha}, as the intermediary in progestin activation of Golf was examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
LHRH analog (LHRHa; des-Gly10,[d-Ala6]LHRH 91–90 ethylamide) was purchased from Bachem (Torrance, CA). All steroids were purchased from Steraloids (Newport, RI). The synthetic progestin R5020 and [35S]GTP{gamma}S (guanosine 5'-(3-O-thio)triphosphate; ~12,000 Ci/mmol) were purchased from Amersham Pharmacia (Piscataway, NJ). Pertussis toxin (PTX) was purchased from List Biological Laboratories (Campbell, CA). Synthesis of [3H]17,20β,21-trihydroxy-4-pregnen-3-one ([3H]20β-S) was performed by enzymatic conversion of [3H]11-deoxycortisol (Amersham) by 3{alpha},20β-hydroxysteroid dehydrogenase as described previously (32). Antibodies against Gs and Gi were purchased from Sigma (St. Louis, MO). An antibody against Gq was purchased from Biomol (Plymouth Meeting, PA) and the Gi,o,t,z, and Golf antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). SQ22536 and 2',5'-dideoxyadenosine (dd-Ado) were purchased from Calbiochem (La Jolla, CA). All other chemicals and reagents were purchased from Sigma unless otherwise noted.

Animals
Adult Atlantic croaker were captured by shrimp trawl and purchased from local bait shops. Fish were acclimated to the laboratory for 2 months before use in experiments in 12,000 liters recirculating tanks at 22–24 C and a photoperiod of 11 h light, 13 h dark to promote and maintain gonadal development. All procedures used in this study were approved by the Institutional Animal Care and Use Committee of the University of Texas at Austin.

Sperm collection and membrane isolation
Sperm were collected with a syringe from the cloaca of fully mature male Atlantic croaker as described previously (33). For all experiments, sperm were pooled from multiple (n = 2–6) donors. Sperm membranes were isolated as described previously (34) with minor modifications. Briefly, 3–5 ml of sperm were diluted in 10 ml of cold homogenization buffer [HAED; 25 mM HEPES, 10 mM NaCl, 10 mM MgCl2, 1 mM dithioerythritol, 1 mM EDTA (pH 7.6)] and centrifuged at 1000 x g to isolate sperm from seminal fluid. Sperm were resuspended in 10 ml HAED with HALT protease inhibitor cocktail (Pierce, Rockford, IL). Sperm suspensions were twice forced through a 23.5-gauge needle and sonicated at medium power for 6 sec on ice. Samples were then centrifuged at 500 x g for 20 min at 4 C to remove nuclear material. The resulting supernatants were transferred to a clean tube and centrifuged at 17,000 x g to obtain the cell membrane fraction. Isolated sperm membranes were used immediately or stored at –80 C. Croaker tissues were collected from fish that were humanely killed by anesthesia followed by cervical dislocation. Testicular, ovarian, and olfactory epithelial cell membranes were prepared in the same manner as above after 10 passes through a glass homogenizer. Protein concentrations of membrane preparations were determined using a Bradford protein assay (Bio-Rad, Hercules, CA).

Sperm motility analyses
Sperm were collected and sperm motility experiments were performed as described previously (35). Briefly, croaker sperm were diluted 100-fold and preincubated in physiological saline with steroid (20β-S, R5020, or corticosterone) or vehicle (EtOH, 0.02%) for 1 min at room temperature. A 2-µl aliquot of each sperm suspension was then placed on a microscope slide and diluted with 25 µl of artificial seawater. A coverslip was placed on the slide and sperm were viewed using dark field microscopy. For mAC experiments, sperm were diluted and preincubated in physiological saline with mAC inhibitors; dd-Ado (50 or 100 µM) or SQ22536 (500 µM) for 20 min at room temperature. Control samples were incubated with vehicle alone (dimethylsulfoxide; 0.1%). Sperm were then treated with 20 nM 20β-S or vehicle (EtOH, 0.02%) for 1 min or with forskolin (10 µM final concentration) for 5 min. Each experiment was recorded using a charge-coupled device camera (Cohu Electronics, San Diego, CA) and a VHS recorder and the percent of sperm displaying hypermotility (characterized by rapid velocity, an increased rate of turning and rapid flagellar beating) for each treatment was determined. For sperm velocity measurements, videos were digitized using a VP110 video digitizer and ExpertVision software (Motion Analysis Corp., Santa Rosa, CA).

Western blot analyses
Approximately 10 µg of membrane protein were added to loading buffer [0.5 M Tris-HCl, 10% sodium dodecyl sulfate (SDS), 0.5% bromophenol blue, 10% glycerol] and resolved on 10% SDS-PAGE gels. After transfer to polyvinyl difluoride membranes, membranes were blocked in a solution containing 5% nonfat milk, 0.1% Tween 20 in PBS [136 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 10 mM KH2PO4 (pH 7.4)]. Membranes were rinsed in PBS and incubated overnight at 4 C with antibodies directed against Gi1–2, Gq, or Golf at a concentration of (1:500–1:1000). Because the Golf antibody used could potentially cross-react with vertebrate Gs, Western blots were performed with croaker sperm and testicular membranes using a Gs antibody to determine whether the two antibodies recognize similar proteins. This Gs antibody has previously been shown to recognize proteins in the ovary of a closely related species to croaker, the spotted seatrout (36). Membranes were rinsed in PBS and incubated with a goat antirabbit horseradish peroxidase-linked secondary antibody (1:5000; Abcam, Cambridge, UK) in blocking solution. Proteins were then visualized using SuperSignal WestPico chemiluminescent substrate (Pierce). For Golf Western blots, the specificity of the reaction was confirmed by preabsorbing the antibody against the peptide antigen (Santa Cruz Biotechnology).

Fertilization experiments
Female Atlantic croaker with fully grown oocytes (diameter >0.5 mm) were injected with LHRHa (100 µg/kg body weight) to induce oocyte maturation and ovulation. After 30 h, fish were anesthetized and the eggs were expelled into a beaker containing seawater by applying gentle pressure to the abdomen. Milt was then collected and diluted 1:10 in physiological saline with or without 100 nM 20β-S. Sperm were then diluted in activating solution (1:10,000 and 1:20,000 final) and mixed with the egg suspensions using a feather. Fertilization was allowed to proceed for 90 sec, and then seawater was added to the beaker and the eggs were incubated with gentle aeration at 21–23 C for 2 h. The percentage of fertilized oocytes was determined by counting the number of eggs showing a perivitelline space.

Activation of G proteins in croaker sperm membranes
Activation of G proteins by hormonal treatment was assayed by measuring increased [35S]GTP{gamma}S binding to croaker sperm membranes as described previously with minor modifications (36, 37). Sperm membranes were suspended in binding buffer [50 mM Tris, 100 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.6 mM EDTA, 0.1% BSA (pH 7.6)] and preincubated with 10 µM GDP and 1 nM [35S]GTP{gamma}S in the presence (nonspecific binding) or absence (total binding) of 1 µM nonradioactive GTP{gamma}S. Aliquots (250 µl) of sperm membrane preparations were then added to tubes containing 100 nM steroid (20β-S, cortisol, or R5020) or vehicle alone. Reactions were allowed to proceed for 20 min at room temperature with light shaking and were terminated by the addition of 750 µl of binding buffer containing 100 µM GDP/GTP{gamma}S (stop solution). A 200-µl volume of sperm membrane preparations were filtered through Whatman GF/B glass fiber filters using a vacuum manifold and washed with 25 ml of wash buffer [50 mM Tris, 100 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.6 mM EDTA (pH 7.6)]. Total and nonspecific [35S]GTP{gamma}S binding was counted using a Beckman LS 6000SC scintillation counter (Fullerton, CA) and specific binding calculated by subtracting nonspecific from total binding. The 20β-S response relative to the values of the no-treatment controls was enhanced in some assays by decreasing the [35S]GTP{gamma}S concentration to 0.25 nM.

Identification of G proteins activated in croaker sperm membranes
Immunoprecipitation of activated G protein {alpha}-subunits bound to [35S]GTP{gamma}S from croaker sperm membranes with specific G protein {alpha}-subunit antibodies was performed as described previously (36). Sperm membranes were preincubated with 4 nM [35S]GTP{gamma}S in the presence (nonspecific binding) or absence (total binding) of 4 µM nonradioactive GTP{gamma}S. After incubation with 100 nM 20β-S or vehicle, stop solution was added and sperm membranes were centrifuged at 14,000 x g for 15 min at 4 C. Membrane pellets were resuspended in solubilization buffer [25 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS (pH 7.6)] with protease inhibitor cocktail and incubated for 1 h at 4 C with gentle shaking. Solubilized sperm membranes were then centrifuged at 14,000 x g for 10 min and the supernatants were collected. Polyclonal antibodies directed against Gi,o,t,z, Gi1–2, Gq, Gs, Golf, or preimmune rabbit serum (negative control) were added to the supernatants at a concentration of 1:100 and incubated for 8 h at 4 C. Protein A/G plus agarose beads (50 µl; Santa Cruz Biotechnology) were then added, and samples were incubated for 8 h at 4 C. Beads were washed three times with wash buffer [50 mM HEPES, 100 mM NaCl, 50 mM Na2HPO4, 100 µM NaF, 1% Triton X-100, 0.1% SDS (pH 7.6)] and boiled in 0.5% SDS for 10 min. Samples were centrifuged, the supernatants collected and specific [35S]GTP{gamma}S binding determined. For steroid-specificity experiments, the same procedure was performed using a 1:50 concentration of the Golf antibody after treatment of sperm membranes with 100 nM of 20β-S, 17β-estradiol, 11-ketotestosterone, R5020, or corticosterone.

Measurement of cAMP production by croaker sperm membranes
Sperm cell membranes were resuspended in buffer [75 mM Tris-HCl, 5 mM MgCl2, 2 mM EDTA (pH 7.6)] to a total protein concentration of about 1 mg/ml. Membrane preparations (100 µl) were added to assay buffer [0.2 mM ATP, 10 nM GTP, 0.5 mM phosphoenolpyruvate, 20 µg pyruvate kinase, 2 mM 3-isobutyl-1-methylxanthine (IBMX)] containing 20β-S (20 or 100 nM final concentration) or vehicle (0.01% EtOH). Reactions were allowed to proceed for 1 min at room temperature. Samples were then boiled for 5 min and centrifuged at 14,000 x g for 10 min. Supernatants were collected, diluted 10- to 20-fold, and cAMP concentrations determined using a commercial cAMP enzyme immunoassay kit following the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI).

To investigate the potential role of stimulatory Golf in 20β-S-stimulated cAMP production, sperm cell membrane preparations were preincubated with cholera toxin (CTX), which activates members of the stimulatory G protein family, including Golf (38). CTX was activated by incubation at 37 C for 30 min or heat-inactivated (iCTX) by boiling for 30 min before addition to sperm membranes. Membrane preparations were then preincubated with 12.5 µg/ml CTX or iCTX for 20 min at room temperature before addition to assay buffer containing 20β-S (20 nM final concentration) or vehicle (0.01% EtOH) and incubated for 1 min as described above. To confirm that CTX can activate stimulatory G proteins in croaker sperm, membranes were incubated with 12.5 µg/ml CTX for 5 min at room temperature. A similar experimental design was used with the mAC inhibitors [50 or 100 µM dd-Ado, 500 µM SQ22536, or vehicle (0.01% dimethylsulfoxide)]. In addition to these treatments, membrane preparations were incubated with the mAC activator forskolin to confirm mAC activity in croaker sperm. For most experiments, data were normalized to mean control values due to the variation of cAMP produced between samples, which is a noted problem in evaluating cAMP concentrations of multiple sperm donors (15).

20β-S binding to croaker sperm membranes
To determine whether the 20β-S receptor on croaker sperm membranes is coupled to a G protein and whether the coupled G protein is PTX sensitive, specific [3H]20β-S binding in the presence of GTP{gamma}S and PTX was examined as described previously (36). Briefly, sperm membranes were preincubated with 12.5 µM GTP{gamma}S or 3 µg PTX or heat-inactivated PTX for 20 min at 18 C. Membranes were then incubated with either 5 or 10 nM [3H]20β-S for the GTP{gamma}S experiments or 20 nM [3H]20β-S for the PTX experiments, in the presence (nonspecific binding) or absence (total binding) of 1000-fold excess of nonradioactive 20β-S for 30 min at 4 C. For PTX experiments, [3H]20β-S binding to croaker ovarian membrane preparations with and without preincubation with PTX (3 µg for 20 min at 18 C) was performed as a positive control. Membrane-bound [3H]20β-S was separated from free steroid by filtration of sperm membrane incubations through Whatman GF/B fiber filters using a Brandel Semi-Auto Harvester (Gaithersburg, MD). Filters were rinsed three times with 5 ml of ice-cold wash buffer [25 mM HEPES, 10 mM NaCl, 1 mM EDTA (pH 7.6)]. Specific [3H]20β-S binding to croaker sperm membranes was calculated by subtraction of nonspecific from total binding.

Immunocytochemistry
Sperm were collected and diluted 1000-fold in cold PBS. Aliquots of sperm suspensions were spread on poly-L-lysine-coated slides and air dried for 30 min. Sperm were fixed in 4% paraformaldehyde for 20 min on ice and rinsed with PBS. Slides were then blocked in 2% BSA for 2 h. For immunocytochemical localization of Golf proteins, 0.3% Triton X-100 was added to the blocking buffer to permeabilize sperm. After three rinses with PBS, slides were incubated with a rabbit anti-Golf antibody (1:500) or a rabbit anti-mPR{alpha} antibody (1:1000) overnight at 4 C. The mPR{alpha} antibody was generated against a peptide sequence within the N-terminal region of mPR{alpha} from a closely related species, the spotted seatrout (Cynoscion nebulosus; GenBank Q801D8), which is identical in Atlantic croaker (GenBank EU095257). As a negative control, both antibodies were preabsorbed against the peptide antigens. Slides were rinsed with PBS and incubated for 2 h at room temperature with an AlexaFluor 488 goat antirabbit secondary antibody (1:1000; Molecular Probes, Eugene, OR). To visualize sperm nuclei, slides were treated with 300 nM 4',6-diamidino-2-phenylindole for 10 min and rinsed with PBS, and coverslips were mounted using ProLong gold antifade reagent (Molecular Probes). Sperm were visualized using a Eclipse E600 fluorescent microscope (Nikon, Tokyo, Japan).

Coimmunoprecipitation of Golf proteins and mPR{alpha}
Sperm cell membrane pellets were solubilized by resuspension in HAED buffer with 1% Triton X-100 at a final protein concentration of about 1 mg/ml. Solubilized membrane proteins were then immunoprecipitated with the Golf antibody or normal rabbit IgG (negative control) using a Seize immunoprecipitation kit (Pierce) following the manufacturer’s instructions. After elution from the column, immunoprecipitated proteins were run on a 10% SDS polyacrylamide gel, and Western blot analyses were performed as described above using an antibody against mPR{alpha} (18). The presence of Golf in the immunoprecipitates was confirmed by Western blot analysis. A freshly prepared croaker sperm membrane sample was run on each gel as a positive control for Golf and mPR{alpha}.

In vivo hormonal regulation of mPR{alpha} in croaker sperm
Croaker early in testicular development, which were producing low quantities of sperm, were divided into groups of three and sperm was collected from each group and pooled. An aliquot of sperm was placed on ice and used immediately for sperm motility analyses. The remainder of the sperm was frozen in liquid nitrogen for subsequent measurement of mPR{alpha} protein abundance. Groups of croaker were then given an ip injection of 100 µg/kg of LHRHa (n = 6) in 0.8% saline or saline alone (control groups; n = 6). After 24 h, sperm were collected, and sperm motility analyses were performed immediately. The remainder of the sperm was frozen for analysis of mPR{alpha} protein abundance. The abundance of mPR{alpha} protein on croaker sperm membranes before and after LHRHa treatment was determined by Western blot analyses as described above.

Statistical analyses
For all experiments, data are presented as means ± SEM. Statistical significance was determined using a Student’s t test or one-way ANOVA and Dunnett’s or Tukey’s multiple comparison posttests using GraphPad Prism Software (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of progestin-stimulated sperm hypermotility
Treatment with both 20 and 100 nM 20β-S resulted in a significant increase in the percentage of hypermotile croaker sperm (Fig. 1AGo) and in sperm velocity (results not shown). Treatment of a single pool of croaker sperm membranes with 20 nM 20β-S in three replicate experiments caused a significant 2-fold increase in cAMP concentrations (Fig. 1BGo). A similar trend was observed after treatment of croaker sperm membranes with 100 nM 20β-S. Steroid induction of sperm hypermotility was specific for 20β-S. Treatment with 20 nM 20β-S caused a significant increase in the percent of sperm that were hypermotile, whereas treatments with the same concentration of the nuclear progestin receptor agonist R5020 or corticosterone were ineffective (Fig. 1CGo). To determine whether this 20β-S-induced increase in sperm hypermotility is of physiological importance, the effect of in vitro 20β-S pretreatment on the ability of sperm to fertilize croaker oocytes was examined. Pretreatment of croaker sperm with 100 nM 20β-S resulted in a significant increase in the percentage of oocytes that were fertilized at both dilutions of sperm tested (Fig. 1DGo).


Figure 1
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  		FIG. 1. Characteristics of progestin induction of sperm hypermotility in Atlantic croaker. A, Changes in percentage of hypermotile sperm (n = 18–31 measurements of individual sperm from four donors) after 1 min treatment with 20 or 100 nM 20β-S or EtOH alone (control). B, Changes in cAMP production by croaker sperm membranes (n = 3) after 1 min treatment with 20 or 100 nM 20β-S or EtOH alone (control). C, Specificity of progestin induction of Atlantic croaker sperm hypermotility. Croaker sperm were treated with 20 nM of 20β-S, R5020 or corticosterone (CORT) for 1 min (n = 5). D, Differences in fertilization success of croaker sperm after treatment with or without (control) 100 nM 20β-S (n = 6). All data represent mean ± SEM. Significant differences (*, P < 0.05) from control means were determined using one-way ANOVA and Dunnett’s multiple comparison posttest. For D, significant differences (*, P < 0.05) were determined by Student’s t test between 100 nM 20β-S and untreated control groups for each dilution tested.

 
Expression of Golf in croaker sperm
Western blot analyses demonstrated the presence of Gi1–2, Gq, Gs, and Golf proteins in sperm and testicular membranes (Fig. 2AGo). Bands of approximately 45 kDa were detected in croaker sperm and testicular membranes with the Golf protein antibody, which corresponds to the predicted size of this protein as well as the reported size of this protein in other fish species using the same antibody (29, 30). In both sperm and testes, the Gs antibody used recognized two proteins, neither of which were the same size as the protein detected using the Golf antibody. To further confirm that the Golf antibody did not cross-react with Gs, Western blot analyses were performed on croaker olfactory epithelium and ovary, which should express high concentrations of Golf and Gs, respectively. These experiments showed that the Golf antibody recognized a 45-kDa protein in olfactory epithelium but detected no proteins in the ovary (Fig. 2BGo).


Figure 2
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  		FIG. 2. Identification of G proteins in Atlantic croaker sperm membranes. A, Representative Western blot analyses of G proteins in membrane preparations of croaker sperm and testes using antibodies directed against Gi1–2, Gq, Gs, and Golf {alpha}-subunits (gel loading = 10 µg). B, Western blot analyses of membrane preparations of croaker sperm, olfactory epithelium (OE), and ovary using an anti-Golf antibody (gel loading = 3 µg). Results are typical of four independent experiments.

 
Activation of G proteins by 20β-S in croaker sperm membranes
Activation of G proteins in croaker sperm by 20β-S was assessed by measuring binding of the nonhydrolyzable GTP analog, [35S]GTP{gamma}S, to sperm membranes. Binding of [35S]GTP{gamma}S to sperm membranes significantly increased after treatment with 100 nM 20β-S (Fig. 3AGo) and showed a 25% greater increase in the modified binding assay (Fig. 3BGo). Treatment of sperm membranes with 100 nM of another C21 steroid, cortisol and the nuclear progestin receptor agonist, R5020, did not activate G proteins (Fig. 3AGo).


Figure 3
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  		FIG. 3. G protein activation by 20β-S in Atlantic croaker sperm membranes. A, Effects of treatment with 100 nM 20β-S, 100 nM cortisol, 100 nM R5020, or 0.01% EtOH (control) for 20 min on specific binding of [35S]GTP{gamma}S to croaker sperm membranes. B, Enhanced G protein activation by 20β-S in Atlantic croaker sperm membranes using a modified assay protocol. Croaker sperm membrane preparations were treated with 100 nM 20β-S, 100 nM R5020, or 0.01% EtOH (control) for 20 min in the presence of 0.25 nM [35S]GTP{gamma}S (compared with 1 nM in Fig. 2AGo). C, Immunoprecipitation of [35S]GTP{gamma}S bound to G proteins on croaker sperm membranes with specific antibodies directed against Gi,o,t,z, Gi1–2, Gq, Gs, or Golf protein {alpha}-subunits or rabbit serum (RS; negative control). D, [35S]GTP{gamma}S immunoprecipitation by the anti-Golf antibody after treatment with 100 nM 20β-S, 17β-estradiol, 11-ketotestosterone (11-KT), R5020, or corticosterone (CORT). Data represent mean ± SEM of specific [35S]GTP{gamma}S binding compared with untreated controls. Statistically significant differences between steroid treatments and control treatments (*, P < 0.05) are denoted by asterisks and were determined using a one-way ANOVA and Dunnett’s multiple comparison posttest for [35S]GTP{gamma}S binding experiments (n = 4–5) (A and B) and steroid specificity experiments (n = 4) (D). Significant differences (*, P < 0.05), determined by Student’s t test, between the 100 nM 20β-S treatments and their respective controls for each G protein antibody tested in the immunoprecipitation studies (C) are shown as asterisks (n = 4). Entire experiments were repeated at least three times.

 
The identity of the G protein activated by 20β-S in croaker sperm membranes was determined by immunoprecipitating [35S]GTP{gamma}S bound to G proteins in sperm membranes with specific antibodies directed against different G protein {alpha}-subunits. Treatment with 100 nM 20β-S resulted in Golf protein activation as shown by a significant increase in the amount of [35S]GTP{gamma}S immunoprecipitated with the Golf protein antibody compared with controls (Fig. 3CGo). In contrast, 20β-S treatment did not activate Gs proteins. Similarly, the amounts of [35S]GTP{gamma}S immunoprecipitated with preimmune rabbit serum and the Gi,o,t,z, Gi1–2, and Gq protein antibodies were unaltered by 20β-S treatment (Fig. 3CGo).

Steroid activation of Golf in croaker sperm membranes was specific for 20β-S. Treatment with 100 nM 20β-S resulted in a significant activation of Golf (Fig. 3DGo), whereas the same concentration of 17β-estradiol, 11-ketotestosterone, R5020, and corticosterone caused no Golf activation in croaker sperm membrane preparations.

Inhibition of 20β-S-stimulated cAMP synthesis by croaker sperm membranes with cholera toxin
Treatment of sperm membranes with 20 nM 20β-S for 1 min caused a significant increase in cAMP concentrations compared with vehicle controls (Fig. 4AGo), consistent with activation of the stimulatory G protein, Golf. If 20β-S is acting through this pathway, pretreatment with an activator of Golf protein, CTX (38), should decrease the cAMP response to 20β-S because CTX would deplete the pool of unactivated Golf proteins coupled to the receptor. The results show that pretreatment with 12.5 µg/ml CTX prevented the 20β-S-stimulated increase in cAMP levels, whereas iCTX was ineffective (Fig. 4AGo). Pretreatment of sperm membranes with 12.5 µg/ml CTX or iCTX alone did not result in a significant change in cAMP concentrations. To verify that CTX can activate croaker stimulatory G-proteins, resulting in increased cAMP concentrations, sperm membranes were treated with CTX in the presence of reagents necessary for cAMP synthesis (0.1 mM ATP, 5 nM GTP, 0.25 mM phosphoenolpyruvate, 10 µg/100 µl pyruvate kinase, 1 mM IBMX). This treatment caused a significant increase in cAMP concentrations after 5 min incubation of the croaker sperm membranes (Fig. 4AGo). Changes in sperm membrane cAMP production after 20β-S treatment for an individual fish are shown in Fig. 4BGo.


Figure 4
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  		FIG. 4. Inhibition of 20β-S-stimulated cAMP production by Atlantic croaker sperm membranes by CTX. A, Production of cAMP was measured after 1 min treatment with 20 nM 20β-S after preincubation with 12.5 µg/ml CTX or iCTX. To confirm activation of G proteins by CTX, sperm membranes were incubated with CTX in the presence of 0.1 mM ATP, 5 nM GTP, 0.25 mM phosphoenolpyruvate, 10 µg pyruvate kinase, or 1 mM IBMX for 5 min [CTX(+)]. B, Representative data from an individual fish. Data represent means ± SEM (A). Statistically significant differences between treatments (P < 0.05) were determined using a one-way ANOVA and Tukey’s multiple comparison test (n = 5) and are denoted by different letters where a = significantly different from control and b = significantly different from 20 nM 20β-S treatments. Entire experiments were repeated at least three times.

 
Involvement of mAC in 20β-S-stimulated cAMP production by croaker sperm membranes
To determine whether mAC is potentially activated by 20β-S, the effects of mAC inhibitors on 20β-S-stimulated cAMP production by croaker sperm membranes was examined. Pretreatment of sperm membranes with 100 µM dd-Ado significantly decreased cAMP production in response to 20 nM 20β-S (Fig. 5AGo). Production of cAMP after treatment with 10 µM forskolin was used to confirm the presence of mAC in croaker sperm membranes and resulted in an increase in cAMP synthesis, which was blocked by preincubation of sperm membranes with 100 µM dd-Ado (Fig. 5AGo). A second mAC inhibitor, SQ22536 (500 µM), was also effective in inhibiting cAMP production in response to 20β-S in sperm membranes (Fig. 5BGo).


Figure 5
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  		FIG. 5. Inhibition of 20β-S-stimulated cAMP production by sperm membranes and hypermotility in Atlantic croaker sperm by the mAC inhibitors, dd-Ado and SQ22536. Sperm membrane preparations (A and B) or live sperm (C and D) were treated with 20 nM 20β-S for 1 min after a 20-min preincubation with 50 or 100 µM dd-Ado or 500 µM SQ22536. For dd-Ado experiments, a group of sperm membranes or live sperm were incubated for 5 min with 10 µM forskolin. Data represent means ± SEM. Statistically significant differences between treatments (P < 0.05) were determined using a one-way ANOVA and Tukey’s multiple comparison test (n = 5) and are denoted by different letters where a = significantly different from control, b = significantly different from 20 nM 20β-S for all experiments, and c = significantly different from 10 µM forskolin treatments for dd-Ado experiments. Entire experiments were repeated at least three times.

 
Involvement of mAC in 20β-S-induced sperm hypermotility
To verify that mAC is involved in 20β-S-induced croaker sperm hypermotility, the ability of two cell-permeable mAC inhibitors to block this action of 20β-S was investigated in an in vitro sperm motility bioassay. Pretreatment of live sperm with both 100 µM dd-Ado and 500 µM SQ22536 was effective in blocking induction of sperm hypermotility by 20 nM 20β-S (Fig. 5Go, C and D). Similar to the effects of treatment with 20β-S, incubation of sperm with 10 µM forskolin resulted in an increase in the percentage of hypermotile sperm, which was blocked by pretreatment of sperm with 100 µM dd-Ado (Fig. 5CGo).

20β-S binding to croaker sperm membranes
To determine whether the 20β-S receptor on croaker sperm membrane is coupled to a G protein, sperm membranes were preincubated with GTP{gamma}S as described previously (18, 36). Treatment of GPCRs and mPR{alpha} with excess GTP{gamma}S causes activation and dissociation of the G protein-coupled to the receptor, resulting in decreased receptor binding (18, 36). The reduction in binding is thought to be due to the receptor reverting to a low-affinity ligand binding state when it is uncoupled from the G protein and also possibly through a reduction in the number of binding sites on the cell surface (39). In this case, preincubation of croaker sperm membranes with 12.5 µM GTP{gamma}S resulted in a significant decrease in binding of 10 nM [3H]20β-S compared with the controls (Fig. 6AGo). A similar trend was observed in membrane preparations incubated with 5 nM [3H]20β-S. Possible coupling of the progestin membrane receptor to a PTX-sensitive inhibitory G protein was investigated by determining the effects of pretreatment of sperm membranes with PTX on [3H]20β-S binding. Preincubation with 3 µg/ml activated PTX, and inactivated PTX for 20 min at 18 C did not alter [3H]20β-S binding to sperm membranes (Fig. 6BGo), suggesting the progestin membrane receptor is not coupled to a PTX-sensitive Gi protein in sperm. In contrast, the same PTX treatment, but not inactivated PTX, caused a significant decrease in [3H]20β-S binding to croaker ovarian membranes (Fig. 6CGo), consistent with previous studies, showing that PTX treatment blocks 20β-S-induced oocyte maturation in this species (40).


Figure 6
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  		FIG. 6. Effects of treatment with GTP{gamma}S and PTX on binding of [3H]20β-S to Atlantic croaker sperm membranes. Specific [3H]20β-S binding to croaker membranes was assessed after pretreatment of croaker sperm membranes with or without (control) 12.5 µM GTP{gamma}S (A) or whole sperm with 3 µg/ml PTX or heat-inactivated PTX (iPTX) (B). C, Effects of the same PTX treatments on specific [3H]20β-S binding to croaker ovarian membranes as a positive control. Data represent means ± SEM. Statistically significant differences from control samples (*, P < 0.05) were determined using a Student’s t test for each concentration of [3H]20β-S tested (n = 5) for GTP{gamma}S experiments and a one-way ANOVA and Dunnett’s multiple comparison posttest for PTX experiments (n = 3). Entire experiments were repeated at least three times.

 
Coexpression and association of Golf proteins and mPR{alpha} in croaker sperm
Immunocytochemical studies show that Golf and mPR{alpha} are coexpressed in the sperm midpiece and flagella (Fig. 7Go, A and B). In the studies performed, the percentage of sperm expressing Golf and mPR{alpha} on the midpiece and flagella were 92.8 ± 0.5 and 95.6 ± 2.6%, respectively. The specificity of the immunoreactions was confirmed by preabsorbing the antibodies against peptide antigens (Fig. 7Go, C and D). In addition, coimmunoprecipitation experiments demonstrated that mPR{alpha} and Golf are associated with one another. Immunoprecipitation of solubilized sperm membranes with the anti-Golf antibody, and subsequent Western blot analyses with the mPR{alpha} antibody, resulted in detection of a 73-kDa protein which corresponds to the size of the major mPR{alpha} protein (72.6 kDa) that has previously been demonstrated in croaker sperm membranes (17) (Fig. 7EGo). Protein bands of approximately this size are commonly observed with the mPR family of receptors and are thought to represent a mPR{alpha} dimer (18). Control blots confirmed the presence of Golf in sperm membranes and the proteins immunoprecipitated with the anti-Golf antibody (Fig. 7FGo). As a negative control, sperm membranes were incubated with normal rabbit IgG, which did not immunoprecipitate mPR{alpha} or Golf.


Figure 7
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  		FIG. 7. Coexpression and coimmunoprecipitation of Golf and mPR{alpha} in Atlantic croaker sperm. Immunocytochemical localization of Golf (A) and mPR{alpha} (B) (both in green) in croaker sperm using anti-Golf and anti-mPR{alpha} antibodies, respectively. C and D, Experiments performed after preabsorption of Golf and mPR{alpha} antibodies with peptide antigens. Nuclei (blue) were counterstained with 4',6-diamidino-2-phenylindole. Bar, 5 µm. Both experiments were repeated three times. E, Coimmunoprecipitation of Golf and mPR{alpha}. Solubilized sperm membranes were incubated with rabbit anti-Golf antibody (IP:Golf) immobilized on a commercial column. Immunoprecipitated proteins (3 µg) were eluted from the column and run on a 10% SDS-PAGE gel and analyzed by Western blot analyses using an antibody directed against mPR{alpha} (WB:mPR{alpha}). As a negative control, solubilized sperm membranes were incubated in a column with normal rabbit IgG (IP:IgG). As a positive control, freshly prepared sperm membranes (SM) were run on the same gel (SM). F, Control immunoblot confirming immunoprecipitation of the Golf protein. Results are typical of three independent experiments.

 
In vivo hormonal regulation of mPRa
Treatment of croaker with 100 µg/kg LHRHa for 24 h resulted in a significant increase in both basal and 20β-S-induced sperm hypermotility (Fig. 8Go, lower panel). This increase in the percent of sperm that were hypermotile was associated with an increase in mPR{alpha} protein abundance in croaker sperm membranes (Fig. 8Go, upper panel). Control groups receiving only an injection of saline showed no overall change in the percent hypermotile sperm or mPR{alpha} protein abundance.


Figure 8
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  		FIG. 8. In vivo hormonal regulation of Atlantic croaker sperm hypermotility and mPR{alpha} protein abundance. Sperm was collected from croaker before and after receiving an ip injection of LHRHa (100 µg/kg body weight) or 0.8% saline. Sperm was then analyzed for mPR{alpha} protein abundance by Western blot analyses and percent hypermotility after treatment with artificial seawater with 0.02% EtOH (control) or artificial seawater with 20 nM 20β-S. Upper panel, mPR{alpha} protein abundance in sperm membrane preparations before (0 h) and after (24 h) treatment with LHRHa or saline for three representative groups of croaker (each group comprises pooled sperm samples from three donors). Sperm hypermotility data in lower panel represent means ± SEM for all groups of croaker before (0 h) and after (24 h) control or LHRHa treatments. Statistically significant differences from 0 h control (A) or 0 h 20 nM 20β-S (B) for each treatment (P < 0.05) were determined using a one-way ANOVA and Dunnett’s posttest (n = 6).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we demonstrate that progestins activate a stimulatory G protein, Golf, in Atlantic croaker sperm membranes and that this is associated with progestin induction of sperm hypermotility. To our knowledge, the results of this study provide the first evidence that some progestin hormone actions in vertebrate sperm are mediated through activation of G proteins and that hormones can induce responses in target cells through activation of Golf. The finding that cAMP concentrations in croaker sperm membrane incubations were increased by 20β-S and that this effect was blocked by pretreatment with CTX and two mAC inhibitors, dd-Ado and SQ22536, suggests the signaling pathway induced by the progestin hormone involves activation of a stimulatory G protein and mAC. Moreover, mAC appears to be involved in progestin induction of sperm hypermotility because preincubation of croaker sperm with both dd-Ado and SQ22536 effectively blocked this stimulatory effect of 20β-S. Increases in cAMP concentrations by 20β-S were associated with activation of Golf, and no evidence was obtained for activation of Gs or Gi proteins in sperm membranes by this progestin. Golf and mPR{alpha} were coexpressed on the midpiece of croaker sperm and coimmunoprecipitated from sperm membranes. Finally, increased mPR{alpha} protein abundance was associated with increased 20β-S-induced hypermotility in croaker sperm. Taken together, these findings suggest that 20β-S activates Golf via mPR{alpha} to activate mAC and induce hypermotility in Atlantic croaker sperm.

The teleost progestin hormone, 20β-S, has previously been shown to act directly on sperm from Atlantic croaker and several other marine fishes in a concentration-dependent manner to induce hypermotility in vitro (6, 12, 41). 20β-S is thought to act through specific receptors that have been characterized biochemically on croaker and spotted seatrout sperm membranes (17, 34), resulting in rapid increases in intracellular cAMP and Ca2+ concentrations (6). Thus, the basic mechanism of progestin induction of sperm hypermotility in these teleosts is similar to that in humans and other mammals (13, 14, 15). A major advantage of teleost sperm models is that the signaling pathways involved in progestin-induced hypermotility can be studied in isolation without the complication of progestin pathways associated with the acrosome reaction because teleost sperm does not undergo this process (16). Hormonal treatment of teleost sperm in vitro also permits direct progestin effects on sperm motility to be distinguished from indirect genomic actions involving alterations in seminal fluid composition, which has been demonstrated in several fish species (42, 43).

Several lines of evidence suggest that the G protein activated by 20β-S and detected by the Golf antibody is Golf, not Gs, which share 88% amino acid sequence homology in mammals (21). The Golf antibody recognizes a 45-kDa protein on Western blots of olfactory epithelia from croaker and three other fish species: the sea lamprey, goldfish, and channel catfish (28, 29, 30). A similar 45-kDa protein was also detected in croaker sperm and testes, which corresponds to the size of the human Golf (44). In contrast, the Golf antibody did not cross-react with any of the protein bands on Western blots of croaker sperm and testicular tissue detected with the Gs antibody. Similarly, the Golf antibody failed to recognize any protein on Western blots of croaker ovarian tissue, which expresses Gs in croaker and a closely related species, the spotted seatrout (36, 45). Finally, the immunoprecipitation experiments showing that a Golf protein, but not a Gs protein, was activated by 20β-S provide further evidence for the specificity of the Golf antibody and the identity of the G protein activated by 20β-S.

The identification and localization of Golf in croaker sperm is in agreement with the results of previous studies in mammals. Golf has been found in human, rat, and mouse male germ cells and, as shown here with croaker sperm, is expressed in the midpiece and flagella of human and mouse sperm (23, 24, 46). The presence of various mACs has also been demonstrated in human, rat, and mouse male germ cells, and in particular, mACIII has been shown to colocalize with Golf in these species (23, 24, 31, 46). Interestingly, disruption of mACIII, which may be a target of activated Golf proteins, results in impaired sperm function in mice, including decreases in both sperm motility and fertilization success (47). Indirect evidence for the presence of mAC in fish sperm was obtained in the present study using forskolin and the inhibitors, dd-Ado and SQ22536. Thus, the presence of both Golf and mAC in sperm appears to be conserved in vertebrates. However, the precise physiological roles of this pathway in vertebrate sperm physiology remain to be clarified because in addition to progestin induction of sperm hypermotility in fish, Golf and mAC have been proposed to mediate sperm chemotaxis in response to odorants in mammals (23, 48, 49, 50, 51).

The relatively modest effect of 20β-S on motility of croaker sperm in vitro is likely to be due, at least partially, to prior activation of sperm hypermotility in response to progestin exposure in vivo. Croaker sperm seminal fluid contains nanomolar concentrations of 20β-S (Detweiler, C., and P. Thomas, unpublished observations), and circulating levels of 20β-S vary during the reproductive cycle in male croaker (52). Indeed, during the peak of spawning season when 20β-S levels are maximal, croaker sperm have greater than 80% hypermotile sperm after release into seawater (12), and treatment with 20β-S does not further stimulate sperm hypermotility (Thomas, P., unpublished observations). Experimental protocols were selected for the signaling studies that were physiologically relevant, rather than those that maximized the response. For example, increases in cAMP concentrations and induction of hypermotility occurred within 1 min in vitro, which is the period of time croaker sperm are viable once released into seawater (33). Furthermore, low nanomolar concentrations of 20β-S, which are similar to endogenous levels during the peak of spawning (52), were sufficient to induce sperm hypermotility (12) and changes in cAMP concentrations. The moderate increase in sperm motility in response to 20β-S appears to be physiologically important, however, because it results in increased fertilization success of croaker oocytes. Thus, these findings suggest an important role for 20β-S during spawning. At present, the source of 20β-S is not clear. One possibility is circulating 20β-S concentrations, which increase during spawning in male croaker, act on sperm in vivo to ensure a high degree of motility on release. However, females are also a source of 20β-S, which may be present in sufficient concentrations in ovarian fluid to induce sperm hypermotility during spawning.

The finding that the nuclear progestin receptor agonist, R5020, did not stimulate croaker sperm hypermotility or activate Golf in croaker sperm membranes suggests the involvement of a nonclassical progestin receptor in progestin induction of sperm hypermotility. Thus, the nuclear progesterone receptor, which has been proposed to mediate rapid actions of progestins in human sperm (53, 54), does not appear to be an intermediary in progestin induction of hypermotility of croaker sperm. Similarly, the finding that corticosterone, which binds the putative receptor, progesterone receptor membrane component 1 with high affinity (55, 56), was ineffective in stimulating sperm hypermotility and did not activate Golf in croaker sperm membranes suggests that it is not involved in this progestin action in croaker sperm. Moreover, both of these proteins are localized to the posterior head and acrosomal region of mammalian sperm, suggesting that they may be involved in progestin stimulation of the acrosome reaction. In contrast, several lines of evidence indicate that mPR{alpha} is the likely intermediary in progestin (20β-S) stimulation of croaker sperm hypermotility. First, a clear association was found between the abundance of the receptor protein on croaker sperm and the percent of hypermotile sperm. Donors with lower abundance of mPR{alpha} displayed a lower percentage of sperm that were hypermotile. Second, 20β-S was shown to activate a G protein in croaker sperm, as has been shown for other fish (3) and human mPR{alpha}s (57). The finding that 20β-S activates Golf in croaker sperm and that this G protein coimmunoprecipitated with mPR{alpha} and is also coexpressed with the receptor on the sperm midpiece is also consistent with an involvement of mPR{alpha} in progestin stimulation of sperm hypermotility.

The present results provide the first evidence that mPRs can activate multiple classes of G proteins. Previous studies have shown that mPR{alpha}s are coupled to PTX-sensitive inhibitory G proteins in a variety of target cells including fish oocytes (31), human myometrial cells (16), and human T cells in women (51). Moreover, recombinant seatrout and human mPR{alpha} also activate Gi in an eukaryotic breast cancer cell expression system and coimmunoprecipitate with the G protein (15). In contrast, activation of Golf through mPR{alpha} has been demonstrated only in fish sperm to date. Therefore, it remains unclear whether an association of mPR{alpha} with Golf is restricted to sperm or whether it also occurs in other cells in the male reproductive system or in nonreproductive tissues.

Perhaps one of the most significant findings of the present study is that steroid receptors activate Golf at low physiological concentrations to elicit a biological response. This finding further expands the repertoire of signal transduction pathways through which steroid hormones can act and may explain some of the pleiotropic actions of steroids, particularly their intraspecific actions as pheromones. It is noteworthy that urinary metabolites of 20β-S and other progestins secreted by a variety of female fish species are detected in the olfactory epithelia of conspecific males and act as pheromones, inducing both behavioral and hormonal responses (58, 59). Activation of Golf proteins has been reported previously only in the neuroepithelium (21) and sperm (23) in response to odorants and in the central nervous system in response to the neurotransmitter dopamine (60). The present results indicate that Golf proteins have a more widespread role in transducing extracellular chemical signals into intracellular ones and are also activated by steroid hormones. The functions of other tissues that express Golf, such as the pancreatic islets (22), are also influenced by steroid hormones. Therefore, steroids could potentially act in these tissues via a similar mechanism to that identified in croaker sperm through activation of Golf.


   Acknowledgments
 
We are especially grateful to Susan Lawson, Kelly Doughty, and Jing Dong for assistance with fish care, sperm motility analyses, and the PTX experiments, respectively. We also thank all who provided critical review and comments regarding the manuscript.


   Footnotes
 
This work was supported by the National Research Initiative Competitive Grant 2006-35203-17130 from the U.S. Department of Agriculture Cooperative State Research, Education and Extension Service (to P.T.) and the E. J. Lund Fellowship in Marine Science and the Laura Brooks Flawn, M.D., Endowment (to C.T.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 18, 2008

Abbreviations: CTX, Cholera toxin; dd-Ado, 2',5'-dideoxyadenosine; Gi, inhibitory G proteins; Golf, olfactory G protein; GPCR, G protein-coupled receptor; Gs, stimulatory family of G proteins; HAED, homogenization buffer of HEPES, NaCl, MgCl2, dithioerythritol, and EDTA; [3H]20β-S, [3H]17,20β,21-trihydroxy-4-pregnen-3-one; IBMX, 3-isobutyl-1-methylxanthine; iCTX, heat-inactivated CTX; LHRHa, LHRH analog; mAC, membrane adenylyl cyclase; mPR{alpha}, membrane progestin receptor-{alpha}; PTX, pertussis toxin; 20β-S, 17,20β,21-trihydroxy-4-pregnen-3-one; SDS, sodium dodecyl sulfate.

Received April 11, 2008.

Accepted for publication September 8, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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