Activation of sperm motility in striped bass via Shuyang HeKaren Jenkins-KeeranL. Curry Woods III aDepartment of Animal and Avian Sciences, University of Maryland, bNational Institutes of Health, Bethesda, MD 20892, USA Received 12 March 2003; accepted 26 August 2003 The objective of the present study was to identify the effect of osmolality, ions (Kþ, Hþ, Ca2þ, Mg2þ) and cAMP on the initiation of sperm motility in striped bass (Morone saxatilis). Striped bassspermatozoa remained motile in solutions isotonic to seminal plasma (350 mOsm/kg) until osmol-ality reached 600 mOsm/kg. Kþ (0–100 mM) had no effect (P > 0:05) on sperm motility, and spermdisplayed a high percentage of motility over a wide range of pH (6.0–8.5). Sperm motility could beinitiated in Ca2þ-free solutions. In contrast, sperm motility was inhibited (P < 0:01) by solutionscontaining !10 mM Ca2þ, and sperm could not be reactivated by a Ca2þ-free solution. This Ca2þinhibition was not affected by verapamil, a Ca2þ channel blocker. However, if sperm motility was firstinitiated in a Ca2þ-free solution, the addition of Ca2þ solutions, up to 80 mM, failed to inhibit spermmotility, suggesting that Ca2þ inhibited the initiation of motility, but had no control of motilespermatozoa. Mg2þ solutions had similar inhibitory effects on sperm motility as Ca2þ solutions.
Therefore, initiation of motility in striped bass sperm may be related to voltage-gated channels acrossthe cell’s plasma membrane. Membrane permeable cAMP did not initiate motility of quiescent, intactstriped bass spermatozoa, and motility of demembranated sperm could be activated in the absenceof cAMP.
# 2003 Elsevier Inc. All rights reserved.
Keywords: Sperm motility; cAMP; Signal transduction; Striped bass Fish spermatozoa remain quiescent in gonadal seminal plasma in most species that have external fertilization. They become motile at spawning when expelled into the surrounding * Corresponding author. Tel.: þ1-301-405-7974; fax: þ1-301-314-9059.
E-mail address: [email protected] (L.C. Woods III).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2003.08.015 S. He et al. / Theriogenology 61 (2004) 1487–1498 water. Changes in the ionic and osmotic environment of the sperm cells have beenidentified as two critical external factors that may be responsible for initiating motilityin fish spermatozoa . Sperm motility of rainbow trout (Oncorhynchus mykiss) isregulated by Kþ concentration, and a Kþ concentration of 40 mM effectively inhibitedmotility In contrast to rainbow trout, many species examined appear to be regulatedin part by osmolality. Spermatozoa that are quiescent in solutions isotonic to theseminal plasma become motile when exposed to hypotonic environments in freshwaterteleosts and hypertonic environments in marine teleosts However, forstriped bass (Morone saxatilis), an anadromous species, we observed that sperm wereactivated by hypo-, iso-, and hypertonic (up to 600 mOsm/kg) sodium chloride solutions Numerous studies have shown that second messengers, such as cyclic adenosine monophosphate (cAMP) and Ca2þ, play key roles in sperm motility expression in manyanimal groups, such as mammals sea urchins and salmonid fish Intracellular cAMP concentration controls the net level of phosphorylation of certainspecific proteins, especially protein kinase A (PKA) , that directly leads to initiationof axoneme movement. Calcium increased cAMP through activation of adenylyl cyclase inthe spermatozoa of sea urchins and salmonid fish It has been generallyaccepted that external ionic and/or osmotic changes induce the alternation inintracellular concentration of calcium and/or cAMP, and the inhibition of motility mayoften be due to the inability of the spermatozoa to produce and/or maintain sufficient levelsof cAMP to stimulate PKA. However, it has been reported that cAMP and Ca2þ are notnecessary for the hypotonic induced initiation of sperm motility in the common carp(Cyprinus carpio), and no inhibitory effect of Ca2þ fluxes at the plasma membrane havebeen observed with sperm from this species Striped bass spermatozoa are used to fertilize the eggs of white bass (Morone chrysops) to produce hybrid striped bass for the striped bass aquaculture industry. This industryincreased almost 10-fold from 1986 to 1995 , and is now the fourth largest finfishspecies by value in the US Cryopreservation of striped bass spermatozoa can aid theindustry by amelioration of problems that striped bass and white bass spawning seasons aswell as geographic locations are asynchronous. Banked sperm from striped bass can beshipped and stored on site where white bass females are environmentally conditioned tospawn year-round in hatcheries.
It is critical to determine what controls the activation of striped bass spermatozoa. Since the duration of motility in high quality striped bass spermatozoa ranges only from 30 to60 s , it is critical that sperm are not prematurely activated prior to fertilization orcryopreservation. Because striped bass sperm are activated by isotonic solutions, wecurrently utilize hypertonic cryomedia solutions prior to freezing. However, such solutionscarry great risk associated with severe cellular dehydration during periods of equilibrationprior to freezing . Development of isotonic solutions that will not activate striped basssperm would provide good opportunity, through increased time, to manipulate and improvecryopreservation protocols and post-thaw gamete quality at fertilization.
The plasma membranes of fish spermatozoa can be partially removed by detergents, such as Triton X-100 This process is defined as demembranation, which allowsthe axoneme to come directly into contact with the external environment. In the present S. He et al. / Theriogenology 61 (2004) 1487–1498 study, both fresh and demembranated striped bass spermatozoa were applied to evaluate theeffects of osmolality, various ions (Kþ, Hþ, Ca2þ, Mg2þ) and dibutyryl cAMP (dbcAMP)on the initiation of sperm motility, in our efforts to develop an isotonic extender that mightkeep striped bass sperm immotile prior to freezing.
All reagents, unless otherwise stated, were purchased from Fisher Scientific Interna- tional Inc. (Atlanta, GA). Three-year-old striped bass males were randomly selected from apopulation maintained under ambient photothermal conditions of the previously describedflow-through, 40,000-l circular tank system at the University of Maryland’s CraneAquaculture Facility. Water temperature ranged from 5 to 30 8C during the year. In thespring, males were moved into a 6400-l circular tank, part of a recirculating water system,and held at 15 Æ 1 8C for the remainder of the 5-week study. Each fish was given acholesterol cellulose implant containing 150 mg of mammalian gonadotropin-releas-ing hormone (Sigma Chemical Co., St. Louis, MO) inserted into the dorsal lymphatic sinus,as previously described for striped bass Three days after administering theimplant, the fish were anesthetized in a 70 mg/l quinaldine bath and urine wasremoved by applying gentle pressure around the urogenital vent. Semen was expresseddirectly into 50 ml sterile conical tubes and placed immediately on ice. In each experiment,only striped bass sperm samples (n ¼ 5) exhibiting !90% motility were used. Bloodsamples were drawn from the caudal vasculature using heparinized needles (21 gauge) andsyringes (3 ml).
To estimate the percentage of motile sperm, aliquots of semen (approximate 0.01 ml) were placed into a Makler counting chamber (TS Scientific Inc. Perkasie, PA) and quicklymixed with 10 ml of the treatment activating solution. The loaded Makler chamber wasimmediately placed under a Zeiss model D-7082 compound microscope (Berlin, Germany)at 400Â. The activation of each sample was recorded on videotape using a Hitachi ModelKP-140 video camera (Tokyo, Japan). The percentage of motile sperm was determined byreviewing videotapes and counting spermatozoa (approximately 200–800 cells), thendividing the number of motile sperm by the total number of sperm cells in the field of view.
Spermatozoa that simply vibrated or did not show progressive forward movement werenot considered in the estimates of motility, as recommended by Billard and Cosson To test the effect of Kþ, Mg2þ, or dbcAMP (Sigma Chemical Co.), fresh sperm wereactivated by the solution containing Kþ, Mg2þ (0–100 mM) or 0.1 mM dbcAMP. Totest the effect of pH, fresh sperm were activated by NaCl solution (100 mOsm/kg) withvarious pH values from 4.0 to 9.0, adjusted with 1N HCl. To test the effect of Ca2þ on themotility of intact sperm, two methods were used. (i) The semen was diluted 50-fold in aCa2þ-free solution (240 mM NaCl, 10 mM KCl, 3 mM NaHCO3, 0.5 mM EDTA, pH 7.6) S. He et al. / Theriogenology 61 (2004) 1487–1498 and centrifuged at 800 Â g for 5 min. The precipitate was suspended in the same volumeof Ca2þ-free solution, and 1 ml of this suspension was mixed with 10 ml activatingsolution containing various concentrations of Ca2þ (0–100 mM). (ii) The semen wasdiluted 50-fold in a calcium solution (240 mM NaCl, 10 mM KCl, 1 mM CaCl2, 3 mMNaHCO3, pH 7.6) with or without 100 mM of the calcium channel blocker verapamil(Sigma Chemical Co.) and incubated for 30 min, then the suspension was mixed withactivating solution containing the same concentration of verapamil and 20 mM Ca2þ.
Sperm motility was estimated in each test as described above. The duration of spermmotility was timed beginning with the addition of activation solutions to the sperm sampleand ending when the majority (approximately 90%) of sperm in the field of view hadstopped moving.
2.3. Demembranation and reactivation of spermatozoa To partially remove the plasma membranes of the spermatozoa, 10 ml of semen was added to 990 ml of demembranation solution containing 240 mM NaCl, 10 mMKCl, 0.5 mM EDTA, 5 mM Tris–HCl, and 0.04% (w/v) Triton X-100, and havinga pH of 7.6. After 30 s, 1 ml of the demembranated spermatozoa were placed into theMakler counting chamber and quickly mixed either with 10 ml reactivating solutioncontaining 0.5 mM EDTA, 5 mM Tris–HCl and 1 mM Mg-ATP (Sigma Chemical Co.)as well as various concentrations of NaCl and/or KCl (pH of 7.6) or with the samereactivating solution but without Mg-ATP. To verify that the plasma membranes ofspermatozoa were partially broken by the demembranation solution, propidium iodide(12 mM), a fluorescent dye, was used. Spermatozoa stained red with propidium iodideindicated that plasma membranes were not intact. Stained samples were analyzedwith a Zeiss Model Axioplan 2 imaging epifluorescent microscope (Berlin, Germany)at 400Â.
Semen and blood samples were centrifuged at 8000 Â g for 30 min, using a Heraeus Model 400-R centrifuge (Hanau, Germany) at 4 8C. Plasmas were decanted, placed in1.5 ml snap-cap vials, and kept on ice for 30 min before analyses were performed.
Osmolality and pH were measured using a Wescor Model 5400 vapor pressure osmometer(Logan, UT) and a Hach Model Sension 2 pH electrode (Loveland, CO), respectively.
Sodium, potassium, and calcium were measured using a Perkin-Elmer Model 5100 PCatomic absorption spectrophotometer (Shelton, CT).
Statistical analysis was performed with SAS version 8.0 (SAS Institute, Inc., Cary, NC) Data are presented as means Æ standard error of the mean (S.E.M.). The assumptionsof normality and homogeneity of variances were verified before the parametric tests. Datawere analyzed by one-way analysis of variance (ANOVA), and pairwise contrasts wereused to identify between means (5% level).
S. He et al. / Theriogenology 61 (2004) 1487–1498 3.1. Effect of osmolality on intact or demembranated spermatozoa Striped bass spermatozoa were fully motile (!90%) immediately after dilution in NaCl or KCl solutions with an osmolality between 40 and 300 mOsm/kg (Increasing theosmolality >300 mOsm/kg decreased the percentage of motile sperm, although approxi-mately 60% spermatozoa were motile in solutions isotonic to striped bass seminal plasma(350 mOsm/kg). Sperm motility was not completely inhibited until osmolality reached600 mOsm/kg. After measurement of the compositions of Naþ and Kþ in seminal plasmaof striped bass (a solution containing both NaCl and KCl at a comparable 2:1 ratioof Naþ:Kþ was tested. This solution exhibited the same effect on striped bass spermmotility as the NaCl or KCl solutions alone.
Fluorescent staining using propidium iodide confirmed that striped bass spermatozoa were demembranated by Triton X-100 (data not shown). In the absence of ATP,demembranated spermatozoa could not be activated. When ATP was added to thereactivating solution, demembranated spermatozoa motility showed the same trend asintact spermatozoa when exposed to increasing osmolality (). However, theduration of movement lasted only 15–30 s, compared with 30–60 s in intact sperma-tozoa.
3.2. Effect of Hþ, Ca2þ, Mg2þ on sperm motility The percentage of motile striped bass sperm was high (!90%) over a broad range of pH values ). Sperm motility decreased (P < 0:05) when pH was <6.0 or >8.5. At a pH of4.5, only 20% of sperm were motile and those moved primarily in small circulartrajectories, compared with primarily linear and to a lesser degree, large circular trajec-tories when the pH was between 6.0 and 8.5. Additionally at a pH of 4.5, sperm movementdecreased rapidly and all cells were immotile after 15 s.
The Ca2þ concentration of seminal plasma in striped bass was very low (0:5 Æ 0:2 mM), compared to its concentration in blood plasma (2:8 Æ 0:1 mM; ), and seminalplasma in other species such as rainbow trout (2:6 Æ 0:19 mM) and common carp(2:0 Æ 0:18 mM) . Sperm motility was significantly inhibited with 10 mM or higherconcentration of Ca2þ ion in the activating solution ). Only 20–30% of sperm were Table 1Ion concentration (means Æ S:E:M:) in striped bass seminal and blood plasma Within a column, values with different superscripts (a and b) are different (P < 0:05).
S. He et al. / Theriogenology 61 (2004) 1487–1498 Fig. 1. (a) The percentage of motile sperm (means Æ S:E:M:) as a function of osmolality and composition of theactivating solution. Activating solution contained NaCl, KCl, and the mixture of NaCl and KCl with a ratio of2:1 (Naþ:Kþ), that mimicked the ratio of Naþ:Kþ in seminal plasma. (b) The percentage of motiledemembranated sperm (means Æ S:E:M:) as a function of osmolality and composition of the activating solution.
Activating solution contained NaCl, KCl, and the mixture of NaCl and KCl with a ratio of 2:1 (Naþ:Kþ), thatmimicked the ratio of Naþ:Kþ in seminal plasma.
S. He et al. / Theriogenology 61 (2004) 1487–1498 Fig. 2. The percentage of motile sperm (means Æ S:E:M:) as a function of extracellular pH. Sperm wereactivated by distilled water.
motile at a concentration of 40 mM Ca2þ; furthermore, cells that were moving appearedvery slow or weak. The pattern of movement also changed in the presence of a highconcentration of extracellular Ca2þ. Sperm swam in small circular trajectories with veryshort durations (average, 10–15 s), similar to those described above for low pH. Thiscalcium inhibition of motility was not reversible. Addition of distilled water failed toactivate sperm previously diluted in the 40 mM Ca2þ solution (However, even theaddition of Ca2þ concentrations as high as 80 mM did not inhibit sperm motility, oncesperm were activated by distilled water (Verapamil, a voltage-dependent calciumchannel blocker, had no effect on sperm motility in the presence of 20 mM Ca2þ (data notshown). High concentrations of Mg2þ ions also inhibited sperm motility and thisinhibition could not be overcome by dilution with distilled water.
Spermatozoa held under various osmolalities or Ca2þ concentrations were not affected by the addition of dbcAMP The dbcAMP did not induce the initiation of spermmotility in the NaCl solution with an osmolality of 600 mOsm/kg. No dbcAMP effect(P > 0:05) on sperm motility was detected in NaCl solutions with osmolalities of 350 or500 mOsm/kg, or in the 40 mM Ca2þ solution.
S. He et al. / Theriogenology 61 (2004) 1487–1498 Fig. 3. The percentage of motile sperm (means Æ S:E:M:) as a function of extracellular Ca2þ or Mg2þconcentration. To evaluate the effect of Ca2þ, sperm suspended in Ca2þ-free Solution were activated by theactivating solution containing various Ca2þ concentrations. To evaluate the effect of Mg2þ, sperm were activatedby the activating solution containing various Mg2þ concentrations.
Table 2Effect of dbcAMP on sperm motility (means Æ S:E:M:) Fresh semen was activated in solutions with or without (control) 0.1 mM cAMP. Activation solutions includedNaCl solution with various osmolalities (350, 500, or 600 mOsm/kg), and CaCl2 solution with 40 mM Ca2þ.
Within a column, values with different superscripts (a and b) are different (P < 0:05).
In the present study, motility was triggered in >50% spermatozoa by experimental solutions isotonic to seminal plasma, regardless of the Kþ concentration of thosesolutions. However, striped bass spermatozoa are immotile in seminal plasma Thissuggests that inhibition of motility in striped bass seminal plasma may not be controlledby Kþ ion concentration, nor by osmolality. The broad range of osmolalities that can S. He et al. / Theriogenology 61 (2004) 1487–1498 Fig. 4. The relationship between extracellular Ca2þ concentration and initiation/continuity of sperm motility(means Æ S:E:M:). In line a, sperm suspended in Ca2þ-free solution were diluted with 40 mM CaCl2 at t ¼ 0 s.
At t ¼ 15 s, distilled water was added to the suspension. In line b, sperm suspended in Ca2þ-free solution werediluted with distilled water at t ¼ 0 s. At t ¼ 15 s, 80 mM CaCl2 was added to the suspension.
activate striped bass sperm may be explained by the fact that striped bass is an anadromousspecies, which migrates from saltwater to freshwater to spawn. To adapt to environmentalconditions with such a large variation in osmolality, striped bass sperm may havedeveloped a mechanism by which activation of sperm motility could be initiated in eitherfresh or brackish waters. A similar effect of osmolality on sperm motility, where spermremained motile in isotonic solutions (approximately 300 mOsm/kg) was observed insalmonids, and that motility was not completely inhibited until the osmolality reached400 mOsm/kg In the present study, demembranated striped bass spermatozoa exhibited the same characteristic patterns of motility as intact spermatozoa, when both were exposed tosolutions of various osmolalities. Furthermore, axoneme motility was inhibited in thesolution with an osmolality of 600 mOsm/kg. Axoneme motility in demembranatedcommon carp spermatozoa was also resistant to inhibition until osmolality approached600 mOsm/kg, even though intact spermatozoa remained immotile when osmolality washigher than isotonic values of 250–300 mOsm/kg It has been reported that highosmolality could directly inhibit axoneme motility in common carp through increased S. He et al. / Theriogenology 61 (2004) 1487–1498 viscosity of the solution This suggests that variations in the extracellular osmolalitywere not directly detected by the common carp spermatozoa axoneme because the plasmamembrane acted as a barrier. However, the barrier function of the plasma membrane ofstriped bass spermatozoa appears to be bypassed, since the axoneme of intact spermatozoareacted to osmolality the same as that of demembranated spermatozoa.
It has been shown that Ca2þ is a key factor in sperm motility activation in many species.
In trout, when the concentration of free Ca2þ was reduced below 10À9 M (due to additionof EDTA), motility was completely inhibited, but it could be restored by the addition ofCa2þ . In common carp, the results were less clear. While Perchec-Poupard et al. indicated that initiation of motility in common carp spermatozoa was independent of Ca2þions, Krasznai et al. concluded that the Ca2þ influx from the extracellular environmentwas the trigger of the initiation of motility in the same species. In the present study, weinferred that the initiation of motility in striped bass spermatozoa does not require thepresence of extracellular Ca2þ. However, since the intracellular Ca2þ concentration wasnot monitored, we cannot rule out the potential role of intracellular Ca2þ for the initiationof sperm motility. Instead of stimulating sperm motility, high concentrations of extra-cellular Ca2þ inhibited motility, shortened the duration and modulated the swimmingpattern. The sperm of sea bass (Dicentrarchus labrax) and bull (Bos taurus) hadthe same swimming pattern of asymmetrical flagellar beating and circular trajectorymovement that was directly induced by accumulation of intracellular Ca2þ. This, togetherwith the observation that diluting the Ca2þ concentration in the activation solution failed toreactivate striped bass sperm motility, indicated that inhibition may be related to Ca2þ ioninflux into spermatozoa. Mg2þ concentration also had the same patterns of sperm motilityinhibition. Therefore, inhibition may be associated with divalent cations and the associatedvoltage-gated channels in the plasma membrane. Similar membrane channels may bepresent in trout spermatozoa. Membrane channels for divalent cations, including Ca2þMg2þ and Sr2þ may be present in trout spermatozoa. It was unclear whyCa2þ concentration in striped bass seminal plasma was so low. We inferred that striped bassspermatozoa may transport extracellular Ca2þ into the cells to maintain a certain level ofintracellular Ca2þ concentration, which may be important for normal sperm function.
In addition to the Ca2þ, cAMP has been implicated as an activator of sperm through its protein phosphorylation pathway. Morisawa and Okuno first demonstrated that cAMPwas required before ATP can trigger the initiation of sperm motility in rainbow trout.
However, present results provided evidence that cAMP did not play a major role in theinitiation of sperm motility in striped bass, because the membrane permeable cAMPanalog, dbcAMP, was not effective for initiating striped bass sperm motility. Furthermore,demembranated sperm could be reactivated using solution that did not contain dbcAMP.
In conclusion, the mechanism for the initiation of striped bass sperm motility differs from those previously described for other fish such as the rainbow trout and the commoncarp. Further experimentation is needed to develop isotonic solutions that will not activatestriped bass sperm. For example, measuring ionic influx into the sperm cells could providemore information on ionic effects. Future studies to identify plasma membrane channelsin striped bass spermatozoa, such as mechanically-activated channels may alsoprovide useful information that may elucidate the mechanism for activation of striped bassspermatozoa.
S. He et al. / Theriogenology 61 (2004) 1487–1498 We thank Dr. William King for his assistance in the critical review of this manuscript.
Thanks also go to Daniel Theisen, Chongmin Wang, and Daniel Castranova of ourlaboratory for providing care and husbandry of the mature striped bass.
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