- Email: [email protected]
Temperature adaptation changes ion concentrations in spermatozoa and seminal plasma of common carp without affecting sperm motility
Aquaculture 167 Ž1998. 85–94
Temperature adaptation changes ion concentrations in spermatozoa and seminal plasma of common carp without affecting sperm motility M. Emri b, T. Marian ´ ´ b, L. Tron ´ b, L. Balkay b, Z. Krasznai a
a,)
Department of Biophysics and Cell Biology, UniÕersity Medical School, PO Box 39, H-4012 Debrecen, Hungary b Positron Emission Tomography Centre, UniÕersity Medical School, Debrecen, Hungary Accepted 20 June 1998
Abstract Cold or warm adaptation usually results in changes of the cellular parameters of poikilothermic animals. However, no data are available about the changes in cellular parameters of sperm samples from cold or warm adapted animals. Here the effects of warm and cold adaptation on the spermation of common carp Ž Cyprinus carpio L.. and the changes in the characteristics of the individual sperm cells are described. Measurements were carried out on semen samples from 10 warm adapted and 10 cold adapted animals. The sperm cells from the cold adapted animals had a higher intracellular pH Ž7.4 " 0.1. than those from the warm adapted ones Ž7.1 " 0.1.. The pH of the seminal plasma of the cold adapted animals Ž8.6 " 0.2. was also higher than that of the warm adapted animals Ž8.3 " 0.1.. The concentration of spermatozoa in the semen of cold adapted animals was about half that for the warm adapted animals Ž0.7 " 0.1 = 10 10 vs. 1.4 " 0.2 = 10 10 cellsrml.. The Naq concentration of the seminal plasma of the cold adapted animals Ž83 " 12 mM. was higher, while the Kq concentration in these samples Ž64 " 11 mM. was lower than the corresponding data for the warm adapted animals Ž63 " 10 mM and 87 " 16 mM, respectively.. All of these differences proved to be significant at 5% level of significance using the Student’s t-test. In contrast, there was no significant difference between the intracellular free Kq concentrations in the spermatozoa from cold and warm adapted animals Ž58 " 8 mM vs. 60 " 7 mM.. The ion compositions and concentrations of the blood sera of cold and warm adapted animals were similar. Also, the motile fraction and duration of motility of the spermatozoa from cold and warm adapted animals were identical. An increase by 0.2 pH unit occurred in the intracellular pH during hypoosmotic shock induced motility of sperm cells from the cold and warm adapted animals. This
)
Corresponding author. Tel.: q36-52-412-623; Fax: q36-52-412-623; E-mail: [email protected]
0044-8486r98r$ - see front matter q 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 3 0 9 - 3
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pH increase could be blocked by the NaqrHq exchange inhibitor amiloride in a concentration of 100 mM. Based on the kinetics of the processes involved and on additional experimental evidence it is suggested that the hypoosmotic shock induced immediate hyperpolarization of the sperm under usual spawning conditions. Thus, it may be a regulatory step in the motility activation of common carp sperm but not in the relatively slowly occurring intracellular alkalinization. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Common carp sperm; Intracellular pH; Intracellular ion concentration; NaqrHq exchanger; Temperature adaptation; Seminal plasma composition; Sperm motility
1. Introduction Molecular mechanisms of fish spermatozoa activation represent one of the central research objectives in the field of developmental and reproductive biology. Although a large body of experimental data has been collected relating to fish sperm motility and its dependence on intracellular and extracellular parameters, many details of the underlying mechanisms are still unknown. Changes in the sperm environment have long been recognised to initiate sperm motility. It has been shown that intracellular and extracellular pH ŽMarian ´ ´ et al., 1997., as well as the ionic composition of the activating solution influenced the initiation and duration of the sperm motility ŽMorisawa et al., 1983; Morisawa, 1994; Redondo-Muller et al., 1991; Billard et al., 1995; Minamikawa and ¨ Morisawa, 1996.. Several groups reported similar effects of transmembrane potential ŽBoitano and Omoto, 1991; Zeng et al., 1995.. 4AP, a potassium channel blocker was shown to inhibit carp sperm motility ŽKrasznai et al., 1995.. A recently published review on the ionic composition data of the seminal fluid of carps ŽBillard et al., 1995. reported substantial variation in these concentrations. In addition to deviations between data by different authors an expressed difference was observed in sodium concentration data measured at different times throughout the year. These experimental findings directed our interest to the potassium, sodium and hydrogen ion distributions across the cytoplasmic membrane of carp sperm under physiological conditions and their relations to sperm motility. Keeping in mind the importance of providing good sperm motility for fish culture throughout the year, it was decided to carry out these investigations with both cold-adapted and warm-adapted carps.
2. Materials and methods 2.1. Chemicals and reagents The tetraacetoxymethyl ester of the dye 2X ,7-bis-Ž2-carboxyethyl.-5Žand-6.-carboxyfluorescein ŽBCECF-AM., propidium iodide ŽPI., nigericin and digitonin were from Molecular Probes ŽEugene, OR, USA.; amiloride and inorganic chemicals were from Sigma ŽSt. Louis, MO, USA..
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2.2. Buffers Prior to hypoosmotic shock, the semen was diluted with fish physiological solution ŽFPS: 140 mM NaCl, 5 mM KCl, 10 mM phosphate buffer, pH 7.4, osmolality: 305 mOsm. andror seminal plasma like solution ŽSPS: 70 mM NaCl, 70 mM KCl, 10 mM HEPES, pH 7.2, osmolality: 300 mOsm.. To activate the prediluted sperm cells, hypoosmotic sodium buffer ŽHSB: 50 mM NaCl buffered with 5 mM phosphate buffer, pH 7.4, osmolality: 110 mOsm. or hypoosmotic salt solution ŽHSS: 25 mM NaCl q 25 mM KCl buffered with 5 mM HEPES, pH 7.2, osmolality: 110 mOsm. was used. Tap water Ž0.08 mM Kq, 1 mM Naq, 1.8 mM Ca2q . was used for the hypoosmotic dilution of prediluted samples in routine motility tests of native carp sperm. 2.3. Semen collection Three-year-old male common carp Ž Cyprinus carpio L.. were cultured at Bocskai Fisheries Cooperative, Hajduszoboszlo ´ ´ according to standard technology ŽBakos and Gorda, 1995.. Sperm was collected in spring ŽMay–June., when the water temperature was above 208C for more than 14 days Žin a range of 20–228C., and in autumn ŽNovember., with the water temperature below 108C for more than 14 days Žin a range of 8–108C.. The semen was collected by gently pressing the abdomen 24 h after intramuscular injection of acetone-dried common carp pituitaries dissolved in FPS at a dose of 4.5 mgrkg body weight ŽMarian ´ ´ et al., 1993.. Contamination of the semen with water or urine was carefully prevented. Altogether, 40 male common carps were used in the experiments reported. 2.4. Motility testing The motile fraction of the activated sperm cells Žviability. and the duration of flagellar motion were determined by light microscopy. The semen was diluted 1:100 in SPS prior to microscopic motility testing. The prediluted sperm cells were activated by adding HSS in a 30-fold volume. The duration of fast movement was measured ŽBillard et al., 1995.. The reported data are the means of at least 3 independent measurements. 2.5. Ion concentration of seminal plasma and blood serum of common carp Semen was centrifuged at 1500 = g for 25 min and the supernatant was stored frozen until use. Blood samples were drawn from the caudal vein of the animals into heparinized tubes and centrifuged at 800 y g for 25 min, and the supernatant was stored frozen until use. Ion concentrations were determined in a Zeiss atomic absorption spectrophotometer. 2.6. Loading cells with pH indicator dye The stock solution of 1 mM BCECF-AM Žin DMSO. was kept at y208C. Sperm cells diluted in FPS or SPS Ž10–30 = 10 6 sperm cellsrml. were loaded with the pH indicator BCECF-AM Žat 5–7 mM final concentration. at 378C. After a 20-min
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incubation, the cells were pelleted and resuspended in FPS or SPS, followed by an additional incubation period at 378C for 20 min. After this step, the loaded cells were kept at room temperature until use within 1.5 to 2 h. BCECF loading did not result in any measurable change in the viability of the sperm ŽMarian ´ ´ et al., 1997.. 2.7. Flow cytometry A Becton Dickinson FACSstar Plus flow cytometer equipped with a Spectra Physics 164-08 argon-ion laser tuned to 488 nm and 500 mW power was used in the measurements. The fluorescence signal was electronically gated on the forward angle light scatter signal to exclude dead cells and cell debris from the analysis. The green fluorescence of the BCECF-loaded cells was measured through a combination of a 515-nm long pass and a 540-nm bandpass filter. Cells were run at a concentration of 1 = 10 6rml, at room temperature. 2.8. pHi measurement Intracellular pH ŽpH i . was determined by measuring the ratio of fluorescence intensities detected in two spectral regions ŽBalkay et al., 1992.. This method allows accurate pH i determinations on a cell-by-cell basis, and the data are not distorted by the dye contents of the suspending medium. Measurements by this method were normalized for standard cell volume and corrected for changes Žif any. in the geometry of the input–output optics of the flow cytometer. The method applied a standard calibration curve, which did not have to be reconstructed for each new set of measurements The optical system comprised a 515-nm long pass filter followed by a 50r50 beam splitter the former being used to block the excitation light. The first and second spectral regions of emission were further defined by a 540-nm broadband interference filter and a 620-nm long pass filter, respectively. Output signals from the red and green phototubes were routed into analog to digital converters and digitized data were stored in the memory of a computer in a correlated way ŽTron ´ et al., 1990.. pH i was calibrated versus Ž . Ž fluorescence ratio by using nigericin Thomas et al., 1979; Balkay et al., 1992.. 2.9. Calculation of intracellular free K q x was determined by measuring pH i in The intracellular free Kq concentration wKq i the presence of nigericin as described by Balkay et al. Ž1992, 1997. and Marian ´ ´ et al. Ž1997.. The ionophore adjusts pH i according to the relation: q q q Hq i rH e s K i rK e q q Žwhere Kq and Kq and Heq the i e are the intra-and extracellular free K , and Hi q q q intra-and extracellular H concentrations, respectively.. As w He x and w K e x are known parameters and pH i can be measured, Kq I can be calculated via the above equation. 2.10. Cell concentration Semen was prediluted in FPS to a final concentration of 2–4 = 10 6 sperm cellsrml. The membrane of the sperm cells was permeabilized with digitonin Ž10 mgrml. and the
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fluorescent DNA dye PI Ž30 mgrml. was added. Samples were then run on a Becton Dickinson FACSstar flow cytometer. The sperm concentration in the prediluted semen was determined from cell counts containing haploid DNA. The volume of the related fraction of the sample was calculated from the mass decrease during flow cytometric counting. 2.11. Statistical data eÕaluation Differences in pH as well as the intra-and extra-cellular concentrations of potassium and sodium ions of the cold and warm adapted animals were determined using Student’s t-test.
3. Results 3.1. Ion concentrations and pH of seminal plasma and blood serum Measured Naq and Kq ion concentrations in the seminal plasma and blood serum are listed in Table 1. An expressed increase in the Naq contents of the seminal fluid was found as a result of cold adaptation Žrange for spring samples: 50–80 mM; range for late autumn samples: 68–101 mM., accompanied by a similar decrease in Kq concentration. The pH of the seminal plasma of the cold adapted animals Ž8.6 " 0.16. was higher than that of the warm adapted ones Ž8.3 " 0.12.. All of these changes proved to be significant at a 5% level of significance, determined independently on semen from 10 cold adapted and 10 warm adapted animals using Student’s t-test. In contrast, cold or warm adaptation did not bring about any changes in pH or Naq or Kq concentrations in the blood serum: below 108C and above 208C, the pH was constant at 7.0, while the Naq and Kq concentrations were close to 145 and 2.8 mM, respectively.
Table 1 Ion concentrations and pH of seminal plasma and blood serum and intracellular pH and potassium concentration of sperm cells of cold and warm adapted common carp Sample
Warm-adapted
Cold-adapted
Seminal plasma wNaq x mM Seminal plasma wKq x mM Seminal plasma pH Blood serum wNaq x mM Blood serum wKq x mM Blood serum pH Intracellular wKq x of sperm cells Intracellular pH of sperm cells
63"10) 87"16) 8.3"0.12) 145"3 2.8"0.8 7.0"0.12 60"7 7.1"0.1)
83"12) 64"11) 8.6"0.16) 144"3 2.8"0.3 7.0"0.16 58"8 7.4"0.1)
Data are means"S.D. of results on samples from 10 animals. )Data belonging to warm- and cold-adapted animals are significantly different at a 5% level of significance using Student’s t-test.
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3.2. Spermatozoon concentration The spermatozoon concentrations of the ejaculates were measured by flow cytometry on PI-stained samples of digitonin-permeabilized prediluted sperm cells. The histograms exhibited a single sharp maximum, arguing for the homogeneous fluorescence distribution of a small coefficient of variation ŽFig. 1.. The semen samples of cold and warm adapted carp were found to contain 0.7 " 0.1 = 10 10 and 1.4 " 0.2 = 10 10 cellsrml, respectively. In addition, the semen of the cold adapted animals displayed a marked Ž5-fold. decrease in volume Ždata not shown..
Fig. 1. Three-dimensional fluorescence and light scatter histogram of PI-stained spermatozoa before digitonin treatment Žpanel A. and after permeabilization Žpanel B.. The sample was made from sperm of warm adapted common carp.
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3.3. K i and pHi of sperm cells There was no difference in the Kq i contents of the sperm cells from the cold and warm adapted animals ŽTable 1.. The pH i of common carp sperm was determined with a flow cytometric method ŽBalkay et al., 1992. originally developed for measurements of the pH i of mammalian cells with the fluorescent pH indicator dye BCECF. This method yielded pH i values for sperm cell samples which were significantly lower Ž p s 0.05. in the warm adapted animals than in the cold adapted ones ŽTable 1.. During hypoosmotic shock-induced sperm activation, a slight increase in pH i was measured ŽFig. 2.. The kinetics of the change was very similar in the cold-adapted and in the warm-adapted animals, but the amplitude of the changes was somewhat larger in the cold-adapted fish. This rise in pH proved to be significant at p s 0.05 level of significance using Student’s t-test Ž n s 10.. It could, in all cases, be blocked by the NaqrHq exchange inhibitor amiloride at a concentration of 100 mM, while hypoosmosis induced sperm motility was not affected by this concentration of the drug. There were no significant differences in the motile fraction of spermatozoa or the duration of motility of the sperm of cold and warm adapted animals. The duration of motility and motile fraction of hypoosmosis activated sperm of cold- and warm-adapted common carp were found to be 33.4 " 4.8 and 31.1 " 5.6 s and 82.6 " 8.6 and 82.4 " 7.7%, respectively.
Fig. 2. Rate of osmotic shock-induced change in intracellular pH of common carp spermatozoa. BCECF-loaded cells were diluted in HSS solution at time t s 0.
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4. Discussion Our data for the Naq and Kq concentrations Ž63 mM and 87 mM. in the seminal plasma of warm adapted carp ŽTable 1. are close to those of Morisawa et al. Ž1983.: Ž75 mM and 72 mM, respectively.. There were significant differences in the Naq and Kq concentrations of samples collected in the spring and in the autumn period ŽTable 1.. There have been reports on seasonal changes in the ion concentrations of the seminal plasma other species also. Munikittrick and Moccia Ž1987. observed a gradual halving of the Naq, Kq and Cly concentrations of the seminal plasma of rainbow trout during 3 months from the beginning of the spawning season. Redondo-Muller et al. Ž1991. ¨ examined the osmolality and the spermatozoa concentration Žspermatocrit value. of the seminal plasma of the common carp. Twenty samples collected from 6 different animals Žincluding repeated semen collection from the same male. under hormonally induced spermation revealed substantial differences in both the volume of semen and the spermatocrit value. The animals used in the present study were of the same genetic origin and had been cultured and matured under standard technological conditions ŽBakos and Gorda, 1995.. Thus, the differences observed in the ionic composition and spermatozoa concentrations of the seminal plasma would appear to arise primarily because of the different environmental conditions in the spring and late autumn seasons. The seminal plasma tended to be slightly alkaline. The cytoplasm was virtually neutral, but with a slight alkaline shift in the cold adapted animals. It has recently been reported that extreme intracellular and extracellular pH values Žbelow pH s 5.0 and above pH s 9.5 and extracellular Žbelow pH s 5.5 and above pH s 9.0, respectively. inhibit common carp sperm motility ŽMarian ´ ´ et al., 1997.. The slightly alkaline pH i for the cold adapted animals ŽpH i s 7.4. was far from the pH i range in which flagellar motion was inhibited ŽMarian ´ ´ et al., 1997.. This was in accordance with the very similar motility parameters measured for sperm collected from cold- or warm-adapted carps. The difference between the pH i values of spring and autumn carp sperm samples might be due to the lower metabolic activity at lower temperature or it could result from the temperature dependence of the cytoplasm ion channels and active transport mechanisms ŽNarK-ATP-ase.. Takai and Morisawa Ž1995. found osmolality dependent pH i values in the sperm cells of puffer fish using the null point pH method of Babcock et al. Ž1983.. They detected pH s 7.35 in isoosmotic buffer, and pH i s 6.9 under hyperosmotic conditions. In the carp, the sperm maintains pH i below the extracellular pH ŽpH e . in both the warm adapted and cold adapted state. Although seminal plasma pH values of 8.6 and 8.3 were measured for cold- and warm-adapted animals, they were not inhibitory, as the sperm tolerated extreme pH values from the extracellular side and started to decrease motility in the pH e range between 8.6 and 9.0 ŽMarian ´ ´ et al., 1997; Morisawa et al., 1983; Linhart et al., 1991 and Billard et al., 1995.. The available experimental evidence was insufficient to elicit the physiological role of the more alkaline pH i of cold-adapted relative to warm adapted sperm. Several groups have published data on sperm activation by intracellular alkalinization ŽWong et al., 1981; Babcock et al., 1983; Lee et al., 1983. in different species. Based on these findings alkalinization, it was suggested to be a conservative mechanism of activation of sperm motility ŽBabcock and Pfeiffer, 1987; Tombes and Shapiro, 1989.. This hypothesis has
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not been supported by the present findings. Although pH i increased during the activation of the sperm collected from both cold- and warm-adapted carp, this rise was a consequence of the activation, rather than the triggering signal itself as it was complete only after the motile phase had been over. Moreover, the pH i of resting sperm in cold adapted animals was at least as alkaline as the plateau value of the activated sperm collected from warm adapted animals ŽFig. 2.. Additional evidence against this mechanism as a conservative step in the activation of sperm motility was that artificial increase of pH i in common carp sperm did not result in activation ŽMarian ´ ´ et al., 1997.. Thus, a pH i change alone could clearly not regulate sperm motility in common carp. Our ion concentration measurements revealed almost identical Kq concentrations on the two sides of the cytoplasmic membrane of the immotile carp sperm in the seminal plasma. These data suggested a fully depolarised state of the spermatozoa, provided that the membrane potential is basically determined by the Kq distribution across the membrane. During motility activation the extracellular concentrations of Kq and Naq dropped immediately to the submillimolar range, resulting in prompt hyperpolarization, unless the Naq and Kq permeability of the cytoplasmic membrane was blocked to a high extent. This hyperpolarization could be one of the elementary steps in the sequence of events of sperm activation. The initial small effect could be amplified by the positive feedback of the consequent opening-up of voltage-regulated Kq channels. The observation that 4-AP, a potassium channel blocker, inhibited hypoosmosis-induced sperm motility ŽKrasznai et al., 1995., supported the above assumption. Further flagellar motion was an event of short duration and was initiated immediately after the dilution of the sperm in a hypoosmotic environment. This was not consistent with the slow alkalinization taking place on the 2–5 min time scale ŽFig. 2.. This conclusion was also in agreement with the suggestion that alkalinization was produced by a unique voltage-sensitive NaqrHq antiport mechanism ŽBabcock et al., 1992; Florman et al., 1992.. Boitano and Omoto Ž1991. found that membrane hyperpolarization activated trout sperm without an increase in pH i , a finding supporting the statement that the increase in the pH i was only a result of primary events triggering sperm activation.
Acknowledgements This work was partially supported by OTKA grants T 22435, T 6184, 13947, T 16149 and OMFB-JAP-5r98 grant.
References Babcock, D.F., Rufo, G.A., Lardy, H.A., 1983. Potassium-dependent increases in cytosolic pH stimulate metabolism end motility of mammalian sperm. Proc. Natl. Acad. Sci. U.S.A. 80, 1327–1331. Babcock, D.F., Pfeiffer, D.R., 1987. Independent elevation of cytosolic wCaq x and pH of mammalian sperm by voltage dependent and pH sensitive mechanism. J. Biol. Chem. 262, 15041–15047. Babcock, D.F., Bosma, M.M., Battaglia, D.E., Darszon, A., 1992. Early persistent activation of sperm Kq channels by the egg peptide speract. Proc. Natl. Acad. Sci. U.S.A. 89, 6001–6005.
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Bakos, J., Gorda, S., 1995. Genetic-improvement of common carp starin using intraspecific hybridization. Aquaculture 129, 183–186. Balkay, L., Marian, ´ ´ T., Emri, M., Tron, ´ L., 1992. A novel method to measure intracellular pH. Effect of neutron irradiation on pHi of transformed cells. J. Photochem. Photobiol. B Biol. 16, 367–375. Balkay, L., Marian, ´ ´ T., Emri, M., Krasznai, Z., Tron, ´ L., 1997. Flow cytometric determination of intracellular free potassium concentration. Cytometry 28, 42–49. Billard, R., Cosson, J., Perches, G., Linhart, O., 1995. Biology of sperm and artificial reproduction in carp. Aquaculture 129, 95–122. Boitano, S., Omoto, C.K., 1991. Membrane hyperpolarization activates trout sperm without an increase in intracellular pH. J. Cell Sci. 98, 343–349. Florman, H.M., Corron, M.E., Kim, T.D., Babcock, D.F., 1992. Activation of voltage dependent calcium channels of mammalian sperm is required for zona pellucida-induced acrosomal exocytosis. Dev. Biol. 125, 301–314. Krasznai, Z., Marian, ´ ´ T., Balkay, L., Gaspar, ´ ´ R. Jr., Tron, ´ L., 1995. Potassium channels regulate hypo-osmotic shock-induced motility of common carp Ž Cyprinus carpio . sperm. Aquaculture 129, 123–128. Lee, H.C., Epel, D., Johnson, C., 1983. Changes in internal pH associated with initiation of motility and acrosome reaction of sea-urchin sperm. Dev. Biol. 95, 31–45. Linhart, O., Slechta, V., Slavik, T., 1991. Fish sperm composition and biochemistry. Bull. Inst. Zoology, Ac. Sinica, Monograph 16, 285–331. Marian, ´ ´ T., Krasznai, Z., Balkay, L., Balazs, ´ M., Emri, M., Bene, L., Tron, ´ L., 1993. Hypoosmotic shock induces an osmolality dependent permeabilization and structural changes in the membrane of carp sperm. J. Histochem. Cytochem. 41, 291–298. Marian, ´ ´ T., Krasznai, Z., Balkay, L., Emri, M., Tron, ´ L., 1997. Role of extra-and intracellular pH in the sperm motility. Hyperosmosis modifies regulation of the NaqrHq exchanger. Cytometry 27, 374–382. Minamikawa, S., Morisawa, M., 1996. Acquisition, initiation and Maintenance of Sperm motility in the Shark, Triakis Scyllid. Comp. Biochem. Physiol. 113 Ž4., 387–392. Morisawa, M., 1994. Cell signalling mechanism for sperm motility. Zool. Sci. 11, 647–662. Morisawa, M., Suzuki, K., Shimizu, H., Morisawa, S., Yasuda, K., 1983. Effects of osmolality and potassium on motility of spermatozoa from freshwater cyprinid fishes. J. Exp. Biol. 107, 95–103. Munikittrick, K.R., Moccia, R.D., 1987. Seasonal changes in the quality of rainbow trout Ž Salmo gairdneri . semen: effect of a delay in stripping on spermatocrit, motility, volume and seminal plasma constitutions. Aquaculture 64, 147–156. Redondo-Muller, G., Cosson, M.P., Cosson, J., Billard, L., 1991. In vitro maturation of potential for ¨ movement of carp spermatozoa. Mol. Reprod. Dev. 29, 259–270. Takai, H., Morisawa, M., 1995. Change in intracellular Kq concentration caused by external osmolality change regulates sperm motility of marine and freshwater teleosts. J. Cell Sci. 108, 1175–1181. Thomas, J.A., Buchsbaum, R.N., Zimniak, A., Racker, E., 1979. Intracellular pH measurement in Erlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18, 2210–2218. Tombes, R.M., Shapiro, B.M., 1989. Energy transport and cell polarity: relationship and phosphagen kinase activity to sperm function. J. Exp. Zool. 251, 82–90. Tron, ´ L., Pieri, C., Marian, ´ ´ T., Balkay, L., Emri, M., Damjanovich, S., 1990. Bretilium causes a Kq-Naq pump activation that is independent of NaqrHq exchange in depolarized rat, mouse and human lymphocytes. Mol. Immunol. 27, 1307–1311. Wong, P.Y.D., Lee, W.M., Tsang, A.Y.F., 1981. The effects of sodium and amiloride on the motility of the caudal epidimal spermatozoa of the rat. Experientia 37, 69–71. Zeng, Y., Clark, E.N., Florman, H.M., 1995. Sperm membrane potential: hyperpolarization during capacitation regulates zona pellucida-dependent acrosomal secretion. Dev. Biol. 171, 554–563.
Temperature adaptation changes ion concentrations in spermatozoa and seminal plasma of common carp without affecting sperm motility M. Emri b, T. Marian ´ ´ b, L. Tron ´ b, L. Balkay b, Z. Krasznai a
a,)
Department of Biophysics and Cell Biology, UniÕersity Medical School, PO Box 39, H-4012 Debrecen, Hungary b Positron Emission Tomography Centre, UniÕersity Medical School, Debrecen, Hungary Accepted 20 June 1998
Abstract Cold or warm adaptation usually results in changes of the cellular parameters of poikilothermic animals. However, no data are available about the changes in cellular parameters of sperm samples from cold or warm adapted animals. Here the effects of warm and cold adaptation on the spermation of common carp Ž Cyprinus carpio L.. and the changes in the characteristics of the individual sperm cells are described. Measurements were carried out on semen samples from 10 warm adapted and 10 cold adapted animals. The sperm cells from the cold adapted animals had a higher intracellular pH Ž7.4 " 0.1. than those from the warm adapted ones Ž7.1 " 0.1.. The pH of the seminal plasma of the cold adapted animals Ž8.6 " 0.2. was also higher than that of the warm adapted animals Ž8.3 " 0.1.. The concentration of spermatozoa in the semen of cold adapted animals was about half that for the warm adapted animals Ž0.7 " 0.1 = 10 10 vs. 1.4 " 0.2 = 10 10 cellsrml.. The Naq concentration of the seminal plasma of the cold adapted animals Ž83 " 12 mM. was higher, while the Kq concentration in these samples Ž64 " 11 mM. was lower than the corresponding data for the warm adapted animals Ž63 " 10 mM and 87 " 16 mM, respectively.. All of these differences proved to be significant at 5% level of significance using the Student’s t-test. In contrast, there was no significant difference between the intracellular free Kq concentrations in the spermatozoa from cold and warm adapted animals Ž58 " 8 mM vs. 60 " 7 mM.. The ion compositions and concentrations of the blood sera of cold and warm adapted animals were similar. Also, the motile fraction and duration of motility of the spermatozoa from cold and warm adapted animals were identical. An increase by 0.2 pH unit occurred in the intracellular pH during hypoosmotic shock induced motility of sperm cells from the cold and warm adapted animals. This
)
Corresponding author. Tel.: q36-52-412-623; Fax: q36-52-412-623; E-mail: [email protected]
0044-8486r98r$ - see front matter q 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 3 0 9 - 3
86
M. Emri et al.r Aquaculture 167 (1998) 85–94
pH increase could be blocked by the NaqrHq exchange inhibitor amiloride in a concentration of 100 mM. Based on the kinetics of the processes involved and on additional experimental evidence it is suggested that the hypoosmotic shock induced immediate hyperpolarization of the sperm under usual spawning conditions. Thus, it may be a regulatory step in the motility activation of common carp sperm but not in the relatively slowly occurring intracellular alkalinization. q 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Common carp sperm; Intracellular pH; Intracellular ion concentration; NaqrHq exchanger; Temperature adaptation; Seminal plasma composition; Sperm motility
1. Introduction Molecular mechanisms of fish spermatozoa activation represent one of the central research objectives in the field of developmental and reproductive biology. Although a large body of experimental data has been collected relating to fish sperm motility and its dependence on intracellular and extracellular parameters, many details of the underlying mechanisms are still unknown. Changes in the sperm environment have long been recognised to initiate sperm motility. It has been shown that intracellular and extracellular pH ŽMarian ´ ´ et al., 1997., as well as the ionic composition of the activating solution influenced the initiation and duration of the sperm motility ŽMorisawa et al., 1983; Morisawa, 1994; Redondo-Muller et al., 1991; Billard et al., 1995; Minamikawa and ¨ Morisawa, 1996.. Several groups reported similar effects of transmembrane potential ŽBoitano and Omoto, 1991; Zeng et al., 1995.. 4AP, a potassium channel blocker was shown to inhibit carp sperm motility ŽKrasznai et al., 1995.. A recently published review on the ionic composition data of the seminal fluid of carps ŽBillard et al., 1995. reported substantial variation in these concentrations. In addition to deviations between data by different authors an expressed difference was observed in sodium concentration data measured at different times throughout the year. These experimental findings directed our interest to the potassium, sodium and hydrogen ion distributions across the cytoplasmic membrane of carp sperm under physiological conditions and their relations to sperm motility. Keeping in mind the importance of providing good sperm motility for fish culture throughout the year, it was decided to carry out these investigations with both cold-adapted and warm-adapted carps.
2. Materials and methods 2.1. Chemicals and reagents The tetraacetoxymethyl ester of the dye 2X ,7-bis-Ž2-carboxyethyl.-5Žand-6.-carboxyfluorescein ŽBCECF-AM., propidium iodide ŽPI., nigericin and digitonin were from Molecular Probes ŽEugene, OR, USA.; amiloride and inorganic chemicals were from Sigma ŽSt. Louis, MO, USA..
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2.2. Buffers Prior to hypoosmotic shock, the semen was diluted with fish physiological solution ŽFPS: 140 mM NaCl, 5 mM KCl, 10 mM phosphate buffer, pH 7.4, osmolality: 305 mOsm. andror seminal plasma like solution ŽSPS: 70 mM NaCl, 70 mM KCl, 10 mM HEPES, pH 7.2, osmolality: 300 mOsm.. To activate the prediluted sperm cells, hypoosmotic sodium buffer ŽHSB: 50 mM NaCl buffered with 5 mM phosphate buffer, pH 7.4, osmolality: 110 mOsm. or hypoosmotic salt solution ŽHSS: 25 mM NaCl q 25 mM KCl buffered with 5 mM HEPES, pH 7.2, osmolality: 110 mOsm. was used. Tap water Ž0.08 mM Kq, 1 mM Naq, 1.8 mM Ca2q . was used for the hypoosmotic dilution of prediluted samples in routine motility tests of native carp sperm. 2.3. Semen collection Three-year-old male common carp Ž Cyprinus carpio L.. were cultured at Bocskai Fisheries Cooperative, Hajduszoboszlo ´ ´ according to standard technology ŽBakos and Gorda, 1995.. Sperm was collected in spring ŽMay–June., when the water temperature was above 208C for more than 14 days Žin a range of 20–228C., and in autumn ŽNovember., with the water temperature below 108C for more than 14 days Žin a range of 8–108C.. The semen was collected by gently pressing the abdomen 24 h after intramuscular injection of acetone-dried common carp pituitaries dissolved in FPS at a dose of 4.5 mgrkg body weight ŽMarian ´ ´ et al., 1993.. Contamination of the semen with water or urine was carefully prevented. Altogether, 40 male common carps were used in the experiments reported. 2.4. Motility testing The motile fraction of the activated sperm cells Žviability. and the duration of flagellar motion were determined by light microscopy. The semen was diluted 1:100 in SPS prior to microscopic motility testing. The prediluted sperm cells were activated by adding HSS in a 30-fold volume. The duration of fast movement was measured ŽBillard et al., 1995.. The reported data are the means of at least 3 independent measurements. 2.5. Ion concentration of seminal plasma and blood serum of common carp Semen was centrifuged at 1500 = g for 25 min and the supernatant was stored frozen until use. Blood samples were drawn from the caudal vein of the animals into heparinized tubes and centrifuged at 800 y g for 25 min, and the supernatant was stored frozen until use. Ion concentrations were determined in a Zeiss atomic absorption spectrophotometer. 2.6. Loading cells with pH indicator dye The stock solution of 1 mM BCECF-AM Žin DMSO. was kept at y208C. Sperm cells diluted in FPS or SPS Ž10–30 = 10 6 sperm cellsrml. were loaded with the pH indicator BCECF-AM Žat 5–7 mM final concentration. at 378C. After a 20-min
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incubation, the cells were pelleted and resuspended in FPS or SPS, followed by an additional incubation period at 378C for 20 min. After this step, the loaded cells were kept at room temperature until use within 1.5 to 2 h. BCECF loading did not result in any measurable change in the viability of the sperm ŽMarian ´ ´ et al., 1997.. 2.7. Flow cytometry A Becton Dickinson FACSstar Plus flow cytometer equipped with a Spectra Physics 164-08 argon-ion laser tuned to 488 nm and 500 mW power was used in the measurements. The fluorescence signal was electronically gated on the forward angle light scatter signal to exclude dead cells and cell debris from the analysis. The green fluorescence of the BCECF-loaded cells was measured through a combination of a 515-nm long pass and a 540-nm bandpass filter. Cells were run at a concentration of 1 = 10 6rml, at room temperature. 2.8. pHi measurement Intracellular pH ŽpH i . was determined by measuring the ratio of fluorescence intensities detected in two spectral regions ŽBalkay et al., 1992.. This method allows accurate pH i determinations on a cell-by-cell basis, and the data are not distorted by the dye contents of the suspending medium. Measurements by this method were normalized for standard cell volume and corrected for changes Žif any. in the geometry of the input–output optics of the flow cytometer. The method applied a standard calibration curve, which did not have to be reconstructed for each new set of measurements The optical system comprised a 515-nm long pass filter followed by a 50r50 beam splitter the former being used to block the excitation light. The first and second spectral regions of emission were further defined by a 540-nm broadband interference filter and a 620-nm long pass filter, respectively. Output signals from the red and green phototubes were routed into analog to digital converters and digitized data were stored in the memory of a computer in a correlated way ŽTron ´ et al., 1990.. pH i was calibrated versus Ž . Ž fluorescence ratio by using nigericin Thomas et al., 1979; Balkay et al., 1992.. 2.9. Calculation of intracellular free K q x was determined by measuring pH i in The intracellular free Kq concentration wKq i the presence of nigericin as described by Balkay et al. Ž1992, 1997. and Marian ´ ´ et al. Ž1997.. The ionophore adjusts pH i according to the relation: q q q Hq i rH e s K i rK e q q Žwhere Kq and Kq and Heq the i e are the intra-and extracellular free K , and Hi q q q intra-and extracellular H concentrations, respectively.. As w He x and w K e x are known parameters and pH i can be measured, Kq I can be calculated via the above equation. 2.10. Cell concentration Semen was prediluted in FPS to a final concentration of 2–4 = 10 6 sperm cellsrml. The membrane of the sperm cells was permeabilized with digitonin Ž10 mgrml. and the
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fluorescent DNA dye PI Ž30 mgrml. was added. Samples were then run on a Becton Dickinson FACSstar flow cytometer. The sperm concentration in the prediluted semen was determined from cell counts containing haploid DNA. The volume of the related fraction of the sample was calculated from the mass decrease during flow cytometric counting. 2.11. Statistical data eÕaluation Differences in pH as well as the intra-and extra-cellular concentrations of potassium and sodium ions of the cold and warm adapted animals were determined using Student’s t-test.
3. Results 3.1. Ion concentrations and pH of seminal plasma and blood serum Measured Naq and Kq ion concentrations in the seminal plasma and blood serum are listed in Table 1. An expressed increase in the Naq contents of the seminal fluid was found as a result of cold adaptation Žrange for spring samples: 50–80 mM; range for late autumn samples: 68–101 mM., accompanied by a similar decrease in Kq concentration. The pH of the seminal plasma of the cold adapted animals Ž8.6 " 0.16. was higher than that of the warm adapted ones Ž8.3 " 0.12.. All of these changes proved to be significant at a 5% level of significance, determined independently on semen from 10 cold adapted and 10 warm adapted animals using Student’s t-test. In contrast, cold or warm adaptation did not bring about any changes in pH or Naq or Kq concentrations in the blood serum: below 108C and above 208C, the pH was constant at 7.0, while the Naq and Kq concentrations were close to 145 and 2.8 mM, respectively.
Table 1 Ion concentrations and pH of seminal plasma and blood serum and intracellular pH and potassium concentration of sperm cells of cold and warm adapted common carp Sample
Warm-adapted
Cold-adapted
Seminal plasma wNaq x mM Seminal plasma wKq x mM Seminal plasma pH Blood serum wNaq x mM Blood serum wKq x mM Blood serum pH Intracellular wKq x of sperm cells Intracellular pH of sperm cells
63"10) 87"16) 8.3"0.12) 145"3 2.8"0.8 7.0"0.12 60"7 7.1"0.1)
83"12) 64"11) 8.6"0.16) 144"3 2.8"0.3 7.0"0.16 58"8 7.4"0.1)
Data are means"S.D. of results on samples from 10 animals. )Data belonging to warm- and cold-adapted animals are significantly different at a 5% level of significance using Student’s t-test.
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3.2. Spermatozoon concentration The spermatozoon concentrations of the ejaculates were measured by flow cytometry on PI-stained samples of digitonin-permeabilized prediluted sperm cells. The histograms exhibited a single sharp maximum, arguing for the homogeneous fluorescence distribution of a small coefficient of variation ŽFig. 1.. The semen samples of cold and warm adapted carp were found to contain 0.7 " 0.1 = 10 10 and 1.4 " 0.2 = 10 10 cellsrml, respectively. In addition, the semen of the cold adapted animals displayed a marked Ž5-fold. decrease in volume Ždata not shown..
Fig. 1. Three-dimensional fluorescence and light scatter histogram of PI-stained spermatozoa before digitonin treatment Žpanel A. and after permeabilization Žpanel B.. The sample was made from sperm of warm adapted common carp.
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3.3. K i and pHi of sperm cells There was no difference in the Kq i contents of the sperm cells from the cold and warm adapted animals ŽTable 1.. The pH i of common carp sperm was determined with a flow cytometric method ŽBalkay et al., 1992. originally developed for measurements of the pH i of mammalian cells with the fluorescent pH indicator dye BCECF. This method yielded pH i values for sperm cell samples which were significantly lower Ž p s 0.05. in the warm adapted animals than in the cold adapted ones ŽTable 1.. During hypoosmotic shock-induced sperm activation, a slight increase in pH i was measured ŽFig. 2.. The kinetics of the change was very similar in the cold-adapted and in the warm-adapted animals, but the amplitude of the changes was somewhat larger in the cold-adapted fish. This rise in pH proved to be significant at p s 0.05 level of significance using Student’s t-test Ž n s 10.. It could, in all cases, be blocked by the NaqrHq exchange inhibitor amiloride at a concentration of 100 mM, while hypoosmosis induced sperm motility was not affected by this concentration of the drug. There were no significant differences in the motile fraction of spermatozoa or the duration of motility of the sperm of cold and warm adapted animals. The duration of motility and motile fraction of hypoosmosis activated sperm of cold- and warm-adapted common carp were found to be 33.4 " 4.8 and 31.1 " 5.6 s and 82.6 " 8.6 and 82.4 " 7.7%, respectively.
Fig. 2. Rate of osmotic shock-induced change in intracellular pH of common carp spermatozoa. BCECF-loaded cells were diluted in HSS solution at time t s 0.
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4. Discussion Our data for the Naq and Kq concentrations Ž63 mM and 87 mM. in the seminal plasma of warm adapted carp ŽTable 1. are close to those of Morisawa et al. Ž1983.: Ž75 mM and 72 mM, respectively.. There were significant differences in the Naq and Kq concentrations of samples collected in the spring and in the autumn period ŽTable 1.. There have been reports on seasonal changes in the ion concentrations of the seminal plasma other species also. Munikittrick and Moccia Ž1987. observed a gradual halving of the Naq, Kq and Cly concentrations of the seminal plasma of rainbow trout during 3 months from the beginning of the spawning season. Redondo-Muller et al. Ž1991. ¨ examined the osmolality and the spermatozoa concentration Žspermatocrit value. of the seminal plasma of the common carp. Twenty samples collected from 6 different animals Žincluding repeated semen collection from the same male. under hormonally induced spermation revealed substantial differences in both the volume of semen and the spermatocrit value. The animals used in the present study were of the same genetic origin and had been cultured and matured under standard technological conditions ŽBakos and Gorda, 1995.. Thus, the differences observed in the ionic composition and spermatozoa concentrations of the seminal plasma would appear to arise primarily because of the different environmental conditions in the spring and late autumn seasons. The seminal plasma tended to be slightly alkaline. The cytoplasm was virtually neutral, but with a slight alkaline shift in the cold adapted animals. It has recently been reported that extreme intracellular and extracellular pH values Žbelow pH s 5.0 and above pH s 9.5 and extracellular Žbelow pH s 5.5 and above pH s 9.0, respectively. inhibit common carp sperm motility ŽMarian ´ ´ et al., 1997.. The slightly alkaline pH i for the cold adapted animals ŽpH i s 7.4. was far from the pH i range in which flagellar motion was inhibited ŽMarian ´ ´ et al., 1997.. This was in accordance with the very similar motility parameters measured for sperm collected from cold- or warm-adapted carps. The difference between the pH i values of spring and autumn carp sperm samples might be due to the lower metabolic activity at lower temperature or it could result from the temperature dependence of the cytoplasm ion channels and active transport mechanisms ŽNarK-ATP-ase.. Takai and Morisawa Ž1995. found osmolality dependent pH i values in the sperm cells of puffer fish using the null point pH method of Babcock et al. Ž1983.. They detected pH s 7.35 in isoosmotic buffer, and pH i s 6.9 under hyperosmotic conditions. In the carp, the sperm maintains pH i below the extracellular pH ŽpH e . in both the warm adapted and cold adapted state. Although seminal plasma pH values of 8.6 and 8.3 were measured for cold- and warm-adapted animals, they were not inhibitory, as the sperm tolerated extreme pH values from the extracellular side and started to decrease motility in the pH e range between 8.6 and 9.0 ŽMarian ´ ´ et al., 1997; Morisawa et al., 1983; Linhart et al., 1991 and Billard et al., 1995.. The available experimental evidence was insufficient to elicit the physiological role of the more alkaline pH i of cold-adapted relative to warm adapted sperm. Several groups have published data on sperm activation by intracellular alkalinization ŽWong et al., 1981; Babcock et al., 1983; Lee et al., 1983. in different species. Based on these findings alkalinization, it was suggested to be a conservative mechanism of activation of sperm motility ŽBabcock and Pfeiffer, 1987; Tombes and Shapiro, 1989.. This hypothesis has
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not been supported by the present findings. Although pH i increased during the activation of the sperm collected from both cold- and warm-adapted carp, this rise was a consequence of the activation, rather than the triggering signal itself as it was complete only after the motile phase had been over. Moreover, the pH i of resting sperm in cold adapted animals was at least as alkaline as the plateau value of the activated sperm collected from warm adapted animals ŽFig. 2.. Additional evidence against this mechanism as a conservative step in the activation of sperm motility was that artificial increase of pH i in common carp sperm did not result in activation ŽMarian ´ ´ et al., 1997.. Thus, a pH i change alone could clearly not regulate sperm motility in common carp. Our ion concentration measurements revealed almost identical Kq concentrations on the two sides of the cytoplasmic membrane of the immotile carp sperm in the seminal plasma. These data suggested a fully depolarised state of the spermatozoa, provided that the membrane potential is basically determined by the Kq distribution across the membrane. During motility activation the extracellular concentrations of Kq and Naq dropped immediately to the submillimolar range, resulting in prompt hyperpolarization, unless the Naq and Kq permeability of the cytoplasmic membrane was blocked to a high extent. This hyperpolarization could be one of the elementary steps in the sequence of events of sperm activation. The initial small effect could be amplified by the positive feedback of the consequent opening-up of voltage-regulated Kq channels. The observation that 4-AP, a potassium channel blocker, inhibited hypoosmosis-induced sperm motility ŽKrasznai et al., 1995., supported the above assumption. Further flagellar motion was an event of short duration and was initiated immediately after the dilution of the sperm in a hypoosmotic environment. This was not consistent with the slow alkalinization taking place on the 2–5 min time scale ŽFig. 2.. This conclusion was also in agreement with the suggestion that alkalinization was produced by a unique voltage-sensitive NaqrHq antiport mechanism ŽBabcock et al., 1992; Florman et al., 1992.. Boitano and Omoto Ž1991. found that membrane hyperpolarization activated trout sperm without an increase in pH i , a finding supporting the statement that the increase in the pH i was only a result of primary events triggering sperm activation.
Acknowledgements This work was partially supported by OTKA grants T 22435, T 6184, 13947, T 16149 and OMFB-JAP-5r98 grant.
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