Ivan Aranha, M Bhagya * & H N Yajurvedi

Transcription

1 Indian Journal of Experimental Biology Vol. 46, October 2008, pp Concentration of cations in different parts of male reproductive system and their influence on in vitro sperm motility in lizard, Mabuya carinata Schneider. Ivan Aranha, M Bhagya * & H N Yajurvedi Department of Zoology, University of Mysore, Manasagangotri, Mysore , India Received 15 February 2007; revised 18 August 2008 Concentrations of mono- and divalent cations in the testis, three different regions of the epididymis and the vas deferens of lizard, M. carinata were determined. The concentrations of Na +, K + and Ca 2+ increased gradually from testis to vas deferens. However, no significant variation in the levels of Mg 2+ was observed between testis and anterior epididymis, and between posterior epididymis and vas deferens. Further, in an in vitro sperm motility study, wherein different concentrations of the cations were tested, the maximum forward progressive motility of posterior epididymal spermatozoa was observed following treatment with 140 mm NaCl, 25 mm KCl, and 50 mm CaCl 2 whereas, treatment with magnesium brought about a decrease in sperm motility. The results indicate that Na +, K +, and Ca 2+ have stimulatory effect at an optimum dose, whereas Mg 2+ has an inhibitory effect even at very low concentration, on sperm motility in the lizard M. Keywords: Cations, Lizard, Mabuya carinata, Sperm motility Testicular spermatozoa lack motility and fertilizability, which they acquire by the time they reach the tail of the epididymis 1. The acquisition of motility by the spermatozoa is associated with changes in composition of epididymal fluid from caput to cauda regions of the epididymis in mammals 2. Like mammals, lizard spermatozoa, which are non-motile while in the testis, develop the abilities of fertilizability and motility as they pass through the excurrent duct system 3. Ions are important constituents of epididymal fluid and exhibit regional variations in their concentrations and influence the motility of spermatozoa in vertebrates 4-7. In reptiles, though the epididymis is not morpho-logically differentiated as in mammals, anterior, middle and posterior regions of the epididymis in lizards Lacerta vivipara 3, Hemidactylus flaviviridis 6, Mabuya carinata 8 and Sitana ponticeriana 9 have been reported and these regions are histologically comparable to caput, corpus and cauda of mammalian epididymis, respectively. The fact that spermatozoa exhibit region-specific flagellar movements indicates the influence of intraluminal factors in inducing sperm motility 10. To date, regional differences in ion concentration and the contribution of epididymal *Correspondent author Telephone: mbhagya9@rediffmail.com luminal ions in inducing motility in testicular spermatozoa are reported only in one species of reptile, the wall lizard H. flaviviridis 11. The vas deferens is another accessory reproductive structure, which has been endowed with role in sperm maturation in the mammals. However, the ionic composition of luminal fluid of vas deferens and its comparison with that of epididymis and testis have not been studied in reptiles. Comparison of ionic concentration of luminal fluids of testis, epididymis and vas deferens, and in vitro demonstration of effects of different concentrations of different ions on sperm motility may reveal the role of different cations in inducing motility. Since, reptiles form pivotal groups in vertebrate phylogeny, studies on these lines are necessary to understand evolution of sperm maturation mechanism in the vertebrates. Hence, the present study has been aimed at investigating differences, if any, in the concentration of Na +, K +, Ca 2+ and Mg 2+ in the testis, three different regions of the epididymis and vas deferens of the lizard Mabuya carinata Schneider, and the effects of concentrations of different ions on posterior epididymal sperm motility in vitro. Materials and Methods Estimation of mono- and divalent cations Presence of two dark reddish orange coloured bands on ventral body surface of male lizards Mabuya

2 ARANHA et al.: CATIONS AND SPERMATOZOA MOTILITY IN LIZARD MABUYA CARINATA 721 carinata Schneider (Family: Scincidae) is the indication of sexual maturity. Five such lizards were collected from in and around Mysore city (12 18' N latitude, 76 12' E longitude), India, in the month of November (breeding season 8 ). Lizards were sacrificed under light ether anaesthesia. Testis, three regions of epididymis, and vas deferens were dissected out, weighed separately and their luminal fluids were collected by flushing the tissues in deionized water (1 ml/mg tissue). The tunica albugenia of the testis was removed, the seminiferous tubules were punctured and the contents were released into deionized water. The suspensions were then centrifuged at 600 g for 15 min at 4 C. The supernatants were used for the estimation of Na +, K +, Ca 2+ and Mg 2+ using an Atomic Absorption Spectrophotometer (Perkin Elmer, Norwalk, Connecticut, USA). Effect of cations on sperm motility in vitro To study the influence of ions on sperm motility, the spermatozoa collected from the posterior segment of the epididymis were used. As in the pilot study the maximum number of spermatozoa from this region showed forward progressive motility after in vitro activation compared to those from other regions of the epididymis. Sperm suspension was made at the final concentration of sperm/ml following the method of Nirmal and Rai 10. Sperm suspension was made in K + free phosphate buffered saline (PBS: 120 mm NaCl, 1.67 mm NaH 2 PO 4, 8.1 mm Na 2 HO 4 ; ph 7.4) and was used to observe the in vitro effect of K +, Ca 2+ and Mg 2+ on forward progressive motility of spermatozoa. The motility was recorded after the addition of varying concentrations of KCl (0-100 mm), CaCl 2 (0-100 mm) and MgCl 2 (0-100 mm) separately to sperm suspension. For the evaluation of influence of Na + on sperm motility, 0.6% HEPES buffer (Na + -free) was used in place of PBS for the preparation of sperm suspension, and sperm motility in Na + -free medium was recorded. Motility of spermatozoa was visually assessed after 1 hr incubation in respective concentrations of ion by placing a drop of suspension on a micro slide, and observing in a light microscope ( 100). The number of spermatozoa exhibiting progressive forward motility per 100 spermatozoa was recorded for each treatment group and expressed as % of spermatozoa showing forward progressive motility. The observation was repeated by taking five random samples per treatment group, and the mean values were computed using these values. The mean values of various parameters studied were analyzed using one way analysis of variance followed by Duncan s multiple range test to test the significance of the differences. Results The concentrations of different cations in the luminal fluid of testis, three different regions of epididymis and vas deferens are shown in Table 1. The concentration of sodium ions increased gradually, yet significantly (P<0.01), from testis through vas deferens, with the lowest concentration in the testis and the highest in the vas deferens. Potassium and calcium ions indicated the same pattern as sodium ions in their distribution with the lowest in the testis and the highest in the vas deferens. The concentration of Mg 2+ of testis and anterior epididymis did not differ significantly, whereas that of middle epididymis was significantly higher than these two regions. The concentration of Mg 2+ of posterior epididymis and vas deferens was significantly higher than other regions. Influence of ions on sperm motility (in vitro study) The testicular spermatozoa remained immotile when prepared in physiological saline. Cauda epididymal spermatozoa remained immotile in sodium-free medium, whereas became highly motile when suspended in physiological saline (120 mm NaCl). Initiation of sperm motility was found first at 20 mm NaCl concentration, the percent motility Table 1 Concentration of Na +, K +, Ca 2+ and Mg 2+ ions in the luminal fluids of testis, anterior (Ant), middle (Mid) and posterior (Post) epididymis (Epd) and vas deferens of the lizard M. carinata [Values are mean ± SE from 100 observations in each group] Cations (mg/l) Testis Ant Epd Mid Epd Post Epd Vas deferens Na ±0.08 a 3.50±0.005 b 5.01±0.005 c 5.96±0.007 d 6.12±0.01 e K ±0.005 a 2.82±0.009 b 3.71±0.006 c 3.96±0.007 d 4.19±0.01 e Ca ±0.008 a 1.24±0.005 b 1.84±0.007 c 1.91±0.01 d 2.23±0.008 e Mg ±0.005 a 0.53±0.007 a 0.58±0.003 b 0.61±0.005 c 0.63±0.009 c Values bearing the same superscripts (a-e) in a given row do not significantly differ.

3 722 INDIAN J EXP BIOL, OCTOBER 2008 increased with further increase in NaCl concentration in sodium-free medium and reached the maximum motility at 140 mm (Fig. 1A). However, further increase in NaCl concentration caused a decrease in motility of spermatozoa (Fig. 1A). The motility of spermatozoa in K + -free medium containing Na + was 55.78%. The maximum motility (59.01%) was observed at 25 mm concentration of KCl. However, further increase in concentration of KCl in the medium decreased the forward progressive motility and was 47.05, and 13.05% at 50, 75 and 100 mm concentration of KCl, respectively (Fig. 1B). In Ca 2+ -free medium containing Na +, the sperm motility was 55.65%, and following addition of exogenous 25 and 50 mm CaCl 2 the percent motility increased to 61 and 64.07% respectively (Fig. 1C). When the CaCl 2 concentration was further increased to 75 and 100 mm the motility decreased to and %, respectively (Fig. 1C). Sperm motility was 54.34% in Mg 2+ free medium, and it decreased significantly with increase in concentration of MgCl 2. At concentrations of 20, 40, 80 and 100 mm, the motility decreased to 32.08, 21.05, and 7.04%, respectively (Fig. 1D). Discussion Lizard spermatozoa, which are non-motile in the testis, acquire motility as they pass through the excurrent duct systems 3. The metabolism, flagellar beating and acrosome reaction of spermatozoa are regulated by ion flux across the plasma membrane in mammals. Ionic content in luminal fluid indicates characteristic changes from the testis to cauda region of the epididymis 2 and cations play an important role in inducing sperm motility 7,12. In the present study the concentration of sodium ions was the highest compared to other three cations, viz., K +, Ca 2+ and Mg 2+, in all the organs studied. The concentration of sodium revealed a gradual but significant increase from anterior to posterior epididymis in M. carinata, whereas in the wall lizard H. flaviviridis its concentration decreased remarkably in the posterior epididymis compared to anterior epididymis 11. The present study is the first report in reptiles showing a pattern in the concentration of Na +, K + and Ca 2+ in the Fig. 1 Influence of different concentrations of NaCl (A), KCl (B), CaCl 2 (C) and MgCl 2 (D) on motility of M. carinata spermatozoa in vitro [values with same superscript alphabets are not significantly different, whereas those with different superscript alphabets are significantly (P<0.01) different as judged by DMRT. Values are mean ±SE from 100 observations in each group].

4 ARANHA et al.: CATIONS AND SPERMATOZOA MOTILITY IN LIZARD MABUYA CARINATA 723 different parts of the male reproductive system i.e., testis<anterior epididymis< middle epididymis< posterior epididymis< vas deferens, and such a pattern in the concentration of Mg 2+ was absent. The facts that, concentrations of Na +, K + and Ca 2+ increase significantly in the epididymis and vas deferens, compared to testis, and spermatozoa attain motility while in these organs, indicate the involvement of one or all these cations in attainment of motility by spermatozoa, and our in vitro study supports this view. Spermatozoa incubated in Na + -free medium did not show any motility, while addition of NaCl induced motility as shown by dose-dependent increase in percent motility in the present study. These observations concur with those found in rat 4,5,13, ram 12 and wall lizard 11. The Na + induced motility in rat spermatozoa is attributed to the activation of Na + -H + antiport, which causes Na + -dependent release of H + from sperm cells and consequent rise in the intracellular ph 5. Although increasing concentrations of Na + enhance sperm motility, a dose higher than certain concentration was inhibitory on sperm motility in M. carinata and also in the wall lizard 11. These observations indicate requirement of optimal concentration of Na + for inducing sperm motility, which appears to differ from species to species, for instance, 120 mm in wall lizard 11 and 140 mm in M. The concentration of K +, another monovalent cation, showed a significant increase from the testis to the vas deferens in the present study in M. carinata, which corresponds to the situation prevailing in the rat, from caput to cauda 14. However, in the wall lizard the K + concentration decreased significantly in the posterior epididymis compared to other two regions 11. In the present in vitro study, 25 mm K + concentration increased the spermatozoa motility, but at higher concentrations K + had inhibitory effect. Similar results of inhibitory effect of K + are reported in wall lizard 11, and rat 13. In human spermatozoa preserved in the electrolyte free solution at 4 C, the reinitiation of motility was induced by potassium ion and extracellular alkalization 15. These results indicate differential responses of spermatozoa to K + in different animal groups. Apart from monovalent ions Na + and K +, the divalent cations, especially Ca 2+, are a major regulatory factor in controlling the motility of mammalian spermatozoa, though its effect may be inhibitory or stimulatory depending on concentrations and species In the present study in M. carinata there was a significant increase in the concentration of Ca 2+ ion from testis to vas deferens, and it concurs with the observation in the wall lizard wherein there is an increase in Ca 2+ concentration from anterior to posterior epididymis 11. In vitro observations in M. carinata in the present study show that calcium at a lower concentration (<50 mm), can induce sperm motility but at higher levels (>50 mm) it acts as an inhibitor. However, in the wall lizard H. flaviviridis calcium ions, even at very low concentration (0.025 mm), reduced the forward progressive motility 11. Calcium at an appropriate level in the medium can induce sperm motility in mammals 17-19, as found in M. Another divalent cation, Mg 2+, although showed significant difference from testis to vas deferens in its concentration in M. carinata, the in vitro effect of Mg 2+ on sperm motility showed inhibitory effect at all doses tested. Similar results have been reported for wall lizard 11. In human spermatozoa also concentration of magnesium ions above 0.8 mm decreased the motility 20. It is to be noted that along with cations, ph, temperature, osmolality of seminal plasma and albumin also play an important role in the motility of spermatozoa In conclusion, the results of the present study reveal that concentration of several mono- and divalent cations (Na +, K +, Ca 2+ and Mg 2+) differs between testis and accessory reproductive organs. Further, the results show that mere increase in concentration of certain ions in the epididymis and the vas deferens, compared to the testis, correlating with attainment of motility of spermatozoa, does not implicate the roles of all these ions in inducing spermatozoa motility, because it is evident from the present results, that although Mg 2+ showed a significant increase in the middle and posterior epididymis and vas deferens compared to the testis and anterior epididymis, it did not have stimulatory effect on spermatozoa motility. Further, sodium ion is the principal cation that induces motility of spermatozoa, and potassium and calcium ions have additive effects depending on the concentration, higher concentrations being inhibitory. However, other non-ionic components also induce motility of the spermatozoa, which are normally found in the luminal fluids and are released along with sodium ion during ejaculation into the female reproductive tract.

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