Indian Journal of Experimental Biology
Vol. 46, October 2008, pp. 720-724
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 570 006, 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 Ca2+ increased gradually from testis to
vas deferens. However, no significant variation in the levels of Mg2+ 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 CaCl2 whereas, treatment with magnesium
brought about a decrease in sperm motility. The results indicate that Na+, K+, and Ca2+ have stimulatory effect at an
optimum dose, whereas Mg2+ has an inhibitory effect even at very low concentration, on sperm motility in the lizard M.
carinata.
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 epididymis1. 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
mammals2.
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 system3. Ions are important
constituents of epididymal fluid and exhibit regional
variations in their concentrations and influence the
motility of spermatozoa in vertebrates4-7. In reptiles,
though the epididymis is not morpho-logically
differentiated as in mammals, anterior, middle and
posterior regions of the epididymis in lizards Lacerta
vivipara3, Hemidactylus flaviviridis6, Mabuya
carinata8 and Sitana ponticeriana9 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
motility10. To date, regional differences in ion
concentration and the contribution of epididymal
__________
*Correspondent author
Telephone: 0821 2411217
E-mail: [email protected]
luminal ions in inducing motility in testicular
spermatozoa are reported only in one species of
reptile, the wall lizard H. flaviviridis11. 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+,
Ca2+ and Mg2+ 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
ARANHA et al.: CATIONS AND SPERMATOZOA MOTILITY IN LIZARD MABUYA CARINATA
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
Lizards were
November (breeding season8).
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+,
Ca2+ and Mg2+ 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 4×108 sperm/ml following the
method of Nirmal and Rai10. Sperm suspension was
made in K+ free phosphate buffered saline (PBS: 120
mM NaCl, 1.67 mM NaH2PO4, 8.1 mM Na2HO4; pH
7.4) and was used to observe the in vitro effect of K+,
Ca2+ and Mg2+ on forward progressive motility of
spermatozoa. The motility was recorded after the
addition of varying concentrations of KCl (0-100
mM), CaCl2 (0-100 mM) and MgCl2 (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
721
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 Mg2+ of testis and anterior epididymis did not differ
significantly, whereas that of middle epididymis was
significantly higher than these two regions. The
concentration of Mg2+ 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+ , Ca2+ and Mg2+ 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+
K+
Ca2+
Mg2+
2.33±0.08a
2.01±0.005a
0.96±0.008a
0.51±0.005a
3.50±0.005b
2.82±0.009b
1.24±0.005b
0.53±0.007a
5.01±0.005c
3.71±0.006c
1.84±0.007c
0.58±0.003b
5.96±0.007d
3.96±0.007d
1.91±0.01d
0.61±0.005c
6.12±0.01e
4.19±0.01e
2.23±0.008e
0.63±0.009c
Values bearing the same superscripts (a-e) in a given row do not significantly differ.
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, 32.08 and 13.05% at 50, 75
and 100 mM concentration of KCl, respectively
(Fig. 1B).
In Ca2+-free medium containing Na+, the sperm
motility was 55.65%, and following addition of
exogenous 25 and 50 mM CaCl2 the percent motility
increased to 61 and 64.07% respectively (Fig. 1C).
When the CaCl2 concentration was further increased
to 75 and 100 mM the motility decreased to 58.01 and
41.08 %, respectively (Fig. 1C).
Sperm motility was 54.34% in Mg2+ free medium,
and it decreased significantly with increase in
concentration of MgCl2. At concentrations of 20, 40,
80 and 100 mM, the motility decreased to 32.08,
21.05, 13.03 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 systems3. 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 epididymis2 and cations play an important role
in inducing sperm motility7,12. In the present study
the concentration of sodium ions was the highest
compared to other three cations, viz., K+, Ca2+ and
Mg2+, 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 epididymis11. The
present study is the first report in reptiles showing a
pattern in the concentration of Na+, K+ and Ca2+ in the
Fig. 1―Influence of different concentrations of NaCl (A), KCl (B), CaCl2 (C) and MgCl2 (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].
ARANHA et al.: CATIONS AND SPERMATOZOA MOTILITY IN LIZARD MABUYA CARINATA
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 Mg2+ was absent. The facts
that, concentrations of Na+, K+ and Ca2+ 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 rat4,5,13, ram12
and wall lizard11. 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 pH5. 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 lizard11. 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 lizard11 and 140 mM in M.
carinata.
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 cauda14. However, in the wall
lizard the K+ concentration decreased significantly in
the posterior epididymis compared to other two
regions11. 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 lizard11, and rat13. In human spermatozoa
preserved in the electrolyte–free solution at 4°C, the
reinitiation of motility was induced by potassium ion
and extracellular alkalization15. These results indicate
differential responses of spermatozoa to K+ in
different animal groups.
Apart from monovalent ions Na+ and K+, the
divalent cations, especially Ca2+, are a major
regulatory factor in controlling the motility of
mammalian spermatozoa, though its effect may be
723
inhibitory or stimulatory depending on concentrations
and species16-19. In the present study in M. carinata
there was a significant increase in the concentration of
Ca2+ ion from testis to vas deferens, and it concurs
with the observation in the wall lizard wherein there is
an increase in Ca2+ concentration from anterior to
posterior epididymis11. 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 motility11.
Calcium at an appropriate level in the medium can
induce sperm motility in mammals17-19, as found in M.
carinata.
Another divalent cation, Mg2+, although showed
significant difference from testis to vas deferens in its
concentration in M. carinata, the in vitro effect of
Mg2+ on sperm motility showed inhibitory effect at all
doses tested. Similar results have been reported for
wall lizard11. In human spermatozoa also
concentration of magnesium ions above 0.8 mM
decreased the motility20. 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 spermatozoa21-26.
In conclusion, the results of the present study reveal
that concentration of several mono- and divalent
cations (Na+, K+, Ca2+ and Mg2+) 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 Mg2+ 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.
724
INDIAN J EXP BIOL, OCTOBER 2008
Acknowledgment
Thanks are due to Department of Science and
Technology, New Delhi for financial support and to
Dr. Umesh Rai, Department of Zoology, University of
Delhi for atomic absorption spectrophotometer
facility.
12
13
14
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