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Collapsed mitochondrial cristae
in goat spermatozoa due
to mercury result in lethality
and compromised motility
along with altered kinematic
patterns
Bhawna Kushawaha1,4,6*
, Rajkumar Singh Yadav2,4
, Dilip Kumar Swain3,4
,
Priyambada Kumari1,4
, Akhilesh Kumar1,4
, Brijesh Yadav3,4
, Mukul Anand3,4
,
Sarvajeet Yadav3,4
, Dipty Singh5
 & Satish Kumar Garg2,4
Earlier we have reported mercury-induced alterations in functional dynamics of buck spermatozoa
through free radicals-mediated oxidative stress and spontaneous acrosome reaction. Based on
our earlier findings, we aimed to investigate the effect of mercury exposure on motility, kinematic
patterns, DNA damage, apoptosis and ultra-structural alterations in goat spermatozoa following
in vitro exposure to different concentrations (0.031–1.25 µg/ml) of mercuric chloride for 15 min
and 3 h. Following exposure of sperm cells to 0.031 µg/ml of mercuric chloride for 3 h, livability
and motility of sperms was significantly reduced along with altered kinematic patterns, significant
increase in per cent necrotic sperm cells and number of cells showing DNA damage; and this effect
was dose- and time-dependent. Contrary to up-regulation of Bax gene after 3 h in control group,
there was significant increase in expression of Bcl-2 in mercury-treated groups.Transmission electron
microscopy studies revealed rifts and nicks in plasma and acrosomal membrane, mitochondrial
sheath, and collapsed mitochondria with loss of helical organization of mitochondria in the middle
piece of spermatozoa. Our findings evidently suggest that mercury induces necrosis instead
of apoptosis and targets the membrane, acrosome, mid piece of sperms; and the damage to
mitochondria seems to be responsible for alterations in functional and kinematic attributes of
spermatozoa.
Lead, mercury, cadmium, nickel and arsenic have been reported to adversely affect male fertility including
fertilizing competence of ­spermatozoa1
. In-vivo toxicological studies have shown that heavy metals have the
tendency to accumulate in testis and disrupt endocrine and regenerative capacity of testicular ­cells2
. Mercury
is extensively used in day-to-day life like in dental amalgam, thermometers, batteries, logical gadgets, electrical
switches, semiconductors, additives, pesticides, and pharmaceutical ­formulations3,4
. Toxicity of mercury in
males, especially its effect on testicular functions, following in vivo exposure of animals has been ­reported5–7
.
Certain in-vitro studies have revealed adverse effects of mercury on functional dynamics of spermatozoa of ­bulls8
,
­humans9
, ­rats10
, ­monkeys11
, and ­fish12
. We too have recently reported mercury-induced significant alterations
in functional dynamics of goat spermatozoa following in vitro exposure due to significant decrease in total antioxidant
defense, increase in intracellular calcium and cAMP levels along with spontaneous acrosomal reaction,
inhibition of tyrosine phosphorylation and capacitation like events in sperm ­cells13
.
OPEN
1
College of Biotechnology, Mathura, India. 2
Department of Veterinary Pharmacology and Toxicology, Mathura,
India. 3
Department of Veterinary Physiology, Mathura, India. 4
UP Pandit Deen Dayal Upadhyaya Pashu Chikitsa
Vigyan Vishwavidyalaya (Veterinary University), Mathura 281001, Uttar Pradesh, India. 5
ICMR-National Institute
for Research in Reproductive Health (NIRRH), Mumbai, India. 6
National Institute of Animal Biotechnology (NIAB),
Hyderabad, India.*
email: bhawnarajput31jan@gmail.com
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Various pathways of mercury-induced cell death have been reported in different ­cells14–18
. Biochemical mechanisms
of sperm cell death, especially the genes involved in this process are currently under intense study. Ionderegulation,
particularly ­Ca2+
, plays an important role in cell death following either apoptosis or ­necrosis19
.
In contrast to apoptosis, accidental cell death leading to necrosis with karyorhexis, cell shrinkage, dilatation
of the endoplasmic reticulum (ER), swelling of cytosol and condensed mitochondria has also been ­reported20
.
Necroptosis cell death pathway is mediated by changes in functional status of the mitochondria e.g., change in
mitochondrial membrane permeability transition, loss of mitochondrial trans-membrane potential (MTP), ROS
generation, ­Ca2+
overload and release of cytochrome-c15
. Intrinsic variability of semen samples as well as individual
variations or differences arising as a result of treatments can be better studied by using computer-assisted
sperm analysis (CASA) system that allows simultaneous generation of huge data-sets consisting of kinematic
trajectories from thousands of ­spermatozoa21
. Therefore, CASA systems have evolved rapidly during the last
decade due to major innovations in ­technology22
.
In view of the above and also our own recent findings about the mechanism of mercury-induced alterations
in functional dynamics of buck-spermatozoa13
, we envisaged to unravel the precise target-site and mechanism
of mercury-induced alterations in functional dynamics and kinematics of goat sperm cells to substantiate our
hypothesis. Therefore, the present study was undertaken to investigate mercury-induced alterations in motility
and motion kinematics of goat spermatozoa along with ultra-structural changes and mechanism(s) of sperm cell
death. Effect of mercury on motility and kinematic patterns will help also us in determining the quality and its
suitability for cryopreservation, and also delineating the molecular mechanism(s) of its toxic effects apart from
providing some insights in evolving suitable cyto-protective measures to counter mercury-induced cell damage,
and improve sperm competence for successful fertilization.
Material and methods
Experimental animals.  Present study was conducted on six healthy adult male goats (bucks) of Barbari
breed aging between two and four years and weighing from 25 to 35 kg. Animals were maintained under standard
semi-intensive system of management in goat sheds of the Department of Veterinary Physiology of the
Institute. Guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals,
Govt. of India were followed and the study was undertaken after approval of the experimental protocols by the
Institutional Animal Ethics Committee (IAEC) of U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan
Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU) (Approval No. 110/IAEC/16).
Semen collection and dilution.  Total ninety semen ejaculates were collected from six different bucks
during the months of March to May and September to November using artificial vagina (AV) with graduated
semen collection cups. The frequency of semen-collection from each buck was twice a week. Immediately after
collection, semen samples were kept in ­CO2 incubator at 37 °C, then processed over thermostatically regulated
stage at 37 °C and examined under phase-contrast microscope.
Semen ejaculates of good quality, as manifested by mass motility of ≥ 3, sperm motility of ≥ 80% and abnormal
sperms morphology of ≤ 10%, were used for detailed investigation as established earlier in our ­laboratory23
and
rest of the semen samples were discarded. Concentration of spermatozoa in semen samples were determined
using hemocytometer (Improved Neubauer’s chamber). Semen samples were diluted using semen dilution
medium (phosphate buffer solution containing 0.5% glucose and of pH 7) as described ­earlier13
to obtain the
final working concentration of 50 × 106
spermatozoa/ml.
Experimental design.  Stock solution (1 mg/ml) of mercuric chloride ­(HgCl2 having 73.89% Hg) was prepared
in PBS (pH 7.4). The study was undertaken in four treatment groups using four different concentrations
of mercury chloride (0.031, 0.125, 0.25 and 1.25 μg/ml) and one control group (PBS + 0.5% glucose). These
concentrations of mercuric chloride were 1/40th, 1/10th, 1/5th and equivalent to the ­LC50 value of ­HgCl2 for
goat ­spermatozoa13
.
Effect on livability.  To determine the percentage of live spermatozoa in semen samples following in vitro
exposure to different concentrations of mercury chloride (0.031, 0.125, 0.25 and 1.25 μg/ml) for different time
intervals (15 min, 1 h and 3 h), smears from semen samples of different groups were prepared, air-dried and
then stained with Eosin-Nigrosin employing the method of ­Hancock24
as briefly described by us in our recent
­publication13
.
Effect on motility and motion kinematics.  Effect of mercuric chloride (0.031, 0.125, 0.25 and 1.25 μg/
ml) on motility percentage, percent of total motile cells and rapid progressive motility (%), and kinematic patterns
of spermatozoa-curvilinear velocity (VCL; µm ­s−1
), straight-line velocity (VSL; µm ­s−1
), average path velocity
(VAP; µm ­s−1
), linearity (LIN; %), straightness (STR; %), beat cross frequency (BCF; Hz), wobble (WOB;
%), and maximum amplitude of head lateral displacement (maxALH; µm), compared to the control group,
was determined using computer-assisted semen analyser (CASA; Biovis-2000, Version V 4.59, developed by
Expert Vision Labs. Pvt. Ltd., Mumbai, India, URL:-http://www.exper​tvisi​onlab​s.com/Biovi​sCASA​.html),
sperm counting chamber, negative phase contrast and 10X objective after different times of in vitro exposure
(15 min, 1 h and 3 h) as per the method described by Anand et al.25
. The CASA system was programmed using
algorithm based on the size, shape, and detection of sperm head as follows: Frames/s—60, number of frames
acquired—61, max velocity (μm/s) for tracking 150 motility min, curvilinear velocity (VCL; μm/s) > 25 motility
min, average path velocity (μm/s) > 10 motility min, straight-line velocity (μm/s) > 1 min, track length (% of
frames) 51, aspect 0–99,999, area 2–20, axis (major) 4–20, axis (minor) 2–10, compactness 0–50, perimeter ratio
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0–99,999, minimum cell size on major axis 20, minimum cell size on min axis 10, magnification × 10 phase, cali-
bration × (pixels/unit) − 1.905 μ, Y (pixels/unit) 1.905 μ, and size of the image 1280 × 960 pixels (58). Spermatozoa
were maintained for up to 3 h at 37 °C in dry bath. 5 μl sample from each diluted semen sample (50 × 106
/ml)
was loaded in metallic sperm counting chamber with surface graticule of 100 × 0.01 sq mm (Sperm processor,
Welcomenagar, India) and six fields from each ejaculate and at each time point were measured, and the average
values were computed to minimize the measurement errors.
Effect on DNA fragmentation.  The APO-BrdU TUNEL Assay Kit (A23210, Invitrogen) was used to
detect mercury-induced DNA fragmentation in spermatozoa following in vitro exposure to mercuric chloride
(0.031, 0.125, 0.25 and 1.25 μg/ml) for 15 min and 3 h. After treatment, semen samples was centrifuged at
1500 rpm for 5 min in DPBS (twice) to remove free mercury from the media by removing the supernatant. DNA
fragmentation was assessed as per manufacturer’s instructions. Briefly, 5 ml 1% (w/v) paraformaldehyde in PBS
was added to 1–2 × 106
sperm cells (in 0.5 ml of phosphate-buffered saline) and placed on ice for 15 min. Samples
were centrifuged for 5 min at 300×g and the supernatant were discarded. Cells were washed in 5 ml PBS, and
then centrifuged to make the pellet and this step was once again repeated to obtain the pellet of treated-cells.
Cells were re-suspended in 0.5 ml of PBS. To this, 5 ml of ice-cold 70% (v/v) ethanol was added and the cells
were allowed to stand for a minimum period of 30 min in − 20 °C freezer. Total 50 µl DNA-labelling solution
was prepared for each sample by adding 10 µl of the reaction buffer, 0.75 µl of TdT enzyme, 8.0 µl of BrdUTP
and 31.25 µl of distilled water in tube and mixed well. Re-suspended the cell pellets of each tube in 50 µl of the
DNA-labelling solution. The DNA-labelling reaction was carried out at 22–24 °C overnight. After incubation,
1.0 ml rinse buffer was added to each tube and centrifuged at 300×g for 5 min. Supernatant was removed by
aspiration and this step was repeated by rinsing the cells with 1.0 ml of rinse buffer. Centrifuged the samples at
300×g and removed the supernatant by aspiration. 100 µl of the antibody staining solution for each sample was
prepared by mixing 5.0 µl of the Alexa Fluor 488 dye-labelled anti-BrdU antibody with 95 µl of the rinse buffer.
Cells pellet was re-suspended in 100 µl of the antibody solution and kept at room temperature for 30 min, and
during incubation samples were protected from light. 0.5 ml of propidium iodide, a staining buffer, was added
to each sample and the cells were incubated again at room temperature for an additional period of 30 min while
protecting from light exposure. At least 400 cells were evaluated under fluorescent microscope within 3 h of
completing the staining procedure.
Effect on apoptosis/necrosis.  The standard protocol as described by manufacture of Annexin V-FITC
(APOAF, Sigma, USA) kit was followed. Briefly, after treatment of spermatozoa with mercuric chloride, sperm
cells were washed twice with DPBS and centrifuged at 1500 rpm for 5 min to remove free mercury, if any, present
in the media. Then the cells were re-suspended in 1X binding buffer at a concentration of ~ 1 × 106
cells/ml.
500 µl of the cell’s suspension was transferred to plastic test tube (12 × 75 mm) to which 5 µl of Annexin V-FITC
conjugate and 10 µl of propidium-iodide (PI) solution were added. The tubes were incubated at room temperature
for exactly 10 min while protecting from light. Same procedure was followed for camptothecin (10 μM)
treated group sample, the positive control, as well. Fluorescence of cells was immediately captured using fluorescent
microscope (excitation at 400–440 nm and emission at 470 nm using 40× objective). At least 400 cells were
counted. Live cells showed no staining either by propidium-iodide solution or by annexin V-FITC conjugate. But
the cells which were in early apoptotic process were stained with Annexin V-FITC conjugate while the necrotic
cells were stained by propidium-iodide. Annexin V-FITC staining was detected as green fluorescence while
propidium iodide staining as red fluorescence.
Expression of apoptotic and anti‑apoptotic genes.  After exposure to mercury, semen samples were
centrifuged at 2000 rpm for 10 min to remove free mercury present in the media, if any, and then the samples
were transferred in DEPC-treated micro-centrifuge tubes (Eppendorf). For lysis of the sperm cells, 1 ml of 0.5%
Triton X-100 was added to the pelleted spermatozoa for 10 min and then tubes were briefly vortexed and centrifuged
at 3000 rpm for 10 min. After that, pelleted spermatozoa were used for RNA isolation.
RNA isolation.  RNA extraction was carried out using RNA isolation kit (Gene JET K0871, ThermoFischer
Scientific, USA) as per the procedure described by manufacturer and stored at − 80 °C for further analysis. Purity
of the total RNA was checked by using Biophotometer (Eppendorf, Germany). RNA samples having A260/A280
values equal to or more than 1.8 were used for cDNA synthesis.
Synthesis of cDNA.  The cDNA was synthesized from the isolated RNA samples using Revert Aid First Strand
cDNA Synthesis Kit (ThermoFischer Scientific, USA) following manufacturer’s procedure in thermal cycler
(Bio-Rad). The cDNA obtained was stored at − 20 °C. End point PCR conditions were optimized to amplify the
Bax, Bcl-2 and β-actin genes sequences by gradient PCR (Bio-Rad) using Dream Taq PCR master mix (K1071,
ThermoFischer Scientific, USA). To amplify the desired genes, the already published primer sequences were
used (60). These primers were further aligned by using PRIMER BLAST at NCBI; details of which have been
given in Table 1.
Real‑time PCR.  Reaction mixture was prepared by adding 1 μl of cDNA template (100 ng/μl), 1 μl each of the
forward and reverse primers (10 pmol), 10 μl of PowerUp SYBR Green qPCR Mix (ThermoFischer Scientific,
USA), and 7 μl of nuclease-free water to make up the final volume of 20 μl. Initial denaturation was performed at
95 °C for 10 min, annealing at 61 °C for Bcl-2, 60 °C for Bax and β-actin for 30 s, extension at 72 °C for 30 s for 40
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cycles, and 72 °C for 5 min for final extension. Light Cycler 480 (Applied Biosystems, USA) was used to analyse
the gene expression. Each gene sample was run in duplicate. Non-template control (NTC) was also run simultaneously.
Pipetting was done with sterile DEPC-treated tips without creating bubbles to avoid any interference in
reading of fluorescence by the instrument. Confirmation of amplification of the specific sequence was done by
using agarose gel electrophoresis. 2.5% agarose was mixed with 30 ml of 1X TAE buffer at 60 V/cm. Gene ruler
of 50 bp was electrophoresed in parallel to the amplicons (Figs. S1–S6 Supplementary Data).
Electron‑microscopic studies.  After treatment of semen samples with the lowest (0.031 μg/ml) and highest
(1.25 μg/ml) used concentrations of mercuric chloride for 15 min and 3 h, semen samples of the control and
mercury-treated groups were centrifuged at 1500 rpm for 5 min in DPBS (twice) to remove the free mercury, if
any, from the media. Then the pellets were re-suspended in the mixture of 2% paraformaldehyde and 2.5% glutaraldehyde
in 0.1 M phosphate buffer (PB; pH 7.4) and kept for 12 h at 4 °C for fixation. Then the samples were
again centrifuged at 1500 rpm for 5 min and the supernatant was discarded to remove the fixative. The pellet was
suspended in 0.1 M PB, and again centrifuged. Thereafter, the samples (pellets) were fixed for 1 h in 1% osmium
tetroxide at 40 °C and then dehydrated in acetone, infiltrated and embedded in araldite CY 212 (TAAB, UK).
Sections (0.5 µm) were cut with an ultra-microtome, mounted on to the glass slides, stained with aqueous toluidine
blue and observed under light microscope for gross observation of the area and quality of the tissue fixation.
For scanning electron microscopy, after fixation of samples as mentioned above, the samples were centrifuged
at 1000 rpm for 10 min and supernatant was discarded. The pellet was suspended in buffer, centrifuged and
washed. It was re-suspended in buffer and a drop of it was spread on a cover slip. The samples were air-dried,
sputter-coated with colloidal gold and observed under an EVO 18 Zeiss scanning electron microscope at an
operating voltage of 20 kV. Images were digitally acquired by using the SmartSEM software.
Thin sections of grey-silver colour interference (70–80 nm thick) were cut and mounted onto 300 mesh
copper. The sections were stained with uranyl acetate and alkaline lead citrate, then gently washed with distilled
water and observed at Sophisticated Analytical Instrumentation Facility (SAIF), All India Institute of Medical
Sciences, New Delhi under Tecnai G2 20 S-Twin transmission electron microscope (Fei Company, The Netherlands)
at an operating voltage of 200 kV. Images were digitally acquired by using camera and Digital Micrograph
software attached to the microscope.
Statistical analysis.  Data generated were subjected to two-way ANOVA and Tukey’s test using SPSS Version
23.0 where p value of 0 < 0.05 was considered significant. Data presented in tables are mean ± SE of the
observations on semen samples of six different male goats. Separate multiple linear regression models were used
to examine the effect of different concentrations of mercury and times of exposure on each of the CASA parameters
after controlling for potential confounding factors. All the values for kinematic variables were standardized
to avoid any scale effect. Correlation was also calculated between the motility and other kinematic patterns.
Analysis of the relative mRNA expression data between different groups was based on the crossing point (Cp)
values. The Cp value of each gene was subtracted from the arithmetic mean of Cp value of the β-actin gene to
calculate the ΔCt. The relative expression of PCR product was determined by the Eq. (2) (− ΔΔCt) as per Livak
­method26
and the level of significance was set at 0.01% (p < 0.01).
Ethics committee approval.  All experiments involved non-invasive procedures on animals including on
large animals are approved by the Institutional Animal Ethics Committee (IAEC) of U.P. Pandit Deen Dayal
Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU) as per the
guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) of
Govt of India. (Approval No. 110/IAEC/16, dated: 16-09-2016).
Results
Effect on live spermatozoa count.  Compared to the control, there were no significant alterations in per
cent count of the live spermatozoa following exposure to different concentrations of mercuric chloride (0.031,
0.125, 0.25 and 1.25 μg/ml) for 15 min. But compared to the control (90.07%), significant (p < 0.05) decrease
in live spermatozoa percentage count (80.20 and 79.14%) was observed in semen samples exposed to higher
concentrations (0.25 and 1.25 µg/ml) of ­HgCl2 after 1 h. After 3 h of exposure to different concentrations of
mercury also, compared to the control (85.44%), significant (p < 0.05) reduction in percentage counts of the
live spermatozoa (75.65, 72.82, 65.62%) were observed in groups treated with 0.125, 0.25 and 1.25 µg/ml ­HgCl2,
respectively (Table 2, Fig. 1a).
Table 1.  Primers sequences of Bcl-2, Bax and β-actin genes.
Primers sequences EMBL/reference Product size (bp) Annealing temp (°C)
Bcl-2-F 5′-TGC​TGC​TGT​TTC​TGC​CTA​CA-3′
Bcl-2-R 5′-GCA​CTT​TTG​CAT​GGG​TCA​A-3′
NM_001166486.1 143 61
Bax-F 5′-CAT​GGA​GCT​GCA​GAG​GAT​GA-3′
Bax-R 5′-GTT​GAA​GTT​GCC​GTC​GGA​AA-3′
XM_002701934.1 101 60
β-actin F 5′-AGT​TCG​CCA​TGG​ATG​ATG​A-3′
β-actin R 5′-TGC​CGG​AGC​CGT​TGT-3′
Dangi et al.61
54 60
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Effect on total motility and progressive motility.  Data presented in Fig. 1b,c and Table 3 revealed
that compared to the control, there were no significant (p > 0.05) differences in the percentage count of total and
progressive motility of spermatozoa in the semen samples exposed to different concentrations of ­HgCl2 after
15 min. But significant (p < 0.05) decrease in total and progressive motile spermatozoa percentage count was
observed after 1 h in 1.25 µg/ml ­HgCl2-treated group (65.79 and 58.78%, respectively) compared to the control
group after 1 h (84.47 and 71.53%, respectively). After 3 h of exposure to ­HgCl2 at 0.25 and 1.25 µg/ml, there was
further decrease in count of the total motile and progressive spermatozoa as these values were found to be 39.84
and 19.9%, respectively.
Effect on kinematic patterns.  Data presented in Table 3 revealed significant differences in kinematic
patterns of the spermatozoa in semen samples treated with different concentrations of mercuric chloride (0.031,
0.125, 0.25 and 1.25 μg/ml) compared to the control group. Compared to the control group after 1 h, slow
and non-progressive motility in semen samples treated with different concentrations of mercury were found
to be significantly (p < 0.05) increased. However, slow and non-progressive motility were found to significantly
(p < 0.05) increase in dose-dependent manner compared to control after 3 h. Different velocity parameters of
spermatozoa (VCL, VSL, and VAP) were significantly (p < 0.05) decreased in concentration- and time dependent
manner (Fig. 1d–f). Data presented in Fig. 2a–c showed significant (p < 0.05) increase in values of LIN and
STR after 3 h exposure to different concentration of mercury as compared to the control group. WOB per cent
count was also significantly (p < 0.05) increased as the time of exposure to 0.125, 0.25 and 1.25 µg/ml of ­HgCl2
Table 2.  Effect of in-vitro exposure of spermatozoa to different concentrations of mercuric chloride (0.031–
1.25 µg/ml) on live spermatozoa count at different time intervals. Data presented are mean ± SE of the semen
samples of six bucks. Different capital superscripts in the rows indicate significant (p < 0.05) differences
between the different time intervals. Different small superscripts in the columns indicate significant (p < 0.05)
differences between the different treatment groups.
Treatments
% live spermatozoa at different time intervals
15 min 1 h 3 h
Control 91.61 ± 2.59A
90.07 ± 1.97aA
85.44 ± 1.66aA
0.031 µg/ml mercuric chloride 90.49 ± 1.37A
86.71 ± 1.78abA
79.58 ± 2.20abB
0.125 µg/ml mercuric chloride 88.72 ± 1.36A
83.13 ± 1.73abAB
75.65 ± 0.83bB
0.25 µg/ml mercuric chloride 87.33 ± 1.85A
80.20 ± 1.67bA
72.82 ± 1.38bB
1.25 µg/ml mercuric chloride 85.77 ± 1.27A
79.14 ± 1.43bAB
65.62 ± 0.04cB
Figure 1.  The relationship between different concentrations of ­HgCl2 and computer-assisted semen analysis
(CASA) kinematic parameters for per cent live (a), per cent total motility (b), per cent rapid progressive (C),
VCL (d), VSL (e), VAP (f). Linear regression lines are shown along with the coefficients of determination (R2
)
for 15 min and 3 h. Each data point represents the mean ± SE value of repeated measurements from six male
goats.
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Table 3.  Effect of in-vitro exposure of spermatozoa to different concentrations of mercuric chloride
(0.031–1.25 µg/ml) on motile spermatozoa count at different time intervals. Data presented are mean ± SE of
the semen samples of six bucks. VCL curvilinear velocity (µm ­s−1
), VSL straight-line velocity (µm ­s−1
), VAP
average path velocity (µm ­s−1
), LIN linearity of forward progression (%), STR straightness (%), WOB wobble
(%), maxALH maximum amplitude of lateral head displacement (µm), BCF beat-cross frequency (Hz).
A = 0.031 µg/ml, B = 0.125 µg/ml, C = 0.25 µg/ml, and D = 1.25 µg/ml ­HgCl2. Different small superscripts in
the columns indicate significant (p < 0.05) differences in kinematic patterns between control and different
treatment groups at different time intervals.
Motility
Rapid
progressive
Slow
progressive
Nonprogressive
VCL VAP VSL LIN% STR% WOB% BCF maxALH
15 min
Control 85.94 ± 1.2a
74.98 ± 0.16a
2.23 ± 0.33a
6.72 ± 1.45a
161.69 ± 0.23a
129.26 ± 0.17a
123.6 ± 0.61a
77.44 ± 0.53a
95.62 ± 1.36ab
80.74 ± 0.45 41.27 ± 0.31a
3.8 ± 0.28a
A 88.50 ± 1.2a
78.39 ± 1.26a
2.96 ± 0.27a
6.96 ± 1.23a
160.08 ± 0.51a
128.21 ± 0.48a
121.3 ± 0.42a
78.46 ± 1.30a
94.61 ± 1.32ab
81.51 ± 0.82 40.26 ± 0.44a
3.7 ± 0.22a
B 86.95 ± 0.5a
78.18 ± 1.22a
2.82 ± 0.61a
4.08 ± 1.7a
159.32 ± 0.61a
125.18 ± 1.29ab
120.02 ± 1.03a
77.21 ± 1.40a
93.68 ± 1.44a
81.42 ± 1.48 39.67 ± 1.11ab
3.51 ± 0.41a
C 85.62 ± 0.6a
76.20 ± 0.72a
3.40 ± 0.39a
6.48 ± 1.02a
156.25 ± 1.71ab
122.9 ± 0.36b
117.52 ± 1.20a
76.28 ± 1.58a
94.04 ± 1.03ab
82.14 ± 1.20 37.28 ± 0.49ab
3.3 ± 0.20a
D 84.75 ± 0.3ab
74.42 ± 0.81a
2.48 ± 0.61a
6.44 ± 1.31a
150.11 ± 1.43bc
120.1 ± 0.81b
111.17 ± 1.25b
78.01 ± 1.20a
95.07 ± 1.05ab
82.74 ± 1.06 34.25 ± 0.55b
2.91 ± 0.11ab
1 h
Control 84.47 ± 1.7a
71.53 ± 0.27ab
2.22 ± 0.29a
7.47 ± 1.03a
160 ± 0.38a
129.03 ± 1.49a
123.03 ± 1.04a
77.275 ± 1.06a
95.34 ± 1.30ab
80.64 ± 1.72 40.52 ± 0.31a
3.8 ± 0.21a
A 78.06 ± 0.3ab
68.02 ± 0.41b
4.21 ± 1.37ab
6.24 ± 1.36a
158.13 ± 1.45a
125.1 ± 1.48a
118.27 ± 0.36a
78.76 ± 0.91a
94.34 ± 1.20ab
81.47 ± 1.25 39.01 ± 0.32ab
3.2 ± 0.22a
B 77.73 ± 1.2b
67.55 ± 1.59b
7.36 ± 1.82ab
7.81 ± 1.05a
152.45 ± 1.62b
122.11 ± 1.72b
113.31 ± 0.47b
79.58 ± 1.22a
93.33 ± 1.03a
80.53 ± 1.34 39.08 ± 0.33ab
2.81 ± 0.10a
C 72.73 ± 1.0b
65.04 ± 0.61b
5.23 ± 2.21ab
8.18 ± 1.51a
150.94 ± 0.91bc
119.42 ± 1.04b
106.16 ± 0.62c
80.03 ± 1.42a
94.7 ± 1.20ab
82.04 ± 1.44 36.28 ± 0.29ab
2.52 ± 0.31ab
D 65.79 ± 0.4c
58.78 ± 0.39c
2.38 ± 1.40a
9.20 ± 0.91a
148.7 ± 0.79bc
116.08 ± 1.81c
99.32 ± 0.82d
80.21 ± 1.39a
95.37 ± 1.25ab
83.06 ± 1.53 33.28 ± 0.71b
2.4 ± 0.13ab
3 h
Control 83.61 ± 0.12a
70.57 ± 0.18ab
4.46 ± 0.41ab
8.54 ± 1.61a
159.14 ± 1.28a
128.31 ± 0.15a
122.17 ± 0.41a
76.06 ± 1.04a
95.12 ± 1.7ab
80.27 ± 0.88 39.74 ± 0.48ab
3.7 ± 0.29a
A 68.23 ± 1.16c
52.29 ± 1.26c
9.86 ± 1.6b
10.78 ± 1.29a
145.28 ± 1.32c
120.17 ± 1.51b
114.07 ± 1.48b
77.03 ± 1.42a
97.3 ± 1.5ab
82.46 ± 0.94 36.04 ± 0.29ab
2.8 ± 0.21a
B 63.56 ± 1.11c
39.02 ± 1.51d
20.59 ± 0.26c
19.07 ± 1.36b
132.02 ± 2.01d
110.28 ± 1.04d
107.06 ± 1.61c
78.85 ± 0.81a
97.19 ± 1.07ab
82.53 ± 1.37 35.24 ± 0.71b
2.4 ± 0.28ab
C 47.78 ± 0.31d
28.49 ± 1.21e
24.67 ± 1.30 cd
28.55 ± 1.37c
128.17 ± 1.41d
106.02 ± 1.01d
95.06 ± 1.03d
80.92 ± 1.48a
98.62 ± 1.40b
83.41 ± 1.40 27.93 ± 0.66c
2 ± 0.41ab
D 39.84 ± 0.28e
19.9 ± 0.82f. 29.62 ± 1.7d
41.31 ± 1.49d
120.27 ± 0.27e
97.27 ± 1.06e
84.25 ± 0.62e
81.19 ± 1.71a
98.51 ± 1.03b
84.37 ± 1.29 20.61 ± 0.10d
1.7 ± 0.66
p-value 0.005 0.0001 0.0123 0.0432 0.0031 0.004 0.022 0.714 0.041 0.844 0.041 0.022
Figure 2.  The relationship between different concentrations of ­HgCl2 and computer-assisted semen analysis
(CASA) kinematic parameters for per cent linearity (LIN; (a)), per cent straightness (STR: (b)), per cent WOB
(c), maximum amplitude of head lateral displacement (maxALH; µm; (d)), and beat cross frequency (BCF; hz;
(e)). Linear regression lines are shown along with the coefficients of determination (R2
) for 15 min and 3 h. Each
data point represents the mean ± SE value of repeated measurements from six male goats.
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increased. This suggests that LIN, STR and WOB of spermatozoa were increased and the progressive motility
and different velocity parameters (VCL, VAP & VSL) were decreased with increase in exposure time and concentration
of mercury; thus, indicating that mercury-induced alterations in sperm progressiveness had relatively
greater effect on all the kinematic patterns. However, dose- and time-dependent significant (p < 0.05) decrease in
maxALH and BCF, compared to the control group, were observed for up to 3 h (Fig. 2d,e).
DNA damage.  Data presented in Table 4 and shown in Fig. 3 revealed that mercury even at the lower concentration
(0.031 µg/ml) did not induce any DNA damage even after 3 h of exposure. Similarly, compared to
the PBS control, even highest used concentration of mercury (1.25 µg/ml) also failed in inflicting any significant
DNA damage in sperm cells after 15 min (0.71%). However, compared to the control (0%), significant (p < 0.05)
increase in number of sperm cells showing DNA damage (29.04%) was observed after exposure to 1.25 µg/ml
­HgCl2 for 3 h.
Scanning electron microscopy (SEM).  Evaluation of SEM images of the spermatozoa of control group
revealed that spermatozoa had normal surface morphology (Fig. 4). Head of the spermatozoa was normal and
oval in shape with intact acrosome (Fig. 4). Middle piece showed intact plasma membrane and there were no
obvious deformities near the neck and tail joining regions (Fig. 4). Control group spermatozoa had over all
homogenous plasma membrane. Following in-vitro exposure of sperm cells to lower concentration of ­HgCl2
(0.031 µg/ml) for 15 min, apparently no adverse effect was observed on the surface morphology of spermatozoa
(Fig. 5a) and no obvious changes were observed in sperm head, acrosome and tail parts at this time point and
it compared well with the spermatozoa of control group (Fig. 5b,c). When spermatozoa were exposed to the
same concentration of mercuric chloride (0.031 µg/ml) for longer period of time i.e. 3 h, several defects were
noticed (Fig. 5d) as revealed by deformities in the head and middle piece regions of sperm cells at this time-point
Table 4.  Effect of in-vitro exposure of buck spermatozoa to different concentrations of mercuric chloride
(0.031–1.25 µg/ml) on DNA damage in sperm cells at different time intervals. Data presented are mean ± SE
of the semen samples of six bucks. Different capital superscripts in the rows indicate significant (p < 0.05)
differences between the different time intervals. Different small superscripts in the columns indicate significant
(p < 0.05) differences between control and different treatment groups.
Treatments
% spermatozoa showing DNA
damage at different time intervals
15 min 3 h
PBS control 0.00 ± 0.00Aa
0.00 ± 0.00Aa
0.031 µg/ml mercuric chloride 0.00 ± 0.00Aa
0.00 ± 0.00Aa
0.125 µg/ml mercuric chloride 0.00 ± 0.00Aa
0.00 ± 0.00Aa
0.25 µg/ml mercuric chloride 0.00 ± 0.00Aa
2.03 ± 1.01Aa
1.25 µg/ml mercuric chloride 0.71 ± 0.33Aa
29.04 ± 1.06Bb
Figure 3.  DNA damage in goats’ spermatozoa. (a) Showing no-fluorescence in spermatozoa (TUNEL-negative)
in PBS control and 0.031, 1.25 and 0.25 µg/ml ­HgCl2 groups after 3 h. (b) Showing bright green-fluorescence in
head of the spermatozoa (TUNEL-positive; arrow) of mercuric chloride (1.25 µg/ml) treated group after 3 h of
exposure.
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(Fig. 5e,f). In some of the spermatozoa, wrinkled plasma membrane in the head region (Fig. 5e) and focal areas
of membrane rupture at the middle piece were observed (Fig. 5f). However, exposure to higher concentration of
­HgCl2 (1.25 µg/ml) resulted in deformities in spermatozoa head plasma membrane and middle piece even after
15 min of exposure (Fig. 6a). The SEM analysis also showed that at this time point, 1.25 µg/ml ­HgCl2 hampered
the head plasma membrane integrity and there were focal points of membrane damage (Fig. 6b). Middle piece of
sperm cells harbouring mitochondria also appeared wavy depicting the harmful effects of higher concentration
Figure 4.  Buck spermatozoa of control group showing normal surface morphology. Scanning Electron
Microscope images of buck spermatozoa of control group shows normal surface morphology (a), intact
acrosome (b) and intact midpiece (c) after 15 and 3 h.
Figure 5.  Scanning Electron Microscope images of goat spermatozoa in-vitro treated with Hg (0.031 µg/
ml ­HgCl2) showing intact head and tail and normal surface morphology (a), intact acrosome (b) and intact
midpiece (c) after 15 min exposure. While wrinkled plasma membrane on head region (d,e) and middle piece
deformities and focal area of membrane rupture at middle piece were more obvious after 3 h exposure of
mercury (f).
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of mercury on sperm cells (Fig. 6a,c). Further, the morphological defects in sperm cells were more pronounced
after 3 h of exposure to 1.25 µg/ml of ­HgCl2 (Fig. 6d) as the head plasma membrane was found to be wrinkled
and acrosome was swollen (Fig. 6e). Tail part of the sperm cell also appeared damaged and the neck appeared to
have irregular plasma membrane (Fig. 6f).
Transmission electron microscopy (TEM).  Evaluation of TEM images of the spermatozoa of control
group did not show any notable abnormalities in sperm cells after 15 min or 3 h. Spermatozoa of the control
group (15 min) showed intact plasma membrane and well-organized axonemal components (Fig. 7a,b).
The longitudinal section of spermatozoa tail showed normal axoneme arrangements and intact mitochondrial
ultrastructure at the same time point (Fig. 7c). Even after 3 h, it showed intact membrane, acrosome, axonemal
components (*) and well-organized mitochondria (Fig. 7a,b), intact head (Fig. 7d), mitochondria and axonemal
arrangements (Fig. 7e). Spermatozoa of 0.031 µg/ml ­HgCl2 treated group after 15 min of exposure showed
almost normal ultra-structures (Fig. 8a–c) and the TEM observations were in accordance with the SEM observations.
However, few spermatozoa at this time point showed irregular head plasma membrane (Fig. 8a). But
no notable changes were observed in the longitudinal sections of sperm tail at this time point (Fig. 8c). But following
exposure to ­HgCl2 (0.031 µg/ml) for 3 h, certain adverse effects were observed on ultra-structures of the
buck spermatozoa as revealed by ruptured plasma membrane and premature acrosome reaction in longitudinal
sections of spermatozoa (Fig. 8d). The longitudinal sections of the middle piece revealed disorganized and swollen
mitochondria having collapsed cristae in the middle piece of some of the spermatozoa (Fig. 8e,f). Disorganized
outer dense fibres were also observed at this time point in few of the spermatozoa (Fig. 8f). However, higher
concentration (1.25 µg/ml) of ­HgCl2 damaged spermatozoa head and tail integrity even after 15 min of exposure
(Fig. 9a–c). Some of the spermatozoa showed disorganized axonemal components and bent neck (Fig. 9b). The
distorted outer dense fibres in tail part were also observed in few of the spermatozoa (Fig. 9c). Compared to the
ultrastructural damages observed in sperm cells after exposure to 1.25 µg/ml ­HgCl2 for 15 min, more severe
and adverse ultra-structural defects were observed after 3 h of exposure (Fig. 9d–f) and these were in agreement
with the SEM observations. Longitudinal sections of the spermatozoa head showed fragmented plasma
membrane and focal areas of lysis (Fig. 9d). The middle piece region had swollen mitochondria with collapsed
cristae (Fig. 9e,f). Additionally, disorganized axoneme and disorganised outer dense fibres were also noticed at
this time point in some of the spermatozoa (Fig. 9f). Based on the observed lesions observed in TEM images, it
is apparent that the toxic effect of mercury on spermatozoa was both time- and concentration-dependent and
mercury damaged almost all parts of the sperm cells including mitochondria.
Apoptosis and necrosis.  No apoptosis was observed in the spermatozoa of PBS control and mercurytreated
groups after 15 min of exposure. After 3 h, compared to just 0.67% apoptotic sperm cells in semen
samples of the control group, 41.67% apoptotic spermatozoa were observed in the positive control group (Camptothecin)
as evident from the data summarized in Table 5 and shown in Fig. 10. But compared to the untreated
control or positive control (Camptothecin) groups, no apoptosis was observed in sperm cells of any of the mer-
Figure 6.  Scanning Electron Microscope images of goat spermatozoa in-vitro treated with Hg (1.25 µg/ml
­HgCl2) showing deformities in overall spermatozoa (a), membrane damage (b), wavy middle piece (a,c) after
15 min exposure. Swollen acrosome and wrinkled head plasma membrane (d,e), damaged tail and the neck
having irregular plasma membrane (f) after 3 h exposure of 1.25 µg/ml ­HgCl2.
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Figure 7.  Transmission electron micrographs of the goat spermatozoa of control group after 15 min and 3 h
showing intact ultrastructures. (a) Longitudinal section of the sperm nucleus; head (arrow; (a)); axonemal
components (star; (a,b)), (b) intact sperm head (arrow) and intact acrosome (AC; (b)), organized mitochondria
(arrow; (c)), sperm head (arrow; (d,e), axonemal components (star; (e)); organized mitochondria (triangle; (e)),
intact plasma membrane (arrow; (e)).
Figure 8.  Transmission electron micrographs of goat spermatozoa exposed to 0.031 µg/ml ­HgCl2 showing
ultra-structural defects. (a) sperm head (arrow); damage membrane (triangle), (b) sperm head (arrow);
axonemal components (star), (c) outer dense fibers (arrow), (d) reacted acrosome (arrow), (e) mitochondria
(arrow); collapsed mitochondria (arrow), (f) disorganized outer dense fiber(star); collapsed mitochondria
(arrow) after 15 min and 3 h.
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cury-treated groups even after 3 h of incubation. Rather, large number of sperm cells in mercury-treated groups
were found to be necrotic (clearly visible under red filter; Fig. 10d) and their counts were 18.31, 23.45, 32.13
and 39% in 0.031, 0.125, 0.25 and 1.25 µg/ml ­HgCl2-treated groups, respectively after 3 h of exposure (Table 5).
Correlation between DNA damage, apoptosis and necrosis.  Data presented in Table 6 although
indicated negative correlation between apoptosis and necrosis after 15 min of exposure to mercury, but it was
statistically insignificant, Likewise, apoptosis was negatively corelated with necrosis in ­HgCl2-treated groups
after 3 h of exposure as well. But DNA damage was significantly corelated with necrosis while negatively correlated
with apoptosis, although it was statistically insignificant.
Figure 9.  Transmission electron microscopic images of goat spermatozoa exposed to 1.25 µg/ml ­HgCl2
showing ultra-structural defects. (a) swollen sperm head membrane (arrow), (b) collapsed mitochondria
(arrow); bent neck (star); disrupted connection between nucleus and midpiece by centriole (c,b,f), (c) outer
dense fibers (arrow), (d) damaged head membrane (arrow); damaged axoneme membrane (triangle; (d,f)), (e)
mitochondria (arrow); axoneme (AX; arrow); damaged mitochondrial sheath (MS; (e)),collapsed mitochondria
(arrow; (f)); disorganized exoneme (star; (f)) after 15 and 3 h.
Table 5.  Effect of in-vitro exposure of buck-spermatozoa to different concentrations of mercuric chloride
(0.031–1.25 µg/ml) on early apoptosis and necrosis at different time intervals. Data presented are mean ± SE
of the semen samples of six bucks. Different capital superscripts in the columns indicate significant (p < 0.05)
differences between the different treatment groups. *Camptothecin (10 µM) treated buck semen sample.
Treatments
% Apoptotic and necrotic spermatozoa at different time intervals
15 min 3 h
Necrosis Apoptosis Necrosis Apoptosis
Positive control* 5.00 ± 0.58 29.67 ± 0.33 8.33 ± 0.88 41.67 ± 2.40
PBS Control 6.00 ± 1.53D
0.00 ± 0.00 11.00 ± 2.65D
0.67 ± 0.00
0.031 µg/ml Mercuric chloride 10.00 ± 1.15C
0.00 ± 0.00 18.31 ± 2.85CD
0.00 ± 0.00
0.125 µg/ml Mercuric chloride 14.67 ± 0.33B
0.00 ± 0.00 23.45 ± 3.38BC
0.00 ± 0.00
0.25 µg/ml Mercuric chloride 18.68 ± 0.33AB
0.00 ± 0.00 32.13 ± 3.38AB
0.00 ± 0.00
1.25 µg/ml Mercuric chloride 22.00 ± 0.58A
0.00 ± 0.00 39.00 ± 2.65A
0.00 ± 0.00
p-value 0.000 0.0 0.000 0.0
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Comparative expression of apoptotic and anti‑apoptotic genes.  Data presented in Fig. 11 revealed
that compared to the control group, no significant (p > 0.01) change was observed in the relative mRNA expression
of Bcl-2 gene in semen samples exposed to different concentrations of ­HgCl2 (0.031, 0.125, 0.25 and 1.25 µg/
ml) for 15 min. But after 3 h, compared to the control, there was significant (p < 0.01) increase in the relative
mRNA expression of Bcl-2 in 0.25 and 1.25 µg/ml mercuric chloride-treated groups. Compared to β-actin and
Bcl-2 genes, relative mRNA expression of Bax gene was not altered either in the control or in ­HgCl2 treated
groups after 15 min of exposure. But after 3 h, although significant (p < 0.01) increase in the relative expression
of Bax gene was observed in the control group but there was no change in the relative mRNA expression of Bax
gene in any of the mercury-treated groups even after 3 h. Rather significant (p < 0.01) increase in the relative
mRNA expression of Bcl-2 gene was observed in ­HgCl2 (0.25 and 1.25 µg/ml) treated groups.
Figure 10.  Detection of apoptosis and necrosis using Annexin˗V staining in goat spermatozoa. (a)
Spermatozoa with no sign of phosphatidylserine translocation [Annexin-V-FITC (negative) and PI (negative)]
in control group. (b) Annexin-V-FITC (green) and PI (red) positive spermatozoa in positive control
(Camptothecin 10 µM). (c) Annexin-V-FITC (green; negative) and PI (positive; red) spermatozoa in mercuric
chloride treatment groups after 3 h (0.031–1.25 µg/ml) in green filter. (d) Annexin-V-FITC (green; negative)
and PI (positive; red) spermatozoa in mercuric chloride treatment groups after 3 h (0.031–1.25 µg/ml) in red
filter. (e) DIC image of (c,d); mercury treated groups.
Table 6.  Correlation between necrosis, apoptosis and DNA damage of different concentrations of mercuric
chloride (0.031–1.25 µg/ml) at different time intervals (*p ≤ 0.05).
15 min Necrosis Apoptosis DNA damage
Necrosis 1
Apoptosis  − 0.42 1
DNA damage 0.53*  − 0.149 1
3 h Necrosis Apoptosis DNA damage
Necrosis 1
Apoptosis  − 0.86* 1
DNA damage 0.75*  − 0.27 1
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Discussion
According to U.S. Geological Survey and Harvard ­University27
, mercury levels in the northern Pacific Ocean
have increased by about 30 per cent over the past 20 years and it is expected to rise further by 50 per cent by 2050
with increase in industrial mercury emissions. Mercury levels in air are in the range 2–10 ng/m328
. Although
naturally occurring levels of mercury in groundwater and surface water are less than 0.5 µg/l, but local mineral
deposits may result in higher levels of mercury in ­groundwater28
. The permissible limit for mercury in drinking
water is 0.01 ppm28
. Average daily intake of mercury from food is in the range 2–20 µg, but may be much higher
in regions where ambient waters are more contaminated with mercury and where fish constitute major proportion
of human’s ­diet29
. Fish mostly contain varying levels of mercury like 0.031 ppm in pollock, 0.126 ppm in
tuna, 0.144 ppm in fresh/frozen skipjack, 0.150 ppm in freshwater perch, and 1.123 ppm in tilefish in Gulf of
­Mexico29,30
. United Nations Environment Programme’s (UNEP) Chemicals Working Group has pointed out that
mercury contamination in India is reaching at alarming levels largely due to the discharge of mercury-bearing
industrial effluents ranging from 0.058 to 0.268 mg/l31
; and this is several times more than the prescribed Indian
and WHO standards for drinking water and for industrial effluents. Thus, all the above-mentioned reports suggest
that human beings and animals in India and other countries are being exposed to higher concentrations
of mercury.
In our recent publication, we have reported mercury-induced significant increase in ROS and MDA, significant
decrease in TAC and SOD activity, significant decrease in spontaneous acrosome reaction, and inhibited
capacitation in sperm cells despite increase in cAMP and intracellular ­Ca2+
levels even at 0.031 µg/ml compared
to the control group after 15 min exposure. Based on these findings, we proposed that oxidative stress causes
alterations in sperm ­functions13
possibly by affecting sperm membrane as well as sub-cellular structures. To
substantiate our proposed mechanism of mercury-induced alterations in functional dynamics of spermatozoa,
present study was undertaken to understand how cell death takes place and what changes take place in sperms
at molecular and ultra-microscopic levels, especially at organelle level, that affect the motility and kinematic
patterns in an endeavour to give a fillip to our understanding about mercury-induced cell death and alterations
in functional dynamics of spermatozoa.
Mercury has been associated with compromised sperm functions and reproductive ­failures32
. Men having
mean mercury level of 8.1 ng/ml have been reported to be infertile compared to those having 6.3 ng/ml in
­control33
. Infertile males have been reported to have higher level of mercury (0.048 µg/l) in their seminal plasma
compared to the fertile males having 0.032 µg/l in seminal ­plasma34
. Decreased sperm motility, sperm swimming
speed, and increased abnormal sperm tail morphology have been reported in monkeys having blood mercury
levels around 2000 ng/ml35
. Rats having mean blood mercury levels of 30.8 ng/ml36
, and 94.3 and 176.5 ng/ml37
have also been reported to have significant variations in testosterone levels and adverse effects on male reproduction.
In all these studies, spermatozoa were exposed to comparatively higher concentrations of mercury than used
by us in the present study. Several reports on toxic effects of mercury on semen quality and functional dynamics
of sperm cells in humans and experimental animal are also ­available8–10,12,33,38
. But the specific target site(s) for
action of mercury and its mechanistic pathway(s) are yet to be precisely delineated.
Computer assisted semen analyser CASA provides quantitative data of sperms motility (rapid-, slow- and
non-progressive) and specific kinematic measures. Our data provides quantitative evidence in favour of mercuryinduced
reduction in proportion of the motile sperms and their straight-line motion. Major effects of mercury
on motility parameters (rapid, slow and non-progressive) and kinematic motion parameters (VCL, VSL, VAP
and LIN) were highly correlated. Total motility, rapid and non-progressive were associated with the exposure
level of mercuric chloride (0.31 to 1.25 µg/ml) and time of exposure. The VCL, VSL, VAP and LIN of sperms are
bioindicators of the fertilizing ability and have been correlated with in-vivo fertilization rates in ­humans39
. After
3 h, we observed dose-dependent decline (∼ 31% shorter) in VSL of the sperms of 1.25 µg/ml ­HgCl2 exposed
Figure 11.  Relative mRNA expression of the apoptotic (Bax), anti-apoptotic genes (Bcl-2) and housekeeping
gene (β-actin) in the spermatozoa following treatment of semen samples with different concentrations of
mercuric chloride at different time intervals. Vertical bars represent the mean ± SE. Asterisk (*) on bars represent
the significant differences between the PBS control and different treatment groups at different time intervals.
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group as the spermatozoa travelled at the rate of 84.25 μm/s compared to 122.17 μm/s in the control group. The
VAP and VCL data also suggest that sperm of 1.25 µg/ml ­HgCl2 treated group moved ∼ 24% slowly along their
average path and point to point path of travel compared to control (Table 3). The concentration-dependent
significant decline in ALH and BCF suggest that mercury alters the frequency at which sperms cross the average
path (∼ 25 times per second) and maximum amplitude of deviation around that average path (∼ 3.8 μm) in
dose- and time-dependent manner. Taken together, with mercury concentration-dependent increase in LIN,
STR and WOB trend, our findings suggest that sperms following exposure to higher concentration of mercury
follow more linear path (i.e. less curved) and possess less beat cross frequency than the sperms of control group.
The ALH measures the vigour of flagellar beating in conjunction with the frequency of cell ­rotation40
, and
both these are associated with the ability of sperm to penetrate cervical mucus and fuse with oocytes. Therefore,
ALH is often used to assess the outcome of in vitro fertilization ­programmes41
. Higher concentration (1.25 µg/
ml) of ­HgCl2 drastically reduced the sperm population that is likely to reach the oocyte as a consequence of
the reduced percentage of rapid motile sperms and reduced BCF and maxALH motion kinetic in the remaining
motile sperm cells. Thus, our CASA data evidently suggests that mercury directly diminishes the ability of
sperms to penetrate and fuse with oocytes. Our observations of the kinematic patterns also validate our earlier
­finding13
in which we did not observe any capacitation in sperms following exposure to mercuric chloride at
1.25 µg/ml for 15 min and 3 h.
Mercury (217.216 ppm; 800 μΜ) has been reported to significantly (p < 0.001) increase (12.0% compared to
2% in control) percentage of DNA breaks in human ­sperms42
. Positive correlation between sperm DNA fragmentation
and levels of ROS has been reported in testicular tissue of ­mice43
and semen of ­humans44,45
. TUNEL
assay in the present study revealed that mercuric chloride at lower concentration (0.031 µg/ml) did not induce
any DNA damage in sperm cells even after 3 h of exposure. But DNA damage was observed in 2.03 ± 1.01 and
29.04 ± 1.06% sperm cells following exposure to 0.25 µg/ml and 1.25 µg/ml concentrations of mercuric chloride,
respectively, for 3 h. Our findings on DNA damage at higher concentrations are in agreement with the effect of
150, 350 and 550 μM ­HgCl2 induced DNA breaks in bull sperm nuclei where interestingly 92% of the DNA breaks
were double-stranded8
. Hayati et al.46
also reported mercury-induced increase in DNA fragmentation in fish
sperms after in-vitro exposure to different concentrations of ­HgCl2 (0.5, 1, 2.5 and 5 ppm) for 5 s. DNA damage
observed in sperm cells in the present study can be attributed to mercury-induced significant increase in oxidative
stress biomarkers in goat ­semen13
. But possibly such a low concentration of inorganic mercury (0.031 µg/ml) was
not sufficient to induce DNA damage in goat spermatozoa despite significant oxidative stress and viability losses
in goat ­spermatozoa13
. It seems interesting and but difficult to put forth any plausible explanation for the same.
Arabi et al.1
reported 64% viability losses in bull sperm cells after exposure to mercuric chloride (13.576 µg/
ml) for 2 h. Similar viability loss in of goat spermatozoa was also observed and it was found to be reduced by
50% after 3 h of exposure but at comparatively much lower concentration (1.25 µg/ml). Thus, goat sperm cells
seem to be much more sensitive to mercury-induced cellular insult than bull spermatozoa. Higher vulnerability
of goat sperms vis-à-vis bull sperms at the moment can be only attributed to species-difference, however, to
unravel the precise possible reason for such a variability in response to mercury requires comparative studies
on sperm cells of both these species.
Scanning and transmission electron microscopy images of mercury treated spermatozoa revealed loss of
plasma membrane integrity along with acrosomal membrane damage, damage of the head region, damaged
outer dense mitochondrial sheath along with collapsed cristae, disorganized and swollen mitochondria, reacted
acrosome, disorganized axonemal components with altered implantation fossa. These defects in mitochondria
seem to be responsible for reduced mitochondrial transmembrane potential and motility of the spermatozoa.
Abnormalities in head and neck region may be responsible for detachment of the head and tail leading to instant
death of some spermatozoa. Our observations on ultra-structural alterations in goat spermatozoa are almost
similar to those reported in rabbit sperms after treatment with higher than 1 µM mercury, arsenic, cadmium,
and platinum as in rabbit sperms also damage to sperm head membranes and acrosome breakage with formation
of various sized micro-vesicles was ­observed47
.
In the present study, we observed that mercury did not induce apoptosis even at the highest used concentration
(1.25 µg/ml); rather it induced necrosis even at the lowest used concentration (0.031 µg/ml). Generally,
little percentage of labelling with annexin is observed in every basal sample but in the present study, compared
to the control, none of the spermatozoa from mercury-treated groups showed any exteriorization of phosphatidyl
serine. This also substantiates that mercury did not induce early apoptosis like changes in mercury-treated
sperm cells. Rather, mercury resulted in instant cell death as substantiated by propidium iodide positive ­(PI+
)
cells data during Annexin V assay as the sperm cells were mainly labelled with PI and very less/no with annexin
which indicates that sperm membrane ruptures as indicated by electron microscope images might be responsible
for necrosis. Rather mercury appears to be protective against P-serine externalization, however, to substantiate
the protective role of mercury against P-serine externalization, further studies are required specifically taking
spermatozoa of some other species as well from comparative perspective.
Theory of apoptosis-induced sperm DNA damage has been challenged by several ­researchers48–50
. Apoptosisindependent
DNA damage observed in the present study is in agreement with the similar observations of Pereira
et al.51
who have reported mercury induced DNA damage in blood cells by a nonapoptotic mechanism in wild
and caged fish. On U-937 cells also, mercury was found to damage DNA, but apoptosis was not involved. Similarly,
Muratori et al.48
have also reported that sperm DNA fragmentation did not correspond to the apoptosislike
phenomenon and the impaired motility of sperms in human ejaculate was associated with ultrastructural
damages. Sakkas et al.49
also observed that although DNA damage was initiated in some of the spermatozoa by
mercury but subsequently this escaped apoptosis, and they termed it as “abortive apoptosis”. Lack of mercuryinduced
apoptosis in goat spermatozoa is also substantiated by our real time quantification data of the expression
of pre-apoptotic (Bax) and anti-apoptotic (Bcl-2) genes where the former gene was expressed only in sperm
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samples of the control group after 3 h but not at all in the mercury-treated groups; thus implying routine and
time-dependent apoptosis in sperm cells of control group while inhibition of Bax gene and significant concentration-
and time-dependent increase in expression of Bcl-2 gene in mercury-treated groups. Thus, our findings
are evidently indicative of the anti-apoptotic effect of mercury.
Mammalian Bcl-2 and related anti-apoptotic family member (Bcl-xL, Bcl-w, A1, Mcl-1, Boo/DIVA/Bcl-2,
L-10, Bcl-B) proteins are localized on the cytoplasmic aspect of nuclear envelope, endoplasmic reticulum and
outer mitochondrial ­membrane52
. The fate of a cell depends on the balance of Bcl-2 family protein levels through
a dynamic process by which cellular signals modulate the expression of pro- or anti-apoptotic proteins to tip the
equilibrium towards survival or ­death53,54
. When the balance favours death, the oligomerization of pro-apoptotic
effector proteins on outer mitochondrial membrane leads to mitochondrial outer membrane permeabilization
and release of cytochrome-c. This triggers activation of the cascade of caspases which cleave downstream substrates
and ultimately cell ­death53
. Tissue homoeostasis in eukaryotes requires multiple level regulation in tissue
specific manner for fine-tuning of the apoptotic pathway. However, functional dynamics of Bax and Bcl-2 family
members makes it extremely difficult to establish the physiological relevance of different modes of regulation
reported either in cultured cell lines or in animal ­models53,54
.
Our Bcl-2 gene expression data is in agreement with the observation of up-regulation of Bcl-2 mRNA expression
in rat kidney ­cells55
and in neuronal and glial ­cells56
following exposure to mercury. Mercury (62.7 to
81.1 µM)-induced macrophage death has been reported through both- apoptosis and necrosis ­pathways15
. But
contrary to the observation of Kim and ­Raghubir15
, loss of viability and alterations in motility and motionkinematics
in mercury-treated goats sperm cells was due to damaged ultra-structures in spermatozoa but this
process is independent of apoptosis pathway.
Sperm motility is related to mitochondrial activity within the sperm ­midpiece57
. In our earlier study, almost
40% goat sperm cells showed low mitochondrial transmembrane potential (MTP) following exposure to 1.25 µg/
ml ­HgCl2
13
and reduction in MTP correlated very well with the resultant reduction in sperm motility. Mercury
has high affinity to bind with the sulfhydryl groups (–SH) of tubulins, the main component of sperm
axonemal microtubules, and disrupts interactions between axonemal microtubular proteins and dyne in motors
which are essential for sperm flagellar ­motility11,35,58
. Therefore, reduction in sperm motility in mercury-treated
sperms in the present study may be attributed to disorganized and collapsed cristae in mitochondria, release
of cytochrome-c release due to permeabilization of mitochondrial membrane or inhibition of mitochondrial
enzymes and uncoupling of oxidative ­phosphorylation59,60
. However, to precisely understand the mechanism of
Figure 12.  Proposed mechanistic pathways of mercury-induced toxicity to spermatozoa. Mercury-induced
necrosis and death of spermatozoa seems to involve two independent pathways; mercury results in increase in
levels of ROS and MDA and reduces total antioxidant capacity (TAC) and superoxide dismutase (SOD) activity
and these ultimately result in decreased membrane intactness and cell death. Oxidative stress also results in
lowered mitochondrial transmembrane potential (MTP) that may lead to increase in cAMP level and increased
intracellular ­Ca++
release from spermatozoa that increase spontaneous acrosome reactions (AR) and reduction
of capacitation which ultimately lead to reduction of sperm fertilizing ability. Altogether, mercury leads ROSinduced
cell death via necrosis rather than apoptosis pathway. Necrosis seems to be the dominant signaling
pathway in mercury-induced sperm death, and it may be due to major damage to ultra-structures of sperm cells.
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mercury-induced sperm cell death by necrosis along with inhibition of Bax and upregulation of expression of
Bcl-2, further studies are warranted.
Since ultra-structural damage in sperm cells was observed even after 15 min of exposure to mercury, therefore,
changes in motility and kinematic patterns, and necrosis in sperm cells may be due to abrupt stoppage of the
functional machinery of sperm cell, especially mitochondria. Sperm DNA damage and apoptosis do not seem
to be the main cause of instant sperm cell death as DNA damage was observed only after 3 h of exposure. DNA
fragmentation coupled with severe ultrastructural damage seem to be the important mechanisms for delayed
toxic effects including viability and altered functional dynamics of goat spermatozoa. Accordingly, based on our
findings in the present study and those reported by us ­earlier13
, we propose the possible mechanistic pathways of
mercury-induced alterations in functional dynamics and death of goat spermatozoa (Fig. 12). However, possibility
of involvement of certain other pathway(s) cannot be ruled out which may include cytochrome-c or TNF-α
dependent and most likely direct activation of the caspase(s) that lead to necrosis.
Conclusions
Exposure of goat spermatozoa to mercury resulted in instant damage to sperm plasma membrane, acrosome cap,
mitochondrial sheath and microtubules of spermatozoa and thus cell death while DNA damage was observed
only after 3 h. Mercury seems to primarily target sperm mitochondria and plasma membrane and thereby
results in spontaneous cell death independent to apoptosis. Mercury-induced alterations in cellular integrity
and functionality of sperm cells will affect the fertilizing competence of mercury-exposed animals. Therefore,
to ensure the quality control of cryopreserved semen, it should be ensured that the semen of bucks does not
contain mercury or other toxic metals.
Received: 24 August 2020; Accepted: 18 December 2020
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Acknowledgements
We are thankful to Head, Department of Veterinary Physiology for providing the necessary laboratory facilities
to carry out this research. We are also thankful to Professor and Head, Department of Pharmacology and Toxicology
for providing the necessary facilities in ICAR Niche Area of Excellence Laboratories. We are thankful to
Dr Deepak Modi, Scientist-F, National Institute for Research in Reproductive Health, Mumbai for allowing and
helping us in interpretation of the electron microscopy data.
Author contributions
Conceptualization—S.K.G. and D.K.S.; Methodology—B.K., R.S.Y., P.K. and A.K.; Software—B.K. and M.A.; Validation—S.K.G.
and D.K.S.; Electron microscopy-D.S.; Data Curation—B.K. and S.K.G.; Writing of MS: Original
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Draft Preparation—B.K.; Review & Editing—S.K.G.; Supervision—S.K.G.; Lab: S.Y. and B.Y.; Resources—College
of Biotechnology; Funding Acquisition—University funding.
Competing interests 
The authors declare no competing interests.
Additional information
Supplementary Information The online version contains supplementary material available at https​://doi.
org/10.1038/s4159​8-020-80235​-y.
Correspondence and requests for materials should be addressed to B.K.
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