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IN VITRO ACTION OF COBRA VENOM ON GOAT SPERMATOZOA ULTRASTRUCTURE BY TRANSMISSION AND SCANNING ELECTRON MICROSCOPY

Abstract

Goat spermatozoa were incubated in vitro in Tris-citrate buffer, pH 7.2, containing 0, 40, 80, or 160 mug of Naja haje venom/mL buffer for 4 hours. During incubation, the percentages of sperm motility were decreased, while percentages of dead spermatozoa were increased in a time and concentration-dependent manner. The effect of venom concentrations on the ultrastructure of incubated spermatozoa was examined hourly by scanning (SEM) and transmission (TEM) electron microscopy. SEM results showed plasma membrane wrinkling at heads of some spermatozoa after 2 h incubation with 40mug venom. Most spermatozoa suffered membrane wrinkling after 4 h incubation. However, incubation with 80 mug venom caused membrane fractures in most sperm heads after 1 h incubation. The extent and depth of these fractures were increased after 2-3 h incubation. After 4 h incubation, plasma membrane focal erosion of many spermatozoa heads was common. Incubation with 160 mg venom induced sperm head swollen plasma membranes after 1 h incubation. Ruptured and disintegrated membranes were seen after 2 h; lysis and removal of external surface of spermatozoa head plasma membranes were recorded after 3-4 h incubation. TEM indicated slightly swollen areas on the sperm head plasma membrane, but showed normal nuclei, acrosomes, and tail regions after 2 h incubation in 40 mug cobra venom. The swollen areas were accompanied by sperm head membrane disintegration as well as membrane irregularities and distortion of tail mitochondrial cristae after 3-4 h incubation. However, incubation with 80 mug venom showed focal areas of membrane lysis and discontinuity in the sperm heads and tails increasing with incubation time. Severe axoneme and tail longitudinal fiber degeneration and increased numbers of distorted mitochondrial cristae were also observed after 3-4 h incubation. Spermatozoa incubation with 160 mug venom increased severity of plasma membrane dissolution, disintegration, and rupture. Axoneme disorganization and tail longitudinal fiber fusion were seen after 1-2 h; complete deformation of the motility system was recorded after 3-4 h incubation. Nuclei were normal, but acrosomes were partially damaged, and distorted mitochondrial cristae were increased after 4 h incubation. The results indicated that cobra venom induced a concentration and time-dependent alterations on spermatozoa plasma membrane as well as obvious deformation on the motility system and tail mitochondria, which are responsible for sperm viability and fertilizing ability.

cobra venom; ultrastructure; goat spermatozoa; motility; Naja haje


IN VITRO ACTION OF COBRA VENOM ON GOAT SPERMATOZOA ULTRASTRUCTURE BY TRANSMISSION AND SCANNING ELECTRON MICROSCOPY

T. R. RAHMY1 T. R. Rahmy - Department of Biology, Faculty of Science - United Arab Emirates University, Al-Ain, 17551, UAE. E-mail: TarekR@uaeu.ac.ae , M. A. AYOUB2

1 Department of Biology, Faculty of Science, 2 Department of Animal Production, Faculty of Agricultural Sciences, United Arab Emirates University, Al-Ain, 17551, UAE.

ABSTRACT: Goat spermatozoa were incubated in vitro in Tris-citrate buffer, pH 7.2, containing 0, 40, 80, or 160 mg of Naja haje venom/mL buffer for 4 hours. During incubation, the percentages of sperm motility were decreased, while percentages of dead spermatozoa were increased in a time and concentration-dependent manner. The effect of venom concentrations on the ultrastructure of incubated spermatozoa was examined hourly by scanning (SEM) and transmission (TEM) electron microscopy.

SEM results showed plasma membrane wrinkling at heads of some spermatozoa after 2 h incubation with 40mg venom. Most spermatozoa suffered membrane wrinkling after 4 h incubation. However, incubation with 80 mg venom caused membrane fractures in most sperm heads after 1 h incubation. The extent and depth of these fractures were increased after 2-3 h incubation. After 4 h incubation, plasma membrane focal erosion of many spermatozoa heads was common. Incubation with 160 mg venom induced sperm head swollen plasma membranes after 1 h incubation. Ruptured and disintegrated membranes were seen after 2 h; lysis and removal of external surface of spermatozoa head plasma membranes were recorded after 3-4 h incubation.

TEM indicated slightly swollen areas on the sperm head plasma membrane, but showed normal nuclei, acrosomes, and tail regions after 2 h incubation in 40 mg cobra venom. The swollen areas were accompanied by sperm head membrane disintegration as well as membrane irregularities and distortion of tail mitochondrial cristae after 3-4 h incubation. However, incubation with 80 mg venom showed focal areas of membrane lysis and discontinuity in the sperm heads and tails increasing with incubation time. Severe axoneme and tail longitudinal fiber degeneration and increased numbers of distorted mitochondrial cristae were also observed after 3-4 h incubation. Spermatozoa incubation with 160 mg venom increased severity of plasma membrane dissolution, disintegration, and rupture. Axoneme disorganization and tail longitudinal fiber fusion were seen after 1-2 h; complete deformation of the motility system was recorded after 3-4 h incubation. Nuclei were normal, but acrosomes were partially damaged, and distorted mitochondrial cristae were increased after 4 h incubation.

The results indicated that cobra venom induced a concentration and time-dependent alterations on spermatozoa plasma membrane as well as obvious deformation on the motility system and tail mitochondria, which are responsible for sperm viability and fertilizing ability.

KEY WORDS: cobra venom, ultrastructure, goat spermatozoa, motility, Naja haje.

INTRODUCTION

Snakebites are the most important cause of morbidity and death in humans (24). Most cobra bites in Africa and Asia are suffered by grazing animals and rural agricultural workers while working barefoot in fields (37). Hemmaid (8) mentioned that the testes seem to be one of the organs prone to cobra venom action. Xu et al. (38) studied the in vitro effect of a nerve growth factor isolated from Chinese cobra venom on rat epididymal sperm motility.

The in vivo effects of other snake venoms on testicular tissues of experimental animals were also reported by some investigators (18,19,25,26). They showed various testicular alterations, which resulted in reduction of spermatogenesis, and consequently, led to possible reduction of victim fertility or complete sterility (26). However, Mohallal (18) and Rahmy (26) mentioned the presence of morphologically normal spermatozoa within the lumina of some seminiferous tubules during envenomation. Rahmy (26 ) suggested that these spermatozoa could lose their efficiency and motility. Levy et al. (15) added that male infertility could be related to defects in motility, capacitation, acrosome reaction, binding, and penetration of the zona pellucida of the ova.

It is well known that the blood testis barrier protects spermatogenesis from direct contact with blood and could prevent most blood toxins from entering spermatogenic cells and spermatozoa. However, toxins could be in direct contact with spermatozoa when they are transferred to the epididymus or seminal vesicle. For this reason, the purpose of this study is to shed some light on the direct effect of cobra venom on goat spermatozoa ultrastructure by in vitro incubation at different concentrations.

MATERIALS AND METHODS

Snake Venom

Lyophilized venom of the Egyptian cobra Naja haje was reconstituted in saline solution (0.85% NaCl) for the preparation of the concentrations required in the experiment.

In vitro treatment

Semen was collected from normal healthy bucks by using artificial vagina. Two to three ejaculates were pooled from each buck and checked for advanced motility. Samples of less than 60% motility were discarded. Semen was diluted (1:5) in warm Tris-citrate buffer, pH 7.2. Semen samples were washed twice in Tris-citrate buffer at 1000g for 5 minutes. Spermatozoa were suspended at 3-4 X 108/mL in 0 (Control), 40 (Low concentration), 80 (Medium concentration), and 160 g (High concentration) venom in Tris-citrate buffer and incubated at 37°C for 4 h. These concentrations were selected from many assayed in this study for their ability to keep spermatozoa alive over 4 hours.

Test of motility and dead/live spermatozoa

Sperm advanced motility (%) and smears for dead/live counts (stained with 0.5 % eosin; the routine semen evaluation method) were determined hourly for 4 h, with four repetitions.

Statistical Analysis

Complete randomized design was employed for data analysis using Costat statistical program, as per Snedecor and Cochran (29). Duncan's (6) multiple range test was used to compare means at P<0.05.

Electron microscopic preparations

After in vitro incubation of different sperm samples, 0.5 mL was taken from each sample after 1, 2, 3, and 4 h. Samples were centrifuged and supernatants were removed. The remaining pellets containing sperm were then rapidly fixed in 0.1M phosphate buffered fixative solution (4% formaldehyde + 1% glutaraldehyde, pH 7.4) overnight at 4°C (16). Sperm were then washed in 0.1M phosphate buffer, fixed for 1 h in 0.1M phosphate buffered 1% osmium tetroxide (at room temperature) and washed several times in the buffer. Dehydration was then performed in ascending series of ethyl alcohol. Each sample was divided into two equal halves for scanning (SEM) and transmission (TEM) electron microscope preparations.

SEM preparations

Dehydrated samples were loaded on the holders and left to dry for two hours. The holders were then electronically coated with a thin layer of gold and then examined by SEM (JSM-5600) at the Central Laboratories Unit (CLU) of the United Arab Emirates University.

TEM preparations

The dehydrated samples were treated with two changes of propylene oxide and transported to a mixture of propylene oxide and resin. The samples were embedded in a mixture of resin (Agar 100 Epoxy resin + DDSA + MNA + DMP-30) and left overnight. Two changes of resin were then applied and the samples were left in the oven at 60°C for 24 h for polymerization into plastic blocks. The blocks were sectioned at a thickness of 60-70 nm and mounted on copper grids. The grids were stained with uranyl acetate and lead citrate and left to dry. The grids were examined and photographed using a CM10 Philips Electron Microscope at the Faculty of Medicine and Health Sciences, the United Arab Emirates University.

RESULTS

Determination of motility and dead spermatozoa

Data in Table 1 and Figure 1A show that sperm motility decreased significantly (P<0.05) over incubation time. However, the rate of decrease was more pronounced with increased venom concentration in incubation media. The opposite was observed in dead spermatozoa percentage during incubation (Table 2 and Figure 1B). Spermatozoa death rate in the envenomed groups was obviously increased by the increase of the venom concentration and time of incubation and was less than the death rate in the control group.

Table 1.
Rate of goat spermatozoa motility during 4 h incubation at different concentrations of cobra venom (mean+S.E).
Figure 1a.
Rate of goat spermatozoa advanced motility during 4 h incubation at different concentrations of cobra venom.
Figure 1b.
Rate of dead goat spermatozoa during 4 h incubation at different concentrations of cobra venom.
Table 2
. Rate of dead goat spermatozoa during 4 h incubation at different concentrations of cobra venom (mean+S.E).

SEM studies

1 Control group

No surface changes were observed in control samples at zero time and subsequent sampling times. Control group spermatozoa revealed normal oval heads with homogenous, flat, and intact bounding plasma membranes. Neck and different parts of the tail were morphologically normal (Figure 2).

Figure 2.
Control spermatozoa showing intact heads (H) and tail regions.

2 Low concentration group

In vitro spermatozoa incubation for 1 h at low cobra venom concentration showed normal surface morphology of enclosing plasma membranes. Few sperm head plasma membranes showed few swollen spots. However, an obvious plasma membrane wrinkling was observed in the heads of some spermatozoa after 2 h incubation (Figure 3). The number of spermatozoa suffering membrane wrinkling was increased at 3 h (Figure 4) and included most sperm after 4 h incubation (Figure 5). The neck and tails revealed normal surface morphology in almost all spermatozoa in this group.

Figure 3.
Wrinkling (arrows) in the head plasma membrane of some spermatozoa incubated for 2 h with 40 mg venom.
Figure 4.
Spermatozoa incubated for 3 h with 40 mg venom showing wrinkled surrounding plasma membranes.
Figure 5.
Increased membrane wrinkling (arrows) of sperm head plasma membranes after 4 h incubation with 40 mg venom.

3 Medium concentration group

Membrane wrinkling and surface fractures were present on the heads of most sperm incubated for 1 h in the medium concentration venom (Figure 6). After 2 h incubation with the same concentration, irregular fractures were observed in different ways all over the sperm head plasma membranes (Figure 7). Deeper plasma membrane fractures extending in different directions were noticed in most spermatozoa heads was common after 4 h incubation (Figure 9). Fractured and wrinkled plasma membranes were also seen on many sperm tail regions (Figures 8 and 9).

Figure 6.
Fine membrane fracture (arrow) and wrinkling (double arrow) in a sperm head incubated for 1 h with 80 μg venom.
Figure 7.
Membrane fractures (arrows) in different directions along the sperm head after 2 h incubation with 80 mg venom.
Figure 8.
Incubation with 80 mg venom for 3 h showing deeper fractures (arrows) in the plasma membrane enclosing the sperm head.
Figure 9.
Focal areas of erosion (arrows) in the sperm head plasma membrane after 4 h incubation with 80 mg venom.

4 High concentration group

The most severe action was recorded after incubation with highest venom concentration in a time-dependent manner. Most spermatozoa heads showed swollen irregular plasma membranes that lost their continuity in certain places after 1 h incubation (Figure 10). Focal areas of membrane rupture, disintegration, and wrinkling were obvious in many spermatozoa heads after 2 h incubation (Figure 11). After 3 h incubation, lysis and removal of the plasma membranes external surface were seen in particular areas on most sperm heads. The extent and severity of membrane removal differ from one spermatozoon to another (Figure 12). The depth and degree of removal of the plasma membrane outer surfaces on different sperm heads were severely increased after 4 h incubation. Focal lyses of different parts of sperm heads were also noticed (Figure 13). The membranes enclosing different tail regions were also fractured and wrinkled in many sperm (Figures 12 and 13).

Figure 10.
Swelling and irregularity of the sperm head plasma membrane after 1 h incubation with 160 mg venom. Note membrane fracture (arrow).
Figure 11.
Focal area of membrane rupture (arrows) after 2 h incubation with 160 mg venom.
Figure 12.
Removal of the plasma membrane external surface (arrows) of sperm heads after 3 h incubation with 160 mg venom.
Figure 13.
Severe form of membrane rupture and removal of the external plasma membrane layer (arrows) in the head of sperm incubated for 4 h with 160 mg venom. Note focal damage in sperm heads (double arrow).

TEM studies

1 Control group

The control group showed common characteristic features of head, neck, and tail in most spermatozoa. The head was elongated with a nucleus and with an acrosome at its tip surrounded by intact plasma membrane (Figure 14). The neck and the three common tail pieces were also observed. All tail pieces contained intact axonemes surrounded by nine coarse outer fibrils in the middle and principle pieces. The fibrils were surrounded by a sheath of mitochondria in the middle piece (Figure 15) and a fibrous sheath in the principle piece.

Figure 14.
Normal sperm heads showing intact plasma membrane (arrow) and acrosome (A). (X 28000).
Figure 15.
Neck (N) and tail middle piece in control sperm showing the common axoneme (A), coarse fibers (arrow), and normal mitochondria (double arrow). (X 28000).

2 Low concentration group

The spermatozoa incubated with 40 mg cobra venom for 1 h revealed similar characteristics to those of the control group. After 2 h incubation, the sperm head plasma membranes were intact with few and slightly swollen areas (Figure 16). The nuclei and acrosomal regions were also intact and tail regions revealed common characteristics with normal mitochondria in the middle piece.

Figure 16.
Slightly swollen areas (arrows) in a spermatozoon surrounding plasma membrane incubated for 2 h with 40 mg venom. (X 29300).

The swollen areas increased in number and size in the sperm head plasma membranes and included larger numbers of sperm after 3-4 h incubation. Plasma membrane disintegration was observed in some sperm heads after 4 h incubation (Figure 18). All tail regions showed normal structural appearance except for irregularities in their enclosing plasma membranes (Figure 18). In addition, distortion of the mitochondrial cristae was seen in the tail middle piece l after 3-4 h incubation (Figure 17).

Figure 17.
Distortion of mitochondrial cristae (arrows) in the sperm tail middle piece after 4 h incubation with 40 mg venom. (X 22800).
Figure 18.
Swelling (arrows) and disintegration (double arrows) of sperm head plasma membranes after 4 h incubation with 40 mg venom. Note intact nuclei (N), acrosomes (A), axoneme (arrow head), and membrane irregularities around tail regions (double arrow head).(X 15500)

3 Medium concentration group

Lysis and rupture of the sperm head cell membrane are common alterations in spermatozoa incubated for different durations with 80 mg cobra venom. However, variable time-dependent alterations can be seen in tail regions. After 1 h incubation, irregular swollen sperm head and tail plasma membranes were noticed. The head plasma membranes lost their common electron density and appeared to contain focal areas of membrane lysis and disintegration (Figure 19). After 2 h incubation, areas of disintegration and rupture were very common in both the head (Figure 20) and tail (Figure 21) region plasma membranes. Sperm nuclei, acrosomes, axonemes, and tail fibers were all normal in appearance except for mitochondrial cristae distortion in the tail middle pieces (Figure 21).

Figure 19.
Loss of electron density and focal areas of lysis and disintegration (arrows) in a sperm head plasma membrane after 1 h incubation with 80 mg venom. (X 29300).
Figure 20.
Lysis (arrow) and rupture (double arrow) of sperm head plasma membrane after 2 h incubation with 80 mg venom. Note normal axoneme and tail regions surrounded by irregular membranes (arrow head). (X 19600).
Figure 21.
Increased number of mitochondria with distorted cristae (arrow) and lysis of a sperm tail plasma membrane (double arrow) after incubation for 2 h with 80 mg venom. (X 22800).

All these alterations were more severe after 3 h incubation (Figure 22). Plasma membrane lysis and discontinuity in the head regions were very severe after 4 h incubation (Figure 23). Complete lyses of surrounding membranes in certain tail pieces were also common (Figure 24). Axoneme and longitudinal fiber microtubules in different tail regions of many sperm were degenerated and lost their architectural appearance. Completely distorted mitochondrial cristae were also observed in the middle pieces of many sperm (Figure 24).

Figure 22.
Various forms of rupture and lysis (arrows) in sperm head plasma membranes incubated for 3 h with 80 mg venom. (X 16000).
Figure 23.
Head of a spermatozoon incubated for 4 h with 80 mg venom showing a plasma membrane gap (arrow) and membrane dissolution (double arrow). A: Acrosome, N: Nucleus. (X 30000).
Figure 24.
Cross section in different tail pieces showing complete lysis of the surrounding plasma membrane (arrow), degenerated axoneme (arrow head), and distorted mitochondrial cristae (M) after 4 h incubation with 80 mg venom. (X 29300).

4 High concentration group

The most severe alterations were seen in sperm incubated with 160 mg cobra venom. After 1 h incubation, the head region plasma membranes were dissolved and could be seen as fine dusty materials that irregularly extended around the sperm heads (Figure 25). Gaps in such materials were observed after 2 h incubation (Figure 27). However, after 3-4 h incubation, the plasma membranes of all sperm heads were either dissolved in particular areas or converted into minute vesicles irregularly arranged around sperm heads (Figure 29). The nuclei still presented normal appearance, but acrosomes were damaged in certain areas after 3-4 h incubation (Figure 29). The plasma membranes surrounding the neck and tail regions showed similar alterations to those of the head (Figures 26 and 30 Figure 30. Complete deformation of the axoneme and surrounding fibers (arrow) after 4 h incubation with 160 mg venom. Note damaged mitochondria (M), membrane discontinuity (double arrow), and dissolution (arrow head). (X 44800). DISCUSSION ). The axoneme in different tail pieces revealed obvious microtubule disorganization, distortion, and damage. The longitudinal fibers around the tail axoneme showed irregular shape and fused with each other in certain cases after 1-2 h incubation (Figure 26). Complete deformation of the motility system, including both the axonemes and their surrounding fibers, were recorded after 3-4 h incubation. In this case, the axoneme microtubules were completely damaged or lost their doublet appearance as they were fused with each other, with irregularly shaped longitudinal fibers (Figure 30 Figure 30. Complete deformation of the axoneme and surrounding fibers (arrow) after 4 h incubation with 160 mg venom. Note damaged mitochondria (M), membrane discontinuity (double arrow), and dissolution (arrow head). (X 44800). DISCUSSION ). In addition, the number of mitochondria with distorted cristae was increased with incubation time (Figures 26, 28 Figure 28. Most of the mitochondria showing distorted cristae (arrows) after 3 h incubation with 160 mg venom. (X 22800). , and 30 Figure 30. Complete deformation of the axoneme and surrounding fibers (arrow) after 4 h incubation with 160 mg venom. Note damaged mitochondria (M), membrane discontinuity (double arrow), and dissolution (arrow head). (X 44800). DISCUSSION ).

Figure 25.
The plasma membrane as fine dusty materials irregularly extended around the sperm head (arrow) after 1h incubation with 160 mg venom. Note sites of membrane lysis (double arrow).(X 29300).
Figure 26.
Disorganization and distortion of the axoneme (arrow) and tail irregular fused longitudinal fibers (double arrow) after 1 h incubation with 160 mg venom. Note complete lysis of plasma membranes (arrow heads) and distorted mitochondria (M). (X 59800).
Figure 27.
A gap (arrow) in degenerated materials of the plasma membrane after 2 h incubation with 160 mg venom. (X 12700).

Figure 29.
The most severe form of membrane discontinuity (arrows) after 4 h incubation with160 mg venom. Note vesiculated plasma membranes (double arrow) and damaged acrosomal areas (arrow head). (X 22800)

Incubation of goat spermatozoa with cobra venom showed decreases in sperm motility and increases in dead spermatozoa with increased incubation time and venom concentration. These changes were accompanied by severe alterations of spermatozoa plasma membranes, mitochondrial distortion, and motility system deformation.

Membrane alterations could be attributed to the action of cobra venom phospholipase A2. This can interact and penetrate the membrane as it has a side chain fully inserted into the membrane hydrophobic core, and another in the membrane-water interface (32 ). Fletcher and Jiang (7,36) showed that the addition of phospholipase A2, isolated from snake venoms, to cell cultures catalyses hydrolysis of fatty acid ester linkage in the lipid bilayer of the cell membrane phospholipids. Membrane toxins from cobra venom are known to have a lytic synergism with phospholipase on cell membranes. The in vitro lytic activity of Naja naja atra cytotoxin on different cell types was reported by Stevens-Truss et al. (30). Cobra cardiotoxins also possess a strong membrane lytic property on membrane bi-lipid layers and cause cell membrane damage (22,23,31).

The presence of such an array of membrane toxins in cobra venoms could explain the alterations observed in spermatozoa plasma membranes. Vernon and Bell (34) added that disruption of cellular membranes might expose new substrates to other venom toxins. This could explain the recorded partial damage of the acrosomes accompanying the removal and lysis of sperm head plasma membranes in case of incubation with the highest venom concentration. Morini et al. (20) reported that a snake venom phospholipase causes disturbance of acid phosphatase, which is one of the main components of the acrosomes.

Despite such recorded membrane alterations, no changes were detected in spermatozoa nuclei. This could be explained thus; the genetic materials are highly packed during spermatozoa differentiation to prevent any genetic mutations (2). Mckelveymartin et al. (17) mentioned that fertile sperm are more resistant to damage by agents that induce DNA breakdown than infertile samples. Rahmy (26 ) added that DNA depletion due to Echis carinatus envenomation included most spermatogenic cells but not luminal spermatozoa.

On the other hand, distortion of mitochondrial cristae in sperm tail middle pieces after incubation with venom was found to be time and concentration-dependent. Distorted mitochondrial cristae were also observed by Rahmy (27) in renal cells of cobra envenomed rats. Huang and Gopalakrishnakone (10) reported that cobra venom phospholipase A2 is involved in causing mitochondria degeneration and distortion. It also uncouples oxidative phosphorylation in the mitochondria due to inhibition of electron transfer in the sub-mitochondrial system at cytochrome C level followed by other sites in the respiratory chain (33). Oron et al. (21) added that cobra cytotoxin may cause mitochondrial deformation either directly by interacting with mitochondria after cell penetration or indirectly by stimulating intracellular processes after binding to the cell membrane. It was also found that cobra cardiotoxin could damage mitochondria (23). Moreover, Salgueiropagadigorria et al. (28) added that venom cytotoxic effect led to the inhibition of mitochondrial ATP production. Snake venom phospholipase also causes disturbance of ATPase and succinate dehydrogenase (20,27). It is believed that such damaging effect of venom components on the mitochondrial enzymatic system could be responsible for inducing the observed decreases in spermatozoa motility as mitochondria are concerned with providing energy required for sperm motility.

Mitochondrial distortion was also accompanied by deformation in the motility system indicated by axoneme disorganization and damage and fusion and irregularities of tail coarse fibers. This could also directly participate in the reduction of sperm motility seen in this study. It could also be attributed to venom phospholipase action, which is known to affect the cytoskeleton function of certain cell types (5).

These membrane alterations, mitochondrial distortion, and motility system deformation could be responsible for inducing the observed increased death rate in incubated spermatozoa. Butler et al. (3) reported that venoms of seven elapid snakes exhibited a general cytotoxicity and relative in vitro potency for causing cell death of different cell types in mixed cell cultures. Cobra venom cytotoxicity was reported by Wang and Huang (35).

On the other hand, it is believed that cobra venom neurotoxin (13) could also affect the sperm activity though its action on sperm acetylcholine receptor-like molecules. These receptors are correlated with a sperm-egg interaction and the regulation of sperm propulsion (1).

From another point of view, it has been reported that some compounds present in the spermatozoa that participate in their sperm-egg binding activities are homologues to certain venom compounds. Kreil (12) reported that hyaluronidase from hornet venom and hyaluronidase of mammalian spermatozoa, which plays a key role in sperm adhesion to zona pellucida, are homologous proteins and could exert similar activities. Similarly, phospholipase A2 in the acrosomal and postacrosomal regions of human spermatozoa (4), sperm integrins (9), and protein active molecules (11) are homologous to snake venom components. It is believed that these snake venom compounds could lead to lysis of spermatozoa head plasma membranes to accelerate exocytosis of the acrosomes, as they are similar to the sperm compounds involved in this function. On the other hand, homology between compounds released by the zona pellucida of the ova to initiate the process of acrosomal reaction and those in the venom (14) could be responsible for the removal of plasma membranes.

It could be concluded that Egyptian cobra venom induced a direct action on sperm viability and fertilizing ability by affecting spermatozoa plasma membrane and motility system in a time and concentration-dependent manner. This could indicate an expected reduction in fertility of cobra victims.

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30 STEVENS-TRUSS R., MESSER WS., HINMAN CL Heart and T-lymphocyte cell surfaces both exhibit positive cooperativity in binding a membrane-lytic toxin. J. Membr. Biol., 1996, 150, 113-22.

31 SUE SC., RAJAN PK., CHEN TS., HSIEH CH., WU W. Action of Taiwan cobra cardiotoxin on membranes: binding modes of a beta-sheet polypeptide with phosphatidylcholine bilayers. Biochemistry, 1997, 36, 9826-36.

32 SUMANDEA M., DAS S., SUMANDEA C., CHO W. Roles of aromatic residues in high interfacial activity of Naja naja atra phospholipase A2. Biochemistry, 1999, 38, 16290-7.

33 UENO E., ROSENBERG P. Inhibition of phosphorylation of synapsin I and other synaptosomal proteins by beta-bungarotoxin, a phospholipase A2 neurotoxin. J. Neurochem., 1992, 59, 2030-9.

34 VERNON LP., BELL JD. Membrane structure, toxins and phospholipase A2 activity. Pharmacol. Ther., 1992, 54, 269-95.

35 WANG XM., HUANG SJ. The selective cytotoxicity of cobra venom factor immunoconjugate on cultured human nasopharyngeal carcinoma cell line. Hum. Exp. Toxicol., 1999, 18, 71-6.

36 WANG WY., LU QM., ZHANG Y., MENG QX. Cobra (Naja naja atra) membrane toxin isoforms: Structure and function. J. Toxicol. Toxin. Rev., 1998, 17, 525-32.

37 WARRELL DA. Clinical toxicology of snakebite in Africa and the Middle East and Asia. Clin. Toxicol. Anim. Venoms Poisons, 1995, 433-594.

38 XU TR., WANG WY., HUANG YH., MENG QX., LI DS., LU QM., XIONG YL. A nerve growth factor from the venom of Chinese cobra (Naja naja atra) and its effects on male reproductive system in rats. Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol., 1999, 124, 149-56.

Received July 3, 2000

Accepted September 4, 2001

CORRESPONDENCE TO:

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    SUE SC., RAJAN PK., CHEN TS., HSIEH CH., WU W.  Action of Taiwan cobra cardiotoxin on membranes: binding modes of a beta-sheet polypeptide with phosphatidylcholine bilayers.  Biochemistry, 1997, 36, 9826-36.
  • 32
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  • 33
    UENO E., ROSENBERG P.  Inhibition of phosphorylation of synapsin I and other synaptosomal proteins by beta-bungarotoxin, a phospholipase A2 neurotoxin.  J. Neurochem, 1992, 59, 2030-9.
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    VERNON LP., BELL JD.  Membrane structure, toxins and phospholipase A2 activity.  Pharmacol. Ther., 1992, 54, 269-95.
  • 35
    WANG XM., HUANG SJ.  The selective cytotoxicity of cobra venom factor immunoconjugate on cultured human nasopharyngeal carcinoma cell line.  Hum. Exp. Toxicol, 1999, 18, 71-6.
  • 36
    WANG WY., LU QM., ZHANG Y., MENG QX.  Cobra (Naja naja atra) membrane toxin isoforms: Structure and function.  J. Toxicol Toxin. Rev, 1998, 17, 525-32.
  • 37
    WARRELL DA.  Clinical toxicology of snakebite in Africa and the Middle East and Asia. Clin. Toxicol. Anim. Venoms Poisons, 1995, 433-594.
  • 38
    XU TR., WANG WY., HUANG YH., MENG QX., LI DS., LU QM., XIONG YL.  A nerve growth factor from the venom of Chinese cobra (Naja naja atra) and its effects on male reproductive system in rats.  Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol., 1999, 124, 149-56.
  • Figure 28. Most of the mitochondria showing distorted cristae (arrows) after 3 h incubation with 160 mg venom. (X 22800).
  • Figure 30. Complete deformation of the axoneme and surrounding fibers (arrow) after 4 h incubation with 160 mg venom. Note damaged mitochondria (M), membrane discontinuity (double arrow), and dissolution (arrow head). (X 44800).
    DISCUSSION
  • T. R. Rahmy - Department of Biology, Faculty of Science - United Arab Emirates University, Al-Ain, 17551, UAE.
    E-mail:
  • Publication Dates

    • Publication in this collection
      13 May 2002
    • Date of issue
      2002

    History

    • Accepted
      04 Sept 2001
    • Received
      03 July 2000
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