Evaluation of effects of 1,3-dinitrobenzene on sperm motility of hamster using computer assisted semen analysis (CASA)

L.D.C. Peiris, H.D.M. Moore¹

Dept. of Zoology, University of Colombo, Colombo 03, Sri Lanka  
¹Dept. of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S 10 2TN, UK

Asian J Androl  2001 Jun; 3:  109-114

Keywords: mDNB; sperm motility; CASA; sperm fertility

Abstract

Aim: To evaluate the effects of 1,3-dinitrobenznene (mDNB) on sperm motility of hamster and to correlate the results with the fertility.  Methods: Adult male hamsters were gavaged with one of the 3 dose regimes of mDNB (1.5 mg daily for 4 weeks, 1.5 mg one day a week for 4 weeks and 1.0 mg 3 days a week for 4 weeks). Computer assisted semen analysis (CASA) was used to analyse the sperm motility parameters, curvilinear velocity (VCL) and straight line velocity (VSL) of sperm in distal corpus epididymides and distal cauda epididymides. In vitro fertilisation was carried out only for 1.5 mg mDNB daily group to determine the sperm fertilising capacity. Results: There was a significant reduction in sperm velocity parameters at weeks 3 and 4 after treatment, which was correlated with a decline in sperm fertility. Conclusion: Sperm velocity parameters may be used to determine the effect of a toxic insult on the sperm function.

1 Introduction

1,3-dinitrobenzne (mDNB) is used as an intermediate in many industries, including chemicals, pesticides and plastics. Studies have revealed that mDNB is a potent testicular toxicant and its primary target of action is the Sertoli cell. mDNB is known to cause reduction in testicular weight^[1]. It can also cause a reduction in fertility in male rats with a decrease in cauda epididymal sperm reserve and sperm motility, which is not completely recovered even 12 weeks after cessation of chronic^[2] or acute^[3] exposure.

Since lowered fertilising ability is often associated with a poor semen quality, sperm motility is considered as one of the most important parameters in evaluating the fertilising ability of sperm in the human or other mammal species^[4]. In the past, assessment of sperm motility was based mainly on subjective evaluation, which is prone to man-made errors. More recently, computer-assisted sperm analysis (CASA) has been developed as a sensitive and reliable method to analyse various motility parameters. In the human, it has been shown that the rate of fertilisation correlates with the curvilinear velocity (VCL); lateral head displacement had been shown to also correlate with the efficiency of cervical mucus penetration. All these motility parameters affect sperm penetration of mucus or oocyte vestments and thus the fertilising ability^[5].

2 Materials and methods

2.1 Animal treatment

Adult golden male hamsters (Mesocricetus auratus), weighing 120-140 g, were purchased from the Harlen (UK) Limited. The animals were divided at random into 3 groups of 6 animals each. Group 1 gavaged with 1.5 mgkg^-1d^-1 of mDNB (dissolved in 60% PEG) for 4 weeks. After completion of treatment one animal was killed at weekly intervals. A second group were given an oral dose of 1.5 mg/kg mDNB, 1 day per week for 4 weeks, and killed at weeks 3 and 4 after the completion of treatment. A third group were dosed with 1.0 mg/kg mDNB, 3 days a week (Monday, Wednesday and Friday) for 4 weeks and killed at week 3 and week 4 after cessation of dosing. At all the dose levels, control animals (n=6/group) received the vehicle only.

2.2  Medium for sperm

Modified Krebs-Ringers solutions was used for CASA analysis. The medium was supplemented with 12 mg/mL crystalline bovine serum albumin, filter sterilised (0.2 m, Glean Sciences, UK), and equilibrated overnight in an atmosphere of 5% CO₂ in air at 37in a humidified incubator. The final pH of the medium was approximately 7.4 while the osmotic pressure was between 280 and 300 mOsm.

2.3 Collection and preparation of sperm

Hamsters were killed with an overdose of pentobarbitone sodium (J.M. Loveridge, Southampton, UK). The distal corpus and the distal cauda regions of the epididymides were excised quickly and rinsed in BWW medium (kept at 37), blotted on tissue paper to remove excess blood. Pieces of tissue were immersed in 500L of medium and incubated at 37 for a few minutes to enable sperm to swim out into the solution. An aliquot of 150 L of BWW medium was placed in a pre-warmed culture dish (3510 mm, Corning, New York, USA) and covered with a prewarmed 2222 mm coverslip. The edges of the cover slip were covered with pre-warmed paraffin oil (Boots Co., Nottingham, UK). An aliquot of 20 L sperm sample was added to a corner of the coverslip giving a chamber depth about 35 m. The petri dish was placed in an inverted microscope (Diaphot, Nikon, London, UK) fitted with a thermostatically controlled air heated cabinet  (Nikon, London, UK) equilibrated at 37. The microscope was fitted with a video camera (0.5 lux, Pulnix Ltd. London, UK) and a video recorder (Panasonic Ltd., London, UK).

2.4 Videomicroscopy of sperm

Upon placing the petri dish containing the sperm sample on the microscopic stage, the motility pattern of the sperm from the distal corpus and the distal cauda epididymides were video-recorded for about 4-5 min with a4.0 negative phase-contrast objective. The recordings were made immediately after sample preparation.

2.5 CASA

The motility parameters were analysed using the Hobson Sperm Tracker sperm motility analyser (Hobson Tracking System Ltd., Sheffield. UK). The optimum parameter settings for the Hobson Sperm Tracker for hamster sperm were obtained as recommended by the manual and empirical alterations that optimised the tracking as shown by the trail draw facility. Table 1 summarises the various conditions and parameters set up for the motility analyser used in the present study. The trail draw was used to plot out the trails for 3-4 seconds for the inspection of tracking so that the necessary adjustments could be made. The distance in the field of view was calibrated using a calibrated slide (Graticules Ltd., Kent, UK) and the final setting were saved and used for each analysis.

Table 1.  Settings used by the Hobson Tracker for sperm analysis.

2.6 In vitro fertilisation

The method described by Bavister^[8] was used. Briefly, the mature female hamsters were superovulated with 40 IU pregnant mare serum gonadotropin and human chorionic gonadotropin. Eggs were recovered by rupturing the oviducts and inseminated with the sperm recovered from the distal cauda of male hamster. In vitro fertilisation was carried out only for the males in Group 1.

2.7 Statistical analysis

3 Results

3.1 Motility patterns of sperm

Figure 1(a and b) represents the sperm tracks obtained for both distal corpus and distal cauda epididymides. Not all the sperm were motile in the distal corpus and the most characteristic pattern was the angular movement. Some sperm were seen agglutinated head to head. The highest percentage of motility was observed in sperm from the distal cauda region. The sperm were extremely active and exhibited linear progressive motility. Agglutination of cauda sperm was not observed. The sperm exhibited higher VCL and VSL values than those from the distal corpus region. 

Figure 1.  Sperm tracks obtained from distal corpus (A) and distal cauda (B) epididymal sperm using the trial draw facility of Hobson Sperm Tracker. Tracking time: 4 sec.

3.2 Effect of 1.5 mg mDNB daily dose on movement parameters of epididymal sperm 

3.2.1 Effect on distal corpus sperm

Figure 2 (a and b) illustrates the mean VCL and VSL observed from the control and treated hamster sperm samples. Compared to the controls there was a sudden decrease in mean VCL up to week 3 and followed by a gradual recovery with significant reductions occurring at week 3 (P<0.01), week 4 (P<0.01) and week 5 (P<0.05). Similar pattern was observed for mean VSL with marked reductions at weeks 3 and 4 (both P<0.01).

3.2.2 Effects on distal cauda sperm

Similarly, a decrease in the mean VCL was observed from week 1 onwards with minimum values occurring at week 3 (P<0.01). There was a gradual increase in mean VCL after week 3 with values reaching control values at week 5. There was a gradual decrease in mean VSL from weeks 2 to 4, followed by a gradual increase, returning to normal levels at week 6. The minimum mean VSL values were observed at weeks 3 and 4 (P<0.01). Results are presented in Figure 2 (c and d).

Figure 2.  Line graphs of mean curvilinear velocities (VCL) and mean straight line velocities (VSL) of distal corpus (a-b) and distal cauda (c-d) sperm in hamsters treated with  1.5 mg mDNB daily for 4 weeks.  Sperm samples are taken from 6 animals at weekly intervals up to 6 weeks. ^bP<0.05, ^cP<0.01,  compared with the controls.

3.3 In vitro fertilisation

Hamsters exhibited impaired sperm fertilising capacity from week 1 to 5, and was reduced to almost zero at  weeks 3 and 4 after dosing (Figure 3).

Figure 3.    Fertilization rates of male hamsters treated with chronic doses of mDNB. ^bP<0.05, ^cP<0.01,  compared with the controls.

3.4 Relationship between sperm motility and fertilising ability

In the 1.5 mg mDNB daily-dosed treated animals, the moment correlation coefficient between the mean VSL and the sperm fertilising capacity was highly significant (r=0.882, P<0.01). There was a similar although less significant relationship between mean VCL and sperm fertilising capacity (r=0.75, P<0.05).

3.5 Effects of 1.5 mg mDNB, 1 day week and 1.0 mg mDNB, 3 days/week doses on distal corpus and distal cauda sperm motility

4 Discussion

Sperm released from the testis are immotile and incapable of fertilising an egg. During their passage through the epididymis sperm acquire both the ability to swim progressively in a straight direction and the ability to fertilise eggs^[9]. Any detrimental alteration to sperm during epididymal passage, may affect their motility^[10]. Chemicals might also affect flagellum development during spermiogenesis so that epididymal sperm maturation cannot occur properly.

The VCL values obtained for distal cauda epididymal sperm was similar to values (254-344 m/sec) obtained by Kann and Serres^[11] for hamster cauda epididymal sperm. However, it was very difficult to analyse the motility of sperm from either the caput or proximal corpus epididymides quantitatively due to the short duration of sperm survival. The results may vary with the depth of the chamber, the optic source, the medium used, the tracking settings and the temperature^[12]. Therefore, in order to provide constant conditions the inverted microscope was fitted with an automatic stage and the cabinet was thermostatically controlled. Furthermore, the chamber depth and tracking settings were kept constant throughout the experiment.

In the present study, when male hamsters were treated with 1.5 mg mDNB daily, alterations in both the mean VCL and VSL were observed for sperm obtained from both the distal corpus and distal cauda epididymides. The decrease in VCL was due to a reduction in both the angle of lateral head displacement (ALH) and the beat cross frequency (BCF). A decrease in ALH means the sperm head is moving less from side to side while a decrease in BCF means these actions are slower. All these changes will ultimately lead to a reduced ability to penetrate the egg vestments with a reduced fertility rates in vitro^[5]. This was evident from the present study where there was a positive correlation between the VCL and the sperm fertilising ability. It was clear that 1.5 mg mDNB daily dose level can alter the sperm function up to 4 weeks as manifested by both impaired fertility and motion parameters.

Similarly, progressive sperm motility is essential for efficient penetration and it has been shown that a reduction or absence of VSL is associated with male infertility even when other sperm parameters are normal. Wright et al^[6] observed a reduction in VSL of rat sperm from males treated with (cholorohydrin. There was a good correlation between the ability of sperm to fertilise the egg and VSL. Confirming this point, in this study there was a significant correlation between the sperm VSL and fertilising capacity. Similar correlations have been obtained in the rat by Moore and Akhondi^[13] and in the hamster by Slott et al^[14]. Toth et al^[7] have evaluated the relationship between fertility and motion parameters of rats exposed to epichlorohydrin and found a significant correlation between the fertility and all the sperm motion parameters except the BCF.

During passage along the epididymis, not only are the thiol groups in the nuclear protamine of the sperm head oxidised but also are the proteins of the sperm axoneme. This hardening of the axoneme strengthens the structure of the sperm tail^[15] so that it plays a major role in the maturation of sperm motility^[16]. This is the main reason for obtaining reduced VSL values for sperm from the distal corpus when compared to distal cauda epididymidis where disulphide bonds are more stabilised. Cornwall et al^[17] were able to induce progressive motility of hamster sperm by sulphydryl oxidising agents. Therefore, the reduction in sperm motility observed with 1.5 mg mDNB daily dose could be due partly to alteration in sulphydryl oxidation during sperm passage along the epididymis.

In contrast to males dosed daily with 1.5 mg mDNB, those in the other low dose groups did not show any alterations in their sperm motility. Whether mDNB does act on tail axoneme remains to be determined and to clarify this point the exact mechanism of action of mDNB must be determined. Toxicants can alter the sperm motility by inhibiting sperm metabolism and/or by altering the epididymal function^[18]. Toxicants like mDNB can alter sperm motility as a consequence of their testicular toxicity altering the flagellum development and function.

In conclusion, CASA was sensitive enough to analyse the effects of reproductive toxicants on sperm motility in a quantitative manner and it was shown that 1.5 mg mDNB given daily to hamsters for 4 weeks resulted in alteration in sperm motion parameters (VSL, VCL) which were correlated with sperm fertilising capacity.

Acknowledgements

The authors thank the Commonwealth Scholarship Commission in the UK for the financial support and Mr. Nick Jenkins for gavaging the hamsters.

References

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Correspondence to: Dr. L.D.C. Peiris, Dept. of Zoology, University of Colombo, Colombo 03, Sri Lanka.  
Tel: +94-1-503 399  E-mail: dinithsamay@eureka.lk 
Dr. H.D.M. Moore,   E-mail:  h.d.moore@sheffield.ac.uk
Received 2001-03-29     Accepted 2001-05-21