Keywords: lead chloride; seminiferous tubules; epididymis; semen analysis

Abstract

Aim: To evaluate the effect of acute lead chloride exposure on testis and sperm parameters in mice. Methods: PbCl₂, 74 mg/kg, was daily administered to sexually mature male mice for 3 days and the effects on the testicular histology and ultrastructure as well as the motility and density of spermatozoa in cauda epididymis were observed. An additional group of mice were treated for 1-3 days and were allowed to recover for 32 days to determine the reversibility of lead-induced changes. Results: The testicular weight, seminiferous tubular diameter and sperm counts were significantly decreased following 3 days of PbCl₂ treatment, but were unaffected by shorter-term exposures. The changes caused by lead are mostly reversible. Conclusion: Acute lead chloride exposure injures the fertility parameters of male mice and the effects are partially reversible.

1 Introduction

Although preventive measures aimed at reducing lead exposure have been implemented, exposure to inorganic lead remains one of the most prominent occupational and environmental health problems. Our previous studies have demonstrated that lead chloride had some deleterious effects on mice spermatogenesis (1st Post-Graduation Symposium in Biosciences, University of Aveiro, 2002). The present study was designed to further investigate this effect and, specifically, to determine the effects of acute PbCl2 exposure on spermiogenesis, testicular and epididymal structure and the quality and quantity of epidydimal sperm.

2 Materials and methods

A total of 40 male ICR-CD1 mice (2 months of age), purchased from Harlan Interfauna, Iberica (Spain), were used. Animals were housed (4 animals per cage) at 24 , relative humidity 60 % and a 12-hour alternate light-dark cycle. Free access to diet and tap water was provided. PbCl₂ was obtained from Merck (Germany). Animals were divided at random into 8 groups (4 treated and 4 control groups, n = 5), and subcutaneously injected with 0.5 mL of sterilized PbCl2 (74 mg^.kg^-1.day^-1) in saline for 1 day (group I), 2 days ( group II), 3 days (group III) and 3 days followed by a 32-day recovery period (group IV). Controls were set for all groups, substituting lead by only the vehicle. After that, animals were sacrificed by cervical dislocation and the testis, the epididymis and the epididymal sperm were obtained for analyses. All the animal assays were conducted in accordance with the Institutional Guidelines for Ethics in Animal Experiments (based on Rule No. 86/609/CEE - 24/11/92).

Fragments of tissue were fixed in Bouin's solution and routinely prepared for light microscopy. The mean diameter of 60 randomly selected circular tubules in transverse cross sections was determined using an ocular micrometer. For transmission electron microscopy small pieces of testis were fixed in 2.5 % glutaraldeheyde and 2 % phosphate-buffered OsO₄, dehydrated and embedded in Epon. Thin sections prepared with a diamond knife in a ultramicrotome (Leica, Germany) were mounted on formvar-coated 300-mesh grids and stained with uranyl acetate followed by lead citrate. Observations were made using a transmission electron microscope (Hitachi, Japan) at 100 kV accelerating voltage.

2. 3 Sperm analysis

3 Results

No treatment-related effects on the survival and behaviour of animals were observed. No significant differences were observed in body and organ weights or seminiferous tubular diameter between groups I & II and their controls (Table 1). Animals of the group III (PbCl₂ administered for 3 days) exhibited a significant reduction in the body weight (P<0.01) and an increase in the testis weight (P<0.05). The most significant decrease in tubular diameter was found in the seminiferous tubules of this group (P<0.01). For group IV animals (PbCl₂ administered for 3 days, followed by a 32-day recovery period), no differences were observed on body and organ weights. The seminiferous tubular diameters were increased (P<0.01).

Table 1. Effect of PbCl₂ on body and organ weights and seminiferous tubular diameter in mice. ^bP<0.05, ^cP<0.01, compared with controls; ^fP<0.01 compared with that at beginning of experiment.

Figure 1. A). Histological section of the testis from a control animal; B) Lumen of seminiferous tubules from lead-chloride treated animal (group III), showing accumulation of immature germ cells in the lumen (250).

Figure 2. A). Fine structure of a round spermatid from a control animal, evidencing a typical acrosome and nucleus (?6 000); B-C. Fine structure of young spermatids from a PbCl₂-treated animal (group III), where alterations within the acrosome and nucleus (arrows) are noted; B 13 400, C 12 000.

Group III mice had a significantly lower epididymal sperm density, a higher percentage of immotile spermatozoa and more sperm with detached head compared with the controls (P<0.05, Table 2). Interestingly, the percentage of sperm with an intact acrosome was also higher in the PbCl2-treated animals. The differences were no longer detected following the 32 day recovery period (group IV), with the exception of the increased percentage of acrosome-intact sperm in the treated animals (Table 2). There were no visible changes in other sperm parameters (data not shown).

Table 2. Effect of PbCl₂ on epididymal sperm count, percentage of immotile sperm, acrosome-intact sperm and detached sperm heads in mice. *300 spermatozoa counted for each animal; #500 spermatozoa counted for each animal: ^bP<0.05, ^cP<0.001, compared with controls.

The significance of the differences encountered was confirmed by ANOVA (P<0.01).

The exfoliation and the release of immature germ cells within the tubular lumen in PbCl₂-treated animals reported here also represents a degenerative process attributable to lead; the cytological alterations observed within the acrosomal cap may interfere with the potential of these cells to mature into functional sperm. The restoration the morphological features of the seminiferous tubules in the recovery group indicated an apparent reversibility of lead chloride effects on this system.

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Correspondence to: Dr. Maria de Lourdes Pereira, Department of Biology, University of Aveiro, 3810 Aveiro, Portugal.
Tel: +351-234-370 770, Fax: +351-234-426 408
Email: lpereira@bio.ua.pt
Received 2003-08-19     Accepted 2004-04-27