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Effect of lead chloride on spermatogenesis and sperm parameters in mice

Antnio Graça1, 2, João Ramalho-Santos1, Maria de Lourdes Pereira2

1Department of Zoology, University of Coimbra, Coimbra, Portugal
2Department of Biology, University of Aveiro, 3810 Aveiro, Portugal

Asian J Androl 2004 Sep; 6: 237-241

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

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

Recently, there has been substantial interest in the potential adverse effects of exposure to environmental hazardous chemicals on male reproduction. Lead is one of the most widespread contaminants among the myriad of xenobiotics. Lead chloride is used in the manufacture of other lead salts, as the mordant in cotton dyes, lead coating for metals, a drier in paints, varnishes and pigment inks and as a colorant in hair dyes [1].

Evidence of the harmful effects of lead on the public health in general and on male reproduction in different species of rodents in particular have been widely studied. Male occupational exposure to lead compounds results in a general suppression of the hypothalamus-pituitary-testicular axis [2-4]. Semen parameter abnormalities and decreases in testicular weight, serum testosterone level and fertility have also been reported [5, 6]. The use of different animal models for the evaluation of the toxicity by lead compounds have multiple potential mechanisms of action [7-9]. Some authors emphasized that lead targets spermatogenesis and sperm within the epididymis rather than acting on the hypothalamic-pituitary-testicular axis as previously described [10]. Other studies have found an association between the exposure to inorganic lead compounds and increased risks of carcinogenesis[11].

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

2.1 Animals and dosing procedure

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).

2.2 Histological and ultrastructural studies

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

One testis and one epididymis from each mouse of groups III and IV, as well as related controls, were removed and placed in the Tyrode's modified medium (MT6). The epididymis was cut and sperm were allowed to swim out into the medium for 30 min at 35 . One drop of the sperm suspension was placed on a slide for light microscope observation of motility at a magnification of 100. A total of 300 sperm were evaluated for each sample. The concentration of spermatozoa was estimated using a haemocytometer with a double Neubauer ruling at a magnification of 400.

For the analysis of acrosomal status, the sperm suspension was fixed in 5 % formaldehyde, centrifuged and re-suspended in ammonium acetate as previously described [12]. Sperm were stained with Comassie G250 and a total of 500 cells were counted under a light microscope (magnification 400). In addition, at least 500 cells per sample were evaluated for the percentage of detached sperm heads.

2. 4 Statistical analysis

Data are presented in meanSD. Statistical analyses were carried out using Student's t test and complemented with ANOVA.

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.


Group III
(PbCl2 administration for 3 days)

Group IV
(PbCl2 administration for 3 days,
followed by 32 day recovery period)





Body weight at beginning of experiment (g)

34.70.1 (n=3)

38.10.4 (n=5)

33.10.4 (n=5)

33.30.4 (n=10)

Body weight before sacrifice (g)

35.21.4 (n=3)

31.60.4 (n=5)b, f

33.30.5 (n=5)

34.01.0 (n=10)

Testis weight (mg)

121.96.2 (n=5)

122.51.4 (n=10)b

122.11.1 (n=5)

127.34.1 (n=10)

Epididymis weight (mg)

18.22.9 (n=5)

19.62.9 (n=10)

19.10.4 (n=5)

19.10.9 (n=10)

Seminiferous tubular diameter (µm)

186.12.7 (n=3)

153.44.1 (n=3)c

169.21.1 (n=7)

174.61.2 (n=7)c

No gross or histological abnormalities of the testis or epididymis were observed in tissue from any of the control animals. Lead chloride administered to animals for 1 or 2 days failed to produce any observable morphological alterations in either of these tissues. However, the histological sections of testis from group III mice revealed degenerative changes within the seminiferous tubules as compared with the controls (Figure 1). These changes include vacuolation of tubules and sloughing of immature germ cells into the lumen. The micrographs of both the basal and the upper compartments of the seminiferous tubules showed normal histomorphology as the controls (data not shown). However, certain changes were noted in the acrosome and nucleus of the round spermatids (Figure 2A); they were abundant vesicles scattered throughout the acrosomal cap and a clear area devoid of chromatin facing the nucleus in some spermatids (Figure 2B). Other cells displayed an apparently well-developed acrosome, although some clear areas were also evident (Figure 2C). The cytological feature of the seminiferous tubules in animals of group IV was normal. The epididymis in all groups exhibited normal morphology as in the controls (data not shown).

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.


Group III
(PbCl2 administration for 3 days)

Group IV
(PbCl2 administration for 3 days,
followed by 32 day recovery period)





Epididymal sperm density (106 mL-1)

5.70.6 (n=11)

4.20.3 (n=11)b

5.80.3 (n=13)

5.20.2 (n=10)

Percentage immotile sperm*

26.81.5 (n=4)

34.92.7 (n=7)b

26.61.9 (n=7)

21.51.8 (n=6)

Percentage acrosome-intact sperm#

84.910.8 (n=9)


85.46.7 (n=8)

92.23.8 (n=9)c

Percentage detached sperm head#

1.93.5 (n=5)

7.16.4 (n=10)b

2.24.8 (n=5)

2.83.2 (n=10)

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

4 Discussion

The present study showed that 3 daily doses of lead chloride causes a significant decrease in the average body weight and significant alterations in the histological and ultrastructural patterns in the testis. Similar changes accompanied by the accumulation of immature cells within the tubular lumen were also observed in rats under the influence of lead acetate [13]. More conspicuous degenerative changes in testicular tissues and an increase in sperm head abnormalities were observed in mice exposed to lead acetate [14].

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.

Several sperm parameters were severely affected following lead treatment. This is of special interest as the exposure to lead was quite short in terms of the spermatogenic cycle and the affected spermatozoa were either already in the epididymis at the time of injection or were released there shortly thereafter. The lead effects included clearly deleterious consequences, such as lower sperm motility and count and an increase in detached sperm heads, that could compromise the fertility as described for other toxicants [15, 16]. However, these effects were again reversed after a brief recovery period, suggesting that the changes caused by lead are mostly reversible, provided the exposure to the metal is short.

More puzzling and of potential interest is the finding that the percentage of acrosome-intact sperm is increased following lead treatment and that this effect does not seem to be readily reversible. It may suggest that lead interferes with sperm capacitation, therefore rendering the cells more resistant to undergo the acrosome reaction [17-19]. If this is indeed the case, the present results suggest that this effect may be long lasting and may potentially affect fertility at a longer time despite otherwise normal sperm parameters. This hypothesis will be tested in future studies.


The work was supported in part by grants from the Research Centre on Ceramic and Composite Materials (CICECO) from the Aveiro University, Portugal and partly by a grant from FCT, Portugal (POCTI/ESP/38049/2001).


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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