Effect of sodium arsenite on spermatogenesis, plasma gonadotrophins and testosterone in rats

Asian J Androl 2003 Mar; 5: 27-31             

Keywords: arsenite; spermatogenesis; gonadotrophins; testosterone; testis

Abstract

Aim: To investigate the effect of arsenic on spermatogenesis. Methods: Mature (4 months old) Wistar rats were intraperitoneally administered sodium arsenite at doses of 4, 5 or 6 mg.kg-1.day-1 for 26 days. Different varieties of germ cells at stage VII seminiferous epithelium cycle, namely, type A spermatogonia (ASg), preleptotene spermatocytes (pLSc), midpachytene spermatocytes (mPSc) and step 7 spermatids (7Sd) were quantitatively evaluated, along with radioimmunoassay of plasma follicle-stimulating hormone (FSH), lutuneizing hormone (LH), testosterone and assessment of the epididymal sperm count. Results: In the 5 and 6 mg/kg groups, there were significant dose-dependent decreases in the accessory sex organ weights, epididymal sperm count and plasma concentrations of LH, FSH and testosterone with massive degeneration of all the germ cells at stage VII. The changes were insignificant in the 4 mg/kg group. Conclusion: Arsenite has a suppressive influence on spermatogenesis and gonadotrophin and testosterone release in rats.

1 Introduction

Arsenicals are used as herbicides, fungicides and rodenticides and may cause air, soil and water pollution. Arsenical exposure through drinking water is common in many areas of the world [1-3].

Exposure to arsenic is associated with metabolic disorders, hypertrophy of adrenal glands [4] and anemia [5]. A number of sulfhydryl containing proteins and enzyme systems have been found to be altered by exposure to arsenite [6]. Arsenite affects mitochondrial enzymes and impairs tissue respiration, which seem to be related to the cellular toxicity of arsenic [7]. Gonadal effects of arsenic were first evaluated in mice, then in fishes [8-10]. Sodium arsenite has been found to have an inhibitory effect on the activity of testicular steroidogenic enzymes, D⁵-3b-hydroxysteroid dehydrogenase (D⁵-3b-HSD) and 17b-hydroxysteroid dehydrogenase (17b-HSD) and to reduce the weight of testes and accessory sex glands [11].

Most of the available data on arsenic reproductive toxicity indicate that the main concern is with the developmental toxicity on the fetus [12]. Till date there is very few evidence on its male reproductively effects [11,13]. The present study was designed to quantitative study the effect of arsenite on spermatogenesis at stage VII of the seminiferous epithelial cycle and plasma hormone concentrations, which has not been reported so far in the literature.

2 Materials and methods

Adult male Wistar rats weighing 16010 g (120~140 days of age) were maintained in a 12 h light and 12 h dark and 26 ~28 animal house and standard laboratory chew and tap water were available ad libitum. Sodium arsenite was purchased from Sigma (USA) and dissolved in sterile distilled water.

Thirty-two rats were divided into 4 groups of 8 animals each. Three groups of animals were injected with either 4, 5 or 6 mg.kg^-1.day^-1 sodium arsenite per 1 mL sterile distilled water for 26 days (Group II, III and IV, respectively). Animals of Group I were injected the same amount of distilled water for 26 days and served as the controls. Treatment for 26 days was selected as the duration of one seminiferous cycle is 13.2 days in Wistar rats.

On the 27th day between 08:00 to 10:00, blood samples were collected from the hepatic vein under light ether anesthesia and after that the rats were killed following ethical procedure. Heparinized plasma was prepared and stored at -20 until hormone radioimmuno-assay.

The body weights were recorded on the first day before injection (initial) and the day of sacrifice (final). The testicles and accessory sex organs were dissected out, trimmed off the attached tissues and weighed. The relative weight of organs was expressed per 100g body weight. The left testis of each rat was used for histological study and the right for estimation of elementary arsenic content.

Immediately after removal, the testis was fixed in Bouin's fluid and embedded in paraffin. Sections of 5 mm thickness were taken from the mid portion of each testis and stained with periodic acid Schiff (PAS)-hematoxylin and examined under a light microscope. Quantitative analysis of spermatogenesis was carried out by counting the relative number of each variety of germ-cells at stage VII of the seminiferous epithelium cycle, i.e. type-A spermatogonia (ASg), preleptotene spermatocytes (pLSc), mid pachytene spermatocytes (mPSc) and step 7 spermatids (7Sd), according to the method of Leblond and Clermont [14]. The nuclei of different germ cells (except step 19 spermatids which cannot be enumerated precisely) were counted in 20 round tubules of each rat. All the counts (crude counts) of the germ cells were corrected for differences in the nuclear diameter by the formula of Abercrombie [15]: true count = (crude count section thickness)/ (section thickness - nuclear diameter of germ cell). The nuclear diameter of each variety of germ cell was measured with a Leitz micrometer. The possibility of variable tubular shrinkage in the sections of both arsenite and vehicle injected groups were eliminated by the index of tubular shrinkage which was obtained from the average number of Sertoli cell nuclei containing prominent nucleoli in the sections of the treated rats divided by that of the controls [16]. Stage VII spermatogenesis was analyzed because this stage is highly susceptible to testosterone deficiency [17] and also reflects the final stages of spermatid maturation and thus provides evidence of spermatogenesis as a whole [18].

The sperm count was determined by counting in a haemocytometer. Sperm samples were collected from the cauda epididymis. To minimize the count was repeated at least five times for each rat.

2.5 Hormone assay

Plasma follicle-stimulating hormone (FSH) and lutuneizing hormone (LH) were measured by radioimmunoassay (RIA) as described in the instructions provided with the kits (NIADDK, USA). Carrier free ¹²⁵I for hormone iodination was obtained from the Bhaba Atomic Research Centre, Bombay, India. Pure rat FSH (NIADDK-r FSH-1-6) and LH (NIADDK-rLH-1-6) were iodinated using the chloramine T (Sigma, USA) method of Greenwood et al [19]. The antisera to FSH and LH were NIADDK anti-r FSH-S-11 and NIADDK-anti-rLH-S-9, and were used at a final dilution of 1:100000 and 1:150 000, respectively. Goat anti-rabbit g-globulin used as the second antibody was obtained from the Indo Medix Inc., USA. The sensitivity of the assay was 2.0 mg/L for FSH and 0.15 mg/L for LH. Each sample was assayed at two concentrations, each in duplicate at a time. The intra assay coefficient of variation in each assay was 7.5 % for FSH and 6.0 % for LH. Hormone concentrations were expressed in terms of NIH reference preparation RP-2.

Plasma testosterone was assayed according to Auletta et al [20]. Methodological loss was monitored and accounted for by adding 1000 c. p. m. [1b, 2b³ - H (N)] testosterone (sp. act. 50.4 Ci/m mol; New England Nuclear, Boston, MA, USA) before extraction with diethyl ether. Samples were assayed at two concentrations, each in duplicate. The antisera to testosterone was purchased from the Endocrine Science, USA, and had a 44 % cross reactivity with dihydrotestosterone. Free and bound testosterone were separated by using dextrancoated charcoal. The recovery of plasma testosterone after ether extraction was estimated to be 88.5 %. The sensitivity of the assay was 69.5 % pmol/L and the intraassay variance was 6.4 %. All samples were measured in a single assay. Since chromatographic purification of the samples was not performed, the testosterone values reported are the sum of testosterone and dihydrotestosterone.

The flameless atomic absorption spectrophotometric technique [21] was used for the determination of arsenic concentration. Animals were scarified 24h after the last arsenite injection and one testis from each animal was collected and digested with a mixture of nitric acid, sulfuric acid and perchloric acid (3:1:1). Values are expressed in µg of arsenic/g of testicular wet tissue.

Data were expressed in meanSEM. Statistical analysis was performed by analysis of variance (ANOVA) followed by multiple comparison by two-tailed t-test.

3 Results

In all the treated groups, the body weight was not significantly different from that of the controls. The relative weights of the testis, seminal vesicle and ventral prostate were significantly decreased (P<0.05) after 5 mg or 6 mg treatment, but not after 4 mg of sodium arsenite treatment (Table 1).

Table 1. Effect of sodium arsenite on body weight (g) and organ weights (mg % body weight) (meanSEM, n=8) in rats. ^bP<0.05, compared with controls. ANOVA followed by multiple comparison two-tailed t-test.

Sodium arsenite treatment at the dose of 5 mg/kg significantly reduced the number of ASg, pLSc, mPSc and 7Sd when compared with those of the controls. Six mg/kg caused a more prominent spermatogenic arrest. No significant change was found in the cellular counts in the 4 mg/kg treated group (Table 2). Theoretically, the ratio of mPSc:7Sd is 1: 4. This ratio was 1:2.76 and 1:2.57 after 5 and 6 mg/kg treatment, respectively; the ratio of the control group was 1:3.41. The percentage spermatid degeneration (35.75 %) as calculated from the above ratio, was highly significant after 6 mg of sodium arsenite treatment (Table 2).

Table 2. Effect of sodium arsenite on number of germ cells per tubular cross section at stage VII of the seminiferous epithelial cycle in rats (meanSEM, n=8). ASg = spermatogonia A; pLSc = preleptotene spermatocytes; mPSc = mid pachytene spermatocytes; 7Sd = step 7 spermatid. ^bP<0.05, compared with controls. ANOVA followed by multiple comparison two-tailed t-test.

The sperm count was significantly reduced in both the 5 and 6 mg/kg, but not in the 4 mg/kg treated groups compared with the controls (Table 3).

Table 3. Effect of sodium arsenite on the sperm count and testicular arsenic content in rats (n=8). ^bP<0.05, compared with controls.

At the dose of 5 and 6 but not 4 mg/kg, the plasma levels of FSH and LH were significantly decreased compared with the controls. The changes were more prominent in the 6 mg/kg group (Figures 1 and 2).

Figure 1. Effect of sodium arsenite on plasma level of FSH. ^bP<0.05, compared with controls (n=8).

Figure 2. Effect of sodium arsenite on plasma level of LH. ^bP<0.05, compared with controls (n=8).

Plasma testosterone was significantly decreased (P<0.01) in rats of the 5 and 6 mg/kg group and was more prominent in the latter compared with the controls. It was not significantly changed in the 4 mg/kg group (Figure 3).

Figure 3. Effect of sodium arsenite on plasma level of testosterone. ^bP<0.05, compared with controls (n=8).

The arsenic concentration was significantly increased in the testes in all the treated animals in a dose-dependent manner (Table 3).

4 Discussion

A significant decrease in the plasma FSH, LH and testosterone levels and degenerative changes in testicular histology in arsenite treated rats are in agreement with previous findings of the inhibitory effect of arsenite on the gonadal structure and function in mice [13] and fishes [8-10] and the testicular D⁵-3b-HSD and 17b-HSD activities in rats [11]. As these enzymes are gonadotrophin dependent [22], a decrease in their levels may reflect reduced pituitary gonadotrophin secretion.

It was indicated that in parallel with the decrease in 7Sd and increased 7Sd degeneration, the sperm count was markedly reduced. Earlier reports have revealed degenerative changes in the testicular histology in fish and mice treated with arsenite [8, 13].

LH and FSH are required for the initiation and maintenance of spermatogenesis in prepubertal and pubertal rats [23] and for quantitatively normal spermatogenesis in pubertal rats [17]. The reduction of FSH and LH and a consequent reduction in testosterone production may, therefore, be held responsible for this arsenite-induced changes in spermatogenesis. The reduction in the number of ASg in arsenite treated rats is possibly due to the increased rate of degeneration of Asg as FSH inhibits the normal degeneration of ASg and reduced FSH secretion may promote ASg degeneration.

We found that higher doses of sodium arsenite increase adrenocortical activity and elevated serum corticosterone level [4], which in turn may reduce the serum gonadotriphin and testosterone levels [24]. Inhibitory effects of glucocorticoids on LH secretion have been reported in cultured pituitary [25]. Glucocorticoids also directly suppress testosterone production and secretion by decreasing the testicular LH receptors [26], resulting in the reduction of spermatogenesis and sperm count.

It appears that the primary site of arsenic action may be on the brain or pituitary, however, a direct action on the germ cells cannot be ruled out and further studies are required to clarify these points.

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Correspondence to: Dr. Mahitosh Sarkar, Reproductive Physiology Unit, Dept. of Physiology, University Colleges of Science and Technology, Calcutta University, 92 Acharya Prafulla Chandra Road, Calcutta 700009, India.
Tel: +91-33-563 0365
E-mail: mohimono@yahoo.com.in
Received 2002-05-20      Accepted 2002-12-12