Testicular dysfunction in BALB C mice with Schistosoma intercalatum bilharziasis

Hermine Boukeng Jatsa^1,3, Pierre Kamtchouing¹, Innocent Takougang², Selestin Dongmo Sokeng⁴

Asian J Androl  2002 Jun; 4:  143-147             

Keywords: Schistosoma intercalatum; bilharziasis; testis; testosterone; seminal vesicles

Abstract

Aim: To evaluate the effect of Schistosoma intercalatum infestation on the testicular function of mice. Methods: Male BALB C mice were infested by immersion of the tail and hind feet into the water with 50 or 100 cercariae of Cameroon strain S. intercalatum. Sixty days later the animals were killed, blood was collected and the testis, epididymis and seminal vesicles were dissected and weighed. The plasma and testicular testosterone were evaluated with radioimmunoassay, the seminal vesicular fructose with colorimetric method, and the histology of testis and cauda epididymis observed under light microscope. The intensity of infestation was estimated in terms of S. intercalatum egg load in the liver. Results: In infested mice, the testicular weight did not change significantly while the epididymal and seminal vesicular weights were significantly lowered compared to the controls. Furthermore, the fructose levels in the seminal vesicle fluid were significantly (P<0.01) reduced in about 50 % of infested mice. S. intercalatum infestation also decreased the plasma and testicular testosterone concentrations. Histological studies indicated that the spermatogenesis, the testicular interstitial tissue and the cauda epididymis were qualitatively normal. Parasite eggs were not found in these organs. The mean seminiferous tubular diameter did not show significant differences between the infested and control mice. Conclusion: S. intercalatum infestation impairs testicular function.

1 Introduction

Schistosomiasis or bilharziasis is a parasitic disease endemic in 74 countries and attacks more than 200 million people. Of these, 20 million suffer serious consequences from the disease and 120 million show various symptoms [1]. Schistosoma intercalatum causes rectal bilharziasis in humans. It first occurred in Cameroon since the 1960s [2,3] and presently three endemic foci are known: Yaound, Eda and Eseka [4]. The key pathogenic event in this disease is the formation of granulomas around the schistosome eggs trapped in host tissues. The development of these granulomas is stimulated by the cytokine tumour necrosis factor alpha produced by the host [5-7]. Schistosomiasis of the male reproductive tract was reported as early as 1911 in Egypt [8]. Post mortem studies revealed the presence of S. haematobium and S. mansoni eggs in the prostate gland, seminal vesicles and vas deferens [9,10]. Semen analysis and testicular biopsy have shown azoospermia and spermatogenic arrest associated with deposition of ova in the interstitium [11]. Bilharzial liver disease is known to affect the serum levels of estriol, estrone and estradiol in the male [12], while conflicting results exist concerning the testosterone level [13-15]. Regarding S. intercalatum infestation in the genital organs, eggs were occasionally found in the prostate, seminal vesicles and testis of gibbon and chimpanzee [16].

Although schistosomiasis was found to be associated with histopathological changes in the male genital and endocrine systems, the effect of S. intercalatum on the reproduction of the final host has drawn little attention of the researchers [17-20]. The present study was undertaken to evaluate the effect of S. intercalatum infestation on testicular function in mice.

2 Materials and methods

2.1 Mice and infestation

BALB C mice were obtained from the Centre Pasteur of Yaound in Cameroon and raised in the animal house of the Faculty of Sciences, University of Yaound I with mouse chow and tap water ad libitum. Approximately ninety-day-old mice were infested by immersing their tail and hind feet in the water with 50 or 100 cercariae of the Cameroon strain S. intercalatum maintained in laboratory bred Bulinus forskalii from Cameroon. Infestation of this intermediate host was carried out using miracidia obtained from the eggs found in S. intercalatum-infested children faeces. Informed consent was obtained from parents of the children. Faecal test was strictly confidential and bilharzial treatment (praziquantel) was provided for the sick. The institutional Ethical Committee of the University of Yaound I approved this research.

Sixty days after infestation, animals were killed and blood collected in heparinized tubes. Plasma were separated by centrifugation and stored at -20 until assayed.

The testis, epididymis, seminal vesicles and liver were removed and weighed. Testes and seminal vesicles were homogenised separately in 1.5 mL saline phosphate buffer (0.1mol/L, pH 7.3) and 1.5 mL of distilled water, respectively. The supernatant were used for biochemical analyses. Pieces of liver weighed above 20 mg were digested in 10 % KOH at 37 for 5 h. After centri-fugation, the eggs in the deposit were counted to estimate the number of eggs per gram liver. Three experimental groups were then obtained in terms of egg load:

E_1-699: 1 to 699 eggs/g liver
E_700-1499: 700 to 1499 eggs/g liver
E_{» 1500}: 1500 or more eggs/g liver

Testis and cauda epididymis were fixed in an alcoholic Bouin solution and sections of 10 mm stained with haematoxylin of Carazi and alcian blue. The mean diameter of round seminiferous tubules was measured by a micrometric reticule on 20 fields per testis at magnification 160.

Testosterone levels were measured by radioimmunoassay with ³H-labeled testosterone tracer after extraction of samples with cyclohexane-ethyl acetate solution. All samples were assayed in duplicate; the intra-assay coefficient of variation was 3.08 %.

Estradiol was radio-immunoassayed on pools of testis (3 animals/pool) using the estradiol assay kit (cat # 2464, IMMUNOTECH, Marseille) with ¹²⁵I-labelled estradiol tracer.

Seminal vesicle fructose content was determined as described in the WHO manual for semen analysis [17].

The results are expressed as meanSD. Analysis of variance and Student's t-test were used to determine the significance of differences between various groups. P<0.05 was set as significant.

No variation was observed in the testis weight of infested animals compared to the controls. Epididymis weight of infested mice was significantly lower in groups E_700-1499 (-16%, P < 0.05 compared with the controls) and E_»1500 (-24 %, P < 0.01). The weight of seminal vesicles was significantly (P < 0.01) lower in all the experimental groups: -46 % for E_1-699, -72 % for E_700-1499 and -61 % for E_»1500 (Table 1).

Table 1. Testis, epididymis and seminal vesicle weights of S. intercalatum-infested mice sixty days after inoculation.

Data in meanSD. ^bP < 0.05, ^cP < 0.01, compared with controls. E_1-699: infested with 1 to 699 S. intercalatum eggs/g liver; E_700-1499: infested with 700 to 1499 S. intercalatum eggs/g liver; E_{» 1500} : infested with 1500 or more S. intercalatum eggs/g liver.

Figure 1 shows that in all groups of infested mice, seminal vesicles fructose content was 44 % (E_1-699), 49 % (E_700-1499) and 56 % (E_»1500) lower than that of normal mice (P < 0.01).

Testosterone: Plasma testosterone levels in mice with an egg load range from 700 to 1499 were 3/5 (0.86 0.35 ng/mL) of that of uninfested mice (2.150.65 ng/mL), but not in mice with more than 1500 eggs/g of liver (Figure 2a). Testicular testosterone concentrations were lower in all infested mice (-35 % for E_1-699, -41 % for E_700-1499, -36 % for E_»1500) compared to unin-fested mice (Figure 2b).

Figure 2. Plasma (A) and testis (B) testosterone concentrations in S. intercalatum-infested mice sixty days after inoculation. (Data in meanSD, ^cP<0.01 vs control.)

Estradiol: Testicular estradiol concentrations were significantly higher (+58 %, P<0.01) in infested mice compared to uninfested ones (0.190.02 vs 0.12 0.01 pg/mg testis).

Interstitial tissue and cauda epididymis sections were qualitatively not affected by S. intercalatum. All types of germ cells, spermatogonia, spermatocytes, spermatids and spermatozoa, were present. No parasite ova were seen in these organs.

Diameters of seminiferous tubules showed no clear difference between different groups (Figure 3).

Figure 3. Seminiferous tubular diameter of S. intercalatum-infested mice sixty days after inoculation. (Data in meanSD, no significant difference between different groups.)

The study indicated that S. intercalatum induced a considerable decrease in testicular testosterone levels of all infected mice and plasma testosterone levels of mice with moderate egg counts. A low level of testosterone was previously described in patients with schistosomal hepatic disease [13]. Bilharziasis is usually associated with the formation of granulomas around schistosome eggs trapped in the liver. The schistosome egg-laying and the related granulomatous inflammation are influenced by the cytokine tumour necrosis factor, the concentration is increased in schistosomiasis [5,7]. In our study, bilharzial granulomas were present in the liver of all infested mice, suggesting that liver damage induced by S. inter-calatum apparently impairs testosterone synthesis through the intermediary of tumour necrosis factor produced by schistosome-infested animals. This cytokine inhibits in the mouse Leydig cells the expression of enzyme complex 17a-hydroxylase/C_17,20lyase which converts progesterone to testosterone during steroidogenesis [21]. It is thus evident that S. intercalatum bilharziasis interferes with testosterone synthesis. The consequence of this inhibition might be an accumulation of progesterone and a reduction of testosterone. S. inter-calatum eggs can also induce portal vein occlusion which as well as portal bypass is associated with increased aromatisation of testosterone to estradiol [22]. This can explain the low levels of testosterone and the increased testicular estradiol concentration in these mice. In heavily infested mice, plasma testosterone levels were slightly above controls, probably due to the accumulation of plasma testosterone and the inadequate binding to proteins such as albumin. In our previous study,we have shown that the total plasmatic protein levels were reduced in heavily infested mice, while it remained unchanged in mice with low infestation (Jatsa, personal communication). Moreover, the average albumin level in patients with hepatic disease as bilharziasis is considerably lowered [23].

Although serum testosterone concentration was unchanged in some S. intercalatum-infested mice, the total testosterone production was reduced, with a decrease in epididymis and seminal vesicle weights [24] and a reduction in the fructose content in seminal vesicle fluid. Testosterone controls the development of epidydimis and seminal vesicles and the activity of seminal vesicle columnar and basal cells where fructogenesis takes place [25].

Although a few eggs of S. intercalatum have been previously discovered in the testis of a chimpanzee [16], no ovum was seen in the testis and epididymis of our animals. It may be due to the variation in relative distribution of S. intercalatum eggs in different host species and parasite strain [26].

In contrast to the decreased testosterone levels, the interstitial tissue and the cauda epididymis sections were qualitatively normal, as well as the diameters of seminiferous tubules. Low testosterone concentrations may be adequate for spermatogenesis [27,28], but the latter may lead to oligospermia and asthenospermia as often observed in infertile men. The decreased epididymis weight of infested mice is the consequence of low testosterone levels, which may lead to epididymal dysfunction. It is well known that testosterone stimulates the synthesis of specific epididymal proteins essential for post-testicular sperm differentiation, capacitation and acquisition of fecundity [29]. A low level of testosterone would not adequate for epididymal maturation of spermatozoa. Thus in infested mice, spermatozoa released from the seminiferous tubules would not acquire progressive motility and fertilizing ability during their passage through the epididymis. In addition, the low levels of fructose, the energetic nutrient of spermatozoa, would not allow them to be energetically motile.

Acknowledgements

This research was supported by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR), grant M8/181/4/J.89. We thank Dr. John Williams and Dr. Marie Thrse Hoche-reau de Reviers for the supervision of Jatsa BH training course in INRA Nouzilly in France, and Ms. Marie Thrse Durand and Christine Perreau for their technical as-sistance.

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Correspondence to: Dr. Hermine Boukeng Jatsa, Animal Physiology Laboratory, Faculty of Sciences, University of Yaound?I, P.O. Box 812, Yaound? Cameroon.
Tel: +237-2-312 375
E-mail: h.jatsa_@caramail.com
Received 2001-09-02      Accepted 2002-02-04