Acta Tropica, 47(1990)177-184
177
Elsevier ACTROP 00065
Gonadotropic dysfunction produced by Trypanosoma brucei brucei in the rat M. Hublart 1, D. Tetaert 1, D. Croix 2, F. Boutignon 1, P. Degand 1 a n d A. B o e r s m a 1 1Unitk I N S E R M No. 16, Place de Verdun, Lille, France, and 2Unitk I N S E R M No. 156, Place de Verdun, Lille, France
(Received 18 July 1989; accepted 2 November 1989) Hormonal disorders have been frequently observed in humans and animals infected with tsetsetransmitted (African) trypanosomes. We studied the pituitary gonadal axis (plasma concentrations of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and the pituitary gonadotropin (LH, FSH) concentrations) in rats as an experimental model infected with an acute stock of Trypanosoma brucei brucei (AnTat 1.1A). The same investigations in vivo were carried out with rats inoculated by trypanosomal preparations: surface coat components slowly released at pH 5.5 and the parasitic cellular pellet. The releasing procedure as firstly described by Baltz et al. (1976) was performed in the presence or the absence of protease inhibitors. We noted a testicular hypogonadism produced by the acute infection with the decrease of the testosterone level and an increase of the pituitary LH concentration, although the other circulating FSH and LH hormone levelswere stable. The injection of the trypanosomal pellet, obtained in the presence of antiproteases, generated a similar clinical hormonal picture: decrease of testosterone level; increase in pituitary LH, FSH content; absence of significant variation of circulating FSH and LH rates. When the trypanosomal pellet was prepared in absence of antiproteases the circulating gonadostimuline levels were significantly decreased. In the same conditions (absence of antiproteases) the trypanosomal supernatant pH 5.5 induced the decrease of the testosterone and plasma LH levels. These results suggested that component(s) from trypanosomes generated hormonal perturbations. Key words: Trypanosorna brucei brucei; Gonadotrope axis; Luteinizing hormone; Testosterone
Introduction W i t h the increasing reports o f the occurrence o f severe degenerative lesions of the reproductive organs in m a n a n d a n i m a l s suffering from t r y p a n o s o m i a s i s (Apted, 1970; Ikede et al., 1988), it has become necessary to examine the degree of the e n d o c r i n e disorders a n d especially the g o n a d a l dysfunctions. F o r example, due to the g o n a d o s t i m u l i n e secretion disorders, it has been reported by Ridet in 1953, that w o m e n infected with t r y p a n o s o m e s showed a n irregular estrous cycle a n d m a y be infertile or sterile. T h e studies c o n c e r n i n g the h u m a n h o r m o n a l p a r a m e t e r s have been emphasized m o r e recently: the g o n a d a l repercussions ( E m e h a n d N d u k a , 1983; B o e r s m a et al., 1989b) a n d the t h y r o i d ones (Boersma et al., 1989a). Correspondence address: Pr. A. Boersma, Unit6 INSERM No. 16, Place de Verdun, 59045 Lille Cedex,
France. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
178
The hormonal perturbations appeared to be the translation of the host-parasite relationship within the endocrine homeostasis of the organism. The experimental animal trypanosomiasis was an adequate model to investigate the mechanisms implicated in the endocrine disorders. In particular, Ikede and Losos (1972) have shown the damages of the testicular tissues in sheep experimentally infected with Trypanosoma brucei brucei and, thereafter, several reports on the changes in reproductive functions produced by different stocks of african trypanosomes have been presented (review by Ikede et al., 1988). The more recent investigations (Waindi et al., 1986; Mutayoba et al., 1988) have dealt with variations respectively for the testosterone level in rams and for progesterone, 17fl-estradiol levels in goats infected by T. congolense. These variations induced hypogonadism which was comparable to that found in men or women (Boersma et al., 1989b). Among the hypotheses concerning the direct relationship between the hormonal disorders and the trypanosomiasis, Knowles et al. (1987) suggested the involvement of factors released from the parasite during their development in the host circulating blood. In the present work, we report the effect of an acute stock of T.b. brucei and of trypanosomal component preparations on the pituitary-gonadal axis in the male rat.
Materials and Methods
Trypanosomes The antigenic variant Antat 1.1. A was cloned at the Institute of Tropical Medicine (Antwerp) (Drs. N. Vanmeirvenne and T. Vervoort) from the EATRO 1125 T.b. brucei stock. From stabilate stored at -80°C, the trypanosomes (180x 10 6 approx.) were diluted with phosphate-saline-glucose buffer (PSG) (NaC1 44 mM, NazHPO 4 57 mM, NaHzPO 4 3 mM, glucose 1%, pH 8.0). The viability of the parasites was checked and passaged once in Swiss mice (inoculum of 60 to 75 x 106 cells). After no longer than two days (to prevent antigenic variation), the parasitemia reached approximately 500 to 600 × l 0 6 cells, and the infected blood was withdrawn by cardiac puncture. The blood was diluted with PSG (1:18; v/v) in order to obtain approx. 10 x 106 trypanosomes/ml. Infection in rats Male Sprague-Dawley rats (250 days, 400-600 g) (Iffa-Credo, l'Arbresle, France) were used. The Antat 1.1 A trypanosomes (50 x 106) were expanded by intraperitoneal injection in 8 rats. As controls, 8 rats were inoculated with 5 ml of PSG containing healthy mouse blood (1:18; v/v). After 3 days, the rats were decapitated in the morning (9 to 10 a.m.). The blood was centrifuged (1000xg, 30 min) after formation of the clot, and the serum obtained was stored at - 20°C until needed for hormonal radioimmunoassays. The pituitary was removed and the gland was frozen at - 20°C until the assays (follicle stimulating hormone (FSH), luteinizing hormone (LH)) were carried out.
179
Trypanosomal component preparations Bloodstream forms of T.b. brucei Antat 1.1 A were grown in 48 Sprague-Dawley rats. The parasites were harvested after 3 days by cardiac puncture and prepared by DEAE-cellulose chromatography as described by Lanham and Godfrey (1970). The organisms were washed and then incubated overnight at 4°C in the slightly modified buffer described by Baltz et al. (1976): 0.125 M sodium phosphate buffer, pH 5.5, containing 1% glucose supplemented with 0.2 mM antiproteases phenylmethylsulfonylfluoride (PMSF), N-~-tosyl-L-lysine chloromethyl ketone (TLCK), N-ethylmaleimide (NEM). Following centrifugation (3000 × g, 10 min), the supernatant and the respective pellet were obtained. The supernatant was further ultracentrifuged (165 000 × g, 1 h). Under these conditions, two trypanosomal component preparations were defined: (i) supernatant p H 5.5, mainly corresponding to the released components of the trypanosome cell surface; (ii) cellular pellet p H 5.5, corresponding to intact cells devoid of their surface coat. The preparations were also obtained from 24 rats with the buffer pH 5.5 except that antiproteases were not added.
Inoculation in rats with the cellular-pellet and supernatant p H 5.5 preparations obtained with and without antiproteases The rats were inoculated intraperitoneally either by 5 ml of supernatant pH 5.5 ml or by 5 ml of cellular pellet suspended in phosphate saline buffer (PBS) (0.125 M NaC1; 0.012 M NaHPO4; 0.003 M KH2PO 4, pH 7.25). The 'test-quantities' inoculated (supernatant and pellet) corresponded to four fold the parasitemia. In the first experiment, using the trypanosomal component preparations containing antiproteases, a series of 12 animals received the cellular pellet and groups of 6 rats were killed after 1 or 5 h to collect the blood. A series of 12 animals received similarly the supernatant pH 5.5 and two groups of rats were exsanguinated as previously. For each series, control animals were used: respectively, 6 rats were inoculated (i) with 5 ml of pH 5.5 phosphate buffer containing antiproteases, (ii) 6 rats with 5 ml of PBS pH 7.25; they were killed 5 h later. In a second experiment using the trypanosomal component preparations without antiproteases, the procedure was followed as described above: animals were inoculated (i) 6 rats with cellular-pellet, (ii) 6 rats with supernatant pH 5.5, (iii) 6 controls with PBS, (iv) 6 controls with pH 5.5 phosphate buffer without antiproteases. Each time, the rats were decapitated at about the same time of day (9-10 a.m.) and the samples (blood and tissues) were immediately collected.
The hormone assays From the rat sera, the following gonadal hormone assays were carried out for testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH). FSH and LH levels were determined from the pituitaries. Each pituitary was homogenized in 500 Ixl of 0.01 M sodium phosphate buffer, pH 7.5, 0.15 M NaC1, 0.02 mM Bacitracine, 0.1% NaN 3 (w/v). After centrifugation at 4°C (2500 x g, 10 min), the supernatant was diluted 200 fold in the sodium phosphate buffer pH 7.5. Assays were performed using 3 aliquots of respectively 20, 50 and 100 p.l.
180 Analysis for FSH and L H contents by radioimmunoassay using rat L H and FSH RP-2 standards, provided by N I H Pituitary Hormone Distribution Program, were carried out when the hormones were labelled by 125I according to the procedure using the chloramine T as described by Greenwood et al. (1963). RIA SB-testo kit (Oris, Gif sur Yvette, France) was used to determine the testosterone levels. Statistical analyses were performed by Student's t test.
Results
We report firstly the effect of an acute infection and secondly the effects of trypanosomal component preparations on gonadal function in rats.
Acute trypanosomiasis effect on rat gonadal function The variations of testosterone, FSH and LH levels from sera of rats infected with T.b. brucei Antat 1.1 A were followed and the results are shown in Fig. 1. After 3 days of acute parasitemia, we noted a significant decrease (p
177
Elsevier ACTROP 00065
Gonadotropic dysfunction produced by Trypanosoma brucei brucei in the rat M. Hublart 1, D. Tetaert 1, D. Croix 2, F. Boutignon 1, P. Degand 1 a n d A. B o e r s m a 1 1Unitk I N S E R M No. 16, Place de Verdun, Lille, France, and 2Unitk I N S E R M No. 156, Place de Verdun, Lille, France
(Received 18 July 1989; accepted 2 November 1989) Hormonal disorders have been frequently observed in humans and animals infected with tsetsetransmitted (African) trypanosomes. We studied the pituitary gonadal axis (plasma concentrations of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and the pituitary gonadotropin (LH, FSH) concentrations) in rats as an experimental model infected with an acute stock of Trypanosoma brucei brucei (AnTat 1.1A). The same investigations in vivo were carried out with rats inoculated by trypanosomal preparations: surface coat components slowly released at pH 5.5 and the parasitic cellular pellet. The releasing procedure as firstly described by Baltz et al. (1976) was performed in the presence or the absence of protease inhibitors. We noted a testicular hypogonadism produced by the acute infection with the decrease of the testosterone level and an increase of the pituitary LH concentration, although the other circulating FSH and LH hormone levelswere stable. The injection of the trypanosomal pellet, obtained in the presence of antiproteases, generated a similar clinical hormonal picture: decrease of testosterone level; increase in pituitary LH, FSH content; absence of significant variation of circulating FSH and LH rates. When the trypanosomal pellet was prepared in absence of antiproteases the circulating gonadostimuline levels were significantly decreased. In the same conditions (absence of antiproteases) the trypanosomal supernatant pH 5.5 induced the decrease of the testosterone and plasma LH levels. These results suggested that component(s) from trypanosomes generated hormonal perturbations. Key words: Trypanosorna brucei brucei; Gonadotrope axis; Luteinizing hormone; Testosterone
Introduction W i t h the increasing reports o f the occurrence o f severe degenerative lesions of the reproductive organs in m a n a n d a n i m a l s suffering from t r y p a n o s o m i a s i s (Apted, 1970; Ikede et al., 1988), it has become necessary to examine the degree of the e n d o c r i n e disorders a n d especially the g o n a d a l dysfunctions. F o r example, due to the g o n a d o s t i m u l i n e secretion disorders, it has been reported by Ridet in 1953, that w o m e n infected with t r y p a n o s o m e s showed a n irregular estrous cycle a n d m a y be infertile or sterile. T h e studies c o n c e r n i n g the h u m a n h o r m o n a l p a r a m e t e r s have been emphasized m o r e recently: the g o n a d a l repercussions ( E m e h a n d N d u k a , 1983; B o e r s m a et al., 1989b) a n d the t h y r o i d ones (Boersma et al., 1989a). Correspondence address: Pr. A. Boersma, Unit6 INSERM No. 16, Place de Verdun, 59045 Lille Cedex,
France. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
178
The hormonal perturbations appeared to be the translation of the host-parasite relationship within the endocrine homeostasis of the organism. The experimental animal trypanosomiasis was an adequate model to investigate the mechanisms implicated in the endocrine disorders. In particular, Ikede and Losos (1972) have shown the damages of the testicular tissues in sheep experimentally infected with Trypanosoma brucei brucei and, thereafter, several reports on the changes in reproductive functions produced by different stocks of african trypanosomes have been presented (review by Ikede et al., 1988). The more recent investigations (Waindi et al., 1986; Mutayoba et al., 1988) have dealt with variations respectively for the testosterone level in rams and for progesterone, 17fl-estradiol levels in goats infected by T. congolense. These variations induced hypogonadism which was comparable to that found in men or women (Boersma et al., 1989b). Among the hypotheses concerning the direct relationship between the hormonal disorders and the trypanosomiasis, Knowles et al. (1987) suggested the involvement of factors released from the parasite during their development in the host circulating blood. In the present work, we report the effect of an acute stock of T.b. brucei and of trypanosomal component preparations on the pituitary-gonadal axis in the male rat.
Materials and Methods
Trypanosomes The antigenic variant Antat 1.1. A was cloned at the Institute of Tropical Medicine (Antwerp) (Drs. N. Vanmeirvenne and T. Vervoort) from the EATRO 1125 T.b. brucei stock. From stabilate stored at -80°C, the trypanosomes (180x 10 6 approx.) were diluted with phosphate-saline-glucose buffer (PSG) (NaC1 44 mM, NazHPO 4 57 mM, NaHzPO 4 3 mM, glucose 1%, pH 8.0). The viability of the parasites was checked and passaged once in Swiss mice (inoculum of 60 to 75 x 106 cells). After no longer than two days (to prevent antigenic variation), the parasitemia reached approximately 500 to 600 × l 0 6 cells, and the infected blood was withdrawn by cardiac puncture. The blood was diluted with PSG (1:18; v/v) in order to obtain approx. 10 x 106 trypanosomes/ml. Infection in rats Male Sprague-Dawley rats (250 days, 400-600 g) (Iffa-Credo, l'Arbresle, France) were used. The Antat 1.1 A trypanosomes (50 x 106) were expanded by intraperitoneal injection in 8 rats. As controls, 8 rats were inoculated with 5 ml of PSG containing healthy mouse blood (1:18; v/v). After 3 days, the rats were decapitated in the morning (9 to 10 a.m.). The blood was centrifuged (1000xg, 30 min) after formation of the clot, and the serum obtained was stored at - 20°C until needed for hormonal radioimmunoassays. The pituitary was removed and the gland was frozen at - 20°C until the assays (follicle stimulating hormone (FSH), luteinizing hormone (LH)) were carried out.
179
Trypanosomal component preparations Bloodstream forms of T.b. brucei Antat 1.1 A were grown in 48 Sprague-Dawley rats. The parasites were harvested after 3 days by cardiac puncture and prepared by DEAE-cellulose chromatography as described by Lanham and Godfrey (1970). The organisms were washed and then incubated overnight at 4°C in the slightly modified buffer described by Baltz et al. (1976): 0.125 M sodium phosphate buffer, pH 5.5, containing 1% glucose supplemented with 0.2 mM antiproteases phenylmethylsulfonylfluoride (PMSF), N-~-tosyl-L-lysine chloromethyl ketone (TLCK), N-ethylmaleimide (NEM). Following centrifugation (3000 × g, 10 min), the supernatant and the respective pellet were obtained. The supernatant was further ultracentrifuged (165 000 × g, 1 h). Under these conditions, two trypanosomal component preparations were defined: (i) supernatant p H 5.5, mainly corresponding to the released components of the trypanosome cell surface; (ii) cellular pellet p H 5.5, corresponding to intact cells devoid of their surface coat. The preparations were also obtained from 24 rats with the buffer pH 5.5 except that antiproteases were not added.
Inoculation in rats with the cellular-pellet and supernatant p H 5.5 preparations obtained with and without antiproteases The rats were inoculated intraperitoneally either by 5 ml of supernatant pH 5.5 ml or by 5 ml of cellular pellet suspended in phosphate saline buffer (PBS) (0.125 M NaC1; 0.012 M NaHPO4; 0.003 M KH2PO 4, pH 7.25). The 'test-quantities' inoculated (supernatant and pellet) corresponded to four fold the parasitemia. In the first experiment, using the trypanosomal component preparations containing antiproteases, a series of 12 animals received the cellular pellet and groups of 6 rats were killed after 1 or 5 h to collect the blood. A series of 12 animals received similarly the supernatant pH 5.5 and two groups of rats were exsanguinated as previously. For each series, control animals were used: respectively, 6 rats were inoculated (i) with 5 ml of pH 5.5 phosphate buffer containing antiproteases, (ii) 6 rats with 5 ml of PBS pH 7.25; they were killed 5 h later. In a second experiment using the trypanosomal component preparations without antiproteases, the procedure was followed as described above: animals were inoculated (i) 6 rats with cellular-pellet, (ii) 6 rats with supernatant pH 5.5, (iii) 6 controls with PBS, (iv) 6 controls with pH 5.5 phosphate buffer without antiproteases. Each time, the rats were decapitated at about the same time of day (9-10 a.m.) and the samples (blood and tissues) were immediately collected.
The hormone assays From the rat sera, the following gonadal hormone assays were carried out for testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH). FSH and LH levels were determined from the pituitaries. Each pituitary was homogenized in 500 Ixl of 0.01 M sodium phosphate buffer, pH 7.5, 0.15 M NaC1, 0.02 mM Bacitracine, 0.1% NaN 3 (w/v). After centrifugation at 4°C (2500 x g, 10 min), the supernatant was diluted 200 fold in the sodium phosphate buffer pH 7.5. Assays were performed using 3 aliquots of respectively 20, 50 and 100 p.l.
180 Analysis for FSH and L H contents by radioimmunoassay using rat L H and FSH RP-2 standards, provided by N I H Pituitary Hormone Distribution Program, were carried out when the hormones were labelled by 125I according to the procedure using the chloramine T as described by Greenwood et al. (1963). RIA SB-testo kit (Oris, Gif sur Yvette, France) was used to determine the testosterone levels. Statistical analyses were performed by Student's t test.
Results
We report firstly the effect of an acute infection and secondly the effects of trypanosomal component preparations on gonadal function in rats.
Acute trypanosomiasis effect on rat gonadal function The variations of testosterone, FSH and LH levels from sera of rats infected with T.b. brucei Antat 1.1 A were followed and the results are shown in Fig. 1. After 3 days of acute parasitemia, we noted a significant decrease (p
