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Effects of Trypanosoma congolense on pituitary and adrenocortical function in sheep: responses to exogenous corticotrophin-releasing hormone
Research in Veterinary Science 1995, 58, 180-185
Effects of Trypanosomacongolense on pituitary and adrenocortical function in sheep: responses to exogenous corticotrophin-releasing hormone B. M. MUTAYOBA*, P. D. ECKERSALL, C. SEELY, Department of Veterinary Clinical Biochemistry, University of Glasgow Veterinary School, Bearsden, Glasgow G61 1QH, C. E. GRAY, Institute of Biochemistry, Royal Infirmary, Glasgow G40SF, V. CESTNIKt, Department of Veterinary Clinical Biochemistry, I. A. JEFFCOATE, P. H. HOLMES,
Department of Veterinary Physiology, University of Glasgow Veterinary School, Bearsden, Glasgow G61 1QH
SUMMARY To investigate whether the aberrations in adrenocortical and gonadal activity observed in trypanosomiasis may be induced by the refractoriness of the pituitary to hypothalamic liberins, the responses of the pituitary and adrenal glands and the testes to stimulation with ovine corticotrophin-releasing hormone (oCRH)were studied in rams 23 days (acute phase) and 65 days (chronic phase) after they were infected with Trypanosomacongolense. On both occasions a peak of plasma ACTHwas observed within 20 minutes of the injection of CRH but the rate of increase in ACTHand the mean peak values in the infected rams were significantly lower (P<0.001) on day 23 but higher (P<0.05) on day 65 than in the uninfected control rams. Plasma cortisol concentration increased in all the rams after the injection of CRH.The rate of increase in plasma cortisol and the mean peak values were not significantly different between the control and infected rams on day 23 but were significantly (P<0.001) higher in the infected rams on day 65. However, the post peak concentrations of ACTHdeclined more rapidly in the infected rams than in the controls on both days 23 and 65. The plasma concentration of luteinising hormone (LH) did not change after the injection of CRH, whereas the testosterone levels showed a delayed response and its concentration increased when plasma ACTH and cortisol concentrations declined in both groups. On day 23, there was a greater increase in testosterone in the infected than in the control rams. These results demonstrate that the responsiveness of the pituitary corticotrophs to CRH is depressed during the acute phase and enhanced during the chronic phase of T congolenseinfection in rams, whereas the adrenal cortisol response is less affected. The results are also consistent with the hypothesis that the modulation of the pituitary-adrenal axis by infective trypanosomes may exacerbate the changes in testicular steroidogenesis frequently observed in trypanosomiasis. F O L L O W I N G the elucidation of the molecular structure of ovine corticotrophin-releasing hormone (OCRH) by Vale et al (1981) its physiological significance in regulating the secretion of adrenocorticotrophin hormone (ACTH) and cortisol has been recognised (Donald et al 1983, Plotsky and Vale 1984, Rivier and Plotsky 1986). It has been shown that through CRH, the hypothalamus plays a fundamental role in coordinating the stress response by integrating psychoneuroendocrine and autonomic functions (Moberg 1985, Lenz et al 1987, Fisher 1989). T h e injection of CRH elicits increases in plasma ACTH and cortisol and these have been used to test pituitary-adrenal responsiveness under various conditions (Pradier et al 1988, Shutt et al 1989, Parrot 1990). Although the primary focus of most neuroendocrine studies has been on the stress response of the pituitary and adrenal glands there is an increasing body of evidence which links the stress-induced stimulation of ACTH and glucocorticoids with changes in other pituitary hormones including growth hormone, prolactin, thyroid stimulating hormone and the gonadotrophins (Moberg 1985). Furthermore, there is now evidence that the stress-induced activation of the hypothalamo-pituitary-adrenal (HPA) axis might be responsible for the subsequent infertility observed in male rats (Charpenet et al 1981, Rivier et al 1986), bulls * Present address: Depam~nent of Veterinary Physiology, Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Sokoine University of Agriculture, PO Box 3017, Morogoro, Tanzania ? Institute of Physiology, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Ljubljana, Gerbiceva 60, 61000, Slovenia
(Johnson et al 1982), boars (Litrap and Raeside 1975) and rams (Naylor et al 1990). Trypanosomiasis is associated with changes in the activity of several endocrine glands (Ikede et al 1988, Mutayoba and Gombe 1989). For example, plasma cortisol concentrations are increased in goats (Mutayoba and Gombe 1989) and sheep, and in the latter the increases are associated with adrenal hyperplasia (Mutayoba et al 1995). On the other hand plasma testosterone concentration is decreased in sheep (Adeyemo et al 1990), and it seems possible that the infertility observed in trypanosomiasis could be associated with changes in the HPA axis. The aims of this study were to investigate the interactions between the HPA axis and the gonadal axes in trypanosome-infected rams. CRI-Iwas used' to provoke a pituitary-adrenal endocrine response and the effect on the pituitary-testicular axis was observed simultaneously.
MATERIALS AND METHODS
Animals and trypanosome infection Ten Scottish blackface rams aged six months and weighing approximately 34 kg were used. These rams were part of a group used to study the effects of trypanosomiasis on adrenal pathophysiology, and their management before and during infection has. been described by Mutayoba et al (1995). Five rams were infected intravenously with approximately 4 × 105 trypanosomes as
180
Effects ofT congolense infection in sheep
already described and together with the five uninfected control rams they were monitored for a period of 79 days. As part of larger control and infected groups these animals contributed to the group mean haematology, bodyweight and rectal temperature data presented by Mutayoba et al (1995).
181
antibody RIA using the method described by Jeffcoate (1992). The limit of detection of the assay was 0.12 ng m1-1 (n = 10). The intra-assay c v was less than 5 per cent at 2-99 and 10.71 ng m1-1 and the interassay c v was less than 13 per cent (n = 5) at 2.86 and 11.32 ng m1-1.
Statistics CRH
injection
On day 23 and day 65 after infection, a jugular catheter was inserted into each of the rams between 08.00 and 10.00 and kept patent with heparinised saline (200 iu m1-1 in 0.9 per cent w/v sodium chloride). Beginning one hour later, 3 ml blood samples were taken every 20 minutes into heparinised tubes for a period of one hour. Each animal then received oCRH via the cannula (25 gg/2 ml saline, C-3167, Sigma), and blood samples were taken five, 10, 20, 30, 40, 60, 90, 120 and 150 minutes after the injection. The samples were centrifuged immediately and the plasma snap frozen in dry ice and stored at -20°C for measurements of cortisol, ACTH, luteinising hormone (LH) and testosterone. The dose of CRH was selected to give a pituitary-adrenal response lasting for two to three hours (Pradier et al 1988).
Hormonal measurements Plasma cortisol concentration was measured by radioimmunoassay (R/A) according to McConway and Chapman (1986) and the reagents used for the assay have been described by Mutayoba et al (1995). Plasma testosterone concentration was measured by the RIA described by Cook and Beastall (1987). Testosterone standards were purchased from Steraloids and 125I-histamine-testosterone was synthesised by C. E. Gray. Rabbit-anti-testosterone (AB-1030) was purchased from Bioclinical Services, Cardiff; according to the suppliers this antibody shows the following cross-reactivity: testosterone 100 per cent, 5Gt-dihydrotestosterone 16 per cent, 5ct-androstane-3a17~3-diol 5.8 per cent, 5a-androstane-313-1713-diol 3.7 per cent, androstenedione 2.1 per cent, dihydroepiandrosterone 0.04 per cent and cortisol <0-01 per cent. The limit of detection (2 x SD of total binding) for cortisol was 3.0 nmol litre -1 (n = 12) and for testosterone 0.1 nmol litre -1 (n = 12). The intra-assay coefficient of variation (cv) for the cortisol assay was less than 11 per cent (n = 13) at 14.6 and 136.6 nmol litre -1 and the interassay c v was less than 15 per cent (n = 7) at 11.4 and 77.7 nmol litre -1. The intraassay c v for the testosterone assay was less than 8 per cent (n = 16) at 3.3 and 12.4 nmol litre -1 and the interassay c v was less than 12 per cent (n = 9) at 3.0 and 22,6 nmol litre-1. Plasma ACTH was measured by a direct second antibody R~A standardised against NIBSC hACTH 74•555 (Holly Hill, Hampstead) and using rabbit anti-human ACTH serum described by Nicholson et al (1984). This antiserum is directed against a non-species specific amino-terminal (1-18) sequence of ACTH. 125I-hACTH (1-39) prepared by the chloramine-T method was used as a label. Second antibody (normal rabbit serum and donkey anti-rabbit) for all the assays was provided by the Scottish Antibody Production Unit. The limit of detection of the assay was 3 mU litre -1 (1 mU is approximately 4,03 ng ACTH) and the intra- and interassay batch cvs were 8 per cent and 16 per cent, respectively. Plasma LH concentration was determined by a second
The hormonal data obtained after the injection of CRH were subjected to an analysis of variance with a repeated measures design followed, where applicable, by betweengroup comparisons using Newman-Keuls test. To test for differences between groups in the magnitude of the hormonal response to CRH, the data were also calculated as the areas under the response curves of individual hormone concentrations against time, calculated by trapezoid rule (Schreiber et al 1988). The results are presented as mean
(SEre.
RESULTS
Clinical observations The detailed clinical observations of these rams after infection with T congolense has been described by Mutayoba et al (1995). Briefly, all five infected rams became parasitaemic within seven to nine days after infection and the first peak of parasitaemia occurred one week later and the parasitaemia fluctuated thereafter. In the first 40 days after infection the rams developed a highly fluctuating pyrexia and there were rapid declines in pcv and the rate of liveweight gain. From 41 to 79 days after infection the pcv and liveweights of the infected rams changed only slightly. All the infected rams remained parasitaemic throughout the study.
Plasma ACTH The injection of CRH resulted in a significant increase in plasma ACTH concentrations in the infected and control rams on day 23 and on day 65 after infection (Figs la and 2a, respectively). The ACTH response to CRH on day 23 was initially similar in the control and infected rams (Fig la) and peaked 20 minutes after the injection of CRH (peak ACTH concentration was not significantly different between the groups). Relative to the peak value, the mean ACTtt levels had declined by 46 per cent 30 minutes after the injection and by 68 per cent 150 minutes after the CRH injection in the infected rams. The corresponding figures for the control rams were 9 per cent and 58 per cent, respectively. This accounted for a significantly lower (P<0.05) area under the response curve (Table 1) of the infected groups compared with the controls. The ACTH responses to CRH on day 65 are shown in Fig 2a. The response of one infected ram was delayed until 90 minutes after the injection and these data have been excluded; the concentration of ACTH in the other four infected rams increased rapidly from a mean of 5.6 (1.6) mU litre -1 at the time of the injection to a peak of 38.6 (14.7) mU litre -1 within 10 minutes. The concentration of ACTH in the control rams increased from 4.6 (0.7) mU litre -1 to a mean peak concentration of 21.3 (3.9) mU litre -1 15 minutes after the injection of CRH. The post peak decline in plasma ACTH concentration was more rapid in the infected rams than in the controls. The areas under the response curves (Table 1)
182
B. M. Mutayoba, P. D. Eckersall, C. Seely, C. E. Gray, V. Cestnik, L A. Jeffcoate, P. H. Holmes
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FIG 1: Mean plasma ACTH (a), cortisol (b), LH (c) and testosterone (d) responses after the injection of CRH (indicated by arrow) on day 23 after infection
FIG 2: Mean plasma ACTH (a), cortisol (b), LH (c) and testosterone (d) responses after the injection of CRH (indicated by arrow) on day 65 after infection
of the infected and control groups were not significantly different at this stage (day 65) of infection.
after there was a similar gradual decline in the plasma cordsol levels in both groups. Neither the areas under the response curves nor the peak cortisol concentrations of the infected and control groups were significantly different (Table 1). The mean plasma cortisol changes after the injection of CRI-I65 days after infection are shown in Fig 2b. The infected ram which had a delayed ACTH response also had a slow cortisol response and these data have been excluded. The cortisol concentration increased rapidly in the other four infected animals and between 15 to 30 minutes after the injection, the mean levels were significantly higher (P<0.05) than in the control rams. A sharp decline in plasma cortisol concentration was observed from 40 to 80 minutes after the injection of CRH, by which time the mean levels were similar in both groups. The areas under the response curves of the infected and control rams were not significantly different.
Plasma cortisol
The injection of CRI-Ion day 23 after infection produced a rapid rise in plasma cortisol concentration within five to 10 minutes in all the animals (Fig 2a), coinciding with the rise in plasma ACTH concentration. The mean peak levels were observed within 30 minutes in both groups and thereT A B L E 1 : ' M e a n a r e a u n d e r t h e r e s p o n s e c u r v e s for ACTH, cortisol, LH a n d t e s t o s t e r o n e in r a m s infected with Trypanosoma congolense a n d u n i n f e c t e d r a m s after t h e a d m i n i s t r a t i o n of c o r t i c o t r o p h i n - r e l e a s i n g hormone intravenously
Days after infection 23 Measurement
Control (n=5)
65 infected (n=5)
ACTH 61.9 (8-7) 29.8 (mU 2.5 h litre -1) Cortisol 125.6 (17.9)116.9 (nmol 2.5 h litre -1) LH 1.32 (0.13) 1.41 (ng 2.5 h m1-1) Testosterone 3.45 (1.56) 9.45 (nmol 2.5 h litre-1)
Control (n=5)
Infected (n=4)
(8.2)*
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35.2 (8.0)
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119.5 (26-4)
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2.97 (0.76)
2.13 (0.24)
(1.09)*
7.78 (2.88)
3.57 (1.48)
* Significantly different (P
Plasma LH
The changes in the mean plasma LH concentrations on day 23 in the infected and control groups are shown in Fig lc. Before the injection of CRH the levels fluctuated between 0.2 and 1.0 ng m1-1 in both groups. In the control rams the plasma LH concentrations tended to decline
Effects ofT congolense infection in sheep
between 10 and 30 minutes after the injection of CRH in the infected group they declined between five and 10 minutes after the injection. This difference was not statistically significant but it did occur coincidentally with the ACTH peak and the rapid rise in cortisol concentration. There was no significant effect of CRH on plasma LH concentration in either group (Fig lc, Table 1). The mean plasma LH responses to the injection of CRH on day 65 in both groups are shown in Fig 2c. The control rams tended to have a higher mean basal plasma LH concentration than the infected rams before and after the injection. No significant effect of CRH on plasma LH concentration was observed in either group (Fig 2c, Table 1).
Plasma testosterone The changes in plasma testosterone concentrations after the injection of CRH on day 23 are shown in Fig ld. The plasma testosterone concentration remained basal in the infected and control rams up to 40 minutes after the injection and then increased sharply (P<0.001) in both groups. The amplitude of this increase and the mean area under the response curves (Table 1) were significantly higher (P<0.05) in the infected rams than in the control rams. On day 65 the mean plasma testosterone concentrations from 20 minutes before to 60 minutes after the injection of CRH were significantly lower (P<0.05) in the infected than in the control rams (less than 1.0 nmol litre -1 compared with less than 3.5 nmol litre -1) (Fig 2d). However, in both groups, the plasma testosterone concentration did not change significantly from its value immediately before the injection for 60 minutes after the injection. Subsequently there was an increase in plasma testosterone concentration between 80 and 120 minutes after the injection of CRH in both the infected and control groups. The areas under the response curves of the two groups were not significantly different.
DISCUSSION The changes in the plasma concentrations of ACTH, cortiSO1, LH and testosterone in the rams in response to the injec-
tion of CRH were determined at two stages of T congolense infection. The period up to 40 days after infection, which was characterised by rapid decline in pcv, impaired growth and very variable rectal temperatures, will be termed the acute phase of infection, and the period from 41 to 79 days after infection, which was characterised by stable pcv and liveweight, will be termed the chronic phase of infection. After the injection of CRH, increases in plasma ACTH and cortisol concentrations were observed in both the infected and control groups during the acute phase (day 23) and the chronic phase (day 65), peaking within 10 to 20 minutes for ACTH and within 30 to 40 minutes for cortisol. These results confirm the efficacy of CRH for inducing the release of ACTH and cortisol, as has been demonstrated in sheep by Pradier et al (1988) and in cattle by Abebe (1991). There were, however, quantitative differences between the effects of CRH on day 23 and day 65 after infection. The ACTH response was lower in the infected rams than in the control rams on day 23. This result accords with the observations of Abebe (1991), Abebe et al (1993) and Abebe and Eley (1992) who reported a significant reduction in the plasma ACTH response to the intravenous injection of CRH or insulin during the acute phase of T congolense infection in
183
cattle. However, the concomitant reduction in the cortisol response to the injection of CRH which was reported in cattle (Abebe 1991, Abebe et al 1993) was not observed in the infected rams at this stage of infection because the mean cortisol responses of the infected and uninfected rams were similar. Hence, whereas in cattle the reduction in plasma cortisol levels during the acute phase of T congolense infection is likely to be associated with both pituitary and adrenal defects (Abebe 1991, Ogwu et al 1992, Abebe et al 1993), the results of the present study suggest that in sheep the infection results only in a reduced pituitary responsiveness to CRH, as evidenced by the reduced pituitary ACTH secretion, but that the adrenal response to ACTH was the same as in the control sheep. During the chronic phase of T congolense infection, the responsiveness of the pituitary-adrenal axis to CRH was greater than in the uninfected rams. Thus the plasma ACTH and cortisol concentrations increased rapidly and peaked earlier at a significantly higher concentration in the infected than in the control rams. These findings indicate a biphasic effect of T congolense on the responsiveness of the HPA axis to CRH in that, after a period of suppression during the acute phase of the infection, its responsiveness is markedly enhanced during the chronic phase. This observation is in agreement with the plasma cortisol concentrations and adrenal morphology of T congolense-infected rams described by Mutayoba et al (1995); their mean plasma cortisol concentration was reduced during the acute phase of the infection but markedly enhanced during the chronic phase, when adrenal hyperplasia was also observed. A marked increase in cortisol concentrations during the chronic phase has also been reported in T congolenseinfected goats (Mutayoba and Gombe 1989). The suppression of the HPA axis during the acute phase of the disease may be a response to the onset of parasitaemia, fever and the rapidly developing anaemia which were observed during this stage. It seems, however, that sheep can partially recover from the changes associated with the acute infection, and that the activity of the HPA recovers during the chronic phase. Previous results obtained in various animal species (Litrap and Raeside 1975, Johnson et al 1982, Rivier et al 1986, Naylor et al 1990) have shown that factors which activate the HPA axis and increase cortisol secretion may impair reproductive activity. During the present study, the injection of CRH on days 23 and 65 after infection had no significant effect on the release of LH in either the infected or control rams, but on both occasions there was a decrease in LH concentration in some animals within five to 15 minutes. However, there was too much variation between individual animals for this decrease to be taken as a specific response to increases in ACTH and/or cortisol. In other animals, including rats and cattle, it has been established that stress-induced activation of the HPA axis impairs the pituitary secretion of LH (Johnson et al 1982, Rivier et al 1986). However, experiments on such endocrine interactions have yielded conflicting data. For example, the injection of CRH into sheep either intravenously (Parrot et al 1988) or intracerebroventricularly (Horton et al 1988) had no inhibitory effect on LH secretion and Naylor et al (1990) reported that CRH may have a dose-dependent stimulatory effect on LH secretion. A common feature in the infected and control rams after the injection of CRH was that for 40 to 60 minutes there were no significant changes in testosterone concentration but that after the plasma ACTH concentration had declined
184
B. M. Mutayoba, P. D. Eckersall, C. Seely, C. E. Gray, V. Cestnik, L A. Jeffcoate, P. H. Holmes
there was an increase in testosterone during both the acute and chronic phases of infection. The mechanism of this delayed response may be related to the effects of CRH or cortisol, acting either singly or in concert, on testicular steroidogenesis. CRH is known to inhibit the Leydig cell gonadotrophin-induced generation of cAMP and the subsequent production of androgens by acting on a specific high affinity receptor on the Leydig cell's membrane (Ulisse et al 1990, Dufau et al 1993), and cortisol is known to inhibit gonadal steroidogenesis by the inhibition of testicular LH receptors (Bambino and Hsueh 1981). Although there was no immediate reduction in plasma testosterone after the injection of CRH, the eventual increase in testosterone could be a result of CRrt or cortisol initially limiting the secretion but not the synthesis of androgens so that testosterone accumulated in the Leydig cells. The disappearance of an inhibitor from the plasma could then coordinate the release of testosterone. This explanation of the results is supported by the observation that the delayed increase in plasma testosterone concentration coincided with the decrease in the plasma concentrations of ACTH and cortisol, implying that the latter are important in vivo mediators in this process. The greater magnitude of the delayed testosterone response in the infected rams than the control rams during the acute phase of the infection suggests that their Leydig cells synthesised and accumulated more testosterone during the period when cortisol was limiting the secretion of testosterone. During the chronic phase, there was no difference between the responses to CRH of the infected and control rams, although the concentrations of testosterone before and after the injection were lower in the infected rams. Decreased plasma testosterone levels have also been reported during chronic trypanosomiasis in sheep (Mutayoba 1994, Adeyemo et al 1990) and goats (Waindi et al 1986). These results demonstrate that the CRH-induced secretion of ACTH is impaired during the acute phase and enhanced during the chronic phase of T congolense infection in rams. Such an enhancement of pituitary ACTH secretion may explain the adrenal hyperplasia observed in chronically infected sheep by Mutayoba et al (1995). In addition, these experiments demonstrate the effects of short term exposure of the pituitary gonadal axis to hormones of the pituitary adrenal axis. Chronic exposure of the pituitary gonadal axis to adrenal hormones, which is likely to be one of the effects of trypanosomiasis, may be one factor involved in the changes in testicular steroidogenesis and the infertility of infected animals. ACKNOWLEDGEMENTS The assistance of technical staff of the Department of Veterinary Physiology, University of Glasgow, is greatly acknowledged. B.M.M. was funded by the Association of Commonwealth Universities and V.C. by the Commission of the European Communities.
REFERENCES ABEBE, G. (1991) The integrity of hypothalamic-pituitary-adrenal axis in boran (Bos indicus) cattle infected with Trypanosoma congolense. PhD thesis, Brunel University ABEBE, G. & ELEY, R. M. (1992) Hypothalamic-pituitary-adrenalaxis responsiveness to insulin-induced hypoglycaemia is modified by trypanosome infection in Boran (Bos indicus) cattle. Research in Veterinary Science 53, 68-72 ABEBE, G., ELEY, R. M. & OLEMOIYOI, O. K. (1993) Reduced responsiveness of the hypothalamo-pituitary-adrenal axis in boran (Bos indicus) cattle infected with
Trypanosoma congolense. Acta Endocrinologica (Copenhagen) 129, 75-80 ADEYEMO, O., OYEDIPE, A. & AGBEGANA, O. (1990) Plasma testosterone in Trypanosoma congolense and Trypanosoma brucei-infected West African dwarf rams. Animal Reproduction Science 22, 21-26 BAMBINO, T. H. & HSUEH, A. J. W. (1981) Direct inhibitory effect of glucocorticolds upon testicular luteinizing hormone receptors and steroidogenesis in vivo and in vitro. Endocrinology 108, 2142-2148 CHARPENET, G., TACHE, Y., FOREST, M. G., HAOUR, F., SAEZ, J. M., BERNIER, M., DUCHARME, J. R. & COLLU, R. (1981) Effect of chronic intermittent immobilization stress on rat testicular androgen function. Endocrinology 109, 1254-1258 COOK, B. & BEASTALL, H. B. (1987) Measurement of steroid hormone concentrations in blood, urine and tissue. In Steroid Hormones: A Practical Approach. Eds B. Green and R. E. Leake. Oxford, IRL Press. pp 1-65 DONALD, R. A., REDEKOPP, C., CAMERON, V., NICHOLLS, M. G., BOSTON, J., LIVESEY, J., ESPINER, A., RIVIER, J. & VALE, W. (1983) The hormonal action of corticotropin-releasing factor in sheep: Effect of intravenous and intracerebroventdcular injection. Endocrinology 113, 866-870 DUFAU, M. L , TINAJERO, J. C. & FABBRI, A. (1993) Corticotropin-releasing factor: an antireproductive hormone of the testis. The Faceb Journal 7, 299-307 FISHER, L. A. (1989) Corticotropin-releasing factor: endocrine and autonomic integration of responses to stress. Trends in Pharmacological Sciences 10, 189-193 HORTON, R. J. E., FRANCIS, H. & CLARKE, I. J., (1988) Intracerebroventricular administration of ~-endorphin, but not CRF inhibits LH secretion in ovariectomised ewes. Proceedings of the 8th International Congress of Endocrinology Tokyo, Japan. Abstract number 03-21-102. p 146 IKEDE, B. O., ELHASSAN, E. & AKPAVIE, S. O. (1988) Reproductive disorders in African trypanosomiasis: a review. Acta Tropica 45, 5-10 JEFFCOATE, I. (1992) Concentrations of luteinizing hormone and oestradiol in plasma and response to injection of gonadotrophin-releasing hormone analogue at selected stages of anoestrns in domestic bitches. Journal of Reproduction and Fertility 94, 423-429 JOHNSON, B. H., WELSH, T. H. & JUNIEWICZ, P. E. (1982) Suppression of luteinizing hormone and testosterone secretion in bulls following adrenocorticotropin hormone treatment. Biology of Reproduction 26, 305-310 LENZ, H. J., READLER, A., GRETEN, H. & BROWN, M. R. (1987) CRF initiates biological actions within the brain that are observed in response to stress. American Journal of Physiology 252, R34-R39 LITRAP, R. M. & RAESIDE, J. I. (1975) Increase in plasma testosterone concentration after injection of adrenocorticotrophin into the boar. Journal of Endocrinology 66, 123-131 McCONWAY, M. G. & CHAPMAN, R. S. (1986) Development and evaluation of a simple, direct solid phase radinimmunoassay of serum cortisol from readily available reagents. 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(1990) Central administration of corticotrophin-releasing factor in sheep: effects on secretion of gonadotrophins, prolactin and cortisol. Journal of Endocrinology 124, 117-125 NICHOLSON, W. E., DAVIS, D. R., SHERRELL, B. J. & ORTH, D. N. (1984) Rapid mdioimmunoassay for corticotropin in unextracted human plasma. Clinical Chemistry 30, 259-265 OGWU, D., NJOKU, C. O. & OGBOGU, V. C. (1992) Adrenal and thyroid dysfunctions in experimental Trypanosoma congolense infection in cattle. Veterinary Parasitology 42, 15-26 PARROT, R. F. (1990) Cordsol release in pigs following peripheral and central administration of ovine and human corticotropin-releasing hormone. Acta Endocrinologica (Copenhagen) 123, 108-112 PARROT, R. F., ROBINSON, J. E. & THORNTON, S. M. (1988) No changes in LH pulse frequency or plasma oxytocin in castrated rams treated with corticotropinreleasing hormone. Journal of Endocrinology 199 (Supplement) Abstract number 130 PLOTSKY, P. M. & VALE, W. (1984) Haemolrhage-induced secretion of corticotropin-releasing factor-like immunoreactivity into the rat portal circulation and its inb_ibitionby glucocorticoids. Endocrinology 114, 164-169 PRADIER, P., DALLE, M., TOURNAIRE, C. & DELOST, P. (1988) Plasma concentrations of adrenocorticotropin-related peptides after corticotropin-releasing hormone and vasopressin injections in sheep. Acta Endocrinologica (Copenhagen) 119, 391-396 RIVIER, C. L. & PLOTSKY, P. M. (1986) Mediation by corticotropin-releasing factor (CRH) of adenohypophyseal hormone secretion. Annual Reviews of Physiology 48, 475-494 RIVIER, C. L., RIVIER, J. & VALE, W. (1986) Stress-induced inhibition of reproductive function: Role of endogenous corticotropin-releasing factor. Science 231, 607 -609 SCHREIBER, W., KRIEG, J.-C., BOSSERT, S., JUNKER, M., RAUSCHHUBER, R., STALLA, G. R., MULLER, O. A. & BERGER, M. (1988) Methodological aspects of hCRH-stimulated ACTH and cortisol secretion in healthy subjects. Psychoneuroendocrinology 13, 287-495
Effects o f T c o n g o l e n s e infection in sheep
SHUTT, D. A., CONNELL, R. & FELL, L. R. (1989) Effects of ovine corticotropinreleasing factor and vasopressin on plasma [3-endorphin, cortisol and behaviour after minor surgery in sheep. Life Science 45, 257-262 ULISSE, S., FABBRI, A. & DUFAU, M. L. (1990) A novel mechanism of action of corticotropin-releasing factor in the rat Leydig cell. Journal of Biological Chemistry 265, 1964-1971 VALE, W., SPIESS, C., RIVIER, C. & RIV1ER, J. (1981) Characterization of a 41-
185
residue ovine hypothalamic peptide that stimulates secretion of corticotzopin and J3-endorphin. Science 213, 1394-1397 WAINDI, E. N., GOMBE, S. & ODUOR-OKELO, D. (1986) Plasma testosterone in Trypanosoma congolense-infected Toggenbnrg goats. Archives of Andrology 17, 7-17
Received July 8, 1993 Accepted October 17, 1994
Effects of Trypanosomacongolense on pituitary and adrenocortical function in sheep: responses to exogenous corticotrophin-releasing hormone B. M. MUTAYOBA*, P. D. ECKERSALL, C. SEELY, Department of Veterinary Clinical Biochemistry, University of Glasgow Veterinary School, Bearsden, Glasgow G61 1QH, C. E. GRAY, Institute of Biochemistry, Royal Infirmary, Glasgow G40SF, V. CESTNIKt, Department of Veterinary Clinical Biochemistry, I. A. JEFFCOATE, P. H. HOLMES,
Department of Veterinary Physiology, University of Glasgow Veterinary School, Bearsden, Glasgow G61 1QH
SUMMARY To investigate whether the aberrations in adrenocortical and gonadal activity observed in trypanosomiasis may be induced by the refractoriness of the pituitary to hypothalamic liberins, the responses of the pituitary and adrenal glands and the testes to stimulation with ovine corticotrophin-releasing hormone (oCRH)were studied in rams 23 days (acute phase) and 65 days (chronic phase) after they were infected with Trypanosomacongolense. On both occasions a peak of plasma ACTHwas observed within 20 minutes of the injection of CRH but the rate of increase in ACTHand the mean peak values in the infected rams were significantly lower (P<0.001) on day 23 but higher (P<0.05) on day 65 than in the uninfected control rams. Plasma cortisol concentration increased in all the rams after the injection of CRH.The rate of increase in plasma cortisol and the mean peak values were not significantly different between the control and infected rams on day 23 but were significantly (P<0.001) higher in the infected rams on day 65. However, the post peak concentrations of ACTHdeclined more rapidly in the infected rams than in the controls on both days 23 and 65. The plasma concentration of luteinising hormone (LH) did not change after the injection of CRH, whereas the testosterone levels showed a delayed response and its concentration increased when plasma ACTH and cortisol concentrations declined in both groups. On day 23, there was a greater increase in testosterone in the infected than in the control rams. These results demonstrate that the responsiveness of the pituitary corticotrophs to CRH is depressed during the acute phase and enhanced during the chronic phase of T congolenseinfection in rams, whereas the adrenal cortisol response is less affected. The results are also consistent with the hypothesis that the modulation of the pituitary-adrenal axis by infective trypanosomes may exacerbate the changes in testicular steroidogenesis frequently observed in trypanosomiasis. F O L L O W I N G the elucidation of the molecular structure of ovine corticotrophin-releasing hormone (OCRH) by Vale et al (1981) its physiological significance in regulating the secretion of adrenocorticotrophin hormone (ACTH) and cortisol has been recognised (Donald et al 1983, Plotsky and Vale 1984, Rivier and Plotsky 1986). It has been shown that through CRH, the hypothalamus plays a fundamental role in coordinating the stress response by integrating psychoneuroendocrine and autonomic functions (Moberg 1985, Lenz et al 1987, Fisher 1989). T h e injection of CRH elicits increases in plasma ACTH and cortisol and these have been used to test pituitary-adrenal responsiveness under various conditions (Pradier et al 1988, Shutt et al 1989, Parrot 1990). Although the primary focus of most neuroendocrine studies has been on the stress response of the pituitary and adrenal glands there is an increasing body of evidence which links the stress-induced stimulation of ACTH and glucocorticoids with changes in other pituitary hormones including growth hormone, prolactin, thyroid stimulating hormone and the gonadotrophins (Moberg 1985). Furthermore, there is now evidence that the stress-induced activation of the hypothalamo-pituitary-adrenal (HPA) axis might be responsible for the subsequent infertility observed in male rats (Charpenet et al 1981, Rivier et al 1986), bulls * Present address: Depam~nent of Veterinary Physiology, Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Sokoine University of Agriculture, PO Box 3017, Morogoro, Tanzania ? Institute of Physiology, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Ljubljana, Gerbiceva 60, 61000, Slovenia
(Johnson et al 1982), boars (Litrap and Raeside 1975) and rams (Naylor et al 1990). Trypanosomiasis is associated with changes in the activity of several endocrine glands (Ikede et al 1988, Mutayoba and Gombe 1989). For example, plasma cortisol concentrations are increased in goats (Mutayoba and Gombe 1989) and sheep, and in the latter the increases are associated with adrenal hyperplasia (Mutayoba et al 1995). On the other hand plasma testosterone concentration is decreased in sheep (Adeyemo et al 1990), and it seems possible that the infertility observed in trypanosomiasis could be associated with changes in the HPA axis. The aims of this study were to investigate the interactions between the HPA axis and the gonadal axes in trypanosome-infected rams. CRI-Iwas used' to provoke a pituitary-adrenal endocrine response and the effect on the pituitary-testicular axis was observed simultaneously.
MATERIALS AND METHODS
Animals and trypanosome infection Ten Scottish blackface rams aged six months and weighing approximately 34 kg were used. These rams were part of a group used to study the effects of trypanosomiasis on adrenal pathophysiology, and their management before and during infection has. been described by Mutayoba et al (1995). Five rams were infected intravenously with approximately 4 × 105 trypanosomes as
180
Effects ofT congolense infection in sheep
already described and together with the five uninfected control rams they were monitored for a period of 79 days. As part of larger control and infected groups these animals contributed to the group mean haematology, bodyweight and rectal temperature data presented by Mutayoba et al (1995).
181
antibody RIA using the method described by Jeffcoate (1992). The limit of detection of the assay was 0.12 ng m1-1 (n = 10). The intra-assay c v was less than 5 per cent at 2-99 and 10.71 ng m1-1 and the interassay c v was less than 13 per cent (n = 5) at 2.86 and 11.32 ng m1-1.
Statistics CRH
injection
On day 23 and day 65 after infection, a jugular catheter was inserted into each of the rams between 08.00 and 10.00 and kept patent with heparinised saline (200 iu m1-1 in 0.9 per cent w/v sodium chloride). Beginning one hour later, 3 ml blood samples were taken every 20 minutes into heparinised tubes for a period of one hour. Each animal then received oCRH via the cannula (25 gg/2 ml saline, C-3167, Sigma), and blood samples were taken five, 10, 20, 30, 40, 60, 90, 120 and 150 minutes after the injection. The samples were centrifuged immediately and the plasma snap frozen in dry ice and stored at -20°C for measurements of cortisol, ACTH, luteinising hormone (LH) and testosterone. The dose of CRH was selected to give a pituitary-adrenal response lasting for two to three hours (Pradier et al 1988).
Hormonal measurements Plasma cortisol concentration was measured by radioimmunoassay (R/A) according to McConway and Chapman (1986) and the reagents used for the assay have been described by Mutayoba et al (1995). Plasma testosterone concentration was measured by the RIA described by Cook and Beastall (1987). Testosterone standards were purchased from Steraloids and 125I-histamine-testosterone was synthesised by C. E. Gray. Rabbit-anti-testosterone (AB-1030) was purchased from Bioclinical Services, Cardiff; according to the suppliers this antibody shows the following cross-reactivity: testosterone 100 per cent, 5Gt-dihydrotestosterone 16 per cent, 5ct-androstane-3a17~3-diol 5.8 per cent, 5a-androstane-313-1713-diol 3.7 per cent, androstenedione 2.1 per cent, dihydroepiandrosterone 0.04 per cent and cortisol <0-01 per cent. The limit of detection (2 x SD of total binding) for cortisol was 3.0 nmol litre -1 (n = 12) and for testosterone 0.1 nmol litre -1 (n = 12). The intra-assay coefficient of variation (cv) for the cortisol assay was less than 11 per cent (n = 13) at 14.6 and 136.6 nmol litre -1 and the interassay c v was less than 15 per cent (n = 7) at 11.4 and 77.7 nmol litre -1. The intraassay c v for the testosterone assay was less than 8 per cent (n = 16) at 3.3 and 12.4 nmol litre -1 and the interassay c v was less than 12 per cent (n = 9) at 3.0 and 22,6 nmol litre-1. Plasma ACTH was measured by a direct second antibody R~A standardised against NIBSC hACTH 74•555 (Holly Hill, Hampstead) and using rabbit anti-human ACTH serum described by Nicholson et al (1984). This antiserum is directed against a non-species specific amino-terminal (1-18) sequence of ACTH. 125I-hACTH (1-39) prepared by the chloramine-T method was used as a label. Second antibody (normal rabbit serum and donkey anti-rabbit) for all the assays was provided by the Scottish Antibody Production Unit. The limit of detection of the assay was 3 mU litre -1 (1 mU is approximately 4,03 ng ACTH) and the intra- and interassay batch cvs were 8 per cent and 16 per cent, respectively. Plasma LH concentration was determined by a second
The hormonal data obtained after the injection of CRH were subjected to an analysis of variance with a repeated measures design followed, where applicable, by betweengroup comparisons using Newman-Keuls test. To test for differences between groups in the magnitude of the hormonal response to CRH, the data were also calculated as the areas under the response curves of individual hormone concentrations against time, calculated by trapezoid rule (Schreiber et al 1988). The results are presented as mean
(SEre.
RESULTS
Clinical observations The detailed clinical observations of these rams after infection with T congolense has been described by Mutayoba et al (1995). Briefly, all five infected rams became parasitaemic within seven to nine days after infection and the first peak of parasitaemia occurred one week later and the parasitaemia fluctuated thereafter. In the first 40 days after infection the rams developed a highly fluctuating pyrexia and there were rapid declines in pcv and the rate of liveweight gain. From 41 to 79 days after infection the pcv and liveweights of the infected rams changed only slightly. All the infected rams remained parasitaemic throughout the study.
Plasma ACTH The injection of CRH resulted in a significant increase in plasma ACTH concentrations in the infected and control rams on day 23 and on day 65 after infection (Figs la and 2a, respectively). The ACTH response to CRH on day 23 was initially similar in the control and infected rams (Fig la) and peaked 20 minutes after the injection of CRH (peak ACTH concentration was not significantly different between the groups). Relative to the peak value, the mean ACTtt levels had declined by 46 per cent 30 minutes after the injection and by 68 per cent 150 minutes after the CRH injection in the infected rams. The corresponding figures for the control rams were 9 per cent and 58 per cent, respectively. This accounted for a significantly lower (P<0.05) area under the response curve (Table 1) of the infected groups compared with the controls. The ACTH responses to CRH on day 65 are shown in Fig 2a. The response of one infected ram was delayed until 90 minutes after the injection and these data have been excluded; the concentration of ACTH in the other four infected rams increased rapidly from a mean of 5.6 (1.6) mU litre -1 at the time of the injection to a peak of 38.6 (14.7) mU litre -1 within 10 minutes. The concentration of ACTH in the control rams increased from 4.6 (0.7) mU litre -1 to a mean peak concentration of 21.3 (3.9) mU litre -1 15 minutes after the injection of CRH. The post peak decline in plasma ACTH concentration was more rapid in the infected rams than in the controls. The areas under the response curves (Table 1)
182
B. M. Mutayoba, P. D. Eckersall, C. Seely, C. E. Gray, V. Cestnik, L A. Jeffcoate, P. H. Holmes
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FIG 1: Mean plasma ACTH (a), cortisol (b), LH (c) and testosterone (d) responses after the injection of CRH (indicated by arrow) on day 23 after infection
FIG 2: Mean plasma ACTH (a), cortisol (b), LH (c) and testosterone (d) responses after the injection of CRH (indicated by arrow) on day 65 after infection
of the infected and control groups were not significantly different at this stage (day 65) of infection.
after there was a similar gradual decline in the plasma cordsol levels in both groups. Neither the areas under the response curves nor the peak cortisol concentrations of the infected and control groups were significantly different (Table 1). The mean plasma cortisol changes after the injection of CRI-I65 days after infection are shown in Fig 2b. The infected ram which had a delayed ACTH response also had a slow cortisol response and these data have been excluded. The cortisol concentration increased rapidly in the other four infected animals and between 15 to 30 minutes after the injection, the mean levels were significantly higher (P<0.05) than in the control rams. A sharp decline in plasma cortisol concentration was observed from 40 to 80 minutes after the injection of CRH, by which time the mean levels were similar in both groups. The areas under the response curves of the infected and control rams were not significantly different.
Plasma cortisol
The injection of CRI-Ion day 23 after infection produced a rapid rise in plasma cortisol concentration within five to 10 minutes in all the animals (Fig 2a), coinciding with the rise in plasma ACTH concentration. The mean peak levels were observed within 30 minutes in both groups and thereT A B L E 1 : ' M e a n a r e a u n d e r t h e r e s p o n s e c u r v e s for ACTH, cortisol, LH a n d t e s t o s t e r o n e in r a m s infected with Trypanosoma congolense a n d u n i n f e c t e d r a m s after t h e a d m i n i s t r a t i o n of c o r t i c o t r o p h i n - r e l e a s i n g hormone intravenously
Days after infection 23 Measurement
Control (n=5)
65 infected (n=5)
ACTH 61.9 (8-7) 29.8 (mU 2.5 h litre -1) Cortisol 125.6 (17.9)116.9 (nmol 2.5 h litre -1) LH 1.32 (0.13) 1.41 (ng 2.5 h m1-1) Testosterone 3.45 (1.56) 9.45 (nmol 2.5 h litre-1)
Control (n=5)
Infected (n=4)
(8.2)*
24.9 (4.9)
35.2 (8.0)
(1.7)
85.8 (7.9)
119.5 (26-4)
(0.29)
2.97 (0.76)
2.13 (0.24)
(1.09)*
7.78 (2.88)
3.57 (1.48)
* Significantly different (P
Plasma LH
The changes in the mean plasma LH concentrations on day 23 in the infected and control groups are shown in Fig lc. Before the injection of CRH the levels fluctuated between 0.2 and 1.0 ng m1-1 in both groups. In the control rams the plasma LH concentrations tended to decline
Effects ofT congolense infection in sheep
between 10 and 30 minutes after the injection of CRH in the infected group they declined between five and 10 minutes after the injection. This difference was not statistically significant but it did occur coincidentally with the ACTH peak and the rapid rise in cortisol concentration. There was no significant effect of CRH on plasma LH concentration in either group (Fig lc, Table 1). The mean plasma LH responses to the injection of CRH on day 65 in both groups are shown in Fig 2c. The control rams tended to have a higher mean basal plasma LH concentration than the infected rams before and after the injection. No significant effect of CRH on plasma LH concentration was observed in either group (Fig 2c, Table 1).
Plasma testosterone The changes in plasma testosterone concentrations after the injection of CRH on day 23 are shown in Fig ld. The plasma testosterone concentration remained basal in the infected and control rams up to 40 minutes after the injection and then increased sharply (P<0.001) in both groups. The amplitude of this increase and the mean area under the response curves (Table 1) were significantly higher (P<0.05) in the infected rams than in the control rams. On day 65 the mean plasma testosterone concentrations from 20 minutes before to 60 minutes after the injection of CRH were significantly lower (P<0.05) in the infected than in the control rams (less than 1.0 nmol litre -1 compared with less than 3.5 nmol litre -1) (Fig 2d). However, in both groups, the plasma testosterone concentration did not change significantly from its value immediately before the injection for 60 minutes after the injection. Subsequently there was an increase in plasma testosterone concentration between 80 and 120 minutes after the injection of CRH in both the infected and control groups. The areas under the response curves of the two groups were not significantly different.
DISCUSSION The changes in the plasma concentrations of ACTH, cortiSO1, LH and testosterone in the rams in response to the injec-
tion of CRH were determined at two stages of T congolense infection. The period up to 40 days after infection, which was characterised by rapid decline in pcv, impaired growth and very variable rectal temperatures, will be termed the acute phase of infection, and the period from 41 to 79 days after infection, which was characterised by stable pcv and liveweight, will be termed the chronic phase of infection. After the injection of CRH, increases in plasma ACTH and cortisol concentrations were observed in both the infected and control groups during the acute phase (day 23) and the chronic phase (day 65), peaking within 10 to 20 minutes for ACTH and within 30 to 40 minutes for cortisol. These results confirm the efficacy of CRH for inducing the release of ACTH and cortisol, as has been demonstrated in sheep by Pradier et al (1988) and in cattle by Abebe (1991). There were, however, quantitative differences between the effects of CRH on day 23 and day 65 after infection. The ACTH response was lower in the infected rams than in the control rams on day 23. This result accords with the observations of Abebe (1991), Abebe et al (1993) and Abebe and Eley (1992) who reported a significant reduction in the plasma ACTH response to the intravenous injection of CRH or insulin during the acute phase of T congolense infection in
183
cattle. However, the concomitant reduction in the cortisol response to the injection of CRH which was reported in cattle (Abebe 1991, Abebe et al 1993) was not observed in the infected rams at this stage of infection because the mean cortisol responses of the infected and uninfected rams were similar. Hence, whereas in cattle the reduction in plasma cortisol levels during the acute phase of T congolense infection is likely to be associated with both pituitary and adrenal defects (Abebe 1991, Ogwu et al 1992, Abebe et al 1993), the results of the present study suggest that in sheep the infection results only in a reduced pituitary responsiveness to CRH, as evidenced by the reduced pituitary ACTH secretion, but that the adrenal response to ACTH was the same as in the control sheep. During the chronic phase of T congolense infection, the responsiveness of the pituitary-adrenal axis to CRH was greater than in the uninfected rams. Thus the plasma ACTH and cortisol concentrations increased rapidly and peaked earlier at a significantly higher concentration in the infected than in the control rams. These findings indicate a biphasic effect of T congolense on the responsiveness of the HPA axis to CRH in that, after a period of suppression during the acute phase of the infection, its responsiveness is markedly enhanced during the chronic phase. This observation is in agreement with the plasma cortisol concentrations and adrenal morphology of T congolense-infected rams described by Mutayoba et al (1995); their mean plasma cortisol concentration was reduced during the acute phase of the infection but markedly enhanced during the chronic phase, when adrenal hyperplasia was also observed. A marked increase in cortisol concentrations during the chronic phase has also been reported in T congolenseinfected goats (Mutayoba and Gombe 1989). The suppression of the HPA axis during the acute phase of the disease may be a response to the onset of parasitaemia, fever and the rapidly developing anaemia which were observed during this stage. It seems, however, that sheep can partially recover from the changes associated with the acute infection, and that the activity of the HPA recovers during the chronic phase. Previous results obtained in various animal species (Litrap and Raeside 1975, Johnson et al 1982, Rivier et al 1986, Naylor et al 1990) have shown that factors which activate the HPA axis and increase cortisol secretion may impair reproductive activity. During the present study, the injection of CRH on days 23 and 65 after infection had no significant effect on the release of LH in either the infected or control rams, but on both occasions there was a decrease in LH concentration in some animals within five to 15 minutes. However, there was too much variation between individual animals for this decrease to be taken as a specific response to increases in ACTH and/or cortisol. In other animals, including rats and cattle, it has been established that stress-induced activation of the HPA axis impairs the pituitary secretion of LH (Johnson et al 1982, Rivier et al 1986). However, experiments on such endocrine interactions have yielded conflicting data. For example, the injection of CRH into sheep either intravenously (Parrot et al 1988) or intracerebroventricularly (Horton et al 1988) had no inhibitory effect on LH secretion and Naylor et al (1990) reported that CRH may have a dose-dependent stimulatory effect on LH secretion. A common feature in the infected and control rams after the injection of CRH was that for 40 to 60 minutes there were no significant changes in testosterone concentration but that after the plasma ACTH concentration had declined
184
B. M. Mutayoba, P. D. Eckersall, C. Seely, C. E. Gray, V. Cestnik, L A. Jeffcoate, P. H. Holmes
there was an increase in testosterone during both the acute and chronic phases of infection. The mechanism of this delayed response may be related to the effects of CRH or cortisol, acting either singly or in concert, on testicular steroidogenesis. CRH is known to inhibit the Leydig cell gonadotrophin-induced generation of cAMP and the subsequent production of androgens by acting on a specific high affinity receptor on the Leydig cell's membrane (Ulisse et al 1990, Dufau et al 1993), and cortisol is known to inhibit gonadal steroidogenesis by the inhibition of testicular LH receptors (Bambino and Hsueh 1981). Although there was no immediate reduction in plasma testosterone after the injection of CRH, the eventual increase in testosterone could be a result of CRrt or cortisol initially limiting the secretion but not the synthesis of androgens so that testosterone accumulated in the Leydig cells. The disappearance of an inhibitor from the plasma could then coordinate the release of testosterone. This explanation of the results is supported by the observation that the delayed increase in plasma testosterone concentration coincided with the decrease in the plasma concentrations of ACTH and cortisol, implying that the latter are important in vivo mediators in this process. The greater magnitude of the delayed testosterone response in the infected rams than the control rams during the acute phase of the infection suggests that their Leydig cells synthesised and accumulated more testosterone during the period when cortisol was limiting the secretion of testosterone. During the chronic phase, there was no difference between the responses to CRH of the infected and control rams, although the concentrations of testosterone before and after the injection were lower in the infected rams. Decreased plasma testosterone levels have also been reported during chronic trypanosomiasis in sheep (Mutayoba 1994, Adeyemo et al 1990) and goats (Waindi et al 1986). These results demonstrate that the CRH-induced secretion of ACTH is impaired during the acute phase and enhanced during the chronic phase of T congolense infection in rams. Such an enhancement of pituitary ACTH secretion may explain the adrenal hyperplasia observed in chronically infected sheep by Mutayoba et al (1995). In addition, these experiments demonstrate the effects of short term exposure of the pituitary gonadal axis to hormones of the pituitary adrenal axis. Chronic exposure of the pituitary gonadal axis to adrenal hormones, which is likely to be one of the effects of trypanosomiasis, may be one factor involved in the changes in testicular steroidogenesis and the infertility of infected animals. ACKNOWLEDGEMENTS The assistance of technical staff of the Department of Veterinary Physiology, University of Glasgow, is greatly acknowledged. B.M.M. was funded by the Association of Commonwealth Universities and V.C. by the Commission of the European Communities.
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(1992) Concentrations of luteinizing hormone and oestradiol in plasma and response to injection of gonadotrophin-releasing hormone analogue at selected stages of anoestrns in domestic bitches. Journal of Reproduction and Fertility 94, 423-429 JOHNSON, B. H., WELSH, T. H. & JUNIEWICZ, P. E. (1982) Suppression of luteinizing hormone and testosterone secretion in bulls following adrenocorticotropin hormone treatment. Biology of Reproduction 26, 305-310 LENZ, H. J., READLER, A., GRETEN, H. & BROWN, M. R. (1987) CRF initiates biological actions within the brain that are observed in response to stress. American Journal of Physiology 252, R34-R39 LITRAP, R. M. & RAESIDE, J. I. (1975) Increase in plasma testosterone concentration after injection of adrenocorticotrophin into the boar. Journal of Endocrinology 66, 123-131 McCONWAY, M. G. & CHAPMAN, R. S. (1986) Development and evaluation of a simple, direct solid phase radinimmunoassay of serum cortisol from readily available reagents. 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Received July 8, 1993 Accepted October 17, 1994