Effect of experimental infection with Trypanosoma congolense and scrotal insulation on leydig cell steroidogenesis in the ram

ELSEVIER

EFFECT OF EXPERIMENTAL INFECTION WITH TNoanosomaconnolenseAND SCROTAL INSULATION ON LEYDIG CELL STEROIDOGENESIS IN THE RAM B.M. Mutayoba,lyb P.J. 0’Shaugfnessy,2 I.A. Jeffcoate,2a P.D. Eckersall,’ V. Cestnik pcand P.H. Holmes2 Departments of lClinical Veterinary Biochemistry and 2Veterinary Physiology University of Glasgow Veterinary School, Glasgow, G61 lQH, UK Received for publication: July 9, Accepted: November

1996 20.

1996

ABSTRACT Testicular steroid content and Leydig cell steroidogenesis in vitro were investigated in connolense infection and were compared with those rams on Days 28 and 58 afler w of rams in which testicular temperature had been raised artificially by insulation of the scrotum for 58 d. Testicular testosterone content increased signitlcantly on Day 28 after infection but was lower than that of controls on Day 58 while it increased in scrotal-insulated rams compared with that of controls by Day 58. Testicular progesterone was undetectable in the control and trypanosome-infected groups throughout the experiment, but it increased in the insulated rams by day 58. Basal (unstimulated) Leydig cell testosterone production in the infected rams was similar to that of control rams on Day 28 but was significantly lower on Day 58. Stimulation of Leydig cell testosterone production with hCG or 22R-hydroxycholesterol(22ROHC) significantly reduced in infected rams at both 28 and 58 d after infection as well as in scrotal-insulated rams on Day 58. It is concluded that the increase in testicular testosterone content in the infected and scrotal-insulated rams on Days 28 and 58, respectively, was induced by elevation of testicular temperature by trypanosome infection, perhaps through an effect on testicular blood flow. Reduced testosterone production by Leydig cells from infected and scrotal-insulated rams in response to hCG and 22ROHC suggests that trypanosome-induced pyrexia might be involved in reducing Leydig cell steroidogenesis and subsequent plasma testosterone levels, possibly by affecting enzymes involved in steroid biosynthesis. 0 1997 by ElsevierScience Inc. Keywords: trypanosome-infection, testosterone-secretion Acknowledgments B.M. Mutayoba was funded by the Association of Commonwealth Universities and V. Cestnik by the Commission of the European Community aCorrespondence. bPermanent address: Department of Veterinary Physiology, Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Sokoine University of Agriculture, P.O. Box 3 0 17, Morogoro, Tanzania. ‘Permanent address: Institute of Physiology, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Ljubljana, Gerbiceva, 60, 6 1000, Slovenia

Thenogenology 4C411-422, 1997 0 1997 by ElsevlerScience Inc.

0093.691W97/$17.00 PIISOO93-691X(97)00251-3

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412 INTRODUCTION

Reduced libido, oligospermia and azospermia are well-known clinical signs of w m infection in male ruminants (14), and they probably result from the progressive noninflammatory degenerative lesions seen in the seminiferous tubules, epididymides and accessory tissues (21,26), and declining plasma testosterone concentration (1,29,). The latter, and the reduced responsiveness to hCG stimulation in trypanosome-infected animals might be associated with a decline in the number of LH receptors and/or a change in their equilibrium association constant (27). However, other factors are also likely to be involved in reducing testicular steroidogenesis a&r infection, since only a small percentage of the LH receptor population is apparently required to elicit a normal steroidogenic response (5,25). Hyperthermia, which occurs in bouts at peaks of parasitaemia, is another factor which may contribute to degeneration of the testicular germ cells and the decline in androgen secretion in twanosome-infected animals (2,26). Testicular degenerative changes similar to those observed during T, m infection occur in other febrile conditions (30) and after elevation of testicular temperature in nonfebrile conditions such as cryptorchidism (17) and after scrotal insulation (7) and heat treatment (12). The present experiments were designed to investigate the effects of T. e infection on testicular steroid content and Leydig cell steroidogenesis and to determine whether these effects are simply due to elevated scrotal temperature. MATERIALS AND METHODS Animals and Trypanosome Infection Scottish Blackface rams approximately 8 mo of age at the start of the study in November were divided into groups of noninfected controls (n=lO), trypanosome-infected (n=S) rams and noninfected, scrotal-insulated (n=6) rams. All were housed together in a fly-proof unit under artificial light comprising 8-h &$/16-h dark cycles. 5 Rams in the infected group were each injected intravenously with approximately 4 x lo5 T, coneolense derived from stabilate IL 1180 (8). The scrotal sac of each ram in the scrotal-insulated group was wrapped in cotton wool and held in place by cotton gauze and adhesive bandage. Five control and four infected rams were removed from the experiment and castrated 28 d after infection. The remaining rams (5 control, 4 infected and 6 scrotal-insulated) were castrated on Day 58. Rectal and scrotal skin temperatures were measured twice weekly, the latter with a copper-constantan thermocouple (38 to 40-swg wire) mounted in a polythene probe wired to a 2 channel potentiometric recorder (Smiths Industries Ltd, London, UK, Type RE524.20, Model Servoscribe 2). Sample Collection and Preparation Blood samples were collected 3 times a week into heparinized tubes for monitoring the onset of parasitaemia by counting organisms in the huffy coat from blood centritiged in microhaematocrit tubes. Single blood samples were obtained from each ram before castration and at 20-min intervals over a 6-h period using a jugular vein cannula on Days 26 and 52 after

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infection or insulation Plasma was separated by centrifugation at 1500 g for 10 min and stored at -2OOCfor testosterone assay The testes removed on Day 28 or 58 were immediately cooled on ice then weighed and halved transversely within 30 min. Transverse slices from the testis of each animal were weighed (approximately 1 to 200 mg) and cut into smaller pieces for cell dispersal The remaining tissue was frozen in a methanol-dry ice bath and stored at -2OOC. Testicular cells were dispersed by incubation with gentle agitation (15 min) in Medium 199 (M199; Gibco, Life Laboratories, Paisley, Strathclyde, UK) containing 1 mg/ml crude collagenase (Worthington, type CLS; Lome Diagnostics, Reading UK), 0.01% deoxyribonuclease II (D.8764; Sigma) and 1% (w/v) bovine serum albumin (BSA; Sigma) for 30 min as previously described (19). The dispersed cells were collected by centrifugation at 500 g and 4OC for 5 min, and the cell pellets were washed once with Ml 99 containing 0.1% BSA. After centritigation the cells were resuspended in 2 ml M 199 containing 0.1 % BSA, and cell numbers were counted using a haemocytometer. The percentage of Leydig cells in each fraction was determined by histochemical staining for 3P-hydroxysteroid dehydrogenase activity (24). To measure steroid production by dispersed cells, triplicate fractions of each cell suspension were incubated in Ml99 for 3 h at 32OC under 5% CO2 in air either alone (basal) or in the presence of a maximum stimulating dose of hCG (200 mIU; Sigma) or 22R-hydroxycholesterol (22ROHC, 25 mM; Sigma) as previously described (22). At the end of the incubation period the cells were centrifuged at 1500 g for 5 min and the medium was removed and stored cozen at -2O’C for hormone analyses. Hormone Analyses To measure testicular steroid content, weighed testis slices (1 to 200 mg) cut in duplicate from the frozen samples were homogenized in 5 ml absolute ethanol (AnalaR grade, BDH) containing approximately 2000 cpm 14C-testosterone (Amersham) for recovery estimation. The homogenates were transferred into lo-ml glass tubes (Ciba-Corning, Essex, UK) and centrifbged at 1500 g for 25 min. The supematant was removed then dried under air. The dried extracts were redissolved in 500 ml assay buffer (0.1M phosphate buffered saline containing 0.1% (w/v) gelatin, pH 7.0), vortexed and then lefi overnight at room temperature to ensure maximal dissolution in the assay buffer. The extraction mixture was assayed for testosterone and progesterone by radioimmunoassay, and the results are expressed both as steroid concentration per gram of testis and per testis (wet weights). Recovery was approximately 100% and no corrections for procedural losses were made. Plasma testosterone concentration was measured by radioimmunoassay @IA) according to Cook and Beastall (4), and progesterone was measured using an enzymeimmunoassay kit (Ridgeway Science Ltd, Alvington, UK). The assay limits of detection for testosterone and progesterone were 0.1 nmol/L and 0.6 nmol/L, respectively. Testosterone and progesterone concentration in medium from Leydig cell incubations and in extracted samples of testicular tissue were also measured by RIA as described previously (23). Antibody cross reactivity with 22ROHC was cl%. The assay limits of detection at 2 times the standard deviation of the zero

414

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standard were 0.1 pmol/ml for testosterone and 0.24 pmol/ml for progesterone. Intra- and interassay coefficients of variation were < 8 and < 1O%, respectively, for testosterone and < 10% for progesterone. Statistical Analysis Group means + SE are presented. Results were analyzed using a 2 sample t-test or analysis of variance followed by Newman-Keul’s Multiple Range test. Scrotal temperatures were analyzed using repeated measures analysis of variance. RESULTS Animals Infected rams became parasitaemic within 5 to 9 d of trypanosome inoculation and remained so throughout the experimental period. Infected rams developed a low but fluctuating pyrexia (rectal temperature range 39.5 + 0. l°C to 40.2 + 0.2OC) from Day 13 onwards, which was significantly higher (P
8

13

15

20

27

29

34

36

Days after infection / insulation

22

41

43

48

Figure 1 Scrotal skin temperatures (group mean +SEM) recorded from T. conrrolense infected rams ( controls (+J ) and rams with thermal insulation of the scrotum (0 ).

31

37

) , uninfected

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Table 1. Plasma and testicular testosterone concentrations in the uninfected control, w conpolense-infected and scrotal-insulated rams 28 and 58 days a&r trypanosome inoculation or scrotal insulation

Day

Sample

Control (n = 5)

Infected (n = 4)

28

Plasma (mnoW

6.7 + 1.8

3.4 + 0.4

28

Testis @noVg)

551.0+50.4

1084.6 + 52.2b

me-

28

Testis (untoikestis)

45.0 + 7.5

100.6 + 10.3Ce

__

58

Plasma (nmoW

4.6 + 1.6

1.7+0.6

3.3 + 1.4

58

Testis (nmol/g)

395.4 + 40.4

261.2 + 14.2a

58

Testis

37.1 + 5.1

15.1 +2.8 ad

Scrotalinsulated rams =

1134.0 + 134.oc

81.9 + 7.9’

--- = Samples not collected at this time. Values are means Ih SEM. for group number (same on Days 28 and 58) in parentheses. Croup statistical differences compared with control values within a row are denoted as follows: a P
The testes of infected rams tended to be smaller than control rams 28 Days after infection, this difference becoming significant (P
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Steroid Production by Isolated Leydig Cells Leydig cells isolated from control and infected rams produced similar basal levels of testosterone 28 d atIer infection (Figure 2). Both hCG and 22ROHC stimulated Leydig cell testosterone secretion in the infected rams, but the responses were significantly smaller than in control rams (P
4

I

-

Basal

I

-.I

I

I

C

I

hCG

r-I-5

a

22ROHC

Figure 2. Testosterone production by Leydig cells isolated from testes of uninfected controls (n=5, clear histogram) and Tc congfk~infected rams (n=4, shaded histogram) 28 days after infection. Cells were incubated in Ml99 under basal conditions or in the presence of hCG or 22R-hydroxcholesterol(22ROHC) for 3 hours and the medium was collected for hormonal assay. Values are means &-SEM) of triplicate incubations. Values with different superscripts within each treatment (i.e., basal and hCG or 22ROHC-stimulated) are significantly diierent (P
0

200

400

600

3 (D

420

Theriogenology

activity could not be overcome by incubation of the cells with the steroid precursor 22ROHC The reduced capabilities of Leydig cells collected from both infected and scrotal-insulated rams to synthesize testosterone in the presence of hCG and 22ROHC (Figure 3) suggests a common effect caused by the elevated testicular temperature seen in both groups. Raised testicular temperature has been shown to reduce both testicular blood flow and testosterone secretion (6,11) and to reduce the testicular LH receptor content (13), perhaps explaining the reduced ability of hCG to stimulate testosterone synthesis in Leydig cells from scrotal-insulated and infected rams in vitro in this study and from trypanosome-infected rats (27). Temperature also affects several Leydig cell organelles associated with steroidogenesis (9,16), thereby perhaps contributing to the poorer responses to 22ROHC in the scrotal-insulated and infected rams. Several enzymes involved in the A4 and A5 testosterone biosynthetic pathways are known to be particularly heat-sensitive, including 17cr-hydroxylase, 17P-hydroxysteroid dehydrogenase (3), 3S-hydroxysteroid hydrogenase (15) and 17,20-lyase (10). Elevation of testicular temperature, whether by infecting trypanosomes or by scrotal insulation, could have reduced the levels of cytochrome P450 17&hydroxylase which mediates the activities of both 17a-hydroxylase and 17,20-lyase, 2 enzymes that are involved in the conversion of progesterone to 17u-hydroxyprogesterone and androstenedione (18) in the A4 testosterone biosynthetic pathway. This supposition is supported by the presence of a higher testicular progesterone content in the scrotal-insulated rams compared with that of controls. Impairment of the conversion of progesterone to testosterone caused by reduced 17a-hydroxylase activity occurs under other conditions, such as cryptorchidism which induce elevation of testicular temperature (19). Alternatively, the changes in Leydig cell function after infection may have been due to changes in cytokine levels. It is likely that the increased rectal temperature in the infected rams occurred as part of a systemic febrile response to endogenous pyrogenic cytokines (3 1) secreted during the trypanosome infection (32). Recent studies show, for example, that TNF-a (33) and IL-1 (28) can affect Leydig cell steroidogenesis directly, and thus any change in endogenous cytokines after infection could atibct gonadal steroidogenesis. In conclusion, the results of this study show that testosterone production by Leydig cells declines after L congo&e infection. This can be attributed to a number of trypanosome effects such as reductions in pituitary LH secretion and Leydig cell sensitivity to LH, impaired transfer of testosterone to the systemic circulation and reduced steroiddgenic capacity depending upon the stage of infection. Increasing testis temperature by scrotal-insulation mimics some of these effects, suggesting that trypanosome infection may also impair Leydig cell function through pyrexia. REFERENCES 1. Adeyemo 0, Oyedipe A, Adbegana 0. Plasma testosterone in Trvoanosomaconnolenseand w hntcei-infected West African dwarf rams. Anim Reprod Sci 1990- 22: 21-26. 2. Anosa VO, Isoun TT. Further observations of the testicular pathology in w Y&Z infection in sheep and goats. Res Vet Sci 1980; 28: 151-161.

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