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Journal of the Anatomical Society of India

Testosterone Hormone Level in Albino Rats Following Restraint Stress of Long Duration

Author(s): Rai J*, Pandey SN, Srivastava RK.

Vol. 53, No. 1 (2004-01 - 2004-06)

HIMS, Dehradun*, GSVM Medical College, Kanpur


The present study reports the effect of chronic restraint stress on the serum testosterone level. The rats were exposed to restraint stress for 4hrs. a day for 2 months. After that they were sacrificed, blood sample was taken directly from the heart and serum testosterone level was estimated which revealed marked suppression.

Histological study of testis was also done which showed marked suppression of spermatogenesis. Leyding cells showed increase of cytoplasmic vacoulation that indicates the suppressed activity of these cells.

Keywords: restraints, testosterone, leydig cells, immobilization.


It has been proved through count less study that our mental attitude has powerful influence on our physical health. All kinds of mental and physical stressors simultaneously influence the body and the mind and leads to psychosomatic disease. Walter B. Cannon was one of the first research workers to recognize the role of emotional reaction in the development of illness (Cannon WB, 1909).

In the present study we have used rat model to evaluate the effect of immobilization stress on serum testosterone level and have tried to correlate it with the histological appearance of leydig cells. Norman & Smith (1992) showed that restraint is a potent stimulus for activation of hypothalamo-pituitary-adrenocortical axis.

Material and method:

The study was carried out on 40 male albino rats. The animals were divided into two groups of 20 each.

Group A: served as control.

Group B: rats were immobilized by keeping them into transparent plastic jars with holes, for ventilation, for 4 hrs. a day.

All the male rats of both groups were kept separately with females for whole day except for the stress period. The female rats were taken from the animal house of the department. After 2 months the rats were anaesthetized by giving ether. The thoracic cavity was opened and blood sample was taken directly from the heart. The sample was taken in a plain vial. All the samples were collected in the morning in order to minimize the diurnal variation of hormone levels. Testosterone estimation was done by ELISA method by Ranbaxy Limited, Mumbai. The testis was also taken out and was preserved in Bouins' fluid and 10% formal saline. Haematoxylin & Eosin and Masson's trichrome staining was done.


All the rats of control group showed normal fertility where as in stress group none of the rat showed fertility.

Table 1 shows the serum testosterone level of control and stressed group rats, most of the rats of the control group had serum testosterone level between 3.62 ng/ml - 3.96 ng/ml with an average of 3.79 ± 0.05 ng/ml. Serum testosterone level in stress group ranged between 3.02 ng/ml - 3.92 ng/ml, with an average of 3.43 ± 0.01 ng/ml. Thus statistically it was clear that immobilization stress in rats caused significant fall (p<0.001) in serum testosterone level as compared to control group, with 't' value of 4.10.

Histological Appearance:

In control group the seminiferous tubules were bounded together by loose intertubular connective tissue, which contained fibroblasts, collagen fibers, blood vessels and groups of interstitial cells or leydig cells. These cells were large and polyhedral with euchromatic nucleus, containing nucleoli. The cytoplasm was scanty and poorly stained. The capillaries were infiltrated among the clumps of leydig cells (fig. 1).

In stress group there was marked reduction in spermatogenesis. The architecture of the testis was maintained but the germinal epithelium showed disorganization as well as marked degenerative changes. As the semniferous tubules were reduced in diameter, the interstitial spaces were increased. The numbers of fibroblasts as well as of collagen fibers were also increased. The cell membrane of leydig cells were poorly defined, with cytoplasmic vacuolations in many cells. The nuclei were circular to oval in shape and were smaller as compared to control groups. The capillary network was well defined. Fairly good number of fat cells with large clear spaces and small eccentric nucleus were observed among the clumps of leydig cells (fig. 2).


SL. No. Control Group Stress Group
1. 3.96 3.52
2. 3.72 3.24
3. 3.80 3.36
4. 3.62 3.02
5. 3.88 3.72
6. 3.72 3.80
7. 3.76 3.08
8. 3.84 3.02
9. 3.82 3.44
10. 3.96 3.36
11. 3.64 3.64
12. 3.84 3.82
13. 3.90 3.60
14. 3.80 3.52
15. 3.68 3.40
16. 3.72 3.64
17. 3.78 3.72
18. 3.62 3.20
19. 3.82 3.22
20. 3.90 3.18
Mean 3.79 3.43
S.D. 0.10 0.26
Limit 3.79+0.05 3.43+0.11


In this study we have found that immobilization stress has resulted in impairment of both endocrine and spermatogenic functions of the testis. This alteration in function was clearly reflected by impaired fertility.

Plasma testosterone levels have been reported to alter following exposure to stressful situations, but the data appear to be contradictory. Moreover, in most of the studies only endocrinological effect of stress have been reported but in the present study we have tried to correlate the endocrinological effect with the histological findings as was done by Pollard et al (1980).

During intense physical exercise plasma testosterone level have been reported to increase Sutton et al,(1973), Wheeler et al, (1994). Acceleration stress, Obminski et al, (1997) though of very short duration causes significant increase in serum testosterone level. Pollard et al (1980) also reported significant elevation in serum testosterone level following prolonged exposure to psychological stress. He believed that decrease hepatic clearance of testosterone could be responsible for it. Demura et al (1989) studied the endocrinological response of immobilization stress and reported decrease in plasma leutinizing hormone (LH) and plasma testosterone level. Taylor et al (1994) also reported decrease in plasma testosterone level after immobilization stress. Kosti et al (1997) reported that immobilization stress causes significant decrease in serum testosterone level but serum LH level was not affected. In the present study we also found significant fall (p<0.001) in serum testosterone level following immobilization stress.

Various hypotheses have been proposed to explain the fall in serum testosterone level following immobilization stress. Mayfield (1980) explained that neuroendocrine effect of stress is mediated by hypothalamus. Corticotrophin releasing hormone (CRH) neurons present in hypothalamus summate a large variety of neuronal and hormonal signals which arise in various regions of nervous system and eventually this specific hypothalamic neurohormone either stimulate or inhibit the hypophyseal activity in response to stress. Knol (1991) proposed that stressors generally induce depression of hypothalamo-pituitary-testis system, mediated by activated hypothalamo-pituitary-adrenocortical system, resulting in fall in plasma LH and testosterone levels. CRH induces the release of endo genous opioids from hypothalamus, which along with corticosteroids suppresses the secretion of hypothalamic gonadotrophin releasing hormone (GNRH). Suppression in secretion of GNRH causes reduced secretion of LH & FSH from pituitary, which in turn causes decrease in testosterone level and spermatogenesis.

Orr et al (1992) found that restraint stress causes increase in plasma level of glucocorticoids, decrease in testosterone level without any effect on LH level. He suggested that increase in plasma level of glucocorticoids act via glucocorticoid receptors on testicular interstitial cells to suppress the testicular response to gonadotropins. Mc Givern & Reddi (1994) supported the primary role of glucocorticoids in stress-induced inhibition of reproductive function in rats.

We also observed that the size of the leydig cells were small. Reviers et al (1985) also reported reduction in the size of leydig cells in Rams, when exposed to long days. We found that the cell membrane of leydig cells were not well defined, the shape of the nucleus was from circular to oval and the cytoplasmic vacuolations were prominent. Stadnikov et al (1996) reported that emotional stress impairs the endocrine function of testis. He observed that leydig cell were smaller in size, membrane structure was destroyed and cytoplasm showed vacuolation. Pollard et al (1980) also observed increased number of lipid droplets in leydig cells after prolonged exposure to stress. The lipid droplets function as storage depots of precursors used in androgen synthesis. Thus the increased number of lipid droplets indicates that the androgen synthesis in the leydig cells has been suppressed. Thus the stress-induced changes in testicular function, as reflected by significant decrease (p<0.001) in plasma level of testosterone, correlate well with the morphological changes in leydig cells.


Author wishes to acknowledge Dr. S.L. Jethani, Head of Department of Anatomy, HIMS, Dehradun for his inspiring attitude, valuable suggestions & guidance.


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