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Gonadotrophic secretion in male rats subjected to an acute noxious stimulus
Life Sciences Vol . 10, Part I, pp . 333-340, 1971 . Printed in Great Britain
Pergamon Press
GONADOTROPHIC SECRETION IN MALE RATS SUBJECTED TO AN ACUTE NOXIOUS STIMULUSI Raul C . Schiavi and Daniel White Department of Psychiatry, State University of New York, Dawnatate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203
(Received 28 November 1970; in final form 4 February 1971) Abstract This study evaluated the effect of an acute noxious stimulus, electric shock, on the pituitary concentration of gonadotrophirs and on plasma luteinizing hormone (LH) activity in intact and orchidectomized rats . The owrian ascorbic acid depletion method of Parlow was used for the assay of pituitary and plasma LH . The adenohypophyaeal concentration of follicle stimulating hormone (FSH) was determined by means of the augmentation method of Steelman and Pohley . Administration of an electric shock of moderate intensity which was effective in inducing a marked increase in plasma cortiooaterone did not result in significant changes in pituitary LH and FSH in intact and orchidectomized animals . Plasma LH significantly incroased 5 min after the stressful proceduro in orchi dectamized rats and roturned to control levels at 20 min . No significant differences in plasma LH activity were detected in intact rats . Acute stressful procedures have been shown to alter the secretion of several anterior pituitary hormones in rats . Acute strass results in an incn3ase in blood levels of ACTH followed by a reduction in the pituitary concentration of the hormone (1) . Decreases in pituitary concentration of growth hormone (GH), prolactin, melanocyte-stimulating hormone (MSH) and inhibition of thyrotropin (TSB) secretion (2,3,4,5) have also been reported in rata exposed to various noxious stimuli wch as cold, ether, restraint and fornaline injections . Although there is coruideroble evidence that stress influences gonadal function, little information is available on the effect of acute noxious stimulation on pituitary wntent and release of gonadotrophic hormones . The purpose of this study is to evaluate the effect of an acute noxious stimulus, electric shock, on pituitary concentration of gonadotrophins and on plasma LH in intact and gonadectomized male rah . Method Approximately 350 male rah of the Sprague Dawley strain were included in this
333
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study. The animals were housed 3 io 4/cage in a controlled environment of 24° C with 12 hr of light and 12 hr of darkness for at least one month prior to experimentation . The cages were located in a room solely used for this study and nobody was allowed to enter ít for 16 hr before the initiation of the stressing procedure . The rots were fed ad libitum with Purina Lab Chow and tap water . One half of the rats were orchidectamized 8 weeks prior to the day of exposure to stress which took place when the animals were approximately 5 months of age . The stressing procedure consisted of administering to the animals, placed singly in separated compartments, an electric shock with a current of 1 .5 mA delivered to their paws through an electrified grid for a duration of 60 sec . The animals received the electric shock at 10 :30 AM and were decapitated at 5 min, 20 min, 1 hr, 8 hr and 24 hr following stress . There were three control groups of rats sacrificed before the stressing procedure, and at 8 hr and 24 hr following it . The anterior pituitaries were immediately removed, dissected and weighed on o torsion balance to the nearest 0 .1 mg, rapidly frozen in an acetone bath and stored at -20° C . On the day of bioassay for gonadotrophins the pituitary tissue was homogenized with 0 .996 NaCI solution and adequate dilutions were prepared for I .V . administration to the test animals. Blood was collected from the trunk intro heparinized tubes and spun in a refrigerated centrifuge at 5° C for 30 min . The plauma from each group was pooled and kept at 4° C for determination of LH activity which was carried out within a day of blood collection . Aliquots of the pooled plasma samples were frozen for wbsequent determination of wrticosteroids . Gonadotrophin bioassays: The ovarian ascorbic acid depletion (OAAD) method of .Parlow was used for the assay of pituitary LH (6) . The adenohypophyseal concentration of FSH was determined by means of the augmentation method of 5teelman and Pohley (7) . The procedures used in our laboratory for the bioassay of pituitary LH and FSH have been described in detail in a previous publication (8) . Hormonal estimates were obtained by standard statistical methods for parallel line assays after covariance anclysis (9) . A programmed digüal computer was used for these calculations . The gonadotrophic potency was measured whenever possible in several pools of pituitaries from each control and experimental group of animals and the estimates were combined using a weighting technique after testing for homogeneity. The adenohypophyseal concentration of LH and FSH of the stressed animals and the appropriate non stressed controls were always measured in the
same bioassay in order io reduce the effect of variations in assay sensitivity . Differences between groups were considered significant if the level of statistical probability was 596
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995
or less . The effect of electric shock on plasma LH in intact and orchidectomized rats was meawrod by the one-hour OAAD quay in rocipient rats protreated with prognant maro serum (PMS) and human chorionic gonadotrophin (HCG) . One ovary was romoved prior to intravenous administration of saline, 0 .1 Ng LH, 0.4 Ng LH or 2 .5 cc of plasrrra . One hr after the injection the 2nd ovary was romoved and the percentage depletion of ovarian ascorbic acid concentration in the second ovary as compared to the finit was calculated as an index of plasma LH . The significance of differonoes between groups was determined by Duncan's Multiple Range Test (10) . Measuro of plasma carticosterone : Corticosterone was measurod in pools of plasma from intact and orchidectomized rats by means of the fluorometric method of Guillemin et al (11) . Resul ts The effectiveness of the procedure for the induction of a steu response was independently evaluated by assessing blood corticosterone levels in intact and orchidectomized raft . The mean basal levels of free corticosterone wero 12 .2 Ng/100 ml in 5 plauna pools from intact rats and 18 .2/100 ml in 3 pools of orchidectomized animals. In the intact rats the mean corticasterone values wero 27 .0, 39 .2 and 25 .5 mecsurod in 3 pools from animals sacrificed at 5 min, 20 min and 1 hr rospectively after electric shock . At the same time intervals the mean plasma levels in gonadectomized animals wero 30 .0, 49 .5 and 29 .3 . The effect of stress on the OAA depleting activity of plasma of intact and orchidectomized rats is wmmarized in Table 1 . As can be seen, LH standard induced a highly significant depleting effect at 0 .4 and 0 .1 Ng levels . No significant depleting activity could be detected in the plasma of non stressed intact rats and in infect animals sacrificed 20 min and 60 min after electric shock . Five minutes following steu them was an increase on OAA depletion activity which was significantly different from the saline injected animals (p<0 .01) . The difference in the percentage of OAA depletion between non stressed rots and the animals sacrificed 5 min after electric shock was, however, not significant .
The lack of detectable levels of plasma LH in non stressed male rots prevented
the evaluation of a pouible fall in blood LH following stress in intact animals . It was decided theroforo to study the effect of steu on the higher blood LH levels present in
orchidectomized rots . Plasma from orchidectomized animals showed a significant OAA depleting activity 2 months after surgery. Exposure of gonadectomized rots to strew
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resulted in a significant increase in OAA depleting activity 5 min after electric shock .
The depleting activity of rats decapitated 20 min and 1 hr after stress did not differ from the non stressed rots . TABLE 1 Effect of Acute $trou on Plasma LH Activity in Intact and Orchidectomized Rats; Ovarian Aswrbic Acid Depletion (OAAD) Induced by 2 .5 ml of Plasma Substance injected Intact rots
Orchidectomized roh
No . of plasma donors
No . of recipients
% DAAD
P
Saline LH 0 .4 Ng LH 0 .1 Ng Non stressed 5 min post stress 20 min post stress 1 hr post stress
23 22 9 9
25 26 25 31 28 11 12
1 .82 11 .13 7 .11 5 .34 8 .12 4 .68 4 .36
<0 .001 <0 .01 NS <0 .01 NS NS
Saline LH 0 .4 Ng LH 0 .1 F+g Non stressed 5 min post stress 20 min post stress 1 hr post stress
15 16 15 10
22 25 25 20 19 14 9
2 .59 16 .76 8 .60 9 .24 14 .74 11 .36 11 .94
<0 .001 <0 .01 <0 .01 <0 .001+ <0 .01 <0 .01
P probability of significant difference from saline values ; NS not significant ; + differ significant from the non stress value at <0 .05 level . Table 2 shows the pituitary concentration of LH and FSH in intact rots sacrificed 5 min, 20 min, 1 hr, 8 hrs and 24 hrs after stress . There were no significant
differences in pituitary gonadotrophic concentration between the stressed and cantrol
groups of rots at any of the time periods studied . Similarly, measurement of pituitary
gonadotrophins in orchtdectomized rats did not provide evidence of significant changes in LH and FSH following electric shock stress (Table 3) .
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TABLE 2 Adenohypophyseal Gonadotrophic Concentration in Intact Male Rats Exposed to Acute Stress No . of Pools
LH Bioassay+ Mean 9596 Confid . LH Conc . m * Limits
No . of Pools
FSH Bioassay FSH 95% Conc . Confid . m ** Limits
Non strossed rats 5 min post across 20 min post strass 1 hr post stress
4 4 4 4
1 1 1 1
.30 .32 .20 .36
0 .90-2 .00 0 .84-2 .11 0 .76-1 .91 0 .87-2 .18
1 1 1 1
25 .41 25 .15 25 .54 26 .66
18 .41-34 .82 18 .30-34 .58 18 .60tii5 .16 19 .466 .80
8 hr non stressed 8 hr post stras:
2 2
1 .22 1 .59
0 .74-2 .00 0 .99-2 .61
1 1
23 .33 26 .15
16 .79 1 .05 19 .076 .05
24 hr non atrossed 24 hr post stress
2 2
1 .45 1 .63
0.93-2 .31 0.99-2 .79
+ The mean lambda value for al I LH Bioassays is 0.26 ++ The lambda value for the FSH Bioassay is 0 .16 * Exprossed as Ng NIH-LH-S14/mg net weight of anterior pituitary ** Exproued as Ng NIH-FSH-S3/mg net weight of anterior pituitary TABLE 3 Adenohypophyseal Gonadotrophic Concentration in Orchideciamized Rats Exposed ro Acute Stress No . of Pools
LH Bioassay+ Mean 95% LH Conc . Confid . m * Limits
No . of Pools
FSH Bioassoy~ 95% FSH Conc . Confid . m ** Limits
Non stressed rats 5 min post stress 20 min post streu 1 hr post stress
3 3 3 3
9 .59 10 .46 8 .80 9.90
5 .93-16 .40 6 .61-17 .47 5 .50-14 .76 6 .26-16 .31
1 1 1 1
34 .68 33 .12 35 .37 30 .35
28 .25,42 .95 26 .99,41 .04 28 .9143 .85 24 .83~i7 .37
8 hr non stressed 8 hr post strass
1 1
10 .02 8 .22
6 .34-16 .08 5 .20-12 .95
1 1
40 .12 30 .30
32 .71-50 .10 24 .79~i7 .31
24 hr non stressed 24 hr post streu
1 1
11 .67 7.46
7 .45-18 .67 4 .66-11 .83
1 1
41 .58 40 .82
33 .88-52 .04 33 .27-51 .03
+ The mean lambda value for all LH Bioassays is 0.23 ++ The lambda value for the FSH Bioassay is 0 .11 * Exprossed as pg NIH-LH - S14/mg net weight of anterior pituitary ** Exprossed as pg NIH-FSH-S3/mg nef weight of anterior pituüary
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Discussion Several investigators have reported that chronic exposure of male rodents to stressful procedures involving visual, auditory and vibratory stimulation, restraint or crowding result in decreased testicular and seminal vesicles weights and abnormal spermato genesis (12,13,14) . Bardi and Peterson (15) have recently observed a fall in testosterone secretion in testicular venous blood of rats 2 hr after ether anesthesia . In another study (16) a reduction in testicular testosterone lasting for 24 hr has also been observed within 2 min following exposure of rats to ether stress . Although direct evidence is lacking, it has been hypothesized that the reproductive changes observed in response to chronic stress procedures (12,14) or the fall'in testosterone secretion following acute stress (15,16) may be related to an inhibition of pituitary release of gonadotrophins . However, evidence has been gathered primarily on female animals, that under certain conditions gonadotrophins are released rather than inhibited in response to stressful stimuli . It has been observed for instance that low temperature (17) or traumatic stimulation (18) advances the onset of puberty in rats ; audiogenic stimulation significantly increases ovarian and uterine weights in animals (19) and exposure to stressful procedures involving several stimulus modalities result in increased urinary gonadotrophins (20) . Recently the effect of convulsive electric shock on serum concentrations of FSH and LH were studied in humans (21) . Small but significant increases in serum FSH and LH occurred 2 hr after electric shock in some but not all the men studied . An increase in urinary gonadotrophins has also been reported to occur in men following electro-convulsive troatment (22) . Investigations on the effect of various stressors on GTH secretion have usually been made without concurrent evaluation of adrenal activity . In the present study administration of an electric shock to rats which was effective in activating the pituitary adrenal axis as shown by the increased plasma corticosterone did not induce measurable changes in adenohypophyseal LH and FSH in both intact and gonadectomized animals during a 24 hr period following stress . The lack of effect of an acute noxious stimulus on pituitary gonadotrophins is in contrast with the depletion of adenohypophyseal ACTH, GH, MSH and prolactin which has been reported to occur within the first 24 hr after an acute stress . Acute changes in GTH secretion have been observed in male rots in response
to environmental stimulation as shown by the increase in plasma LH levels measured by the OAAD method within 5 min after coitus (23) . In the present study a small but not
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significant increase in plasma OAAD activity was noted in intact rah 5 min after stress . The use of a more sensitive procedure, wch as rodioimmunoassay of plasma LH, may
permit the study of possible changes in intact rats . Orchidectomy appeared to enhance
the responsiveness of the animals ro electric shock since a significant increase in plasma LH activity was detected 5 min after stress . The increase in plasma LH activity suggests that in orchidectamized rah electric shock not only activates the pituitary adrenocortical axis but also induces an acute release of LH . There was no evidence in the present study of an inhibition of pituitary gonadotrophins in response to electric shock . There is reason to believe that the stimulus characteristic of the stressor as well as ih intensity and duration are determining factors in the naturo and degree of gonadotrophin responsiveness to stress . It has been suggested (24) that in the rat, electric shock, a stimulus frequently used in behavioral research and a potent activator of the adrenal cortex, may not be as effective as other stresson in influencing gonadal function . Although it is unlikely, the possibility exists that the resulh of electric shock in orchidectamized animals reflech the direct effect of the electric current on brain structures responsible for the control of LH release . The investigation of the effect of other stresson on the pituitary release of gonadotrophins would be of interest in this regard . Acknowledgmenh This investigation was wpported by Research Grant MH-13408 of the National Institute of Mental Health, U . S . Public Health Service, Bethesda, Maryland . The authors gratefully acknowledge the supply of NIH-LH-S 14 and NIH-FSH-S3 provided by the Endocrinology Study Section of NIH . Referonces 1.
J . VERNIKOS-0ANELLIS, Endocrin ol~ 75, 574 (1963) .
2.
E . E . MULLER, A . ARIMURA, S . SAWANO, T . SAITO and A . V . SCHALLY, Proc . Soc . Biol . Med . 125, 874 (1967) .
3.
C . E . GROSVENOR, S . M . MC CANN and R . NALLAR, Endocrinology 76, 883 (1965) .
4.
A . J . KASTIN, A . ARIMURA, S . VIOSCA, L . BARRETT and A . V . SCHALLY, Neuroendocrinology 2, 200 (1%7) .
5.
P. DUCOMMUN, E . SAKIZ and R . GUILLEMIN, Proc . Soc . Biol . Med . __121, 921 (1966) .
6.
A . F . PARLOW, in Human Pituita Goradotro ins, p . 300 . A . Albert, Ed . Charles C . Thomas, Springfis d, I . 196
340
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Gonadotrophic Secretion
7.
S . L . STEELMAN and F . M . POHLEY, Endocrinol~ 53, 604 (1953) .
8.
R . C. SCHIAVI, Endocrinol~ t~2, 983 (1968) .
9.
C . I . BLISS, in The Statistics of Bioassay, Academic Press, New York (1952) .
10 .
B . D . DUNCAN, Biometrics 11, 1 (1955) .
11 .
R . GUILLEMIN, G . W . CLAYTON, J . I Endocrinology 63, 349 (1958) .
12 .
E . JÄNKÄLÄ and E . K . NAA~íKNEN, Ann . Med . Eimer. Fenn . 33, 231 (1955) .
13 .
R . MILINE and T. CHTCHÉPOVITCH, C . R . l' Assoc. Anat . 40, 173 (1954) .
14 .
J . J . CHRISTIAN, Am . J . Physiol . ~, 292 (1955) .
15 .
C . W. BARDIN and R . E . PETERSON, Endocrinology 8~, 38 (1967) .
16 .
B . L . FARISS, T. J . HURLEY, S . HANE and P . H . FORSHAM, Endocrinalogy 84, 940 (1969) .
17 .
A . M . MANDL and S. ZUCKERMAN, J . of Endocrinology 8, 357 (1952) .
18 .
A . ÂRVAY and T. NAGY, Acro Medica Scient . Hung . 11, 4 (1958) .
19 .
B . ZONDEK and I . TAMARI, Acro Endoainol . 90, 227 (1964) .
20 .
K . SOIVA, M. GRÓNROOS, U . K . RINNE and E . 1~(ÂA~T~CNEN, Acta Obs tetric . Gynecolog. Scandinav. 38, 5 (1959) .
21 .
R . J . RYAN, D . W . SWANSON, C. FAIMAN, W . E . MAYBERRY and A . J . SPADONI, J . Clin . End . 3_Q, 51 (1970) .
22 .
K . ITO, Y. HOSHIMURA, I . YAMASHITA, T . MOROJI and M. ENDO, Folio. Psychiat . Neural . Jap. 18, 287 (1A64) .
23 .
S . TALEISNIK, L. CALIGARIS and J . J . ASTRADA, Endocrinolo~ 79, 49 (1966) .
24 .
G . GIULIANI, in Ph iolo and Pbtholo of Ada rotíon Mechanisms . Eors Bajusz, Ed ., Pergamon Presa, O rd 1968 .
11TH and H . S. LIPSCOMB,
Y
Pergamon Press
GONADOTROPHIC SECRETION IN MALE RATS SUBJECTED TO AN ACUTE NOXIOUS STIMULUSI Raul C . Schiavi and Daniel White Department of Psychiatry, State University of New York, Dawnatate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203
(Received 28 November 1970; in final form 4 February 1971) Abstract This study evaluated the effect of an acute noxious stimulus, electric shock, on the pituitary concentration of gonadotrophirs and on plasma luteinizing hormone (LH) activity in intact and orchidectomized rats . The owrian ascorbic acid depletion method of Parlow was used for the assay of pituitary and plasma LH . The adenohypophyaeal concentration of follicle stimulating hormone (FSH) was determined by means of the augmentation method of Steelman and Pohley . Administration of an electric shock of moderate intensity which was effective in inducing a marked increase in plasma cortiooaterone did not result in significant changes in pituitary LH and FSH in intact and orchidectomized animals . Plasma LH significantly incroased 5 min after the stressful proceduro in orchi dectamized rats and roturned to control levels at 20 min . No significant differences in plasma LH activity were detected in intact rats . Acute stressful procedures have been shown to alter the secretion of several anterior pituitary hormones in rats . Acute strass results in an incn3ase in blood levels of ACTH followed by a reduction in the pituitary concentration of the hormone (1) . Decreases in pituitary concentration of growth hormone (GH), prolactin, melanocyte-stimulating hormone (MSH) and inhibition of thyrotropin (TSB) secretion (2,3,4,5) have also been reported in rata exposed to various noxious stimuli wch as cold, ether, restraint and fornaline injections . Although there is coruideroble evidence that stress influences gonadal function, little information is available on the effect of acute noxious stimulation on pituitary wntent and release of gonadotrophic hormones . The purpose of this study is to evaluate the effect of an acute noxious stimulus, electric shock, on pituitary concentration of gonadotrophins and on plasma LH in intact and gonadectomized male rah . Method Approximately 350 male rah of the Sprague Dawley strain were included in this
333
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study. The animals were housed 3 io 4/cage in a controlled environment of 24° C with 12 hr of light and 12 hr of darkness for at least one month prior to experimentation . The cages were located in a room solely used for this study and nobody was allowed to enter ít for 16 hr before the initiation of the stressing procedure . The rots were fed ad libitum with Purina Lab Chow and tap water . One half of the rats were orchidectamized 8 weeks prior to the day of exposure to stress which took place when the animals were approximately 5 months of age . The stressing procedure consisted of administering to the animals, placed singly in separated compartments, an electric shock with a current of 1 .5 mA delivered to their paws through an electrified grid for a duration of 60 sec . The animals received the electric shock at 10 :30 AM and were decapitated at 5 min, 20 min, 1 hr, 8 hr and 24 hr following stress . There were three control groups of rats sacrificed before the stressing procedure, and at 8 hr and 24 hr following it . The anterior pituitaries were immediately removed, dissected and weighed on o torsion balance to the nearest 0 .1 mg, rapidly frozen in an acetone bath and stored at -20° C . On the day of bioassay for gonadotrophins the pituitary tissue was homogenized with 0 .996 NaCI solution and adequate dilutions were prepared for I .V . administration to the test animals. Blood was collected from the trunk intro heparinized tubes and spun in a refrigerated centrifuge at 5° C for 30 min . The plauma from each group was pooled and kept at 4° C for determination of LH activity which was carried out within a day of blood collection . Aliquots of the pooled plasma samples were frozen for wbsequent determination of wrticosteroids . Gonadotrophin bioassays: The ovarian ascorbic acid depletion (OAAD) method of .Parlow was used for the assay of pituitary LH (6) . The adenohypophyseal concentration of FSH was determined by means of the augmentation method of 5teelman and Pohley (7) . The procedures used in our laboratory for the bioassay of pituitary LH and FSH have been described in detail in a previous publication (8) . Hormonal estimates were obtained by standard statistical methods for parallel line assays after covariance anclysis (9) . A programmed digüal computer was used for these calculations . The gonadotrophic potency was measured whenever possible in several pools of pituitaries from each control and experimental group of animals and the estimates were combined using a weighting technique after testing for homogeneity. The adenohypophyseal concentration of LH and FSH of the stressed animals and the appropriate non stressed controls were always measured in the
same bioassay in order io reduce the effect of variations in assay sensitivity . Differences between groups were considered significant if the level of statistical probability was 596
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995
or less . The effect of electric shock on plasma LH in intact and orchidectomized rats was meawrod by the one-hour OAAD quay in rocipient rats protreated with prognant maro serum (PMS) and human chorionic gonadotrophin (HCG) . One ovary was romoved prior to intravenous administration of saline, 0 .1 Ng LH, 0.4 Ng LH or 2 .5 cc of plasrrra . One hr after the injection the 2nd ovary was romoved and the percentage depletion of ovarian ascorbic acid concentration in the second ovary as compared to the finit was calculated as an index of plasma LH . The significance of differonoes between groups was determined by Duncan's Multiple Range Test (10) . Measuro of plasma carticosterone : Corticosterone was measurod in pools of plasma from intact and orchidectomized rats by means of the fluorometric method of Guillemin et al (11) . Resul ts The effectiveness of the procedure for the induction of a steu response was independently evaluated by assessing blood corticosterone levels in intact and orchidectomized raft . The mean basal levels of free corticosterone wero 12 .2 Ng/100 ml in 5 plauna pools from intact rats and 18 .2/100 ml in 3 pools of orchidectomized animals. In the intact rats the mean corticasterone values wero 27 .0, 39 .2 and 25 .5 mecsurod in 3 pools from animals sacrificed at 5 min, 20 min and 1 hr rospectively after electric shock . At the same time intervals the mean plasma levels in gonadectomized animals wero 30 .0, 49 .5 and 29 .3 . The effect of stress on the OAA depleting activity of plasma of intact and orchidectomized rats is wmmarized in Table 1 . As can be seen, LH standard induced a highly significant depleting effect at 0 .4 and 0 .1 Ng levels . No significant depleting activity could be detected in the plasma of non stressed intact rats and in infect animals sacrificed 20 min and 60 min after electric shock . Five minutes following steu them was an increase on OAA depletion activity which was significantly different from the saline injected animals (p<0 .01) . The difference in the percentage of OAA depletion between non stressed rots and the animals sacrificed 5 min after electric shock was, however, not significant .
The lack of detectable levels of plasma LH in non stressed male rots prevented
the evaluation of a pouible fall in blood LH following stress in intact animals . It was decided theroforo to study the effect of steu on the higher blood LH levels present in
orchidectomized rots . Plasma from orchidectomized animals showed a significant OAA depleting activity 2 months after surgery. Exposure of gonadectomized rots to strew
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resulted in a significant increase in OAA depleting activity 5 min after electric shock .
The depleting activity of rats decapitated 20 min and 1 hr after stress did not differ from the non stressed rots . TABLE 1 Effect of Acute $trou on Plasma LH Activity in Intact and Orchidectomized Rats; Ovarian Aswrbic Acid Depletion (OAAD) Induced by 2 .5 ml of Plasma Substance injected Intact rots
Orchidectomized roh
No . of plasma donors
No . of recipients
% DAAD
P
Saline LH 0 .4 Ng LH 0 .1 Ng Non stressed 5 min post stress 20 min post stress 1 hr post stress
23 22 9 9
25 26 25 31 28 11 12
1 .82 11 .13 7 .11 5 .34 8 .12 4 .68 4 .36
<0 .001 <0 .01 NS <0 .01 NS NS
Saline LH 0 .4 Ng LH 0 .1 F+g Non stressed 5 min post stress 20 min post stress 1 hr post stress
15 16 15 10
22 25 25 20 19 14 9
2 .59 16 .76 8 .60 9 .24 14 .74 11 .36 11 .94
<0 .001 <0 .01 <0 .01 <0 .001+ <0 .01 <0 .01
P probability of significant difference from saline values ; NS not significant ; + differ significant from the non stress value at <0 .05 level . Table 2 shows the pituitary concentration of LH and FSH in intact rots sacrificed 5 min, 20 min, 1 hr, 8 hrs and 24 hrs after stress . There were no significant
differences in pituitary gonadotrophic concentration between the stressed and cantrol
groups of rots at any of the time periods studied . Similarly, measurement of pituitary
gonadotrophins in orchtdectomized rats did not provide evidence of significant changes in LH and FSH following electric shock stress (Table 3) .
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TABLE 2 Adenohypophyseal Gonadotrophic Concentration in Intact Male Rats Exposed to Acute Stress No . of Pools
LH Bioassay+ Mean 9596 Confid . LH Conc . m * Limits
No . of Pools
FSH Bioassay FSH 95% Conc . Confid . m ** Limits
Non strossed rats 5 min post across 20 min post strass 1 hr post stress
4 4 4 4
1 1 1 1
.30 .32 .20 .36
0 .90-2 .00 0 .84-2 .11 0 .76-1 .91 0 .87-2 .18
1 1 1 1
25 .41 25 .15 25 .54 26 .66
18 .41-34 .82 18 .30-34 .58 18 .60tii5 .16 19 .466 .80
8 hr non stressed 8 hr post stras:
2 2
1 .22 1 .59
0 .74-2 .00 0 .99-2 .61
1 1
23 .33 26 .15
16 .79 1 .05 19 .076 .05
24 hr non atrossed 24 hr post stress
2 2
1 .45 1 .63
0.93-2 .31 0.99-2 .79
+ The mean lambda value for al I LH Bioassays is 0.26 ++ The lambda value for the FSH Bioassay is 0 .16 * Exprossed as Ng NIH-LH-S14/mg net weight of anterior pituitary ** Exproued as Ng NIH-FSH-S3/mg net weight of anterior pituitary TABLE 3 Adenohypophyseal Gonadotrophic Concentration in Orchideciamized Rats Exposed ro Acute Stress No . of Pools
LH Bioassay+ Mean 95% LH Conc . Confid . m * Limits
No . of Pools
FSH Bioassoy~ 95% FSH Conc . Confid . m ** Limits
Non stressed rats 5 min post stress 20 min post streu 1 hr post stress
3 3 3 3
9 .59 10 .46 8 .80 9.90
5 .93-16 .40 6 .61-17 .47 5 .50-14 .76 6 .26-16 .31
1 1 1 1
34 .68 33 .12 35 .37 30 .35
28 .25,42 .95 26 .99,41 .04 28 .9143 .85 24 .83~i7 .37
8 hr non stressed 8 hr post strass
1 1
10 .02 8 .22
6 .34-16 .08 5 .20-12 .95
1 1
40 .12 30 .30
32 .71-50 .10 24 .79~i7 .31
24 hr non stressed 24 hr post streu
1 1
11 .67 7.46
7 .45-18 .67 4 .66-11 .83
1 1
41 .58 40 .82
33 .88-52 .04 33 .27-51 .03
+ The mean lambda value for all LH Bioassays is 0.23 ++ The lambda value for the FSH Bioassay is 0 .11 * Exprossed as pg NIH-LH - S14/mg net weight of anterior pituitary ** Exprossed as pg NIH-FSH-S3/mg nef weight of anterior pituüary
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Discussion Several investigators have reported that chronic exposure of male rodents to stressful procedures involving visual, auditory and vibratory stimulation, restraint or crowding result in decreased testicular and seminal vesicles weights and abnormal spermato genesis (12,13,14) . Bardi and Peterson (15) have recently observed a fall in testosterone secretion in testicular venous blood of rats 2 hr after ether anesthesia . In another study (16) a reduction in testicular testosterone lasting for 24 hr has also been observed within 2 min following exposure of rats to ether stress . Although direct evidence is lacking, it has been hypothesized that the reproductive changes observed in response to chronic stress procedures (12,14) or the fall'in testosterone secretion following acute stress (15,16) may be related to an inhibition of pituitary release of gonadotrophins . However, evidence has been gathered primarily on female animals, that under certain conditions gonadotrophins are released rather than inhibited in response to stressful stimuli . It has been observed for instance that low temperature (17) or traumatic stimulation (18) advances the onset of puberty in rats ; audiogenic stimulation significantly increases ovarian and uterine weights in animals (19) and exposure to stressful procedures involving several stimulus modalities result in increased urinary gonadotrophins (20) . Recently the effect of convulsive electric shock on serum concentrations of FSH and LH were studied in humans (21) . Small but significant increases in serum FSH and LH occurred 2 hr after electric shock in some but not all the men studied . An increase in urinary gonadotrophins has also been reported to occur in men following electro-convulsive troatment (22) . Investigations on the effect of various stressors on GTH secretion have usually been made without concurrent evaluation of adrenal activity . In the present study administration of an electric shock to rats which was effective in activating the pituitary adrenal axis as shown by the increased plasma corticosterone did not induce measurable changes in adenohypophyseal LH and FSH in both intact and gonadectomized animals during a 24 hr period following stress . The lack of effect of an acute noxious stimulus on pituitary gonadotrophins is in contrast with the depletion of adenohypophyseal ACTH, GH, MSH and prolactin which has been reported to occur within the first 24 hr after an acute stress . Acute changes in GTH secretion have been observed in male rots in response
to environmental stimulation as shown by the increase in plasma LH levels measured by the OAAD method within 5 min after coitus (23) . In the present study a small but not
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significant increase in plasma OAAD activity was noted in intact rah 5 min after stress . The use of a more sensitive procedure, wch as rodioimmunoassay of plasma LH, may
permit the study of possible changes in intact rats . Orchidectomy appeared to enhance
the responsiveness of the animals ro electric shock since a significant increase in plasma LH activity was detected 5 min after stress . The increase in plasma LH activity suggests that in orchidectamized rah electric shock not only activates the pituitary adrenocortical axis but also induces an acute release of LH . There was no evidence in the present study of an inhibition of pituitary gonadotrophins in response to electric shock . There is reason to believe that the stimulus characteristic of the stressor as well as ih intensity and duration are determining factors in the naturo and degree of gonadotrophin responsiveness to stress . It has been suggested (24) that in the rat, electric shock, a stimulus frequently used in behavioral research and a potent activator of the adrenal cortex, may not be as effective as other stresson in influencing gonadal function . Although it is unlikely, the possibility exists that the resulh of electric shock in orchidectamized animals reflech the direct effect of the electric current on brain structures responsible for the control of LH release . The investigation of the effect of other stresson on the pituitary release of gonadotrophins would be of interest in this regard . Acknowledgmenh This investigation was wpported by Research Grant MH-13408 of the National Institute of Mental Health, U . S . Public Health Service, Bethesda, Maryland . The authors gratefully acknowledge the supply of NIH-LH-S 14 and NIH-FSH-S3 provided by the Endocrinology Study Section of NIH . Referonces 1.
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11TH and H . S. LIPSCOMB,
Y