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The effect of pinealectomy on rat plasma corticosterone levels under various conditions of light
Lüe Sciences Vol . 10, Part I, pp . 317-324, 1971 . Printed in Great Britain
Pergamon Press
THE EFFECT OF PINEALECTOMY ON RAT PLASMA CORTICOSTERONE LEVELS UNDER VARIOUS CONDITIONS OF LIGHT I. Nír, U. Schmidt, N. Hirschmann and F.G . Sulman Department of Applied Pharmacology, School of Pharmacy, Hebrew University, Jerusalem, Israel (Received 9 December 1970 ; in final form 27 January 1971) TAE results of several studies have suggested a functional relationship between the pineal gland and the pituitary-adrenal axis . reported by Wurtman et al .
1
It has been
that pinealectomy results in hypertrophy of the
adrenal and pituitary glands in young female rats, and that these changes can be reversed by giving pineal extracts .
Kinson et al .
2
too, concluded that the
pineal exerts an inhibitory effect on the adrenal cortex .
Furthermore, sub-
cutaneous administration of melatonin, as well as hypothalamic implants, brought about considerable atrophy of the adrenal glands and a reduction in plasma On corticosterone levels of rats .3 the other hand, pinealectomy was not found to result in significant adrenal enlargement or change in plasma or adrenal corticosteroids six weeks after operation and treatment with bovine pineal extracts caused adrenal hypertrophy with a rise in plasma and adrenal corticosterone . 4 Similarly, melatonin was claimed to increase the production of corticosterone 5, and in vitro pineal extracts were reported to augment corticoidgenesis .
ô
Equally confusing are the reports on the effect of pineal-
ectomy and mels.tonin on ACTH levels in pituitary and plasma .
7
Exposure to continuous light induces changes in the rat hypothalamus and ovary similar io those caused by ablation of the pineal gland. $ Moreover, depressed pineal metabolism 317
9 . 10,
and abnormally small pineal
318
Pineal and Plasma Corticosterone
Vol . 10, No . 8
glands ll , were found in rats kept under experimental conditions of continuous light. These and other findings suggested a link between light and the pineal gland and that the changes induced by light may be operating via the same neuroendocrine pathway as does the availability of pineal hormone.
8, 12
We attempted to elucidate whether and how the adrenal cortex is involved with pineal function, and the possible role light may be playing in this connection, by subjecting pinealectomized and control maturing female rats to constant light, constant darkness, or an alternate light-dark schedule, and then measuring their blood corticosterone levels . Materials and Methods A total of 420 female rats of the Hebrew University's "Sabra" strain were used .
The animals were 21 days old and weighed 35-45 g at the start
of the experiment .
Pinealectomy was performed on 165 randomly selected
rata under sodium pentobarbital anaesthesia by the method of Hoffman and Reiter l3 and sham-operation was carried out on 155.
One hundred intact
rate were included in the study as an additional control. The animals were housed six per cage and placed in one of three experimental lighting environments : constant light, and alternating light-dark .
constant darkness,
Food and water were supplied ad libitum
and the temperature was maintained at 24°C ± 1° in all lighting conditions . Light was provided by overhead fluorescent 40 W "daylight" tubes . Alternating light was automatically controlled with a 12-hr light period beginning at 7 a. m.
Animals maintained ín darkness were placed in a
light-tight dark room and food and water changes were carried out at
Vol . 10, No, ß
Pineal aid Plasma Corticosterone
319
random hours under a dim red lamp placed high in the corner of the room to the back of the cages . After exposure to various lighting environments for 10 or 30 days animals were sacrificed by decapitation between 10 and 11 a. m. and their blood was collected within half a minute in heparinized teat tubes. Plasma was separated by centrifugation, measured into test tubes in 0 . 5 ml aliquots and stored frozen until assay.
Determination of the blood corticosterone was
carried out in duplicate by the fluorometric method of Guillemin et al . 14 Autopsy of the operated animals indicated complete pineal ablations without lesions to the adjacent areas and healing of the sites of surgery. Results Table 1 shows the effect of pinealectomy on plasma corticosterone levels of rats after 10 days' exposure to the various conditions of light.
It can be
seen that plasma corticosterone levels were elevated in the pinealectomized rats kept in alternating light and constant darkness .
However, only in the
light-deprived animals did the increase reach significant proportions .
No
difference whatsoever was found between the pinealectomized and shamoperated control animals subjected to continuous light. TABLE 1 Effect of Pinealectomy on Plasma Corticoeterone Levels (in ytg % ± SE of the Mean) of Female Rats Maintained in Various Conditions of Light for Ten Days . Pinealectomized
Sham-operated
A 15 . 1 ± 3. 3 (24)
A1 14 . 6 t 2. 0 (17)
Continuous darkness
B 16 . 9 ± 4. 2 (24)*
B1
8 . 6 ± 1 . 6 (23)
Alternating light
C
C1
6 . 5 ± 0 . 9 (15)
Light exposure Continuous light
9. 1 ± 2. 5 (15)
p <0 . O1 when compared with group Bl p sp, O1 when compared with groups B1 and C1 . Figures in parentheses represent number of animals .
320
Vol. 10, No. 8
Pineal and Plasma. Corticosterone
It can be further seen from Table 1 that continuous light significantly increased the plasma corticosterone levels of the sham-operated controls compared with those of their counterparts kept in alternating light and constant darkness . Table 2 demonstrates what happens when the animals are exposed to these same lighting environments for 30 days .
The difference between the
pínealectomized and sham-operated control rats, so marked in those kept in constant darkness for 10 days, has completely disappeared and no disparity exists between the pinealectomized and control groups maintained in any form of lighting . Continuous light, however, still acts as a stress factor and the plasma corticoeterone levels of all groups of rata, pinealectomized, sham-operated and intact controls, exposed to constant light are significantly higher than those of the animals kept in alternating light or constant darkness (Table 2) . TABLE 2 Effect of Pinealectomy on Plasma Corticosterone Levels (in pg % ± SE of the Mean) of Female Rata Maintained in Various Conditions of Light for 30 Days . Light exposure Continuous light
Pinealectomized
Sham-operated
Intact
A 24 . 5 ± 2 . 2 (28)* A 1 23 . 9 ± 2 . 0 (27)~ A2 20 . 7 ± 3. 1 (20)*
Continuous darkness B 11 .0 ± 1 . 1 (38)
Bl 11 . 2 ± 1, 1 (39)
B2
Al to rnating light
C 1 10 . 5 ± 1 . 0 (34)
C2 11 . 3 ± 1 . 2 (57)
#
C 10 . 1 ± 1 . 1 (34)
9 . 8 ± 1. 4 (23)
p < 0. O1 when compared with all B and C groups . Figures in parentheses represent number of animals.
Vol. 10, No. 8
Pineal and Plasma Corticosterone
321
Discussion The data presented indicate that the pineal gland in maturing female rate, especially those deprived of light, ezerts an inhibitory effect on adrenocortical function, as evidenced by increased plasma corticoaterone levels in animals from which the pineal glands have been removed . It can also be seen that light affects the pituitary-adrenal azie by stimulating corticoaterone secretion throughout the experiment in both pinealectomized and control animals, which also indicates that this stimulatory process does not neceesarüy depend upon the presence oP the pineal . Moreover, it appears that pinealectomy and continuous light induce their effects through the same neuroendocrine pathway, since each produces the same degree of increase in plasma corticoaterone levela, but the two factors combined do not have an additive effect . These findings concur with those of Wurtman et al . 1 who observed that pinealectomy and light cause adrenal hypertrophy, and of Motta et al .
3
who reported that melatonín causes atrophy of the adrenal glands and reduced plasma corticoaterone levela .
Furthermore, increased plasma corticoaterone
levels following pinealectomy have been found by Kinaon et a1 . 2 However, our results demonstrate increased plasma corticoaterone levela 10 days after operation but not on the 30th day, the time Kinaon et al .
2
found theirs .
An
ezplanation for this disparity could be sezual and age differences, as we used maturing female rata and they were working with adult melee . Indeed, female rata do ezhibit a greater adrenal cortical aecretory response to stress than males.
15
Moreover, Kinaon et al .
2
determined the corticoaterone in blood
samples from rata which were under surgical stress and haemorrhaging, and the levels registered were constantly much higher than normal .
322
Pineal and Plasma Corticosterone
Vol . 10, No. 6
Our experiments were performed on maturing female rats, at the age when pineal metabolism is most active .9 The blood samples were collected during the morning, when physiological levels of blood corticosterone are at their lowestis, on the assumption that any increments which may occur in the concentrations of corticosterone in the plasma would be more evident at that hour . This supposition had substantiation in the findings of Zimmerman and Critchlowl7, that stress-induced increments in ACTH were smaller at the time of the diurnal peak in pituitary-adrenal function than at the trough . 18, The results obtained parallel those of Tronchetti et al . who found changes in adrenal activity and plasma corticosterone values in animals which had been blinded for seven days but no difference between blinded and control animals when the experimental period was extended to 30 days . They also agree with the findings of Houssay and Pazol9 of a significant increase in adrenal weight 10 and 14 days after pinealectomy but not after 21 days or more . It would appear that pinealectomy causes a transitional increase in plasma corticosterone levels of rats and the ability to detect this may depend on such factors as strain, sex, age of animal, environmental conditions under which it has been kept, and the time at which determination is carried out. Judging from our results we are inclined to believe that an antagonism exists between the pineal and adrenals, or adreno-pituitary axis, which may operate only under certain environmental conditions . This postulation is in accord with the well proven antagonism between pineal, hypothalamic-pituitary and gonadal function .
Vol. 10, No . g
Pineal and Plasma Corticosterone
923
Summary The interrelationship between the pineal gland and adrenal cortex and possible role light may be playing in this connection has been studied by subjecting pinealectomized and control maturing female rate to constant light, constant darkness or an alternate light-dark schedule and then measuring their blood corticosterone levels . Elevated plasma corticosterone levels could be seen in pinealectomized animals maintained for 10 days in alternating light and constant darkness . However, only in the light-deprived rats did the increase reach significant proportions.
Thirty days after pinealectomy no disparity existed between
the various groups . These data indicate that the pineal gland exerts an inhibitory effect on adrenocortical function especially in rate kept in darkness . Light was Pound to affect the pituitary-adrenal axis by stimulating corticosterone secretion, but this was not dependent upon presence of the pineal .
Pinealectomy combined with continuous light had no additive effect,
the same degree of increase in plasma corticosterone being produced by each factor alone or the two together . References 1,
R . J. WURTMAN, M. D. ALTSCHULE and U. HOLMGREN, Am . J. Physiol. 197, 108 (1959) .
2.
G. A. KINSON, B. SINGER and L. GRANT, J. Endocr . 10, 447 (1968) .
3.
M. MOTTA, F. FRASCHINI, F. PIVA and L. MARTINI, Memoirs of the Societ for Endocrinolo , No . 17 p. 3 Cambridge University Press 1968) .
4.
R . E . DILL, Anat . Record 139, 222 (1961) .
5.
E . A . GROMOVA (1967) .
s
V
M. KRAUS and J. KRECEK,
J. Endocr . 39, 345
324
Pineal and Plasma Cortícoeterone
Vol. 10, No. ß
B.
P. JOUAN and S. SAMPEREZ, Research on Steroids . Transactions of the First Meeting of the International Study Group for Steroid Hormones Rome, p. 243 (1963) .
7.
J. BARCHAS, R . CONNER, S. LEVINE and J. VERNIKOS-DANELLIS, Experientia 25, 413 (1989) .
8.
R . J. WURTMAN, W. ROTH, M. D. ALTSCHULE and J. WURTMAN, Acta Endocrin . 36, 617 (1961) . a
9.
I. NIR, N. HIRSCHMANN, J. MISHKINSKY and F. G. SULMAN, Life Sciences 8, 279 (1969) .
10 .
I. NIR, N. HIRSCHMANN and F. G. SULMAN, Proc . Soc . Ex~. Biol. & Med. 133, 452 (1970) .
11 .
F. FLSKE, G . K. BRYANT and J. PUTNAM, Endocrinology _86, 489 (1960) .
12 .
V. CRITCHLOW, Advances in Neuroendocrinology p. 377. of IIlinois Press, Urbana (1983) .
13 .
H. A . HOFFMAN and R. J. REITER, Anat . Record 153, 19 (1965) .
14 .
R . GUILLEMIN, G . W. CLAYTON H. S. LIPSGOMB and J. R . SMITH, J. Lab. Clin. Med. 53, 830 (1959) .
15 .
J. I. KITAY, Endocrinology 88, 818 (1961) .
16 .
V, GRITCHLOW, R .A . LIEBELT, M. BAR-SELA, W. MOUNTCASTLE and H. S . LIPSCOMB, Am . J. Phyeiol. 205, 807 (1983) . a
17 .
B. ZIMMERMAN and V. CRITCHLOW, Proc . Soc. Exp. Biol . & Med. 125, 658 (1967) .
18 .
F. TRONCHETTI, V. MARESCOTTI and P. SABA, Excerpts Medics International Congress Series No . 132 . Proceedings of the Second ïnternational Congress on Hormonal Steroids - Milan, p . 983 (1966) .
19 .
A. B. HOUSSAY and J. H. PAZO, Experientia 24,- 813 (1968) .
University
Pergamon Press
THE EFFECT OF PINEALECTOMY ON RAT PLASMA CORTICOSTERONE LEVELS UNDER VARIOUS CONDITIONS OF LIGHT I. Nír, U. Schmidt, N. Hirschmann and F.G . Sulman Department of Applied Pharmacology, School of Pharmacy, Hebrew University, Jerusalem, Israel (Received 9 December 1970 ; in final form 27 January 1971) TAE results of several studies have suggested a functional relationship between the pineal gland and the pituitary-adrenal axis . reported by Wurtman et al .
1
It has been
that pinealectomy results in hypertrophy of the
adrenal and pituitary glands in young female rats, and that these changes can be reversed by giving pineal extracts .
Kinson et al .
2
too, concluded that the
pineal exerts an inhibitory effect on the adrenal cortex .
Furthermore, sub-
cutaneous administration of melatonin, as well as hypothalamic implants, brought about considerable atrophy of the adrenal glands and a reduction in plasma On corticosterone levels of rats .3 the other hand, pinealectomy was not found to result in significant adrenal enlargement or change in plasma or adrenal corticosteroids six weeks after operation and treatment with bovine pineal extracts caused adrenal hypertrophy with a rise in plasma and adrenal corticosterone . 4 Similarly, melatonin was claimed to increase the production of corticosterone 5, and in vitro pineal extracts were reported to augment corticoidgenesis .
ô
Equally confusing are the reports on the effect of pineal-
ectomy and mels.tonin on ACTH levels in pituitary and plasma .
7
Exposure to continuous light induces changes in the rat hypothalamus and ovary similar io those caused by ablation of the pineal gland. $ Moreover, depressed pineal metabolism 317
9 . 10,
and abnormally small pineal
318
Pineal and Plasma Corticosterone
Vol . 10, No . 8
glands ll , were found in rats kept under experimental conditions of continuous light. These and other findings suggested a link between light and the pineal gland and that the changes induced by light may be operating via the same neuroendocrine pathway as does the availability of pineal hormone.
8, 12
We attempted to elucidate whether and how the adrenal cortex is involved with pineal function, and the possible role light may be playing in this connection, by subjecting pinealectomized and control maturing female rats to constant light, constant darkness, or an alternate light-dark schedule, and then measuring their blood corticosterone levels . Materials and Methods A total of 420 female rats of the Hebrew University's "Sabra" strain were used .
The animals were 21 days old and weighed 35-45 g at the start
of the experiment .
Pinealectomy was performed on 165 randomly selected
rata under sodium pentobarbital anaesthesia by the method of Hoffman and Reiter l3 and sham-operation was carried out on 155.
One hundred intact
rate were included in the study as an additional control. The animals were housed six per cage and placed in one of three experimental lighting environments : constant light, and alternating light-dark .
constant darkness,
Food and water were supplied ad libitum
and the temperature was maintained at 24°C ± 1° in all lighting conditions . Light was provided by overhead fluorescent 40 W "daylight" tubes . Alternating light was automatically controlled with a 12-hr light period beginning at 7 a. m.
Animals maintained ín darkness were placed in a
light-tight dark room and food and water changes were carried out at
Vol . 10, No, ß
Pineal aid Plasma Corticosterone
319
random hours under a dim red lamp placed high in the corner of the room to the back of the cages . After exposure to various lighting environments for 10 or 30 days animals were sacrificed by decapitation between 10 and 11 a. m. and their blood was collected within half a minute in heparinized teat tubes. Plasma was separated by centrifugation, measured into test tubes in 0 . 5 ml aliquots and stored frozen until assay.
Determination of the blood corticosterone was
carried out in duplicate by the fluorometric method of Guillemin et al . 14 Autopsy of the operated animals indicated complete pineal ablations without lesions to the adjacent areas and healing of the sites of surgery. Results Table 1 shows the effect of pinealectomy on plasma corticosterone levels of rats after 10 days' exposure to the various conditions of light.
It can be
seen that plasma corticosterone levels were elevated in the pinealectomized rats kept in alternating light and constant darkness .
However, only in the
light-deprived animals did the increase reach significant proportions .
No
difference whatsoever was found between the pinealectomized and shamoperated control animals subjected to continuous light. TABLE 1 Effect of Pinealectomy on Plasma Corticoeterone Levels (in ytg % ± SE of the Mean) of Female Rats Maintained in Various Conditions of Light for Ten Days . Pinealectomized
Sham-operated
A 15 . 1 ± 3. 3 (24)
A1 14 . 6 t 2. 0 (17)
Continuous darkness
B 16 . 9 ± 4. 2 (24)*
B1
8 . 6 ± 1 . 6 (23)
Alternating light
C
C1
6 . 5 ± 0 . 9 (15)
Light exposure Continuous light
9. 1 ± 2. 5 (15)
p <0 . O1 when compared with group Bl p sp, O1 when compared with groups B1 and C1 . Figures in parentheses represent number of animals .
320
Vol. 10, No. 8
Pineal and Plasma. Corticosterone
It can be further seen from Table 1 that continuous light significantly increased the plasma corticosterone levels of the sham-operated controls compared with those of their counterparts kept in alternating light and constant darkness . Table 2 demonstrates what happens when the animals are exposed to these same lighting environments for 30 days .
The difference between the
pínealectomized and sham-operated control rats, so marked in those kept in constant darkness for 10 days, has completely disappeared and no disparity exists between the pinealectomized and control groups maintained in any form of lighting . Continuous light, however, still acts as a stress factor and the plasma corticoeterone levels of all groups of rata, pinealectomized, sham-operated and intact controls, exposed to constant light are significantly higher than those of the animals kept in alternating light or constant darkness (Table 2) . TABLE 2 Effect of Pinealectomy on Plasma Corticosterone Levels (in pg % ± SE of the Mean) of Female Rata Maintained in Various Conditions of Light for 30 Days . Light exposure Continuous light
Pinealectomized
Sham-operated
Intact
A 24 . 5 ± 2 . 2 (28)* A 1 23 . 9 ± 2 . 0 (27)~ A2 20 . 7 ± 3. 1 (20)*
Continuous darkness B 11 .0 ± 1 . 1 (38)
Bl 11 . 2 ± 1, 1 (39)
B2
Al to rnating light
C 1 10 . 5 ± 1 . 0 (34)
C2 11 . 3 ± 1 . 2 (57)
#
C 10 . 1 ± 1 . 1 (34)
9 . 8 ± 1. 4 (23)
p < 0. O1 when compared with all B and C groups . Figures in parentheses represent number of animals.
Vol. 10, No. 8
Pineal and Plasma Corticosterone
321
Discussion The data presented indicate that the pineal gland in maturing female rate, especially those deprived of light, ezerts an inhibitory effect on adrenocortical function, as evidenced by increased plasma corticoaterone levels in animals from which the pineal glands have been removed . It can also be seen that light affects the pituitary-adrenal azie by stimulating corticoaterone secretion throughout the experiment in both pinealectomized and control animals, which also indicates that this stimulatory process does not neceesarüy depend upon the presence oP the pineal . Moreover, it appears that pinealectomy and continuous light induce their effects through the same neuroendocrine pathway, since each produces the same degree of increase in plasma corticoaterone levela, but the two factors combined do not have an additive effect . These findings concur with those of Wurtman et al . 1 who observed that pinealectomy and light cause adrenal hypertrophy, and of Motta et al .
3
who reported that melatonín causes atrophy of the adrenal glands and reduced plasma corticoaterone levela .
Furthermore, increased plasma corticoaterone
levels following pinealectomy have been found by Kinaon et a1 . 2 However, our results demonstrate increased plasma corticoaterone levela 10 days after operation but not on the 30th day, the time Kinaon et al .
2
found theirs .
An
ezplanation for this disparity could be sezual and age differences, as we used maturing female rata and they were working with adult melee . Indeed, female rata do ezhibit a greater adrenal cortical aecretory response to stress than males.
15
Moreover, Kinaon et al .
2
determined the corticoaterone in blood
samples from rata which were under surgical stress and haemorrhaging, and the levels registered were constantly much higher than normal .
322
Pineal and Plasma Corticosterone
Vol . 10, No. 6
Our experiments were performed on maturing female rats, at the age when pineal metabolism is most active .9 The blood samples were collected during the morning, when physiological levels of blood corticosterone are at their lowestis, on the assumption that any increments which may occur in the concentrations of corticosterone in the plasma would be more evident at that hour . This supposition had substantiation in the findings of Zimmerman and Critchlowl7, that stress-induced increments in ACTH were smaller at the time of the diurnal peak in pituitary-adrenal function than at the trough . 18, The results obtained parallel those of Tronchetti et al . who found changes in adrenal activity and plasma corticosterone values in animals which had been blinded for seven days but no difference between blinded and control animals when the experimental period was extended to 30 days . They also agree with the findings of Houssay and Pazol9 of a significant increase in adrenal weight 10 and 14 days after pinealectomy but not after 21 days or more . It would appear that pinealectomy causes a transitional increase in plasma corticosterone levels of rats and the ability to detect this may depend on such factors as strain, sex, age of animal, environmental conditions under which it has been kept, and the time at which determination is carried out. Judging from our results we are inclined to believe that an antagonism exists between the pineal and adrenals, or adreno-pituitary axis, which may operate only under certain environmental conditions . This postulation is in accord with the well proven antagonism between pineal, hypothalamic-pituitary and gonadal function .
Vol. 10, No . g
Pineal and Plasma Corticosterone
923
Summary The interrelationship between the pineal gland and adrenal cortex and possible role light may be playing in this connection has been studied by subjecting pinealectomized and control maturing female rate to constant light, constant darkness or an alternate light-dark schedule and then measuring their blood corticosterone levels . Elevated plasma corticosterone levels could be seen in pinealectomized animals maintained for 10 days in alternating light and constant darkness . However, only in the light-deprived rats did the increase reach significant proportions.
Thirty days after pinealectomy no disparity existed between
the various groups . These data indicate that the pineal gland exerts an inhibitory effect on adrenocortical function especially in rate kept in darkness . Light was Pound to affect the pituitary-adrenal axis by stimulating corticosterone secretion, but this was not dependent upon presence of the pineal .
Pinealectomy combined with continuous light had no additive effect,
the same degree of increase in plasma corticosterone being produced by each factor alone or the two together . References 1,
R . J. WURTMAN, M. D. ALTSCHULE and U. HOLMGREN, Am . J. Physiol. 197, 108 (1959) .
2.
G. A. KINSON, B. SINGER and L. GRANT, J. Endocr . 10, 447 (1968) .
3.
M. MOTTA, F. FRASCHINI, F. PIVA and L. MARTINI, Memoirs of the Societ for Endocrinolo , No . 17 p. 3 Cambridge University Press 1968) .
4.
R . E . DILL, Anat . Record 139, 222 (1961) .
5.
E . A . GROMOVA (1967) .
s
V
M. KRAUS and J. KRECEK,
J. Endocr . 39, 345
324
Pineal and Plasma Cortícoeterone
Vol. 10, No. ß
B.
P. JOUAN and S. SAMPEREZ, Research on Steroids . Transactions of the First Meeting of the International Study Group for Steroid Hormones Rome, p. 243 (1963) .
7.
J. BARCHAS, R . CONNER, S. LEVINE and J. VERNIKOS-DANELLIS, Experientia 25, 413 (1989) .
8.
R . J. WURTMAN, W. ROTH, M. D. ALTSCHULE and J. WURTMAN, Acta Endocrin . 36, 617 (1961) . a
9.
I. NIR, N. HIRSCHMANN, J. MISHKINSKY and F. G. SULMAN, Life Sciences 8, 279 (1969) .
10 .
I. NIR, N. HIRSCHMANN and F. G. SULMAN, Proc . Soc . Ex~. Biol. & Med. 133, 452 (1970) .
11 .
F. FLSKE, G . K. BRYANT and J. PUTNAM, Endocrinology _86, 489 (1960) .
12 .
V. CRITCHLOW, Advances in Neuroendocrinology p. 377. of IIlinois Press, Urbana (1983) .
13 .
H. A . HOFFMAN and R. J. REITER, Anat . Record 153, 19 (1965) .
14 .
R . GUILLEMIN, G . W. CLAYTON H. S. LIPSGOMB and J. R . SMITH, J. Lab. Clin. Med. 53, 830 (1959) .
15 .
J. I. KITAY, Endocrinology 88, 818 (1961) .
16 .
V, GRITCHLOW, R .A . LIEBELT, M. BAR-SELA, W. MOUNTCASTLE and H. S . LIPSCOMB, Am . J. Phyeiol. 205, 807 (1983) . a
17 .
B. ZIMMERMAN and V. CRITCHLOW, Proc . Soc. Exp. Biol . & Med. 125, 658 (1967) .
18 .
F. TRONCHETTI, V. MARESCOTTI and P. SABA, Excerpts Medics International Congress Series No . 132 . Proceedings of the Second ïnternational Congress on Hormonal Steroids - Milan, p . 983 (1966) .
19 .
A. B. HOUSSAY and J. H. PAZO, Experientia 24,- 813 (1968) .
University