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Heat Stress Effects During Pregnancy. II. Pituitary Gonadotropins in Intact, Adrenalectomized, and Ovariectomized Rats*
Vol. 22. No.8, August 1971 Printed in U.S.A.
FERTll.ITY AND STERll.ITY
Copyright' 1971 by The Williams & Wilkins Co.
HEAT STRESS EFFECTS DURING PREGNANCY. II. PITUITARY GONADOTROPINS IN INTACT, ADRENALECTOMIZED, AND OVARIECTOMIZED RATS* PAUL A. HENSLEIGH, M.D., PH.D.,t
AND
DONALD C. JOHNSON, PH.D.
Departments of Obstetrics and Gynecology, and Physiology, University of Kansas Medical Center, Kansas City, Kansas
The detrimental effects of stress on fetal development may be directly related to increased production of adrenal corticoids, since adrenalectomy prior to certain experimental stress exposure blocks the fetal effects. 1 Other evidence suggests that a concomitant decrease in ovarian function may also play a role. Even before the gonadotropin requirements of pregnancy in the rat were known, Selye 2 had noted that stress in nonpregnant animals resulted in regression of the gonads. This finding led to a concept that under stress conditions a shift in anterior pituitary hormone production occurs, such that a marked increase in adrenocorticotropic hormone (ACTH) production is necessarily accomplished at the expense of other trophic hormones. Since ovarian function in the first half of rat pregnancy is known to be dependent on pituitary gonadotropin stimulation, one might expect at least some of the detrimental effects of stress during this period to result from diminished pituitary gonadotropin production. A necessary relationship -between pituitary production of ACTH and gonadotropins has not been established by more recent investigations. Moore 3 had argued that drugs such as dibenamine, dibenzyline, and pathilon block ovulation, not because of their specific pharmacologic effects, but rather because they act as nonspecific stres* Supported by grants from the National Science Foundation and the National Institutes of Health. t Postdoctoral fellow, United States Public Health Training Grant TL-HD-25.
sor agents which cause increased ACTH release and a concomitant limited luteinizing hormone (LH) release. Likewise, the plasma luteinizing hormone (LH) concentration in ovariectomized rats was found to be sharply decreased in rats which were stressed by formalin injections. 4 Edgren and Peterson 5 found that the compensatory ovarian hypertrophy, which normally follows unilateral ovariectomy, could be prevented by removal of the adrenals. The expected compensatory hypertrophy occurred if the rats were given replacement corticoids. However, the lack of response was attributed to disruption of cellular metabolism at the ovarian level in the absence of corticoids, rather than alteration of pituitary function. Other studies have shown elevations of plasma LH under experimental conditions causing increased ACTH production. Ramirez, Moore, and McCann 6 showed that either adrenalectomy (which presumably increased ACTH production) or the administration of large doses of cortisol, to block ACTH production resulted in slightly increased plasma LH. Eleftheriou and Church 7 found that mice which were stressed by exposure to aggression and defeat showed increased LH production. The effects of stress on follicle-stimulating hormone (FSH) and prolactin have not been well documented. Charters, Odell, and Thompson 8 found that following surgical stress, women showed a slight and transient decrease in plasma FSH and LH. Brown, Thorburn, and Crooks 9 found FSH
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HEAT STRESS EFFECTS. II
to be elevated in menopausal women during the first 2 days of treatment with large doses of ACTH. Stressed rats were shown by Grosvenor 1 0 to release increased amounts of prolactin as the result of decreased hypothalamic prolactin inhibitor. The purpose of the present study was to determine the changes which occur in serum FSH and LH, when pregnant rats were subjected to hyperthermic stress in the period immediately following implantation. Serum corticosterone was measured as an indicator of ACTH secretion under the experimental conditions in intact or ovariectomized rats, and this also confirmed the completeness of adrenal removal in adrenalectomized rats. The effects of heat stress on fetal growth will be presented in a separate report. 1 MATERIAL AND METHODS
Pregnant rats of the Holtzman strain were obtained on, or before, Day 5 of pregnancy (vaginal sperm = Day 1). Animals were maintained in air-conditioned quarters with 14 hr. of light/day (6 A.M. and 8 P.M.) and had free access to Purina laboratory chow and tap water. Serum FSH and LH levels for normal pregnant rats were determined by sacrificing groups of 5-9 rats on Days 5-12 and on Day 20 of gestation. Groups of animals to be stressed by hyperthermia were placed in an incubator (38.1 ± 0.3° C ambient temperature with 58.5 ± 2.8% relative humidity) for 4 hr. daily. Under these conditions the body (rectal) temperature was elevated from control values of 37.8 ± 0.1 ° C to 39.7 ± 0.1 ° C at the end of the 4 hr. Rats were stressed daily from Days 7 through Day 11 of gestation. In stress experiments the groups of rats were autopsied on either Day 12 or Day 20, by first giving ether anesthesia to facilitate collection of blood from the abdominal aorta for hormone assays. Nembutal anes-
529
thesia was used on rats whose corticosterone levels were to be determined. Blood was allowed to clot and the serum stored in aliquots at - 20° C until the time of assay. After collection of blood and recording maternal body weight the rats were decapitated and the pituitaries removed, weighed to the nearest milligram on a torsion balance, pooled, and frozen in saline at - 20° C. Ovarian and adrenal weights were then recorded. Corticosterone Assays. Corticosterone assays were done on 0.5-ml. aliquots of serum by a modification of the microfluorometric method of Guillemin and co-workers. 11 The modification utilized was that suggested by Givner and Rochefort 12 of substituting ethanol-sulfuric acid reagent for 30N sulfuric acid, which provides more stable fluorescence of the corticosterone solution. The Farrand Model A fluorometer was used with Corning filters 33895113 as primary, and Kodak wratten gelatin 16-74 as secondary filters. With each group of unknowns a reagent blank and a three point standard curve was run. The fluorescence of the unknowns was then converted by a graphic plot of the standards and expressed as micrograms of corticosterone per 100 ml. of serum. Serum Gonadotropin Assays. Serum samples were analyzed for FSH and LH utilizing a liquid phase double antibody radioimmunoassay technic. Reagents for these assays were supplied by the National Institute of Arthritis and Metabolic Diseases (NIAMD), National Institutes of Health (NIH), Rat Pituitary (RP) Hormone Distribution Program. The methodology employed for iodination of hormones was a modification of that suggested by NIAMD. The modification consisted of substituting 1.0 M sodium phosphate buffer (pH 7.6) for the iodination and shortening the iodination reaction to 50 sec. Carrier free P 31 suitable for iodination of proteins with specific activity
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HENSLEIGH AND JOHNSON
ranging from 400-700 mc./ml. was obtained from Cambridge Nuclear Corporation, Billerica, Mass. The assay procedure used was essentially that described in the NIAMD literature, but incorporated the simplifications in counting activity and calculation of hormone content described by Odell, Rayford, and Ross. 13 The second antibody (sheep antirabbit 'Y globulin) was obtained from Antibodies Incorporated, Davis, Calif. A Nuclear-Chicago automatic 'Y well counter was used for all counting procedures. Serum concentrations of hormone in nanograms/milliliter were calculated on the basis of standards supplied by NIAMD (Rat FSH- RP-I = 2.1 X NIH-FSH-SI by augmentation assay and Rat LH-RP-I = 0.03 X NIH-LH-SI by ovarian ascorbic acid depletion assay (OAAD assay). Bioassay of Rat Pituitary FSH Content. A simplified augmented ovarian weight assay for FSH was used. 14 The standard was ovine FSH-S7 supplied by the Endocrinology Study Section of the NIH with a potency of 1.15 X NIH-FSH-S1. Three doses of standard and 1 or 2 doses of pituitary extract were used in each assay. Routine statistical procedures were used 15 in programming the IBM 1130 computer to convert the results into micrograms of NIHFSH-S7/pituitary. RESULTS
The levels of FSH and LH on Day 5-12 and on Day 20 of gestation are shown in Fig. 1. Both hormones were elevated during the first 11 days of gestation when compared to Day 20 values. There was an elevation of serum FSH on Day 8, which is significant (p < 0.05) when compared to Day 7 or Day 9. Serum LH also appeared to be elevated on Day 8, but it is not significantly different from LH levels on the preceeding or following day. A second (statistically insignificant) increase in serum FSH was observed on Day 11, followed by a large drop on Day 12.
90
i
70
~
40
10
10
DAYS
OF
12
i
20
GESTATION
FIG. 1. Levels of FSH (open bars) and LH (solid bars) in normal pregnant rat. Reference preparations were NIH-rat FSH RP-l and rat LH 1-1. Vertical lines indicate standard errors, with 5-9 animals for each group.
Stress Experiments with Intact Animals. Data from these experiments are shown in Tables 1 and 2. Stress had no effect on body weight, but adrenal weight on Day 12 (Table 1), as well as on Day 20 (Table 2), was increased in the stressed animals. Analysis of serum corticosterone indicated, that although quite variable, the enlarged adrenals produced more of this steroid than those on controls (Table 1). The difference in corticosterone level is particularly evident on Day 20 (Table 2) despite the fact that this is 9 days after their last heat exposure. Ovarian weight, pituitary FSH content and serum LH concentration showed no differences between stressed and control groups. However, serum FSH concentration in stressed rats (IH) was elevated on Day 12 (p < 0.02). By Day 20 both groups hEld serum FSH concentrations which were below the sensitivity of the assay. Stress Experiments with Adrenalectomized Animals. These rats had their adrenals removed on Day 6 immediately after receiving their first daily dose of replacement steroids. Data from groups of rats on two dosages of replacement corticoids and killed on Day 12 are given in Table 1. The lower dose, which has been reported 16
August 1971
531
HEAT STRESS EFFECTS. II
TABLE l. Effect of Heat Stress on Pregnancy: Autopsy on Day 12 Group*
Group No.
~o.
of No. of litters fetuses
Adrenals+
Body weightt
41 26 29 20 15 17 21 16 15 15 9 11 11
10 11 12 13 14 15 16
11 12
441 280 277 205 166 187 237 166 179 151 96 118 108 54 102 136
261 260 259 249 267 245 268 260 263 252 265 239 274 268 263 268
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4 6 4' 4' 411 511 4 4 3 2 4 311 4 3 6 4
Ovaries+
m{f.
141./100 mi.
mI!.
W1./{liand
15.2 ± 0.7 20.0 ± 4.5§
69.6 ± 1.5 69.0 ± 1.4 66.9 ± 2.:3 67.6 ::1: 1.7 65.6 ± 1.7 70.0 ± 1.9
98.5 ± 21 92.9 ± 13 166 ± 38 196 ± 35
63.8 69.0 62.2 67.5 65.6 67.0 72.0 69.8 67.4 75.2
± ± ± ± ± ± ± ±
1.4 2.4' 1.4 2.3' 1.6 2.2 1.9 1.8 ± 2.7 ± 3.6'
LHtj:
FSHtt
60.5 ± 0.9 65.2 ± 0.9
{1m.
Intact I plus Heat A 0.5 AHO.5 Al.O AHI.O 01 :2 OH 1:2 01:4 OH 1:4 01:6 OH 1:6 02:4 5H 2:4 52:8 5H 2:8
Corticosterone+
18.2 ± 4.4§ 20.0 ± 4.5§
ng./ml.
n{l.lml.
:34.8 69.9 53.5 68.5 45.4 69.4 518 478 388 491 638 456 321 254 254 242
412 ± 38 508 ± 58
18.8 26.6 24.3 31.3
'" ± ± ± ± ± ± ± ± ±
4.8 12§ 3.9 5.2§ 4.3 1011 31 44 39 29§
28.6 ± 25.8 ± 28.9 ± 30.0 ± :30.6 31.8 66.6 ± :38.4 37.8 ± 38.6± 32.7 35.6 28.0 24.8 25.4 24.2
4 4.7 3.4 4.0
23 1.6 3.3
* X, adrenalectomy; 0, ovariectomy; numbers in group indicate dose ofrepiacement steroids (explained in text). t Standard error, ±.
4: FSH in gland expressed in terms of NIH-FSH-S7, serum level is nanogram/milliliter of serum in terms of § p < 0.02 compared with unstressed group . • p < 0.05 compared with unstressed group. II p < 0.01 compared to unstressed group.
~IH-FSH-RP-I;
LH is ::-.JIH-LH-Rl.
TABLE 2. Effect of Heat Stress on Pregnancy: Autopsy on Day 20 Group*
Intact I plus Heat A 0.5 AH 0.5 61:4 OH 1:4
Group
2 3 4 5 6
No. of litters
9 7 8 8 9 7
No. of fetuses
68 47 85 81 90 55
Maternal body wt.t
306 304 337 321 337 293
± ± ± ± ± ±
8 10 8 9 10 9§
Adrenalst
Corticosteronet
mg.
WI./IOO mi.
60.2 ± 2.4 68.4 ± 1.9§
14.6 ± 0.8 27.0 ± 2.8§
67.6 ± 2.1 62.9 ± 1.7
13.7 ± 1.2 27.3 ± 5.5§
Ovariest
FSHj:
LHj:
mf,f.
n{l. 1m !.
np.lml.
101.6 106.6 113.2 110.0
± ± ± ±
5.6 9.3 7.6 9
25 25 25 25 278 252
25.4 38.2 28.8 15.6 32.8 73.8
* A, adrenalectomy; 0, ovariectomy; group numbers indicate dose of replacement steroid (explained in text). t Standard error, ±. :j: FSH and LH expressed as nanogram of NIH-Rat FSH-RP-1 and Rat LH-l. § p < 0.01 compared with unstressed group.
to be optimal for fetal growth, consisted of a daily (evening) injection of 0.5 mg of corticosterone and 0.25 mg of deoxycorticosterone acetate. These steroids were given as a single injection of 0.2 ml. in a saline suspension using 20 drops of "Tween 80" /100 ml of saline. The higher replacement dose consisted of 1.0 mg. of corticosterone and 0.5 mg. of deoxycorticosterone prepared and injected in the same manner. These two groups are designated as adrenalectomy (A) 0.5 and A 1.0 in
Table 1: the corresponding heat-stressed groups are designated AH 0.5 and AH 1.0. Control adrenalectomized rats (A 0.5) differ from intact controls in the level of serum FSH (p < 0.01) and pituitary FSH content (p < 0.05), both of which were elevated in the A 0.5, Day-12 group. The ovaries of adrenalectomized controls on the other hand, were smaller (insignificant statistically) than those of intact controls. Pituitary FSH content was slightly higher in the stressed group '(AH 0.5) than in the
532
HENSLEIGH AND JOHNSON
control (A 0.5), and it was nearly double the content of intact stressed animals (p < 0.01, Table 1). Serum FSH concentrations in stressed groups, on either replacement dosage, were significantly higher than their corresponding adrenalectomized controls. By Day 20 of gestation, serum FSH in these animals was at or below the sensitivity of the radioimmunoassay, which was true also for the intact animals. Serum LH was not changed in any of the adrenalectomy groups although the limited numbers of assay done in the higher dose Day-12 or in the Day-20 rats, prevents statistical analysis. Stress Experiments with Ovariectomized Animals. Data obtained with groups of rats ovariectomized on Day 6 and treated with estrone and progesterone are shown in Table 1. The estrone and progesterone were combined and given in a single subcutaneous injection of 0.2 m!. of sesame oil once daily. The adrenals of unstressed, ovariectomized rats in each treatment group tended to be larger than in intact, unstressed rats. The increase was not statistically significant in the groups that received 1 Jig. of estrone plus either 2 or 4 mg. of progesterone (Groups 7 and 9, Table 1). Stress caused a further adrenal enlargement in all groups except the animals treated with 2 Jig. of estrone plus 4 mg. of progesterone (Group 14, Table 1). However, the increase was statistically significant only for Groups 8, 10, and 16. As in intact animals, the adrenal weight changes were reflected in serum corticosterone levels, with stressed animals (Group 10) showing slightly higher values than unstressed rats (Group 9). Corticosterone levels in Groups 7, 8, 11, and 12 ovariectomy (0) 1: 2, OH 1: 2, 0 1: 6, and OH 1: 6) are excluded from Table 1. Animals in these groups were anesthetized with ether prior to autopsy, and corticosterone release was consequently markedly stimulated. Serum from rats
Vol. 22
autopsied in this manner was analyzed for corticosterone nevertheless, and the values were: 0 1: 2-60; OH-32; 0 1: 4-58; OH-33; 1: 6-60; OH-37. As expected, serum corticosterone was tripled by the ether, but it was lower in stressed groups than in their corresponding unstressed groups. This finding suggests a limited responsiveness by the adrenal in previously stressed animals, which could be interpreted as an exhaustion, or perhaps an acclimatization, of the pituitary-adrenal axis of the rats previously exposed to heat. Serum gonadotropin levels were markedly elevated in all the castrated animals despite the fact that "replacement" steroids were given. A significant difference in FSH between stressed and unstressed groups was found in the 1: 4 dose groups (Table 1). Stress appeared to increase pituitary and serum FSH only in animals on the 1: 4 combination, (p < 0.05 and 0.02, respectively). There were no significant differences in plasma LH between control and stressed groups. Groups 13-16 received 2 Jig. of estrone and either 4 or 8 mg. of progesterone. The adrenal weight and corticosterone changes are essentially the same as in the ovariectomized rats on the lower estrone dose. Serum FSH and LH are decreased by replacement with higher estrone dose, but too few samples were assayed for statistical analysis. Groups of ovariectomized rats which received replacement therapy of 1 Jig. of estrone and 4 mg. of progesterone were also autopsied on Day 20 (Table 2). Adrenal weights were not significantly affected by stress in Day-20 ovariectomized rats, but in spite of that, the serum corticosterone remained elevated on Day 20 in stressed rats (Group 6) as it was in intact, stressed rats (Group 2). Serum FSH was essentially the same in both groups on Day 20: it was about 10 times the level in intact animals on Day 20, but distinctly lower than ovari-
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August 1971
HEAT STRESS EFFECTS. II
ectomized animals on Day 12. Serum LH appeared to be elevated in both intact and ovariectomized rats which were stressed, but limited assays of these sera prevents determination of significance. DISCUSSION
During the first half of pregnancy in the rat the pituitary FSH content is very low and only begins to rise during the phase of placental control of the ovaries. 17 Considerable controversy surrounds the pituitary requirements for pregnancy, but experiments involving replacement of gonadotropins in hypophysectomized rats have shown that FSH is a very necessary component. IS Normal fetal-placental weight could be maintained with FSH plus prolactin. In the untreated controls (Fig. 1) we found that serum FSH and LH were easily detectable from Day 5 until Day 11, but there was a drastic drop on Day 12: removal of the pituitary on this day has no effect on the outcome of pregnancy. FSH and LH were both very low on Day 20. Midgley and co-workers 19 reported that LH was only occasionally detectable in the serum during the last 7 days of gestation in the rat. The question arises: Could the retardation of fetal development associated with maternal heat stress 1 be accounted for on the basis of reduced FSH output? According to Selye's2 concept, an increase in ACTH output could cause a drop in gonadotropin output. In order to come to any conclusion we must first consider the evidence that the stress altered pituitary function in regard to ACTH. Adrenal weight was increased on Day 12 as well as on Day 20 (Table 1). Corticosterone in the serum was quite variable on Day 12, but tended to be elevated in all stressed animals. The elevation was much more consistently found on Day 20. Further indication that the animals were responding to the stress by increased release of ACTH is manifested by the in-
533
ability of animals previously stressed for 5 days to respond to ether stress. This suggests that the animals had depleted stores of ACTH. If we can safely assume that ACTH output was increased, then did plasma gonadotropin change? The results demonstrated that FSH, but no LH, increased in the stressed animals when measured on Day 12. There was no elevation on Day 20, hormones being barely detectable at this time as in controls. The significance of the elevation of FSH in these animals is questionable because ovarian stimulation is not apparent from the ovarian weights reported here, nor in subsequent histologic studies of these ovaries. The evidence indicates however, that the immunoreactive gonadotropins in the serum were adequate for maintenance of a normal pregnancy and were not diminished at any time in stressed animals. Prolactin levels were not measured. In previous studies prolactin has been shown to increase during stress. IO Furthermore, the fetal effects of stress appear to be at least equally damaging during the placental phase of ovarian control when prolactin is ineffective. This was demonstrated by the lack of rebound fetal growth in last half of pregnancy after withdrawal of stress at Day 11. If stress, which increases ACTH release, also increases FSH release, then we might expect that adrenalectomy, which drastically increases ACTH output, might also increase FSH output. This was found to be the case: FSH was up 53% in unstressed adrenalectomized animals on the lowest dose of replacement steroids (Table 1). Stress in these animals raised serum FSH to exactly the same level as was found in the intact stressed animals. Increasing the dose of corticoids resulted in a slight drop in serum FSH, but stress returned it to the level found in other stressed animals. Again, there was no elevation of FSH on Day 20. As in the intact animals, the in-
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HENS LEIGH AND JOHNSON
crease in FSH did not appear to have any significant effect upon the ovary. We cannot however overlook the drastic change in pituitary FSH in these animals. This could be the result of a qualitative and/or quantitative change in ovarian steroidogenesis, but since gonadal steroids were not measured we have no direct information regarding effects on ovarian function. Heat stress does not interfere with fetal development in animals that are ovariectomized and treated with estrone and progesterone. 1 Placentae of these animals are smaller on Day 20 than those in unstressed animals, but even these reduced placentae are much larger than controls. Measurement of corticosterone in ovariectomized rats showed that ovariectomy increased ACTH a small amount, and that stress caused only a slight further increase. On Day 20, corticosterone was the same in ovariectomized rats as in intact controls and it was the same in stressed ovariectomized animals as in stressed intact animals (Table 2). We can therefore assume that the pituitary-adrenal responses to stress were about the same in ovariectomized as in intact animals. However, serum FSH was greatly increased in ovariectomized animals: LH changes were much less noticable. Pituitary FSH was about five times the normal level, and therefore, the production and release was relatively little affected by the exogenous steroids. This points up the difficulty of understanding the feedback control of FSH. Higher doses of estrone and progesterone (Table 2) appeared to lower the level of serum FSH a little, but even in these animals it was much higher than that found in intact pregnant rats. The effects of high FSH in ovariectomized pregnant rats is unknown. Since adrenal function is the same in ovariectomized stressed as it is in intact stressed animals, and yet the former do not demonstrate any fetal growth retardation, it is tempting to assign some protective role to
the gonadotropin. If the placenta (growth and/or steroidogenesis) could be stimulated by the FSH, this might explain the protective effect of ovariectomy, but this has not as yet been demonstrated. SUMMARY AND CONCLUSIONS
FSH and LH can easily be detected, by radioimmunoassay, in the serum ()f pregnant rats from the day of implantation until midterm. There is a significant fall in levels of both hormones on Day 12. The amount of FSH and LH on Day 20 is hardly detectable. Intermittent heat stress caused an increase in ACTH output as manifested by increased adrenal size and serum corticosterone level. The latter hormone was elevated on Day 20 also, 9 days after the last stress exposure. Animals with increased adrenal activity had elevated serum FSH on Day 12 of pregnancy. Adrenalectomy resulted in increased serum and pituitary FSH. These values were not nearly as high as those of ovariectomized animals receiving estrone and progesterone. Retarded fetal growth associated with the heat stress used in these experiments cannot be accounted for on the basis of reduced pituitary gonadotropin function. REFERENCES 1. HENSLEIGH, P. A., AND JOHNSON, D. C. Heat stress effects during pregnancy. I. Retardation of fetal rat growth. Fertil Steril22: 522, 1971. 2. SELYE, H. A syndrome produced by diverse nocuous agents. Nature (London) 138:32, 1936. 3. MOORE, W. W. Failure of adrenergic and cholinergic blocking agents to block ovulation in the rat. Amer J Physiol 200:1293, 1961. 4. MOORE, W. W. Effects of stress on plasma LH activity in ovariectomized rats. Physiologist 9:248, 1966. 5. EDGREN, R. A., AND PETERSON, D. L. The influence of adrenalectomy and corticoid replacement on ovarian compensatory hypertrophy in rats. Acta Endocr (Kovenhavn) 47:485, 1964. 6. RAMIREZ, V. D., MOORE, D., AND MCCANN, S. M. Independence of luteinizing hormone and
~.
.
"'
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7.
8.
9.
10.
11.
12.
13.
HEAT STRESS EFFECTS. II
adrenocorticotropin secretion in the rat. Proc Soc Exp Bioi Med 118:169, 1964. ELEFfHERIOU, B. E., AND CHURCH, R. L. Effects of repeated exposure to agression and defeat on plasma and pituitary levels of luteinizing hormone in C57BL/6J mice. Gen Comp Endocr 9: 263, 1967. CHARTERS, A. C., ODELL, W. D., AND THOMPSON, J. C. Anterior pituitary function during surgical stress and convalescence. Radioimmunoassay measurements of TSH, LH, FSH, and growth hormone. J Clin Endocr 29:63, 1969. BROWN, P. S., THORBURN, A. R., AND CROOKS, J. Urinary gonadotrophin excretion during treatment with corticotropin. J Endocr 28:125, 1963. GROSVENOR, C. E. Effect of nursing and stress on prolactin inhibiting activity of rat hypothalamus. Endocrinology 77:1037,1965. GUILLEMIN, R., CLAYTON, G. W., LIpSCOMB, H. S., AND SMITH, J. D. Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J Lab Clin Med 53:830, 1959. GIVNER, M. L., AND RoCHEFORT, J. G. An improved assay of corticosterone in rat serum and adrenal tissue. Steroids 6:485, 1965. ODELL, W. D., RAYFORD, P. L., AND Ross, G.T .
14.
15. 16.
17.
18.
19.
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Simplified, partially automated method for radioimmunoassay of human thyroid-stimulating growth, luteinizing and follicle stimulating hormones. J Lab Clin Med 70:973, 1967. JOHNSON, D. C., AND NAQVI, R. H. A simplified augmented ovarian weight assay for follicle stimulating hormone. Proc Soc Exp Bioi Med 133:536, 1970. GADDUM, J. H. Simplified mathematics for bioassays. J Pharm PharmacoI5:345, 1953. ANDERSON, R. R., AND TURNER, C. W. Maintenance of adrenalectomized rats through conception, pregnancy, and lactation. Amer J Physiol 205: 1077, 1963. GREENWALD, G. S. Ovarian follicular development and pituitary FSH and LH content in the pregnant rat. Endocrinology 79:572, 1966. GREENWALD, G. S., AND JOHNSON, D. C. Gonadotropic requirements for the maintenance of pregnancy in the hypophysectomized rat. Endocrinology 83:1052, 1968. MIDGLEY, A. R., GAY, V. L., CALIGARIS, L. C. S., REGAR, R. W., MONROE, S. E., AND NISWENDER, G. D. Gonodotropins 1968. Rosemberg, E. Ed. Geron-X, Inc. Los Altos, Calif. 1968, p. 307.
FERTll.ITY AND STERll.ITY
Copyright' 1971 by The Williams & Wilkins Co.
HEAT STRESS EFFECTS DURING PREGNANCY. II. PITUITARY GONADOTROPINS IN INTACT, ADRENALECTOMIZED, AND OVARIECTOMIZED RATS* PAUL A. HENSLEIGH, M.D., PH.D.,t
AND
DONALD C. JOHNSON, PH.D.
Departments of Obstetrics and Gynecology, and Physiology, University of Kansas Medical Center, Kansas City, Kansas
The detrimental effects of stress on fetal development may be directly related to increased production of adrenal corticoids, since adrenalectomy prior to certain experimental stress exposure blocks the fetal effects. 1 Other evidence suggests that a concomitant decrease in ovarian function may also play a role. Even before the gonadotropin requirements of pregnancy in the rat were known, Selye 2 had noted that stress in nonpregnant animals resulted in regression of the gonads. This finding led to a concept that under stress conditions a shift in anterior pituitary hormone production occurs, such that a marked increase in adrenocorticotropic hormone (ACTH) production is necessarily accomplished at the expense of other trophic hormones. Since ovarian function in the first half of rat pregnancy is known to be dependent on pituitary gonadotropin stimulation, one might expect at least some of the detrimental effects of stress during this period to result from diminished pituitary gonadotropin production. A necessary relationship -between pituitary production of ACTH and gonadotropins has not been established by more recent investigations. Moore 3 had argued that drugs such as dibenamine, dibenzyline, and pathilon block ovulation, not because of their specific pharmacologic effects, but rather because they act as nonspecific stres* Supported by grants from the National Science Foundation and the National Institutes of Health. t Postdoctoral fellow, United States Public Health Training Grant TL-HD-25.
sor agents which cause increased ACTH release and a concomitant limited luteinizing hormone (LH) release. Likewise, the plasma luteinizing hormone (LH) concentration in ovariectomized rats was found to be sharply decreased in rats which were stressed by formalin injections. 4 Edgren and Peterson 5 found that the compensatory ovarian hypertrophy, which normally follows unilateral ovariectomy, could be prevented by removal of the adrenals. The expected compensatory hypertrophy occurred if the rats were given replacement corticoids. However, the lack of response was attributed to disruption of cellular metabolism at the ovarian level in the absence of corticoids, rather than alteration of pituitary function. Other studies have shown elevations of plasma LH under experimental conditions causing increased ACTH production. Ramirez, Moore, and McCann 6 showed that either adrenalectomy (which presumably increased ACTH production) or the administration of large doses of cortisol, to block ACTH production resulted in slightly increased plasma LH. Eleftheriou and Church 7 found that mice which were stressed by exposure to aggression and defeat showed increased LH production. The effects of stress on follicle-stimulating hormone (FSH) and prolactin have not been well documented. Charters, Odell, and Thompson 8 found that following surgical stress, women showed a slight and transient decrease in plasma FSH and LH. Brown, Thorburn, and Crooks 9 found FSH
528
August 1971
HEAT STRESS EFFECTS. II
to be elevated in menopausal women during the first 2 days of treatment with large doses of ACTH. Stressed rats were shown by Grosvenor 1 0 to release increased amounts of prolactin as the result of decreased hypothalamic prolactin inhibitor. The purpose of the present study was to determine the changes which occur in serum FSH and LH, when pregnant rats were subjected to hyperthermic stress in the period immediately following implantation. Serum corticosterone was measured as an indicator of ACTH secretion under the experimental conditions in intact or ovariectomized rats, and this also confirmed the completeness of adrenal removal in adrenalectomized rats. The effects of heat stress on fetal growth will be presented in a separate report. 1 MATERIAL AND METHODS
Pregnant rats of the Holtzman strain were obtained on, or before, Day 5 of pregnancy (vaginal sperm = Day 1). Animals were maintained in air-conditioned quarters with 14 hr. of light/day (6 A.M. and 8 P.M.) and had free access to Purina laboratory chow and tap water. Serum FSH and LH levels for normal pregnant rats were determined by sacrificing groups of 5-9 rats on Days 5-12 and on Day 20 of gestation. Groups of animals to be stressed by hyperthermia were placed in an incubator (38.1 ± 0.3° C ambient temperature with 58.5 ± 2.8% relative humidity) for 4 hr. daily. Under these conditions the body (rectal) temperature was elevated from control values of 37.8 ± 0.1 ° C to 39.7 ± 0.1 ° C at the end of the 4 hr. Rats were stressed daily from Days 7 through Day 11 of gestation. In stress experiments the groups of rats were autopsied on either Day 12 or Day 20, by first giving ether anesthesia to facilitate collection of blood from the abdominal aorta for hormone assays. Nembutal anes-
529
thesia was used on rats whose corticosterone levels were to be determined. Blood was allowed to clot and the serum stored in aliquots at - 20° C until the time of assay. After collection of blood and recording maternal body weight the rats were decapitated and the pituitaries removed, weighed to the nearest milligram on a torsion balance, pooled, and frozen in saline at - 20° C. Ovarian and adrenal weights were then recorded. Corticosterone Assays. Corticosterone assays were done on 0.5-ml. aliquots of serum by a modification of the microfluorometric method of Guillemin and co-workers. 11 The modification utilized was that suggested by Givner and Rochefort 12 of substituting ethanol-sulfuric acid reagent for 30N sulfuric acid, which provides more stable fluorescence of the corticosterone solution. The Farrand Model A fluorometer was used with Corning filters 33895113 as primary, and Kodak wratten gelatin 16-74 as secondary filters. With each group of unknowns a reagent blank and a three point standard curve was run. The fluorescence of the unknowns was then converted by a graphic plot of the standards and expressed as micrograms of corticosterone per 100 ml. of serum. Serum Gonadotropin Assays. Serum samples were analyzed for FSH and LH utilizing a liquid phase double antibody radioimmunoassay technic. Reagents for these assays were supplied by the National Institute of Arthritis and Metabolic Diseases (NIAMD), National Institutes of Health (NIH), Rat Pituitary (RP) Hormone Distribution Program. The methodology employed for iodination of hormones was a modification of that suggested by NIAMD. The modification consisted of substituting 1.0 M sodium phosphate buffer (pH 7.6) for the iodination and shortening the iodination reaction to 50 sec. Carrier free P 31 suitable for iodination of proteins with specific activity
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ranging from 400-700 mc./ml. was obtained from Cambridge Nuclear Corporation, Billerica, Mass. The assay procedure used was essentially that described in the NIAMD literature, but incorporated the simplifications in counting activity and calculation of hormone content described by Odell, Rayford, and Ross. 13 The second antibody (sheep antirabbit 'Y globulin) was obtained from Antibodies Incorporated, Davis, Calif. A Nuclear-Chicago automatic 'Y well counter was used for all counting procedures. Serum concentrations of hormone in nanograms/milliliter were calculated on the basis of standards supplied by NIAMD (Rat FSH- RP-I = 2.1 X NIH-FSH-SI by augmentation assay and Rat LH-RP-I = 0.03 X NIH-LH-SI by ovarian ascorbic acid depletion assay (OAAD assay). Bioassay of Rat Pituitary FSH Content. A simplified augmented ovarian weight assay for FSH was used. 14 The standard was ovine FSH-S7 supplied by the Endocrinology Study Section of the NIH with a potency of 1.15 X NIH-FSH-S1. Three doses of standard and 1 or 2 doses of pituitary extract were used in each assay. Routine statistical procedures were used 15 in programming the IBM 1130 computer to convert the results into micrograms of NIHFSH-S7/pituitary. RESULTS
The levels of FSH and LH on Day 5-12 and on Day 20 of gestation are shown in Fig. 1. Both hormones were elevated during the first 11 days of gestation when compared to Day 20 values. There was an elevation of serum FSH on Day 8, which is significant (p < 0.05) when compared to Day 7 or Day 9. Serum LH also appeared to be elevated on Day 8, but it is not significantly different from LH levels on the preceeding or following day. A second (statistically insignificant) increase in serum FSH was observed on Day 11, followed by a large drop on Day 12.
90
i
70
~
40
10
10
DAYS
OF
12
i
20
GESTATION
FIG. 1. Levels of FSH (open bars) and LH (solid bars) in normal pregnant rat. Reference preparations were NIH-rat FSH RP-l and rat LH 1-1. Vertical lines indicate standard errors, with 5-9 animals for each group.
Stress Experiments with Intact Animals. Data from these experiments are shown in Tables 1 and 2. Stress had no effect on body weight, but adrenal weight on Day 12 (Table 1), as well as on Day 20 (Table 2), was increased in the stressed animals. Analysis of serum corticosterone indicated, that although quite variable, the enlarged adrenals produced more of this steroid than those on controls (Table 1). The difference in corticosterone level is particularly evident on Day 20 (Table 2) despite the fact that this is 9 days after their last heat exposure. Ovarian weight, pituitary FSH content and serum LH concentration showed no differences between stressed and control groups. However, serum FSH concentration in stressed rats (IH) was elevated on Day 12 (p < 0.02). By Day 20 both groups hEld serum FSH concentrations which were below the sensitivity of the assay. Stress Experiments with Adrenalectomized Animals. These rats had their adrenals removed on Day 6 immediately after receiving their first daily dose of replacement steroids. Data from groups of rats on two dosages of replacement corticoids and killed on Day 12 are given in Table 1. The lower dose, which has been reported 16
August 1971
531
HEAT STRESS EFFECTS. II
TABLE l. Effect of Heat Stress on Pregnancy: Autopsy on Day 12 Group*
Group No.
~o.
of No. of litters fetuses
Adrenals+
Body weightt
41 26 29 20 15 17 21 16 15 15 9 11 11
10 11 12 13 14 15 16
11 12
441 280 277 205 166 187 237 166 179 151 96 118 108 54 102 136
261 260 259 249 267 245 268 260 263 252 265 239 274 268 263 268
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4 6 4' 4' 411 511 4 4 3 2 4 311 4 3 6 4
Ovaries+
m{f.
141./100 mi.
mI!.
W1./{liand
15.2 ± 0.7 20.0 ± 4.5§
69.6 ± 1.5 69.0 ± 1.4 66.9 ± 2.:3 67.6 ::1: 1.7 65.6 ± 1.7 70.0 ± 1.9
98.5 ± 21 92.9 ± 13 166 ± 38 196 ± 35
63.8 69.0 62.2 67.5 65.6 67.0 72.0 69.8 67.4 75.2
± ± ± ± ± ± ± ±
1.4 2.4' 1.4 2.3' 1.6 2.2 1.9 1.8 ± 2.7 ± 3.6'
LHtj:
FSHtt
60.5 ± 0.9 65.2 ± 0.9
{1m.
Intact I plus Heat A 0.5 AHO.5 Al.O AHI.O 01 :2 OH 1:2 01:4 OH 1:4 01:6 OH 1:6 02:4 5H 2:4 52:8 5H 2:8
Corticosterone+
18.2 ± 4.4§ 20.0 ± 4.5§
ng./ml.
n{l.lml.
:34.8 69.9 53.5 68.5 45.4 69.4 518 478 388 491 638 456 321 254 254 242
412 ± 38 508 ± 58
18.8 26.6 24.3 31.3
'" ± ± ± ± ± ± ± ± ±
4.8 12§ 3.9 5.2§ 4.3 1011 31 44 39 29§
28.6 ± 25.8 ± 28.9 ± 30.0 ± :30.6 31.8 66.6 ± :38.4 37.8 ± 38.6± 32.7 35.6 28.0 24.8 25.4 24.2
4 4.7 3.4 4.0
23 1.6 3.3
* X, adrenalectomy; 0, ovariectomy; numbers in group indicate dose ofrepiacement steroids (explained in text). t Standard error, ±.
4: FSH in gland expressed in terms of NIH-FSH-S7, serum level is nanogram/milliliter of serum in terms of § p < 0.02 compared with unstressed group . • p < 0.05 compared with unstressed group. II p < 0.01 compared to unstressed group.
~IH-FSH-RP-I;
LH is ::-.JIH-LH-Rl.
TABLE 2. Effect of Heat Stress on Pregnancy: Autopsy on Day 20 Group*
Intact I plus Heat A 0.5 AH 0.5 61:4 OH 1:4
Group
2 3 4 5 6
No. of litters
9 7 8 8 9 7
No. of fetuses
68 47 85 81 90 55
Maternal body wt.t
306 304 337 321 337 293
± ± ± ± ± ±
8 10 8 9 10 9§
Adrenalst
Corticosteronet
mg.
WI./IOO mi.
60.2 ± 2.4 68.4 ± 1.9§
14.6 ± 0.8 27.0 ± 2.8§
67.6 ± 2.1 62.9 ± 1.7
13.7 ± 1.2 27.3 ± 5.5§
Ovariest
FSHj:
LHj:
mf,f.
n{l. 1m !.
np.lml.
101.6 106.6 113.2 110.0
± ± ± ±
5.6 9.3 7.6 9
25 25 25 25 278 252
25.4 38.2 28.8 15.6 32.8 73.8
* A, adrenalectomy; 0, ovariectomy; group numbers indicate dose of replacement steroid (explained in text). t Standard error, ±. :j: FSH and LH expressed as nanogram of NIH-Rat FSH-RP-1 and Rat LH-l. § p < 0.01 compared with unstressed group.
to be optimal for fetal growth, consisted of a daily (evening) injection of 0.5 mg of corticosterone and 0.25 mg of deoxycorticosterone acetate. These steroids were given as a single injection of 0.2 ml. in a saline suspension using 20 drops of "Tween 80" /100 ml of saline. The higher replacement dose consisted of 1.0 mg. of corticosterone and 0.5 mg. of deoxycorticosterone prepared and injected in the same manner. These two groups are designated as adrenalectomy (A) 0.5 and A 1.0 in
Table 1: the corresponding heat-stressed groups are designated AH 0.5 and AH 1.0. Control adrenalectomized rats (A 0.5) differ from intact controls in the level of serum FSH (p < 0.01) and pituitary FSH content (p < 0.05), both of which were elevated in the A 0.5, Day-12 group. The ovaries of adrenalectomized controls on the other hand, were smaller (insignificant statistically) than those of intact controls. Pituitary FSH content was slightly higher in the stressed group '(AH 0.5) than in the
532
HENSLEIGH AND JOHNSON
control (A 0.5), and it was nearly double the content of intact stressed animals (p < 0.01, Table 1). Serum FSH concentrations in stressed groups, on either replacement dosage, were significantly higher than their corresponding adrenalectomized controls. By Day 20 of gestation, serum FSH in these animals was at or below the sensitivity of the radioimmunoassay, which was true also for the intact animals. Serum LH was not changed in any of the adrenalectomy groups although the limited numbers of assay done in the higher dose Day-12 or in the Day-20 rats, prevents statistical analysis. Stress Experiments with Ovariectomized Animals. Data obtained with groups of rats ovariectomized on Day 6 and treated with estrone and progesterone are shown in Table 1. The estrone and progesterone were combined and given in a single subcutaneous injection of 0.2 m!. of sesame oil once daily. The adrenals of unstressed, ovariectomized rats in each treatment group tended to be larger than in intact, unstressed rats. The increase was not statistically significant in the groups that received 1 Jig. of estrone plus either 2 or 4 mg. of progesterone (Groups 7 and 9, Table 1). Stress caused a further adrenal enlargement in all groups except the animals treated with 2 Jig. of estrone plus 4 mg. of progesterone (Group 14, Table 1). However, the increase was statistically significant only for Groups 8, 10, and 16. As in intact animals, the adrenal weight changes were reflected in serum corticosterone levels, with stressed animals (Group 10) showing slightly higher values than unstressed rats (Group 9). Corticosterone levels in Groups 7, 8, 11, and 12 ovariectomy (0) 1: 2, OH 1: 2, 0 1: 6, and OH 1: 6) are excluded from Table 1. Animals in these groups were anesthetized with ether prior to autopsy, and corticosterone release was consequently markedly stimulated. Serum from rats
Vol. 22
autopsied in this manner was analyzed for corticosterone nevertheless, and the values were: 0 1: 2-60; OH-32; 0 1: 4-58; OH-33; 1: 6-60; OH-37. As expected, serum corticosterone was tripled by the ether, but it was lower in stressed groups than in their corresponding unstressed groups. This finding suggests a limited responsiveness by the adrenal in previously stressed animals, which could be interpreted as an exhaustion, or perhaps an acclimatization, of the pituitary-adrenal axis of the rats previously exposed to heat. Serum gonadotropin levels were markedly elevated in all the castrated animals despite the fact that "replacement" steroids were given. A significant difference in FSH between stressed and unstressed groups was found in the 1: 4 dose groups (Table 1). Stress appeared to increase pituitary and serum FSH only in animals on the 1: 4 combination, (p < 0.05 and 0.02, respectively). There were no significant differences in plasma LH between control and stressed groups. Groups 13-16 received 2 Jig. of estrone and either 4 or 8 mg. of progesterone. The adrenal weight and corticosterone changes are essentially the same as in the ovariectomized rats on the lower estrone dose. Serum FSH and LH are decreased by replacement with higher estrone dose, but too few samples were assayed for statistical analysis. Groups of ovariectomized rats which received replacement therapy of 1 Jig. of estrone and 4 mg. of progesterone were also autopsied on Day 20 (Table 2). Adrenal weights were not significantly affected by stress in Day-20 ovariectomized rats, but in spite of that, the serum corticosterone remained elevated on Day 20 in stressed rats (Group 6) as it was in intact, stressed rats (Group 2). Serum FSH was essentially the same in both groups on Day 20: it was about 10 times the level in intact animals on Day 20, but distinctly lower than ovari-
o
August 1971
HEAT STRESS EFFECTS. II
ectomized animals on Day 12. Serum LH appeared to be elevated in both intact and ovariectomized rats which were stressed, but limited assays of these sera prevents determination of significance. DISCUSSION
During the first half of pregnancy in the rat the pituitary FSH content is very low and only begins to rise during the phase of placental control of the ovaries. 17 Considerable controversy surrounds the pituitary requirements for pregnancy, but experiments involving replacement of gonadotropins in hypophysectomized rats have shown that FSH is a very necessary component. IS Normal fetal-placental weight could be maintained with FSH plus prolactin. In the untreated controls (Fig. 1) we found that serum FSH and LH were easily detectable from Day 5 until Day 11, but there was a drastic drop on Day 12: removal of the pituitary on this day has no effect on the outcome of pregnancy. FSH and LH were both very low on Day 20. Midgley and co-workers 19 reported that LH was only occasionally detectable in the serum during the last 7 days of gestation in the rat. The question arises: Could the retardation of fetal development associated with maternal heat stress 1 be accounted for on the basis of reduced FSH output? According to Selye's2 concept, an increase in ACTH output could cause a drop in gonadotropin output. In order to come to any conclusion we must first consider the evidence that the stress altered pituitary function in regard to ACTH. Adrenal weight was increased on Day 12 as well as on Day 20 (Table 1). Corticosterone in the serum was quite variable on Day 12, but tended to be elevated in all stressed animals. The elevation was much more consistently found on Day 20. Further indication that the animals were responding to the stress by increased release of ACTH is manifested by the in-
533
ability of animals previously stressed for 5 days to respond to ether stress. This suggests that the animals had depleted stores of ACTH. If we can safely assume that ACTH output was increased, then did plasma gonadotropin change? The results demonstrated that FSH, but no LH, increased in the stressed animals when measured on Day 12. There was no elevation on Day 20, hormones being barely detectable at this time as in controls. The significance of the elevation of FSH in these animals is questionable because ovarian stimulation is not apparent from the ovarian weights reported here, nor in subsequent histologic studies of these ovaries. The evidence indicates however, that the immunoreactive gonadotropins in the serum were adequate for maintenance of a normal pregnancy and were not diminished at any time in stressed animals. Prolactin levels were not measured. In previous studies prolactin has been shown to increase during stress. IO Furthermore, the fetal effects of stress appear to be at least equally damaging during the placental phase of ovarian control when prolactin is ineffective. This was demonstrated by the lack of rebound fetal growth in last half of pregnancy after withdrawal of stress at Day 11. If stress, which increases ACTH release, also increases FSH release, then we might expect that adrenalectomy, which drastically increases ACTH output, might also increase FSH output. This was found to be the case: FSH was up 53% in unstressed adrenalectomized animals on the lowest dose of replacement steroids (Table 1). Stress in these animals raised serum FSH to exactly the same level as was found in the intact stressed animals. Increasing the dose of corticoids resulted in a slight drop in serum FSH, but stress returned it to the level found in other stressed animals. Again, there was no elevation of FSH on Day 20. As in the intact animals, the in-
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HENS LEIGH AND JOHNSON
crease in FSH did not appear to have any significant effect upon the ovary. We cannot however overlook the drastic change in pituitary FSH in these animals. This could be the result of a qualitative and/or quantitative change in ovarian steroidogenesis, but since gonadal steroids were not measured we have no direct information regarding effects on ovarian function. Heat stress does not interfere with fetal development in animals that are ovariectomized and treated with estrone and progesterone. 1 Placentae of these animals are smaller on Day 20 than those in unstressed animals, but even these reduced placentae are much larger than controls. Measurement of corticosterone in ovariectomized rats showed that ovariectomy increased ACTH a small amount, and that stress caused only a slight further increase. On Day 20, corticosterone was the same in ovariectomized rats as in intact controls and it was the same in stressed ovariectomized animals as in stressed intact animals (Table 2). We can therefore assume that the pituitary-adrenal responses to stress were about the same in ovariectomized as in intact animals. However, serum FSH was greatly increased in ovariectomized animals: LH changes were much less noticable. Pituitary FSH was about five times the normal level, and therefore, the production and release was relatively little affected by the exogenous steroids. This points up the difficulty of understanding the feedback control of FSH. Higher doses of estrone and progesterone (Table 2) appeared to lower the level of serum FSH a little, but even in these animals it was much higher than that found in intact pregnant rats. The effects of high FSH in ovariectomized pregnant rats is unknown. Since adrenal function is the same in ovariectomized stressed as it is in intact stressed animals, and yet the former do not demonstrate any fetal growth retardation, it is tempting to assign some protective role to
the gonadotropin. If the placenta (growth and/or steroidogenesis) could be stimulated by the FSH, this might explain the protective effect of ovariectomy, but this has not as yet been demonstrated. SUMMARY AND CONCLUSIONS
FSH and LH can easily be detected, by radioimmunoassay, in the serum ()f pregnant rats from the day of implantation until midterm. There is a significant fall in levels of both hormones on Day 12. The amount of FSH and LH on Day 20 is hardly detectable. Intermittent heat stress caused an increase in ACTH output as manifested by increased adrenal size and serum corticosterone level. The latter hormone was elevated on Day 20 also, 9 days after the last stress exposure. Animals with increased adrenal activity had elevated serum FSH on Day 12 of pregnancy. Adrenalectomy resulted in increased serum and pituitary FSH. These values were not nearly as high as those of ovariectomized animals receiving estrone and progesterone. Retarded fetal growth associated with the heat stress used in these experiments cannot be accounted for on the basis of reduced pituitary gonadotropin function. REFERENCES 1. HENSLEIGH, P. A., AND JOHNSON, D. C. Heat stress effects during pregnancy. I. Retardation of fetal rat growth. Fertil Steril22: 522, 1971. 2. SELYE, H. A syndrome produced by diverse nocuous agents. Nature (London) 138:32, 1936. 3. MOORE, W. W. Failure of adrenergic and cholinergic blocking agents to block ovulation in the rat. Amer J Physiol 200:1293, 1961. 4. MOORE, W. W. Effects of stress on plasma LH activity in ovariectomized rats. Physiologist 9:248, 1966. 5. EDGREN, R. A., AND PETERSON, D. L. The influence of adrenalectomy and corticoid replacement on ovarian compensatory hypertrophy in rats. Acta Endocr (Kovenhavn) 47:485, 1964. 6. RAMIREZ, V. D., MOORE, D., AND MCCANN, S. M. Independence of luteinizing hormone and
~.
.
"'
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7.
8.
9.
10.
11.
12.
13.
HEAT STRESS EFFECTS. II
adrenocorticotropin secretion in the rat. Proc Soc Exp Bioi Med 118:169, 1964. ELEFfHERIOU, B. E., AND CHURCH, R. L. Effects of repeated exposure to agression and defeat on plasma and pituitary levels of luteinizing hormone in C57BL/6J mice. Gen Comp Endocr 9: 263, 1967. CHARTERS, A. C., ODELL, W. D., AND THOMPSON, J. C. Anterior pituitary function during surgical stress and convalescence. Radioimmunoassay measurements of TSH, LH, FSH, and growth hormone. J Clin Endocr 29:63, 1969. BROWN, P. S., THORBURN, A. R., AND CROOKS, J. Urinary gonadotrophin excretion during treatment with corticotropin. J Endocr 28:125, 1963. GROSVENOR, C. E. Effect of nursing and stress on prolactin inhibiting activity of rat hypothalamus. Endocrinology 77:1037,1965. GUILLEMIN, R., CLAYTON, G. W., LIpSCOMB, H. S., AND SMITH, J. D. Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J Lab Clin Med 53:830, 1959. GIVNER, M. L., AND RoCHEFORT, J. G. An improved assay of corticosterone in rat serum and adrenal tissue. Steroids 6:485, 1965. ODELL, W. D., RAYFORD, P. L., AND Ross, G.T .
14.
15. 16.
17.
18.
19.
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Simplified, partially automated method for radioimmunoassay of human thyroid-stimulating growth, luteinizing and follicle stimulating hormones. J Lab Clin Med 70:973, 1967. JOHNSON, D. C., AND NAQVI, R. H. A simplified augmented ovarian weight assay for follicle stimulating hormone. Proc Soc Exp Bioi Med 133:536, 1970. GADDUM, J. H. Simplified mathematics for bioassays. J Pharm PharmacoI5:345, 1953. ANDERSON, R. R., AND TURNER, C. W. Maintenance of adrenalectomized rats through conception, pregnancy, and lactation. Amer J Physiol 205: 1077, 1963. GREENWALD, G. S. Ovarian follicular development and pituitary FSH and LH content in the pregnant rat. Endocrinology 79:572, 1966. GREENWALD, G. S., AND JOHNSON, D. C. Gonadotropic requirements for the maintenance of pregnancy in the hypophysectomized rat. Endocrinology 83:1052, 1968. MIDGLEY, A. R., GAY, V. L., CALIGARIS, L. C. S., REGAR, R. W., MONROE, S. E., AND NISWENDER, G. D. Gonodotropins 1968. Rosemberg, E. Ed. Geron-X, Inc. Los Altos, Calif. 1968, p. 307.