- Email: [email protected]
Changes in daily serum corticosterone values in maturing male and female rats
185
CHANGES IN DAILY SERUM CORTICOSTERONE VALUES IN MATURING MALE AND FEMALE RATS1
Judith A. Ramaley Department of Anatomy-Physiology and Medical Sciences Program Indiana University, Bloomington, Indiana 47401 Received: 3123172
ABSTRACT
Weanling age (day 22) Sprague-Dawleymale and female rats were placed on a 14 hour light:10 hour dark cycle (lights on 6 a.m.-8 p.m.). Blood samples were obtained from alternate groups daily at noon (1200 hours) and 8 p.m. (2000 hours) by cardiac puncture. Serum corticosterone (compound B) was assaved by a microassay chemical procedure, sensitive to 2.5 ng B. In female rats, initial values were high, due perhaps to the stress of shipment and subsenuent surgery (13.6t0.2 ug% at 1200 and 28.552.7 ug% at 2000 in controls and 29.9kO.3 up(%at 1200 and 27.2k6.7 ug% at 2000 in ovariectomized [OVX] rats). By day 25 noon values had fallen to the limit of the assay in both control and OVX rats (~2.5 ug%). Values at 2000 fell more rapidly in OVX rats than in controls. By day 27 noon values began to rise in both control and OVX rats, hut the rises were higher in controls. Evening values also rose, again to higher values in controls. On the day of vaginal opening, the noon and evening values were quite high in controls and to a lesser extent in the OVX and had returned to a lower value on the following day. In males significant differences between noon and evening values of R were obtained only on certain days (24-26,32-38,40-41,44-47, 49-50). At other times the means showed a trend toward higher evening values. hut variability obscured the results. There was a tendency for serum B to be higher in the evening in castrated males. It can be concluded that weanling rats already show daily variations in serum B which adjust rapidlv to a new lighting cycle (14:lO). Puberty-related changes in adrenal function are clearly evident in females at puberty. These changes are suppressed but not eliminated by castration at weaning age. Changes are less clear in the male.
'Supported by NSF Grant GB 17438
186
STEROIDS
20:2
INTRODUCTION
It has been clear for several years that gonadal steroids can influence the function of the adrenal cortex. Sex differences in adrenal function have been examined by Kitay (1) and Coyne and Kitay (2) who have reported that the greater size and activity of the female rat adrenal is related to the presence of estrogens. Progestins. on the other hand, appear to inhibit adrenal function as indicated by a decrease in adrenal weight after progesterone administration (although plasma corticosterone levels may not be altered) (3). Heavily luteinized ovaries, resulting from the administrationof large doses of PMS (pregnantmare's serum gonadotrophin) and HCC (human chorionic gonadotrophin) and producing the so-called "Parlow rat," also cause a reduction in adrenal weight (4,5). The rather dramatic changes in circulating gonadal steroids that result from the onset of puberty in males and females might be exnected to alter adrenal cortical function and the interactionsbetween the adrenal and gonadal axis might change radically at this time. The present study was performed in order to determine the pattern of maturational changes in serum corticosterone levels in male and female rats. Allen and Kendall (6) reported that nocturnal rises in plasma corticoids began at about 30 days of age. Ader (7) on the other hand found evidence of daily rhythms in corticosterone output at 21 days of age. Other workers have reported cyclic fluctuations in hypothalamic CRF (corticotrophinreleasing factor) (8) at weanling age. As a preliminary to studies on adrenal gonadal interactions during maturation, we felt it wise to examine the pattern of change in plasma corticosteronemore closely by taking daily samples at the trough and peak times of the adult corticosteronerhythm.
Aug. 1972
STEROIDS
187
MATERIALS AND METHODS
Weanling age Sprague-Dawley derived rats were obtained from Murphy Farms, Plainfield, Indiana, and were placed 5 animals to a cage in a room with a 14 hour light and 10 hour dark cycle (lights on 6 a.m. to 8 p.m.). Food and water were available ad libidum. Temperature was maintained at 22Y+20. The animals were aivT&a at random into groups and 0.5 cc of blood was drawn by cardiac puncture once every three days from each animal. Sufficient groups were maintained so that blood samples could be collected daily at noon and at 8 p.m. A preliminary study indicated that these two times permitted sampling;of the low point (noon) and high point (8 p.m.) of the plasma adrenocorticalvalues in developing rats (Table 1). Experiments were performed during summer 1971. Blood collection Blood was collected by cardiac puncture without anes-__.-__-______~ thesia. Preliminary tests showed that serum corticosteronewas elevated only at 3 minutes after initial handling. Only samples completed within 1 minute after the rat was first handled were used (see Table 2). The blood was allowed to clot at room temperature, the clot was broken up with a small wire loop and the plasma collected by centrifugation at maximum speed in a Sorvall GC-1 clinical centrifuge for 20 minutes at 4'C. The serum was removed and stored at -3O'C until use. Preliminary controls indicated that serum and plasma values of B were comparable. Assav for serum corticosterone (compound B). Serum B was determined in 50 -------.--._~ and 100 ul samples of each sezaliquotusing a modification of the Silber method (91, developed by Dan Akin (10). In this procedure plasma or serum samples are mixed with 1.95 cc iso-octane and mixed on a vortex mixer for 30 seconds. After CentrifuRation for 5 minutes, the aqueous defatted samples are extracted with 6 cc methylene chloride, spun for 1 minute and centrifup;edfor 5 minutes. After the acqueous laver has been discarded, the remainine:acidic and phenolic impurities are removed by 1 ml of 0.1 N NaOH, mixed on a vortex mixer for exactly 5 seconds to avoid excessive loss of c0tiic0ia and centrifuged for 5 minutes. Then 5 ml of the solution is added to 0.5 ml of 70% ethanolic sulfuric acid. Exactly 95 minutes later, fluorescence is measured on an Aminco-Bowman spectrophotometerusing an exciting wavelength of 470 mu and an emitted wavelength of 530 mu on a Turner model 111 Fluorometer using filters #3~48 and 16. The sensitivity of the assay is shown in Figure 1. Values a0m to 5 ng B can be measured routinely and with extra care readings down to 2.5 ng F can be obtained. Lambda for the assay was 0.08+0.01 and slope of the standard curve was 271.7513.6. Slope of the unknown values did not differ significantly from the standard curve. The characteristicsof each assay were determined by means of a FOCAL-1969 program designed by John W. Smith and run on a PDP/~I computer. Recovery of corticosterone. To determine what percentage of corticosterone was actually measured by the fluorescence assay, standard solutions of corticosterone were made up in methylene chloride and added to a series of assay tubes. After the solvent was evaporated under cotton-filterednitrogen, a 100 ul serum sample consisting of pooled noon serum from 10 immature female 22 day old rats was added. These serum samples were treated like any other serum samples. Average recovery (also checked by addition of corticosterone--
STEROIDS
188
4-14C, obtained from New England Nuclear) was 94-971. The same values were obtained whether standards were added to serum or to distilled water blanks indicating that serum contained no interfering compounds. In our procedure, the serum of adrenalectomized animals showed no corticosterone (i.e., values were below limits of the assay, less than 5 ng/lOO ~1). Surgical procedures. Gonadectomy in males was performed via a single midline ventral abdominal approach under ether anesthesia. Gonadectomy in females was by two dorsal incisions. Sham surgery consisted of the same incision without manipulation of the abdominal organs. Puberty in females was determined by daily inspection for vaginal opening. Animals were necropsied the following day to verify ovulation. Animals with hemorrhagic ovarian follicles or a dilated tubal smpulla were considered to have ovulated. Chemicals Corticosteronewas purchased from Sigma chemicals. Other chemi---' cals were reagent grade. Water used in the assay was double-distilled,deionized.
8 -
Figure 1. Relationship between steroid concentration in standard solutions and spectrophotometerreadings on the Amineo-Bowman. Lambda for 10 assays, Each data point repre0.08+0.01; slope of the standard curve, 271..7~~.6. sents mean of 30 readings ztstandard error of the mean.
Aug. 1972
S T ER?AIsD 1
189
S
Circadian Variations in Serum Corticosterone (Table 1) --Daily variations in serum B (corticosterone)were already present,the day after the animals arrived from the supplier but the resting (baseline) levels at 0400, 0800 and 1200 were higher than they were four days later (7.1 ug B/100 ml serum, overall mean for these three times of day compared with ~5 ug B/100 ml serum at 26 days of age). The maximum values reached at the sampling times chosen were the same for both the newly arrived animals and the 26-day-old animals (17.3t2.4 vs. 15.7k4.1 ug B/100 ml serum). The midnight values suggested that the Pdl-off time of the peak was delayed in the 23-day-old animals as compared with the 26-day-old animals (12.Ot1.4 vs. 8.7kO.3 ug B/100 ml serum). Although newly arrived rats showed the same cycle of serum B, they were clearly disturbed by shipment and handling.
TABLE 1
SERUM CORTICOSTERONEVALUES AT VARIOUS TIMES OF DAY IN MATURING FEMALE RATSave
-_ Time of Day
PL 22 days
23
days
26
days
1200 1600 2000 2400 0400 0800
1200 1600 2000 2400 0400 0800
Corticosterone ug/lOO ml serum 6.4r0.gb 12.8k0.2~ 17.3t2.4' 12.0+1.4' 6.OkO.7 8.852.4 3.5t0.2 4.3ko.6 15.7?4.1C
asix rats per group bmean t standard error of the mean 'probability < 0.01 compared to 0400 value for that age group 'limit of the assay 2.5 ng/lOO ul serum or 2.5 ug/lOO ml serum edata obtained December 1970
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20:2
Effect of Cardiac Puncture upon serum corticosteronevalues in adults Since it was felt desirable to obtain blood samples by cardiac puncture so that individual animals could be followed during maturation, it was necessary to determine "safe interval" for obtaining blood in this manner. Table 2 shows that in both male and female adult rats, serum B does not begin to rise until at least 3 minutes after the initial handling of the animal. All blood samples reported in this paper were obtained within that interval. To determine whether disturbance caused by the handling of other animals in the ssme cage or nearby cages might alter baseline values, the order of each sample was also recorded. No correlation was found between the order in which a ssmpl was taken and the value of serum B obtained (r = 0.2). As previously reported (11,12), resting values of B were found to be lower in males than in females both at 1200 hours and at 2000 hours. When serum B was measured at vsribus
TABLE 2
SERUM CORTICOSTERONEVALUES AT VARIOUS TIMES AFTER CARDIAC PUNCTURE IN ADULTSa Male
1200 hrs Resting levels 1 min 3 min 5 min 30 min
Female Serum Corticosterone in ug/lOO ml 1200 hrs 2000 hrs 2000 hrs
3.9k1.2 18.3k3.4' 18.R+2.1C
6.4k3.0 6.421.2 19.7k2.0' 19.~2.6~
16.1k4.0 17.6k7.0 25.6di.0d 38.7+5.0c
19.7+2.0'
17.7fl.0C
60.4+3.3'
2.7r2.4b
27.0i6.6 28.055.0 29.1k3.2 43.7+3.3c 62.~1.6~
ba,ixrats per group mean f standard error of the mean 'significantlydifferent from resting level PcO.01 dsignificantly different from resting level PgO.05 Second serum sasqlc obtained by a second cardiac puncture.
f
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rats (<5 ug% at 1200 hours and 6.821.2 groups (<5 ug% vs.
14.1k3.1 ~3%).
20:2
ug% at 2000 hours) than in the other
The initial values obtained for the rats
at 23 days were lower in the pilot study of rhythmicity (Table 1) which was performed the preceding winter. This may reflect a seasonal variation in adrenal function (17). During;the remainder of the interval between weaning and puberty, evening values of plasma B in ovariectomized rats tended to remain lower than that of the other groups, although this was not consistently the case. Sham operated and intact animals showed similar values. As puberty approached, the noon values of B beRan to rise again to 13.8 52.3 ug% on day 27 and comparable values on other days until day 32 when they fell again to 6.2k3.4 ug%.
Serum B in ovariectomizedvalues remained some-
what lower than control or sham operated animals at noon but did show a rise to levels of 10.4tl.3 ug% on dav 26.
As in the controls, resting;levels of
B at noon dropped in ovariectomized animals by day 31 (to 4.8t1.3 ug%). Evening values were consistently higher than noon values in both intact and ovariectomized rats, the average increase ranging from 5 to 8 ug% from 1200 hours to 2000 hours in ovariectomized rats and 7 to 10 UK% in intact rats before day 31. At 31 days when resting noon values fell in both groups, the average increase in evening values rose to 14-20 ug% in intact rats and B-11 ug% in ovariectomized rats.
In the older prepuberal rat, evening
values were only sliqhtly elevated above those in the younger prepuberal rat. The most interesting result was that on the day of vaginal opening (day 36.020.4 for both groups in this experiment [done in June 1971I), there was a sudden increase in both noon and evening values of B in the intact and sham operated group.
In ovariectomized animals, vaginal open-
ing was delayed and partial (several days of puckering, partial opening
STEROIDS
Aug. 1972
193
along the margins but not centrally) and evening values of B rose somewhat but much less dramatically. Daily variations in pJ.asmacorticosterone (compound B) in males Figure 3 shows the levels of plasma B in male serum at noon and 8 p.m. during the interval between weaning and puberty. The first important finding is that male values of B were consistently less than female values at 8 p.m., but generally not at noon. Males did not show initial high values on days 23-25 as did females, but controls did show elevated evening values on days 24-25. Beginning on say 26 the evening values of B were not significantly different from noon values (both about 3 ug%).
Evening values were high on
day 29 (13.U2.4 ug$) in all animals, probably due to a disturbing banging in the overhead pipes on that day and then began to rise so that significant
DAILY *5 20 c
PLASMA
CORTICOSTERONE MALES
VALUES 6
Figure 3. Daily Variations in Corticosterone Values in Maturing Males. Each data point represents mean of 6 animals t standard error of the mean. Samples taken daily by cardiac puncture at 1130-1200 and 2000-2030 hours. Each rat was sampled once every three days.
20:2
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194
differences between noon and evening values of B were seen on days 32-38. Noon and evening values then returned to a low level (~5 u&)
on days 40-41
and then began to rise again. No consistent pattern of differences between intact or sham operated controls and gonadectomized animals was apparent, although whenever an evening rise in plasma B occurred, values in gonadectomized animals tended to be higher (day 29,31,34,43,44,48,52), Control values for another group of 60-day-old males are included for comparison. These latter values were obtained a month later. All of these experiments were run during the late summer (July, August 1971).
CONCLUSIONS
The ma,jorresults of these experiments are (1) In female rats, there is a daily variation in noon and 8 p.m. values of serum corticosterone (comnound R) levels before puberty. This is detectable on the day of weaning and remains relatively constant with some shifts in baseline (noon) values until the day before vaginal opening when the evening value is elevated. On the day of vaginal opening the evening value is considerably elevated. (2) Puberal-related changes in adrenal function as indicated by values of B are dependent upon the presence
of the ovaries.
(3) There is a less consistent pattern of plasma B in males. There are significant differences between noon and evening samples at several times during the puberal process (days 30-38) and again at days 43-44 and to some extent in older rats (day 48 on), Conadectomy does not alter plasma B consistently, although on certain days plasma B was highest in gonadectomized males.
Aug. 1972
195
STEROIDS discussion
The finding of daily changes in serum B at weaning age confirms the report of Ader (7)
but is in disagreement with that of Allen and Kendall
(6). Factors which might account for the discrepancy include a difference in light cycle used (14:lO in these experiments and 12:12 in Allen and Kendall's experiments),the time of sampling, early morning and mid-afternoon in Allen and Kendall's study, noon and mid-evening in this report. The low values for plasma B found by Ader (7) and Allen and Kendall (6)
agree
very
well with our results (4.8kO.8 ug% for males and 6.1kO.8 ug% for females at 8:30 a.m. in Allen and Kendall's work and comparable values in Ader's work). Evening values were also roughly comparable, although we found a greater sex difference than that reported by Allen and Kendall (6) and earlier changes in evening values. As in Ader's work, we saw an increase in evening values as puberty approached in both males and females. Our study has the advantage that it is a daily sample rather than a sample every five days (6) or every 15 days (7) and reveals more about the changes in adrenal function in relation to sexual maturation. A particularly interesting finding is that adrenal function seems to change rather suddenly at puberty in females and also during the initiation of gonadotrophin output (11) and changes in accessory gland function (12) in males. It has been reported that estrogen can stimulate adrenalcorticalfunction in gonadectomizedmales and females f1,2) but the time course of the response was not determined. A more recent study of adrenocorticalfunction during the estrus cycle suggests that fluctuations in ovarian hormone output can alter adrenal function on a day-to-day basis (13). In that study, both a.m. and p.m. values of plasma B were elevated in proestrous rats in
196
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20:2
comparison to estrous rats. The events preceeding the first ovulation in the puberal animals appear comparable to the proestrous changes found by Raps ..-_ et. _-_ al. (13) except that the values obtained in the young animal are considerably less. The depression of adrenal function after ovulation also has a parallel in the puberal animal. Precocious ovulation induced by PMS results in hyperluteinizationof the immature ovary and a clear depression of adrenal weight (4,5). It is possible that this depression is caused by some product of the luteinized tissue, perhaps progesterone or dehydroepiandrosterone(4,5).
Although the luteal tissue of the pre-
cociously ovulated immature rat appears different functionally from normal adult corpora lutea (lb), the first cycle in precociously ovulated rats seems to be similar to the adult cycle in its overall pattern. We are currently investigating the nature of the hormonal signals that alternately stimulate and depress the adrenal cortex in puberal and cycling rats.
Changes in plasma B in males proved less revealing. The first real changes occurred beginning on day 28, approximately the time at which significant alterations in pituitary function appear in prepuberal rats (11). Later changes in plasma B occur at the time that mature sperm first appear in the seminiferous tubules of the rat (around day 40) (15). After about day 45 the male rat is puberal but not yet fertile since sperm require about two weeks to pass through the male duct system (16). It is not surprising that no further changes in plasma B are seen during this interval. It has been shown that testosterone depresses adrenal function somewhat in males and that gonadectomy in males results in an increase in plasma B (1,2), measured some time after gonadectomy. In prepuberal males, gonadectornyresults in some enhancement of plasma B levels,
Aug. 1972
STEROIDS
197
but the results are not consistent. It appears that the relationship between gonads1 function and adrenal function in males will be harder to investigate due to the lack of clearcut external indices of the onset of puberty.
ACKNOWLEDGEMENTS
El&n Lee Bunn and Pat Kelley, undergraduate work-study students at IU, provided essential technical help in this study,
REFERENCES
1. 2. 3. 4. 2: 7. a.
9. 10. 11. 12. 13. 14. 15. 16. 17.
Kitay, J.I., ENDOCR. 73_, 253 (1963). Coyne, M.D. and J.I. Kitay, ENDOCRIN. E, 1097 (1969). Steinetz, B.G., V.L. Beach, G.D. Pasquale, and J.V. Baltista, Jr., STEROIDS 2, 93 (1965). Rennels, E.G., ANAT. REC. I&, 321 (1969). Rennels, E.G. and E.L. Singer, TEX. REP. BIOL. MED. 28, 303 (1970). Allen, C. and J.W. Kendall, ENDOCR. 80, 926 (1967). Ader, R., SCIENCE 63, 1225 (1.969). Hiroshige, T. and T. Sato, ENDOCR. JAP. l.7_, 1 (1970). Silber, R. H., R.D. Busch, and R. Solapas, CLIN. CHEM. 4, 278 (1958). Akin, Dan, Ph.D. dissertation, Indiana University, 1972. Kragt, C.L. and W. Ganong, PROC. MP. BIOL. MED. 128, 965 (1968). Rajalakshmi, M. and M.R.N. Prasad, J. ENDOCR. 44, 379 (1969). Raps, D., P.L. Barthe, and P.A. Desaulles, EXPERIENTIA 27, 337 (1971). Horikoshih and W.G. Wiest, ENDOCR. 3, 807 (1971). Clermont, Y. and B. Percy, AM. J. ANAT. 100, 241 (1957). Clermont, Y., ADV. EXP. MED. BIOL., Editors E. Rosemberg and C.A. Paulsen, vol. 10, Plenum Press, New York, 1970. Naumenko, E.V. and A.G. StaryKin, DOKLADY ACADEMIIA NAUK. SSSR 195, 729 (1970).
CHANGES IN DAILY SERUM CORTICOSTERONE VALUES IN MATURING MALE AND FEMALE RATS1
Judith A. Ramaley Department of Anatomy-Physiology and Medical Sciences Program Indiana University, Bloomington, Indiana 47401 Received: 3123172
ABSTRACT
Weanling age (day 22) Sprague-Dawleymale and female rats were placed on a 14 hour light:10 hour dark cycle (lights on 6 a.m.-8 p.m.). Blood samples were obtained from alternate groups daily at noon (1200 hours) and 8 p.m. (2000 hours) by cardiac puncture. Serum corticosterone (compound B) was assaved by a microassay chemical procedure, sensitive to 2.5 ng B. In female rats, initial values were high, due perhaps to the stress of shipment and subsenuent surgery (13.6t0.2 ug% at 1200 and 28.552.7 ug% at 2000 in controls and 29.9kO.3 up(%at 1200 and 27.2k6.7 ug% at 2000 in ovariectomized [OVX] rats). By day 25 noon values had fallen to the limit of the assay in both control and OVX rats (~2.5 ug%). Values at 2000 fell more rapidly in OVX rats than in controls. By day 27 noon values began to rise in both control and OVX rats, hut the rises were higher in controls. Evening values also rose, again to higher values in controls. On the day of vaginal opening, the noon and evening values were quite high in controls and to a lesser extent in the OVX and had returned to a lower value on the following day. In males significant differences between noon and evening values of R were obtained only on certain days (24-26,32-38,40-41,44-47, 49-50). At other times the means showed a trend toward higher evening values. hut variability obscured the results. There was a tendency for serum B to be higher in the evening in castrated males. It can be concluded that weanling rats already show daily variations in serum B which adjust rapidlv to a new lighting cycle (14:lO). Puberty-related changes in adrenal function are clearly evident in females at puberty. These changes are suppressed but not eliminated by castration at weaning age. Changes are less clear in the male.
'Supported by NSF Grant GB 17438
186
STEROIDS
20:2
INTRODUCTION
It has been clear for several years that gonadal steroids can influence the function of the adrenal cortex. Sex differences in adrenal function have been examined by Kitay (1) and Coyne and Kitay (2) who have reported that the greater size and activity of the female rat adrenal is related to the presence of estrogens. Progestins. on the other hand, appear to inhibit adrenal function as indicated by a decrease in adrenal weight after progesterone administration (although plasma corticosterone levels may not be altered) (3). Heavily luteinized ovaries, resulting from the administrationof large doses of PMS (pregnantmare's serum gonadotrophin) and HCC (human chorionic gonadotrophin) and producing the so-called "Parlow rat," also cause a reduction in adrenal weight (4,5). The rather dramatic changes in circulating gonadal steroids that result from the onset of puberty in males and females might be exnected to alter adrenal cortical function and the interactionsbetween the adrenal and gonadal axis might change radically at this time. The present study was performed in order to determine the pattern of maturational changes in serum corticosterone levels in male and female rats. Allen and Kendall (6) reported that nocturnal rises in plasma corticoids began at about 30 days of age. Ader (7) on the other hand found evidence of daily rhythms in corticosterone output at 21 days of age. Other workers have reported cyclic fluctuations in hypothalamic CRF (corticotrophinreleasing factor) (8) at weanling age. As a preliminary to studies on adrenal gonadal interactions during maturation, we felt it wise to examine the pattern of change in plasma corticosteronemore closely by taking daily samples at the trough and peak times of the adult corticosteronerhythm.
Aug. 1972
STEROIDS
187
MATERIALS AND METHODS
Weanling age Sprague-Dawley derived rats were obtained from Murphy Farms, Plainfield, Indiana, and were placed 5 animals to a cage in a room with a 14 hour light and 10 hour dark cycle (lights on 6 a.m. to 8 p.m.). Food and water were available ad libidum. Temperature was maintained at 22Y+20. The animals were aivT&a at random into groups and 0.5 cc of blood was drawn by cardiac puncture once every three days from each animal. Sufficient groups were maintained so that blood samples could be collected daily at noon and at 8 p.m. A preliminary study indicated that these two times permitted sampling;of the low point (noon) and high point (8 p.m.) of the plasma adrenocorticalvalues in developing rats (Table 1). Experiments were performed during summer 1971. Blood collection Blood was collected by cardiac puncture without anes-__.-__-______~ thesia. Preliminary tests showed that serum corticosteronewas elevated only at 3 minutes after initial handling. Only samples completed within 1 minute after the rat was first handled were used (see Table 2). The blood was allowed to clot at room temperature, the clot was broken up with a small wire loop and the plasma collected by centrifugation at maximum speed in a Sorvall GC-1 clinical centrifuge for 20 minutes at 4'C. The serum was removed and stored at -3O'C until use. Preliminary controls indicated that serum and plasma values of B were comparable. Assav for serum corticosterone (compound B). Serum B was determined in 50 -------.--._~ and 100 ul samples of each sezaliquotusing a modification of the Silber method (91, developed by Dan Akin (10). In this procedure plasma or serum samples are mixed with 1.95 cc iso-octane and mixed on a vortex mixer for 30 seconds. After CentrifuRation for 5 minutes, the aqueous defatted samples are extracted with 6 cc methylene chloride, spun for 1 minute and centrifup;edfor 5 minutes. After the acqueous laver has been discarded, the remainine:acidic and phenolic impurities are removed by 1 ml of 0.1 N NaOH, mixed on a vortex mixer for exactly 5 seconds to avoid excessive loss of c0tiic0ia and centrifuged for 5 minutes. Then 5 ml of the solution is added to 0.5 ml of 70% ethanolic sulfuric acid. Exactly 95 minutes later, fluorescence is measured on an Aminco-Bowman spectrophotometerusing an exciting wavelength of 470 mu and an emitted wavelength of 530 mu on a Turner model 111 Fluorometer using filters #3~48 and 16. The sensitivity of the assay is shown in Figure 1. Values a0m to 5 ng B can be measured routinely and with extra care readings down to 2.5 ng F can be obtained. Lambda for the assay was 0.08+0.01 and slope of the standard curve was 271.7513.6. Slope of the unknown values did not differ significantly from the standard curve. The characteristicsof each assay were determined by means of a FOCAL-1969 program designed by John W. Smith and run on a PDP/~I computer. Recovery of corticosterone. To determine what percentage of corticosterone was actually measured by the fluorescence assay, standard solutions of corticosterone were made up in methylene chloride and added to a series of assay tubes. After the solvent was evaporated under cotton-filterednitrogen, a 100 ul serum sample consisting of pooled noon serum from 10 immature female 22 day old rats was added. These serum samples were treated like any other serum samples. Average recovery (also checked by addition of corticosterone--
STEROIDS
188
4-14C, obtained from New England Nuclear) was 94-971. The same values were obtained whether standards were added to serum or to distilled water blanks indicating that serum contained no interfering compounds. In our procedure, the serum of adrenalectomized animals showed no corticosterone (i.e., values were below limits of the assay, less than 5 ng/lOO ~1). Surgical procedures. Gonadectomy in males was performed via a single midline ventral abdominal approach under ether anesthesia. Gonadectomy in females was by two dorsal incisions. Sham surgery consisted of the same incision without manipulation of the abdominal organs. Puberty in females was determined by daily inspection for vaginal opening. Animals were necropsied the following day to verify ovulation. Animals with hemorrhagic ovarian follicles or a dilated tubal smpulla were considered to have ovulated. Chemicals Corticosteronewas purchased from Sigma chemicals. Other chemi---' cals were reagent grade. Water used in the assay was double-distilled,deionized.
8 -
Figure 1. Relationship between steroid concentration in standard solutions and spectrophotometerreadings on the Amineo-Bowman. Lambda for 10 assays, Each data point repre0.08+0.01; slope of the standard curve, 271..7~~.6. sents mean of 30 readings ztstandard error of the mean.
Aug. 1972
S T ER?AIsD 1
189
S
Circadian Variations in Serum Corticosterone (Table 1) --Daily variations in serum B (corticosterone)were already present,the day after the animals arrived from the supplier but the resting (baseline) levels at 0400, 0800 and 1200 were higher than they were four days later (7.1 ug B/100 ml serum, overall mean for these three times of day compared with ~5 ug B/100 ml serum at 26 days of age). The maximum values reached at the sampling times chosen were the same for both the newly arrived animals and the 26-day-old animals (17.3t2.4 vs. 15.7k4.1 ug B/100 ml serum). The midnight values suggested that the Pdl-off time of the peak was delayed in the 23-day-old animals as compared with the 26-day-old animals (12.Ot1.4 vs. 8.7kO.3 ug B/100 ml serum). Although newly arrived rats showed the same cycle of serum B, they were clearly disturbed by shipment and handling.
TABLE 1
SERUM CORTICOSTERONEVALUES AT VARIOUS TIMES OF DAY IN MATURING FEMALE RATSave
-_ Time of Day
PL 22 days
23
days
26
days
1200 1600 2000 2400 0400 0800
1200 1600 2000 2400 0400 0800
Corticosterone ug/lOO ml serum 6.4r0.gb 12.8k0.2~ 17.3t2.4' 12.0+1.4' 6.OkO.7 8.852.4 3.5t0.2 4.3ko.6 15.7?4.1C
asix rats per group bmean t standard error of the mean 'probability < 0.01 compared to 0400 value for that age group 'limit of the assay 2.5 ng/lOO ul serum or 2.5 ug/lOO ml serum edata obtained December 1970
STEROIDS
190
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Effect of Cardiac Puncture upon serum corticosteronevalues in adults Since it was felt desirable to obtain blood samples by cardiac puncture so that individual animals could be followed during maturation, it was necessary to determine "safe interval" for obtaining blood in this manner. Table 2 shows that in both male and female adult rats, serum B does not begin to rise until at least 3 minutes after the initial handling of the animal. All blood samples reported in this paper were obtained within that interval. To determine whether disturbance caused by the handling of other animals in the ssme cage or nearby cages might alter baseline values, the order of each sample was also recorded. No correlation was found between the order in which a ssmpl was taken and the value of serum B obtained (r = 0.2). As previously reported (11,12), resting values of B were found to be lower in males than in females both at 1200 hours and at 2000 hours. When serum B was measured at vsribus
TABLE 2
SERUM CORTICOSTERONEVALUES AT VARIOUS TIMES AFTER CARDIAC PUNCTURE IN ADULTSa Male
1200 hrs Resting levels 1 min 3 min 5 min 30 min
Female Serum Corticosterone in ug/lOO ml 1200 hrs 2000 hrs 2000 hrs
3.9k1.2 18.3k3.4' 18.R+2.1C
6.4k3.0 6.421.2 19.7k2.0' 19.~2.6~
16.1k4.0 17.6k7.0 25.6di.0d 38.7+5.0c
19.7+2.0'
17.7fl.0C
60.4+3.3'
2.7r2.4b
27.0i6.6 28.055.0 29.1k3.2 43.7+3.3c 62.~1.6~
ba,ixrats per group mean f standard error of the mean 'significantlydifferent from resting level PcO.01 dsignificantly different from resting level PgO.05 Second serum sasqlc obtained by a second cardiac puncture.
f
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192
rats (<5 ug% at 1200 hours and 6.821.2 groups (<5 ug% vs.
14.1k3.1 ~3%).
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ug% at 2000 hours) than in the other
The initial values obtained for the rats
at 23 days were lower in the pilot study of rhythmicity (Table 1) which was performed the preceding winter. This may reflect a seasonal variation in adrenal function (17). During;the remainder of the interval between weaning and puberty, evening values of plasma B in ovariectomized rats tended to remain lower than that of the other groups, although this was not consistently the case. Sham operated and intact animals showed similar values. As puberty approached, the noon values of B beRan to rise again to 13.8 52.3 ug% on day 27 and comparable values on other days until day 32 when they fell again to 6.2k3.4 ug%.
Serum B in ovariectomizedvalues remained some-
what lower than control or sham operated animals at noon but did show a rise to levels of 10.4tl.3 ug% on dav 26.
As in the controls, resting;levels of
B at noon dropped in ovariectomized animals by day 31 (to 4.8t1.3 ug%). Evening values were consistently higher than noon values in both intact and ovariectomized rats, the average increase ranging from 5 to 8 ug% from 1200 hours to 2000 hours in ovariectomized rats and 7 to 10 UK% in intact rats before day 31. At 31 days when resting noon values fell in both groups, the average increase in evening values rose to 14-20 ug% in intact rats and B-11 ug% in ovariectomized rats.
In the older prepuberal rat, evening
values were only sliqhtly elevated above those in the younger prepuberal rat. The most interesting result was that on the day of vaginal opening (day 36.020.4 for both groups in this experiment [done in June 1971I), there was a sudden increase in both noon and evening values of B in the intact and sham operated group.
In ovariectomized animals, vaginal open-
ing was delayed and partial (several days of puckering, partial opening
STEROIDS
Aug. 1972
193
along the margins but not centrally) and evening values of B rose somewhat but much less dramatically. Daily variations in pJ.asmacorticosterone (compound B) in males Figure 3 shows the levels of plasma B in male serum at noon and 8 p.m. during the interval between weaning and puberty. The first important finding is that male values of B were consistently less than female values at 8 p.m., but generally not at noon. Males did not show initial high values on days 23-25 as did females, but controls did show elevated evening values on days 24-25. Beginning on say 26 the evening values of B were not significantly different from noon values (both about 3 ug%).
Evening values were high on
day 29 (13.U2.4 ug$) in all animals, probably due to a disturbing banging in the overhead pipes on that day and then began to rise so that significant
DAILY *5 20 c
PLASMA
CORTICOSTERONE MALES
VALUES 6
Figure 3. Daily Variations in Corticosterone Values in Maturing Males. Each data point represents mean of 6 animals t standard error of the mean. Samples taken daily by cardiac puncture at 1130-1200 and 2000-2030 hours. Each rat was sampled once every three days.
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differences between noon and evening values of B were seen on days 32-38. Noon and evening values then returned to a low level (~5 u&)
on days 40-41
and then began to rise again. No consistent pattern of differences between intact or sham operated controls and gonadectomized animals was apparent, although whenever an evening rise in plasma B occurred, values in gonadectomized animals tended to be higher (day 29,31,34,43,44,48,52), Control values for another group of 60-day-old males are included for comparison. These latter values were obtained a month later. All of these experiments were run during the late summer (July, August 1971).
CONCLUSIONS
The ma,jorresults of these experiments are (1) In female rats, there is a daily variation in noon and 8 p.m. values of serum corticosterone (comnound R) levels before puberty. This is detectable on the day of weaning and remains relatively constant with some shifts in baseline (noon) values until the day before vaginal opening when the evening value is elevated. On the day of vaginal opening the evening value is considerably elevated. (2) Puberal-related changes in adrenal function as indicated by values of B are dependent upon the presence
of the ovaries.
(3) There is a less consistent pattern of plasma B in males. There are significant differences between noon and evening samples at several times during the puberal process (days 30-38) and again at days 43-44 and to some extent in older rats (day 48 on), Conadectomy does not alter plasma B consistently, although on certain days plasma B was highest in gonadectomized males.
Aug. 1972
195
STEROIDS discussion
The finding of daily changes in serum B at weaning age confirms the report of Ader (7)
but is in disagreement with that of Allen and Kendall
(6). Factors which might account for the discrepancy include a difference in light cycle used (14:lO in these experiments and 12:12 in Allen and Kendall's experiments),the time of sampling, early morning and mid-afternoon in Allen and Kendall's study, noon and mid-evening in this report. The low values for plasma B found by Ader (7) and Allen and Kendall (6)
agree
very
well with our results (4.8kO.8 ug% for males and 6.1kO.8 ug% for females at 8:30 a.m. in Allen and Kendall's work and comparable values in Ader's work). Evening values were also roughly comparable, although we found a greater sex difference than that reported by Allen and Kendall (6) and earlier changes in evening values. As in Ader's work, we saw an increase in evening values as puberty approached in both males and females. Our study has the advantage that it is a daily sample rather than a sample every five days (6) or every 15 days (7) and reveals more about the changes in adrenal function in relation to sexual maturation. A particularly interesting finding is that adrenal function seems to change rather suddenly at puberty in females and also during the initiation of gonadotrophin output (11) and changes in accessory gland function (12) in males. It has been reported that estrogen can stimulate adrenalcorticalfunction in gonadectomizedmales and females f1,2) but the time course of the response was not determined. A more recent study of adrenocorticalfunction during the estrus cycle suggests that fluctuations in ovarian hormone output can alter adrenal function on a day-to-day basis (13). In that study, both a.m. and p.m. values of plasma B were elevated in proestrous rats in
196
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comparison to estrous rats. The events preceeding the first ovulation in the puberal animals appear comparable to the proestrous changes found by Raps ..-_ et. _-_ al. (13) except that the values obtained in the young animal are considerably less. The depression of adrenal function after ovulation also has a parallel in the puberal animal. Precocious ovulation induced by PMS results in hyperluteinizationof the immature ovary and a clear depression of adrenal weight (4,5). It is possible that this depression is caused by some product of the luteinized tissue, perhaps progesterone or dehydroepiandrosterone(4,5).
Although the luteal tissue of the pre-
cociously ovulated immature rat appears different functionally from normal adult corpora lutea (lb), the first cycle in precociously ovulated rats seems to be similar to the adult cycle in its overall pattern. We are currently investigating the nature of the hormonal signals that alternately stimulate and depress the adrenal cortex in puberal and cycling rats.
Changes in plasma B in males proved less revealing. The first real changes occurred beginning on day 28, approximately the time at which significant alterations in pituitary function appear in prepuberal rats (11). Later changes in plasma B occur at the time that mature sperm first appear in the seminiferous tubules of the rat (around day 40) (15). After about day 45 the male rat is puberal but not yet fertile since sperm require about two weeks to pass through the male duct system (16). It is not surprising that no further changes in plasma B are seen during this interval. It has been shown that testosterone depresses adrenal function somewhat in males and that gonadectomy in males results in an increase in plasma B (1,2), measured some time after gonadectomy. In prepuberal males, gonadectornyresults in some enhancement of plasma B levels,
Aug. 1972
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197
but the results are not consistent. It appears that the relationship between gonads1 function and adrenal function in males will be harder to investigate due to the lack of clearcut external indices of the onset of puberty.
ACKNOWLEDGEMENTS
El&n Lee Bunn and Pat Kelley, undergraduate work-study students at IU, provided essential technical help in this study,
REFERENCES
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Kitay, J.I., ENDOCR. 73_, 253 (1963). Coyne, M.D. and J.I. Kitay, ENDOCRIN. E, 1097 (1969). Steinetz, B.G., V.L. Beach, G.D. Pasquale, and J.V. Baltista, Jr., STEROIDS 2, 93 (1965). Rennels, E.G., ANAT. REC. I&, 321 (1969). Rennels, E.G. and E.L. Singer, TEX. REP. BIOL. MED. 28, 303 (1970). Allen, C. and J.W. Kendall, ENDOCR. 80, 926 (1967). Ader, R., SCIENCE 63, 1225 (1.969). Hiroshige, T. and T. Sato, ENDOCR. JAP. l.7_, 1 (1970). Silber, R. H., R.D. Busch, and R. Solapas, CLIN. CHEM. 4, 278 (1958). Akin, Dan, Ph.D. dissertation, Indiana University, 1972. Kragt, C.L. and W. Ganong, PROC. MP. BIOL. MED. 128, 965 (1968). Rajalakshmi, M. and M.R.N. Prasad, J. ENDOCR. 44, 379 (1969). Raps, D., P.L. Barthe, and P.A. Desaulles, EXPERIENTIA 27, 337 (1971). Horikoshih and W.G. Wiest, ENDOCR. 3, 807 (1971). Clermont, Y. and B. Percy, AM. J. ANAT. 100, 241 (1957). Clermont, Y., ADV. EXP. MED. BIOL., Editors E. Rosemberg and C.A. Paulsen, vol. 10, Plenum Press, New York, 1970. Naumenko, E.V. and A.G. StaryKin, DOKLADY ACADEMIIA NAUK. SSSR 195, 729 (1970).