0013-7227/79/1045-1525$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 104, No. 5 Printed in U.S.A.
Cyclic Changes in Utero-Ovarian Blood Flow and Ovarian Hormone Secretion in the Hamster: Effects of Adrenocorticotropin, Luteinizing Hormone, and Follicle-Stimulating Hormone* BERT ALAN VARGAf AND GILBERT S. GREENWALD Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary; and the Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66103
ABSTRACT. These experiments were designed to determine utero-ovarian vein blood flow and ovarian secretion rates and the ability of ACTH, LH, or FSH to modify these parameters in the cyclic hamster. Hamsters were anesthetized with pentobarbital, and polyethylene cannulae were introduced into the utero-ovarian and femoral veins. Five-minute blood samples were collected from the utero-ovarian vein for 50 min. After the second blood sample, ACTH (10 /ig/100 g BW), LH (2.5 /xg/100 g BW), or FSH (10 jug/ 100 g BW) was injected via the femoral vein. Based on the second 5-min control sample, the pattern of ovarian blood flow and steroid secretion rates during the cycle can be reconstructed. On day 3, utero-ovarian blood flow decreased compared to the day 2 level, but it increased again on day 4. Secretion of estradiol (E2) was low on day 1 (estrus); it increased gradually during the following days and reached a peak on day 4 (proestrus) between 1400-1500 h; thereafter E2 rapidly decreased. The pattern of testosterone (T) secretion paralleled E2 secretion. On days 1 and 2, progesterone (P)
I
NTRAVENOUS administration of ACTH to anesthetized hamsters results in a greater increase in ovarian than in adrenal blood flow (1). This appears to be a direct effect of ACTH on the ovary because it is still observed in dogs which are hypophysectomized (2) or adrenalectomized (3, 4). After ACTH administration, estrogen secretion is elevated in dogs pretreated with hCG (5), which may similarly result from an increase in ovarian blood flow. To determine whether ACTH has an effect on ovarian hormone secretion in the cyclic hamster, in the present study, blood was collected directly from the utero-ovarian vein. Before and after ACTH administration, utero-
secretion was high; it diminished on day 3. There was a further decrease in P on day 4 at 1000 h, but after 1430 h, P increased very rapidly to an even higher level than that found on day 1 or 2. ACTH increased utero-ovarian blood flow on days 1-3. On the morning of day 4, ACTH stimulated E2 and, after 1430 h, it stimulated P secretion. On day 1, ACTH augmented T and P secretion. LH and FSH on day 1 had a slight effect on E2 and P secretion. On day 2, LH and FSH very significantly stimulated E2 and T and slightly increased P secretion. On day 3 and the morning of day 4, the gonadotropins elevated E2, T, and P secretion but were no longer effective on day 4 after 1430 h. At no time did LH or FSH increase utero-ovarian blood flow. ACTH increased utero-ovarian blood flow and ovarian P secretion in adrenalectomized day 1 hamsters. It is concluded that ACTH may directly stimulate ovarian blood flow and, depending on the stage of the cycle, ACTH may participate in ovarian steroid secretion, but its effects are different from LH or FSH. {Endocrinology 104: 1525, 1979)
ovarian blood flow was measured and 17/?-estradiol (E2), testosterone (T), and progesterone (P) were determined in the effluent samples. The effects on ovarian blood flow and hormone secretion of iv ACTH were then compared with the effects observed after iv administration of LH or FSH. Materials and Methods
Received March 10, 1978. Address all correspondence and requests for reprints to: Dr. Gilbert S. Greenwald, Department of Physiology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, Kansas 66103. * This work was supported by grants from the NIH (HD-00596) and The Ford Foundation. t Supported by a grant from the National Academy of Science.
Golden hamsters, weighing 90-130 g, were maintained on a 14-h light, 10-h dark photoperiod with the lights on at 0500 h. Before the experiment, all animals had at least three consecutive 4-day estrous cycles, as determined by a postovulatory vaginal discharge on day 1; proestrus corresponded to day 4. On the various days of the cycle, animals were anesthetized with sodium pentobarbital (8.0 mg/100 g BW; Napentol, Beecham Products, Pittsburgh, PA), and thin polyethylene catheters (1 mm od, 0.5 mm id; Dural Plastics, Australia) were inserted in the femoral and one of the utero-ovarian veins. The length of the utero-ovarian catheter was 180 mm, and the collecting end was 30 mm below the level of the vena cava. One
1525
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 16:31 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1979 Vol 104 • No 5
VARGA AND GREENWALD
1526
group of day 1 hamsters was adrenalectomized 10 min before the cannulation. Blood clotting was prevented by 300 IU/100 g BW heparin (Riker Laboratories Inc., Northridge, CA) injected into the femoral vein. From the utero-ovarian vein, 5-min blood fractions were continuously collected for 50 min. After collection of the second 5-min fraction, 10 jug/100 g BW synthetic ACTH (Cortrosyn, 1-24 Corticotropin; Organon), 2.5 jug/100 g BW LH (NIH-LH-S18, ovine), or 10 /xg/100 g BW FSH (NIHFSH-S-10, ovine) were injected into the femoral vein. The time of injection is referred to as time zero. The volume of each blood fraction was measured, and then the fractions were centrifuged and the plasma from each aliquot was stored separately at -20 C. Plasma (10 /xl) was used for RIA of E2 (6) or T (7), and 0.4 /AI was used for RIA of P (8, 9). The T antibody used cross-reacts 58% with 5a-dihydrotestosterone (DHT) and 2% with androstanedione. However, in the cyclic hamster, the preovulatory surge of androgen represents T exclusively, and no detectable amounts of DHT are present (Connor, J. C, and G. S. Greenwald, unpublished data). Hormone levels determined with this antiserum are referred to in this paper as T or, in some instances, designated as androgens. Details on the RIAs are provided in a recent publication (7). Blood from one group of hamsters was used to record changes in the hematocrit during the 50-min bleeding period (Table 1). These hematocrit values were used to determine the correct plasma volume of each 5-min fraction. It is evident from Table 1 that the hematocrit remained relatively stable until at least 30 min after the beginning of bleeding. Significant changes in
utero-ovarian blood flow and steroid secretion rates were usually initiated within 5-10 min of the injection of ACTH, FSH, or LH. Hence, these observations occurred during the time span when the hematocrit and, consequently, the cardiovascular system were within the normal physiological range. Uteroovarian blood flow is expressed in terms of microliters per min/ 100 g BW. From the blood flow, hematocrit, and plasma concentration of steroids, the hormone secretion rate was calculated and expressed as picograms or nanograms per min/ovary • 100 g BW. The results were expressed per 100 g BW to decrease the variability introduced by actual body weights ranging from 90-130 g. Statistical differences were determined by analysis of variance. The Duncan multiple range test was used to compare the days of the cycle (Table 2), and the Dunnett multiple range test (10) was used to evaluate the effects of treatments. Results Changes during the cycle Ovarian blood flow and steroid secretion rates during the normal estrous cycle can be determined from the values found during the second 5-min control bleeding
immediately before the hormone injection (Table 2). Utero-ovarian blood flow decreased on day 3 compared to day 2 and rose again on the afternoon of day 4. Secretion of E2 and T reached a peak on day 4 between
TABLE 1. Hematocrit values during 50 min of bleeding from the utero-ovarian veins Time (min)
51.7 ± 0.8
10
15
20
25
30
35
40
45
50
50.0 ± 1.2
47 ± 0.9
47 ± 0.6
47 ± 0.6
46.2 ± 0.8
44 ± 1.3
42.8 ± 1.1
41.0 ± 1.1
39.8 ± 1.1
There were six values studied for each time interval. TABLE 2. Utero-ovarian blood flow, E2, T (androgens), and P secretion during the hamster estrous cycle Day (D) of cycle Significance 1000-1400 (a)
1400-1600 (b)
1600-1930 (c)
37.6 + 3.0(10)
46.6 ± 3.7 (11)
49.5 ± 2.8 (11)
D2-D3"; D4a-D4c"
66.3 ± 10.8 (18)
138.6 ± 24.9 (16)
347.1 ± 42.8 (11)
38.2 ± 10.9 (11)
Dl-D4a*; D3-D4b*; D l D4b"; D4a-D4b"; D2D4a°; D4b-D4c 6 ; D2D4b"
49.2 ± 12.1 (13)
86.7 ± 19.4 (15)
261.1 ± 32.5 (11)
45.0 ± 5.8 (10)
Dl-D4b"; D2-D4b"; D3D4b"; D4a-D4b"; D4bD4c*
Blood flow Oil/min100 g BW)
40.7 ± 3.7 (18)
47.0 ± 2.5 (18)
36.9 ± 3.5 (18)
E2
16.7 ± 5.0 (18)
39.9 ± 10.4 (18)
36.3 ± 3.5 (15)
41.5 ± 5.9 (17)
(pg/min-ovary/ 100 gBW)
T (pg/min-ovary/ 100 g B W )
10.00-14.35 (a)
P
(ng/min- ovary/ 100 g B W )
14.3 ± 1.8 (18)
17.6 ± 2.2 (18)
1.3 ± 0.3 (18)
0.5 ± 0.2 (19)
14.35-19.30 (b)
25.9 ± 1.6 (19)
Dl-D4a"; D1-D3*; D2D4a*; D2-D3*; D3-D4a"; D2-D4b*; D4a-D4b"; DlD4b*
Values given are the mean ± SEM; number of animals is in parentheses. " P < 0.05. "Pjg/l00g bw) FSH - . - • - • (lOjjg/WOgbw) 6 :P
Vol. 104, No. 5 Printed in U.S.A.
Cyclic Changes in Utero-Ovarian Blood Flow and Ovarian Hormone Secretion in the Hamster: Effects of Adrenocorticotropin, Luteinizing Hormone, and Follicle-Stimulating Hormone* BERT ALAN VARGAf AND GILBERT S. GREENWALD Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary; and the Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66103
ABSTRACT. These experiments were designed to determine utero-ovarian vein blood flow and ovarian secretion rates and the ability of ACTH, LH, or FSH to modify these parameters in the cyclic hamster. Hamsters were anesthetized with pentobarbital, and polyethylene cannulae were introduced into the utero-ovarian and femoral veins. Five-minute blood samples were collected from the utero-ovarian vein for 50 min. After the second blood sample, ACTH (10 /ig/100 g BW), LH (2.5 /xg/100 g BW), or FSH (10 jug/ 100 g BW) was injected via the femoral vein. Based on the second 5-min control sample, the pattern of ovarian blood flow and steroid secretion rates during the cycle can be reconstructed. On day 3, utero-ovarian blood flow decreased compared to the day 2 level, but it increased again on day 4. Secretion of estradiol (E2) was low on day 1 (estrus); it increased gradually during the following days and reached a peak on day 4 (proestrus) between 1400-1500 h; thereafter E2 rapidly decreased. The pattern of testosterone (T) secretion paralleled E2 secretion. On days 1 and 2, progesterone (P)
I
NTRAVENOUS administration of ACTH to anesthetized hamsters results in a greater increase in ovarian than in adrenal blood flow (1). This appears to be a direct effect of ACTH on the ovary because it is still observed in dogs which are hypophysectomized (2) or adrenalectomized (3, 4). After ACTH administration, estrogen secretion is elevated in dogs pretreated with hCG (5), which may similarly result from an increase in ovarian blood flow. To determine whether ACTH has an effect on ovarian hormone secretion in the cyclic hamster, in the present study, blood was collected directly from the utero-ovarian vein. Before and after ACTH administration, utero-
secretion was high; it diminished on day 3. There was a further decrease in P on day 4 at 1000 h, but after 1430 h, P increased very rapidly to an even higher level than that found on day 1 or 2. ACTH increased utero-ovarian blood flow on days 1-3. On the morning of day 4, ACTH stimulated E2 and, after 1430 h, it stimulated P secretion. On day 1, ACTH augmented T and P secretion. LH and FSH on day 1 had a slight effect on E2 and P secretion. On day 2, LH and FSH very significantly stimulated E2 and T and slightly increased P secretion. On day 3 and the morning of day 4, the gonadotropins elevated E2, T, and P secretion but were no longer effective on day 4 after 1430 h. At no time did LH or FSH increase utero-ovarian blood flow. ACTH increased utero-ovarian blood flow and ovarian P secretion in adrenalectomized day 1 hamsters. It is concluded that ACTH may directly stimulate ovarian blood flow and, depending on the stage of the cycle, ACTH may participate in ovarian steroid secretion, but its effects are different from LH or FSH. {Endocrinology 104: 1525, 1979)
ovarian blood flow was measured and 17/?-estradiol (E2), testosterone (T), and progesterone (P) were determined in the effluent samples. The effects on ovarian blood flow and hormone secretion of iv ACTH were then compared with the effects observed after iv administration of LH or FSH. Materials and Methods
Received March 10, 1978. Address all correspondence and requests for reprints to: Dr. Gilbert S. Greenwald, Department of Physiology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, Kansas 66103. * This work was supported by grants from the NIH (HD-00596) and The Ford Foundation. t Supported by a grant from the National Academy of Science.
Golden hamsters, weighing 90-130 g, were maintained on a 14-h light, 10-h dark photoperiod with the lights on at 0500 h. Before the experiment, all animals had at least three consecutive 4-day estrous cycles, as determined by a postovulatory vaginal discharge on day 1; proestrus corresponded to day 4. On the various days of the cycle, animals were anesthetized with sodium pentobarbital (8.0 mg/100 g BW; Napentol, Beecham Products, Pittsburgh, PA), and thin polyethylene catheters (1 mm od, 0.5 mm id; Dural Plastics, Australia) were inserted in the femoral and one of the utero-ovarian veins. The length of the utero-ovarian catheter was 180 mm, and the collecting end was 30 mm below the level of the vena cava. One
1525
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 16:31 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1979 Vol 104 • No 5
VARGA AND GREENWALD
1526
group of day 1 hamsters was adrenalectomized 10 min before the cannulation. Blood clotting was prevented by 300 IU/100 g BW heparin (Riker Laboratories Inc., Northridge, CA) injected into the femoral vein. From the utero-ovarian vein, 5-min blood fractions were continuously collected for 50 min. After collection of the second 5-min fraction, 10 jug/100 g BW synthetic ACTH (Cortrosyn, 1-24 Corticotropin; Organon), 2.5 jug/100 g BW LH (NIH-LH-S18, ovine), or 10 /xg/100 g BW FSH (NIHFSH-S-10, ovine) were injected into the femoral vein. The time of injection is referred to as time zero. The volume of each blood fraction was measured, and then the fractions were centrifuged and the plasma from each aliquot was stored separately at -20 C. Plasma (10 /xl) was used for RIA of E2 (6) or T (7), and 0.4 /AI was used for RIA of P (8, 9). The T antibody used cross-reacts 58% with 5a-dihydrotestosterone (DHT) and 2% with androstanedione. However, in the cyclic hamster, the preovulatory surge of androgen represents T exclusively, and no detectable amounts of DHT are present (Connor, J. C, and G. S. Greenwald, unpublished data). Hormone levels determined with this antiserum are referred to in this paper as T or, in some instances, designated as androgens. Details on the RIAs are provided in a recent publication (7). Blood from one group of hamsters was used to record changes in the hematocrit during the 50-min bleeding period (Table 1). These hematocrit values were used to determine the correct plasma volume of each 5-min fraction. It is evident from Table 1 that the hematocrit remained relatively stable until at least 30 min after the beginning of bleeding. Significant changes in
utero-ovarian blood flow and steroid secretion rates were usually initiated within 5-10 min of the injection of ACTH, FSH, or LH. Hence, these observations occurred during the time span when the hematocrit and, consequently, the cardiovascular system were within the normal physiological range. Uteroovarian blood flow is expressed in terms of microliters per min/ 100 g BW. From the blood flow, hematocrit, and plasma concentration of steroids, the hormone secretion rate was calculated and expressed as picograms or nanograms per min/ovary • 100 g BW. The results were expressed per 100 g BW to decrease the variability introduced by actual body weights ranging from 90-130 g. Statistical differences were determined by analysis of variance. The Duncan multiple range test was used to compare the days of the cycle (Table 2), and the Dunnett multiple range test (10) was used to evaluate the effects of treatments. Results Changes during the cycle Ovarian blood flow and steroid secretion rates during the normal estrous cycle can be determined from the values found during the second 5-min control bleeding
immediately before the hormone injection (Table 2). Utero-ovarian blood flow decreased on day 3 compared to day 2 and rose again on the afternoon of day 4. Secretion of E2 and T reached a peak on day 4 between
TABLE 1. Hematocrit values during 50 min of bleeding from the utero-ovarian veins Time (min)
51.7 ± 0.8
10
15
20
25
30
35
40
45
50
50.0 ± 1.2
47 ± 0.9
47 ± 0.6
47 ± 0.6
46.2 ± 0.8
44 ± 1.3
42.8 ± 1.1
41.0 ± 1.1
39.8 ± 1.1
There were six values studied for each time interval. TABLE 2. Utero-ovarian blood flow, E2, T (androgens), and P secretion during the hamster estrous cycle Day (D) of cycle Significance 1000-1400 (a)
1400-1600 (b)
1600-1930 (c)
37.6 + 3.0(10)
46.6 ± 3.7 (11)
49.5 ± 2.8 (11)
D2-D3"; D4a-D4c"
66.3 ± 10.8 (18)
138.6 ± 24.9 (16)
347.1 ± 42.8 (11)
38.2 ± 10.9 (11)
Dl-D4a*; D3-D4b*; D l D4b"; D4a-D4b"; D2D4a°; D4b-D4c 6 ; D2D4b"
49.2 ± 12.1 (13)
86.7 ± 19.4 (15)
261.1 ± 32.5 (11)
45.0 ± 5.8 (10)
Dl-D4b"; D2-D4b"; D3D4b"; D4a-D4b"; D4bD4c*
Blood flow Oil/min100 g BW)
40.7 ± 3.7 (18)
47.0 ± 2.5 (18)
36.9 ± 3.5 (18)
E2
16.7 ± 5.0 (18)
39.9 ± 10.4 (18)
36.3 ± 3.5 (15)
41.5 ± 5.9 (17)
(pg/min-ovary/ 100 gBW)
T (pg/min-ovary/ 100 g B W )
10.00-14.35 (a)
P
(ng/min- ovary/ 100 g B W )
14.3 ± 1.8 (18)
17.6 ± 2.2 (18)
1.3 ± 0.3 (18)
0.5 ± 0.2 (19)
14.35-19.30 (b)
25.9 ± 1.6 (19)
Dl-D4a"; D1-D3*; D2D4a*; D2-D3*; D3-D4a"; D2-D4b*; D4a-D4b"; DlD4b*
Values given are the mean ± SEM; number of animals is in parentheses. " P < 0.05. "Pjg/l00g bw) FSH - . - • - • (lOjjg/WOgbw) 6 :P
