Effect of Hysterectomy on Serum Luteinizing Hormone Concentrations and on Corpus Luteum Function in the Rat W. J. DE GREEF, J. DULLAART,+ AND G. H. ZEILMAKER Department of Endocrinology, Growth, and Reproduction, and ^Department of Anatomy, Erasmus University, Rotterdam, The Netherlands ABSTRACT. Serum progesterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were estimated in intact and hysterectomized pseudopregnant rats. Progesterone concentrations increased significantly from day 2 of pseudopregnancy (PSP) onwards, and a plateau was reached on days 4 and 5. No significant differences in progesterone concentrations between intact and longterm hysterectomized animals were observed until day 10 of PSP. In the intact rats, progesterone concentrations decreased after day 8, but in the hysterectomized rats they remained elevated until days 18-20 of PSP. No differences between FSH concentrations in intact and hysterectomized rats were found. In hysterectomized rats, however, LH concentrations were significantly lower than those in the intact rats on days 2-7 of PSP. When hysterectomy was performed on day 2 of PSP (acute hysterectomy) lower LH concentrations were also found in the hysterectomized animals compared with sham-operated animals. No differ-
ences in progesterone concentrations were found between sham-operated controls and acutely hysterectomized animals on days 3-8 of PSP. It was found that LH concentrations in ovariectomized long-term hysterectomized rats were lower than those in ovariectomized, but otherwise intact, animals. In contrast to its effect on tonic LH secretion, long-term hysterectomy had no influence on the surge of LH during the afternoon of proestrus. In both intact and long-term hysterectomized rats the duration of PSP was not significantly altered after transplantation of an ovary under the kidney capsule and the removal of the ovaries in situ. It is concluded that hysterectomy leads to a decreased LH secretion by the pituitary gland, but has no influence on the maximal progesterone concentrations during PSP. The significance of these findings, particularly with regard to the prolongation of PSP after hysterectomy, is discussed. (Endocrinology 98: 1228, 1976)
AS early as 1923, Loeb (2) reported that i l the removal of the uterus resulted in a prolongation of the luteal phase in the guinea pig. This finding suggested that the uterus influences the life span of the corpus luteum, perhaps by the secretion of a luteolytic factor. Hysterectomy also leads to a prolongation of the rat's luteal phase, but, in spite of numerous investigations, the mechanism of action of the uterus on the corpus luteum of the rat is still not understood. In recent years much attention has been focussed on the possibility that a prostaglandin could be the luteolytic factor.
There is some evidence in favor of this view in the sheep (3) and the guinea pig (4). Prostaglandin F 2a may terminate pseudopregnancy as well as pregnancy in the rat (5,6), by greatly reducing the peripheral progesterone concentrations (7,8). In the rat, however, the physiological importance of prostaglandins during luteolysis may be questioned, since Saksena et al. (9) could only demonstrate a slight increase in the circulation in F prostaglandins around day 7 of pseudopregnancy and no increase in E prostaglandins. In the present study the effect of hysterectomy on luteal function was reinvestigated by comparing hormone concentrations in intact and hysterectomized animals. Furthermore, the duration of pseudopregnancy was studied in animals bearing an ovarian graft, in order to investigate whether the uterus
Received October 16, 1975. Supported in part by the Organization for Medical Research in the Netherlands (FUNGO). Part of these results were presented at the X Acta endocrinologica Congress, 1975 (1).
1228
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1229
HYSTERECTOMY AND LH
exerts a local or systemic effect upon coipus luteum function. Materials and Methods Three to 7-month-old female (RxU) F, hybrid rats were used. The animals were kept at a temperature of 22-24 C and exposed to a schedule of 14 h light and 10 h darkness. They received standard laboratory diet and tap water ad libitum. Vaginal smears were taken 6 days per week, and animals were used after three consecutive 5-day cycles. On the evening of proestrus, female rats were caged overnight with vasectomized male rats and were inspected for the presence of a vaginal plug the following morning (day 0 of pseudopregnancy). The duration of pseudopregnancy was determined as described elsewhere (10). Hysterectomy was performed in most of the animals at an age of 6-8 weeks. The uterus was exposed through a mid-line abdominal incision and after ligation it was removed via trans-section just proximal to the cervix and at the uterotubal junctions. In one experiment a group of animals was hysterectomized or sham-operated on day 2 of pseudopregnancy. Ovariectomy was performed through dorsolateral incisions and ligation. Ovaries for transplantation were taken from 28-day-old rats and immediately afterwards transplanted under the kidney capsule of an adult female rat. In one experiment, ovaries were removed on day 6 of pseudopregnancy, and, after processing, sections were made in order to determine the number and maximum diameter of the coipora lutea. Blood was usually collected between 0900 and 1000 h by puncturing the orbital plexus under light ether anesthesia. From each animal 2-4 blood samples of about 0.5 ml were taken. At least 2 days elapsed between successive bleedings. After the removal of the animal from its cage, blood sampling was performed within 1-2 minutes. After collection, the blood was allowed to clot overnight in a refrigerator. The serum samples were stored at —18 C until assayed. The animals which were bled on the afternoon of proestrus were checked for ovulation the following morning. Only blood samples from animals which had ovulated (about 95% of the animals) were assayed for LH. From each
of these rats 2 blood samples were taken during the afternoon of proestrus. Serum LH and FSH were estimated by radioimmunoassays as described previously (11,12). NIAMDD-rat-LH RP-1 and NIAMDD-rat-FSH RP-1 were used as standards. Serum progesterone concentrations were determined with a radioimmunoassay (13), as reported previously (14). All samples were assayed in at least two serum dilutions. The results were analysed using a two-factor analysis of variance for the overall analysis, and the Student's t test for testing data from various days. Differences were considered to be significant if the double-tail probability was =£0.05. The results are given as means ± SEM. Results The duration of pseudopregnancy in intact and long-term hysterectomized rats bearing a transplanted ovary The duration of pseudopregnancy in 8 intact animals was 13.9 ± 0.3 days, and in 8 hysterectomized animals 20.9 ± 0.6 days (P < 0.005). In the same animals an immature ovary was transplanted under the kidney capsule following pseudopregnancy. Ten days later the ovaries in situ were removed, and after two weeks the animals
were mated again with vasectomized males. The duration of this second pseudopregnancy, sustained only by an ovarian graft, was 15.0 ± 0.3 days in the rats with a uterus, and 18.9 ± 0.7 days in the longterm hysterectomized animals (P < 0.005). The duration of pseudopregnancy was not significantly different in the animals with the ovaries in situ compared with the same animals with only an ovarian graft. Serum progesterone, FSH, and LH concentrations in intact and long-term hysterectomized animals during pseudopregnancy In both intact and long-term hysterectomized rats progesterone concentrations increased from day 2 of pseudopregnancy onwards and reached a plateau on days
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1230
DE GREEF, DULLAART AND ZEILMAKER
Endo • 1976 Vol 98 • No 5
90"
FIG. 1. Serum progesterone concentrations (means ± SEM) in intact (n = 6; days 4-8, n = 10) and long-term hysterectomized (n = 6; days 4-8, n = 14) pseudopregnant rats. After day 10 progesterone concentrations were significantly lower in the intact rats than in the longterm hysterectomized animals (t test: day 11, P < 0.05, days 12-16, P < 0.005).
80 7 0
!
« 60 o 41
S 50 2 "40 30 20 10 J
10
20
doy of pseudopregnancy
4 and 5 (Fig. 1). Until day 10 no significant differences were found in progesterone concentrations between intact and hysterectomized rats. In intact rats progesterone concentrations decreased after day 8, but in the hysterectomized rats progesterone concentrations remained elevated until days 18-20. Moreover, it was found that the maximum diameter of the corpora lutea on day 6 of pseudopregnancy was not significantly different between the two groups of animals: 1.11 ± 0.20 mm in 10
intact animals and 1.13 ± 0.05 mm in 13 long-term hysterectomized animals. Also the number of corpora lutea was not significantly different: 11.8 ± 0.3 and 12.2 ± 0.5, respectively. Serum FSH concentrations were measured on days 2, 5, 8, and 11. The mean values were between 80 and 120 ng FSH RP-l/ml serum, and no significant differences were found between intact (n = 8) and long-term hysterectomized (n = 9) animals.
35 -,
30 -
FIG. 2. Serum LH concentrations (means ± SEM) in intact (n = 6-16; white bars) and longterm hysterectomized (n = 6 13; black bars) pseudopregnant rats. On days 2-7, LH concentrations were lower in long-term hysterectomized rats than in intact animals (t test: day 2, P < 0.05; days 4, 6, and 7, P < 0.025; and days 3 and 5, P < 0.005; overall analysis: P < 0.005).
25 E \ 20 -
z ? 10
5-
day of pieudopregnancy
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HYSTERECTOMY AND LH
1231
TABLE 1. Serum LH and progesterone concentrations (means ± SEM) in rats after the removal of the uterus on day 2 of pseudopregnancy ng NIAMDD-rat-LH RP-l/ml serum Day of pseudopregnancy
Sham-operated animals
2 3
4 5 6 7 8
ng progesterone/ml serum Sham-operated animals
Hysterectomized animals
23.7 ± 1.8 (10) t 18.9 21.1 20.8 18.2 17.8 22.3
dt dt dt dt dt dt
2.5 (7) 2.6 (8) 2.6 (8) 3.6 (7) 1.1 (8) 2.1 (8)
Hysterectomized animals
16.0 ± 1.9 (10) a
39.0 67.9 68.3 75.4 62.0 56.9
11.2 ± 2.0 (7) 13.6 ± 1.9 (6)b 12.6 ± 2.0 (7)b 15.9 ± 2.2 (8) 15.1 ± 1.8 (7) 13.5 ± 1.8 (7)c
dt 3.1 (7) dt 9.3 (8) dt 6.2 (8) dt 12.3 (7) dt 5.4 (8) db 5.8 (8)
36.7 ± 4.8 65.3 ± 3.0 83.3 ± 5.7 80.4 ± 8.6 77.4 ± 4.6 73.3 ± 9.4
(7) (6) (7) (8) (7) (7)
t Number of animals in parentheses. Significantly different from sham-operated animals (a P < 0.025; b P < 0.05; c P < 0.005).
n bc
Serum LH concentrations were measured on days 2 through 8 of pseudopregnancy (Fig. 2). In the long-term hysterectomized animals, LH concentrations were significantly lower than those found in the intact rats from day 2 up to and including day 7.
sented in Table 1. As can be seen from this table, no differences in progesterone concentrations were observed between the operated and sham-operated animals. However, an overall analysis revealed that following hysterectomy lower LH concentrations were present (P < 0.01).
Serum progesterone and LH concentrations on days 2-8 of pseudopregnancy after removal of the uterus on day 2 of pseudopregnancy The observed mean serum progesterone and LH concentrations (±SEM) on days 2-8 of pseudopregnancy after hysterectomy or sham-operation on day 2 are pre-
Serum LH concentrations in long-term hysterectomized and intact rats after ovariectomy Intact and long-term hysterectomized rats were ovariectomized, and blood was collected 1, 2, and 3 weeks after ovariectomy. Significantly lower LH concentrations
500-,
2 400 FIG. 3. Serum LH concentrations (means ± SEM) in rats with intact uteri (week 0, n = 8; weeks 1-3, n = 13; white bars) and long-term hysterectomized (week 0, n = 8; weeks 1-3, n = 12; black bars) animals following ovariectomy. Following ovariectomy lower LH concentrations were found in hysterectomized animals (t test: week 1, P < 0 . 0 5 ; weeks 2 and 3, P < 0.005).
I 300-
Q Q
I
200-
ences in progesterone concentrations were found between sham-operated controls and acutely hysterectomized animals on days 3-8 of PSP. It was found that LH concentrations in ovariectomized long-term hysterectomized rats were lower than those in ovariectomized, but otherwise intact, animals. In contrast to its effect on tonic LH secretion, long-term hysterectomy had no influence on the surge of LH during the afternoon of proestrus. In both intact and long-term hysterectomized rats the duration of PSP was not significantly altered after transplantation of an ovary under the kidney capsule and the removal of the ovaries in situ. It is concluded that hysterectomy leads to a decreased LH secretion by the pituitary gland, but has no influence on the maximal progesterone concentrations during PSP. The significance of these findings, particularly with regard to the prolongation of PSP after hysterectomy, is discussed. (Endocrinology 98: 1228, 1976)
AS early as 1923, Loeb (2) reported that i l the removal of the uterus resulted in a prolongation of the luteal phase in the guinea pig. This finding suggested that the uterus influences the life span of the corpus luteum, perhaps by the secretion of a luteolytic factor. Hysterectomy also leads to a prolongation of the rat's luteal phase, but, in spite of numerous investigations, the mechanism of action of the uterus on the corpus luteum of the rat is still not understood. In recent years much attention has been focussed on the possibility that a prostaglandin could be the luteolytic factor.
There is some evidence in favor of this view in the sheep (3) and the guinea pig (4). Prostaglandin F 2a may terminate pseudopregnancy as well as pregnancy in the rat (5,6), by greatly reducing the peripheral progesterone concentrations (7,8). In the rat, however, the physiological importance of prostaglandins during luteolysis may be questioned, since Saksena et al. (9) could only demonstrate a slight increase in the circulation in F prostaglandins around day 7 of pseudopregnancy and no increase in E prostaglandins. In the present study the effect of hysterectomy on luteal function was reinvestigated by comparing hormone concentrations in intact and hysterectomized animals. Furthermore, the duration of pseudopregnancy was studied in animals bearing an ovarian graft, in order to investigate whether the uterus
Received October 16, 1975. Supported in part by the Organization for Medical Research in the Netherlands (FUNGO). Part of these results were presented at the X Acta endocrinologica Congress, 1975 (1).
1228
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1229
HYSTERECTOMY AND LH
exerts a local or systemic effect upon coipus luteum function. Materials and Methods Three to 7-month-old female (RxU) F, hybrid rats were used. The animals were kept at a temperature of 22-24 C and exposed to a schedule of 14 h light and 10 h darkness. They received standard laboratory diet and tap water ad libitum. Vaginal smears were taken 6 days per week, and animals were used after three consecutive 5-day cycles. On the evening of proestrus, female rats were caged overnight with vasectomized male rats and were inspected for the presence of a vaginal plug the following morning (day 0 of pseudopregnancy). The duration of pseudopregnancy was determined as described elsewhere (10). Hysterectomy was performed in most of the animals at an age of 6-8 weeks. The uterus was exposed through a mid-line abdominal incision and after ligation it was removed via trans-section just proximal to the cervix and at the uterotubal junctions. In one experiment a group of animals was hysterectomized or sham-operated on day 2 of pseudopregnancy. Ovariectomy was performed through dorsolateral incisions and ligation. Ovaries for transplantation were taken from 28-day-old rats and immediately afterwards transplanted under the kidney capsule of an adult female rat. In one experiment, ovaries were removed on day 6 of pseudopregnancy, and, after processing, sections were made in order to determine the number and maximum diameter of the coipora lutea. Blood was usually collected between 0900 and 1000 h by puncturing the orbital plexus under light ether anesthesia. From each animal 2-4 blood samples of about 0.5 ml were taken. At least 2 days elapsed between successive bleedings. After the removal of the animal from its cage, blood sampling was performed within 1-2 minutes. After collection, the blood was allowed to clot overnight in a refrigerator. The serum samples were stored at —18 C until assayed. The animals which were bled on the afternoon of proestrus were checked for ovulation the following morning. Only blood samples from animals which had ovulated (about 95% of the animals) were assayed for LH. From each
of these rats 2 blood samples were taken during the afternoon of proestrus. Serum LH and FSH were estimated by radioimmunoassays as described previously (11,12). NIAMDD-rat-LH RP-1 and NIAMDD-rat-FSH RP-1 were used as standards. Serum progesterone concentrations were determined with a radioimmunoassay (13), as reported previously (14). All samples were assayed in at least two serum dilutions. The results were analysed using a two-factor analysis of variance for the overall analysis, and the Student's t test for testing data from various days. Differences were considered to be significant if the double-tail probability was =£0.05. The results are given as means ± SEM. Results The duration of pseudopregnancy in intact and long-term hysterectomized rats bearing a transplanted ovary The duration of pseudopregnancy in 8 intact animals was 13.9 ± 0.3 days, and in 8 hysterectomized animals 20.9 ± 0.6 days (P < 0.005). In the same animals an immature ovary was transplanted under the kidney capsule following pseudopregnancy. Ten days later the ovaries in situ were removed, and after two weeks the animals
were mated again with vasectomized males. The duration of this second pseudopregnancy, sustained only by an ovarian graft, was 15.0 ± 0.3 days in the rats with a uterus, and 18.9 ± 0.7 days in the longterm hysterectomized animals (P < 0.005). The duration of pseudopregnancy was not significantly different in the animals with the ovaries in situ compared with the same animals with only an ovarian graft. Serum progesterone, FSH, and LH concentrations in intact and long-term hysterectomized animals during pseudopregnancy In both intact and long-term hysterectomized rats progesterone concentrations increased from day 2 of pseudopregnancy onwards and reached a plateau on days
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 October 2015. at 00:33 For personal use only. No other uses without permission. . All rights reserved.
1230
DE GREEF, DULLAART AND ZEILMAKER
Endo • 1976 Vol 98 • No 5
90"
FIG. 1. Serum progesterone concentrations (means ± SEM) in intact (n = 6; days 4-8, n = 10) and long-term hysterectomized (n = 6; days 4-8, n = 14) pseudopregnant rats. After day 10 progesterone concentrations were significantly lower in the intact rats than in the longterm hysterectomized animals (t test: day 11, P < 0.05, days 12-16, P < 0.005).
80 7 0
!
« 60 o 41
S 50 2 "40 30 20 10 J
10
20
doy of pseudopregnancy
4 and 5 (Fig. 1). Until day 10 no significant differences were found in progesterone concentrations between intact and hysterectomized rats. In intact rats progesterone concentrations decreased after day 8, but in the hysterectomized rats progesterone concentrations remained elevated until days 18-20. Moreover, it was found that the maximum diameter of the corpora lutea on day 6 of pseudopregnancy was not significantly different between the two groups of animals: 1.11 ± 0.20 mm in 10
intact animals and 1.13 ± 0.05 mm in 13 long-term hysterectomized animals. Also the number of corpora lutea was not significantly different: 11.8 ± 0.3 and 12.2 ± 0.5, respectively. Serum FSH concentrations were measured on days 2, 5, 8, and 11. The mean values were between 80 and 120 ng FSH RP-l/ml serum, and no significant differences were found between intact (n = 8) and long-term hysterectomized (n = 9) animals.
35 -,
30 -
FIG. 2. Serum LH concentrations (means ± SEM) in intact (n = 6-16; white bars) and longterm hysterectomized (n = 6 13; black bars) pseudopregnant rats. On days 2-7, LH concentrations were lower in long-term hysterectomized rats than in intact animals (t test: day 2, P < 0.05; days 4, 6, and 7, P < 0.025; and days 3 and 5, P < 0.005; overall analysis: P < 0.005).
25 E \ 20 -
z ? 10
5-
day of pieudopregnancy
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 08 October 2015. at 00:33 For personal use only. No other uses without permission. . All rights reserved.
HYSTERECTOMY AND LH
1231
TABLE 1. Serum LH and progesterone concentrations (means ± SEM) in rats after the removal of the uterus on day 2 of pseudopregnancy ng NIAMDD-rat-LH RP-l/ml serum Day of pseudopregnancy
Sham-operated animals
2 3
4 5 6 7 8
ng progesterone/ml serum Sham-operated animals
Hysterectomized animals
23.7 ± 1.8 (10) t 18.9 21.1 20.8 18.2 17.8 22.3
dt dt dt dt dt dt
2.5 (7) 2.6 (8) 2.6 (8) 3.6 (7) 1.1 (8) 2.1 (8)
Hysterectomized animals
16.0 ± 1.9 (10) a
39.0 67.9 68.3 75.4 62.0 56.9
11.2 ± 2.0 (7) 13.6 ± 1.9 (6)b 12.6 ± 2.0 (7)b 15.9 ± 2.2 (8) 15.1 ± 1.8 (7) 13.5 ± 1.8 (7)c
dt 3.1 (7) dt 9.3 (8) dt 6.2 (8) dt 12.3 (7) dt 5.4 (8) db 5.8 (8)
36.7 ± 4.8 65.3 ± 3.0 83.3 ± 5.7 80.4 ± 8.6 77.4 ± 4.6 73.3 ± 9.4
(7) (6) (7) (8) (7) (7)
t Number of animals in parentheses. Significantly different from sham-operated animals (a P < 0.025; b P < 0.05; c P < 0.005).
n bc
Serum LH concentrations were measured on days 2 through 8 of pseudopregnancy (Fig. 2). In the long-term hysterectomized animals, LH concentrations were significantly lower than those found in the intact rats from day 2 up to and including day 7.
sented in Table 1. As can be seen from this table, no differences in progesterone concentrations were observed between the operated and sham-operated animals. However, an overall analysis revealed that following hysterectomy lower LH concentrations were present (P < 0.01).
Serum progesterone and LH concentrations on days 2-8 of pseudopregnancy after removal of the uterus on day 2 of pseudopregnancy The observed mean serum progesterone and LH concentrations (±SEM) on days 2-8 of pseudopregnancy after hysterectomy or sham-operation on day 2 are pre-
Serum LH concentrations in long-term hysterectomized and intact rats after ovariectomy Intact and long-term hysterectomized rats were ovariectomized, and blood was collected 1, 2, and 3 weeks after ovariectomy. Significantly lower LH concentrations
500-,
2 400 FIG. 3. Serum LH concentrations (means ± SEM) in rats with intact uteri (week 0, n = 8; weeks 1-3, n = 13; white bars) and long-term hysterectomized (week 0, n = 8; weeks 1-3, n = 12; black bars) animals following ovariectomy. Following ovariectomy lower LH concentrations were found in hysterectomized animals (t test: week 1, P < 0 . 0 5 ; weeks 2 and 3, P < 0.005).
I 300-
Q Q
I
200-
