Pergamon Press
Life Sciences Vol . 21, pp . 43-48 Printed In The U .S .A .
THE INFLUENCE OF LONG-TERM ESTROGEN TREATMENTS ON DAILY PLASMA PROLACTIN LEVELS IN OVARIECTOMIZED RATSI G .T . Goodman 2 and D .M . Lawson Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201 . (Received in final form April 28, 1977) Summar~ Prolactin levels were determined in the plasma of female rats from 4 to 54 days after ovariectort~y or ovariectorgy and treatment with a long acting polymer of estradiol, polyestradiol phosphate (PEP), or Silastic implants of crystalline estradiol-179 . Blood samples were withdrawn from indwelling aortic catheters in the morning (0900-1100) and afternoon (1500-1700) . Both methods of estrogen delivery elevated plasma prolactin in the morning and afternoon compared to ovariectomized controls . However, the increases in the afternoon were significantly greater than those in the morning . The difference from ovariectomized controls and the morning-afternoon differences were maintained for 25-26 days in the polyestradiol phosphate-treated group whereas those differences in the group receiving the implants of estradiol were In significant for the entire length of the experiment (54 days) . addition, there were periodic fluctuations in the morning and afterIt is noon levels of prolactin in the estradiol implanted animals . suggested that the plasma prolactin response to estrogen varies with time of day, time after administration of estrogen and with the method of estrogen delivery . The prolactin content of the anterior pituitary (1,2) and serum (3-6) Further, increases following administration of estrogen to the female rat . estrogen appears to induce a greater increase in plasma prolactin in the afternoon than in the morning (5-7) . The purpose of this study was to determine the temporal effect of longterm estrogen treatment on plasma prolactin in ovariectomized female rats sampled in the morning and in the afternoon . Materials and Methods Fifty-four mature female Sprague-Dawley rats (Spartan Research Animals, Inc ., Haslett, Michigan) weighing 230 to 285 grams were used in this study . After eight days of acclimation to lighting conditions (lights on from 0600 to 2000 hr) the rats were bilaterally ovariectomized and randomly assigned to one of three groups . One group was left untreated, a second was injected upportedôy N.I .H . Grant RR 05384 2 Present address : Department of Dairy Science, Michigan State University, East Lansing, Michigan 48834 . 43
44
Estrogen Therapy And Prolactin Levels
Vol . 21, No . 1,
1977
subcutaneously with 0 .5 mg polyestradiol phosphate (PEP ; 1 .0 mg Estradurin R, Ayerst Laboratories), and the third group received subcutaneous Silastic implants containing 12 .5 mg crystalline estradiol-l7ß . Estrogen administration was initiated at the time of ovariectomy . The implants were similar in construction to those used by Legan, Coon and Karsch (8) and each was incubated for 30 minutes in distilled water at 20°C and wiped with ethanol prior to insertion into a subcutaneous pocket in the nape of the neck . On the day of ovariectomy and estrogen administration (Day 0) 6 animals from each of the three groups had indwelling catheters inserted into the aorta through the left common carotid artery as described previously (7} . Blood sampling was initiated four days after catheterization . At 0900 hr on the first day of sampling, extensions made from polyethylene tubing (PE 50) and containing heparinized saline were connected to the indwelling catheters of three of the six animals from each group . The animals were left undisturbed until 1100 hr at which time a 1 .0 ml blood sample was withdrawn . At 1300 hr catheter extensions were connected to the catheters of the remaining three animals from each of the three experimental groups and blood samples were drawn at 1500 hr . On the morning of the next day, blood samples were drawn from the animals which were sampled during the afternoon of the previous day . Likewise, the animals sampled in the morning of the previous day were sampled in the afternoon of the second day . Samples were collected in this manner from six rats in each treatment group on Days 4-5, 7-8, 11-12, 14-15, and 18-19 . On Day 17 and again on Day 35 after ovariectomy or ovariectomy and estrogen treatment another six animals from each group were catheterized, and after a four day recovery period, were sampled for 10 days in a manner similar to the first group . Following withdrawal each blood sample was immediately placed in a chilled, plastic tube containing an equal volume of heparinized phosphate buffered saline (PBS ; pH 7 .5 ; 50 units heparin/cc), centrifuged, and the plasma frozen at -20°C until assayed . The red blood cells were resuspended in warm, sterile, physiological saline and returned to the animal from which the sample was originally drawn . Plasma prolactin was estimated at two dilutions in duplicate in a doubleantibody radioimmunoassay described by Kuo and Gala (9) with the following modifications . Plasma or standard rat prolactin (NIAMDD-RP-1 ; 11 IU/mg) diluted in 500 ul of phosphate buffered saline (PBS ; pH 7 .6) containing 1% bovine serum albumin was incubated for 6 hours at 22°C with 100 ul of 1251-rat prolactin (NIAMDD-RP-I-2, approx . 0 .25 n ) and 200 u1 of antibody prepared against rat prolactin secreted in vitro (9~ at a dilution of 1 :4000 . At the end of this incubation period 200 ul of goat anti-rabbit gamma globulin (P-4 ; Antibodies Inc ., Davis, California) at a dilution of 1 :64 were added and the tubes incubated at 4°C for 18 hours . After addition of 3 ml cold PBS, the tubes were centrifuged and the precipitate counted in a Packard Autogamma Spectrometer . One way analysis of variance (10) was used to assess statistical significance between plasma prolactin levels of ovariectomized and ovariectomized estrogen treated groups and between morning and afternoon levels within each group . Results In untreated ovariectomized rats, plasma prolactin levels in the afternoon were slightly higher than morning values after Days 4-5 although this difference was not statistically significant (Figure 1, top panel) .
Vol . 21, No . 1, 1977
Estrogen Therapy And Prolactin Levels
45
In contrast, the PEP-injected animals exhibited afternoon levels of prolactin which were significantly higher than the respective morning levels from Day 4-5 until Day 25-26 . Thereafter the afternoon levels decreased sharply and were similar to levels in the morning (Figure l, middle panel) . When compared with ovariectomized controls the PEP-treated animals exhibited significantly greater elevations in both morning and afternoon levels of prolactin ; however, the afternoon difference was maintained for 12-13 days longer than the morning difference (Figure 2) . Estrogen-implanted animals also demonstrated a significant increase in plasma prolactin in the afternoon when compared to the morning, but in contrast to the PEP-treated group, the morning-afternoon difference was maintained Both throughout the duration of the experiment (Figure 1, bottom panel) . morning and afternoon levels in estradiol-implanted animals were significantly greater than the levels in ovariectomized controls except on Days 14-15, 18-19, and 21-22 (Figure 2) . Morning and afternoon levels in the estrogen-implanted group were not significantly different from the levels in PEP-treated rats until the prolactin in this latter group decreased on Days 25-26 . In addition to these general changes in plasma prolactin in the estradiol-implanted group, both morning and afternoon levels fluctuated periodically with peak levels occurring at 2-3 week intervals . The peaks in the morning levels generally lagged 3-4 days behind those seen in the afternoon . Discussion The present study confirms many reports that estrogen stimulates prolactin secretion in the ovariectomized rat and that the degree of stimulation is greater in the afternoon than in the morning (3-7) . In addition, this investiga tion extends these observations to include the duration of the stimulatory effect of estrogen administered by two different methods . The present observation that PEP treatment significantly increased plasma levels of prolactin in the afternoon compared with the levels in the morning only until Days 25-26 differs from the previous report of Subramanian and Gala (11) which demonstrated a significant difference until Day 49 . The reason for the difference between reports is not completely clear although two procedural differences may have contributed . One of these is the time that estrogen was administered after ovariectomy . In the study of Subramanian and Gala (11) 2-3 weeks elapsed between ovariectomy and PEP treatment whereas in the present study PEP was administered on the day of ovariectomy . The second difference in procedure relates to the time after catheterization that samples were taken . In the previous report animals were catheterized and sampled at approximately weekly intervals up to 7 weeks . In the present study rats were sampled for 2-3 weeks after catheterization . A second interesting observation relating to the PEP-treated group was the difference in the duration of the stimulatory effect of estrogen in the morning compared with the afternoon . Morning levels of prolactin in the PEP group were significantly greater than those in ovariectomized rats until Day 11-12, whereas afternoon levels were significantly greater until Day 25-26 . This suggests that the mechanisms responsible for prolactin release in the morning may become less sensitive to estrogen than the mechanisms which control prolactin release in the afternoon . Another possibility, however, is that there may be a difference in the level of estrogen in circulation between the two times of the day . Subramanian and Gala (11) demonstrated a significantly lower level of estradiol in the late afternoon (1500-1700 hr) compared to late morning-early afternoon (1100-1300 hr) . Perhaps the level of prolactin in plasma in the morning reflects the effects of estrogen in circulation during the previous afternoon .
46
Estrogen Therapy Aad Prolactin Levels ~ro -- -a ~r~~
Vol . 21, No . 1, 1977 t r" oao m r" o-ou ~a r. aoi ~+ ra o~eoo
ova eorr~o~o
ooa s~ 000 ..
~r0
-
Y
r,nomr lrruwT
FIG . 1
Effect of long-term estrogen treatment on morning (0--0 ; 1100 hrs) and afternoon (H ; 1500 hrs) levels of plasma prolactin in catheterized ovariectomized rats . Top panel - Untreated controls . Middle panel - Polyestradiol phosphate (PEP ; 0 .5 mg) treated . Bottom panel - Estradiol-implanted (Silastic capsules of 12 .5 mg Estradiol-l7ß) . Statistical comparisons made between morning and afternoon within each group . The differences in the duration of the stimulatory effects of estrogen noted between PEP-treated rats and those receiving estradiol implants may be accounted for by differences in levels of estradiol in circulation although this cannot be proven without simultaneous determination of estradiol in the plasma . Studies are currently in progress to determine if this is true . The periodicity of plasma prolactin in the estradiol-implanted animals confirms an earlier report of van der Gugten and associates (5) for animals receiving subcutaneous cholesterol pellets of estrone . The mechanisms responsible for such periodic fluctuations are obscure at present, but one
Vol . 21, No . 1, 1977
Estrogen Therapy And Prolactin Levels ovX
coNraoL
o----0
PEP INJECTED "-~ ESTROGEN IMPLANT
47
iFD " aoe i;o " O oxa 1"0 < o ol "iG < o ooe *W Wq~W L ~N wHrHn
wp
w ,o stw ao a~ E
W
2 u a
ô â a o.
FIG . 2 Data redrawn from Figure 1 . same time of day .
Statistical comparisons made between groups at the
possible mechanism is a cyclic pattern of estrogen-induced cell division in the pituitary . Cyclic patterns have been demonstrated in the mitotic index of pituitary cells of male rats given a single injection of diethylstibesterol by Lloyd et al . (12) . This same group observed elevations in serum prolactin which paralléi-edthe mitotic changes in the pituitary . Acknowledgements The authors wish to thank Dr . Richard R . Gala for his advice and support of this investigation and to the Rat Pituitary Hormone Distribution Program of NIAMDD for the generous gift of rat prolactin .
48
Estrogen Therapy And Prolactin Levels
Vol . 21, No . 1, 1977
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
R .P . Reece, Proc . Soc . Exp . Biol . Med . 39 : 77-80 (1938) . J . Meites and C .W . Turner, Proc . Soc . Ex. Biol . Med . 49 : 190-193 (1942) . C .L . Chen and J . Meites, Endocrinolo 86 : 503-505 1970 . J .D . Neill, Endocrinolo 87 : 192-1 971970) . A .A . van der ugten, . SaTä and H .G . Kwa, Acta Endocr . 64 : 265-272 (1974) . L . Caligaris, J .J . Astrada and S . Taleisnik, . En ocr . _60: 205-215 (1974) . D .M . Lawson and R .R . Gala, J . Endocr . 62 : 75-83 1974 . 96 : 50-56 (1975) . S .J . Legan, G .A . Coon and F .J . Karsch, Éndocrinolo E .Y .H . Kuo and R .R . Gala, Biochim . Bio h s . cta 264 : 462-571 (1972) . R .G .D . Stee l and J .H . Torrie, _r~nc ples and Procedures of Statistics , New York, McGraw-Hill (1960) . M .G . Subramanian and R .R . Gala, Endocrinolo 98 : 842-848 (1976) . H .M . Lloyd, J .D . Meares and J . Jacobi, J . n ocr. 58 : 227-231 (1973) .
Life Sciences Vol . 21, pp . 43-48 Printed In The U .S .A .
THE INFLUENCE OF LONG-TERM ESTROGEN TREATMENTS ON DAILY PLASMA PROLACTIN LEVELS IN OVARIECTOMIZED RATSI G .T . Goodman 2 and D .M . Lawson Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201 . (Received in final form April 28, 1977) Summar~ Prolactin levels were determined in the plasma of female rats from 4 to 54 days after ovariectort~y or ovariectorgy and treatment with a long acting polymer of estradiol, polyestradiol phosphate (PEP), or Silastic implants of crystalline estradiol-179 . Blood samples were withdrawn from indwelling aortic catheters in the morning (0900-1100) and afternoon (1500-1700) . Both methods of estrogen delivery elevated plasma prolactin in the morning and afternoon compared to ovariectomized controls . However, the increases in the afternoon were significantly greater than those in the morning . The difference from ovariectomized controls and the morning-afternoon differences were maintained for 25-26 days in the polyestradiol phosphate-treated group whereas those differences in the group receiving the implants of estradiol were In significant for the entire length of the experiment (54 days) . addition, there were periodic fluctuations in the morning and afterIt is noon levels of prolactin in the estradiol implanted animals . suggested that the plasma prolactin response to estrogen varies with time of day, time after administration of estrogen and with the method of estrogen delivery . The prolactin content of the anterior pituitary (1,2) and serum (3-6) Further, increases following administration of estrogen to the female rat . estrogen appears to induce a greater increase in plasma prolactin in the afternoon than in the morning (5-7) . The purpose of this study was to determine the temporal effect of longterm estrogen treatment on plasma prolactin in ovariectomized female rats sampled in the morning and in the afternoon . Materials and Methods Fifty-four mature female Sprague-Dawley rats (Spartan Research Animals, Inc ., Haslett, Michigan) weighing 230 to 285 grams were used in this study . After eight days of acclimation to lighting conditions (lights on from 0600 to 2000 hr) the rats were bilaterally ovariectomized and randomly assigned to one of three groups . One group was left untreated, a second was injected upportedôy N.I .H . Grant RR 05384 2 Present address : Department of Dairy Science, Michigan State University, East Lansing, Michigan 48834 . 43
44
Estrogen Therapy And Prolactin Levels
Vol . 21, No . 1,
1977
subcutaneously with 0 .5 mg polyestradiol phosphate (PEP ; 1 .0 mg Estradurin R, Ayerst Laboratories), and the third group received subcutaneous Silastic implants containing 12 .5 mg crystalline estradiol-l7ß . Estrogen administration was initiated at the time of ovariectomy . The implants were similar in construction to those used by Legan, Coon and Karsch (8) and each was incubated for 30 minutes in distilled water at 20°C and wiped with ethanol prior to insertion into a subcutaneous pocket in the nape of the neck . On the day of ovariectomy and estrogen administration (Day 0) 6 animals from each of the three groups had indwelling catheters inserted into the aorta through the left common carotid artery as described previously (7} . Blood sampling was initiated four days after catheterization . At 0900 hr on the first day of sampling, extensions made from polyethylene tubing (PE 50) and containing heparinized saline were connected to the indwelling catheters of three of the six animals from each group . The animals were left undisturbed until 1100 hr at which time a 1 .0 ml blood sample was withdrawn . At 1300 hr catheter extensions were connected to the catheters of the remaining three animals from each of the three experimental groups and blood samples were drawn at 1500 hr . On the morning of the next day, blood samples were drawn from the animals which were sampled during the afternoon of the previous day . Likewise, the animals sampled in the morning of the previous day were sampled in the afternoon of the second day . Samples were collected in this manner from six rats in each treatment group on Days 4-5, 7-8, 11-12, 14-15, and 18-19 . On Day 17 and again on Day 35 after ovariectomy or ovariectomy and estrogen treatment another six animals from each group were catheterized, and after a four day recovery period, were sampled for 10 days in a manner similar to the first group . Following withdrawal each blood sample was immediately placed in a chilled, plastic tube containing an equal volume of heparinized phosphate buffered saline (PBS ; pH 7 .5 ; 50 units heparin/cc), centrifuged, and the plasma frozen at -20°C until assayed . The red blood cells were resuspended in warm, sterile, physiological saline and returned to the animal from which the sample was originally drawn . Plasma prolactin was estimated at two dilutions in duplicate in a doubleantibody radioimmunoassay described by Kuo and Gala (9) with the following modifications . Plasma or standard rat prolactin (NIAMDD-RP-1 ; 11 IU/mg) diluted in 500 ul of phosphate buffered saline (PBS ; pH 7 .6) containing 1% bovine serum albumin was incubated for 6 hours at 22°C with 100 ul of 1251-rat prolactin (NIAMDD-RP-I-2, approx . 0 .25 n ) and 200 u1 of antibody prepared against rat prolactin secreted in vitro (9~ at a dilution of 1 :4000 . At the end of this incubation period 200 ul of goat anti-rabbit gamma globulin (P-4 ; Antibodies Inc ., Davis, California) at a dilution of 1 :64 were added and the tubes incubated at 4°C for 18 hours . After addition of 3 ml cold PBS, the tubes were centrifuged and the precipitate counted in a Packard Autogamma Spectrometer . One way analysis of variance (10) was used to assess statistical significance between plasma prolactin levels of ovariectomized and ovariectomized estrogen treated groups and between morning and afternoon levels within each group . Results In untreated ovariectomized rats, plasma prolactin levels in the afternoon were slightly higher than morning values after Days 4-5 although this difference was not statistically significant (Figure 1, top panel) .
Vol . 21, No . 1, 1977
Estrogen Therapy And Prolactin Levels
45
In contrast, the PEP-injected animals exhibited afternoon levels of prolactin which were significantly higher than the respective morning levels from Day 4-5 until Day 25-26 . Thereafter the afternoon levels decreased sharply and were similar to levels in the morning (Figure l, middle panel) . When compared with ovariectomized controls the PEP-treated animals exhibited significantly greater elevations in both morning and afternoon levels of prolactin ; however, the afternoon difference was maintained for 12-13 days longer than the morning difference (Figure 2) . Estrogen-implanted animals also demonstrated a significant increase in plasma prolactin in the afternoon when compared to the morning, but in contrast to the PEP-treated group, the morning-afternoon difference was maintained Both throughout the duration of the experiment (Figure 1, bottom panel) . morning and afternoon levels in estradiol-implanted animals were significantly greater than the levels in ovariectomized controls except on Days 14-15, 18-19, and 21-22 (Figure 2) . Morning and afternoon levels in the estrogen-implanted group were not significantly different from the levels in PEP-treated rats until the prolactin in this latter group decreased on Days 25-26 . In addition to these general changes in plasma prolactin in the estradiol-implanted group, both morning and afternoon levels fluctuated periodically with peak levels occurring at 2-3 week intervals . The peaks in the morning levels generally lagged 3-4 days behind those seen in the afternoon . Discussion The present study confirms many reports that estrogen stimulates prolactin secretion in the ovariectomized rat and that the degree of stimulation is greater in the afternoon than in the morning (3-7) . In addition, this investiga tion extends these observations to include the duration of the stimulatory effect of estrogen administered by two different methods . The present observation that PEP treatment significantly increased plasma levels of prolactin in the afternoon compared with the levels in the morning only until Days 25-26 differs from the previous report of Subramanian and Gala (11) which demonstrated a significant difference until Day 49 . The reason for the difference between reports is not completely clear although two procedural differences may have contributed . One of these is the time that estrogen was administered after ovariectomy . In the study of Subramanian and Gala (11) 2-3 weeks elapsed between ovariectomy and PEP treatment whereas in the present study PEP was administered on the day of ovariectomy . The second difference in procedure relates to the time after catheterization that samples were taken . In the previous report animals were catheterized and sampled at approximately weekly intervals up to 7 weeks . In the present study rats were sampled for 2-3 weeks after catheterization . A second interesting observation relating to the PEP-treated group was the difference in the duration of the stimulatory effect of estrogen in the morning compared with the afternoon . Morning levels of prolactin in the PEP group were significantly greater than those in ovariectomized rats until Day 11-12, whereas afternoon levels were significantly greater until Day 25-26 . This suggests that the mechanisms responsible for prolactin release in the morning may become less sensitive to estrogen than the mechanisms which control prolactin release in the afternoon . Another possibility, however, is that there may be a difference in the level of estrogen in circulation between the two times of the day . Subramanian and Gala (11) demonstrated a significantly lower level of estradiol in the late afternoon (1500-1700 hr) compared to late morning-early afternoon (1100-1300 hr) . Perhaps the level of prolactin in plasma in the morning reflects the effects of estrogen in circulation during the previous afternoon .
46
Estrogen Therapy Aad Prolactin Levels ~ro -- -a ~r~~
Vol . 21, No . 1, 1977 t r" oao m r" o-ou ~a r. aoi ~+ ra o~eoo
ova eorr~o~o
ooa s~ 000 ..
~r0
-
Y
r,nomr lrruwT
FIG . 1
Effect of long-term estrogen treatment on morning (0--0 ; 1100 hrs) and afternoon (H ; 1500 hrs) levels of plasma prolactin in catheterized ovariectomized rats . Top panel - Untreated controls . Middle panel - Polyestradiol phosphate (PEP ; 0 .5 mg) treated . Bottom panel - Estradiol-implanted (Silastic capsules of 12 .5 mg Estradiol-l7ß) . Statistical comparisons made between morning and afternoon within each group . The differences in the duration of the stimulatory effects of estrogen noted between PEP-treated rats and those receiving estradiol implants may be accounted for by differences in levels of estradiol in circulation although this cannot be proven without simultaneous determination of estradiol in the plasma . Studies are currently in progress to determine if this is true . The periodicity of plasma prolactin in the estradiol-implanted animals confirms an earlier report of van der Gugten and associates (5) for animals receiving subcutaneous cholesterol pellets of estrone . The mechanisms responsible for such periodic fluctuations are obscure at present, but one
Vol . 21, No . 1, 1977
Estrogen Therapy And Prolactin Levels ovX
coNraoL
o----0
PEP INJECTED "-~ ESTROGEN IMPLANT
47
iFD " aoe i;o " O oxa 1"0 < o ol "iG < o ooe *W Wq~W L ~N wHrHn
wp
w ,o stw ao a~ E
W
2 u a
ô â a o.
FIG . 2 Data redrawn from Figure 1 . same time of day .
Statistical comparisons made between groups at the
possible mechanism is a cyclic pattern of estrogen-induced cell division in the pituitary . Cyclic patterns have been demonstrated in the mitotic index of pituitary cells of male rats given a single injection of diethylstibesterol by Lloyd et al . (12) . This same group observed elevations in serum prolactin which paralléi-edthe mitotic changes in the pituitary . Acknowledgements The authors wish to thank Dr . Richard R . Gala for his advice and support of this investigation and to the Rat Pituitary Hormone Distribution Program of NIAMDD for the generous gift of rat prolactin .
48
Estrogen Therapy And Prolactin Levels
Vol . 21, No . 1, 1977
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 .
R .P . Reece, Proc . Soc . Exp . Biol . Med . 39 : 77-80 (1938) . J . Meites and C .W . Turner, Proc . Soc . Ex. Biol . Med . 49 : 190-193 (1942) . C .L . Chen and J . Meites, Endocrinolo 86 : 503-505 1970 . J .D . Neill, Endocrinolo 87 : 192-1 971970) . A .A . van der ugten, . SaTä and H .G . Kwa, Acta Endocr . 64 : 265-272 (1974) . L . Caligaris, J .J . Astrada and S . Taleisnik, . En ocr . _60: 205-215 (1974) . D .M . Lawson and R .R . Gala, J . Endocr . 62 : 75-83 1974 . 96 : 50-56 (1975) . S .J . Legan, G .A . Coon and F .J . Karsch, Éndocrinolo E .Y .H . Kuo and R .R . Gala, Biochim . Bio h s . cta 264 : 462-571 (1972) . R .G .D . Stee l and J .H . Torrie, _r~nc ples and Procedures of Statistics , New York, McGraw-Hill (1960) . M .G . Subramanian and R .R . Gala, Endocrinolo 98 : 842-848 (1976) . H .M . Lloyd, J .D . Meares and J . Jacobi, J . n ocr. 58 : 227-231 (1973) .
