A comparison of the uterotrophic response in cyclic and ovariectomized normotensive and spontaneously
hypertensive rats
M. B. Kerr, K. Marshall and J. Senior Postgraduate Studies in Pharmacology, School of Pharmacy, University of Bradford, Bradford
received
9
bd7
idp, U.K.
January
1992
ABSTRACT
The uterotrophic response in the normotensive (CD) and the hypertensive (SHR) rat was compared in intact cyclic rats and in ovariectomized rats given oestradiol. The parameters measured were blood flow, and uterine wet and dry weights. In the cyclic animals blood flow to the oestogen target tissue varied throughout the oestrous cycle, peak flows being achieved at pro-oestrus ; in the SHR rat, however, the pro-oestrous maximum was significantly attenuated compared with the CD rat. Uterine wet and dry weights were similar. The temporal response to oestradiol in ovariecto-
mized rats showed that in the CD rat the hyperaemic response peaked earlier than in the SHR rat, significant changes in terms of increased water imbibition also occurred more quickly in the CD strain. In both strains, uterine dry weight was the last parameter to be significantly increased, the maximum weight being attained more quickly in the SHR rat. The results of this study indicated that it is the blood flow to the oestrogen target tissues of the uterus and vagina that is most susceptible to change with strain of rat. Journal of Endocrinology (1992) 135, 263\p=n-\269
INTRODUCTION
MATERIALS AND METHODS
In the rat the oestrous
Animals
cycle lasts for 4 days, levels reproductive steroids peak during pro-oestrus decline during oestrus. Uterine blood varies widely throughout the oestrous cycle in
of the and flow
relation to the hormonal status of the animal
(Harvey
&
Owen, 1976). Oestrogens
not
only
induce hyperaemia but also enhance endomet¬ rial capillary permeability (Markee, 1932) and initiate a number of metabolic and biosynthetic processes. In this study the uterine response
(in terms of uter¬ ine blood flow, wet and dry weight) was examined in two different strains, the normotensive (CD) and the
spontaneously hypertensive (SHR) rat (hypertension, the exact aetiology of which is not known, develops several months after birth) throughout the oestrous cycle. The temporal response of the two strains to exogenous oestrogen was then compared after ovariectomy, in an attempt to clarify the relation¬ ship between hypertension and the oestrogenic response.
Mature defined
virgin as
female rats
(sexual maturity
was
the earliest age at which the animal is
capable of producing young) of a CD-derived Sprague-Dawley random bred strain and spontane¬ ously hypertensive rats, SHR strain from the animal house, University of Bradford, were used throughout and were housed in light (07.00-19.00 h)- and tem¬ perature (18°C)-controlled rooms, food and water being available ad libitum. All the animals used in this study were age-matched and some were bilaterally ovariectomized at least 21 days before further experimentation and were assigned to groups at random.
phase of the oestrous cycle Vaginal smears were obtained daily by saline lavage and examined microscopically. Only rats showing two consecutive 4-day cycles were used. Determination of the
Indirect measurements of systolic arterial pressure The tail-cuff method (validated by Pfeffer, Pfeffer & Fröhlich, 1971) was employed to determine systolic arterial pressure in the conscious CD and SHR rats. Measurement of blood flow
The
microsphere technique (previously described by Phaily & Senior, 1978) was used to determine blood flow in the rat anaesthetized with sodium pentobarbitone (60 mg/kg). This dose was selected to ensure anaesthetic control throughout the experimental pro¬ cedure and minimal cardiovascular changes. The microspheres used were 15 pm in diameter (NENTrac, New England Nuclear, Boston, MA, U.S.A.), uniformly labelled with Sc45 and suspended in 10% (w/v) dextran containing 0-1% (w/v) Tween 80. Uter¬ ine blood flow was calculated using wet weight of tissue. In all cases blood flow between the adrenals and kidneys was balanced, indicating a uniform distri¬ bution of microspheres. It is recognized that in some cases blood flow measurements fall below 50 ml/min per 100 g and at this level too few microspheres will be present to determine flow accurately but these measurements are shown for comparative purposes. When dry weights are quoted the tissue was dried until the weight remained constant. Heart rate was calculated from the recording produced after cannulation of the left ventricle.
Drug administration Oestradiol (BDH, Poole, Dorset, U.K.) was dissolved in a 10% (v/v) solution of propylene glycol and was administered intravenously at a dose level of 0- 5 pg/ kg (single dose) into the tail vein of the conscious rat at various times ( 1 5, 2, 3 or 6 h) before measurements
substantially during
the course of the oestrous in strains of rat. both Uterine and vaginal blood cycle flow was at a peak during the pro-oestrous phase. At this time flow in both strains was significantly elevated compared with dioestrus or oestrus. The pro-oestrous flow to both uterus and vagina was significantly greater in the CD than in the SHR groups. Ovarian flow was at a maximum during the pro-oestrous phase, falling off during oestrus and beginning to increase again in dioestrus. vary
Influence of the oestrous cycle intact CD and SHR rats
on
uterine
weight
in
Neither uterine wet nor dry weight fluctuated signifi¬ cantly during the oestrous cycle in either strain. In the CD rat, uterine wet and dry weights ranged between 346 ±30 mg (pro-oestrus) to 405 ±42 mg (oestrus), and 61 ± 5 mg (pro-oestrus) to 75 ± 14 mg (dioestrus) respectively. In the SHR strain, the equivalent ranges were between 295 ±41 mg (dioestrus) to 384 ±48 mg (pro-oestrus) and 563 ± 3 mg (pro-oestrus) to 71 ± 7 mg (dioestrus). Influence of the oestrous cycle on cardiovascular parameters in intact CD and SHR rats
Neither cardiac output nor heart rate was altered dur¬ ing the oestrous cycle in either strain (Table 1). How¬ ever, at any chosen stage of the oestrous cycle, significant differences in cardiac output and blood pressure were observed between the two strains. Car¬ diac output was higher in the CD rat while blood pressure in the SHR was significantly elevated com¬ pared with the CD strain.
·
made. This dose of oestradiol was chosen after preliminary experiments showed it to be the minimum required to produce significant changes in uterine blood flow and weight (doses of up to 2 pg oestradiol/ kg produced no greater response). The solvent used did not have any effect on the parameters measured and these results have been grouped as controls. were
Statistical
analysis The results (expressed as means ± s.e.m.) were pared by analysis of variance and Student's (two-tailed) (Snedecor & Cochran, 1979).
com¬
Mest
RESULTS
Influence of the oestrous cycle on uterine, vaginal and ovarian blood flow in intact CD and SHR rats Figure 1 shows that flow to all three organ systems (uterus, vagina and paired ovaries) was observed to
Influence of the oestrous to other organs
cycle
on
blood flow
Blood flow to the adrenals, kidneys, spleen and stom¬ ach was not affected by the various phases of the oestrous cycle. However, of the organs examined, blood flow to the spleen was significantly higher in the CD rat throughout pro-oestrus (286 ±41 against 114± 12 ml/min per 100 g, P
hypertensive rats
M. B. Kerr, K. Marshall and J. Senior Postgraduate Studies in Pharmacology, School of Pharmacy, University of Bradford, Bradford
received
9
bd7
idp, U.K.
January
1992
ABSTRACT
The uterotrophic response in the normotensive (CD) and the hypertensive (SHR) rat was compared in intact cyclic rats and in ovariectomized rats given oestradiol. The parameters measured were blood flow, and uterine wet and dry weights. In the cyclic animals blood flow to the oestogen target tissue varied throughout the oestrous cycle, peak flows being achieved at pro-oestrus ; in the SHR rat, however, the pro-oestrous maximum was significantly attenuated compared with the CD rat. Uterine wet and dry weights were similar. The temporal response to oestradiol in ovariecto-
mized rats showed that in the CD rat the hyperaemic response peaked earlier than in the SHR rat, significant changes in terms of increased water imbibition also occurred more quickly in the CD strain. In both strains, uterine dry weight was the last parameter to be significantly increased, the maximum weight being attained more quickly in the SHR rat. The results of this study indicated that it is the blood flow to the oestrogen target tissues of the uterus and vagina that is most susceptible to change with strain of rat. Journal of Endocrinology (1992) 135, 263\p=n-\269
INTRODUCTION
MATERIALS AND METHODS
In the rat the oestrous
Animals
cycle lasts for 4 days, levels reproductive steroids peak during pro-oestrus decline during oestrus. Uterine blood varies widely throughout the oestrous cycle in
of the and flow
relation to the hormonal status of the animal
(Harvey
&
Owen, 1976). Oestrogens
not
only
induce hyperaemia but also enhance endomet¬ rial capillary permeability (Markee, 1932) and initiate a number of metabolic and biosynthetic processes. In this study the uterine response
(in terms of uter¬ ine blood flow, wet and dry weight) was examined in two different strains, the normotensive (CD) and the
spontaneously hypertensive (SHR) rat (hypertension, the exact aetiology of which is not known, develops several months after birth) throughout the oestrous cycle. The temporal response of the two strains to exogenous oestrogen was then compared after ovariectomy, in an attempt to clarify the relation¬ ship between hypertension and the oestrogenic response.
Mature defined
virgin as
female rats
(sexual maturity
was
the earliest age at which the animal is
capable of producing young) of a CD-derived Sprague-Dawley random bred strain and spontane¬ ously hypertensive rats, SHR strain from the animal house, University of Bradford, were used throughout and were housed in light (07.00-19.00 h)- and tem¬ perature (18°C)-controlled rooms, food and water being available ad libitum. All the animals used in this study were age-matched and some were bilaterally ovariectomized at least 21 days before further experimentation and were assigned to groups at random.
phase of the oestrous cycle Vaginal smears were obtained daily by saline lavage and examined microscopically. Only rats showing two consecutive 4-day cycles were used. Determination of the
Indirect measurements of systolic arterial pressure The tail-cuff method (validated by Pfeffer, Pfeffer & Fröhlich, 1971) was employed to determine systolic arterial pressure in the conscious CD and SHR rats. Measurement of blood flow
The
microsphere technique (previously described by Phaily & Senior, 1978) was used to determine blood flow in the rat anaesthetized with sodium pentobarbitone (60 mg/kg). This dose was selected to ensure anaesthetic control throughout the experimental pro¬ cedure and minimal cardiovascular changes. The microspheres used were 15 pm in diameter (NENTrac, New England Nuclear, Boston, MA, U.S.A.), uniformly labelled with Sc45 and suspended in 10% (w/v) dextran containing 0-1% (w/v) Tween 80. Uter¬ ine blood flow was calculated using wet weight of tissue. In all cases blood flow between the adrenals and kidneys was balanced, indicating a uniform distri¬ bution of microspheres. It is recognized that in some cases blood flow measurements fall below 50 ml/min per 100 g and at this level too few microspheres will be present to determine flow accurately but these measurements are shown for comparative purposes. When dry weights are quoted the tissue was dried until the weight remained constant. Heart rate was calculated from the recording produced after cannulation of the left ventricle.
Drug administration Oestradiol (BDH, Poole, Dorset, U.K.) was dissolved in a 10% (v/v) solution of propylene glycol and was administered intravenously at a dose level of 0- 5 pg/ kg (single dose) into the tail vein of the conscious rat at various times ( 1 5, 2, 3 or 6 h) before measurements
substantially during
the course of the oestrous in strains of rat. both Uterine and vaginal blood cycle flow was at a peak during the pro-oestrous phase. At this time flow in both strains was significantly elevated compared with dioestrus or oestrus. The pro-oestrous flow to both uterus and vagina was significantly greater in the CD than in the SHR groups. Ovarian flow was at a maximum during the pro-oestrous phase, falling off during oestrus and beginning to increase again in dioestrus. vary
Influence of the oestrous cycle intact CD and SHR rats
on
uterine
weight
in
Neither uterine wet nor dry weight fluctuated signifi¬ cantly during the oestrous cycle in either strain. In the CD rat, uterine wet and dry weights ranged between 346 ±30 mg (pro-oestrus) to 405 ±42 mg (oestrus), and 61 ± 5 mg (pro-oestrus) to 75 ± 14 mg (dioestrus) respectively. In the SHR strain, the equivalent ranges were between 295 ±41 mg (dioestrus) to 384 ±48 mg (pro-oestrus) and 563 ± 3 mg (pro-oestrus) to 71 ± 7 mg (dioestrus). Influence of the oestrous cycle on cardiovascular parameters in intact CD and SHR rats
Neither cardiac output nor heart rate was altered dur¬ ing the oestrous cycle in either strain (Table 1). How¬ ever, at any chosen stage of the oestrous cycle, significant differences in cardiac output and blood pressure were observed between the two strains. Car¬ diac output was higher in the CD rat while blood pressure in the SHR was significantly elevated com¬ pared with the CD strain.
·
made. This dose of oestradiol was chosen after preliminary experiments showed it to be the minimum required to produce significant changes in uterine blood flow and weight (doses of up to 2 pg oestradiol/ kg produced no greater response). The solvent used did not have any effect on the parameters measured and these results have been grouped as controls. were
Statistical
analysis The results (expressed as means ± s.e.m.) were pared by analysis of variance and Student's (two-tailed) (Snedecor & Cochran, 1979).
com¬
Mest
RESULTS
Influence of the oestrous cycle on uterine, vaginal and ovarian blood flow in intact CD and SHR rats Figure 1 shows that flow to all three organ systems (uterus, vagina and paired ovaries) was observed to
Influence of the oestrous to other organs
cycle
on
blood flow
Blood flow to the adrenals, kidneys, spleen and stom¬ ach was not affected by the various phases of the oestrous cycle. However, of the organs examined, blood flow to the spleen was significantly higher in the CD rat throughout pro-oestrus (286 ±41 against 114± 12 ml/min per 100 g, P
