Effect of estradiol-17P on blood flow to reproductive and nonreproductive tissues in pregnant ewes CHARLES
R.
FRANK
H.
MORRISS.
L.
MAKOWSKI
FREDERICK EDGAR
ROSENFELD
C.
GIA 0.20. is not significant at P > 0.10.
Microsphere infusion. Radioactive microspheres* with a specific activity of 10 mCi. per gram and a mean diameter of 25 p -+ 5 S.D. were employed. Approximately 150 PCi of 14’Ce and 350 &i of j’Cr were administered t-o each animal. The mean disintegrations per minute (DPM) and number of microspheres infused with each isotope are presented in Table I. The methods of infusing the microspheres and determining the number infused have been described.13 Prior to the microsphere infusion, reference samples were withdrawn from both femoral arterial catheters into counting vials by a pump? at a rate of 3.88 ml. per minute for three minutes. Simultaneous samples were also withdrawn from one of the uterine vein catheters and the femoral vein catheter for an equal period of
*Minnesota Mining and Manufacturing Co., St. Paul, Minnesota 55101. tHarvard Apparatus Co., 150 Dover Rd., Millis, Massachusetts 02054.
1.5, 1970; Gynecol.
time in order to detect the escape of mic-losphere.\ into the venous circulation from the uterine vascular bed and other vascular beds in the body. The DPM in the3e samples were compared to the DPM in the arterial sample (Table II). There was no significant loss of’ microspheres from the uterine vascular becl either before or after the vasodilatory effects ot I& confirming the results of pre\-ious studies from this laboratorv.7, ” There was a significant shunting of. microspheres across other- vascular beds into the inferior vena cava. equaling approximately 2 per cent of’ the infused number of microspheres. ‘[‘his flgui-e did not change significantly following an infusion of E:?. Tissue preparation. The ewes were weighed and killed jvithin two to four hours aftrl the second microsphere infusion and dissected f’or tissue preparation. All tissues were placed in prcweighetl, numbered counting vials (25 mm. in diameter. X0 mm. in length). The preparation of the ~lo~lp~-egmm~ and pregnant uteri and other tissues was 3s previousI\ described.l-’ Samples of skeletal muscle were taken from the lateral chest \~a11 and hind limb. Skin sample5 \\.ere taken from the foreleg. chest. and the skin overlying the mammary gland. Radioactive assay. The samples were counted in an automatic dual-channel gamma (,ounter*: ivith a threeinch crystal detector and a wide well i 1 !/x inches in diameter). by a method previousl!, reported.” Oxygen assay. Whole-blood oxygen content ~.as measured with a gas chromatographt equipped with a blood gas accessory.9 Date reduction. Individual organ blood flows \\crt calculated with the integrated arterial ref’erencr samples”: Organ blood flow (ml.imin.1 = total DPM in an organ mean DPM in arterial samples x rate of withdrawal. With a similar equation, mined simultaneously:
cardiac
output
1.
was deter-
Cardiac output (ml./min.) = total DPM infused mean DPM in arterial samples x rate of withdrawal. For the purpose
of statistical analysis the animals
2.
are
*Nuclear-Chicago Corp., 2000 Nuclear Dt-., Des PIaims. Illinois 60018. KX-PA, Beckman Instruments, Inc.. 2500 Harbor Blvd.. Fullerton, California 92634.
Volume Number
124 6
E2 effect
Table III. Blood nonpregnant and minutes after the Ez. The hormone two minutes
on blood
flow
to tissues
621
flow to the uterine tissues of pregnant ewes, before and 100 injection of 1 Fg per kilogram of was injected intravenously over Bloodjlow
Tissue
Before
Total uterine blood flow Nonpregnant 23.36 Group I 358.90 Group II 1,099.oo Myometrium Nonpregnant 9.46 Group I 25.20 Group II 35.28 Endometrium Nonpregnant 7.76 Group I 116.00 Group II 157.60 Cotyledons or Caruncles Nonpregnant 7.23 Group I 214.70 Group II 906.20
(ml.lmin.)
EZ
After
k 2.87 + 69.28 * 173.50 ” + t
346.20 625.40 1,575.OO
E2
k 28.27* ” 76.63* ” 231.70*
1.73 4.92 5.10
148.50 113.70 122.70
? f +
23.11* 22.21* 10.94*
k 1.27 ZL 15.78 ” 22.97
110.10 295.30 314.90
” f +
14.86* 41.33* 39.35*
IT 1.26 * 46.23 2 155.30
87.59 252.50 1,138.OO
2 11.09* f 42.8lt * 119.50t
The mean 2 1 S.E.M. is presented. *The change in flow is significant at P< 0.005 (paired t test). t The change in flow is significant at P < 0.05 (paired t test).
divided into three groups in the tables and figures: six nonpregnant ewes, seven ewes at 38 to 78 days of gestation (Group I), and six ewes at 120 and 139 days of gestation (Group II).
Results Effect of E2 on the uterine vascular bed. In previous reports we documented the inhibitory effect of surgical stress on the response of uterine blood flow to E2 in nonpregnant ewe+ I3 and thus performed this series of experiments after the animals had recovered from operation and had resumed a normal diet. In both pregnant and nonpregnant ewes there was a significant increase in uterine blood flow following the systemic infusion
of
average
increase
1 pg
per
kilogram
in
flow
of in
Ez (Table
milliliters
III). per
The minute
GESTATlOlYAL A,OE (DAYS)
Fig. 2. Effect of estradioLl7P on total Graph A shows the flow before and after per minute; B, the per cent change absolute change in flow (milliliters per nant, early pregnant (Group I), and late ewes.
uterine estradiol in flow; minute) pregnant
blood flow. in milliliters and C, the in nonpreg(Group II)
(Fig. 2. C) was not significantly different among the nonpregnant and the two pregnant groups and was equal data
to
351
expressed
ml.
per
as a per
minute cent
f
36
change
demonstrate a mean increase of S.E.M. in the nonpregnant ewes,
1,455 while
S.E.M.
The
same
in flow
(Fig.
2, B)
per cent ? 197 in the pregnant
animals there was a gradual decline in the relative magnitude of the change from 225 per cent at 38 days to a mean of 43 per cent near term (Group II). As shown in Table III and Figs. 3, 4, and 5, all three
major uterine tissues, i.e., myometrium, endometrium, and cotyledons (represented by the caruncles in the nonpregnant uterus), showed significant increases in blood
flow
following
an infusion
of Ez, but
the
pattern
and magnitude of response throughout gestation were different for each tissue. Myometrial blood flow increased after estrogen stimulation to approximately the same maximum (128 ml. per minute ? 11.4
622
March Am. J. Obstet.
Rosenfeld et al.
15, 1976
Gynecol.
o After E.stradbl-R$ . Before Estradkd-17.P
0
0
200
After . eefws
E~tmdkd-17# Edmdiol-17,B
. A 0
20
so QESTATIONAL
100 uo AGE (DAYS)
GESTATRXAL
Fig. 3. Effect of estradioL17P on myometrial Graph A shows the flow before and after estradiol per minute: B, the per cent change in flow: absolute change in flow (milliliters per minute) nant, early pregnant (Group I), and late pregnant ewes.
S.E.M.)
in
significant
pregnant increase
of
tion. On the contrary, estrogen-stimulated crease
during
the response stimulation trial
and
gestation was
and nonpregnant the base-line
both endometrial endometrial blood (Fig.
4. A).
of the placental not as pronounced
endometrial
flow
flows.
In
the
In
blood flow. in milliliters and C, the in nonpreg(Group II)
days
(Group
blood
flow
blood flow
doubled
iate
pregnancy
II), was
25
the per
average cent,
increase whereas
and
myometrial
flow
EZ stimulation flow to the nonpregnant
causes sites of animal,r3
an enormous implantation
blood flow to E, as that of myome-
in
group
effect
at 120 to 139
(MK)
Fig. 4. Effect of estradiol-17/I on endometrial Graph A shows the flow before and after estradiol per minute; B, the per cent change in flow; absolute change in flow (milliliters per minute), nant, early pregnant (Group I), and late pregnant ewes.
ewes despite a during gestaflow and did in-
ME
pregnancy of
there exogenous
estrogen
of
flow
3.5-fold. increase (caruncles)
in on
placental
endometrial
increased
it is apparent is a decrease
blood flow. in milliliters and C, the in nonpreg(Group II)
Since of
blood in the
that at some time the vasodilatory the
placental
ar-
Volume Number
Ez effect on blood flow to tissues
124 6
623
Y=lol5+t8 P-O.01
3ooo.
k
g l
z
t
e $
E A $
e After Estradiol- 17.p l Before Estradiob~l7. p
1600
t
! 0
Fig. 5. Effect of estradiol-17g on blood flow on the placental cotyledons. A is the flow before and after estradiol in milliliters per minute; B is the per cent change in flow, and C is the absolute change in flow (milliliters per minute), in the caruncles of the nonpregnant ewe and in the cotyledons of early (Group I) and late (Group II) pregnancy. terioles. This time can be located at approximately the second month of gestation by considering the experiments within Group I which extended from the thirty-eighth to the seventy-eighth day of pregnancy. At 38 days, estrogen stimulation caused a marked increase of cotyledonary flow from 30.6 to 109.5 ml. per minute, i.e., a 2.6-fold increase. In the experiment at 47 days, Ez caused the cotyledonary flow to double,
. 20
.
-=....o GESTATIONAL
.l
# . . loo 140 AGE (GAYSI .
A
Fig. 6. Effect of estradiol-17P on cervical blood flow. A is the flow before and after estradiol in milliliters per minute; B is the per cent change in flow, and C is the absolute change in flow (milliliters per minute) in the nonpregnant, early pregnant (Group I), and late pregnant (Group II) ewe.
from 45.1 to 89.7 ml. per minute. In the next two experiments at 60 days, the effect of exogenous Ez on cotyledonary blood flow was virtually zero, with a mean increase from 265.2 to 272.8 ml. per minute. In the next group of three experiments performed between the sixty-seventh and seventy-eighth days, there was an average increase of 13 per cent, from 299.0 to 340.9 ml. per minute. Effect of E2 on other reproductive tissues. There was a significant vasodilatory effect of Ez on blood flow to the tissues of the cervix, vagina, ovaries, uterine tubes, and mammary gland throughout pregnancy
624
Rosenfeld et al.
*
B f
March Am. J. Obstet.
15, 1976 Gynecol.
Y=l626+(-6.92)x PcO.05
300
6 ; Y E 200 0.
l
c * *t
1000
* * * t
6
P
f 6.0 ! E .m t
0
c
Y=251.7+(-1.57)x P- ,001
oAfter Estradiol-17,p *Before Estrediol-17,p
20
l --:
\
--
GEST%lONiL
A 0 After
i%
(QAY5; la0
l
6efwe
Estradbk17,B Estradlol-l7.p
2 .
Fig. 7. Effect of estradiol-17/3 on vaginal blood flow plotted against gestational age. A is the flow before and after estradiol in milliliters Per minute . gram, and B is the per cent change in flow.
(Table IV). As in the uterine tissues, as pregnancy progressed toward term, the cervix (Fig. 6). vagina (Fig. 7), and mammary gland (Fig. 8) demonstrated a reduction in the per cent change in flow following an infusion of E,. This phenomenon was not evident in the uterine tubes and ovaries. Effect of E2 on cardiac output. The reliability of simultaneous measurements of cardiac output and tissue or organ blood flows with the microsphere method has been reported.15 This method was employed in these experiments to demonstrate the effect of E2 on cardiac output in four of the nonpregnant animals and all of the pregnant animals included in this study. There was a mean increase in cardiac output of 13.3 ml. per minute . kilogram ? 4.25 S.E.M. (P < 0.005) at 60 to 90 minutes following an infusion of Ez. This is an increase of 14.5 per cent r?r 4.51 S.E.M. A similar increase was observed by Parer and associates lo following the infusion of large
4 :*
GESTA-L
ABE &MS)
Fig. 8. Effect of estradiol-17P on blood flow to the mammary gland. A is the flow before and after estradiol in milliliters per minute: B is the Per cent change in flow, and C is the absolute change in flow (milliliters per minute) in the nonpregnant, early pregnant (Group I), and late pregnant (Group II) ewe.
doses of estrone. No significant change in mean arterial pressure was noted: 97.9 mm. Hg before E2 and 96.8 mm. Hg afterward (P > 0.40). There was a significant change in heart rate, which increased from 100 to 111 beats per minute (P < 0.001). In the last animal studied, the Ez was administered in isotonic saline, 1 pug per milliliter, rather than in the carrier solution previously employed, which contains ethyl alcohol and
Volume Number
124 6
Eg effect on blood flow to tissues
2000m I -
T
Table IV; The effect of estradiol-17/3 (E,) on blood flow to reproductive tissues of pregnant and nonpregnant ewes. Same experimental conditions as Table III Blood Jaw Tissue Cervix Cervic
Before
Vagina
(ml.lmin
4.15 *
tubes (mklmin.
Groups I and II Ovaries with corpus luteum (ml. lmin.) Group I Group II Ovaries without corpu.s luteurn (ml.Imin.)
Group I and II Mammary
Afk
Ez
0.45
3.57
2
0.62
8.01
+
1.62
73.38 56.63 49.94
f 12.45* ” 4.86* 2 5.06*
0.207 0.185
f 0,042 2 0.024
3.31 2.13
2 t
0.77t 0.14*
0.474
f
4.01
f
0.28*
. Gm.)
Nonpregnant Groups I and II Uterine
E2
(ml. /min.) (ml. /min.)
Nonpregnant Group I Group II
L
625
) 0.086
9.12
t
1.14
4.82
f
0.51
14.81 f 8.20
+
l.llt
1.29*
0.252
2
0.029
1.00
f
0.24t
(ml.lmin.)
Nonpregnant Group I Group II
10.58 15.75 151.70
+ 2.79 k 3.51 2 26.34
32.06 39.74 200.70
5 5.58? k 16.2l.f + 23.49t
Fig. 9. Comparison of the per cent change in those tissues in the nonpregnant ewe which demonstrated a significant change in flow (P < 0.01) following a systemic infusion of estradioL17P (1 pg per kilogram). The nonreproductive tissuesrare represented by the shaded bars. The means f 1 S.E.M. are presented.
The pregnant animals are divided into unless there is no significant difference The mean 2 1 S.E.M. is,presented. *The change in flow is significant at P < tThe change in flow is significant at P