BIOLOGY

OF

REPRODUCTION

(1978)

18,434-440

Testosterone, Dehydroepiandrosterone and 4-Androstene-3,1 Concentrations in Maternal and Fetal Plasma in the Last Days of Ovine Gestation1 A. E. COLAS Department

and

L. B. CURET

of Gynecology

and

of Wisconsin Center Madison, Wisconsin

University

7-dione

Obstetrics,

for Health 53706

Sciences,

ABSTRACT were implanted in the femoral artery, uterine vein and umbilical blood vessels of 12 pregnant ewes to obtain sequential plasma samples for the determination of testosterone, dehydroepiandrosterone and 4-androstene-3,17-dione by radioimmunoassay during the last weeks of gestation. Delivery occurred at 147.8 ± 0.7 days of gestation (mean ± SEM). The concentration of dehydroepiandrosterone was 12-27 times greater than that of testosterone or 4-androstene3,1 7-dione which were of the same order of magnitude. The mean dehydroepiandrosterone concentration in all the uterine vein samples, 4.32 ± 0.50, was significantly greater than in the femoral artery, 2.97 ± 0.19 ng/ml (mean ± SEM). An analysis of variance of these data grouped by 1-day intervals showed a progressive decrease in plasma dehydroepiandrosterone in the uterine vein as the time of parturinon approached. A similar analysis for 4-androstene-3,1 7-dione in umbilical vein plasma samples showed a significant increase to 550 ± 108 pg/mi (mean ± SEM) in the last day of gestation. There was a tendency for the values of dehydroepiandrosterone and testosterone to be greater in the fetal than in the maternal samples, while the levels of 4-androstene-3,1 7-dione were greater in the femoral than in the umbilical artery. The results are compatible with the hypothesis that the rate of transformation of extraplacental dehydroepiandrosterone to other C,9 steroids, and perhaps estrogens, increases during the last few days of pregnancy. Catheters

INTRODUCTION

by

Ovine parturition a surge of the

gated 1971).

estrogen in This surge

progressive

levels before

is immediately concentration

in

1970;

the

et

ab.,

1972).

A

lambs

124-130

days

results

in marked

increases

Accepted Received ‘A short

prolonged

1970; ACTH of

is quite Dixon

strong et ab.,

Nathanielsz infusion gestationab in estrogen

microsomes,

fetal

age

these greater

also

concen-

(Mann data effect

22, 1977. 21, 1977. communication of portions of this work was presented at the meeting of the Society for Gynecobogic Investigation, Tucson, Arizona, March 23-25, 1977. This research was supported in part by NIH grants No. 1-RO1-I-ID-05387-05 and No. 5-TOlHD-00104-10, by Ford Foundation grant No. 630-0505B,C

velopment

September

by

departmental

research

and

the pregnancy

and but

precursors for ewes

estrone the

corrected for body same during the (Challis et ab.,

ing,

by

rates

ovine

et al., 1975; Ash et al., would be consistent with of cortisol on the estrogen

a

greater

availability

placental

of aromatization

in vitro were in the last days

system of the placenta. the placental aromatization

February

and funds.

rates

b).

estradiob-1713

et ab., into

clearance are essentially cycle and

which are probably rates, since the

The detailed mechanism linking the increased plasma estrogen in the ewe to the increased secretion of cortisol by the adrenal gland of the fetal lamb is not fully elucidated. Cortisob added to the incubation mixtures had no effect on the conversion of 19 steroids to

et al., 1973). The in cortisol concengland 1974;

metabolic weight estrous 1973a,

plasma

around the 7th day 150 ng/ml over the

(Drost increase

fetal adrenal and Wagner,

Beitins

cortisol

fetal

10 ng/ml to nearly

last 48 h of gestation evidence tracing the tration to (Thompson

preceded unconju-

maternal plasma (Challis, is preceded in turn by a

increase

from about parturition

of

tration (Strott et al., 1974) due to increased production

On

the was of

of

significantly of gestation 1973). These an inducing synthesizing other hand, if not rate limitC1

9

estrogen

precursors might be the determining factor for the surge. Recent observations in vitro and in vivo suggest that glucocorticoids significantly enhance the 17a-hydroxylase and 17,20-byase activities required for the placenta to form the

de-

434

C,9

C1

estrogen

(Anderson

precursors

et

al.,

1975;

Steele

Steele cental)

et ab., source

be

the

fetal

comitantly

ab.,

John

1975;

IN LATE

Flint

Pierrepoint, et al,

adrenal

gland

before evidence,

their

which

1975;

might

secretion

and

parturition. On the possibility

conthat

of

the basis of that such a

source makes a significant contribution to the pool of precursors needed for the estrogen surge is difficult to evaluate, particularly in view C19

of the paucity of steroid levels in the

circulations

during

ovine gestation. the results of drosterone, terone

the

last

made

repeatedly

data on the and umbilical

2 or

In the present measurements

4-androstene-3,1

12 pregnant

3 weeks

of

article we report of dehydroepian7-dione

during

and

that

was

testos-

period

Procedures

AND on

Pregnant

1. Gestational

435

not

always

possible

to

keep

all

the

catheters

Umbilical artery samples were particularly difficult to obtain. Oxytetracycline, 0.5 g i.m., was given to the animal daily for 5 days. The length of gestation was 147.8 ± 0.7 days and the gestation age at the time the catheters were implanted was 134.8 ± 2.5 days (mean ± SEM). Additional information on the ewes used in these experiments is presented in Table 1.

in

METHODS Ewes

Steroid

Twelve pregnant ewes of various breeds with known breeding dates were brought from the farm to the Animal Care Unit at least 4 days before the experiment. The ewes were fasted for 48 h and on the day of surgery, they received 0.4 mg of scopolamine and 1 g of Ketamine [2-(methylamino)-2-(2-chlorophenyl)cyclohexanonei i.m., followed by spinal anesthesia with 10 mg of tetracaine hydrochloride. The animal was placed in the dorsal recumbent position and an endotracheal tube was inserted. During the course of the operation the ewe was allowed to

TABLE

GESTATION

patent.

ewes. MATERIALS

Surgical

published maternal

OVINE

breathe 100% oxygen and sedation was maintained with 5-10 mg of diazepam i.v., repeated as needed. The abdomen was opened under sterile conditions and the uterus was exposed. A small opening was made in the myometrium and branches of the umbilical vein and artery were catheterized with polyyinyl tubing (0.023” I.D., 0.032” O.D.). The catheters were advanced 12 inches to assure positioning in the main vessels. One or both uterine veins were similarly catheterized ascertaining that the tip of the catheter was in the main uterine vein of the occupied horn. All catheters were sewed in place with a suture of 3-0 chromic catgut through the myometruum before closing the abdomen. Another catheter was inserted into the femoral artery and advanced 15 inches so that the tip reached the aorta. All catheters were tunneled subcutaneously, exteriorized through a stab incision in the flank, filled with heparin, sealed with wire brads and maintained in a leather pouch sutured to the side of the animal. Despite daily flushing with heparm, it

progesterone and

1976). An alternate (extraplaof C1 9-steroid precursors could

increase

cortisol existing

from

1975;

et

STEROIDS

and sampling

Blood samples were collected in heparinized containers starting 2 days after the implantation of the catheters. Sampling was carried out once daily until 2-5 days before the eventual time of parturition when 3-4

daily Plasma

samples steroids

were

usually

collected.

were measured by radioimmunoassay. The following rabbit antisera against steroid oxime-bovine serum albumin conjugates were purchased from Endocrine Sciences (18418 Oxnard St., Tarsana, California): No. T3-1 25 for testosterone, No.

data on the pregnant

Days pre gnant

Determinations

ewes.

at:

Catheter Animal

tation

Delivery

No. of lambs

Blood

A4 A5 P3

139 141 128

146 148 151

1

UmA(11),

1 1

UmA (1), UtV(15)

P5

135

150

1

FA(15)

P6 P6A

136 109

146 145

1 2

P7 P11

138 140

148 152

P14 P15

137 139

147 147

1 1 2

UmA (7), FA (7), FA(26) FA(18) FA(7),UtV(7) UmV (13), FA(13) UmV(8), FA(11)

P17 P19

138 138

145

2

UmV(8),FA(10)

148

1

FA(15)

implan-

aUmA

samples

=

umbilical

is in parentheses.

artery;

UmV

=

umbilical

1

vein;

FA

=

femoral

artery;

UtV

samplesa

uterine

UmV(12), UmV

vein.

(22),

UtV

FA(11) FA

(23)

(6)

The number

of

436

COLAS

TABLE

2. Ovine

conce ntrations

plasma

of C,

AND

steroids

CURET

du ring the 25 days preceding

parturition.

4-Androstene-3,1 7-dione

Dehydroepi-

Plasma

Testosterone

androsterone

34(57)

Umbilical

356

±

Umbilical

288 160 152 68

± 51 (10)

vein (UmV) artery (UmA) Uterine vein (UtV) Femoral artery (FA) UmV minus UmA

± 10(22) ± ±

11(117) 83

5P4%) of C,9 steroids with the antiserum for 4-androstene-3,17-dione occurred with 1,4-androstadiene-3,17-dione and So- and 5(1-androstane3,1 7-dione. Plasma extractions, chromatographic fractionations and radioimmunoassays were carried out separately for each steroid following the procedures provided by the supplier; they are based on the method of Furuyama et al. (1970). Briefly, plasma diluted with distilled water was extracted with 8 vol of hexane:ethyl acetate (9:1) for the assays of dehydroepiandrosterone and testosterone. For 4.-androstene3,1 7-none, the extraction solvent was hexane:ethyl acetate (100:1). The corresponding tritiated steroid was added to the diluted plasma before extraction to determine

recoveries.

3,17-dione, were dried androsterone

triplicate aliquots of each plasma extract for the assay. In the case of dehydroepiand testosterone, the plasma extracts

TABLE

case

of

± 0.19

(134)

± ± ±

388

±

113

±

35 (58) 34(14) 33 (28) 27 (142) 73

-56±62

significant. ,1 7-dione

and ng/ml

±

SEM

for dehydroepian-

RESULTS

There to-day

were

considerable

variations

steroids. testosterone

As

in the

greater. to gain

concentrations

individual

and

concentrations

of

Table 2 shows, and 4-androstene-3,1

of the same order dehydroepiandrosterone

4-androstene-

3. Plasma dehydroepiandrosterone

±

2.97

356 243 332

0.47 (58) 0.55 (12) 0.50 (26)

were purified by column chromatography using alumina (Bio-Rad No. AG7, 100-200 mesh) which had been acid-washed and dried at 100#{176} C. The fractions eluted by 0.75% or 0.10% ethanol in hexane were used for the assays of dehydroepiandrosterone or testosterone, respectively. Logit-log plots for serial dilutions of maternal and fetal ovine serum were parallel to those for the steroid standards. The within-assay and between-assays coefficients of variation were under 9 and 14%, respectively. All concentrations reported are corrected for losses. The statistical comparisons of the data were made by analysis of variance and multiple range testing (Zar, 1974).

Dl 7-113 for dehydroepiandrosterone, 22 for 4-androstene-3,1 7-dionc.

the

±

were not statistically

are pg/mI ± SEM for testosterone and 4-androstene-3 The number of samples is in parentheses.

In

±

5.45 4.32

-0.97 ± 1.08 1.35±O.51a

8±25

UtVminusFA

4.48

of

Although testosterone

the

Uterine

veina

the

magnitude were

dayall

3

values for 7-dione were

but those for 12-27 times

umbilical blood appeared and 4-androstene-3,17-di-

(ng/ml).

Days before parturition

Umbilical

>9 8-#{176}9

...

...

7-+8 6-#{176}7 5-’#{243} 4-#{176}5 3-4 2-+3 1-+2

.

.

.

aThe

intervals

were

or nested

.

are mean

±

3.64

5.05

(1)

8.90

5.03

(1)

5.10

± ±

(12)

significantly analysis

SE (the number

± 0.84

2.88

±

3.53 3.85 2.54

± 0.82(10)

± 0.75

(1) (3) (5) (6)

0.82

(7)

(1)

3.18

4.39

± 2.11

6.54

(1)

4.74

± 1.32

2.92 3.18

±

different

of variance

4.07

(1) ± ±

0.87 (2) 1.26(5)

by analysis which

considered

of variance

of samples

(F7

the individui

is in parentheses).

2.48 (2) (1)

(2)

2.15

4.01 6.66 5.39

0.41 (17) 1.28(18)

±

(1) t

4.45

P#{176}0.155).

Values

5.86

13.5

(1) 1.85(5)

± 0.81

.

artery

1.84±0.49(10)

...

(1) ± 0.81(4) ±

.

Femoral

...

..

4.27 3.50 5.17

hierarchical

artery

...

6.53 5.00 4.59 5.22

0-#{176}1

2.134;

Umbilical

vein

lB

=

donor

2.865; sheep

2.94 2.55 P=0.034)

±

(5)

0.97 (6) 0.42 (18) (23)

± 0.26 ± 0.35 ±

(33)

0.62 (27)

but not by a

as subgroups

(F7

=

C,9

4.

TABLE

Plasma

STEROIDS

4-androstene-3,17-dione

IN LATE

OVINE

concentrations

437

GESTATION

(pg/mi).

Days before parturition

Umbilical

>9

...

...

...

8-’9

...

...

...

7-’8

.

6-’7 5-#{176}6 4-+5

O1

aThe intervals Values

one and to lose passage through umbilical

±

282

(1)

445

±

374

±

328 233

*

322 397

±

±

256

±

±

28(12)

218

± 106

273

± ±

40(17) 108 (15)

271 261

*

550

.

significantly

different

..

of samples

dehydroepiandrosterone the placenta, the

means

and

arterial

different

for

samples any

±

53 (2)

(3) 130 (3) 21 (4)

by analysis

SE (the number

venous

significantly

343

307

±

were In

not

then

grouped

samples

concentrations The data for by

1-day

obtained

intervals

more

than

for dehydrothis steroid were (except 9

for

days

the

before

parturition, which were pooled in a single group) (Table 3). Individual one-way analyses of variance for each type of plasma indicated that only the progressive decrease in dehydroepiandrosterone concentration in uterine venous

plasma

as

time

the

proached was statistically if the individual donor sidered plasma

separately. 4-androstene-3

(Table umbilical

4)

A

of

parturition

significant, sheep were similar ,1 7-dione

showed that the venous plasma samples

days

(Fig.

1).

No

significant

apbut not

only con-

analysis for concentrations mean for obtained

the 24 h period before parturition cantly higher than those for the

was

The

umbilical

artery

concentration

ical vein umbilical Finally,

than

in the

of testosterone

±

(2)

(2) (2) (3) (5)

305 359 431 395

±

56 (6) 84(7)

451

±

362

±

192 188 162 74 63

± ±

(F6,5,

=

2.304;

43 (10) 403 (2) (1)

± 224(4)

98 (6) 57(10)

± ±

89(16)

±

61 (25) 77 (34) 34 (34)

P=0.048).

3,17-dione

in

femoral artery the umbilical

either

the

was artery.

umbilical

significantly

vein greater

or

the

than

in

DISCUSSION

As steroid during

mentioned earlier, information on C19 bevels in maternal and fetal plasma ovine pregnancy is scanty. Pomerantz

and Nabbandov (1975) plasma concentration and 866

female ± 141

reported that of testosterone

the mean of

male

lambs near term was, respectively, and 459 ± 223 pg/mI (± SEM). Since

these values were derived from unchromatographed samples, they probably are not significantly higher than the corresponding umbilical concentrations in our experiments (Tables 2, 5).

On

the

hand,

other

the

concentrations

of

the WI

the over

IJ&JC6I.

VWPI

PLA*

signifi-

4 preceding

differences

were

found in the case of testosterone (Table 5). In sheep A4, both arterial and venous samples were obtained from the umbilical circulation (Fig. 2). The mean dehydroepiandrosterone concentration was significantly higher in the umbilical circulation than in the maternal femorab artery; it was also higher in the

±

507 104 345

the

maternal circulation, there was only a significant difference between the uterine venous and femoral arterial epiandrosterone.

268

artery

is in parentheses).

in its of all

steroid.

of variance

Femoral

(1)

(1)

288 229

±

vein

129 99

(1)

33 (2) 68(5) 47 (6)

±

Uterine

artery

...

were

are mean

Umbilical

..

312 462

3-4 2-+3 1-*2

veina

vein

(Table

in the

6).

umbil-

was significantly higher than in the artery or the maternal femoral artery. the concentration of 4-androstene-

I’

#{176}

0

.

-.

I#{149}.

‘ -i

-

-

.

.

-

6

nmn FIG. 1. Plasma 4-androstene-3,17-dione concentrations in ng/ml. The individual values are represented by filled circles while the vertical bars represent the mean ± SEM for the same data grouped at one-day intervals.

COLAS

438 TABLE

testosterone

5. Plasma

concentrations

AND

(pg/mi).

Days before parturition

Umbilical vein

Umbilical

>9

...

...

8-”9

...

.

78

...

3-+4 23 1-#{247}2

Values

are mean

4-androstene-3,1

(1) (2) (5)

± 146 ± 209

56 (5) 60(13) 63 (17) 65 (14)

±

317

±

360

± ±

372

0-.1

SE (the number

±

7-dione

during

in our

samples

days

of

last

the

parturition induced mean utero-ovarian their

.

..

438 390

(1) (1)

375

(1)

618 113 206

(1) (1) ± 17(2)

179

±

of samples

animals

few

collected

pregnancy

between was

356

±

dehydroepiandrosterone

to

dione and testosterone during the last days

before

125

and

73

pg/ml

rapid increases the administration

androsterone

to

fetuses

of

6. Plasma concentrations

of C,

154 144

*

32(5) 34 (6)

±

48(9)

121

±

198

±

141 187

±

15(15) 49(19) 13(28) 18 (24)

120±

± 17(4) ± ±

23 (4) 20 (5)

gestation. The isolated of gestation is capable

±

nenolone

sheep fetus at 130 days of transforming preg-

to dehydroepiandrosterone

progesterone therefore,

(Pierrepoint possible that

not

to

et ab., 1971). It an increased utilization

but

is,

before tation

(±

parturition in the ewe. of our data must be

may estrogen

days

Such an interpreregarded as only

of

these experiments. plausible hypothesis, dence is obtained, increase in the

Nevertheless, until direct

in

vivo

that a glucocorticoid-induced placental biosynthesis of

in a pregnant

ewe (A4)

during

the 6 days

before

4-Androstene-

androsterone

-3,1 7-dione

Umbilical vein (UmV) Umbilical artery (UmA) Femoral artery (FA) UmV minus UmA UmV minus FA UmA minus FA

526 272 167

4.81 5.96 1.18

389 176 416

±

213

±65C

-27 240

± 50 ± 43C

ap