299

~DlOi~UNOAS~Y ESTRADIOL-I? D. A.

AND

OF PROGESTERONE, 17-HYDRO~PROGESTERONE, PROSlAGlANDlN F IN HUMAN CORPUS LUTEUM.

SHUTT, R. P. SHEARMAN, G. R. McMAHON

R.C. LYNEHAM, and P. GOH

A.

H, CLARKE,

Queen Elizabeth 11 Research institute for Mothers and Infants, Department of Obstetricsand Gymecology, University of Sydney, N.S.W., 2006, Australia. Received: 5,/26/75

ABSTRACT

Radioimmunoassay procedures have been adapted for the assay of progesterone, 17-hydroxyprogesterone, estradiol-173, and prostoglandin F in human The method utiliser a single homogenisation and extraction of the corpus luteum. tissue followed by froctiomtion of the steroids on alumina, and separation of the prostaglandins of the F series from the E and A series on silica gel, prior to mdioAn attempt has been made to validate the method for the progesimmuncassay. tins by comparison with results after fractionation of the progestins on Sephadex LH-20, for estmdiol-178 by comparison with values obtained with competitive protein-binding, and for prostaglandin F by comparison with values after additional purification. The results showed that peak concentrations of the three steroids in corpora Iutea from women during the luteal phase of the menstrual cycle were comHowever, parable to those found in corpora lutea from women in early pregmncy, in six out of fourteen corpora lutea from non-pregmnt women, prostaglandin F levels were higher than those found in corpom lutea from seven women in early pregnancy, i.e. 13-46 rig/g compared with 1-7 rig/g.. Of the above six corpom Iutea, four were on days 23-25 of the cycle, at a time when luteolysis would be commencing. The results in this paper support the conclusion that the corpus luteum is a major site of synthesis of the three steroids examined, although the site of synthesis of prostaglandin F is still equivocal l

INTRODUCTION Present evidence suggests that in the sheep and some other mrmmrlian species a uterine luteolysia,

pmstaglandin

F2a,

is secreted from the uterus in a

cyclic folshion and is responsible for the periodic regression of the corpus luteum in those species (?,2). (3).

However,

A similar mechanism has not been demonstmted in the human

when prostaglondin F2~was injected

luteum there was a rapid kll the injection,

in peripheml

directly

into the human corpus

progesterone during the 24 hours after

and this fall in progesterone level in the plasma was associated with

the onset of uterine bleeding(4)

.

in primates increases in intmovarian

Knobil (5) has also indiaPted that estrogen concentmtiont

in the latter

September, 2975

stages of the luteal knowledge different study

phase may have a luteolytic

of steroid

and prostaglandin

stages of the luteal

of luteolysis

in human cluded

corpora

The

index

(6) that

Reagents

: Steroid

compounds,

were

corpora

cycle

lutea

corpus

Mixer, final

luteal

The

:

ovarian

in acidified

esttudiol-17B

saline.

of 18-36

of corpus (Woelm), esterone

5%

the addition was then

Ci/mM)

England

and 5,6

were

Ramat Gan, and 1,2

Nuclear,

(3H)

(3H)

Boston,

prostaglandin

F, a

U.K.

freshly

obtained

gynaecological

air and stored at -1O’C was carried

from surgery.

until

assayed.

out and the stage of the

ml ethyl

tissue

F

cavity

in a Sorvall

dis-

Omni-

17-hydroxyprogesterone

from the homogenate

by the method used by Orczyk

and radioimmunoassay

into

and the

and Behrman

of progesterone,

17-hydroxy-

:

was evaporated

under

H

O&V/W),

in hexane (9),

separated

from tissue.

: 1) for chromatography

described

extmcted

luteum

in the central

homogenised

progesterone,

were

acetate

and the corpus

and any fluid

and then

The steroids

(9

nitrogen

and taken

on a micro-column

(R.

I. Cox,

were

in hexane

personal

then eluted

and esttadiol-17B

of 4 ml 5% ethanol

carried

in plasma

Kalamazoo,U.S.A.

Amersham,

lutea

was thawed

cut in half

and 1 72-hydroxyprogesterone

as previously

levels

(80-100

abdominal

biopsy

was weighed

Pand estradiol-17

luteum

acetate

Centre,

in liquid

of prostaglandins

Chromatography

ethyl

F

was in-

from lkapharm

from New

(85 Ci/mM)

tissue

tissue,

tissue

(8) for the extraction

progesterone

were

for elective

and prostaglandin

volume

and prostaglandin

(7).

Extraction The

to the

the mdioimmuno-

Company,

progesterone

: Corpom

Iuteum

were snap-frozen

determined

carded.

(3H) Ci/mM)

women endometrial

from surrounding

at

METHODS

were obtained

estradiol-178

to the hospital

In non-pregnant

luteum

may be critical

describes

from the Upjohn

from the Radiochemical

Human women admitted The

standards

1, 2,6,7 (40-60

and 2,4,6,7(3H)

(59 Ci/mM)

study

the corpus

of 17-hydroxyprogesterone

AND

were a gift

17-hydroxyprogesterone U.S.A.

cycle

that a

of luteinization.

Pros-s

Radioactive

within

17-hydroxyprogesterone

MATERIALS

Israel.

suggests

estradiol-178

Radioimmunoassay

as it has been suggested

are a useful

present

17-hydroxyprogesterone,

lutea,

This

concentrations

phase of the menstrual

in the human.

assay of progesterone,

action.

up in 6 ml hexane

of 0.5

g alumina

communication).

in ethanol-hexane

Progmixtures

was eluded from the same column (10).

out by the method of Lindner

Radioimmunoassay

et al.

(11).

by

of the steroids

Commercial

antisera

raised in the ewe to 1 h h drox progesterone-l l&-hemisuccinate-HSA(progesCanada) were used for progesterone terone-1 1-HS-HSA) BlOfiA, x\ontreal,

assays

:

s

WnEoxDm

301

and antisera raised in rabbits in this laboratory to 17-hydroxyprogesterone-3-(0carboxy-methyl) oxime-BSA(170H-progesterone-SCMO-BSA) and to 6-ketoestmdiol-17@-6-(O-oarboxymethyl) oxime-BSA(estradiol-17B&MO-BSA) were used for 17-hydroxyprogesterone assays and for estradiol assays respectively. Some estmdiol fmctions were assayed by the method of Shut, & Cox (10) using the estrogen receptor protein in sheep uterine cytosol. The within-assay precision of the steroid mdioimmunoassays measured as the coefficient of variation was 13-15% and the mean recovery of tritiated progesterone, 17-hydroxyprogesterone and estmdiol-17$after extraction and chromatography was 75%, 65% and 54% respectively. When 1Oug progesterone, 10 pg 17-hydroxyprogesterone and 1Opg estmdiol-178were added to 1 g corpus luteum tissue before homogenisation and then assayed, mean recoveryiS.E.( n =4) was 9.8*0.6pg, lO.l& 1.1 yg and 8.6 *O.l pg respectively after correction for procedural losses. Chromatogmphy and mdioimmunoassay of prostaglandin F : A 6 ml aliquot of the ethyl acetate extract was dried under nitrogen, taken up in 10 ml chloroform, acidified with 0.1 - 0.2 ml glacial acetic acid and chromatogmphed on 0.5 g silica gel (100-200 mesh) using chloroform/methanol mixtures to separate prostaglandins of the F series from the E and A series as previously described (12, 13). The E and A series of prostaglandins were eluted with 12 ml 2.5 % methanol in chloroform and the prostaglandin F compounds with 6 ml 7% methanol Badioimmunoassay of prostaglandin F expressed in prostaglandin in chloroform. F units was cnrried out by the method of Clarke et al. (14) except that incuba pmn was overnight at room temperature, and the-and protein-bound prostaglandins were sepamted by overnight incubation with anti-rabbit precipitating serum (Wellcome, Beckenham, England). The mean withinassay precision of the prostaglandin mdiiimmunoassay measured as the coefficient of variation was 15%, and the mean recovery of tritiated prostaglandin after extraction and chromatogmphy was 73%. When 10 ng prostaglandin F2awas added to 1-2 g corpus luteum tissue before homogenisation and then assayed, mean recovery was 11 ng f 1.2 (S.E .) n = 5 after correction for procedural losses. Specificity of the steroid and prostaglandin antisem : The crossreactions of some related compounds with the antisem used in the mdioimmunoassays are shown in Table 1. It can be seen that the antisem against progesterone -11 HS-HSA and estmdiol-178-6CMO-BSA are highly selective for progesterone and estmdiol-178 respectively. Of the compounds tested with the antisem against 170H-progesterone-3 CMO-BSA only 170H-pregnenolone showed appreciable cross-reaction. Antisera against prostaglandin F2a-BSA showed selective crossreaction with the F series of prostaglandins but negligible cross-reaction with the E or A series or with 13, 14 dihydro-15 keto-PGF2,.

S

302

Table

Specificity

1 :

T=EOIDI

of antisera

raised

with

progesterone-l

170H-progesterone-XMO-BSA, or prostaglandin

Iwe Anti

* Steroid

F2a-BSA

-Pro-

gesterone

1 -HS-HSA,

estradiol-17e6CMO-BSA

Rabbit

*

ll-

Steroid

HS-HSA

Progesterone

Anti-l

70H

-Progesterone

-

BCMO-BSA

100

170H-Progesterone

100

Deoxycorticosterone

7

Androstenedione

4

2Oa-Dihydroprogesteron

3

Pregnenolone

2

Testosterone

4

0.1

170H-Progesterone

1

Androstenediotm

Radioimmunoassay of progesterone, 17-hydroxyprogesterone, estradiol-17beta and prostaglandin F in human corpus luteum.

Radioimmunoassay procedures have been adapted for the assay of progesterone, 17-hydroxyprogesterone, estradiol-17beta, and prostaglandin F in human co...
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