Receptors for estrogens and progesterone in the porcine cervix

THERIOGENOLOGY RECEPTORS

FOR

Ph.

ESTROGENS

AND

1)"

Stanchev

A.

a;d Departments Swedish

of

'Clinical

Univeryity

Department

of

of

PROGESTERONE Kunavongkrit

H.

Eriksson

Received

L.-E.

,

CERVIX I)

Edqvist

3)

and

Karolinska

for

PORCINE

2 Obstetrics and Gynaecology, Sciences, Uppsala,Sweden and

Chemistry

Agricultural

Chemistry,

IN THE 2)""

Institutet,

Stockholm,

Sweden.

November 2, 1983 March 15, 1984

publication: Accepted: ABSTRACT

In vitro binding -levels of estradiol fractions of

the

was

of

about

maximum ing

cells

estrous

to

obtained The

level

onset

heat

of

during

sites

was

300 seen

of

the

however,

cycle,

the on the

1200

reached

day

its

to

of

used

phase

on

end

day

3-4. of

determine

at and

The

lowest

stages receptors

increased the

estradiol

luteal

in

and the

of

4700

760

sites

sites/cell

on

days

on

3-4

uterus.

support

a concept

response via

to

steroid

in

which

increased

the

in

the

constriction

concentrations

of

of

Furthermore, the

cervix

circulating

day

and

nisms

levels

of

its cycle proThe nuclear

during the luteal phase. theories on the endocrine

receptor

the

number

a steady level of about 1000 sites/cell The data obtained agree with present regulating

a

decreas-

nuclear

phase

reduction

to

cycle,

3-4.

level

of

nuclear

different

cytosolic receptor and the estradiol receptor.

a value

the

and

cytosolic

1 of

the No

1 and

to

cytosolic

cervix

luteal days

in

estradiol

sites/cell.

between

increased

porcine

sites/cell

The level of the progesterone file was very similar to that of receptor,

the

between to

were

receptors

sites/cell

increased from

from

7600

2700

methods

concentration

sites/cell

a level

nuclear

progesterone

approximately

receptor of

exchange

cycle.

4500 of

and and

mecha-

these

data

occurring

estradiol

1

showed

in

is mediatec

receptors. INTRODUCTION

of and

The

consistency

the

estrous

of

the

cycle.

edematous(l).

cervix

in

During

estrus,

Ovariectomy

causes

the the

pig

is

correlated

cervix

is

a complete

to

the

constricted,

relaxation

of

stage rigid

the

vagi-

Injection of estrogen into ovarnal portion of the porcine cervix(Z). iectomized sows causes cervical constriction. In a recent study, Kunavongkrit et a1.(3) found that the tonicity of the cervix increased from .a;a;:;imately and concluded

4 days softened

before

the

again

that

estrogen

ACKNOWLEDGEMENTS

:This

during

might

work

be

was

onset the

of

heat,was

most

postestrous

implicated

supported

in

by

firm

period. the

the

increased

Swedish

of

Reproduction,

**

be

requested.

From

whom

reprints

may

MAY 1984VOL. 21 NO. 5

Bulgarian

authors tonicity

Council

Forestry and Agricultural Research. The award of a fellowship Vienna, to Dr. Ph. Stanchev is gratefully acknowledged. * Present address: Department of Physiology and Endocrinology, of Biology and Immunology Sofia 1113, Bulgaria.

during

The

Academy

of

for

by

IAEA,

Institute of

Science,

757

THERIOGENOLOGY the cervix information

and on

that

rectal

ovarian

examination

of

the

cervix

provides

valuable

function.

The purpose of the present study was.to further elucidate the role of estrogen and progesterone in regulating the consistency of the cervix by determining the binding of these two hormones to their respective cytosolic and nuclear receptors in porcine cervical cells obtained at different stages of the estrous cycle. MATERIALS AND METHODS Experimental Animals and Sample Collection Eleven Swedish Landrace and Swedish Yorkshire qilts or Landrace X Yorkshire crossbreeds, six to eight months old and with body weights ranging from 80 to 100 kg, were used. The animals were housed in pens with three to four gilts per pen and were fed a commercial pig feed. Estrous detection was performed twice daily in the presence of a boar. After at leastone regular estrous cycle,the animals were slaughtered on day 'T(fourgilts), days 3-4 (four gilts) or days IO-13 (three gilts) of the estrous cycle. The first day of heat was defined arbitrarily as day 1 of the estrous cycle. Blood samples were collected by jugular venipuncture prior to slaughter. The samples were centrifuged at 3000 rpm. Plasma was removed and stored at -20 C until analysed for progesterone (4) and estradiol-17B (5) by radioimmunoassay. All hormone determinations were carried out in duplicate. Tissue samples from the cervices were collected immediately after slaughter. The tissue specimens were taken from the middle part of the cervix and frozen in nitrogen within 20 min after collection and kept at -7O'C until analysed. Chemicals and Buffers Radiolabeled compounds [ 2,4,6,73Hl estradiol-17B(S.A. 104 Ci/mmol), ['H-17a-methyl]prbgesterone(R'5BZO),S.A. 87 Ci/mmol and non-labelled R 50'20were obtained from New England Nuclear Corp.(Boston,MA). Diethystilbestrol(DES) and liquid scintillation cocktail were obtained from Sigma Chemical Corp.(Saint Louis,MO) and Koch-Light Lab Ltd.(Colnbrook, Berks, England),respectively. All other chemicals were of reagent grade or better. Buffer A was IO mM Tris-HCl(pH 7.4), buffer B was IO mM Tris-HCl and 1.5

mM

EDTA(pH

7.4)

and

buffer

C was

buffer

A and

IL

bovine

serum

albu-

min.

The subscript following the letter designation of a buffer( e.g. A ) denotes the percentage glycerol(v/v) in the buffer. Dextran-coated c ;1O arcoal(DCC) consisted of 1 g Norit and 50 mg Dextran T70 in 100 ml buffer( Buffer A ,. for cytosolic receptors of progesterone (PRc) and buffer B for cytosolic receptors of estradiol (ERc) 1. Receptor Assay The tissue samples from the cervix were divided into smaller portions and 300-500 mg were used for determination of progesterone receptors(PR) or of estrogen receptors(ER). The samples were weighed and placed in for PA and B for ER). All subsequent steps were carried 6 ml buffer( A out at 0-4'C e%!ept where otherwise noted. The tissue was homogenized by means of a motor-driven glass-glass homogenizer with three S- to 8-set burstsat 30-set intervals. Homogenate(0.2 ml) was taken and kept at -2O'C

758

MAY 1984VOL. 21 NO. 5

THERIOGENOLOGY for DNA measurement. The remainder was centrifuged at 800~~~ for 20 min. The supernatant was then centrifuged at 105,000xc~for 60 min and used immediately for cytosolic receptor(Rc) determination. The crude nuclear pellet from the first centrifugation was washed twice by rehomogenization in 3-ml buffer(A for PR and B for ER) and centrifuged at 800~~~ for wash. The final pellet was resuspended in 6 ml of the 15 min after eat?I0 samy buffer. [ Hlestradiol-17B and r3H] 17a-methyl(R 5020) were used as the labellcd ligand. The saturytion analyses were performed with six different concgntrations of [ H] estradiol-17B( from 0.125 to 4 nM/l) for ER or of [ HI R 5020(from 0.3 to IO nM/l) for PR. Aliquots(0.25 ml) of the cytosolic or nuclear fraction were added to two parallel series of the tubes (in duplicate), one containing 0.1 ml of the labelled steroid and the other containing 0.1 ml of the same concentration of the labelled steroid plus a 'TOO-foldmolar excess of unlabelled steroid(DE5 for ER and R5020 for PR). The optimal conditions for the DCC- and Nuclear-Exchange assays have been examined in detail and the data are presented elsewhere ( Stanchev, Ph., Edqvist, L.-E. and Eriksson, H., In preparation). The following incubation times were used for ERc,ERn,PRc and PRn: ERc and ERn 18hat 4'C PRc 1 h at 20°C PRn 24hat 4'C. Bound and free steroid in the cytosol was separated by adding 0.5 ml DCC to each tube. After incubation for IO min at O'C, the tubes were centrifuged at 3000~~~for IO min . The supernatant was added to 4 ml of liquid scintillation cocktail for radioactivity determination. Incubations of the nuclgar fractions were terminated by centrifugation at 3000~~~for IO min at 4 C. The pellet was then washed three times with 1 ml buffer (C for PRn and B for ERn). The Rn-bound ligand was extracted with 1 ml absolute ethanol( overnight,room temp.) and counted for radioactivity. Other Analytical Methods DNA concentration was determined by the method of Burton(6) as modified by Richards(7). The dissociation constant and binding data were determined by the method of Scatchard( corrected for nonspecific binding according to Chamness & McGuire(9), as was described previously(l0). The receptor concentration was expressed as number of sites per cell. The conventional statistical methods were used for analysis of the results(l1). RESULTS The dissociat.Jonconstantsc Gj3for cytosolic and nuclear pellet fractions for [ Hlestradiol and-[ HI R 5020 in the cervix were estimated from Scatchard plot analysis. Representative plots are shown in Figure 1. The Scatchard plots were linear and the K_ values differed for the two ligands. These binding data indicated a smgle class of high-affinityllC low-capacity bindinq18ites with $ values ranging from 1.8 to 4.2x IO and from 1.1 to 2.3 for estradiel and R 5020, respectively. Plasma levels of progesterone and estradiol-17B in the slaughtered pigs are shown in Table 1. The plasma progesterone level was low during day 1 (range 0.2-0.8 nmol/l) and somewhat elevated at days 3-4(range 3.1-9.6 nmol/l) of the estrous cycle. On days IO-13 of the estrous

MAY

1984 VOL. 21 NO. 5

759

cycle,progesterone level of estradiol-770 30 pmol/l) on days mean value of 37.5

levels

ranged from 22.0 to 42.0 nmol/l. The plasma was around the basal level(mean value less than 3-4 and days ‘TO-13 of the estrous cycle. A higher pmol/l was obtained on day 1 of the estrous cycle.

0.2

ESTRADIOL

0 0.15 Kd .3.86-lo

/\

lx1o-‘o

5x1 o-l0 BOUND (nM/I

1x1o-g 1

Figure

l.Representative Scatchard plot analysis of r3H]estradiol and [3 HIR 5020 binding in cytosolic(O-----C)) and nuclear(%) pellet fractions of pig cervix. Cytosolic and nuc.Jear pellet(0.25 rns) were incubated with varying concentrations of [ Hlestradiol and [ H] R5020 as described in MATERIALS AND METHODS. Each point is the mean of the duplicate values. 760

MAY 1984 VOL. 21 NO. 5

THERIOGENOLOGY Table

1. Plasma levels of progesterone and estradiol-77D in different stages of the estrous cycle

Day IO-13 Day 1 Day 3-4

(n = 3) (n q 4) (n q 4)

from 11 gilts

Progesterone(nmol/l)

Estradiol-170(pmol/l)

mean

range

mean

range

32.0 .0.3 6.9

22.0-42.0 <0.2- 0.8 3.1- 9.6

23.0 37.5 20.3

16.0-32.0 23.0-51 .O 19.0-23.0

c]

10

Cytosoli c

isl Nuclear

5

(3)

b (3)

0

10-13 DAY

Figure

2.

1

OF

ESTROUS

3-4 CYCLE

Estradiol binding in the cytosolic and nuclear fraction of the pig cervix ( mean 2 SEM ) on day 1, days 3-4 and days IO-13 of the estrous cycle. Numbers of examined samples are given in parentheses.

The estradiol binding to cytosol and nuclear receptors in the cervix in maximum amounts on the is shown in Figure 2. The ERc are present day of estrusjaverage of 7614 sites/cell),decreasing to lower levels (mean of 2670 sites/cell) on days 3-4 of the estrous cycle. The levels of ERn did not differ when the day of estrus is compared with days 3-4 of the estrous cycle. During the luteal phase (day 10-131, the mean ERc was 4595 sites/cell and the mean for ERn was 331 sites/cell.

MAY 1984 VOL. 21 NO. 5

761

THERIOGENOLOGY

q Cytosolic ISl

Nuclear

10

5

L

0 DAY

Figure

3.

OF

ESTROUS

CYCLE

Progesterone binding in the cytosolic and nuclear fractions of the pig cervix ( mean + SEM ) on day 1, days 3-4 and Numbers of examined samplec days ICI-13 of the estrous cycle. are given in parentheses.

The progesterone binding to cytosol and nuclear receptors in the cervix is shown in Figure 3. On the day of estrus , the levels of PRn were low( y = 758 sites/cell), whereas the levels of PRc were high (?? = 9156 sites/cell). On days 3-4 of the estrous cycle, PRn was high (?? q 4723 sites/cell). The mean values for PRn and PRc were lower for pigs slaughtered on days lo-13 than for pigs slaughtered on days 3-4 of the estrous cycle.

762

MAY

1984 VOL. 21 NO. 5

THERIOGENOLOGY DISCUSSION The Scatchard analysis of ligand-receptor affinity for estradiol-17l? and R 5020 displayed linearity and indicated a single class of highThe K_, for estradiol-170 lay in low-capacity binSlAng sites. affinity, for both ERc and ERn. These data were the range 2.1 to 4.2 x 10 similar to those reported for pig endometrium(l2) and rat uteri(l3) but were different from those obtained for endometrium from gilts by Pack et a1.(14). The K for progesterone is similar to data reported for uteri from the ra-e (15,16),from the ewe(l7) and for bovine endometrium reported for some of the hormone(18). The differences in K values as compared to those presented by receptor complexes in thi$-study other authors are most likely due to the different methodology employed. Such differences have been described and discussed(23). It is generally considered that the ERc content increases in the rat uterus during the follicular phase in response to a rise in the circulating estradiol level (13,19), and when the complex is translocated to the cell nucleus (ERn), it initiates the specific metabolic changes which represent the target cell response to estrogen(20). Progesterone provokes a rapid and specific reduction of ERn in the uterus of the hamster(21). Although it is clear that progesterone and estradiol have involved are 1~s:: mutually counteractive effects on ERn, the mechanisms clear. In this study it was found that the level of ERn increased from a luteal phase value of 330 sites/cell to 1200 sites/cell during estrus. This agrees closely with the concomitantly observed elevated plasma level of estradiol and the resulting translocation of ERc to the nucleus. During the same period,no change in plasma progesterone concentration was seen, whereas a small reduction in PRn from IROO sites/cell to 760 These findings offer an explanation for the estrogensites was recorded. mediated constriction of the cervix found during estrus and the soft consistency of the cervix during the luteal phase. However,on days 3-4 of the estrous cycle,a stage in the cycle when the cervix is gradually softening,the levels of both ERn and PRn were high. It is known that estrogens act to increase the tissue content of progesterone receptors (22),whereas progesterone counteracts the action of estrogen by reducing This effect of progesterone the tissue level of estrogen receptors(23). on the estrogen receptor level is brought about by a mechanism in which progesterone blocks the second phase of the replenishment process, i.e. the expression of a relatively soft the -de novo synthesis(23). Hence, cervix during this cycle period might be explained by a progressive reduction in estrogen sensitivity in the cervix, caused by a progesterone mediated lowering of estrogen receptors. The high concentration of ERc anti the comparatively low concentration of ERn on day 1 may reflect the replenishment of ERc. The highest level of estradiol in blood in pigs with normally functioning reproductive cycles usually occurs during the days immediately preceding standing estrus(3). High levels of estradiol will cause a depletion of CRC to the receptor,the complex will bc because upon binding of the steroid translocated to the cell nucleus where it appears as ERn complexes(23). Such a depletion of ERc will be followed by replenishment through reactivation of ERn complexes and -de novo synthesis of ERc molecules.

MAY 1984 VOL. 21 NO. 5

763

THERIOGENOLOGY In oryans merely

that

The

high

plained

the

of

PRc

on

of

day

the of

circulating

of

PRc.

RNA

finding

estrous

and of

to

the

consistency

levels

that

of

the

follicular

the

via

probably

exat

Furthermore,estradiol

protein

this can

synthesis(24,25,26). low

PRc

levels

in

phase(27). the

presence

cervical

occurring

specific

is

progesterone

in

estrogen

of

specific

tissue.

cervix,receptor-binding

constriction

is mediated

replenishment

cycle

relatively

this study demonstrates In conclusion, gen and progesterone receptors in porcine suggest

steroids,EPc

1 of

through the

the

to

levels

depletion

PRc

with

during

response ERn.

low

slow

tallies

uterus

in of

relatively

synthesis

finding rat

grow

subsequent

stimulate the

not

recycling

level

by

stage,with This

do

involves

When

estrorelated

characteristics

response

to

high

estradiol

receptors.

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