Bovine Placentomes Contain Factors Which Decrease Progesterone ...

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Jun 10, 1983 - and JEROME. F. STRAUSS,. Ill4. Department of Hormone ... of Pennsylvania. Philadelphia,. Pennsylvania. 19104. ABSTRACT. We examined.
BIOLOGY

OF

REPRODUCTION

856-862

29,

Bovine

(1983)

Placentomes Contain Decrease Progesterone

MORDECHAI

SHEMESH,”2

and

JEROME

Factors Which Secretion

WILLIAM

HANSEL3

F. STRAUSS,

Department

of Hormone

Kimron

Research2

Veterinary Beit Dagan,

Department

Ill4

Institute Israel

of Physiology3

Cornell University Ithaca, New York 14853 and Departments

of Pathology and

Obstetrics

and

University Philadelphia,

Gynecology4

of Pennsylvania Pennsylvania

19104

ABSTRACT We examined the effects of 20% ammonium sulfate precipitates from cytosolic extracts of whole placental tissue collected between 100-150 days of gestation on progesterone secretion by bovine granulosa cells and dispersed bovine luteal cells. These extracts produced a dose-dependent inhibition (23-92%) of progesterone synthesis by bovine granulosa cells. However, no inhibitory activity could be demonstrated in similarly prepared extracts from term placentae. Inhibitory activity could be extracted from both maternal caruncles and fetal cotyledons. In the presence of 2 mg/mI of maternal caruncle extract, basal progesterone secretion was dramatically reduced (90%), as was steroidogenesis in the presence of bovine lutenizing hormone (bLH) and 8 bromocyclic (Br)-cAMP. Moreover, coincubation of dispersed luteal cells and dispersed fetal or maternal placental cells from 100- to 150-day placentae produced a significant (50%) reduction in progesterone content of the medium. The addition of 2 mg/mI of caruncle or fetal cotyledon extract from 100- to 150-day placentae also produced 100% and 50% inhibitions, respectively, of progesterone secretion by dispersed placental cells. Thus, the inhibitory factor appears to be produced by cells of both the maternal and fetal placenta. It is heat-stable and not extractable by ether, The inhibitory substance eluted as two distinct peaks from Sephadex G-100 columns, one with a molecular weight of about 60,000 daltons and the other about 30,000 daltons. Using isoelectric focusing, several peaks of inhibitory activity were obtained, one with api of 3-5, the others having pls between 6 and 9. A substantial purification was achieved by both column chromatography and isoelectric focusing. This inhibitory substance(s) may play a role in regulating placental steroidogenesis. INTRODUCTION We

have

bovine and

recently

examined

placentomes reported

for the

gonadotropin-like from age

protein

(Ailenberg

of

of and

was detected employing rat lation

gonadotropic

presence

placentomes

steroidogenesis

of days

a

1983). radioreceptor tissue, and by

bovine

cells onic

of

activity a

and dispersed gonadotropin-like

labile

chorionic

in preparations 50-100

Shemesh,

using testicular

extracts

and

60,000 as the course

made gestational

heat-stable

This

factor

placental

by

assay stimu-

describe of this

had

rat a

of

Medicine, University,

molecular

determined of these

The was

weight

by gel filtration. studies we also

inhibitor extracts.

of In

of

choriheatabout

During found a

steroidogenesis

the

the activity and inhibitory substance.

present

some

of the

in study

we

properties

granulosa MATERIALS

Accepted Received ‘Reprint Physiology,

Leydig cells. substance

June 10, 1983. April 4, 1983. requests: Dr. Mordechai Shemesh, Dept. New York State College of Veterinary 827 Veterinary Research Tower, Cornell Ithaca, NY 14853.

Preparation

and Extracts

of Placental

of Culture

AND

METHODS

Extracts

Media

Placental tissues were collected at an abbatoir multiparous Holstein-Friesan cows. Gestation were estimated by crown-rump measurements

856

from ages of the

BOVINE

PLACENTAL

STEROIDOGENESIS

and Preparative

Isoelectric

cooling bath maintained the cooling plate at a temperature of 4#{176}C.A p1-1 gradient of pH 3-10 was prepared using ampholytes purchased from Pharmacia (Uppsala, Sweden), on a support of Ultradex (LKB). Samples to be subjected to preparative isoelectrc focusing (300 mg protein) were mixed with an ampholyte/Ultradex slurry and added to the flat-bed plate. Isoelectric focusing was then carried Out as described in LKB Application Note 198. After isoelectric focusing, a grid was applied to the bed, dividing it into 15 fractions. Each section was removed, mixed in a tube with 1 ml of deionized water and poured into a 5 X 100 mm column (Econo-column, Bio-Rad, Richmond, CA). The gel was then eluted with 5 ml of deionized water. The pH of each eluate was determined; the eluates were then dialyzed against deionized water to remove anipholytes and 0.1-mI aliquots of the fraction were examined for effects on progesterone secretion by bovine granulosa cells. Assay of the Inhibitory Placental Extracts on

1. Inhibitor by bovine

effects granulosa

of 20% cells.

Focusing

ammonium

Net

5.00

Control (Medium Placental 100-150

199

+

sulfate

progesterone

(ng/4

Treatment

X i0

±

Activity Progestin

of Secretion

The inhibitory activity of placental extracts on progesterone secretion was assayed using three different incubation systems: 1) suspensions of bovine granulosa cells; 2) dispersed luteal cells from bovine corpora lutea; and 3) dispersed fetal cotyledon cells. The incubation of bovine granulosa cells has been described previously (Shemesh, 1979). Briefly, 2-5 X io cells were routinely used, with steroid secretion being monitored over a 24-h period. The procedure of Simmons et al. (1976) was used to prepare dispersed bovine luteal cells, which were used in short-term incubations (2 h). Similar methods were used to prepare dispersed cells from fetal cotyledons and maternal caruncles (see below). Medium 199 was used as the basic incubation medium in all studies. Progesterone secreted into the incubation medium was quan-

Gel filtration of the 20% ammonium sulfate precipitates of placental extracts was performed using a column of Sephadex G-100 (2.5 X 97 cm), eluted with 10 mM Tris-HCI buffer, pH 9, containing 0.03 M NaCI. Three hundred mg of the extract in 8 ml of elution buffer were applied to the column. Fractions (5 ml) were collected at 4#{176}C, dialyzed against distilled water, lyophiized and then reconstituted in 1 ml of

TABLE secretion

857

Medium 199 (Gibco). The activites of 0.1 ml aliquots on the steroidogenic activity of bovine granulosa cells were examined as described below. Preparative isoelectric focusing was carried out using a flat-bed LKB system (Multiphor). A circulating

fetuses. Extracts were routinely prepared from placental tissues collected between 100-150 days gestational age and, in some instances, placental tissue obtained at term. The placentae were always collected within 30 mm of slaughter, placed on ice, and processed or frozen within 1 h. The fetal cotyledons were separated from the maternal caruncles and used immediately or frozen until extracted. The fetal tissues were easily separated so that there was no contamination of the fetal cotyledon preparations with maternal caruncle tissue. However, a small number of fetal cotyledon cells may have remained in the maternal caruncle preparations. Twenty different preparations of maternal caruncles and fetal cotyledons from 20 different placentomes were examined. Pools of tissue (300-500 g) were homogenized in 3 vol of chilled 0.9% saline. The homogenates were centrifuged at 10,000 X g for 30 mm. The supernatants were then centrifuged at 108,000 X g for 1 h and the supernatants from this centrifugation were treated with ammonium sulfate (20% w/v) with constant stirring for 45 miss at 4#{176}C.The precipitates were collected by centrifugation at 13,000 X g for 45 mm at 4#{176}C.In some instances, the media from cultures of dispersed cells from maternal caruncles or fetal cotyledons were processed in a similar fashion. The precipitated material was collected by centrifugation, suspended in distilled water and dialyzed for 96 h against distilled water at 4#{176}C.The dialyzed solution was adjusted to pH 7 with 0.1 NaOH and lyophiized. The lyophilized material was tested for inhibitory activity or subjected to further analysis by gel filtration or preparative isoelectric focusing.

Gel Filtration

INHIBITOR

precipitates

of

bovine

placentomes

on

progesterone

secretiona cells

per

24 h)

Percent

inhibition

0.17

BSA)

age days

1mg/mI 3mg/ml 5 mg/ml

3.62±0.18 1.99±0.12 0.37 ± 0.02

23 61 92

Term

3-5

mg/mI

aValues different

5.21 presented

placental

are means preparation.

±

SEM

In each

of

±

0.17

0

progesterone

experiment

each

secretion dose

from

of extract

5 separate was

tested

experiments, on 8 replicates.

each

utilizing

a

SHEMESH

858

ET AL.

tified by radioimmunoassay (Shemesh et al., 1979). The interand intraassay coefficients of variation of the progesterone assay were 11% and 9%, respectively. In all cases, net progesterone secretion into the medium was determined by subtracting progesterone levels in the cells and incubation medium at zero time from levels after the indicated incubation period. Treatment groups consisted of at least five replicate incubations in all experiments.

RESU

The

effects

precipitates

from

placentomes days

Fetal laminar tissues

0.13%

of Dispersed

Placental

and maternal tissues were dissected in a flow hood using sterile instruments. The were then minced and incubated at 37#{176}Cwith collagenase (Sigma, Type 1) in Medium 199 (1

generalized

Analytical

were carried The media

out in an atmopshere were changed every

Statistical

2. Effects of bovine luteal

extracts cells.

of

maternal

caruncles

199) ng/ml) (1 mM) caruncle (2 mg/mI) ng/ml + caruncle

(2 mg/mI)

presented

io

40-50%

±

SEM

for

per

mg

precipitate. next examined

of

viability

the

that

the from

fetal had

cotyledons the greater

20%

ammonium

effects

extracts

secretion by dispersed bovine luteal Results from a representative experiare shown in Table 2. In the presence mg/mI

of

placentae

of

terone cells. ment 2

150-day

all treatment

100-

caruncle

extract,

additional

experiments

2 mg/ml

extract produced >90% progesterone secretion by

on

basal

proges-

as well

a 150-day

placentome

on

progesterone

(Control

±

21

632 835

± ±

30 30