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
