Download Servomechanism of Prolactin and Progesterone in Regulating

Servomechanism of Prolactin and
Progesterone in Regulating Uterine
Gene Expression
Beverly S. Chilton, Shailaja K. Mani, and D. W. Bullock
Department of Cell Biology and Anatomy (B.S.C.)
Texas Tech University Health Sciences Center
Lubbock, Texas 79430
Department of Cell Biology (S.K.M., D.W.B.)
Baylor College of Medicine
Houston, Texas 77030
To investigate the interaction of PRL and progesterone in regulating uterine gene expression, we have
quantitated the concentration of PRL receptor and
of uteroglobin (UG) mRNA in the endometrium of
rabbits of different ages and after treatment with
different hormones. During uterine differentiation in
2- to 4-week old rabbits, a marked increase in unoccupied uterine PRL receptor number was observed, presumably increasing uterine sensitivity to
PRL. Receptor values for 4-week old rabbits were
comparable to values for sexually mature, estrous
females, but were lower than in 5-day pseudopregnant (PSP) animals. When total PRL receptor was
determined by Scatchard analysis after in vitro desaturation with MgCI2, PSP animals again expressed
the highest receptor concentration with no changes
in the dissociation constant (Kd) values. To determine whether progesterone regulates uterine PRL
receptor, long term ovariectomized rabbits (>12
weeks) were treated with various combinations of
hormones, and unoccupied and total uterine PRL
receptors were determined. Progesterone treatment
resulted in the highest concentration of both unoccupied and total PRL receptor after desaturation and
removal of anti-ovine PRL antibodies with MgCI2.
The value for total uterine PRL receptor was equivalent to the value for mammary gland, and the Kd
values (2-4 x 10~10 M) were similar. Treatment of
long term ovariectomized rabbits with progesterone,
with or without estradiol, produced an increase (P <
0.05) in the UG mRNA content, which also occurred
in PSP animals. PRL alone had no effect on UG
mRNA but PRL plus progesterone increased (P <
0.05) UG mRNA in a dose-dependent manner. We
propose a servomechanism by which PRL acts morphologically and biochemically to enhance the uterine response to progesterone. (Molecular Endocrinology 2: 1169-1175, 1988)
INTRODUCTION
PRL receptors have been identified in steroidogenic
organs (testis, ovary, and adrenal glands) and target
tissues for steroid hormones, especially uteri from
sheep (1), pigs (2), rats (3), mink (4), and rabbits (5, 6).
The rabbit uterus is a target organ for progesterone,
which acts directly on uterine epithelial cells (7, 8) to
produce a rise in the steady state level of uteroglobin
(UG) mRNA (9) through an increase in the rate of
transcription of the UG gene (10). Thus UG provides a
useful marker for the molecular mechanism of action of
progesterone (11,12). PRL acts on the rabbit uterus to
cause endometrial hypertrophy and glandular differentiation (13,14). These changes are accompanied by an
increase in the concentration of cytosol estrogen and
progesterone receptor (15) when long term ovariectomized (LTOVX) rabbits are treated with PRL. The sequential treatment of LTOVX rabbits with PRL plus
progesterone induces UG secretion 4-fold higher than
in LTOVX rabbits treated with progesterone alone (15).
Collectively, these data support the hypothesis that a
dynamic relationship exists between PRL and progesterone in the regulation of uterine function (15).
To define the mechanism whereby PRL augments
the uterine response to progesterone, we quantitated
changes in PRL receptor during uterine development
and after hormonal treatment. To investigate whether
PRL modulates the progesterone-induced accumulation of UG mRNA, LTOVX animals were treated with
different hormone regimens, and UG mRNA levels were
quantitated by slot-blot hybridization to a 32P-labeled
genomic DNA probe. We report here that progesterone
regulates the uterine PRL receptor in adult animals and
suggest a mechanism for the mutual effects of these
hormones on UG mRNA.
RESULTS
0888-8809/88/1169-1175$02.00/0
Molecular Endocrinology
Copyright © 1988 by The Endocrine Society
PRL receptor assays were characterized with membranes from rabbit mammary glands after in vivo de1169
Vol2No. 12
MOL ENDO-1988
1170
saturation of receptor with bromocriptine. Maximum
specific binding of [125I]PRL was obtained after 6-8 h
at 25 C and remained constant for 24 h. Thus, assays
were conveniently incubated overnight for 16 h. Specific
binding was linear between 25 and 600 ^g membrane
protein. Scatchard (16) analysis of [125I]PRL receptor
binding data indicated a dissociation constant (K^ of
4.5 ± 0.5 x 10~10 M and a total PRL receptor concentration of 146.3 ± 8.8 fmol/mg protein.
Similar results were obtained with membranes from
uterine endometrium. Specific binding of [125I]PRL to
unoccupied receptor was saturable, and Scatchard
analysis revealed a single set of binding sites. As shown
in Fig. 1, a significant increase in unoccupied uterine
receptor number occurred between 2 and 4 weeks of
age, presumably increasing uterine sensitivity to PRL
during development. This PRL-specific binding could
not be competed with GH (data not shown). Values for
4-wk-old animals were comparable to values for sexually mature estrous females. Five-day pseudopregnant
(PSP) animals, however, had significantly higher values
than estrous controls, suggesting that progesterone
influences PRL receptor content.
Total PRL receptor was measured after in vitro desaturation of receptor with MgCI2 (17). The brief (5-min)
treatment of membranes with MgCI2 resulted in a 90%
loss of total protein for all animal groups. Because of
the linear nature of PRL receptor binding, the initial
protein content was adjusted such that each assay
tube contained a minimum of 60 ng protein. As shown
in Fig. 1, a small increase (P > 0.05) in uterine PRL
receptor occurred between 2 and 4 wk of age, and a
2-fold increase (P < 0.05) was apparent at the time of
sexual maturation (estrous, 6 months of age). Total
PRL receptor increased an additional 33% in PSP animals (Fig. 1). The Kd values of 2-4 x 10~10 M for each
group (±MgCI2) were comparable to the values reported
for the mammary gland. Thus, increases in PRL binding
represented increases in the number of available bind-
100
~
D
•
unoccupied
total
80
| | eon
jE
oc =
40
Q. O
E
r. 20
D
2-WK
B
1
4-WK
ESTROUS
Age or Hormonal Status
8
PSP
Fig. 1. Uterine PRL Receptor Concentrations in 2-Week (2wk) and 4-Week-(4-wk) Old Juveniles, in Sexually Mature
Estrous Rabbits, and in 5-Day PSP Rabbits
Values are expressed as mean ± SEM, and mean values
with the same letter designation are not significantly different
(P > 0.05).
ing sites rather than changes in the affinity of the
receptor.
Ovariectomized rabbits (Fig. 2) showed a significant
reduction in the concentration of unoccupied uterine
receptor (6.8 ± 0.4 fmol/mg protein) compared to estrous controls, suggesting that the concentration of
PRL receptor may depend on ovarian hormones. As
shown in Fig. 2, when LTOVX animals were injected
with various combinations of steroid hormones, progesterone treatment resulted in a significant increase in the
concentration of PRL receptor. This stimulation was
not further enhanced by pretreatment with PRL, and
the Kd values for all treatment groups were similar to
those of the mammary gland and uterine tissue from
estrous control animals. Pretreatment with PRL plus
estradiol resulted in a significant decrease in the stimulation of PRL receptor by progesterone (Fig. 2). In the
absence of PRL, estradiol alone decreased the stimulation of PRL receptor by progesterone, although the
decrease was not significant.
Animals injected with PRL produce anti-ovine PRL
(oPRL) antibodies which may contaminate membrane
preparations and cause an artificial increase in the
determination of PRL receptor (18). There was no evidence of anti-oPRL antibodies in group 1 and 2 animals
that were treated with PRL alone, or in group 5 and 7
animals that were treated with steroid hormones. Animals in groups 3,4, and 6, however, that were injected
sequentially with PRL and steroid hormones, produced
measurable quantities of anti-oPRL antibodies (mean ±
SEM, 18,294 ± 4381 cpm or 36.2 ± 8.7% of the total
counts, compared to estrous controls, 188 ± 12 cpm
or 0.38 ± 0.02% of the total counts).
As the antibodies are removed from membrane preparations by MgCI2 (17), the assay for total PRL receptor
(18) achieves antibody-free conditions. As shown in Fig.
3, progesterone treatment of LTOVX rabbits resulted
in the highest concentration of total uterine endometrial
S£
T
T
30-
I
a.
a
a. co 2 0 -
LTOVX
PR.
1PRL.P
2PRLP
2PRUE«P
E»P
P
Hormonal Status
Fig. 2. Unoccupied PRL Receptor Concentrations in Uterine
Endometrium of LTOVX Rabbits Treated with PRL (1 or 2
mg); 1 mg PRL Followed by Progesterone (1 PRL + P); 2 mg
PRL Followed by Progesterone (2PRL + P); 2 mg PRL,
Followed by Estradiol, Followed by Progesterone (2PRL + E
+ P); Estradiol Followed by Progesterone (E + P); or Progesterone Alone (P)
The injection protocols are described in Table 1. Mean (±
SEM) values with the same letter designation are not significantly different (P > 0.05).
Uterine Gene Expression
1171
PRL receptor (132.5 ± 6.5 fmol/mg protein). This value
was significantly higher than those for animals treated
with PRL (1 or 2 mg) plus progesterone and was
equivalent to that reported above for total receptor in
mammary gland (146.3 ± 8.8 fmol/mg protein). Because the Kc values for all treatment groups were the
same as for mammary gland, the changes observed in
PRL binding after treatment with MgCI2 represented
changes in the number of available binding sites rather
than changes in the affinity of the receptor for the ligand
or bias due to anti-oPRL antibodies. Absent from Fig.
1PRL+P
2PRL+P
2PRL+E+P
E+P
P
Hormonal Status
Fig. 3. Total Uterine PRL Receptor Concentrations in Uterine
Endometrium of LTOVX Rabbits Treated with Various Combinations of Hormones
Abbreviations as in Fig. 2, and mean (±SEM) values with the
same letter designation are not significantly different (P >
0.05). Membrane preparations were extracted with MgCI2 to
remove endogenous PRL and anti-oPRL antibodies.
3 is the determination of total PRL receptor values for
LTOVX animals and for LTOVX animals treated with
PRL. The 90% loss of total protein with MgCI2 treatment
plus extreme atrophy of the uterine endometrium precluded the measurement of total PRL receptor in these
animals, i.e. 8-10 animals would have been required
for the determination of a single value. The significance
of this experiment is also negated by the fact that the
values for total PRL receptor behaved the same as
values for unoccupied PRL receptor.
Quantification of autoradiograms by computer-assisted image analysis indicated that PRL plus progesterone significantly increased the uterine content of UG
mRNA in a dose-dependent fashion (Fig. 4). The concentration of UG mRNA was lowest in LTOVX rabbits.
Treatment of animals with progesterone alone, or with
estradiol followed by progesterone, produced a significant increase in the UG mRNA content, which also
occurred in PSP animals. PRL alone (1 or 2 mg) had no
effect on UG mRNA, but PRL (1 mg) plus progesterone
significantly increased the amount of UG mRNA over
the value for progesterone alone. With a higher dose of
PRL (2 mg), the amount of UG mRNA was further
increased (P < 0.05) over the value for PRL (1 mg) plus
progesterone. PRL also significantly enhanced the uterine response to estradiol plus progesterone. Without
estradiol, the inclusion of PRL in the injection protocol
with progesterone resulted in UG mRNA levels comparable to the value for PSP animals. A significant
increase over this value was achieved when animals
were treated sequentially with PRL plus estradiol plus
progesterone.
4 DE
CD
3 -
w
2-
1 -
0 l|s
"v
Eslrous
LTOVX
P
E*P
PSP
2PRUP
2PRUE+P
Hormonal Status
Fig. 4. Concentrations of UG mRNA in Endometrium of Estrous, LTOVX, and Hormone-Treated Rabbits
Abbreviations as in Fig. 2, and mean (±SEM) values with the same letter designation are not significantly different (P > 0.05).
Total RNA (10 Mg) was applied to nylon membrane and hybridized to a 32P-labeled UG genomic DNA probe. UG mRNA was
quantitated by densitometry of autoradiograms calibrated with known amounts (0-4 ng) of total endometrial RNA from progesteronetreated rabbits (inset).
MOL ENDO-1988
1172
DISCUSSION
We have demonstrated that uterine PRL receptor is
regulated by progesterone and that PRL augments the
progesterone-dependent increase in UG mRNA. To explain the role of PRL in enhancing the action of progesterone we propose that a servomechanism operates
between these two hormones and their receptors (Fig.
5). Pretreatment of LTOVX animals with PRL causes
increased formation of glandular epithelium (13), which
contains more UG mRNA than does luminal epithelium
(8). Acting through its receptor, PRL produces an increase in the concentration of progesterone receptor
(14, 15), enhancing the cellular response to progesterone. Progesterone, acting through its receptor, in turn
promotes the expression of PRL receptor, which enhances cellular sensitivity to PRL and establishes the
servomechanism (Fig. 5). Estrogen can regulate this
system by increasing the concentration of both PRL
(19,20) and progesterone (21,22) receptors. The physiological importance of this mechanism is suggested by
the fact that increased plasma PRL levels (23) occur
coincident with progesterone-dependent increases in
endometrial PRL receptor concentration, preferential
stimulation of UG gene transcription (10), and maximum
UG secretion (9) on days 4-6 of preimplantation pregnancy.
Because PRL does not act through adenylate cyclase
and cAMP (24), other intracellular messengers that
activate protein kinases to phosphorylate intracellular
proteins have been proposed. Moreover, there is increasing evidence that the hormone binding capacity of
the progesterone receptor may be activated by phos-
Fig. 5. Model of the Interactions of PRL and Progesterone in
Regulating Uterine Gene Expression
A servomechanism is proposed, whereby PRL binds to its
receptor (PRL-R) and increases progesterone receptor (PR)
concentration. This in turn enhances the cellular response to
progesterone (P), including the progesterone-dependent stimulation of UG transcription and an increase in PRL receptor
number. The increase in PRL receptor further increases the
sensitivity to PRL and consequently amplifies the response to
progesterone.
Vol2No. 12
phorylation and inactivated by a dephosphorylation
mechanism (25-27). While it remains to be determined
whether the increase in progesterone receptor results
from a receptor phosphorylation, or from the direct
action of PRL on the progesterone receptor gene, which
can be tested, it is noteworthy that PRL treatment
enhances the uterine sensitivity to progesterone
through an increase in progesterone receptor.
As shown in this study, progesterone results in an
increase in both unoccupied and total PRL receptor.
Total receptor was estimated after the treatment of
membranes with MgCI2 (17), to remove bound endogenous PRL (28) and also anti-hormone antibodies that
could result in an artificial up-regulation of receptors
(18). However, if one subtracts the number of unoccupied receptors from the total number of receptors, the
resultant number of so-called occupied receptors does
not correlate with the hormonal status of the animal.
The total number of receptors could thus include cryptic
receptors, i.e. inactive receptors or receptors from
some subcellular location. Posner et al. (29) have demonstrated that liver PRL receptors with the greatest
affinity for ligand are found in the Golgi fractions. Furthermore, during estrogen induction, the most rapid
and marked increase in receptors occurred in the Golgi
fractions. This finding is compatible with the idea that
the Golgi receptors are precursors for those ultimately
found in the plasma membrane (30-32). Alternatively,
cryptic receptors may play a role in the elaboration of
uterine protein secretion that occurs when animals are
treated sequentially with PRL plus progesterone (15).
This idea is underscored by the fact that PRL administration to LTOVX rabbits results in ultrastructural
changes in uterine epithelial cells, including hypertrophy
of the Golgi complexes concomitant with the appearance of electron-opaque material (13). The treatment of
LTOVX animals with PRL plus progesterone results in
the secretion of two new postalbumin proteins each of
which constitutes approximately 4-5% of the total uterine protein (33).
Progesterone increases the steady state concentration of UG mRNA in the endometrium by regulating the
transcriptional activity of the UG gene (10). Depending
upon the developmental stage of the animal, the hormonal status of the animal (estrous or ovariectomized),
and the sequence and dosage of steroid administered,
estrogen alters the accumulation of UG mRNA (for
review see Ref. 34). It is not clear why estrogen reduced
the stimulatory effect of progesterone on PRL receptor,
yet augmented its effect on UG mRNA. Estrogen can
have acute antiprogestational effects in the rabbit
uterus at high doses (35). Possibly the dose dependence or time of administration differs in the effects of
estrogen on these two different responses to progesterone.
As shown in this study, the pretreatment of LTOVX
rabbits with PRL significantly increased the progesterone response of UG mRNA in a dose-dependent fashion, with or without pretreatment with estradiol. This
increase in the concentration of UG mRNA correlates
well with the dose-dependent effect of PRL pretreat-
Uterine Gene Expression
1173
ment on the amount of secreted UG (33). Thus PRL is
involved at the biochemical as well as the morphological
level in modulating the uterine response to progesterone. Whether PRL, in combination with progesterone,
alters the transcriptional regulation of the UG gene and
affects specific trans-acting factors (36), in addition to
the progesterone receptor, is under investigation.
MATERIALS AND METHODS
Reagents and Buffers
Reagents and buffers were obtained from the following
sources: Na125l (14.8-16.9 mC\/fig) from Amersham Corp.
(Arlington Heights, IL); [«-32P]CTP (3528 Ci/mmol) from ICN
Radiochemicals (Irvine, CA); nick translation reagent kit from
Bethesda Research Laboratories (Gaithersburg, MD); BSA
(fraction V), Sephadex G-100, 2-bromo-a-ergocryptine methane sulfonate (bromocriptine mesylate), 17/3-estradiol, and progesterone from Sigma Chemical Co. (St. Louis, MO). For the
membrane receptor assays, oPRL (AFP-7150B) was donated
by the National Hormone and Pituitary Program, NIADDK
(Baltimore, MD). oPRL (NIADDK-o-PRL-17 and NIADDKoPRL-18-AFP-8277E) from the National Hormone and Pituitary
Program, NIADDK, and oPRL from Sigma Chemical Co. were
equally effective for displacement in the binding assays and
for animal injections. For the measurement of serum antibodies
to oPRL, rabbit anti-human PRL was obtained from Accurate
Chemical and Scientific Corporation (Westbury, NY), and goat
anti-mouse immunoglobulin G (IgG) was obtained from E-Y
Labs, Inc. (San Mateo, CA).
Animal Treatment
Beginning on the sixth day of lactation after her first pregnancy,
one New Zealand White doe was injected sc with 2 mg
bromocriptine mesylate 36, 24, and 12 h before death to
produce maximal desaturation of mammary PRL receptor (37).
Day 6 was selected because the number of PRL receptors
reaches a maximum at this time (37).
Adult (3.6-4.5 kg) virgin New Zealand White rabbits were
housed individually for 3 weeks before experimentation. The
estrous condition of these animals was verified by the presence of mature ovarian follicles at the time of laparotomy
(estrous controls, n = 43), or at the time of ovariectomy (n =
42). At 12 weeks or more after ovariectomy, nine animals were
used as experimental controls, i.e. untreated with steroid
hormones. The remaining ovariectomized animals were divided into seven treatment groups (three to seven animals per
group) as shown in Table 1. Group 1 rabbits (1 PRL) received
sc injections of oPRL (1.0 mg) in PBS every 24 h for 5 days.
Group 2 rabbits (2PRL) received sc injections of oPRL (2.0
mg) in PBS every 24 h for 5 days. Group 3 (1 PRL + P) rabbits
were treated with oPRL (1.0 mg) for 5 days followed by
progesterone (3 mg/kgday) for 4 days. Group 4 animals (2
PRL + P) were treated with oPRL (2.0 mg) for 5 days followed
by progesterone for 4 days. Group 5 rabbits (P) were treated
with progesterone alone for 4 days. Group 6 animals (2 PRL
+ E + P) were treated sequentially with oPRL (2 mg) for 5
days, estradiol (10 /ug/kg-day) for 3 days, and progesterone
for 4 days. Group 7 animals (E + P) were treated with estradiol
for 3 days followed by progesterone for 4 days.
A second group of adult, virgin New Zealand White rabbits
(n = 12) were made pseudopregnant, 5 days before death,
with an iv injection of 20 IU human CG followed by cervical
stimulation. These progesterone-dominated animals (13, 15)
were used for a physiologically relevant comparison with progesterone-treated LTOVX animals. Two-week-old (n = 98)
and 4-week-old (n = 80) juvenile New Zealand White females
were also purchased for experimentation. All juveniles were ±
12 h of the stated age. To minimize potential seasonal variability, experiments with adults and juveniles were done from
August through April.
Membrane Preparations
Approximately 100 g mammary gland were removed, quick
frozen on dry ice, stored at - 8 0 C, and used for subsequent
membrane preparations. The remaining mammary tissue (~60
g) was used for the initial membrane preparation according to
Shiu et al. (38). All subsequent steps were performed at 4 C.
Briefly, mammary gland was minced with scissors and homogenized (1 g/5 ml) in 0.3 M sucrose with a Polytron Pt-10
homogenizer (Brinkman Instruments, Westbury, NY) at maximum speed for 60 sec. The homogenates were pooled and
filtered through eight layers of cheese cloth. The filtrate was
centrifuged at 2,000 x g for 20 min. The pellets were discarded, and the supernatants were centrifuged at 15,000 x g
for 20 min. Again, the pellets were discarded, and the supernatants were centrifuged at 105,000 x g for 90 min. The
105,000 x g crude membrane pellets were resuspended in 25
mM Tris-HCI (pH 7.6) and 10 mM MgCI2. Protein contents were
determined according to the method of Lowry et al. (39).
Membrane suspensions were quick-frozen on dry ice and
stored frozen (-80 C) until binding assays were performed.
Mammary membranes were used to characterize the receptor
assay and as a reference standard for each preparation of
iodinated oPRL.
For adult rabbits, uterine endometrium was scraped from
underlying myometrium, weighed, and homogenized in 0.3 M
sucrose with a Polytron Pt-10 homogenizer as described
above. For juvenile animals, whole uteri were homogenized in
5 ml 0.3 M sucrose with a Polytron Pt-10 homogenizer. All
subsequent steps were as described above, except that none
of these homogenates was filtered through cheese cloth.
lodination of oPRL
Ovine PRL was iodinated by the method of Hunter and Greenwood (40) using chloramine-T as the oxidizing agent. Unreacted iodine and damaged hormone were removed by frac-
Table 1. Hormone Injection Schedule
Days of Treatment
Treatment Group
n
1
1. 1PRL
2. 2PRL
3. 1PRL + P
4. 2PRL + P
5. P
7
7
4
5
4
6.2PRL + E + P
7. E + P
3 PRL
3
2
PRL
3
PRL
4
5
6
PRL
PRL
PRL
PRL
PRL
PRL
PRL
PRL
E
E
7
8
9
10
11
12
13
PRL
PRL
PRL
PRL
PRL
PRL
PRL
PRL
P
P
P
PRL
PRL
P
P
P
PRL
PRL
P
P
P
PRL
PRL
P
P
P
PRL
PRL
P
P
P
Animals were killed 12 h after the last injection. PRL, 1 or 2 mg PRL animal; P, 3 mg progesterone/kg; E, 10 ^g estradiol/kg; n,
number of animals.
Vol2No. 12
MOL ENDO-1988
1174
tionation on a Sephadex G-100 column (1.5 cm x 50 cm) with
Tris-HCI buffer (25 mM; pH 7.6). [ 1 2 5 I]OPRL that eluted from
the column at a position where the native hormone eluted was
used for the binding assays. The specific activity of the hormone was determined according to Rose et al. (4). First, an
inhibition curve was established by adding increasing concentrations of oPRL (1-1,000 ng) to tubes containing 400 ^g
mammary membranes and [ 1 2 5 I]OPRL (50,000 cpm). Second,
increasing amounts of [ 1 2 5 I]OPRL (2.5 x 104 to 5.0 x 105 cpm)
were added to tubes containing 400 fig mammary membranes.
Specific activity was calculated by dividing the counts per min
(converted to microcuries) obtained at a bound/total counts
per min (B/T) ratio of 50% by the quantity of oPRL (converted
to micrograms) that displaced 50% of the [ 1 2 5 I]OPRL in the
inhibition curve. The specific radioactivity of the [ 125 I]OPRL was
4-9
PRL Receptor Assay
For the measurement of PRL receptor, crude membrane preparations (400 fig) were incubated in 25 mM Tris-HCI buffer, pH
7.6, containing 10 mM MgCI2 and 0.1% (wt/vol) BSA in a final
volume of 500 fi\ in the presence of increasing amounts of
[ 1 2 5 I]OPRL ±'\ ng oPRL. Incubations were terminated after 16
h at room temperature by adding 2 ml ice-cold buffer, followed
by centrifugation at 2000 x g for 30 min. Precipitated radioactivity was counted in a Beckman 7-counter. Concentrations
of unoccupied receptor were determined by Scatchard (16)
analysis of specific binding data.
Total PRL receptor was determined after in vivo desaturation of crude receptor preparations (17) and the removal of
anti-oPRL antibodies (18) with 4 M MgCI2. Briefly, membrane
preparations (250-750 fi\) were incubated with 5 ml 4 M MgCI2
for 5 min at 4 C. This incubation mixture was diluted 4-fold
with ice-cold Tris-HCI buffer containing 10 mM MgCI2, and
centrifuged at 15,000 x g for 15 min. Each receptor-containing
pellet was resuspended in 2.5 ml of the Tris-MgCI2 buffer, and
the protein concentration was determined according to Lowry
ef al. (39). Receptor assays were performed as described
above, and total receptor concentrations were determined by
Scatchard (16) analysis of specific binding data.
Detection of Serum Antibodies to oPRL
Serum antibodies were determined according to the method
of Klemcke ef al. (41). Briefly, serum samples were diluted
1:10, and 100-|tl aliquots were incubated with 50,000 cpm
[ 125 I]OPRL ± 2 fig oPRL for the determination of nonspecific
binding. The final incubation volume was adjusted to 350 n\
with 25 mM Tris-HCI buffer, pH 7.6, containing 10 mM MgCI2,
0.1% (wt/vol) BSA, and 0.1% sodium azide at room temperature (23 C). After the samples were incubated for 24 h at
room temperature, a 1:10 dilution of goat anti-mouse immunoglobulin G was added, and the tubes were incubated at 4
C for an additional 24 h. The reactions were terminated by the
addition of 2 ml ice-cold incubation buffer and centrifuged at
2000 x g for 30 min at 4 C. Supematants were aspirated, and
precipitated radioactivity was counted in a Beckman 7-counter. The negative control consisted of pooled serum from three
to four estrous animals that did not express anti-oPRL antibodies. The positive control consisted of serum samples from
the same pool plus a 1:1 dilution of rabbit anti-human PRL.
RNA Isolation and Slot-Blot Analysis
About 200 mg of whole uterus from each animal in each
treatment group were frozen in liquid nitrogen. Total cellular
RNA was isolated after homogenization (1:15, wt/vol) in 4 M
guanidine thiocyanate, centrifugation through a 5.7 M CSCI
step-gradient, and chloroform-isoamyl alcohol/butanol extraction (42). After ethanol precipitation and washing, RNA concentration was determined by absorbance at 260 nm. For each
sample, 10 ^g total RNA were diluted with 20x SSC (1 x SSC
= 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0) to a volume
of 100 n\. RNA samples were denatured by heating at 68 C
for 15 min followed by quick cooling on ice. The RNA was
filtered in duplicate onto Biotrans nylon membranes (0.2 fim)
with a Bio-Dot SF (Bio-Rad Laboratories, Richmond, CA)
manifold. The RNA blots were baked (90 min at 80 C) in vacuo,
prehybridized for 6-12 h at 42 C in a solution containing (final
concentration) 50% formamide, 5x SSC, 50 mM sodium phosphate (pH 6.5), 0.1% sodium dodecyl sulfate, 250 fig/m\ sonicated denatured herring sperm DNA, and 0.1% each of BSA,
Ficoll, and polyvinylpyrrolidone. The hybridization buffer contained the following constituents (final concentration): 45%
formamide, 4x SSC, 100 mM sodium phosphate (pH 6.5),
0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate,
100 fig/m\ sonicated denatured herring sperm DNA, 10%
dextran sulfate, and 0.02% each of BSA, Ficoll, and polyvinylpyrrolidone. Blots were hybridized for 12-16 h at 42 C with a
minimum of 2-3 x 107 cpm nick-translated pUGi 1.8 (SA, 1-3
x 108 cpm/^g), a full-length UG genomic clone (43). Our
previous work (43) has shown that this probe detects only one
species of mRNA in total RNA from rabbit uterus.
After hybridization, the blots were washed sequentially for
15 min and for 60 min at room temperature in 2x SSC
containing 0.1% sodium dodecyl sulfate, then for 3 h at 68 C
in 0.2x SSC containing 0.5% sodium dodecyl sulfate. Finally,
blots were washed for 10 min at room temperature in 0.2x
SSC containing 0.5% sodium dodecyl sulfate. Autoradiographic exposure was performed at - 7 0 C using XAR-5 xray film (Eastman Kodak Co., Rochester, NY) with an intensifying screen (DuPont Cronex, Lightning-Plus EH, Dupont Co.,
Wilmington DE). Relative intensities of the resulting mRNA
autoradiograms were quantified with a computer-assisted image analysis system (Bio-Image Visage 2000, Eastman Kodak
Co., Ann Arbor, Ml). All samples were analyzed in duplicate,
and concentrations of UG mRNA were determined from a
standard curve for each blot.
Statistical Analysis
All biochemical data were analyzed by one-way analysis of
variance, followed by Duncan's multiple range test (P < 0.05
significance level) using the SAS Statistics package (44). In
Figs. 1-4, values are expressed as mean ± SEM, and mean
values with the same letter designation are not significantly
different (P > 0.05).
Acknowledgments
We thank Mr. Bill M. Wallace, Mr. Keith N. Wilson, and Ms.
Elizabeth K. Peck for excellent technical assistance, and Ms.
Donna Stuart for preparation of the manuscript. We also thank
Dr. J. C. Daniel, Jr. (Old Dominion University), for stimulating
discussions and review of the manuscript.
Received June 7, 1988. Revision received July 27, 1988.
Accepted August 5,1988.
Address requests for reprints to: Dr. Beverly S. Chilton,
Department of Cell Biology-Anatomy, Texas Tech University
Health Sciences Center, 3601 Fourth Street, Lubbock, Texas
79430.
This work was supported by NIH Grant HD-20271 (to
B.S.C.) and NIH Grant HD-09378 (to D.W.B.) B.S.C. is the
recipient of NIH Research Career Development Award HD00704.
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