BIOLOGY OF REPRODUCTION 48, 741-751 (1993)
Neonatal Age and Period of Estrogen Exposure Affect Porcine Uterine Growth,
Morphogenesis, and Protein Synthesis'
THOMAS E. SPENCER, 3 ANNE A. WILEY, and FRANK F. BARTOL 2
Department of Animal and Daily Sciences, Auburn University, Auburn, Alabama 36849-5415
ABSTRACT
To determine whether neonatal age and estrogen exposure affect uterine growth, morphogenesis, and protein synthesis,
crossbred gilts were randomly assigned at birth (Day O) to receive either corn oil vehicle (CO) or estradiol-17[ valerate (EV;
50 Flg/kg BW/day). Gilts were treated for 7 days, chosen to coincide with specific periods of uterine development, prior to
hysterectomy on Day 7, 14, or 49. Uteri were weighed, and tissues were fixed for histology or explanted with L-4,5-[H]leucine
(5 H-leu) for 24 h. Endometrial and myometrial thicknesses were measured in uterine wall cross sections. Radiolabeled proteins
produced by uterine wall tissues from 3H-leu and released into explant medium were identified by fluorography of two-dimensional SDS-PAGE gels. Proteins for which fluorographic spot intensities were consistently affected by age and/or treatment were
excised from gels, and associated radioactivity was quantified. Normal growth and histogenesis were observed in uteri from COtreated gilts. Exposure to EV increased (p < 0.01) uterine wet weight on all days examined, although effects were most pronounced on Day 49 (day x treatment, p < 0.01). Histologically, uteri of EV-treated gilts exhibited precocious or altered patterns of development of endometrial glands and folds. Endometrial thickness was greater (p < 0.01) in EV-treated gilts, and response was most pronounced on Day 49 (day x treatment, p < 0.01). Treatment with EV increased (p < 0.01) myometrial
thickness on Day 49 only. Twenty-five uterine proteins were identified to be affected consistently by neonatal age, EV, or both.
Production of four of these proteins was affected by age alone, while six were affected exclusively by treatment with EV alone,
and 15 were affected differentially by both age and EV. Treatment with EV affected production of three of these 25 proteins on
Day 7, 8 of 25 on Day 14, and 14 of 25 on Day 49. Results indicate that uterine growth and development of the porcine uterine wall during early neonatal life are accompanied by predictable alterations in patterns of uterine protein synthesis. Data also
demonstrate that the neonatal porcine uterus is estrogen-sensitive and that both physical and biochemical responses of uterine
tissues to estrogen vary with period of exposure. It is suggested that EV may be useful as a tool with which to induce developmental lesions in neonatal porcine uterine tissues. Identification of EV-induced lesions in adult uterine tissues and of the neonatal periods associated with their induction will represent an important step toward identification of developmental determinants of porcine uterine capacity.
INTRODUCTION
Events associated with uterine wall development in the
neonatal pig are similar to those characterized for other
species including the sheep, rat, and mouse, in which remodeling of the uterus proceeds in an autonomous manner, independent of a requirement for ovarian and possibly
even steroid support [10-13]. Interruption of uterine development by exposure of tissues to steroids, including estrogens, during pre- and postnatal periods permanently
modifies adult urogenital tract structure and function [1423]. Rats treated with estrogens during specific periods of
neonatal life develop structural and functional uterine lesions characteristic of the neonatal periods when exposure
occurs [14, 15, 24-26]. Thus, estrogens may be particularly
useful as a tool with which to interrupt normal patterns of
uterine development. Observations also support the hypothesis that interruption of development during specific
critical periods can compromise the success of organizational events associated with perinatal uterine wall development and determine the potential of the adult uterus to
function competently [10, 11].
The extent to which normal patterns of porcine uterine
wall development must be preserved if adult uterine function is to be optimized remains to be defined. However, if
estrogens can be used to interrupt normal patterns of uterine development in the neonatal pig, it may be possible to
employ such compounds as tools with which to induce de-
Growth and development of the neonatal porcine uterus
has been characterized grossly [1-3], histologically [1, 4-7],
and biochemically [7-9]. Neonatal porcine uterine growth,
as reflected by uterine horn length and weight, proceeds
in an ovary-independent manner prior to Day 60 of neonatal life [3]. During this period, the porcine uterine wall
undergoes dramatic remodeling events including appearance and proliferation of endometrial glands, formation of
endometrial folds, and growth and development of the
myometrium [1, 2, 4-7]. Associated, predictable alterations
in endometrial DNA synthesis, glycosaminoglycan distribution, and glycoconjugate biosynthesis and deposition have
been defined as characteristic of infantile (prior to Day 7),
proliferative (Days 7 through 14), and growth (after Day
14) periods of neonatal porcine uterine development [79].
Accepted December 4, 1992.
Received July 27, 1992.
'This work was supported in part by the Alabama NSF/EPSCoR program in Molecular, Cellular and Developmental Biology grant #RII-8610669 and USDA-NRICGP
Agreement No. 91-37203-6605. This manuscript represents contribution no. 4-923305
of the Alabama Agricultural Experiment Station.
ZCorrespondence. FAX: (205) 844-1519.
3Present address: Department of Animal Science, 442 Kleberg Center, Texas A&M
University, College Station, TX 77843-2471.
741
742
SPENCER ET AL.
velopmental lesions identifiable in adult uterine tissues.
Identification of such lesions, and the neonatal periods associated with their induction, would provide a model system with which to investigate and define developmental determinants of porcine uterine capacity [7, 27, 28]. The present
study represents the first step toward development of such
a model system. Effects of estrogen exposure on developmental responses of neonatal porcine uterine tissues have
not been characterized. Consequently, objectives of this study
were (1) to determine whether neonatal porcine uterine
tissues are estrogen-sensitive by assessing effects of treatment with estradiol-1713 valerate (EV) on patterns of uterine
growth, morphogenesis, and protein synthesis; and (2) to
determine whether uterine responses to treatment with EV,
administered during infantile, proliferative, or growth periods of neonatal porcine uterine development, vary with
period of exposure.
MATERIALS AND METHODS
Materials
All components of culture medium, except for insulin
and glucose, were purchased from Grand Island Biological
Company (Grand Island, NY). Insulin, glucose, and EV were
from Sigma Chemical Company (St. Louis, MO). L-4,5[3 H]Leucine (3 H-leu; spec. act. -50 Ci/mmol) was obtained
from Dupont (NEN Research Products, Boston, MA). Spectrapor dialysis tubing was from Spectrum Medical Industries (Los Angeles, CA). Chemicals including N,N'-methylene-bis-acrylamide, ascorbic acid, Coomassie Brilliant blue
R-250 dye, diallyltartardiamide, dithiothreitol, -mercaptoethanol, Nonidet P-40, potassium carbonate, SDS, sodium
salicylate, Tris, and urea were from Sigma. Acrylamide and
glycine were from Serva Biochemicals (Heidelberg, Germany). Ampholines were from Pharmacia LKB (Uppsala,
Sweden). X-Omat AR diagnostic x-ray film was from Eastman Kodak (Rochester, NY). All other reagents and chemicals were obtained from Sigma and were of electrophoretic or molecular biology grade.
Explant Medium Preparation
Explant medium consisted of Eagle's minimum essential
medium (EMEM) supplemented with fungizone (0.5 tzg/
ml), glucose (5 mg/ml), insulin (7.5 mg/ml), nonessential
amino acids (1%, v/v), penicillin (200 U/ml), and streptomycin (200 ptg/ml) as described by Basha et al. [29]. Content of L-leucine was limited to 5.2 [Ig/ml (0.1 standard) to
enhance uptake of 3 H-leu in uterine explant cultures.
Animal Manipulation and Tissue Collection
Crossbred gilts (n = 22) were assigned randomly at birth
(Day 0) to receive treatment with either corn oil vehicle
(CO) or EV (50 pLg/kg BW/day, i.m.) for a period of 7 days
during either infantile (Days 0 through 6; n = 3 CO/4 EV),
proliferative (Days 7 through 13; n = 3 CO/5 EV), or growth
(Days 42 through 48; n= 3 CO/4 EV) periods of neonatal
porcine uterine development [7]. The dose of EV was chosen because it is recognized to be sufficient to induce pseudopregnancy in cyclic, adult gilts and, consequently, is known
to elicit a significant biological response in adult uterine
tissues [30]. Gilts were subsequently hysterectomized on
either Day 7, 14, or 49. No treatment or surgically related
complications were observed in any gilts. All animal manipulations were approved by the Auburn University Institutional Animal Care and Use Committee and conformed
to Guiding Principles for Care and Use of Research Animals
published by the Society for the Study of Reproduction.
Tissue Processingand Organ Culture
At hysterectomy, a piece from the middle portion of each
uterine horn was fixed in Bouin's fixative and processed
for histology and morphometry. Remaining excised tissue
was immediately placed into a small, sterile Erlenmeyer flask
containing approximately 20 ml of warm (37 0C) EMEM and
was transported to the laboratory. Under a laminar flow hood,
tissues were transferred into fresh EMEM in a petri plate,
washed in EMEM, and trimmed free of remaining oviduct,
connective tissues, and cervix. Each uterine horn was then
sliced longitudinally to expose the endometrial surface and
cut into small pieces (-2-3 mm 3). Each piece included endometrial, myometrial, and perimetrial tissue layers. Individual uterine pieces were blotted on sterile gauze and
transferred aseptically into 60 x 15-mm plastic petri dishes
(Falcon 3037, Becton Dickinson Labware, Lincoln Park, NJ)
containing 5.0 ml of fresh EMEM at an explant rate of approximately 50 mg of wet tissue/1.0 ml of EMEM (-250
mg wet tissue/explant). All explants were supplemented with
10 p.Ci of 3 H-leu/ml of EMEM (50 ,uCi/explant).
Explant cultures were then placed into a controlled atmosphere chamber (Bellco Biological Glassware, Vineland,
NJ) maintained under a gas mixture consisting of 45% 02:50%
N2:5% CO2 (v/v/v) and incubated on a rocking platform
(Bellco) at 6 cycles/min and 37°C for 24 h. Procedures for
neonatal porcine uterine explants were essentially the same
as those described by Bartol et al. [31] for bovine endometrium and Basha et al. [29,32] for porcine endometrium.
Explant cultures were terminated by separation of tissue
and medium using centrifugation at 2000 x g and 4°C for
20 min. Medium (supernatant) was stored at -20 0C until
further analysis.
Release of de novo-synthesized macromolecules into explant culture medium was determined by measuring the
incorporation of 3 H-leu into nondialyzable macromolecules. Incorporation was determined by extensively dialyzing explant medium in Spectrapor 7 dialysis tubing (molecular weight cutoff = -1000) for 24 h against several
thousand volumes of Tris-HCl buffer (10 mM, pH 8.2) at
4°C. Radioactivity present in medium before and after dialysis was quantified by liquid scintillation spectrometry (LKB
ESTROGEN EXPOSURE AFFECTS PORCINE UTERINE DEVELOPMENT
Estradiol
[M Corn Oil
Valerate
50
*
40
CD
30
'
20
.5
5
10
4
C
D
*
3
a
2
1
0
7
14
49
Day of Hysterectomy
FIG. 1. Effects of neonatal age and treatment with CO or EV on neonatal porcine uterine weight. Gilts were treated with CO or EV (50 Ipg/kg
BW/day) for 7 days before hysterectomy on Day 7, 14, or 49 (see text). For
CO-treated gilts, uterine weight increased after Day 14 (a vs. b; p < 0.01).
Treatment with EV increased uterine weight on all days (*p < 0.01), although the magnitude of this response was greatest on Day 49 (day x
treatment, p < 0.01). Data are presented as LSM + SE.
Wallac Oy Rackbeta 1214, Turku, Finland), and the percentage of total 3H-leu retained after dialysis was determined for each explant. Percentage of incorporation was
not affected by day or treatment (LSM ± SE: 2.3 + 0.2%)
and fell within the range for this response as reported for
adult bovine and porcine endometrial explants [29, 31, 32].
Dialyzed medium was stored at -20°C until analysis by
electrophoresis.
Histology and Morphometry
Fixed uterine tissues were processed for light microscopy as described previously [7]. Slides were stained with
Mayer's hematoxylin and counterstained with eosin. General histological observations and measurements of endometrial and myometrial thickness were made in four to five
nonsequential cross sections of four to five different areas
from each uterine horn using an Olympus BH-2 microscope (Olympus Optical Company, Ltd., Tokyo, Japan) fitted
with a calibrated ocular micrometer. Morphometric measurements were made at four equidistant points along the
perimeter of uterine cross sections. Endometrial thickness
was measured from the apical border of the lumenal epithelium to the junction of the stroma with the inner circular layer of myometrium. Myometrial thickness was measured from the junction of the stroma with the inner circular
layer of myometrium to the perimetrium.
Electrophoresis and Fluorography
Radiolabeled proteins present in dialyzed explant culture medium were identified by two-dimensional SDS-PAGE
743
(2D-PAGE) and fluorography following procedures described by Roberts et al. [33]. Individual samples containing
75 000 cpm of nondialyzable macromolecules were loaded
onto each gel. Paired samples from each day and treatment
were run together under identical conditions. Proteins were
resolved in the first dimension by isoelectric focussing. Procedures facilitated separation of proteins and polypeptides
in the first dimension by isoelectric point (pl) over a pH
range of approximately 3.4-8.8, determined as described
by Roberts et al. [33].
Second-dimension separation was accomplished by use
of polyacrylamide (12.5%, w/v) slab gels containing SDS
(0.1%, w/v). Molecular weight standards, including phosphorylase b (97 400), bovine albumin (66 200), egg albumin (42 700), glycerol-3-phosphate dehydrogenase (31 000),
trypsin inhibitor (21 500), and ao-lactalbumin (14 400; Sigma
Chemical Co.), were run on each gel, adjacent to sample
proteins. After electrophoresis, gels were stained with
Coomassie Brilliant blue R-250 dye. Fluorographs were then
prepared, with sodium salicylate used as a fluor [33] and
Kodak X-OMAT x-ray film. All dried gels were exposed together for 90 days at -70°C, developed with an automated
developer, and photographed.
Qualitative and Quantitative Evaluation of 2D-PAGE
Fluorographs
Protein profiles generated by fluorography of 2D-PAGE
gels were first compared visually among animals on a within
day and treatment basis to establish that fluorographic patterns were repeatable. Comparisons of fluorographic patterns were facilitated by preparing a tracing of each fluorograph. Tracings were sufficiently translucent to permit
superimposition of all fluorographic patterns on a lighted
background. Fluorographs and tracings were then compared to determine whether either the array of proteins
present or production of individual proteins was affected
by neonatal age or treatment. Procedures revealed 25 proteins for which fluorographic spot intensity changed consistently in association with neonatal age, treatment, or both.
Molecular weight and isoelectric points were then estimated for each of these proteins using linear regression
procedures.
The intensity of a fluorographic spot is proportional to
the amount of radioactive precursor incorporated into, and
the synthesis rate of, the corresponding protein [34]. Therefore, to assess effects of age and treatment on relative production of each of the 25 proteins that were identified by
visual inspection more objectively, radioactivity associated
with each fluorographic spot was quantified by liquid scintillation spectrometry following procedures described by
Malayer et al. [35]. Briefly, fluorographs were realigned with
dried 2D-PAGE gels and fixed in place with tape, and each
spot was excised from the dried gel by punching through
the x-ray film and dried gel simultaneously with a stainless
steel cork borer. If a particular spot was not visible on a
744
SPENCER ET AL.
FIG. 2. Representative photomicrographs, taken at constant magnification (x10), of uteri from gilts treated with CO (left) or EV (50 pig/kg BW/day;
right) for 7 days before hysterectomy on Day 7, 14, or 49 (see text). A and B: Day 7. Note larger cross-sectional area, precocious development of endometrial
glands (G), and endometrial folds (arrows) in tissue from EV-treated gilt. C and D: Day 14. Note distribution and physical appearance of endometrial glands,
development of endometrial folds (arrows), and pattern of stromal (S) organization in tissue from EV- as compared to CO-treated gilt. E and F: Day 49.
Note pronounced effects of EV on both endometrial and myometrial organization and development. (IC,inner circular myometrium; OL, outer longitudinal
myometrium).
set of fluorographs, a punch of the same size used to obtain
the analogous visible area from other gels was used to excise an equivalent amount of material. Thus, a piece of dried
2D-PAGE gel corresponding to the migration position of
each of the 25 proteins in question was obtained from all
gels. In addition, a reference spot that was clearly present
on all fluorographs and did not appear to change in relative
intensity was also punched from each gel. Quantification of
radioactivity in this reference spot was used to confirm that
loading rates were similar among 2D-PAGE gels. Radioactivity associated with each fluorographic spot was quantified according to published procedures [35]. The value (cpm)
obtained for each excised protein spot was corrected mathematically, as described by Malayer and coworkers [35], to
reflect the relative amount of radiolabel representative of
each protein in the total 5-ml explant culture for each gilt.
ESTROGEN EXPOSURE AFFECTS PORCINE UTERINE DEVELOPMENT
Corn
Oil
-
Estradiol
Valerate
1800
-
1600
E
1200
(A)
1200
E
oa
600oo
u
300
ago
0
7
14
49
Day of Hysterectomy
I
1200 r-
1000
(B)
.i
800
E
e
E
:I
600
400
200
0
7
14
49
Day of Hysterectomy
FIG. 3. Effects of neonatal age and treatment with CO or EV on porcine uterine endometrial (A) and myometrial (B) thickness (m). Gilts were
treated with CO or EV (50 g/kg BW/day) for 7 days prior to hysterectomy
on Day 7, 14, or 49 (see text). A) For CO-treated gilts, endometrial thickness
increased after Day 14 (a vs. b; p < 0.01). Treatment with EV increased
endometrial thickness on all days (*p < 0.01), although response was greatest
on Day 49 (day x treatment, p < 0.01). B) For CO-treated gilts, myometrial
thickness increased after Day 14 (p < 0.01). Treatment with EV did not
affect myometrial thickness on Days 7 and 14 (p > 0.10), but increased
myometrial thickness on Day 49 (day x treatment, p < 0.01). Data are presented as LSM + SE.
StatisticalAnalyses
All quantitative data were subjected to least-squares analyses of variance using the General Linear Models procedures of the Statistical Analysis System [36]. Analyses of
uterine wet weight, morphometry, and protein data considered variation due to day, treatment, and their interaction. Preplanned orthogonal contrasts of day (Day 7 vs. Day
14; Days 7 and 14 vs. Day 49) were included in analyses
where appropriate. To identify effects of neonatal age on
production of individual proteins, data derived from COtreated control gilts were analyzed separately with day used
as the only source of variation. To identify effects of EV
treatment on production of individual proteins, responses
for each day were analyzed separately with treatment used
as the only source of variation. Error terms used in all tests
of significance were identified on the basis of the expectations of the mean squares for error. All quantitative data
are presented as least-squares means with standard errors
(LSM
SE).
745
RESULTS
Uterine Weight
Normal uterine growth was observed in CO-treated control gilts. Uterine wet weight was unaffected (p > 0.10) by
age between Day 7 and Day 14 in CO-treated gilts, but increased (p < 0.01) by Day 49 (Fig. 1). Compared to respoi es of CO-treated gilts, EV increased (p < 0.01) uteriie'iet weight on Day 7 (1.1
0.3 vs. 2.8 + 0.3 g), Day
0.3 g), and Day 49 (9.8 + 2.0 vs.
14 (1.0 + 0.3 vs. 3.2
2.0 g). Response to EV was most pronounced on
41.5
Day 49 following exposure during the growth period of
uterine development (day x treatment, p < 0.01).
Histology
As expected, normal porcine uterine wall development
was seen in CO-treated gilts (Fig. 2, A, C, and E). Uterine
diameter appeared to increase after Day 14 in a manner
that paralleled changes in uterine wet weight (Fig. 1). Endometrial glands, apparent in shallow stroma (stratum
compactum) on Day 7 (Fig. 2A), appeared more frequently
in tissues from Day 14 (Fig. 2C) and extended to the inner
circular layer of myometrium in tissues from Day 49 (Fig.
2E). Endometrial folds and the outer longitudinal layer of
myometrium were clearly developed by Day 49 (Fig. 2E).
Treatment of neonatal gilts with EV had marked effects
on uterine wall histoarchitecture. Uteri of all EV-treated gilts
displayed precocious development of endometrial glands
and distinct endometrial folds (Fig. 2). Compared to those
from CO-treated gilts, endometrial glands in uteri from Day
14 and Day 49 EV-treated gilts were sparsely distributed and
less intensely coiled (Fig. 2, D and F). Though not quantified, fewer endometrial gland openings were apparent along
the luminal surface of the endometrium in tissues obtained
on Day 14 and Day 49 from EV-treated gilts (Fig. 2, D and
F).
The increase in uterine size after EV exposure reflected
stromal hypertrophy and edema. The densely cellular, shallow stromal zone (stratum compactum) visible beneath the
luminal epithelium in uteri of CO-treated gilts was not consistently evident in uteri obtained from EV-treated gilts, suggesting EV-induced stromal disorganization (Fig. 2). Myometrial hypertrophy was pronounced only in uteri obtained
from EV-treated gilts on Day 49 (Fig. 2F).
Morphometry
In CO-treated gilts, endometrial thickness was not affected by age (p > 0.10) between Day 7 and Day 14, but
increased (p < 0.01) by Day 49 (Fig. 3A). Treatment with
EV increased (p < 0.01; CO vs. EV) endometrial thickness
on Days 7 (282 + 49 vs. 412 + 49 Im), 14 (281 + 49 vs.
525 + 42 Aim), and 49 (815 + 49 vs. 1535 + 43 pzm). Endometrial response to EV treatment was greatest on Day 49
after exposure during the growth period of uterine development (day x treatment, p < 0.01).
746
SPENCER ET AL.
747
ESTROGEN EXPOSURE AFFECTS PORCINE UTERINE DEVELOPMENT
TABLE 1. Porcine neonatal uterine proteins (pNUP) affected by age and/or treatment with CO or EV.'
Day 7
2
Day 14
SE3
Day 49
pNUP
Mr/pl
CO
EV
CO
EV
CO
EV
CO
EV
pNUP1
pNUP2
pNUP3
pNUP4
pNUP5
pNUP6
pNUP7
pNUP8
pNUP9
76.3/4.2
74.4/3.3
72.3/5.6
72.0/5.5
72.0/5.4
69.6/4.5
60.2/5.9
60.2/5.8
59.8/5.7
20
120
316
635
476
99
911a
986
427
28
139
331
756
439
304*
662+
744
450
29
110a
393
567 a
381
90
942
4660
45
135
346
614
384
146
773+
831
356
17
59b
282
438 b
317
77
528C
4 13 b
92b
115*
211*
438*
688*
471*
125
945*
901*
134
9
14
33
29
39
32
97
70
29
8
12
29
25
34
28
84
61
25
pNUP10
pNUP11
48.7/5.4
47.0/6.4
191
967
250
856
2758
10798
184
455*
833
14 7 b
b
827*
828
54
44
56
38
pNUP12
41.3/6.3
251
315
335
131'
268
pNUP13
pNUP14
38.8/4.7
36.0/6.3
565a
8130
500
881
29.1/6.4
388
370
pNUP16
pNUP17
24.6/4.0
19.6/6.1
122
161a
427*
150
560
864
337
250*
143*
233
c
245
pNUP15
794
931b
4220
47 5 b
b
417
195
c
348
pNUP18
19.1/6.9
1430
175
283
99*
pNUP19
18.5/5.7
506
467
4668
416
pNUP20
pNUP21
17.2/5.2
16.3/6.6
345
202
321
189
283
158
pNUP22
pNUP23
pNUP24
pNUP25
16.2/6.8
15.9/6.3
14.1/4,5
13.4/6.7
1420
1080
1830
a
141
132
136
248
93
343
b
245
b
274
b
266
a
1 28 8 b
a
b
b
b
206
32
28
c
596*
856*
41
49
36
42
b
265
33
29
519*
372
46
29
40
25
202
302
23
20
1 36 b
546*
43
37
261
86
287
81b
482*
68
31
26
26
23
144*
119
177
43*
58
c
66
c
56
327
67
169*
82
330
14
18
21
36
12
16
18
31
200
c
c
'Results are expressed as cpm/explant (LSM).
Values are M, (X10- 3 )±0.05/pl±0.05.
3
Standard errors were calculated across days on a within-treatment (CO or EV) basis; overall, error variances for each
pNUP estimate were homogeneous.
'-"Effects of day were examined for CO-treated gilts on an individual pNUP basis (orthogonal contrasts: Day 7 vs.
Day 14; Days 7 and 14 vs. Day 49); means in a row with different superscripts (a, b, or c) differ (p < 0.01).
*2+Effects of treatment (CO vs. EV) were assessed for each pNUP on a within-day basis; therefore, within each day,
superscripts indicate that pNUP production was affected by EV treatment (* p < 0.01, + p < 0.05).
2
Myometrial thickness was also similar in CO-treated gilts
on Days 7 and 14 (p > 0.10), and increased (p < 0.01) by
Day 49 (Fig. 3B). Treatment with EV did not affect myometrial thickness on Day 7 (CO vs. EV; 222 ± 19 vs. 252
18
16 m). However,
m) or Day 14 (204
18 vs. 221
treatment with EV during the growth period increased myometrial thickness on Day 49 (321
18 vs. 911 + 16 Lm)
when compared to responses of CO-treated gilts (day x
treatment, p < 0.01).
Uterine Protein Synthesis
As illustrated in Figure 4, fluorography of 2D-PAGE gels
revealed a complex array of proteins that were synthesized
FIG. 4. Representative fluorographs illustrating effects of neonatal age
and treatment with CO (left) or EV (right) on patterns of uterine protein
synthesis. Gilts were treated with CO or EV (50 Fig/kg BW/day) for 7 days
prior to hysterectomy on Day 7, 14, or 49 (see text). A and B: Day 7. C and
D: Day 14. E and F: Day 49. A subset of 25 porcine neonatal uterine proteins (pNUP 1-25; arrows) was identified by visual appraisal of fluorographs (see text) for which production (reflected by spot intensity) was affected consistently by neonatal age in CO-treated gilts (compare A, C, and
E) and/or treatment with EV during infantile (A vs. B), proliferative (C vs.
D), or growth periods (E vs. F). Characteristics of the 25 pNUP identified
here are listed in Table 1.
from 3H-leu by neonatal uterine tissues and released into
explant medium. Visual comparison of fluorographic patterns revealed a subset of 25 proteins for which production
appeared to be affected consistently by neonatal age and/
or treatment with EV. Quantification of the radioactivity associated with fluorographic spots representing these 25
proteins corroborated visual appraisals. The 25 porcine
neonatal uterine proteins (pNUP 1-25) that were examined
are listed in Table 1 by molecular weight (Mr x 10 - 3 ) and
pI. Conditions (age, treatment, or both) affecting production of these proteins are also identified in Table 1. Representative fluorographs depicting these relationships are
illustrated in Figure 4. The 2D-PAGE migration positions of
these proteins and conditions that affected their production
are summarized in Figure 5.
Overall, production of 19 of the 25 identified proteins
was affected by neonatal age. However, production of only
4 of these 19 proteins was affected exclusively by neonatal
age (Table 1). In CO-treated gilts, production of pNUP 9,
15, and 21 decreased from Day 14 to Day 49, whereas production of pNUP 24 increased between Days 7 and 14 and
decreased on Day 49 (Table 1 and Fig. 4).
Production of 21 of the 25 proteins was affected by treatment with EV (Table 1). However, production of only 6 of
748
SPENCER ET AL.
pl
9
7
5
2.10
1.90
x
11
*12
012
140
1.70
1.50
o
_j
.
345
Fo
0
3
60
13
+15
.
1.10
9
7
+
Age only
0
Trt only
*
Age and Trt
10
16
18
17 9
021
0 20
22.+ 23
0
1.30
2
25
24
+
5
3
FIG. 5. Schematic illustration summarizing 2D-PAGE migration positions of the 25 porcine neonatal uterine proteins (1-25) identified by fluorography, and the conditions (age = plus sign; treatment = open circle;
age and treatment = closed circle) that affected their production. Protein
migration positions are mapped to molecular weight (Log, 0 M., 10-3 ) and
relative p coordinates. Four proteins (9, 15, 21, and 24) were affected by
age alone and 6 proteins (1, 3, 5, 6, 12, and 20) by estrogen treatment
alone; 15 proteins (2, 4, 7, 8, 10, 11, 13, 14, 16, 17, 18, 19, 22, 23, and 25)
were affected differentially by both age and estrogen treatment (see Table
1 and Fig. 4). Trt = treatment.
these 21 proteins was affected exclusively by EV treatment,
independently of effects of neonatal age. For these 6 proteins, treatment with EV increased production of pNUP 6
by tissues obtained on Day 7, decreased production of pNUP
12 by tissues obtained on Day 14, and increased production
of pNUP 1, 3, 5, and 20 by tissues obtained on Day 49 (Table 1 and Fig. 4). Production of the other 15 proteins in
this subset was affected differentially by neonatal age and
treatment with EV as summarized in Table 1 and illustrated
in Figures 4 and 5.
Overall, the number of proteins in the subset of 25 identified in Table 1 affected by treatment with EV increased
with neonatal age. However, the direction of response to
treatment-that is, whether relative production of a given
protein increased or decreased-varied with period of estrogen exposure (Table 1 and Figs. 4 and 5). Specifically,
treatment during the infantile period of uterine development affected production of three proteins on Day 7 (pNUP
6, 7, and 16; Fig. 4, A and B). Effects of EV on production
of pNUP 6 and 16 were positive, whereas production of
pNUP 7 decreased. Treatment with EV during the proliferative period of uterine development consistently suppressed production of 8 uterine proteins on Day 14 (pNUP
7, 11, 12, 16, 17, 18, 22, and 25; Fig. 4, C and D). However,
treatment of gilts with EV during the growth period consistently enhanced production of 14 proteins (pNUP 1, 2,
3, 4, 5, 7, 8, 10, 13, 14, 16, 19, 20, and 23; Fig. 4, E and F).
DISCUSSION
The porcine uterine wall is incompletely developed at
birth, but undergoes dramatic remodeling during the first
two months of neonatal life [1, 2, 4-7]. Growth of the uterus
during this period occurs normally in ovariectomized gilts
[3]. Spencer and coworkers [7,8] identified age- and tissue
site-specific changes in patterns of stromal and epithelial
DNA synthesis and parallel alterations in the distribution of
glycosaminoglycans, lectin binding sites, and newly synthesized glycoconjugates in neonatal porcine uterine tissues
obtained between birth and Day 56. From these observations, infantile (prior to Day 7), proliferative (Days 7 through
14), and growth (after Day 14) periods of neonatal porcine
uterine development were defined and characterized [7].
However, the extent to which these and other developmental events must occur without interruption in order to
insure normal adult uterine function remains unclear.
The idea that interruption of development during specific periods of time could have lasting affects on tissue form
and function is not new [37]. Accordingly, exposure of fetal
and neonatal rodents to estrogens has been demonstrated
to produce a variety of structural and functional lesions in
adult uterine and urogenital tract tissues [17,19,20,38].
Moreover, it has been recognized that both the type and
degree of severity of such lesions vary depending upon when
exposure occurs [14,15,24-26]. Before this study, developmental responses of porcine uterine tissues to estrogen
exposure during specific periods of early neonatal life had
not been characterized. However, available data provided
support for the idea that estrogen might be useful as a tool
with which to interrupt the normal pattern or program of
uterine development in the neonatal pig.
In the present study, effects of both age and treatment
with EV on uterine development and protein synthesis were
demonstrated clearly. Patterns of uterine response to EV
can be interpreted to suggest that estrogen sensitivity of
these tissues changes with age between the first and seventh weeks of neonatal life. Positive uterine growth responses were observed after estrogen treatment during all
periods. However, the magnitude of response, as reflected
by changes in uterine wet weight, was much more pronounced for gilts treated with estrogen during the growth
period than for gilts treated during either infantile or proliferative periods. Histological and morphometric data indicated that effects of estrogen were evident primarily in
endometrial tissues after treatment during infantile and
proliferative periods. In contrast, treatment during the growth
period induced striking alterations in both endometrial histoarchitecture and myometrial development.
Ontogeny of estrogen receptor expression in developing
neonatal porcine uterine tissues has yet to be evaluated.
However, present data suggest that estrogen receptor
expression may occur progressively in porcine endometrial
tissues during the first two weeks of neonatal life, while
expression in myometrial tissues is delayed. Data are consistent with the idea that acquisition of competence to respond to estrogen occurs progressively in the developing
uterus [39]. Pasqualini and Sumida [40] observed that estrogen receptor synthesis may be a constitutive property of
uterine cells in the neonate.
ESTROGEN EXPOSURE AFFECTS PORCINE UTERINE DEVELOPMENT
The ability of estrogen to affect uterine responses, especially during early neonatal life, could also be affected
by the presence of estrogen binding proteins in the peripheral circulation. Alpha fetoprotein (tFP), a recognized
estrogen binding protein in the rat [41], is present in serum
obtained from neonatal pigs [42]. Serum concentrations and
hepatic synthesis of aFP have been reported to increase
between birth and Day 5 in the pig [42]. However, porcine
oaFP has not been reported to bind estrogen with high affinity and may, therefore, not affect bioavailability of circulating estrogen in this species [43].
As the porcine uterus develops during the first seven
weeks of postnatal life, uterine glands appear, penetrate,
and proliferate within the endometrial stroma [1, 4-7]. Although morphometric data have not been collected, subjective evaluation of endometrial histogenesis during this
period suggests that the ratio of mesenchyme or stroma to
epithelium is comparatively high at birth and decreases
thereafter as glands penetrate the stromal matrix. Tissue recombination studies have been interpreted to suggest that
both patterns of epithelial growth and absolute organ size
reflect the ratio of mesenchyme to epithelium during development [44]. Functional, reciprocal interactions between
uterine epithelium and underlying stroma have been demonstrated to be required to support epithelial development, stromal organization, and myometrial growth
[22, 45, 46]. As suggested above, responses of neonatal uterine tissues to estrogen exposure may indicate development-related changes in sensitivity of endometrial stroma
and epithelium to estrogen. Morphogenetic responses of
neonatal porcine uterine tissues to estrogen may, therefore,
reflect (1) the size and nature of cell populations in the
uterine wall that are competent to respond to estrogen and
(2) the consequences of estrogen-induced aberrations in
the ratio of epithelium to stroma on subsequent events associated with uterine growth and development.
A substantial body of information [47-5,1], including a
recent report from this laboratory [7], supports the idea that
morphogenetic events associated with development of the
uterine wall may be regulated locally via synergistic effects
of insoluble components of the extracellular matrix and locally produced, diffusible polypeptide or protein morphogens on constituent cell populations. Moreover, lesions resulting from short-term exposure of the developing genital
tract to steroids have been suggested to occur, in part, as
a consequence of developmentally inappropriate patterns
of production of growth factors or other inductors [52].
Therefore, it was important to identify proteins produced
by neonatal uterine tissues, as well as the consequences of
estrogen exposure during specific developmental periods
on patterns of production of these proteins. Efforts were
focused on identification and preliminary characterization
of proteins produced by neonatal tissues from 3 H-leu that
were released into explant medium. This set of proteins is
749
likely to represent the array of uterine secretory products
with potential for extracellular diffusion.
Analysis of metabolically labeled macromolecules produced from radiolabeled substrates by tissues maintained
in organotypic culture provides a means for assessing the
state of differentiation of an organ without compromising
its three-dimensional structure. This approach was suggested to provide information lacking from cell culture experiments in which normal cell and tissue interactions are
ignored [53,54]. In vitro metabolic labeling procedures
combined with 2D-PAGE and fluorography have been used
successfully to identify molecular markers of normal and
aberrant uterine and urogenital tract development in a
number of laboratory species [18, 53, 55, 56]. Neonatal porcine uterine tissues produce a complex array of proteins
in vitro. However, patterns of production of 25 of these
proteins (pNUP 1-25, Table 1) were found to be consistently affected by neonatal age and (or) treatment with EV.
Consequently, effects of age and treatment on uterine protein production were examined most carefully for this subset of uterine products. A similar approach was described
by Newbold and coworkers [18] in studies designed to
identify uterine and vaginal proteins that might serve as
molecular markers of exposure to diethylstilbestrol in the
mouse.
Results of 2D-PAGE studies revealed effects of neonatal
age and period of estrogen exposure on uterine development at the molecular level that were complementary to
morphometric and histological data. Patterns of production
of four proteins (pNUP 9, 15, 21, and 24) were affected
exclusively by neonatal age, independently of treatment with
EV (Table 1). These proteins may prove to be useful biologic markers of normal uterine development. Similarly, the
six proteins (pNUP 1, 3, 5, 6, 12, and 20) identified to be
affected exclusively by EV treatment may prove useful as
biologic markers of estrogen exposure during specific periods of neonatal life: pNUP 6 during the infantile period;
pNUP 12 during the proliferative period; and pNUP 1, 3, 5,
and 20 during the growth period.
Treatment of neonatal gilts with EV during infantile, proliferative, and growth periods affected production of three,
eight, and 14 individual proteins, respectively. The present
results are generally consistent with data reported for the
neonatal rat, in which administration of estrogen during the
first two weeks of life stimulates production of a limited
number of uterine proteins compared to those produced
by older animals [57]. Observations by others [39,40] lend
further support to the idea that uterine tissues acquire the
ability to respond to estrogen gradually during the postnatal period.
Further study is needed to identify and clarify the developmental roles of porcine neonatal uterine proteins described here. However, one of these proteins (pNUP 17;
see Table 1) is electrophoretically similar to retinol binding
protein (RBP). Production of this protein increased from
750
SPENCER ET AL.
Day 7 to Day 14, decreased to Day 49, and was enhanced
by EV treatment during the proliferative period (Table 1).
A similar, 3H-leu-labeled protein has been precipitated from
neonatal porcine uterine explant medium with rabbit antihuman RBP antiserum [58]. Consistent with present results, uterine production of this RBP-like protein was found
to increase in association with onset of endometrial gland
proliferation from birth to Day 6 [58].
Collectively, these data indicate that normal patterns of
porcine uterine development can be interrupted by treatment of neonatal gilts with EV. Effects of treatment varied
depending upon whether exposure occurred during (1) the
infantile period, associated with appearance and onset of
rapid development of endometrial glands; (2) the proliferative period, associated with intense glandular epithelial
DNA synthesis; or (3) the growth period, when endometrial
morphogenetic activity decreases and uterine wall histoarchitecture is stabilized [7]. However, uterine responses to
EV were consistent and predictable. Thus, a dependable
method for interruption of normal uterine developmental
events and induction of specific physical and biochemical
responses in neonatal porcine uterine tissues has been
identified.
Whether critical periods [37] of uterine development exist for the neonatal pig, during which interruption of development by exposure of tissues to estrogens will produce
permanent structural and functional lesions in adult tissues,
cannot be determined from this study. However, lesions
recognized to occur in uteri of adult rodents after neonatal
estrogen exposure include reduced levels of estrogen and
progesterone receptors, altered estrogen metabolism and
responsiveness, altered protein synthesis, cystic endometrial hyperplasia, squamous metaplasia, adenomyosis, myometrial hypoplasia, and general uterine hypoplasia
[14, 15, 18, 20, 22, 24, 38, 59]. Clearly, similar consequences
in the pig could compromise the ability of adult uterine
tissues to support cyclicity and pregnancy [60]. The fact that
short-term developmental responses of neonatal porcine
uterine tissues were age-specific suggests that any long-term
effects of EV-induced developmental interruption might be
expected to reflect the period when estrogen exposure occurred. In this respect, efforts to identify such lesions in
the adult porcine uterus, together with the neonatal periods of estrogen exposure associated with their induction,
will represent an important step toward identification of
developmental determinants of uterine function.
ACKNOWLEDGMENTS
The authors wish to thank Mr. David McGee and Ms. Mabel Robinson for technical assistance; Dr. Dale Coleman for assistance with surgical manipulations; Mr.
Thomas Martin and Mr. Bill Robinson for assistance with preparation of illustrations;
and Mr. Mike Carroll and Mr. Clinton Dowdell for assistance with management of
gilts.
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