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Center for Animal Biotechnology and Genomics (H.K., L.A.J., G.A.J., T.E.S., F.W.B.) and Departments of Animal Science (H.K., G.A.J., T.E.S., F.W.B.) and Veterinary Anatomy and Public Health (L.A.J.), Texas A&M University, College Station, Texas 77843-2471
Address all correspondence and requests for reprints to: Dr. Fuller W. Bazer, Department of Animal Science and Center for Animal Biotechnology and Genomics, 442D Kleberg Center, Texas A&M University, College Station, Texas 77843-2471. E-mail: fbazer{at}cvm.tamu.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the pig, KGF expression in the endometrium is highest on day 12 of pregnancy during the period of maternal recognition of pregnancy (4). In male and female reproductive organs, KGF gene expression in rodents is up-regulated by steroid hormones such as androgens, estrogen, and progesterone (P4). The KGF gene has an androgen response element in the promoter region (5), and testosterone increased the expression of KGF in prostate and seminal vesicles (6, 7). In primate endometrium, P4 increases KGF expression during the luteal phase, suggesting that KGF is a mediator of P4 action on epithelial cells or a progestamedin (8). It has also been suggested that KGF is induced by estrogen in the female genital tract of mice during neonatal development (9). 17ß-Estradiol (E2) treatment in vivo increases KGF messenger RNA (mRNA) and protein expression in the mouse mammary gland (10). In the porcine uterus, estrogens are secreted by conceptus trophectoderm beginning on days 11 and 12 of pregnancy and are the signal for maternal recognition of pregnancy (11). Progesterone levels also increase up to 30 ng/ml in plasma during early pregnancy (12). Therefore, estrogens and/or P4 may be responsible for the increased KGF expression in the porcine uterine endometrium during early pregnancy.
During early pregnancy, pig conceptuses undergo dramatic changes in morphology and differentiation in preparation for implantation and placentation (13, 14). It is well known that KGF stimulates the proliferation and migration of various epithelia and also affects cellular differentiation processes (1). The biological activity of KGF is achieved through intracellular signaling activated by KGFR, and KGF activates phosphorylation of KGFR and the mitogen-activated protein kinase (MAPK) pathway (15). Given that KGF is a component of histotroph, it may stimulate the proliferation and differentiation of the conceptus. Therefore, the objectives of this study were to determine: 1) the effects of estrogens and P4 on KGF expression in the porcine uterine endometrium, and 2) the effects of KGF on proliferation and differentiation of porcine conceptus trophectoderm in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Explant culture
Endometrium was dissected from myometrium and placed into warm
phenol red-free DMEM/F-12 culture medium (DMEM/F-12;
Sigma) containing penicillin G (100 IU/ml), streptomycin
(0.1 mg/ml), and amphotericin (0.25 µg/ml; Life Technologies, Inc., Grand Island, NY), as described previously
(17). Endometrium was then minced with scalpel blades into
small pieces (2–3 mm3), and aliquots of 500 mg
were placed into culture dishes (100 x 15 mm) with serum-free
modified DMEM/F-12 containing 10 µg/ml insulin (Sigma,
catalogue no. I5500), 10 µg/ml transferrin (Sigma,
catalogue no. T1428), and 10 ng/ml hydrocortisone (Sigma,
catalogue H0396). Endometrial explants were cultured immediately after
mincing in the presence of E2 (0, 0.05, 0.5, 5,
and 50 ng/ml), P4 (0, 0.03, 0.3, 3, and 30
ng/ml), catechol estrogens [5 ng/ml 2-hydroxy-E2
(2OH-E2) or 5 ng/ml
4OH-E2], estrogen receptor (ER) antagonist [50
ng/ml ICI 182,780 (ICI)], or E2 (5 ng/ml) plus
P4 (3 ng/ml) for 48 h with rocking under an
atmosphere of 45% nitrogen, 5% carbon dioxide, and 50% oxygen.
E2 (catalogue no. E8875),
P4 (catalogue no. P0130),
2OH-E2 (catalogue no. H3131), and
4OH-E2 (catalogue no. H4637) were obtained from
Sigma, and ICI was purchased from Tocris (Ballwin, MO).
Explant tissues were then harvested, and RNA was extracted for slot
blot analysis of KGF expression. These experiments were conducted using
endometrium from three individual gilts. Treatments were performed in
triplicate using tissues obtained from each gilt.
Porcine trophectoderm cells
Porcine trophectoderm cells were isolated using nonenzymatic
dispersion of trophoblast from conceptuses collected on day 12 of
gestation (18). A trophectoderm cell line (pTr) was
established by repeated passage and culture of the cells on Primaria
tissue culture plastic (Falcon, Lincoln Park, NJ). Cells were
maintained in DMEM/F-12 containing 5% charcoal-stripped serum,
antibiotics, 2 mM glutamine (Sigma), and 0.1
U/ml bovine insulin (Sigma).
[3H]Thymidine incorporation assay
Effect of KGF on proliferation of pTr cells was determined as
described previously (19). Briefly, pTr cells were plated
at 20,000 cells/cm2 in DMEM/F-12 containing 5%
FBS, then serum-starved for 24 h in serum-free DMEM/F-12,
containing 2 mM glutamine and 0.1% BSA. Cells were then
treated with recombinant rat KGF (rKGF; 0, 1, 10, or 100 ng/ml) for
24 h at 37C in serum-free DMEM/F-12 containing 5 µCi/ml
[3H]thymidine, precipitated in 10%
trichloroacetic acid for 30 min on ice, and fixed in cold methanol. The
fixed cells were solubilized in 0.6 ml 0.05% trypsin/0.1% SDS for 30
min at 37 C. [3H]Thymidine incorporation was
counted using an LS 3801 liquid scintillation counter (Beckman Coulter, Inc., Palo Alto, CA). The total DNA content was
determined using Picogreen (Molecular Probes, Inc.,
Eugene, OR) as described by the manufacturer. Data are expressed as
disintegrations per min/µg total DNA.
Northern and slot blot hybridization analysis
Total cellular RNA was isolated from endometrial explant tissues
and cultured pTr cells using TRIzol reagent (Life Technologies, Inc.). Expression of KGF in explant tissues and of
urokinase-type plasminogen activator (uPA) in pTr cells was determined
by Northern blot and slot blot hybridization analyses as described
previously (16). Twenty micrograms of total cellular RNA
were hybridized with 32P-radiolabeled antisense
complementary RNA probes generated against a linearized 690-bp porcine
KGF partial complementary DNA (cDNA) (4), 511 bp bovine
uPA partial cDNA (provided by Dr. A. R. Menino, Jr., Oregon State
University, Corvallis, OR), or 18S ribosomal RNA (pT718S, Ambion, Inc., Austin, TX). Autoradiographs of Northern blots to
determine the size of the uPA transcript were prepared using
Kodak X-OMAT x-ray film (Eastman Kodak Co.,
Rochester, NY). The radioactivity in each slot was quantified using a
Packard Instant Imager (Packard, Meriden, CT) and is expressed as total
counts.
RT-PCR
Expression of KGFR and interferon-
(IFN) by pTr was
determined by RT-PCR as described previously (20). Five
micrograms of total RNA from pTr cells were reverse transcribed to
obtain cDNAs using Superscript II reverse transcriptase (Life Technologies, Inc.). Newly synthesized cDNA was acid-ethanol
precipitated, resuspended in 20 µl water, and stored at -20 C. The
cDNAs were then diluted (1:10) with sterile water, and templates were
amplified by PCR using AmpliTaq DNA polymerase (Perkin-Elmer Corp., Foster City, CA) and specific primers based on the human
KGFR (GenBank accession no. M80637; forward, 5'-TCTGTTCAATGTGACCGAGG;
reverse, 5'-GTTTTGGCAGGACAGTGAGC) or the porcine IFN
(GenBank accession no. Z22706; forward, 5'-ATGGATTGTCCCCATGTAGG;
reverse, 5'-CTGAGCTACCAGGGTTACCG). PCR conditions
were 35 cycles of 95 C for 30 sec, 55 C for KGFR or 58 C for IFN for
30 sec, and 72 C for 1 min. PCR products (104 bp for KGFR and 296 bp
for IFN) were separated in 2% agarose gels and visualized by
ethidium bromide staining. The identity of each amplified PCR product
was verified by sequence analysis after cloning into the pCRII vector
(Invitrogen, Carlsbad, CA).
Immunohistochemistry
Expression of immunoreactive proliferating cell nuclear antigen
(PCNA) was evaluated in conceptus and paraformaldehyde-fixed,
paraffin-embedded, uterine tissue cross-sections (5 µm) using 2
µg/ml of a monoclonal antibody to PCNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; catalogue no. sc-56) and a
Super ABC Mouse IgG Kit (Biomeda, Foster City, CA), and procedures
described previously (16). A boiling citrate buffer
antigen retrieval protocol was used to reveal the PCNA according to the
manufacturer’s recommendations. Purified normal mouse IgG
(Sigma) at 2 µg/ml was substituted for mouse anti-PCNA
and served as a negative control.
Immunofluorescence
The epithelial phenotype of pTr cells was confirmed using
immunofluorescence microscopy as described previously
(21). pTr cells were cultured in LabTek four-well chamber
slides (Nunc, Naperville, IL), washed with PBS, fixed with methanol for
10 min at -20 C, air-dried, blocked in 5% normal goat serum, and
incubated in primary antibody overnight at 4 C. Monoclonal antibodies
to cytokeratin (hybridoma 8.13; 1:200 dilution) and vimentin (hybridoma
V9; 1:200 dilution) were obtained from Sigma. Slides were
then washed and incubated with fluorescein-conjugated rabbit antimouse
IgG (1:200 dilution; Sigma) for 1 h at room
temperature. After rinsing, all slides were overlaid with coverslips
and Prolong antifade mounting reagent (Molecular Probes, Inc., Eugene, OR). Fluorescence images were recorded using a
Carl Zeiss Axioplan 2 microscope fitted with a Hamamatsu
C-5810 chilled three-color CCD camera (Carl Zeiss,
Thornwood, NY) with Adobe Photoshop 5.0 (Adobe Systems, Seattle, WA)
image capture software.
Immunoprecipitation and Western blot analyses
To confirm activation of the KGFR in vitro, pTr cells
were treated with rKGF (10 ng/ml), and phosphorylation of the KGFR and
the MAPKs, extracellular signal-regulated kinases 1 and 2 (ERK1/2), was
assessed by immunoprecipitation and Western blot analysis. Briefly,
monolayer cultures of pTr cells were grown to 75% confluence on
75-cm2 tissue culture flasks and then incubated
in serum-free DMEM/F-12 containing 0.1% BSA for 48 h. Whole cell
extracts were prepared as previously described (22). The
protein concentration of the lysate supernatant was determined by
Bradford assay (Bio-Rad Laboratories, Inc., Burlingame,
CA) using BSA as the standard and 1 mg of each extract used for
immunoprecipitation. Five micrograms of KGFR antibody (Santa Cruz Biotechnology, Inc., catalogue no. sc-122) or normal rabbit IgG
were added to each extract, and bound proteins were purified using
protein A/G plus agarose as described previously (22).
Immunoprecipitated proteins were separated by SDS-PAGE and analyzed by
Western blotting (as described below) with antibody to phosphotyrosine
(Santa Cruz Biotechnology, Inc.; catalogue no. sc-7020)
diluted 1:100 in 5% BSA-TBST (Tris-buffered saline/0.1%
Tween-20).
In similar experiments pTr cells were serum-starved and treated with rKGF as described above. In addition to KGF treatment, some cells were pretreated with 0 or 50 µM of the MAPK/ERK kinase 1 (MEK1) inhibitor, PD98059 (New England Biolab, Beverly, MA; catalogue no. 9900L) for 1 h, then treated with 0 or 10 ng/ml rKGF for 60 min. Twenty micrograms of whole cell extract protein from each sample were separated by SDS-PAGE and transferred to nitrocellulose as described previously (21). Blots were blocked for 4 h at 4 C with either 5% BSA-TBST for phospho-specific antibodies or 5% nonfat milk-TBST for other antibodies, and then incubated with primary antibodies overnight at 4 C. Monoclonal antibodies to phospho-ERK1/2 (pERK1/2; 1:400 dilution in 5% BSA-TBST; catalogue no. sc-7383) and phospho-tyrosine (1:100 dilution in 5% BSA-TBST; catalogue no. sc-7020), and goat polyclonal antibody to ERK1/2 (1:400 dilution in 2% milk-TBST; catalogue no. sc-94-g) were obtained from Santa Cruz Biotechnology, Inc. Blots were then incubated with rabbit antigoat IgG or goat antimouse IgG conjugated to peroxidase (Kirkegaard & Perry Laboratories, Bethesda, MD) for 1 h at room temperature, and immunoreactive proteins were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, NY) according to the manufacturer’s recommendations.
Effect of KGF on pTr cell differentiation
To determine whether KGF affects functional cell
differentiation, pTr cells were treated with increasing doses of rKGF
(0, 1, 10, and 100 ng/ml) in serum-free DMEM/F-12 for 24 h.
Expression of uPA was used as a marker for pTr cell differentiation by
Northern and slot blot analyses.
Statistical analysis
All quantitative data were subjected to least squares ANOVA
using the general linear models procedures of the Statistical Analysis
System (SAS Institute, Inc., Cary, NC) (23).
Data from dose-response studies on KGF and uPA expression were analyzed
by least squares regression analysis. Slot blot data (total counts)
were analyzed using the 18S ribosomal RNA data as a covariate to
correct for differences in sample loading. Preplanned contrasts
(control vs. E2 plus ICI;
E2 vs. E2 plus
ICI; control vs. catechol estrogens; control vs.
E2; P4 vs.
E2 plus P4) were used to
test for effects of treatments in slot blot analyses. All tests of
statistical significance were performed using the appropriate error
terms according to the expectation of mean squares. Data are presented
as least squares means with SEs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The increase in KGF expression by
E2 was blocked by addition of the ER antagonist,
ICI (Fig. 1B; E2 vs.
E2 plus ICI, P < 0.05). Catechol
estrogens, 2OH-E2 and
4OH-E2, did not affect KGF expression in
endometrium (Fig. 1C; control vs.
2OH-E2 and control vs.
4OH-E2, P > 0.05).
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). However,
E2 increased (P < 0.01) KGF
expression in both the absence and presence of P4
(Fig. 2B).
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). All TE cells
of conceptuses recovered between days 10 and 15 of pregnancy expressed
PCNA. In contrast, PCNA was not detected in endometrial luminal
epithelium (LE) or glandular epithelium (GE) between days 9 and 12 of
the estrous cycle or pregnancy. Although PCNA was barely detectable on
day 15 of the estrous cycle and pregnancy, it was strongly detectable
in LE and TE from days 30 and 60 of pregnancy, and was abundant in GE
from day 60 of pregnancy (Fig. 3B).
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) was confirmed based on a cobblestone
morphology (a) and by positive cytokeratin (b) and negative vimentin
(c) immunostaining compared with the IgG control (d). KGFR, expressed
in porcine trophectoderm and endometrial epithelial cells
(4), and IFN, expressed by porcine trophectoderm
(24), were detected in pTr cells by RT-PCR (Fig. 4, B and
C).
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).
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, B and C). Pretreatment of cells with
PD98059, a MEK1 inhibitor, blocked the ability of rKGF to induce
phosphorylation of ERK1/2 (Fig. 5D).
Effect of KGF on pTr cell differentiation
The specificity of the bovine uPA probe was confirmed by Northern
blot analysis of total RNA from pTr cells, and a single transcript of
about 2.5 kb was detected (Fig. 6A).
Treatment of pTr cells with increasing doses of rKGF increased
(quadratic, P < 0.01) the expression of uPA (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Catechol estrogens, 2OH-E2 and
4OH-E2, are secreted by porcine conceptuses on
days 12 and 13 (27), but did not affect KGF expression in
porcine endometrium in the present study. This may be due to the low
affinity of catechol estrogens for the ER compared with
E2 (28, 29) or perhaps the inherent
instability of catechol estrogens. It should be noted that various
forms of estrogens are present in the porcine uterine lumen during
pregnancy, such as estrone, estradiol, estriol, catechol estrogens, and
their sulfated forms (27, 30, 31). Levels of estradiol in
the uterine lumen of pregnant pigs (
4.3 nM) are
significantly higher than those in nonpregnant pigs (0.26
nM) between days 9 and 15, and E2
predominates over all other estrogens in the uterine lumen
(30). Therefore, it is likely that
E2 is the predominant estrogen that acts via
ER
to increase endometrial KGF expression in the pregnant pig
uterus. It remains to be determined whether ERß is expressed in the
porcine uterine endometrium.
E2 probably up-regulates KGF mRNA expression in
endometrial epithelial cells through direct interaction with ER present
in these cells. ER protein is localized to LE and GE cells of the
porcine endometrium (32), and up-regulation of KGF
expression by E2 is inhibited by the ER
antagonist, ICI 182,780. Indeed, the promoter region of the human KGF
gene contains an estrogen response element (33). ER
expression in endometrial epithelial cells during early pregnancy is
unique in the pig uterus compared with other species, such as sheep and
primates, in which the endometrial epithelial cells do not express
ER during the P4-dominant period (16, 34). In pigs, ER staining in LE and GE is readily detectable
from days 5–12 of the estrous cycle and pregnancy, then decreases, but
remains detectable until day 15 of the estrous cycle and pregnancy.
ER is absent in stromal cells between days 5 and 15 of the estrous
cycle and pregnancy (32).
P4 is the major hormone responsible for the
establishment and maintenance of pregnancy. During diestrus and early
pregnancy, production of P4 by the corpus luteum
begins to increase (12) and is associated with significant
increases in uterine secretory activity (13).
P4 treatment in the primate uterus increases KGF
expression (8). However, the results of the present study
indicated that P4 decreased KGF expression in
porcine endometrium. The mechanism of the
P4-mediated decrease in KGF expression is not
known. Interestingly, E2 increased KGF expression
in the presence of P4, a situation to which the
endometrial epithelium would be exposed in vivo during
pregnancy. As P4 down-regulates endometrial ER
in ovine uterus (16, 35, 36), the decreased expression of
KGF in endometrial explants treated with P4 alone
and E2 plus P4 may be the
result of a P4-mediated decrease in ER and an
attenuated response to E2. We cannot rule out the
effects of any residual levels of estradiol on decreased KGF expression
by P4 in this system. It is also possible that
P4 inhibits KGF expression through a direct
mechanism or indirectly by modulation of other unknown factors which
then down-regulate KGF expression.
KGF affects epithelial cell proliferation in various tissues
(37). In the present study KGF increased DNA synthesis of
pTr cells, which are of epithelial cell origin and express KGFR, and
IFN, a porcine trophectoderm cell-specific marker (24).
Porcine conceptuses undergo dramatic morphological changes during early
pregnancy (13, 14). Between days 10 and 12 of pregnancy
there is a rapid transition from spherical (9–10 mm in diameter) to
tubular (10–50 mm in length) and elongated filamentous forms (>100 mm
long) (14). In the present study trophectoderm cell
proliferation was detected between days 9 and 15 of conceptus
development as reported previously (14). Thus, in
vitro results of the present study suggest that KGF of endometrial
epithelial origin may increase the proliferation of conceptus
trophectoderm during the periimplantation phase of development. This
hypothesis is supported by results from PCNA staining of conceptuses
in vivo between days 9–15 of pregnancy, indicating high
intensity PCNA staining from day 10 of pregnancy and thereafter. The
PCNA staining in the conceptuses is coordinate with increasing KGF
expression in the endometrium between days 10 and 15 of pregnancy
(4). The lack of detection of PCNA in the LE and GE during
early pregnancy suggests that epithelial cells do not proliferate to
any great degree at this time. Therefore, although KGF is present in
the uterine lumen, and KGFR is expressed in LE and GE, it is unlikely
that KGF affects epithelial cell proliferation in the endometrium
during early pregnancy. However, it is possible that KGF affects
differentiation of endometrial epithelial cells.
In addition to effecting proliferation, KGF was found to alter conceptus trophectoderm cell differentiation in this study. In various cell types, uPA, aromatase, surfactant protein A and D, syndecan-1, and Na+/K+-adenosine triphosphatase are increased by KGF (1, 37, 38). In particular, KGF increases uPA expression and activity in human uterine exocervical epithelial cells (39) and keratinocytes (40). In this study KGF increased uPA expression in pTr cells. Differentiating pig conceptuses produce uPA from trophoblast (41), and pig conceptus trophectoderm produces uPA in a biphasic manner between days 10 and 12 and between days 14 and 16 of early pregnancy (42), coinciding with estrogen production by conceptuses. Therefore, increased expression of uPA by KGF in pTr cells suggests that KGF expression increases within endometrial LE in response to estrogen, is secreted into the uterine lumen, and may be an important regulator of uPA secretion by conceptus trophectoderm.
Like most receptor tyrosine kinase-activating growth factors, KGFR signals through the MAPK pathway (15). In the present study phosphorylation of KGFR and ERK1/2, members of the MAPK family, was detected in KGF-treated pTr cells, suggesting that effects on trophectoderm proliferation and differentiation were mediated by interaction of KGF with the KGFR. Among the several FGFR isoforms, KGF recognizes only KGFR with high affinity and biological activity (43), precluding the possibility that ERK1/2 is activated by other members of the FGFR family. The precise mechanisms of intracellular KGF signaling for proliferation and differentiation in pTr cells remain to be determined.
In summary, the results of the present study indicate that E2, a pregnancy recognition signal in pigs, increases KGF expression in the uterine endometrium, and that KGF increases the proliferation of conceptus trophectoderm and stimulates the expression of uPA, a marker for differentiation. Thus, KGF of endometrial origin affects both the growth and differentiation of trophectoderm during this crucial phase of conceptus development in pigs.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Animal and Veterinary Science,
University of Idaho, Moscow, Idaho 83844-2330.
Received January 8, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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: the first member of a novel
type I interferon family. Biochimie 80:779–788[Medline]
,16[alpha,17beta]-tetrol). Steroids 27:325–333[CrossRef][Medline]
and ß. Endocrinology 138:863–870
and progesterone on estrogen-stimulated
expression of receptors for estrogen, progesterone and oxytocin in the
endometrium of ovariectomized ewes. Reprod Fertil Dev 8:843–853[CrossRef][Medline]
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