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Center for Animal Biotechnology and Genomics, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University System Health Science Center, and Department of Animal Science, Texas A&M University (T.E.S., A.G.S., M.M.J., F.W.B.), College Station, Texas 77843-2471; the Department of Urology, M.D. Anderson Cancer Center, University of Texas (C.A.W., G.J.), Houston, Texas 77030-4095; and the Department of Animal and Dairy Science, Auburn University (A.A.W., F.F.B.), Auburn, Alabama 36849-5415
Address all correspondence and requests for reprints to: Dr. Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 444 Kleberg Center, Texas A&M University, College Station, Texas 77843-2471. E-mail: tspencer{at}ansc.tamu.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The available evidence for both the sheep and pig strongly supports the idea that secretions from endometrial epithelium influence conceptus development, onset of pregnancy recognition signals, and growth of the fetus and placenta (5, 6, 7, 8, 9). Evidence from a variety of mammalian uteri, including those of humans, nonhuman primates, sheep, and pigs, indicates that both endometrial LE and GE are secretory, although their secretory functions differ (2, 10). For example, uteroferrin is secreted by GE, whereas serine protease inhibitors are secreted by LE (11). Differences in gene expression between endometrial LE and GE in ruminants and pigs lend support to the ideas that secretions of LE prevents invasion of the endometrium and promotes conceptus adhesion and epitheliochorial placentation, whereas secretions of GE supports conceptus (embryo and associated extraembryonic membranes) growth and development (2, 8). A few proteins produced by endometrial glands have been identified and characterized in ungulates, but their functions during the estrous cycle and pregnancy are largely unknown.
In farm animals (sheep, cattle, and pigs), endometrial gland development or adenogenesis occurs rapidly after birth and is essentially complete after about 1 month (12, 13, 14). Birth and the subsequent withdrawal from a progesterone-dominated prenatal environment were proposed to be an endocrine cue for development of endometrial glands in the neonatal ovine uterus (13). Subsequently, Bartol et al. (15) ovariectomized neonatal ewe (female) lambs on postnatal day (PND) 0 (birth) and administered a potent 19-norprogestin implant for 13 days. Progestin administration inhibited endometrial gland development, as reflected by the absence of glands on PND 13. Removal of the implant on PND 13 permitted glands to develop by PND 26. However, compared with normal uteri from PND 26, these glands were not well developed and were histologically abnormal in appearance. Collectively, these studies suggested that prenatal endocrine conditions favor the development and differentiation of the uterine wall layers and the caruncular area of the endometrium; however, removal of uterine tissues from inhibitors of endometrial gland development occurred at birth and permitted site-specific proliferation of the endometrial GE in intercaruncular areas (15). The strategy of prolonged progestin exposure of neonatal ewe lambs was used recently to prevent development of endometrial glands in the ovine uterus epigenetically, thereby producing a unique adult endometrial phenotype characterized by the absence of uterine glands, the ovine uterine gland knockout, or UGKO, phenotype (14, 16).
Given that the adult UGKO ewes lack endometrial glands, a molecular comparison of their glandless endometrium to that of normal gland-containing sheep could be used to rapidly identify genes expressed by the endometrial epithelium. The objective of these studies were to use endometrium from normal and UGKO ewes to identify and clone messenger RNAs (mRNAs) expressed in the endometrial epithelium by the molecular techniques of mRNA differential display PCR (DD-PCR) and PCR-based suppression subtraction hybridization (SSH). The results indicate that either technique can be used successfully to identify and clone many known and novel endometrial epithelial mRNAs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of UGKO ewes
Neonatal ewe lambs were assigned at birth (day 0) to one of two
treatments. Control (CX; n = 8) ewe lambs received no implants.
Treated ewes (UGKO; n = 9) received a single, sc administered
implant every 2 weeks for 32 weeks. This progestin implant releases
approximately 350 µg of a nonmetabolizable 19-norprogestin (compound
SC-21009; 17
-acetoxy-11ß-methyl-19-norpreg-4-ene-3,20-dione;
Murial, Athens, GA) per day for at least 14 days (15). Two to 4 weeks
after the last implant was administered, uteri were obtained from CX
and UGKO adult ewes at necropsy. The endometrium was physically
dissected from the myometrium, frozen in liquid nitrogen, and stored at
-80 C for RNA extraction and analysis. For all differential mRNA
analyses, endometrium was used from ewes in the luteal phase, as
ascertained by the presence of a corpus luteum on the ovary.
Cyclic and pregnant ewes
Mature ewes of primarily Rambouillet breeding were observed
daily for estrous behavior using vasectomized rams. Allewes exhibited
at least two estrous cycles of normal duration (
16–18 days) before
use in this study. At estrus (day 0), ewes were assigned randomly to
cyclic or pregnant status. Ewes assigned to pregnant status were bred
at estrus and at 12 h and 24 h postestrus with intact rams.
Fifty-two ewes were ovariohysterectomized (n = 4 ewes/day) on days
1, 3, 5, 7, 9, 11, 13, and 15 of the estrous cycle or days 11, 13, 15,
17, and 19 of pregnancy. In cyclic ewes and in pregnant ewes on days
11–17, the uterine lumen was flushed with 20 ml sterile saline at
hysterectomy. Pregnancy was confirmed by the presence of an apparently
normal conceptus in uterine flushings (days 11–17) or extension of the
interestrous interval (day 19). Several sections (0.5 cm) from the
midportion of each uterine horn were fixed in fresh 4%
paraformaldehyde in PBS (pH 7.2). In monovulatory pregnant ewes, care
was taken to mark uterine tissue samples as contralateral or
ipsilateral to the ovary bearing the corpus luteum. After 24 h,
fixed tissues were changed to 70% ethanol for 24 h and then
embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). Analyses
conducted in the present study used endometrium only from the
ipsilateral pregnant uterine horn.
DD-PCR
Total RNA was prepared from the endometrium of normal CX and
UGKO uteri using the Trizol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s
directions. Total RNA samples were digested with ribonuclease
(RNase)-free deoxyribonuclease I to remove contaminating genomic DNA
(17). The technique of mRNA DD-PCR (18) was then performed according to
the manufacturer’s recommendations using a RNAimage Kit (GenHunter
Corp., Nashville, TN) and [-33P]deoxy-ATP in
combination with a Genomyx LR PAGE system (Genomyx Corp., Foster City,
CA). Autoradiographs of dried gels were prepared by overnight exposure
to BioMAX MR film (Eastman Kodak Co., Rochester, NY).
Bands corresponding to differentially expressed mRNAs were excised from
the acrylamide gel, reamplified using PCR, and then cloned into PCR2.1
using the T/A Cloning Kit (Invitrogen, Carlsbad, CA).
PCR-based subtractive hybridization
As a complimentary approach to DD-PCR, the PCR-Select
complementary DNA (cDNA) subtraction kit (CLONTECH Laboratories, Inc., Palo Alto, CA) was used to compare the two populations of
mRNA and obtain a library of differentially expressed cDNAs. The
technique of PCR-based SSH prevents undesirable amplification of common
cDNAs and permits enrichment of differentially expressed cDNAs (19).
Using the kit according to the manufacturer’s recommendations, two
types of PCR subtracted ovine endometrial cDNA libraries were
generated: 1) a CX library with UGKO cDNAs subtracted (CX-UGKO) using
CX endometrial cDNAs as tester and UGKO as driver, and 2) a UGKO
library with CX cDNAs subtracted (UGKO-CX) using UGKO endometrial
cDNAs as tester and CX as driver. The efficiencies of the SSH
methods were evaluated with kit controls in which bacteriophage
X174/HaeIII DNA was used as tester, and human skeletal
muscle cDNA was used as driver. Skeletal muscle double stranded cDNAs
were spiked with X174 DNA digested with HaeIII to a total
of 0.2%. As shown in Fig. 2A
(lane 3), the X174/HaeIII
DNA (1.3, 1.1, 0.9, 0.6, and 0.3 kb) was efficiently subtracted out.
Subtracted cDNAs were cloned into pCR2.1 using the
Invitrogen T/A cloning kit, and the resulting libraries
were propagated in DH5 bacteria.
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-32P]deoxy-CTP using a DECAprime II kit
(Ambion, Inc., Austin, TX). DNA slot blots were then
probed using radiolabeled CX or UGKO cDNAs and ExpressHybe
(CLONTECH Laboratories, Inc.). After hybridization and
washing, the radioactivity associated with each slot was quantitated by
electronic autoradiography using an Instant Imager (Packard, Meriden,
CT). Autoradiographs were prepared using BioMAX MR film (Eastman Kodak Co.) and cassettes with intensifying screens. Exposure
times varied from 1–3 days. The sizes of true differentially expressed
cDNA clones were determined by restriction enzyme digestion of purified
library plasmid clones and agarose gel electrophoresis.
DNA sequencing and analysis
Sequences of DD-PCR and SSH cDNAs were obtained using an ABI
PRISM Dye Terminator Cycle Sequencing Kit (Perkins-Elmer PE Applied Biosystems, Foster City, CA) and a ABI PRISM model 377
DNA Sequencer (P-E PE Applied Biosystems) at the Gene
Technologies Core Facility (Department of Biology, Texas A&M
University). Nucleic acid similarity searches were performed using the
basic local alignment and search tools (BLAST) at the National Center
for Biotechnology Information (NIH, Bethesda, MD) (20). Nucleotide
sequence structure analyses were performed using the BCM search
launcher (Human Genome Sequence Center, Baylor College of Medicine,
Houston, TX) (21).
RNA isolation and analyses
RNA isolation. Total cellular RNA was isolated from
endometrial samples using the Trizol reagent (Life Technologies, Inc., Grand Island, NY).
Northern blot hybridization analysis. Endometrial total RNA
was denatured, separated using a 1.5% agarose denaturing gel, and then
transferred onto a Nytran Plus positively charged nylon membrane
(Schleicher & Schuell, Inc.) by downward blotting as
described previously (22). Radiolabeled antisense complementary RNA
(cRNA) probes were then generated from linearized plasmid DNA templates
by in vitro transcription with [-32P]UTP
and either SP6 or T7 bacterial RNA polymerases using a MAXIscript
SP6/T7 kit (Ambion, Inc.). Membranes were hybridized with
radiolabeled antisense cRNA probes and washed as described previously
(22). After washing, nonspecific hybridization was removed by RNase A
digestion (17). After digestion and washing, autoradiographs were
produced using X-Omat AR film (Kodak) and cassettes with
intensifying screens.
In situ hybridization analysis. The location of mRNA in
uterine tissue sections was determined by in situ
hybridization analysis as described previously (22). Deparaffinized,
rehydrated, and deproteinated uterine tissue sections (5–7 µm) were
hybridized with radiolabeled antisense or sense cRNA probes generated
from linearized plasmid templates using in vitro
transcription with [-35S]UTP (3000 Ci/mmol;
Amersham Pharmacia Biotech, Aylesbury, UK).
Autoradiographs of slides were prepared using Kodak BioMAX
MR film for a 16-h exposure period. Autoradiography was accomplished
using Kodak NTB-2 liquid photographic emulsion (17).
Slides were stored at 4 C for 1–2 weeks as judged from
autoradiographs, developed in Kodak D-19 developer,
counterstained with Harris’ modified hematoxylin in acetic acid
(Fisher Scientific, Fairlawn, NJ), dehydrated through a
graded series of alcohol to xylene, and coverslipped.
Photomicroscopy. Photomicrographs were taken under brightfield and darkfield illumination using a Carl Zeiss Axioplan2 photomicroscope (New York, NY) fitted with a Hamamatsu chilled 3CCD color camera (Hamamatsu, Japan). Digital images were captured and assembled using Adobe Photoshop 4.0 (Adobe Systems, Seattle, WA) and a MacIntosh PowerMac G3 computer (Apple Computer, Cupertino, CA). Black-and-white prints were printed electronically using a Kodak DS8650 color printer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In striking
contrast, the endometrium of progestin-treated ewes was devoid of
glands (n = 7/9) or contained only a very few glands (n =
2/9). Thus, progestin treatment ablated uterine gland differentiation
and created an adult UGKO endometrial phenotype. A simple, ruffled LE
overlying compact stroma was observed in intercaruncular endometrium
for UGKO ewes, whereas glandularity was intense in these same
endometrial areas in normal uteri from control ewes. The endometrium
from progestin-treated ewes also lacked clear delineation of stromal
zones characteristic of intercaruncular endometrial areas in
uteri from normal control ewes, as defined by a dense stratum
compactrum and loose stratum spongiosum. No distinct differences in
myometrial histology were observed.
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). A
total of 81 cDNAs were reamplified, cloned, and sequenced from CX and
UGKO endometrial total RNA samples using this procedure. The majority
(75 of 81) of the cDNAs were present in CX, but absent from UGKO
samples. However, a few (6 of 81) of these cDNAs were found only in
UGKO samples. All of the reamplified cDNAs were sequenced and analyzed
for homology in the sequence databases using the gapped BLAST (20).
Twelve of the 75 cDNAs reamplified from CX endometrium had
significant sequence similarity (>90%) to known mRNAs,
including glycosylation-dependent cell adhesion molecule-1
(GlyCAM-1) (23), ß-lactoglobulin (24), pregnancy-associated
2-globulin (2-PEG) (25), E-MAP-115
microtubule-associated protein (26), mitogen-activated response kinase
(27), endogenous type B and D sheep retroviruses (28, 29), and
gp330/megalin (30, 31). Fifty-eight cDNAs from the CX endometrium and
all six from the UGKO endometrium displayed low similarity to human
expressed sequences tags (ESTs) or no similarity to any database
sequences. These clones can be classified as novel and probably
represent undiscovered genes transcribed in the endometrium. Only five
DD-PCR clones were identical to a ribosomal RNA sequence that is
expressed in all tissues.
PCR-based SSH
Using a CLONTECH Laboratories, Inc. PCR-Select cDNA
subtraction kit, PCR-based SSH was used to compare the two populations
of endometrial mRNA from normal CX and UGKO uteri and generate
subtracted libraries containing differentially expressed cDNAs.
Subtractions of cDNAs derived from CX and UGKO endometrial
polyadenylated mRNA were performed in parallel with the subtraction of
X174/HaeIII DNA from human skeletal muscle mRNA as an
internal efficiency control. In both types of subtractions (Fig. 2A), many more cDNAs were present in
unsubtracted cDNAs (lanes 4, 6, and 8) than in subtracted cDNAs (lanes
3, 5, and 7), indicating that the subtraction efficiency was high. As
observed with DD-PCR analyses, many more cDNAs were present in
CX-UGKO than in UGKO-CX subtractions (Fig. 2B).
A PCR strategy was also employed to examine the efficiency of
subtraction using primers designed to amplify a partial cDNA for ovine
sulfated glycoprotein-1 (SGP-1), a prosaposin that is expressed
predominantly in the endometrial GE of the ovine uterus (31). Using
ovine SGP-1-specific primers, a cDNA product of expected size (525
bp) was amplified from the subtracted CX cDNAs, but not from the
subtracted UGKO cDNAs (Fig. 2C).
Differential screening of the CX-UGKO endometrial subtracted cDNA
library
A differential screening procedure was performed on 100 random
clones from the CX-UGKO subtracted cDNA library to identify cDNAs
representing true differentially expressed mRNAs (Fig. 3A). Clones that are truly differentially
expressed should hybridize predominantly with CX cDNA probe, whereas
clones that hybridize with both CX and UGKO cDNA probes are considered
background (24). Five SSH clones had no detectable hybridization signal
and probably represent nondifferentially expressed mRNAs present at low
levels in the subtracted library. Forty-five clones hybridized
equally to both probes, suggesting that they probably do not represent
differentially expressed mRNAs. Thirty-four clones hybridized to
both probes, and the hybridization signal was different, as assessed by
electronic autoradiography. However, only three of these clones (SSH
97, 179, and 196) had a difference in signal intensity of more than
3-fold and could be classified as true differentially expressed cDNAs
by Wang and Brown (24). Sixteen clones hybridized only to the CX probe,
but not to the UGKO probe, and were also classified as true
differentially expressed cDNAs.
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, these
similar sequences included two endogenous sheep retroviruses (28, 29),
matrix Gla protein (34), ovine mitochondrial genome (35), alkaline
phosphatase (36), and gp330/megalin (30, 31). Interestingly, cDNAs for
endogenous type B and D sheep retroviruses and gp330/megalin were also
identified and cloned by DD-PCR. The SSH 48 cloned cDNA was similar to
a short interspersed element (SINE) sequence that is present in bovine
and ovine genomes and appears to have originated from the
retrotransposable element class of nonlong terminal repeat
retrotransposable elements (37). The SSH clones 61, 105, and 170 were
homologous to portions of the ovine mitochondrial genome. SSH 61 had
significant identity to cytochrome c-oxidase subunit 3,
whereas SSH 105 and -170 were homologous to NADH subunits 2 and 3.
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In situ hybridization analysis of differentially expressed
cDNAs
In situ hybridization analyses, using probes generated
from DD and SSH clones, identified which uterine cell type(s) produced
the mRNAs in cyclic and pregnant ewes (see Fig. 4). Overall, analyses indicated that all
clones detected mRNAs present in the endometrial LE and/or GE,
although some were also expressed in other endometrial cell types.
Results from SSH clones 48, 61, 100, 105, 134, and 170 are not shown in
Fig. 4.
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DD (no. 54) and SSH clones (no. 29, 39, 133, 134, 135, 179, 188, and 196) were expressed in the endometrial LE and GE. In general, expression of these mRNAs in cyclic ovine uteri was low on day 1, increased between days 1 and 5, reached maximal levels between days 5 and 13, and then declined to day 15. In pregnant ewes, mRNA levels in the LE and GE declined between days 11 and 19, although this varied according to the individual clone. Further, SSH 179 probes also detected mRNA in the conceptus. Expression of four SSH clones (no. 61, 82, 105, and 170) was detected in all uterine cell types, with high levels of expression in endometrial epithelia. Transcriptional activity detected using probes generated from clones 61, 105, and 170 was not affected by day or pregnancy status. For SSH 82, mRNA was detected in LE, GE, stroma, and smooth muscle, including vasculature (media intima) and myometrium, and was affected by the day of the estrous cycle and early pregnancy. In uteri from cyclic ewes, mRNA levels increased in the stratum compactum between days 1 and 15, whereas levels remained high in other uterine cell types. In uteri from pregnant ewes, SSH 82 mRNA increased in the stroma, whereas expression in the LE and GE declined precipitously to low or undetectable levels.
Effects of day and pregnancy status on expression of SSH mRNAs in
the ovine endometrium
Northern blot hybridization analyses were conducted for selected
SSH clones that were specifically expressed in endometrial LE and/or
GE. SSH clones (no. 61, 82, 105, and 170) that hybridized to all cell
types were excluded from these analyses. Representative blots are
presented in Fig. 5. Consistent with the
fact that SSH48 was similar in sequence to a SINE, the SSH48 antisense
cRNA probe detected many mRNAs that appeared as a smear (data not
shown). Two mRNA transcripts (2.5 and 6 kb) were detected using
antisense cRNA probes derived from clones DD54 and similar SSH clones
(no. 29, 39, 135, and 188). Likewise, two mRNA forms (4 and 2.3 kb)
were detected for SSH 133 (data not shown). For SSH clones 97, 100,
117, and 196, single transcripts of approximately 4.9, 2.5, 4.7, and
4.7 kb were detected in endometrial total RNA. As expected by sequence
similarity, a very large (14 kb) mRNA transcript was observed in
endometrial total RNA probed with the SSH 179 clone.
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and data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies presented here demonstrate the utility of the ovine UGKO model as a powerful tool with which to identify mRNAs transcribed in the endometrial epithelium. Using endometrium from normal gland-containing and UGKO glandless sheep, a combination of DD-PCR and SSH techniques was used to clone many known and novel mRNAs that are specifically located in endometrial epithelia of the ovine uterus. Both techniques facilitated the identification and cloning of similar mRNAs, i.e. endogenous sheep retrovirus and gp330/megalin, which illustrates the effectiveness of either technique as a discovery tool used in combination with the ovine UGKO model. More mRNAs were found to be expressed differentially in control than in UGKO total RNA samples by DD-PCR and in the CX-UGKO cDNA subtracted library generated by SSH. This finding was expected, given that the predominant difference between UGKO and normal endometrium is the absence of endometrial glands. However, DD-PCR analyses indicated the presence of differentially expressed mRNAs in UGKO endometrial samples. This finding was confirmed by the presence of cDNAs in the UGKO-CX endometrial cDNA subtraction. Thus, uterine glands may normally suppress the expression of certain genes that provide a basis for functional differences between intercaruncular and caruncular endometrial areas.
In situ hybridization confirmed that all of the mRNAs identified by DD-PCR and SSH were expressed in normal endometrial epithelium, although some were expressed in other uterine cell types. For DD and SSH epithelial-specific mRNAs, three patterns of mRNA expression were detected by in situ hybridization as follows: 1) GE only, 2) LE and superficial GE, and 3) LE and GE in both the stratum compactum and stratum spongiosum. These observations lend support to the idea that true diversity exists in LE and GE function. In ungulates, secretions of GE are hypothesized to support conceptus growth and development, whereas secretions of LE are thought to be involved in mechanisms that prevent invasion of the endometrium by the conceptus and promote adhesion and epitheliochorial placentation (5, 6, 7, 8, 9, 21). Molecular mechanisms regulating tissue and cell type specificity of epithelial gene expression in the endometrium are not well characterized, but are undoubtedly complex and are likely to involve cell type-specific transcription factors.
Northern blot and in situ hybridization analyses indicated that steady state levels of mRNA for most DD and SSH clones were affected by the day of the estrous cycle and/or pregnancy status. In general, expression of cloned mRNAs was low on day 1, increased to maximal levels between days 9 and 11, and then declined in endometrium from normal pregnant ewes. Although the conceptus removes much of the LE during superficial implantation, expression of most DD and SSH cloned mRNAs remained detectable in endometrial LE and GE in early pregnant ewes. These results suggest that transcription of the corresponding epithelial genes may be affected primarily by circulating progesterone and the epithelial content of PR. In cyclic and pregnant sheep, peripheral concentrations of progesterone increase after day 3 with formation of the corpus luteum and are maximal after day 9. In cyclic sheep, progesterone concentrations decrease between days 13 and 17 during luteolysis, whereas progesterone levels are maintained in pregnant sheep (38). Although progesterone levels are maintained in pregnant sheep, progesterone negatively autoregulates the expression of the PR in endometrial epithelium of both cyclic and pregnant sheep (38). In endometrial LE and shallow GE, expression of PR is low on day 1, maximal on day 5, and undetectable after days 11–13. In the deep GE of the stratum spongiosum, PR expression is reduced during late diestrus and early pregnancy, but mRNA and protein remain detectable at low levels until day 19. Neither the identity of novel mRNAs characterized here, nor the nature or function of proteins produced by their translation are known. However, epithelial-specific transcriptional activity regulated by progesterone is likely to result in the production of important maternal mediators of periimplantation conceptus development and survival. Such transcripts, and the proteins derived from them, may also serve as unique markers of progesterone action and uterine receptivity (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 39).
Sequence analyses revealed that some of the cloned mRNAs expressed
by endometrial epithelium displayed significant homology to existing
sequences, whereas some clones had no homology to known mRNAs and were
considered novel, albeit two of the SSH cloned cDNAs were similar to
human ESTs for uncharacterized genes. Identification of mRNAs by DD and
SSH shown previously to be expressed by endometrial glands in other
mammalian uteri validates the utility of these techniques combined with
the UGKO model system for the identification and cloning of
epithelium-specific mRNAs. In this study, several genes known to be
expressed by endometrial glands of other species were identified,
including alkaline phosphatase, 2-PEG,
ß-lactoglobulin, and gp330/megalin. 2-PEG and
ß-lactoglobulin are expressed in the endometrial GE of the human and
baboon uterus and are hypothesized to function in the transport of
retinol and fatty acids (40). In this study, SSH clone 134 was found to
have sequence homology to alkaline phosphatase and was specifically
expressed in the endometrial LE and GE. Alkaline phosphatase has been
localized to the endometrial epithelium in a number of mammalian uteri
(41, 42) and may be involved in protein transport.
Both DD70 and SSH179 cDNA clones displayed high sequence homology to gp330/megalin. Antisense cRNA probes generated from these cDNAs were used to detect a 14-kb transcript in endometrial total RNA. In situ, this transcript was localized in endometrial LE and GE as well as in the conceptus. Using a rabbit antihuman gp330 antibody, positive immunostaining was also observed in apical aspects of these same ovine uterine and placental cell types (Spencer, T. E., unpublished observations). The gp330 glycoprotein has also been localized in human endometrial GE (43) and was detected in a number of murine epithelia (44). The gp330 protein is a member of the low density lipoprotein receptor family (45) and is generally concentrated on and restricted to apical portions of the cell surface. It binds multiple ligands in vitro such as calcium, plasminogen, extracellular matrix components, lactoferrin, clusterin, and lipoprotein lipase (45). Thus, gp330 may be an important mediator of membrane protein and ion trafficking in specialized epithelia found in the endometrium and conceptus.
Interestingly, several of the known DD and SSH cloned mRNAs are expressed in the epithelium of epitheliomesenchymal organs, such as the uterus, lung, and mammary gland. The DD clone 63 was similar to epithelial microtuble-associated protein 115 (E-MAP-115) (26). This microtubule-stabilizing protein appears to play an important role during reorganization of microtubules during polarization and differentiation of epithelial cells. The DD-PCR clone 67 was similar to mitogen-activated response kinase, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption (27). The DD-PCR clone 21 displayed a region of high sequence homology with glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) (23). GlyCAM-1 is an endothelial secreted ligand for L-selectin involved in lymphocyte extravasation. Immunoreactive GlyCAM-1 protein was found in the LE and GE of the ovine uterus and in uterine lumenal fluid (46). The function of this protein in the ovine uterus is not clear, although it may be involved in periattachment processes regulating trophectoderm adhesion to the endometrial epithelium.
The SSH clone 82 shares sequence homology with matrix Gla protein (MGP)
(34). Expression of this gene has not been reported in the uterus. The
antisense cRNA probe generated from the SSH 82 cDNA clone detected
endometrial transcripts of the expected size (0.8 and 1.5 kb) and
revealed transcriptional activity in LE, GE, stroma, and smooth muscle
of the endometrial vasculature and myometrium. The MGP is a vitamin
K-dependent extracellular matrix protein with wide tissue distribution
and is found in the epithelium of most epitheliomesenchymal organs,
such as the kidney (47), as well as in vascular smooth muscle (48). In
the present study, MGP mRNA was located in all uterine cell types.
However, spatial patterns of MGP expression were affected by day and
pregnancy status in a cell type-specific manner. Interestingly, MGP
mRNA first increased in the LE and GE. Strong signal for MGP was not
detected in stroma until days 17 and 19 of pregnancy. The function of
MGP is obscure, but MGP expression is correlated with development and
maturation processes involving cell matrix interactions, such as lung
branching morphogenesis (47, 49). Loeser and Wallin (50) reported that
MGP is a cell adhesion molecule that promotes adhesion to the
extracellular matrix through a receptor that requires
-carboxy-glutamic acid (Gla) residues. Therefore, this protein may
play an important role in the development and differentiation of the
endometrium during superficial implantation and synepitheliochorial
placentation.
Also detected here was evidence of endometrial epithelial mRNAs for endogenous type B and D sheep retroviruses. Seven of the DD-PCR and three of the SSH clones identified by differential screening displayed high sequence homology with this retrovirus. This is not surprising given the abundance of ovine retroviral mRNA detected in the endometrium by Northern blot and in situ hybridization analyses. Indeed, steady state levels of this mRNA in the endometrium increased 24-fold between days 1 and 13 of the estrous cycle (Stagg, A. G., and T. E. Spencer, unpublished results). The JSRV is thought to be a causative agent of ovine pulmonary carcinoma (28) and can be found widely distributed in the genomic DNA of ungulate mammals, including cattle, sheep, and goats (51). The genome of this virus does not appear to contain an oncogene, and the mechanism by which it causes disease is still unknown. The function of this retrovirus in the sheep uterus is unknown, although the retrovirus can be detected in DNA of ovine pulmonary carcinoma-affected and unaffected sheep (51). Endogenous retroviral sequences have also been found in the human and mouse embryo, fetus, and placenta and in mouse testis, but their physiological and functional significance remains unknown (52). Another interesting finding was the identification of differentially expressed mRNAs that corresponded to products of the mitochondrial genome (SSH clones 61, 105, and 170). SSH 61 was homologous to the cytochrome c-oxidase subunit, whereas SSH 105 and 170 were homologous to NADH subunits 2 and 3. In situ hybridization localized these three mRNAs to all uterine cell types, although expression was very high in the endometrial epithelium (Stagg, A. G., and T. E. Spencer, unpublished results). Given that mitochondrial mRNAs have polyadenylated tails, the SSH procedure allowed for identification of these mRNAs by comparison of endometrium from control and UGKO ewes. These results suggest that the glandular epithelial cells of the ovine uterus contain more mitochondria than other uterine cell types.
Five of the SSH clones were found to have no sequence homology in the nonredundant sequence databases and were considered novel. The SSH 97 mRNA was novel and specifically expressed in endometrial GE. Two other novel mRNAs (SSH 100 and 117) were expressed in the endometrial LE and superficial GE. These cDNAs displayed significant sequence homology with several human ESTs. The remaining two novel mRNAs (SSH 133 and 196) were expressed in LE as well as GE in the stratum compactum and stratum spongiosum. The cloning of these novel mRNAs expressed by various types of epithelium in the endometrium is an important first step in the identification and characterization of genes controlling endometrial function during the cycle and pregnancy. The novel epithelial cDNAs cloned in this study also provide functionally cloned ovine ESTs. The ESTs are useful reagents for constructing a detailed comparative and physical map of novel uterine epithelium-expressed genes and can be used to develop polymorphic markers suitable for genetic fine mapping, interval analysis, and marker-assisted selection (53). In addition, the identified and cloned cDNAs/ESTs can be used as reagents for obtaining the complete sequence of the identified mRNAs and regulatory regions of the corresponding genes. Future research will be aimed at defining the nature and function of the known and novel mRNAs in the ovine uterus and should provide new factors and pathways regulating mammalian endometrial function and markers of progesterone action and uterine receptivity.
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Acknowledgments |
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Footnotes |
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Received December 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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