Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print March 23, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.039776
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/2/271    most recent
biolreprod.105.039776v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dunlap, K. A.
Right arrow Articles by Spencer, T. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dunlap, K. A.
Right arrow Articles by Spencer, T. E.
Agricola
Right arrow Articles by Dunlap, K. A.
Right arrow Articles by Spencer, T. E.
BIOLOGY OF REPRODUCTION 73, 271–279 (2005)
DOI: 10.1095/biolreprod.105.039776
© 2005 by the Society for the Study of Reproduction, Inc.

Sheep Endogenous Betaretroviruses (enJSRVs) and the Hyaluronidase 2 (HYAL2) Receptor in the Ovine Uterus and Conceptus

Kathrin A. Dunlap 2, Massimo Palmarini 3, David L. Adelson 2, and Thomas E. Spencer 1 2

Center for Animal Biotechnology and Genomics,2 Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471 Institute of Comparative Medicine,3 Faculty of Veterinary Medicine, University of Glasgow, Scotland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ovine genome contains approximately 20 copies of endogenous betaretroviruses (enJSRVs) that are highly related to two exogenous oncogenic viruses, Jaagsiekte sheep retrovirus (JSRV) and Enzootic nasal tumor virus. The cellular receptor for both JSRV and the enJSRVs is hyaluronidase 2 (HYAL2). In this study, we assessed expression of enJSRVs envelope (env) and HYAL2 mRNAs in the ovine uterus and conceptus (embryo/fetus and extraembryonic membranes) throughout gestation. By reverse transcription-polymerase chain reaction analyses, enJSRVs env were found to be expressed beginning in the Day 12 conceptus, whereas HYAL2 was expressed from Day 16. HYAL2 mRNA was detected throughout gestation in the placentome but not in the endometrium, whereas enJSRVs env expression was detected throughout gestation in endometrium and placentomes. The enJSRVs env mRNA was specifically expressed in the endometrial lumenal epithelium (LE) and glandular epithelium (GE) as well as the trophoblast giant binucleate cells (BNC) and multinucleated syncytia of the placenta. HYAL2 mRNA was only detected in the BNC and multinucleated syncytial plaques of the placentome. Partial sequencing of the transcriptionally active enJSRVs from sheep endometrium, placentomes, and placenta revealed expression of many enJSRV loci. Cloning of the expressed enJSRVs env mRNA from ovine uteroplacental tissues found sequences similar to the previously identified enJS5F16 and enJS56A1 gene with an intact open reading frame, although the polypeptides they encode were not studied. Collectively, results provide further support for our hypothesis that the enJSRVs Env have been beneficial to the host and are involved in protection of the uterus from viral infection and regulators of placental morphogenesis and function.

conceptus, HYAL2, JSRV, ovine, placenta, pregnancy, retroviruses, syncytiotrophoblast, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A unique feature of retroviruses is their presence as inherited elements in the germline of most eukaryotes. These elements, known as endogenous retroviruses (ERVs), are transmitted through the germline as stable Mendelian genes [1]. It is assumed that ERVs derived from integration events during evolution of ancient exogenous retroviruses (e.g., transmitted horizontally) into the germline of host animal species. The biological relevance of ERVs in mammals has been intensely debated. ERVs have been characterized either as dispensable junk DNA or as indispensable for mammalian development and physiological function [2–4]. Generally, endogenous proviruses are transcriptionally silent and are often defective, typically differing from the exogenous counterpart by deletions or point mutations that render them incapable of forming infectious virus. However, several ERVs maintain at least some intact open reading frames that can be expressed and are associated with either beneficial or detrimental effects to the host [1].

Expression of ERVs in the genital tract and placenta of various animal species has been described for at least three decades [5–10], sparking opinions on their significance in the evolution of placental mammals and development of viviparity [11–14]. In humans and primates, several ERVs, including ERV-3 and HERV-W, appear to play a direct role in formation of the syncytiotrophoblast of the placenta [13, 15–17]. The product of the HERV-W envelope (env) gene is a highly fusogenic membrane glycoprotein, termed syncytin, that induces formation of syncytia upon interaction with the type D mammalian retrovirus receptor [15, 18]. Available results support the hypothesis that ERVs have biological functions in placental morphogenesis in humans and primates.

Sheep represent an interesting model to study the biology of ERVs and their interaction with host species [19]. The ovine genome contains approximately 20 copies of endogenous betaretroviruses (enJSRVs) [20–23] that are highly related to two oncogenic exogenous betaretroviruses, Jaagsiekte sheep retrovirus (JSRV) and Enzootic nasal tumor virus (ENTV) [24]. Hyaluronidase 2 (HYAL2) is a glycosylphosphatidylinositol-anchored cell-surface protein with weak hyaluronidase activity that serves as a cellular receptor for JSRV and enJSRV [25, 26]. Interestingly, in vitro assays found that enJSRVs can block JSRV replication at early and late steps of the replication cycle [27, 28], supporting the hypothesis that enJSRVs protect the host during evolution against pathogenic retroviral infections [28].

The enJSRVs are transcriptionally active in the fetal and adult sheep and particularly abundant in the female reproductive tract [22]. In the uterus, enJSRVs RNA and protein was observed exclusively in the endometrial luminal epithelium (LE) and glandular epithelium (GE) [22, 29, 30]. In addition to the endometrium, enJSRVs RNA expression was detected in the trophoblast giant binucleate cells (BNC) of Day 18 and 20 conceptuses, which form the outer layer of the fetal placental cotyledon and give rise to the syncytial plaques [19, 30, 31]. Trophoblast BNCs are thought to arise from the mononuclear trophectoderm cells (MTC), then to migrate through the apical trophectodermal tight junctions of the chorion, and become apposed to the apical surface of the endometrial LE [32, 33]. Endometrial LE and BNC then fuse apically and form a syncytium of trinucleate cells within the endometrial epithelium. Subsequently, the trinucleate cells enlarge by continued BNC migration and fusion that forms multinucleated syncytial plaques linked by tight junctions [33]. In sheep, the size of the plaques is limited to 20–25 nuclei [33]. The syncytial plaques eventually cover the surface of the endometrial carunucles and aid in development of placentomes, which are formed by interdigitation of fetal placental cotyledons and endometrial caruncles and are necessary for the conceptus to obtain hematrophic nutrition from the maternal uterus. The trophoblast BNC in the sheep placenta are, in many respects, analogous to the trophoblast giant cells of the syncytiotrophoblast in humans [34].

Little is known about the cellular and molecular mechanisms that regulate trophoblast differentiation and syncytia formation during synepitheliochorial placentation in sheep. Based on the temporal and spatial alterations in enJSRVs expression in the ovine uterus and placenta, we hypothesize that the enJSRVs have a biological role in protection of the uterus against viral infection and placental morphogenesis [19, 30, 35]. Specific objectives were to i) determine the ontogeny and expression patterns of enJSRVs env and HYAL2 mRNA during gestation in ovine uteroplacental tissues and ii) determine which enJSRVs loci are transcriptionally active in uteroplacental tissues by sequencing and analysis of enJSRVs env mRNA expressed in uteroplacental tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Design

Mature ewes of primarily Suffolk breeding were observed daily for estrus (designated Day 0) using vasectomized rams. All ewes exhibited at least two estrous cycles of normal duration (~16–18 days). At estrus, ewes were bred to intact rams at 12 h and 24 h postestrus. All experimental and surgical procedures involving animals met the Guidelines for the Care and Use of Agricultural Animals in Agricultural Teaching and Research and were approved by the Institutional Agricultural Animal Care and Use Committee of Texas A&M University.

In study 1, pregnant ewes were assigned randomly to be ovariohysterectomized (n = 4 ewes/day) on Days 20, 30, 40, 50, 60, 80, 100, or 120 of pregnancy (Day 0 = mating). At hysterectomy, the uterus was trimmed free of cervix and oviduct and opened along the mesometrial border. Several sections (~0.5 cm) of both intercaruncular and placentomal regions from the midportion of each uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% ethanol for 24 h and then dehydrated and embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). At hysterectomy, remaining placentomes were removed by physical dissection and additional intercaruncular endometrium was dissected from the myometrium. Various placentomes were physically separated into maternal endometrial caruncle and fetal placental cotyledon. All collected samples of uteroplacental tissues were frozen in liquid nitrogen and stored at –80°C for RNA extraction.

In study 2, cyclic ewes were mated at estrus (Day 0) and Day 1 to fertile rams. At mating, the ewes were randomly assigned to have the conceptus (embryo/fetus and associated extraembryonic membranes) recovered by surgical uterine flush on Days 10, 12, 14, 16, or 18 postmating (n = 5 conceptuses/day). The collected conceptuses were snap frozen in liquid nitrogen and stored at –80°C for RNA extraction.

RNA Extraction

Total cellular RNA was isolated from tissues using Trizol (Gibco-BRL, Bethesda, MD) according to the manufacturer's recommendations. The quantity of RNA was assessed spectrophotometrically, and the integrity of RNA was examined by gel electrophoresis in a denaturing 1% agarose gel.

In Situ Hybridization Analysis

The enJSRV env and HYAL2 mRNAs were localized in uterine tissue sections (5 µm) by in situ hybridization analysis as described previously [29]. The ovine HYAL2 cDNA was kindly provided by Dr. Michael Lerman (National Cancer Institute, Frederick, MD) [36]. Deparaffinized, rehydrated, and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes generated from linearized ovine endometrial enJSRV env cDNA (DD54) [29] or HYAL2 cDNA by in vitro transcription with [{alpha}-35S]UTP. After hybridization, washing, and RNase A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Kodak, Rochester, NY), stored at 4°C for 1 wk, and developed in Kodak D-19 developer. Slides were then counterstained with Harris modified hematoxylin (Fisher Scientific, Fairlawn, NJ), dehydrated through a graded series of alcohol to xylene, and protected with a coverslip.

Northern Blot Analysis

Intercaruncular endometrial or placentomal 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 [29]. Radiolabeled antisense complementary enJSRV env (DD54) RNA probes were then generated from linearized plasmid DNA templates by in vitro transcription with [32P-{alpha}]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 [37]. After digestion and washing, autoradiographs were produced using X-Omat AR film (Kodak) and cassettes with intensifying screens.

Reverse Transcription-Polymerase Chain Reaction Analysis

Expression of enJSRVs RNA and the ovine Hyal2 receptor were determined by reverse transcription-polymerase chain reaction (RT-PCR) using methods described previously [38]. Briefly, cDNA was synthesized from total endometrial, placentomal, or conceptus RNA (5 µg) using random and oligo-dT primers and SuperScript II Reverse Transcriptase (Life Technologies, Gaithersburg, MD). Newly synthesized cDNA was acid-ethanol precipitated, resuspended in 20 µl sterile water, and stored at –20°C. The cDNAs were diluted (1:10) with sterile water before use in PCR reactions. The PCR reactions were performed using AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA) and Optimized Buffer H (Invitrogen, Carlsbad, CA) according to manufacturers' recommendations.

PCR primers from sequences flanking the carboxy-terminal portion of the transmembrane (TM) domain of the JSRV Env region were derived from the sequence of the JSRV21 infectious molecular clone [24] (GenBank #AF105220) because this region is well conserved among exogenous and endogenous sheep betaretroviruses. The enJSRV env primers, enJSRVenvF (5'-AACATTTGCAAGGAATTTGG-3') and enJSRVenvR (5'-GCTCCATAAGATGTTGGTGC-3') amplified a cDNA of 336 base pairs (bp). The enJSRV env PCR amplifications were conducted as follows: 35 cycles at 95°C for 30 sec; 53.5°C for 1 min; and 72°C for 1 min. The HYAL2 primers, bLuca2F135 (5'-CCAGCATGTGGACAGGCCTG-3') and bLuca2R600 (5'-TACACATCCTTGTCCTGCCAG-3') were derived from the Ovis aries HYAL2 mRNA (GenBank #AF411974) and amplified a cDNA of 465 bp as described previously [36]. The HYAL2 PCR amplifications were conducted as follows: 35 cycles at 95°C for 30 sec; 60°C for 1 min; and 72°C for 1 min. As a positive control, ß-actin primers (forward: 5'-ATGAAGATCCTCACGGAACG-3'; reverse: 5'-GAAGGTGGTCTCGTGAATGC-3') were used to amplify a 270-bp cDNA. The PCR conditions and amount of template cDNA used in each reaction were optimized for each primer set to ensure linear amplification of the target. PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining using an Alpha Innotech imaging system.

Isolation and Sequence Analysis of enJSRV Env Clones

To determine the identity of uterine enJSRVs Env, a Day 14 pregnant ovine endometrial cDNA library, constructed by Clontech (Palo Alto, CA) using directional cloning into the pTriplEx2 phage vector, was screened using the radiolabeled antisense ovine enJSRV env cRNA probe (DD54 that corresponds to a portion of enJSRVs env) [29]. Five clones, purified by three sequential hybridization screenings, were then excised from the lambda-TriplEx2 phage using BM25.8 cells for subsequent transformation into DH5{alpha} cells before cDNA extraction (Qiagen, Valencia, CA) and sequencing in both directions using an ABI 373XL DNA sequencer (Applied Biosystems, Foster City, CA) and pTriplEX sequencing primers (Clontech). Nucleic acid similarity searches were performed using the basic local alignment and search tools (BLAST) at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD) [39]. Nucleotide sequence structure analyses were performed using the BCM search launcher (Human Genome Sequence Center, Baylor College of Medicine, Houston, TX).

To determine the identity of enJSRV loci that were transcriptionally active in the endometrium, placenta, and placentome (cotyledon and caruncle), RT-PCR analysis was conducted using the enJSRV env primers listed above and methods as previously described [28]. Tissues from Day 120 pregnant ewes (n = 3) were used for RT-PCR. PCR products were cloned into pCR2.1 (Invitrogen), and eight independent clones were analyzed in each tissue type evaluated. Similarity searches were conducted as previously described, with ClustalW alignment and phylogenetic tree assembly conducted using the alignment services of the European Bioinformatics Institute available online at http://www.ebi.ac.uk/services.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of enJSRV RNAs in the Uterus of Pregnant Ewes

RT-PCR analysis using enJSRV env-specific primers allowed the amplification of a 336-bp cDNA product that sequence analysis confirmed to be originated from enJSRVs env (data not shown). In the conceptus, enJSRVs RNA was expressed beginning on Day 12 (Fig. 1A). The enJSRVs env mRNA was also amplified in both the intercaruncular endometrium and placentomes, regardless of the day of gestation (Fig. 1, B and C). As an internal positive control, the ß-actin mRNA was amplified in all conceptus, endometrium, and placentome samples (Fig. 1D).



View larger version (62K):
[in this window]
[in a new window]
  		FIG. 1. Expression of enJSRVs env mRNA in ovine uteroplacental tissues. A–C) Representative RT-PCR analysis of enJSRVs env mRNA in the (A) early conceptus, (B) endometrium, and (C) placentomes from pregnant ewes. PCR products were separated in a 1.5% agarose gel and visualized using ethidium bromide. The 100-bp marker (M) is shown on the left portion of each gel. D) The ß-actin amplification in all samples. E) Northern blot analysis of total RNA from the intercaruncular endometrium and placentome from pregnant ewes. Endometrial total RNA (20 µg) was separated on a 1.2% denaturing agarose gel, transferred to a nylon membrane, and hybridized with radiolabeled antisense enJSRV env cRNA probe (DD54). Positions of the 28S and 18S rRNAs are indicated on the left. The 7.4-kb RNA corresponds to the full-length enJSRV genome, and the 2.4-kb mRNA likely represents the correctly spliced env RNA

Additional information regarding the temporal expression of enJSRVs is illustrated via Northern blot analyses of total RNA isolated from the endometrium and placentomes. As expected, by using a cRNA enJSRVs env probe, Northern blot analyses detected both the full-length enJSRV genome (7.5 kb) as well as the correctly spliced enJSRV env mRNA (2.4 kb) [29, 40] in the intercaruncular endometrium from Day 12 to Day 30 (Fig. 1E). The 2.4-kb enJSRV env mRNA was predominantly detected in the intercaruncular endometrium after Day 30 and in placentomes from Day 40 to Day 120 of gestation.

In situ hybridization analyses revealed that the enJSRVs RNA was expressed exclusively in the endometrial LE and superficial GE of the intercaruncular endometrium from all ewes regardless of gestational day (Fig. 2). Expression of enJSRVs RNA was very low in the middle to deeper endometrial GE. The enJSRV RNAs were abundantly detected in the cotyledonary portion of the placentome (Fig. 3). The enJSRV RNAs were specifically observed in the trophoblast giant BNC and syncytial plaques that form the outer lining of the cotyledonary chorionic villus of the placenta. However, no expression of enJSRV RNAs was detected in the caruncular portion of the placentome.



View larger version (187K):
[in this window]
[in a new window]
  		FIG. 2. In situ hybridization analysis of enJSRVs RNA expression in the intercaruncular endometrium from pregnant ewes. Cross-sections of the intercaruncular endometrium and placentomes of pregnant ewes were hybridized with radiolabeled antisense or sense ovine enJSRV env cRNA probes. Protected transcripts were visualized by liquid emulsion autoradiography for 1 wk and imaged under brightfield or darkfield illumination. BNC, Binucleate cell; GD, gestational day; LE, luminal epithelium; GE, glandular epithelium; S, stroma; Tr, trophectoderm. Bars = 20 µm



View larger version (157K):
[in this window]
[in a new window]
  		FIG. 3. In situ hybridization analysis of enJSRV RNA expression in the placentomes from pregnant ewes. Cross-sections of the intercaruncular endometrium and placentomes of pregnant ewes were hybridized with radiolabeled antisense or sense ovine enJSRV env cRNA probes. Protected transcripts were visualized by liquid emulsion autoradiography for 1 wk and imaged under brightfield or darkfield illumination. BNC, Binucleate cell; Car, endometrial caruncle; Cot, placental cotyledon; GD, gestational day; SP, multinucleate syncytial plaque; Tr, trophectoderm. Bars = 20 µm

Expression of the enJSRV Receptor HYAL2 in the Uterus of Pregnant Ewes

Analysis of HYAL2 mRNA expression in the conceptus, intercaruncular endometrium, and placentome was determined by RT-PCR analysis of total RNA using ovine HYAL2-specific primers (Fig. 4). The 465-bp cDNA RT-PCR product was cloned and sequenced to confirm its identity as HYAL2 (data not shown). In the conceptus, HYAL2 mRNA was not detected on Days 10, 12, or 14, but was evident in the Day 16 and Day 18 conceptuses (Fig. 4A). Total RNA from sheep lung was used as a positive control for HYAL2 [36]. HYAL2 mRNA was not detected in the intercaruncular endometrium, regardless of the day of gestation (Fig. 4B). In contrast with the intercaruncular endometrium, HYAL2 mRNA was detected in the placentomes from Day 40 to Day 120 of gestation (Fig. 4C). The RNA in samples not positive for HYAL2 mRNA was not degraded because ß-actin, a housekeeping gene, could be amplified in all conceptus and endometrium samples (Fig. 1D).



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 4. Expression of HYAL2 mRNA in ovine uteroplacental tissues. A– C) Representative RT-PCR analysis of HYAL2 mRNA in the (A) early conceptus, (B) endometrium, and (C) placentomes from pregnant ewes. The lung (L) was used as a positive control. PCR products were separated in a 1.5% agarose gel and visualized using ethidium bromide. The 100-bp marker (M) is shown on the left portion of each gel

In situ hybridization analysis revealed that HYAL2 mRNA was expressed exclusively by the BNC and syncytial plaques of the placental cotyledons but not in other cell types in the placentome (Fig. 5). As expected from the RT-PCR analyses, expression of HYAL2 mRNA was not observed in the intercaruncular endometrium or myometrium (data not shown).



View larger version (163K):
[in this window]
[in a new window]
  		FIG. 5. In situ hybridization analysis of HYAL2 RNA expression in the placentomes of pregnant ewes. Cross-sections of the placentomes of pregnant ewes were hybridized with radiolabeled antisense or sense ovine HYAL2 cRNA probes. Protected transcripts visualized by liquid-emulsion autoradiography for 1 wk and imaged under brightfield or darkfield illumination. Car, Endometrial caruncle; Cot, placental cotyledon; GD, gestational day; SP, multinucleate syncytial plaque. Bars = 20 µm

Cloning and Analysis of enJSRVs env RNAs Expressed in the Uteroplacenta

There are approximately 20 copies of enJSRVs integrated in the sheep genome, but no information is available on which loci are expressed in the uterus and placenta. As a first step to identify the transcriptionally active enJSRVs, five 1.4-kilobase (kb) cDNAs encoding enJSRV env mRNAs were isolated by screening a Day 14 pregnant ovine endometrial cDNA library with an enJSRV env probe. All five plaque purified clones were full length, with an intact open reading frame. The inferred amino acid sequences were 94–99% identical to each other, 95–99% to enJS56A1 and enJS5F16 Env, and 89–90% identical to the exogenous JSRV Env (Fig. 6). All of the enJSRV env clones lacked the YXXM motif present in the cytoplasmic tail of the TM domain of the JSRV Env that has been found to be critical for transformation [41]. This deleted region is a feature conserved among all the Env sequences of the enJSRVs.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 6. ClustalW analysis of protein sequences from enJSRVs env cDNAs isolated from a Day 14 pregnant endometrial cDNA library. Alignment includes comparison with the known enJSRVs (enJS5F16 and enJS56A1) and the exogenous JSRV (JSRV21). A period represents a conserved substitution and dashed lines represent deletions. The superficial (SU) and transmembrane (TM) areas and variable region 3 (VR3) are denoted in the figure

To determine whether the transcriptionally active enJSRV loci followed a tissue-specific pattern of expression, we cloned, by RT-PCR, partial enJSRV env cDNAs from endometrium, placenta, caruncles, and cotyledons from Day 120 pregnant ewes. The primers used for RT-PCR were designed to amplify a region of the C-terminus of the JSRV Env containing the variable region 3 (VR3) that is lacking in the enJS5F16 and enJS56A1 env genes [22]. Sequence analysis revealed that the clones derived from the pregnant ovine tissues were more than 95% identical to enJS5F16 and enJS56A1. All the enJSRVs env sequences maintained an open reading frame and lacked the YXXM motif that is present in the VR3 region of the TM domain of the exogenous JSRV Env. We obtained 20 unique RT-PCR clones, suggesting that most of the 20 predicted enJSRV loci are transcriptionally active in the endometrium. Phylogenetic analysis of the partial enJSRVs Env is presented in Figure 7.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 7. Phylogenetic tree based on the neighbor joining method using 10 000 bootstraps within the partial enJSRV env cDNAs cloned by RT-PCR from uteroplacental tissues and known sequences of enJSF16, enJS56A1, and JSRV21 Env. The branch lengths are proportional to an estimate of evolutionary change

All of the partial enJSRV Env sequences were aligned and examined for rates of synonymous (dS) and nonsynonymous (dN) substitutions using the method of Nei and Gojobori [42], incorporating a statistic from Ota and Nei [43], implemented in the SNAP program [44]. The sequences alone gave a dN:dS ratio of 0.33. Because a value of dN: dS <1 is usually viewed as proving evidence of purifying selection [42], the enJSRVs appear to be subject to purifying selection, supporting a functional involvement in the ovine uterus and placenta.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The analysis of enJSRVs env isolated from cDNA library screening and RT-PCR analysis of uteroplacental tissues revealed sequences highly homologous to the previously described enJSRVs Env. No tissue-specific expression of any particular locus was observed in the endometrium, placentome, or placenta. Given the heterogeneity of the sequences obtained by RT-PCR cloning, it appears that most, if not all, of the 20 predicted enJSRV loci are transcriptionally active in the ovine uterus and placenta and encode Env with an open reading frame. The analysis of cloned partial enJSRV Env sequences based on rates of dS and dN substitutions found evidence of purifying selection, which supports our working hypothesis that the enJSRVs have important biological roles in the uterine function and placental morphogenesis. Interestingly, Bonnaud and coworkers [45] recently found conserved selective constraints on the env of ERVWE1 that encodes syncytin in humans and primates, suggesting that this retroviral locus has been recruited in the hominoid lineage to become a bona fide gene.

In previous studies, we determined that enJSRVs were expressed in the uterus of neonatal and adult cyclic and early pregnant ewes [27, 30]. In the present study, enJSRV RNAs were found to be expressed throughout gestation in the uterus and specifically in the endometrial LE and GE of the intercaruncular areas. Indeed, enJSRVs env and HYAL2 mRNAs were first detected by RT-PCR in the Day 12 and Day 16 conceptuses, respectively, which coincides with the initial differentiation of the BNC from the MTC that begins on Days 14–16 [33, 46, 47]. In the placenta, enJSRV env mRNA was specifically expressed in the trophoblast giant BNC and multinucleated syncytial plaques. HYAL2 mRNA was also detected specifically in the BNC and syncytial plaques throughout gestation. These novel results, combined with studies of human ERVs [13, 17] and enJSRVs [27, 28], provide strong support for the hypothesis that enJSRVs may have beneficial biological roles for the host, including 1) protection from exogenous infectious viruses, 2) suppression of local immune recognition of the conceptus, and 3) placental morphogenesis.

The enJSRVs expressed in the endometrial LE and GE may have protected the uterus from infection by exogenous and pathogenic infectious betaretroviruses during evolution. This hypothesis is supported by recent evidence that expression of enJSRVs was found to block entry and exit of the exogenous JSRV by receptor interference and the action of a transdominant Gag protein of the enJSRV locus enJS56A1 [19, 27, 28]. In the present study, cloning of the expressed enJSRVs env mRNA from ovine uteroplacental tissues found sequences similar, but not identical, to the previously identified enJS5F16 and enJS56A1 loci. Thus, all of the 20 enJSRV loci need to be cloned and functionally analyzed for the ability to interfere with JSRV entry and replication through known or novel mechanisms.

The metavirus hypothesis states that all mammals must express immunosuppressive ERV proteins in extraembryonic tissues to suppress local immune recognition of the embryo [11]. Sheep and other domestic ruminants exhibit a synepitheliochorial type of placentation in which the endometrial LE persists but is modified to a variable degree, depending on species, into a hybrid fetomaternal syncytium formed by the migration and fusion of the BNC with the LE, which is functionally equivalent to the syncytiotrophoblast layer of the human placenta [46]. Sheep affected by ovine pulmonary adenocarcinoma (OPA) or ENTV do not show an appreciable antibody response to JSRV or ENTV. Available evidence supports the hypothesis that enJSRV expression in the fetal lamb tolerizes sheep to the related exogenous viruses, JSRV and ENTV [19, 27]. However, the potential immunological consequences of enJSRVs have not been investigated, as proposed for several human ERVs [13].

The enJSRVs Env is also hypothesized to have a biological role in placental morphogenesis. The timing and localization of enJSRVs expression support the possibility that enJSRVs play a role in MTC differentiation into BNC as well as BNC fusion with the endometrial LE to form syncytial plaques. For instance, the enJSRVs and HYAL2 expression in the elongating early conceptus coincides with the initial differentiation and of the trophoblast giant BNC. The enJSRVs are expressed throughout pregnancy in the BNC and syncytial plaques of the cotyledonary portion of the placentomes. Indeed, the BNC are thought to continue to differentiate, migrate, and fuse to form multinucleated syncytia throughout most of pregnancy [33, 46]. Studies of ERVs in the human placenta [12, 13, 17] provide substantial support for this hypothesis. In humans, the product of the HERV-W env gene is a highly fusogenic membrane glycoprotein, termed syncytin, that induces trophoblast cell fusion, differentiation, and formation of syncytia on interaction with the type-D mammalian retrovirus receptor [15, 16, 18]. The enJSRVs Env can use HYAL2 as a receptor [27], but the ability of the different enJSRVs Env to induce cell fusion and the formation of syncytia has not been reported.

Although the morphological aspects of BNC differentiation are well documented, the cellular and molecular mechanisms regulating their differentiation and development are not understood. All the enJSRVs Env expressed in uteroplacental tissues lacked the YXXM motif that is present in the VR3 region of the JSRV Env and that is important for transformation in vitro of rodent fibroblasts [48]. Transformation of cells by the Env gene of JSRV can be Hyal2 receptor independent and involve the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway [41, 48–51]. Interestingly, activation of the PI3-K/Akt signaling pathway regulates development of the differentiated trophoblast giant-cell phenotype in other mammals [51]. Expression of enJSRVs in the BNC and syncytial plaques may be involved in expression of other genes due to the transcriptional regulatory properties of their long terminal repeats and/or function of the Env protein. For example, a HERV-E insertion in the 5'-untranslated region of the growth-factor pleiotrophin (PTN) gene creates a trophoblast-specific promoter [52–54]. The placenta-specific expression of the insulin-like growth factor INSL4 also appears to be driven by a HERV insertion [55]. Therefore, it is tempting to speculate that enJSRVs are important regulators of BNC-specific genes, such as placental lactogen [56] and pregnancy-associated glycoproteins [57], in the ovine placenta. In summary, the temporal and spatial alterations in expression of enJSRVs and HYAL2 in the sheep uterus and placenta engender a variety of physiological roles in conceptus implantation and placentation. The sequence and functionality of the candidate enJSRVs identified in the present study will be useful to unravel their physiological roles in the ovine uterus and placenta.


   ACKNOWLEDGMENTS
 
The authors thank Mr. Kendrick LeBlanc for care and management of ewes and Dr. Clare Gill and Mrs. Collette Abbey for assistance with primer design and sequencing analysis.


   FOOTNOTES
 
1 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979 862 2662; tspencer{at}tamu.edu Back

Received: 8 January 2005.

First decision: 3 February 2005.

Accepted: 18 March 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Boeke JD, Stoye JP. Retrotransposons, endogenous retroviruses and the evolution of retroelements. In: Coffin JM, Hughes SH, Varmus HE (eds.), Retroviruses. Plainview, NY: Cold Spring Harbor Laboratory Press; 1997:343–346
  2. Bock M, Stoye JP. Endogenous retroviruses and the human germline. Curr Opin Genet Dev 2000 10:651-655[CrossRef][Medline]
  3. Lower R. The pathogenic potential of endogenous retroviruses: facts and fantasies. Trends Microbiol 1999 7:350-356[CrossRef][Medline]
  4. Lower R, Lower J, Kurth R. The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences. Proc Natl Acad Sci U S A 1996 93:5177-5184[Abstract/Free Full Text]
  5. Kalter SS, Helmke RJ, Heberling RL, Panigel M, Fowler AK, Strickland JE, Hellman A. Brief communication: C-type particles in normal human placentas. J Natl Cancer Inst 1973 50:1081-1084
  6. Kalter SS, Heberling RL, Helmke RJ, Panigel M, Smith GC, Kraemer DC, Hellman A, Fowler AK, Strickland JE. A comparative study on the presence of C-type viral particles in placentas from primates and other animals. Bibl Haematol 1975;391–401
  7. Vernon ML, McMahon JM, Hackett JJ. Additional evidence of type-C particles in human placentas. J Natl Cancer Inst 1974 52:987-989
  8. Smith CA, Moore HD. Expression of C-type viral particles at implantation in the marmoset monkey. Hum Reprod 1988 3:395-398[Abstract/Free Full Text]
  9. Harris JR. The evolution of placental mammals. FEBS Lett 1991 295:3-4[CrossRef][Medline]
  10. DeHaven JE, Schwartz DA, Dahm MW, Hazard ES 3rd, Trifiletti R, Lacy ER, Norris JS. Novel retroviral sequences are expressed in the epididymis and uterus of Syrian hamsters. J Gen Virol 1998 79:pt 112687-2694[Abstract]
  11. Villarreal LP, Villareal LP. On viruses, sex, and motherhood. J Virol 1997 71:859-865[Medline]
  12. Harris JR. Placental endogenous retrovirus (ERV): structural, functional, and evolutionary significance. Bioessays 1998 20:307-316[CrossRef][Medline]
  13. Muir A, Lever A, Moffett A. Expression and functions of human endogenous retroviruses in the placenta: an update. Placenta 2004 25:suppl_AS16-25
  14. Stoye JP, Coffin JM. A provirus put to work. Nature 2000 403:715-717[CrossRef][Medline]
  15. Mi S, Lee X, Li X, Veldman GM, Finnerty H, Racie L, LaVallie E, Tang XY, Edouard P, Howes S, Keith JC Jr, McCoy JM. Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 2000 403:785-789[CrossRef][Medline]
  16. Frendo JL, Olivier D, Cheynet V, Blond JL, Bouton O, Vidaud M, Rabreau M, Evain-Brion D, Mallet F. Direct involvement of HERV-W Env glycoprotein in human trophoblast cell fusion and differentiation. Mol Cell Biol 2003 23:3566-3574[Abstract/Free Full Text]
  17. Rote NS, Chakrabarti S, Stetzer BP. The role of human endogenous retroviruses in trophoblast differentiation and placental development. Placenta 2004 25:673-683[CrossRef][Medline]
  18. Blond JL, Lavillette D, Cheynet V, Bouton O, Oriol G, Chapel-Fernandes S, Mandrand B, Mallet F, Cosset FL. An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J Virol 2000 74:3321-3329[Abstract/Free Full Text]
  19. Palmarini M, Mura M, Spencer TE. Endogenous betaretroviruses of sheep: teaching new lessons in retroviral interference and adaptation. J Gen Virol 2004 85:1-13[Abstract/Free Full Text]
  20. Bai J, Bishop JV, Carlson JO, DeMartini JC. Sequence comparison of JSRV with endogenous proviruses: envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor. Virology 1999 258:333-343[CrossRef][Medline]
  21. Hecht SJ, Stedman KE, Carlson JO, DeMartini JC. Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals. Proc Natl Acad Sci U S A 1996 93:3297-3302[Abstract/Free Full Text]
  22. Palmarini M, Hallwirth C, York D, Murgia C, de Oliveira T, Spencer T, Fan H. Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous Jaagsiekte sheep retrovirus. J Virol 2000 74:8065-8076[Abstract/Free Full Text]
  23. York DF, Vigne R, Verwoerd DW, Querat G. Nucleotide sequence of the Jaagsiekte retrovirus, an exogenous and endogenous type D and B retrovirus of sheep and goats. J Virol 1992 66:4930-4939[Abstract/Free Full Text]
  24. Palmarini M, Sharp JM, de las Heras M, Fan H. Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep. J Virol 1999 73:6964-6972[Abstract/Free Full Text]
  25. Rai SK, Duh FM, Vigdorovich V, Danilkovitch-Miagkova A, Lerman MI, Miller AD. Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for Jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation. Proc Natl Acad Sci U S A 2001 98:4443-4448[Abstract/Free Full Text]
  26. Miller AD. Identification of Hyal2 as the cell-surface receptor for Jaagsiekte sheep retrovirus and ovine nasal adenocarcinoma virus. Curr Top Microbiol Immunol 2003 275:179-199[Medline]
  27. Spencer TE, Mura M, Gray CA, Griebel PJ, Palmarini M. Receptor usage and fetal expression of ovine endogenous betaretroviruses: implications for coevolution of endogenous and exogenous retroviruses. J Virol 2003 77:749-753
  28. Mura M, Murcia P, Caporale M, Spencer TE, Nagashima K, Rein A, Palmarini M. Late viral interference induced by transdominant Gag of an endogenous retrovirus. Proc Natl Acad Sci U S A 2004 101:11117-11122[Abstract/Free Full Text]
  29. Spencer TE, Stagg AG, Joyce MM, Jenster G, Wood CG, Bazer FW, Wiley AA, Bartol FF. Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model. Endocrinology 1999 140:4070-4080[Abstract/Free Full Text]
  30. Palmarini M, Gray CA, Carpenter K, Fan H, Bazer FW, Spencer TE. Expression of endogenous betaretroviruses in the ovine uterus: effects of neonatal age, estrous cycle, pregnancy, and progesterone. J Virol 2001 75:11319-11327[Abstract/Free Full Text]
  31. Sanna E, Sanna MP, Loddo C, Sanna L, Mura M, Cadelano T, Leoni A. Endogenous Jaagsiekte sheep retrovirus RNA is expressed by different cell types in ovine foetus and placenta. Eur J Histochem 2002 46:273-280[Medline]
  32. Wimsatt WA. Observations of the morphogenesis, cytochemistry and significance of the binucleate giant cells of the placenta of ruminants. J Anat 1951 89:233-282[CrossRef]
  33. Wooding FB. Role of binucleate cells in fetomaternal cell fusion at implantation in the sheep. Am J Anat 1984 170:233-250[CrossRef][Medline]
  34. Hoffman LH, Wooding FB. Giant and binucleate trophoblast cells of mammals. J Exp Zool 1993 266:559-577[CrossRef][Medline]
  35. DeMartini JC, Carlson JO, Leroux C, Spencer T, Palmarini M. Endogenous retroviruses related to Jaagsiekte sheep retrovirus. Curr Topics Microbiol Immunol 2003 275:117-137[Medline]
  36. Dirks C, Duh FM, Rai SK, Lerman MI, Miller AD. Mechanism of cell entry and transformation by enzootic nasal tumor virus. J Virol 2002 76:2141-2149[Abstract/Free Full Text]
  37. Spencer TE, Ing NH, Ott TL, Mayes JS, Becker WC, Watson GH, Mirando MA, Brazer FW. Intrauterine injection of ovine interferon-tau alters oestrogen receptor and oxytocin receptor expression in the endometrium of cyclic ewes. J Mol Endocrinol 1995 15:203-220[Abstract]
  38. Stewart MD, Johnson GA, Gray CA, Burghardt RC, Schuler LA, Joyce MM, Bazer FW, Spencer TE. Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy. Biol Reprod 2000 62:1779-1789[Abstract/Free Full Text]
  39. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997 25:3389-3402[Abstract/Free Full Text]
  40. Palmarini M, Murgia C, Fan H. Spliced and prematurely polyadenylated Jaagsiekte sheep retrovirus-specific RNAs from infected or transfected cells. Virology 2002 294:180-188[CrossRef][Medline]
  41. Palmarini M, Maeda N, Murgia C, De-Fraja C, Hofacre A, Fan H. A phosphatidylinositol-3-kinase (PI-3K) docking site in the cytoplasmic tail of the Jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH3T3 cells. J Virol 2001 75:11002-11009[Abstract/Free Full Text]
  42. Nei M, Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 1986 3:418-426[Abstract]
  43. Ota T, Nei M. Variance and covariances of the numbers of synonymous and nonsynonymous substitutions per site. Mol Biol Evol 1994 11:613-619[Abstract]
  44. Korber B. HIV signature and sequence variation analysis. In: Rodrigo AG, Learn GH (eds.), Computational Analysis of HIV Molecular Sequences. Dordrecht, Netherlands: Kluwer Academic Press Publishers; 2000:55–72
  45. Bonnaud B, Bouton O, Oriol G, Cheynet V, Duret L, Mallet F. Evidence of selection on the domesticated ERVWE1 env retroviral element involved in placentation. Mol Biol Evol 2004 21:1895-1901[Abstract/Free Full Text]
  46. Wooding FB. Current topic: the synepitheliochorial placenta of ruminants: binucleate cell fusions and hormone production. Placenta 1992 13:101-113[Medline]
  47. Guillomot M. Cellular interactions during implantation in domestic ruminants. J Reprod Fertil Suppl 1995 49:39-51[Medline]
  48. Maeda N, Inoshima Y, Fruman DA, Brachmann SM, Fan H. Transformation of mouse fibroblasts by Jaagsiekte sheep retrovirus envelope does not require phosphatidylinositol 3-kinase. J Virol 2003 77:9951-9959[Abstract/Free Full Text]
  49. Zavala G, Pretto C, Chow YH, Jones L, Alberti A, Grego E, De las Heras M, Palmarini M. Relevance of Akt phosphorylation in cell transformation induced by Jaagsiekte sheep retrovirus. Virology 2003 312:95-105[CrossRef][Medline]
  50. Chow YH, Alberti A, Mura M, Pretto C, Murcia P, Albritton LM, Palmarini M. Transformation of rodent fibroblasts by the Jaagsiekte sheep retrovirus envelope is receptor independent and does not require the surface domain. J Virol 2003 77:6341-6350[Abstract/Free Full Text]
  51. Kamei T, Jones SR, Chapman BM, McGonigle KL, Dai G, Soares MJ. The phosphatidylinositol 3-kinase/Akt signaling pathway modulates the endocrine differentiation of trophoblast cells. Mol Endocrinol 2002 16:1469-1481[Abstract/Free Full Text]
  52. Schulte AM, Lai S, Kurtz A, Czubayko F, Riegel AT, Wellstein A. Human trophoblast and choriocarcinoma expression of the growth factor pleiotrophin attributable to germ-line insertion of an endogenous retrovirus. Proc Natl Acad Sci U S A 1996 93:14759-14764[Abstract/Free Full Text]
  53. Schulte AM, Wellstein A. Structure and phylogenetic analysis of an endogenous retrovirus inserted into the human growth factor gene pleiotrophin. J Virol 1998 72:6065-6072[Abstract/Free Full Text]
  54. Schulte AM, Malerczyk C, Cabal-Manzano R, Gajarsa JJ, List HJ, Riegel AT, Wellstein A. Influence of the human endogenous retrovirus-like element HERV-E. PTN on the expression of growth factor pleiotrophin: a critical role of a retroviral Sp1-binding site. Oncogene 2000 19:3988-3998[CrossRef][Medline]
  55. Bieche I, Laurent A, Laurendeau I, Duret L, Giovangrandi Y, Frendo JL, Olivi M, Fausser JL, Evain-Brion D, Vidaud M. Placenta-specific INSL4 expression is mediated by a human endogenous retrovirus element. Biol Reprod 2003 68:1422-1429[Abstract/Free Full Text]
  56. Liang R, Limesand SW, Anthony RV. Structure and transcriptional regulation of the ovine placental lactogen gene. Eur J Biochem 1999 265:883-895[Medline]
  57. Hughes AL, Green JA, Garbayo JM, Roberts RM. Adaptive diversification within a large family of recently duplicated, placentally expressed genes. Proc Natl Acad Sci U S A 2000 97:3319-3323[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
J. Kim, G. Song, H. Gao, J. L. Farmer, M. C. Satterfield, R. C. Burghardt, G. Wu, G. A. Johnson, T. E. Spencer, and F. W. Bazer
Insulin-Like Growth Factor II Activates Phosphatidylinositol 3-Kinase-Protooncogenic Protein Kinase 1 and Mitogen-Activated Protein Kinase Cell Signaling Pathways, and Stimulates Migration of Ovine Trophectoderm Cells
Endocrinology, June 1, 2008; 149(6): 3085 - 3094.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
P. R. Murcia, F. Arnaud, and M. Palmarini
The Transdominant Endogenous Retrovirus enJS56A1 Associates with and Blocks Intracellular Trafficking of Jaagsiekte Sheep Retrovirus Gag
J. Virol., February 15, 2007; 81(4): 1762 - 1772.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K. A. Dunlap, M. Palmarini, M. Varela, R. C. Burghardt, K. Hayashi, J. L. Farmer, and T. E. Spencer
Endogenous retroviruses regulate periimplantation placental growth and differentiation
PNAS, September 26, 2006; 103(39): 14390 - 14395.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/2/271    most recent
biolreprod.105.039776v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dunlap, K. A.
Right arrow Articles by Spencer, T. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dunlap, K. A.
Right arrow Articles by Spencer, T. E.
Agricola
Right arrow Articles by Dunlap, K. A.
Right arrow Articles by Spencer, T. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS