Document Detail

Uterine gene expression in the live-bearing lizard, Chalcides ocellatus, reveals convergence of squamate reptile and mammalian pregnancy mechanisms.
Jump to Full Text
MedLine Citation:
PMID:  22333490     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Although the morphological and physiological changes involved in pregnancy in live-bearing reptiles are well studied, the genetic mechanisms that underlie these changes are not known. We used the viviparous African Ocellated Skink, Chalcides ocellatus, as a model to identify a near complete gene expression profile associated with pregnancy using RNA-Seq analyses of uterine transcriptomes. Pregnancy in C. ocellatus is associated with upregulation of uterine genes involved with metabolism, cell proliferation and death, and cellular transport. Moreover, there are clear parallels between the genetic processes associated with pregnancy in mammals and Chalcides in expression of genes related to tissue remodeling, angiogenesis, immune system regulation, and nutrient provisioning to the embryo. In particular, the pregnant uterine transcriptome is dominated by expression of proteolytic enzymes that we speculate are involved both with remodeling the chorioallantoic placenta and histotrophy in the omphaloplacenta. Elements of the maternal innate immune system are downregulated in the pregnant uterus, indicating a potential mechanism to avoid rejection of the embryo. We found a downregulation of major histocompatability complex loci and estrogen and progesterone receptors in the pregnant uterus. This pattern is similar to mammals but cannot be explained by the mammalian model. The latter finding provides evidence that pregnancy is controlled by different endocrinological mechanisms in mammals and reptiles. Finally, 88% of the identified genes are expressed in both the pregnant and the nonpregnant uterus, and thus, morphological and physiological changes associated with C. ocellatus pregnancy are likely a result of regulation of genes continually expressed in the uterus rather than the initiation of expression of unique genes.
Authors:
Matthew C Brandley; Rebecca L Young; Dan L Warren; Michael B Thompson; Günter P Wagner
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2012-02-14
Journal Detail:
Title:  Genome biology and evolution     Volume:  4     ISSN:  1759-6653     ISO Abbreviation:  Genome Biol Evol     Publication Date:  2012  
Date Detail:
Created Date:  2012-04-02     Completed Date:  2012-07-03     Revised Date:  2013-10-24    
Medline Journal Info:
Nlm Unique ID:  101509707     Medline TA:  Genome Biol Evol     Country:  England    
Other Details:
Languages:  eng     Pagination:  394-411     Citation Subset:  IM    
Affiliation:
Department of Ecology and Evolutionary Biology, Yale University, USA. mbrandley@gmail.com
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Animals
Evolution, Molecular
Female
Gene Expression / genetics
Lizards / genetics*
Mammals
Pregnancy
Pregnancy, Animal*
Reptiles / genetics*
Uterus / metabolism*
Viviparity, Nonmammalian / genetics*
Comments/Corrections
Comment In:
Genome Biol Evol. 2012;4(3):372-3   [PMID:  22467917 ]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): Genome Biol Evol
Journal ID (iso-abbrev): Genome Biol Evol
Journal ID (hwp): gbe
Journal ID (publisher-id): gbe
ISSN: 1759-6653
Publisher: Oxford University Press
Article Information
Download PDF
© The Author(s) 2012. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
creative-commons:
Accepted Day: 3 Month: 2 Year: 2012
Print publication date: Year: 2012
Electronic publication date: Day: 14 Month: 2 Year: 2012
collection publication date: Year: 2012
pmc-release publication date: Day: 14 Month: 2 Year: 2012
Volume: 4 Issue: 3
First Page: 394 Last Page: 411
ID: 3318437
PubMed Id: 22333490
DOI: 10.1093/gbe/evs013

Uterine Gene Expression in the Live-Bearing Lizard, Chalcides ocellatus, Reveals Convergence of Squamate Reptile and Mammalian Pregnancy Mechanisms
Matthew C. Brandley14*
Rebecca L. Young1
Dan L. Warren2
Michael B. Thompson3
Günter P. Wagner1
1Department of Ecology and Evolutionary Biology, Yale University
2Section of Integrative Biology, University of Texas
3School of Biological Sciences (A08), University of Sydney, Australia
4Present address: School of Biological Sciences (A08), University of Sydney, Australia
*Corresponding author: E-mail: mbrandley@gmail.com.
Associate editor: Takashi Gojobori

Introduction

Reproductive mode is subject to intense selective pressure and any modification will directly affect an organism's fitness. Thus, the transition between oviparity (egg-laying) and viviparity (live-bearing) is one of the most dramatic changes in the evolution of vertebrate animals. As a result of constraints on reproductive function, most vertebrate clades show a high phylogenetic conservatism in reproductive mode. For example, therian mammals represent only a single (albeit the best-known) origin of viviparity, whereas birds, alligators, and turtles exhibit only oviparity. By contrast, the evolutionary history of squamate reptiles (lizards and snakes) includes over 100 transitions from oviparity to viviparity, more than all other lineages of vertebrates combined (Blackburn 2006), with an extraordinary diversity of placental morphology and physiology (Blackburn et al. 1984; Blackburn 1993; Stewart 1993; Thompson and Speake 2006). Squamates are therefore an ideal model system with which to explore the processes that shape the evolution of viviparity and placentation in vertebrates.

Viviparity and placentation in squamate reptiles have been the subject of extensive morphological and physiological studies (e.g., Weekes 1935; Blackburn 1993; Thompson et al. 2000; Stewart and Thompson 2000; Thompson and Speake 2006). These studies have described a continuum of placental complexity, ranging from simple placentae formed by the apposition of uterine and embryonic tissue (e.g., most Eulamprus quoyii group skinks; Murphy et al. 2012) to highly complex placentae characterized by extensive folding of the uterus associated with blood vessels and containing enlarged uterine and chorionic epithelial cells (e.g., some Mabuya group skinks; Jerez and Ramírez-Pinilla 2001; Ramírez-Pinilla et al. 2006; Vieira et al. 2007; Blackburn and Flemming 2011). For the purposes of this discussion, we refer to these ends of the morphological complexity spectrum as “simple” and “complex,” although we note these categories are an oversimplification of the true underlying diversity (see Blackburn 1993). Subsequent studies have quantified nutrient exchange across these different placental types and have concluded that very little maternal to embryonic transfer occurs via simple placentae, but substantial transfer occurs in species with more complex placentae. In one extreme, the embryos of the South American skink, Mabuya bistrata experience a 47,400% increase in dry mass (Vitt and Blackburn 1991) compared with an average of a 25% decrease in dry mass of oviparous species (Thompson et al. 2000).

Although much is known about the morphological and physiological changes involved in reptilian pregnancy, there has been little examination of the genetic architecture that underlies them (for review, see Murphy and Thompson 2011). Multiple studies have used a targeted gene approach to assess the expression of genes involved with remodeling of embryonic and maternal tissue (Biazik et al. 2007, 2008; Wu et al. 2011), increasing uterine blood supply to facilitate nutrient and gas exchange with the embryo (Murphy et al. 2009), modifying the maternal immune system to prevent rejection of embryos (Paulesu et al. 1995; Romagnoli et al. 2003), and supplying nutrients to the embryo in addition to those provided in the yolk (Biazik et al. 2009).

Although these studies provided important information into genes critical to maintaining squamate reptile pregnancy, the candidate gene approach used in those studies is not adequate to profile the potentially thousands of genes that are regulated during pregnancy in squamate reptiles. Indeed, until recently, it has not been technically feasible to investigate the genetic controls of viviparity and placental development in nonmammalian vertebrates because the necessary baseline genomic data have not been available. However, recent technological advances (RNA-Seq; Marioni et al. 2008) provide the first opportunity to both identify and quantify almost all of the genes expressed in the reptilian uterus. The integration of transcriptomic data with existing morphological and physiological data now allows us to elucidate the genetic mechanisms that underlie reptilian pregnancy, which are subject to drastic change during evolutionary transitions between reproductive modes.

Here, we conduct the first comprehensive examination of gene regulation in a pregnant and nonpregnant viviparous lizard uterus using RNA-Seq. Using the viviparous Ocellated Skink, Chalcides ocellatus as a model system, we provide a near complete gene expression profile associated with maintaining pregnancy (supplementary information 1, Supplementary Material online). Chalcides ocellatus is an ideal subject for this study because it possesses a relatively complex placenta with endometrial folds and histotrophic activity (Corso et al. 1988, 2000; it is roughly in the middle of the continuum of simple to very complex placenta) and because partial gene expression data exist on Chalcides chalcides, a congener used in previous candidate gene analyses (Paulesu et al. 1995, 2001; Romagnoli et al. 2003). We focus particularly on suites of genes associated with growth and remodeling of the uterus, the maternal immune system, transport across the placenta, and uterine metabolism because they are important functions of maintaining pregnancy and have also been the subject of mammalian, and to a lesser extent, squamate reptile gene expression analyses. We compare the gene expression profiles of the uterus of C. ocellatus with mammalian models to determine whether similar genetic mechanisms have evolved to maintain pregnancy mammals and reptiles. Similarities between reptiles and mammals in uterine gene expression would suggest convergent evolution of genetic mechanisms in response to similar natural selection pressures. On the other hand, significant differences in gene expression would be evidence that there are potentially many genetic pathways that permit the transition to viviparity (i.e., “many means to an end”). Both possibilities are inherently interesting because they allow us to interpret the general processes under which pregnancy has evolved in vertebrate animals.


Materials and Methods

Adult C. ocellatus individuals were purchased from a commercial dealer. Phylogenetic comparison of a 396-nt fragment of cytochrome b mitochondrial DNA (mtDNA) from these individuals with a comprehensive Chalcides mtDNA data set (Carranza et al. 2008) revealed them to be from Egyptian populations of C. ocellatus ocellatus (results not shown). The lizards were housed at Yale University in ∼40 l glass aquaria with a 12 h light/dark photoperiod, a 24–37 °C thermal gradient (during the day), free access to water, and fed crickets three times a week. From a euthanized pregnant female, we dissected out a uterine egg chamber, removed the embryonic tissues attached to the uterus, and stored the uterus in RNA-Later (Ambion). The developmental state of the embryo was ∼37 according to the normal development table of Defaure and Hubert (1961). For a sample of nonpregnant uterus, we obtained an additional uterus from a female that was isolated for 6 weeks postpartum. There is no record of sperm retention in C. ocellatus, and the uterus showed no signs of pregnancy.

Total RNA was extracted from the uterus using an RNeasy Mini Kit (Qiagen). cDNA library construction and sequencing was performed by the Yale Center for Genome Analysis. The Illumina Genome Analyzer was used to sequence 72-bp pair-end fragments of cDNA libraries from both the pregnant and the nonpregnant samples.

There is no sequenced complete genome for C. ocellatus; indeed, only one complete genome of any nonavian reptile is currently sequenced (Anolis carolinensis), and it shares a common ancestor with C. ocellatus approximately 175 Ma (Brandley et al. 2008, 2011). To facilitate identification of sequenced transcripts, we therefore used ABySS 1.2.4 to assemble individual raw Illumina reads into larger contigs that are more easily identified. We used the subtractive multiple-k method of Surget-Groba and Montoya-Burgos (2010) and constructed multiple assemblies using a range of k-mer lengths (25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, and 69). We then used CD-HIT-EST (Li and Godzik 2006) to remove redundant contigs and then removed contigs less than 100 bp (table 1).

To identify the contigs, we used BlastX with an e value of 10−5 to compare them with a database including 38 complete sequenced genomes of vertebrate animals (Ensembl build 61). We note that this methodology will not identify recently diverged paralogs and may therefore underestimate the true diversity of genes expressed in the uterus but that this is a potential problem for all transcriptomic studies and does not alter our broad conclusions. The raw Illumina data were filtered to remove any reads less than 30 bp. We then used the short-read aligner Bowtie 0.12.7 (Langmead et al. 2009) to align these filtered reads from both the nonpregnant and the pregnant uterine samples to a database composed of the identified contigs, allowing two nucleotide mismatches. Because of differing sequence quality between the forward and the reverse pair-end reads, we chose to only use the forward reads for alignment. Because both reads were sequenced from the same fragment, this should introduce little or no bias in the gene counts.

We assessed differential gene expression of normalized numbers of transcripts between the nonpregnant and the pregnant samples using the DEGseq (Wang et al. 2010) R package. Preliminary analysis revealed that some genes are massively upregulated in the pregnant uterus (e.g., 497,141 reads of cathepsin L1 in the pregnant uterus compared with 917 reads in the nonpregnant; see Results and Discussion). Because the number of cDNA fragments sequenced is finite, we were concerned that several highly expressed genes were sequenced at the expense of others, thereby artificially lowering the apparent expression of other genes (Robinson and Oshlack 2010). To try to mitigate this sequencing artifact, we used the trimmed mean of M values (TMM) method of Robinson and Oshlack (2010) in the edgeR R package (Robinson et al. 2010) to normalize the data and create an effective library size for both samples. We scaled both the pregnant and the nonpregnant libraries to “counts per 107 reads” using the following formula:


where the TMM estimated normalization factor is 0.698231 for the pregnant library and 1.0 for the nonpregnant library and the original library sizes of 20,060,751 and 23,120,916, respectively. We then estimated differential expression of each gene between the nonpregnant and the pregnant samples using the MA plot–based method with random sampling model (MARS; Wang et al. 2010) in the DEGseq using a P value cutoff of 0.001 and a false discovery rate of 0.1%.

We used Gene Ontology (GO) analyses to broadly infer the biological function of the genes significantly regulated (as identified in the MARS differential expression analysis) in the uterine transcriptomes. We imported gene symbol lists of the significantly regulated genes in both the pregnant and the nonpregnant uteri into the Database for Annotation, Visualization, and Integrated Discovery (DAVID) functional GO annotation program and calculated GO terms corresponding to biological processes (BP-FAT). To more easily interpret the hundreds of individual GO biological functions inferred by DAVID, we aggregated similar processes to the following broad functional groups:

  • Catabolism—processes that breakdown complex molecules. This group contains any GO biological processes that were explicitly catabolic or proteolytic.
  • Cell death and differentiation—processes that regulate cellular proliferation, mitosis, and death.
  • Cell morphogenesis and development—processes involved with cell organization, including localization, cytoskeleton maintenance, tissue development, and motility.
  • Gene expression—processes that regulate transcription, translation, and posttranscriptional modification.
  • Homeostasis and response to stimuli—processes involved with maintaining homeostasis or reaction to stimuli.
  • Metabolism and energy—processes involved with the metabolism of macromolecules such as amino acids, lipids, nucleic acids, and proteins for the production of energy that are not explicitly catabolic (see above). This group also contains those mitochondrial processes involved in ATP production.
  • Molecule biosynthesis—processes that result in the production of macromolecules.
  • Signaling—processes involved with intracellular communication.
  • Transport—processes that transport both organic and inorganic molecules both intracellularly and intercellularly.
  • Other—those processes that do not naturally fit into any of the above categories.

We grouped individual GO biological processes into the above categories provided they were represented by at least 50 unique genes. We note that these categories are not necessarily mutually exclusive as, for example, those processes involved in catabolism are also metabolic. However, we chose to identify separately explicit catabolic functions given the substantial upregulation of proteolytic and hydrolytic enzymes in the pregnant uterus (see Results and Discussion).


Results and Discussion

Our study is the first to survey the almost complete gene expression profile associated with maintaining pregnancy in a live-bearing reptile. Indeed, it is one of few studies that have used RNA-Seq to assess gene expression in any reptile (Gracheva et al. 2010; Schwartz et al. 2010; Wall et al. 2011). Our transcript identification analysis positively identified 12,070,543 of 20,060,751 (60.2%) transcripts in the pregnant and 12,330,621 of 23,120,916 (53.8%) transcripts in the nonpregnant C. ocellatus uterine transcriptome (supplementary information 1, Supplementary Material online). If we assume a minimum cutoff of ≥2 transcripts per million reads as our conservative criterion for calling a gene expressed, we identified 8,846 unique genes expressed in the pregnant and 8,881 unique genes expressed in the nonpregnant uteri, 8,296 (88%) of which are expressed in both the nonpregnant and the pregnant uterus. Our differential expression analysis using the MARS method in DEGseq identified a total of 3,903 upregulated and 2,709 downregulated genes in the pregnant C. ocellatus uterus, respectively. The 50 most upregulated and downregulated genes (as assessed by z-score) in the uterus of the pregnant C. ocellatus uterus, as well as specific genes discussed in the remainder of the paper, are provided in tables 2 and 3.

It is notable that many of the genes downregulated in the pregnant C. ocellatus uterus are genes involved with basic cellular housekeeping processes (e.g., ribosomal subunit proteins, desmin, myosin; table 3), which could indicate that the TMM normalization was inadequate and that these genes were undersampled. However, if these data were improperly normalized, then properly normalizing them would only increase the relative number of highly expressed transcripts in the pregnant uterus (table 2) and therefore does not severely impact our conclusions. We also emphasize that, given the lack of comparative genomic resources, we were unable to identify almost half of the sequenced transcripts and we have likely undersampled particularly quickly evolving genes—a challenge inherent in any RNA-Seq study of an organism with no closely related sequenced genome.

Most of the genes expressed in the pregnant uterus are also expressed in the nonpregnant uterus, which suggests that the morphological and physiological processes that maintain reptilian pregnancy are mostly a function of differential regulation of genes continually expressed in the uterus rather than the new expression of unique genes. If a similar pattern exists in the expression profiles of other oviparous and viviparous squamate reptiles, it would suggest that the evolutionary transition to viviparity is relatively “easy” given that the necessary genes are already expressed in the uterus when not gravid/pregnant and only become modulated in their expression level rather reprogrammed.

The GO biological function analysis revealed that C. ocellatus pregnancy is associated with upregulation of genes involved with catabolism, cell death and differentiation, homeostasis and response to stimuli, cellular signaling, and transport are upregulated in the pregnant uterus. By contrast, the nonpregnant uterus is dominated by genes that regulate cell morphogenesis and development, gene expression, and other processes.

Given the impracticality of examining the expression and function of each gene, we limit our discussion primarily to suites of genes that are associated with the biological functions that are apparently critical to maintaining pregnancy, including growth and remodeling of the uterus, nutrient transport across the placenta, and uterine metabolism and histotrophy. We also focus on the regulation of the maternal immune system and estrogen and progesterone activity, given their importance in maintaining mammalian pregnancy. We particularly focus on the most highly upregulated and downregulated genes identified by the MARS analysis and genes that have been assessed previously in squamate reptiles (table 4). Unless otherwise stated, we limit our discussion to only genes that are significantly upregulated or downregulated (P ≤ 0.001) according to our MARS differential expression analysis. The entire results of the differential expression analysis for all genes are provided in the supplementary information 1 (Supplementary Material online).

Growth and Remodeling of the Uterus

The increasing demands of the growing embryo, including gas exchange, nutrition, and uterine space, require both fundamental destruction and growth of uterine tissue to both increase the size of the uterus and maximize the surface area of the maternal–embryonic interface. Genes likely involved in these remodeling process are some of the most highly upregulated in the pregnant C. ocellatus uterus (table 2) and include cytoskeletal elements (e.g., β-actin and keratins) and gelsolin (a powerful actin regulator), genes that promote (e.g., N-myc downstream regulated 1, Granulin) and inhibit (e.g., B-cell translocation gene 1, DNA damage–inducible transcript four) cell proliferation, and hyaluronoglucosaminidases 3 and 4, that break down the extracellular matrix.

Although there are potentially hundreds or thousands of genes involved in the uterine remodeling process, we focus on the proteolytic enzymes because they are massively upregulated and have been the subjects of multiple studies of mammalian pregnancy. We also evaluate expression of the highly upregulated cell adhesion and angiogenic genes because they have been subject to previous studies in squamate reptiles (Biazik et al. 2007, 2008, 2010; Murphy, Belov, et al. 2010; Wu et al. 2011) and therefore provide a rare opportunity to assess the diversity of molecular mechanisms involved in reptilian pregnancy.

Proteases

Because the close apposition of maternal and fetal tissue facilitates gas and nutrient exchange, uterine and placental tissues are remodeled to minimize this distance, regardless of whether the placental type is epitheliochorial, endothelialchorial, or hemochorial (Enders and Carter 2004). The destruction of this tissue is achieved, in part, via the actions of both cysteine and serine proteases (Salamonsen and Nie 2002). Perhaps the most striking gene expression upregulation in the pregnant C. ocellatus uterine transcriptome is the enormous upregulation of cysteine and serine proteases. Cathepsins (CTS) are lysosomal cysteine proteases that degrade extracellular matrix and catabolize intracellular proteins (Kirschke et al. 1997). Cathepsins make up over 8% of the entire upregulated transcriptome, with CTSL alone representing 5% of the transcriptome. The serine proteases PRSS2 (trypsin) and PRSS3 (mesotrypsin) have the highest log2-fold changes of all the upregulated genes (16.7 and 15.5, respectively; table 2). Taken together with other highly upregulated proteases such as legumain (LGMN) and tripeptidyl peptidase I (TPP1), these results reveal that the breakdown of tissue is one of the most active physiological processes in the C. ocellatus pregnant uterus.

Although the roles of cathepsins, especially CTSB and CTSL, in uterine remodeling has been elucidated in several mammal species (e.g., Afonso et al. 1997; Divya et al. 2002; Song et al. 2006, 2010; Varanou et al. 2006), comparison of cathepsin expression between C. ocellatus lizards and pigs is most instructive because both possess an epitheliochorial placenta. In pigs, CTSB and CTSL are expressed in both uterine and chorionic epithelia, and this expression increases as pregnancy progresses (Song et al. 2010). Both CTSB and CTSL are upregulated in the pregnant C. ocellatus uterus, CTSL being the most abundant transcript in the transcriptome. Although we cannot localize expression of these genes to a specific region of the uterus, we nonetheless speculate that, as in mammals, these genes may have a primary role in degrading uterine tissue during the remodeling process in the C. ocellatus uterus.

Placental remodeling is an interplay between proteases and their inhibitors (Afonso et al. 1997; Salamonsen and Nie 2002; Song et al. 2006, 2007, 2010), especially the reciprocal actions of cathepsins and their inhibitors, cystatins (CST). Cystatin C (CSTC) is a powerful inhibitor of cathepsins L and B and is highly upregulated in C. ocellatus. CTSL and two other lysosomal cysteine proteases, CTSV (also called CTSL2) and LGMN, are also inhibited by cystatin M/E (also called cystatin 6), a very highly upregulated gene in the pregnant C. ocellatus transcriptome. Moreover, multiple serine protease inhibitors, including tissue factor pathway inhibitor 2 (TFPI2), serine peptidase inhibitor, Kunitz types 1 and 2 (SPINT1 and 2), are significantly upregulated, thereby suggesting active regulation of serine protease activity. Thus, there is a clear pattern of protease expression and concomitant regulation by their inhibitors. Moreover, the genes likely responsible for these processes are identical to those used by mammals.

Cell Adhesion

Remodeling of the uterus throughout pregnancy requires the growth and movement of the closely apposed maternal and embryonic surface epithelial cells and presumably the molecules that permit adhesion of these cells to one another. These cellular adhesion proteins, including those critical to the development of desmosomes, tight junctions, and adherens junctions, are some of the few proteins extensively studied in squamate reptiles (Australian skinks; Biazik et al. 2007, 2008, 2010; Wu et al. 2011) and therefore provide a rare opportunity to assess gene expression differences amongst multiple species of viviparous reptiles (table 4).

Desmoglelin 2 (DSG2) is a protein involved in the formation of desmosomes and its expression has been evaluated in the uteri of four scincid lizards representing the extremes of placental development (Biazik et al. 2010). DSG2 expression is not different in pregnant and nonpregnant females in two species with simple placentae (Lerista bougainvillii and Saiphos equalis) but is lower during pregnancy in two species with complex placentae (Pseudemoia entrecasteauxii and Pseudemoia spenceri), which suggests that a decrease in desmosome number may allow for easier deformation and remodeling of the uterus. In contrast, our transcriptome results demonstrate a statistically significant increase in DSG2 expression in the uterus of pregnant C. ocellatus and in the absence of corroborating morphological data, presumably an increasing number of desmosomes.

In addition to simple cell adhesion, tight junctions regulate paracellular transport by controlling the permeability of the interstitial space between adjacent cells to molecules of specific sizes. Occludin (OCLN) and claudins (CLDN) are the dominant proteins in tight junctions. OCLN expression increases as pregnancy progresses in the uteri of two viviparous species of skinks with complex placentation (P. entrecasteauxii and P. spenceri), suggesting decreased paracellular permeability with compensation by transcellular transport (Biazik et al. 2007). OCLN expression was not detected in two species with simple placentae (L. bougainvillii and S. equalis; Biazik et al. 2007). Similarly, our transcriptomic analysis finds no significant difference in OCLN expression between nonpregnant and pregnant C. ocellatus females (P = 0.187), although this species has a more complex placenta. CLDN5 is expressed over the entire surface of the uterine epithelial cell in early pregnancy in four viviparous skink species but becomes localized at the tight junctions, suggesting an increased role of the tight junctions in limiting paracellular transport (Biazik et al. 2008). As no study has examined tight junction distribution in C. ocellatus, we cannot directly compare our results with Biazik et al. (2008), but we do find significant upregulation of nine claudin loci, including CLDN1, 3–8, 12, and 23, suggesting that they have an important role in maintaining pregnancy in C. ocellatus.

Cadherins (CDH) are a family of proteins that are components of adherens junctions between adjacent cells. Cadherin expression is high near ovulation, as revealed using an antibody that labels all cadherins but significantly decreases as pregnancy progresses and is absent in late pregnancy in three species of Australian skinks (Wu et al. 2011). Thus, decreasing cadherin expression, and therefore few adherens junctions, is speculated to allow the uterus to expand to accommodate the growing embryo (Wu et al. 2011). In C. ocellatus, we detected a far more complicated expression profile of cadherins with significant upregulation of CDH1 (epithelial cadherin), 2, 4, 11, 15, 22, and 24 and downregulation of CDH5 and 13. This indicates that, unlike the three Australian skinks studied to date, adherens junctions actually proliferate in C. ocellatus during pregnancy and apparently do not inhibit uterine growth.

Our transcriptomic analyses reveal some significant differences between our study and previous studies of cell adhesion molecules in the uteri of viviparous skinks. These differing results could be due to the higher resolution of gene expression possible with transcript sequencing as opposed to immunological assays or western blots used in previous studies (Biazik et al. 2007, 2008, 2010; Wu et al. 2011). However, rather than any methodological problem, a much more plausible explanation for the difference in uterine cell adhesion gene expression is that the study organisms represent ancient independent derivations of viviparity. Although also a skink, C. ocellatus and Australian lygosomine skinks share a common ancestor approximately 95 Ma (Brandley et al. 2008, 2011), about the same age as the common ancestor of primates, ungulates, carnivores, and bats (Murphy et al. 2007). It is therefore reasonable to hypothesize that these two skink lineages evolved different molecular processes and suggests that there is a diversity of genetic processes that act to maintain pregnancy within viviparous squamate reptiles.

Angiogenesis and Vascular Physiology

As the embryo matures, its growing oxygen demand is accommodated by increasing placental vascularization (Guillette and Jones 1985; Masson and Guillette 1987; Murphy, Belov, et al. 2010; Murphy, Parker, et al. 2010; Parker, Manconi, et al. 2010; Parker, Murphy, et al. 2010). Like mammals, increasing expression of vascular endothelial growth factor A (VEGFA) in pregnant squamate reptiles is correlated with vascular growth in the uterus (Murphy, Belov, et al. 2010). VEGFA is also significantly upregulated in the uterus of pregnant C. ocellatus. VEGFA is upregulated in addition to a suite of other genes with angiogenic properties. Most notable are the endothelial PAS domain protein 1 (EPAS1) and hypoxia-inducible factor 1α (HIF1A) genes that activate VEGF expression in response to hypoxic conditions (Forsythe et al. 1996; Peng et al. 2000; Takeda et al. 2004); interleukin 20 receptor β (IL20RB), an inducer of endothelial cell proliferation (Gaspar et al. 2002; Hsieh et al. 2006); angiogenin (ANG) and angiopoietin 4 (ANGPT4), and general cell proliferation genes with angiogenic properties such as ENPP2/Autotaxin. As in remodeling the structure of the uterus, proteolytic enzymes such as cathepsins are also associated with angiogenesis (Joyce et al. 2004).

In addition to genes that promote the development of blood vessels, several genes that control blood physiology are highly expressed in the C. ocellatus pregnant uterus. Prostacyclin (PTGIS), a vasodilator, is downregulated. Renin, a part of the renin–angiotensin system (RAS) that regulates blood pressure, is highly upregulated suggesting intense vasoconstriction of the uterine vasculature and consequently high blood pressure (perhaps facilitating the diffusion of oxygen and nutrients to the embryo across the placenta). Renin is expressed in the uteroplacental tissues of mammals where it participates in a local RAS (Cooper et al. 1999; Nielsen et al. 2000; Vinson et al. 1997) that may also stimulate cell proliferation and angiogenesis (Hagemann et al. 1994). However, renin does not directly increase blood pressure but rather converts angiotensinogen (AGT) to angiotensin I, which is then converted to the much more potent vasoconstrictor angiotensinogen II via action of angiotesin-converting enzyme (ACE). Angiotensinogen (AGT) is not significantly regulated in the pregnant C. ocellatus uterus, and we found no expression of ACE. Moreover, prolylcarboxypeptidase/angiotensinase C (PRCP) and angiotensin II receptor–associated protein (AGTRAP), two inhibitors of angiotensin II, are upregulated and therefore suggest that blood pressure is locally regulated by the expression of vasoconstrictors and dilators.

Regulation of the Maternal Innate Immune System

The embryo is an allograft of both maternal and paternal tissue and is therefore at risk of attack by the maternal immune system. As such, a variety of mechanisms that “hide” the embryo have evolved in mammals by downregulating elements of the maternal immune system (Moffett and Loke 2006). This immune response is expected to be much higher in mammals with invasive placentation because the maternal epithelia are breached. With a single possible exception (Trachylepis [Mabuya] ivensii; Blackburn and Flemming 2011), all viviparous squamate reptiles studied to date, including C. ocellatus, possess an epitheliochorial placenta with no breaching of the maternal epithelia. Nonetheless, as pregnancy progresses in squamate reptiles, the remnants of the ancestral shell membrane commonly disintegrate, thereby creating direct contact between the embryonic and the maternal epithelia (Blackburn 1993). Although the epithelia remain intact, studies of the mammalian epitheliochorial placentae demonstrate adaptations to hide from the maternal immune system (Moffett and Loke 2006) by regulating the maternal innate immune system and embryonic major histocompatability (MHC) loci.

The innate immune system comprises the general mechanisms by which the body defends against infection in a nonspecific manner. Its components consist of leukocytes (e.g., macrophages, natural killer cells), general antimicrobial proteins (e.g., bactericidal/permeability-increasing protein 2), and molecules that promote inflammation (e.g., cytokines, chemokines). It differs from the adaptive immune system that recognizes and remembers specific pathogens and thereby provides long-term immunity. Inflammation is an innate immune response critical to fighting infection, but excessive uterine inflammation may also lead to spontaneous abortion (Challis et al. 2009). Therefore, regulation of the proinflammatory and anti-inflammatory molecules in the pregnant uterus is essential to the maintenance of pregnancy while maintaining the maternal immune response to pathogens (Walker et al. 2010). Inflammation is regulated by a suite of genes, many of them cytokines—molecules that initiate cascades of intracellular signaling that are particularly active in the innate immune system. In squamate reptiles, the interleukin 1 cytokine system has received particular attention. IL1A and IL1B, two proinflammatory cytokines that also initiate immune response and regulate cellular growth and differentiation, are expressed in the uterine epithelia of C. chalcides, a close relative of C. ocellatus, and this expression continues throughout pregnancy (Paulesu et al. 1995, 2005; Paulesu 1997; Romagnoli et al. 2003; Paulesu, Romagnoli, et al. 2005). Thus, it was assumed that IL1A and IL1B expression was involved in the maintenance of pregnancy in both C. chalcides and mammals.

Surprisingly, our transcriptomic analysis reveals no expression of either IL1A or IL1B in both pregnant and nonpregnant C. ocellatus uteri and only low (but significant) upregulation of an interleukin 1 receptor (IL1R1). If IL1R1 expression indicates IL1 activity, we cannot locate the source of transcripts, and the lack of IL1A or IL1B expression in the uterus C. ocellatus starkly contrasts with that detected in C. chalcides. A potential explanation for these conflicting results could be that we were unable to identify IL1A and IL1B transcripts using our sequence identification strategy. That is, IL1A and IL1B may evolve quickly enough that the Chalcides transcripts of these genes cannot be reliably aligned to those in our reference genomes. An alternate explanation is that uterine IL1 expression was lost in the evolutionary history of the lineage leading to extant C. ocellatus. However, C. chalcides and C. ocellatus share a common ancestor only ∼10.5 Ma (Carranza et al. 2008); that uterine expression of IL1 is also detected in Zootoca (Lacerta) vivipara (Paulesu et al. 2005), a lacertid lizard that shares a common ancestor with scincid lizards (including Chalcides) ∼175 Ma (Brandley et al. 2008, 2011), and mammals, suggests that uterine IL1 expression is evolutionarily conserved. Although we do not detect IL1 expression, we nonetheless find extensive regulation of other genes that regulate the inflammatory response. The anti-inflammatory cytokine IL11 (Zourbas et al. 2001) is upregulated, whereas the proinflammatory IL8 and IL15 (Zourbas et al. 2001; Challis et al. 2009) are significantly downregulated. Finally, the proinflammatory cytokine high-mobility group box 1 (HMGB1) is significantly downregulated.

Significant upregulation and downregulation of other immune system genes also suggest active regulation of the inflammatory response in the uterus. There is significant downregulation of complement component 3 (C3). C3 plays a key role in activating the immune complement system, a component of the innate immune system that has roles in promoting local inflammation and stimulating a cascade of immune response including elements of the adaptive immune system (Sahu and Lambris 2001; Janssen et al. 2005; Walker et al. 2010). Excessive complement activation may lead to inhibited fetal growth or pregnancy termination in mammals (Caucheteux et al. 2003; Girardi et al. 2006).

However, not all inflammatory immune response genes are regulated to prevent inflammation in the pregnant C. ocellatus uterus. For example, phospholipase A2 IB is highly upregulated (PLA2G1B). PLA2G1B is typically expressed in the pancreas to digest dietary phospholipids in the duodenum, but in mammals, it is also expressed in nonpancreatic tissues where it likely plays a role in promoting inflammation by stimulating arachidonic acid release (Nevalainen et al. 2000). Phospholipase A2 expression is also critical for proper implantation in mammals (Dey et al. 2004). On the other hand, function of PLA2G1B may be mediated by the expression of its receptor (Moses et al. 1998) that is not differentially expressed between the pregnant and nonpregnant uteri (P = 0.91; data not shown). Regardless, even if PLAS2G1B expression promotes inflammation, its actions are likely regulated by the highly upregulated annexin A4 (ANXA4), a phospholipase A2 inhibitor.

Major histocompatibility loci encode molecules that present antigens to the host's immune system and are a key component in the body's recognition of “self” from “nonself.” Downregulating MHC expression in the embryonic tissues is therefore one mechanism by which the embryo can hide from the maternal immune system (Moffett and Loke 2006). However, both MHC Class I and II loci are significantly downregulated in the pregnant C. ocellatus uterus (the embryonic tissues were not sampled). Uterine downregulation of MHC Class I expression also occurs in cattle, pigs, and sheep, species with noninvasive epitheliochorial placentae, but in these cases, trophoblast cells merge with those of the uterine epithelia to form binucleate syncytia of embryonic and maternal origin (Davies et al. 2000; Choi et al. 2003; Joyce et al. 2008); downregulation of MHC Class I expression may be a mechanism by which the maternal immune system ignores these allograft cells in mammals. However, there is no evidence of syncytia formation in C. ocellatus (but they may form in the very late stages in pregnancy in C. chalcides [Blackburn et al. 1998] and some Mabuya group skinks [Ramírez-Pinilla et al. 2006; Vieira et al. 2007; Blackburn and Flemming 2011]), and thus, the function of uterine MHC downregulation in C. ocellatus remains unclear.

Transport across the Placenta

A majority of the water, organic and inorganic ions, and nutrients used by the embryo in oviparous and lecithotropic viviparous species is provided as yolk within the ovulated egg. By contrast, there is substantial transport of these molecules from the mother to the embryo via the placenta in lizard species with complex placentation (“placentotrophy”) (Thompson et al. 2000; Ramírez-Pinilla 2006; Thompson and Speake 2006). This resource allocation strategy must therefore involve substantial changes in the expression of molecular transporter genes, and our transcriptomic analysis of C. ocellatus reveals upregulation of many such loci. Placentotrophy has not been directly measured in C. ocellatus, and the species ovulates larger eggs (10 mm; Corso et al. 2000) with a larger yolk mass than highly placentrophic species such as C. chalcides (3 mm; Giacomini 1891), but light and scanning electron microscopic analysis of placental morphology imply that some placentotrophy occurs in C. ocellatus (Corso et al. 2000). Moreover, some placentotrophy has even been documented in primarily lecithotrophic species (Thompson et al. 2000; Thompson and Speake 2006).

Mothers of viviparous species provide much more water to their embryos than oviparous species (Thompson et al. 2000). Water transport is facilitated by aquaporins, cell membrane proteins that selectively regulate water transport. Aquaporins (AQP) are upregulated in the pregnant rat uterus (Lindsay and Murphy 2007) and indeed, AQP1 and AQP3 are also expressed in the uterus of the highly placentotrophic South American scincid lizard, Mabuya sp. (Wooding et al. 2010). AQP3, AQP5, and AQP11 are upregulated in the pregnant C. ocellatus uterus.

The last third of pregnancy in reptiles is particularly crucial for the provision of nutrients and ions such as calcium (Stewart and Thompson 2000; Herbert et al. 2006) and sodium (Thompson et al. 2001), and most of these ions are transported via transcellular routes (Loffing et al. 2001). The upregulation of 136 genes in 35 families of solute carrier proteins (SLCs; a majority not shown in table 2 but see supplementary information 1, Supplementary Material online) in the pregnant C. ocellatus uterus demonstrates a significant shift to increased inorganic and organic molecular transport during pregnancy (see “transport” in fig. 1). These upregulated SLC genes include transporters for inorganic ions (includes Ca2+, Cl, CO32, H+, HCO3, K+, Na+, and PO43), metals (includes Cu2+, Fe2/3+, Mg2+, and Zn2+), and organic molecules, including glucose, amino acids, peptides, fatty acids, and carboxylic acids. The three highest upregulated SLC genes are SLC15A1, an oligopeptide transporter, SLC22A17, an organic cation transporter, and SLC2A3, a glucose transporter, which suggest a great transport of organic molecules by the embryo in late pregnancy—a finding supported by physiological studies of other placentotrophic skinks (Thompson et al. 2000; Thompson and Speake 2006).

Lipids represent the primary source of nutrition to the developing embryo, and physiological studies have inferred a net uptake of lipids across the placenta in numerous viviparous species with relatively complex placentae (Thompson et al. 2000; Speake et al. 2004; Thompson and Speake 2006). Lipid transport is likely facilitated by upregulated fatty acid packaging and transport gene families in the uterus of pregnant C. ocellatus. The primary functions of fatty acid binding proteins are to facilitate intracellular transport of fatty acids (Chmurzyńska 2006) and are active in the mammalian placenta (Haggarty 2002). Similarly, FABP genes (FABP1—5, 7) are upregulated in pregnant C. ocellatus, with FABP1 and FABP7 showing a log2-fold expression change of 15.1 and 12.8, respectively. Apolipoproteins are transporters of fatty acids, cholesterol, and phospholipids, and five apolipoproteins (APOA1, APOA2, APOA4, APOE, and APOM) are significantly upregulated in the pregnant uterus of C. ocellatus.

Uterine Metabolism and Possible Histotrophy Mediated by Lysosomes in the Omphaloplacenta
Uterine Metabolism

As pregnancy progresses, the energy demands to fuel embryonic growth, placental remodeling, and active transport increase sharply, which is reflected in the pregnant Chalcides uterus where genes involved with ATP production are highly upregulated. Most of these genes are associated with carbohydrate metabolism, including genes involved in glycolysis (ALDOA, ALDOB, ALDOC, GAPDH, LDHA, PKM2, PGAM1), the citric acid cycle (MDH1, MDH2), electron transport chain (ATP synthases, cytochromes b and c), gluconeogenesis (PCK2), ketogenesis (HMGCS2), and ATP regeneration (CK). One highly upregulated metabolic gene of particular note is glutamate–ammonia ligase (GLUL, also called glutamine synthetase). Glutamine is critical for the synthesis of nucleic acids and sugars as well as cell growth and differentiation (Self et al. 2004), and upregulation of GLUL mirrors the pattern in numerous mammal species where glutamine concentrations in the fetal plasma are far higher than those in the maternal circulation (e.g., Wu et al. 1995; Cetin 2001; Kwon et al. 2003; Manso Filho et al. 2009). Thus, the upregulation of GLUL is probably critical to maintaining both reptilian and mammalian pregnancies. The peroxidases catalase (CAT) and glutathione peroxidases 1, 3–5 (GPX1, GPX3–5) are upregulated in the pregnant C. ocellatus uterus. These enzymes function to scavenge reactive oxygen species that are the by-product of oxidative metabolism (Myatt and Cui 2004) and are thus markers of increased uterine metabolic activity during pregnancy.

No study has inferred the precise mechanism by which carbon dioxide produced by the embryo is eliminated by the mother, yet the upregulation of carbonic anhydrase II (CA2) offers an intriguing potential mechanism (Sterling et al. 2001). Carbonic anhydrase II catalyzes the reversible reaction of converting CO2 and H2O to HCO3 and H+. In oviparous amniotes, expression of CA2 in the chorioallantois promotes acidification of the eggshell to release calcium carbonate for absorption by the embryo (Stewart and Ecay 2010). Thus, the significant upregulation of this gene in the viviparous C. ocellatus uterus suggests that it may play a different role during pregnancy. We hypothesize that CA2 has been co-opted into the role of eliminating CO2 produced by the embryo in viviparous squamates.

Histotrophy

Viviparous squamate reptiles with complex placentae experience a net uptake of macromolecules during pregnancy (especially lipids; Thompson and Speake 2006). Thus, these species must have the catabolic machinery to digest lipid and protein macromolecules into smaller units that can be actively transported to the embryo via the placenta. Microscopic examination and alkaline phosphatase assays suggest that catabolism and transport of macromolecules occur in the omphaloplacenta (also called the “yolk sac” placenta) of placentrophic lizards (Corso et al. 2000; Adams et al. 2005; Thompson and Speake 2006; Biazik et al. 2009). The omphaloplacenta lies at the abembryonic pole of the embryo/yolk mass and typically consists of well-developed columnar epithelial tissue usually associated with secretory epithelia (Corso et al. 2000; Adams et al. 2005; Thompson and Speake 2006). An extensive array of electron dense structures, interpreted to be lysosomes, occur in the omphaloplacenta of two viviparous Pseudemoia skinks (Biazik et al. 2009) and in C. ocellatus (Corso et al. 2000, fig. 3B in that paper). Although many of the nutrients transported across the placenta to the embryo may derive from the mother's digestive system, the massive upregulation of lysosomal proteases (e.g., cathepsins, LGMN, TPP1) supports the morphological interpretation that histotrophic activity occurs in the omphaloplacenta. We also detected upregulation of lysosomal hydrolases, including hexosaminidases, fucosidases, galactosidases, and sialidase (table 2). Lysosomal hydrolases break down the glycosidic bonds of glycolipids and glycoproteins and frequently process macromolecules digested by proteolytic enzymes (Winchester 2005). The putative presence of lysosomes and the upregulation of lysosomal genes collectively suggest that the omphaloplacenta plays a key role in placentotrophy in C. ocellatus but testing this hypothesis will require studies of tissue and cell-specific expression of these genes.

Steroid Hormones

Concentrations of circulating estrogen and progesterone during gestation are extremely varied across both oviparous and viviparous reptile species (Girling and Jones 2003; Murphy and Thompson 2011). In C. ocellatus, we found significant downregulation of progesterone (PGR) receptor and estrogen receptor 1 (ESR1) (log2-fold change = −3.1 and −4.0, respectively). This finding is puzzling because in C. chalcides, blood serum progesterone concentrations increase over the course of pregnancy with the highest concentration before birth (Guarino et al. 1998). We did not measure serum levels of progesterone in C. ocellatus and thus cannot determine whether the downregulated activity of PGR corresponds to a lower concentration of the hormone during late pregnancy or whether PGR downregulation is a mechanism of functional progesterone withdrawal.

Although serum estrogen has not been measured during pregnancy in any Chalcides species, concentrations of circulating estradiol rise during vitellogenesis, peak at the periovulation stage, and then decrease to previtellogenesis concentrations in late pregnancy in the distantly related skink Niveoscincus metallicus (Jones and Swain 1996). Thus, low ESR1 expression in the pregnant C. ocellatus uterus is not wholly unexpected. The absence or downregulation of PGR and ESR1 in the uterine epithelia is not unprecedented. In mammals, implantation is preceded by a loss of PGR and ESR1 expression in the uterine epithelia (Spencer and Bazer 2002; Spencer et al. 2007; Bazer et al. 2008), and expression in the luminal epithelium and superficial glandular epithelium of PGR and ESR1 is also absent through most of sheep and pig pregnancy, but PR is expressed in endometrial stroma (Geisert et al. 1994; Spencer and Bazer 2002). Mammalian uterine epithelia proliferate in response to progesterone and estrogen, but the actions of these hormones are mediated by paracrine signaling from endometrial stromal cells that do exhibit PGR and ESR1 expression (see Bazer et al. 2008 for review; Cooke et al. 1997). However, this mammalian model of stromal cell–mediated effects of progesterone and estrogen may not be applicable to C. ocellatus because our pregnant uterus sample contained stromal tissue, yet PGR and ESR1 expression were downregulated. Sustained PGR and ESR expression also occur in the myometrium of mice and sheep (Spencer and Bazer 2002), but this too appears to differ with C. ocellatus given that our tissue sample contained both myometrium and endometrium. In summary, PGR and ESR expression are extremely low in the uterine epithelia of mammals and in the entire uterus of C. ocellatus, but the mechanism that explains this phenomenon, whether low progesterone and estrogen levels or some other compensatory mechanism, remains unknown.


Conclusion

This study is the first successful use of RNA-Seq methods to characterize changes in pregnancy-associated gene expression in any squamate reptile and a significant step toward uncovering the genetic mechanisms behind the evolution of viviparity in reptiles. Our results serve as a baseline for future comparative gene expression analyses. Chalcides represents only one of the 100+ origins of viviparity in squamate reptiles, so we are not able assess the generality of our gene expression results. Nonetheless, there are clear parallels between the genetic processes associated with pregnancy in mammals and Chalcides in terms of gene expression related to tissue remodeling, angiogenesis, immune system regulation, and nutrient provisioning to the embryo. Although estrogen and progesterone receptors are downregulated in mammalian uterine epithelia, downregulation of these genes in the entire C. ocellatus uterus is evidence that the gene expression profiles of pregnant mammalian and reptilian uteri are not identical. Future RNA-Seq analyses of other viviparous and oviparous squamate reptiles will reveal the extent to which our results represent the generality of genetic processes associated with pregnancy in mammals and reptiles and potentially explain why the transition to viviparity is so easy in squamates. Moreover, future studies should localize the expression of highly regulated genes in both maternal and embryonic tissues.


Supplementary Material

Supplementary information 1 is available at Genome Biology and Evolution online (http://www.gbe.oxfordjournals.org/).


We thank O. Griffith, V. Lynch, B. Murphy, A. Seago, Y. Surget-Groba, Z. Wang, the MBT laboratory, and three anonymous reviewers for advice and/or comments on the manuscript; A. Dornburg and G. Watkins-Colwell for care of laboratory animals; J. Mai for computational assistance; and Intersect Australia Ltd. for supercomputing resources. This work was supported by the Yale Institute for Biospheric Studies Gaylord Donnelley Postdoctoral Fellowship to M.C.B., the American Association of University Women American Fellowship to R.L.Y., National Science Foundation postdoctoral fellowship DBI-0905701 to D.L.W., an Australian Research Council grant awarded to M.B.T. and C. Murphy, and the John Templeton Foundation grant # 12793 to G.P.W. The opinions expressed in this article are not those of the John Templeton Foundation. Animal care and procedures were approved by the Yale Institutional Animal Care and Use Committee (#2008-11242).


References
Adams SM,Biazik JM,Thompson MB,Murphy CR. Cytoepitheliochorial placenta of the viviparous lizard Pseudemoia entrecasteauxii: a new placental morphotypeJ Morphol.Year: 200526426427615803489
Afonso S,Romagnano L,Babiarz B. The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentationDevelopmentYear: 1997124341534259310336
Bazer FW,Burghardt RC,Johnson GA,Spencer TE,Wu G. Interferons and progesterone for establishment and maintenance of pregnancy: interactions among novel cell signaling pathwaysReprod Biol.Year: 2008817921119092983
Biazik JM,Thompson MB,Murphy CR. The tight junctional protein occludin is found in the uterine epithelium of squamate reptilesJ Comp Physiol B Biochem Syst Environ Physiol.Year: 2007177935943
Biazik JM,Thompson MB,Murphy CR. Claudin-5 is restricted to the tight junction region of uterine epithelial cells in the uterus of pregnant/gravid squamate reptilesAnat Rec.Year: 2008291547556
Biazik JM,Thompson MB,Murphy CR. Lysosomal and alkaline phosphatase activity indicate macromolecule transport across the uterine epithelium in two viviparous skinks with complex placentaJ Exp Zool B Mol Dev Evol.Year: 200931281782619422002
Biazik JM,Thompson MB,Murphy CR. Desmosomes in the uterine epithelium of non-invasive skink placentaeAnat Rec.Year: 2010293502512
Blackburn DG. Chorioallantoic placentation in squamate reptiles: structure, function, development, and evolutionJ Exp Zool A Ecol Genet Physiol.Year: 1993266414430
Blackburn DG. Squamate reptiles as model organisms for the evolution of viviparityHerpetol Monogr.Year: 200620131146
Blackburn DG,Flemming AF. Invasive implantation and the intimate placental associations in a placentotrophic African lizard, Trachylepis ivensi (Scincidae)J Morphol.Year: 201127313715921956253
Blackburn DG,Kleis-San Francisco S,Callard IP. Histology of abortive egg sites in the uterus of a viviparous placentrophic lizard, the skink Chalcides chalcidesJ Morphol.Year: 199823597108
Blackburn DG,Vitt LJ,Beuchat CA. Eutherian-like reproductive specializations in a viviparous reptileProc Natl Acad Sci U S A.Year: 1984814860486316593499
Brandley MC,Huelsenbeck JP,Wiens JJ. Rates and patterns in the evolution of snake-like body form in squamate reptiles: evidence for repeated re-evolution of lost digits and long-term persistence of intermediate body formsEvolutionYear: 2008622042206418507743
Brandley MC,et al. Accommodating heterogenous rates of evolution in molecular divergence dating methods: an example using intercontinental dispersal of Plestiodon (Eumeces) lizardsSyst Biol.Year: 20116031520952756
Carranza S,Arnold EN,Geniez PH,Roca J,Mateo JA. Radiation, multiple dispersal and parallelism in the skinks, Chalcides and Sphenops (Squamata: Scincidae), with comments on Scincus and Scincopus and the age of the Sahara DesertMol Phylogenet Evol.Year: 2008461071109418276164
Caucheteux SM,Kanellopoulos-Langevin C,Ojcius DM. At the innate frontiers between mother and fetus: linking abortion with complement activationImmunityYear: 20031816917212594944
Cetin I. Amino acid interconversions in the fetal-placental unit: the animal model and human studies in vivoPediatr Res.Year: 20014914815411158506
Challis JR,et al. Inflammation and pregnancyReprod Sci.Year: 20091620621519208789
Chmurzyńska A. The multigene family of fatty acid-binding proteins (FABPs): function, structure and polymorphismJ Appl Genet.Year: 200647394816424607
Choi Y,Johnson GA,Spencer TE,Bazer FW. Pregnancy and interferon tau regulate major histocompatibility complex class I and β2-microglobulin expression in the ovine uterusBiol Reprod.Year: 2003681703171012606392
Cooke PS,et al. Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epitheliumProc Natl Acad Sci U S A.Year: 199794653565409177253
Cooper AC,Robinson G,Vinson GP,Cheung WT,Pipkin FB. The localization and expression of the renin-angiotensin system in the human placenta throughout pregnancyPlacentaYear: 19992046747410419812
Corso G,Delitala GM,Carcupino M. Uterine morphology during the annual cycle in Chalcides ocellatus tiligugu (Gmelin) (Squamata: Scincidae)J Morphol.Year: 200024315316510658199
Corso G,Pala M,Pinna AM,Carcupino M. Aspetti morfofunzionali dell'ovidutto di Chalcides ocellatus tiligugu (Gmelin) (Squamata, Scincidae)Arch Ital Anat Embriol.Year: 1988934237251
Davies CJ,Fisher PJ,Schlafer DH. Temporal regional regulation of major histocompatibility complex class I expression in the bovine uterine/placental interfacePlacentaYear: 20002119420210736242
Defaure JP,Hubert J. Table de development de lezard vivipare: Lacerta vivipara JacquinArch Anat Micros Morphol Exp.Year: 196150309328
Dey SK,et al. Molecular cues to implantationEndocr Rev.Year: 20042534137315180948
Divya CP,Mahajan VS,Datta GS,Chauhan SS. Differential activity of cathepsin L in human placenta at two different stages of gestationPlacentaYear: 200223596411869092
Enders AC,Carter AM. What can comparative studies of placental structure tell us?PlacentaYear: 200425S3S915033300
Forsythe JA,et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1Mol Cell Biol.Year: 199616460446138756616
Gaspar J,et al. Opposing functions of the Ets factors NERF and ELF-1 during chicken blood vessel developmentArterioscler Thromb Vasc Biol.Year: 2002221106111212117724
Geisert RD,Pratt TN,Bazer FW,Mayes JS,Watson GH. Immunocytochemical localization and changes in endometrial progestin receptor protein during the porcine oestrous cycle and early pregnancyReprod Fertil Dev.Year: 199467497607624516
Giacomini E. Materiali per la storia dello sviluppo del Seps chalcides (Cuv.) BonapMonitore Zool.Year: 18912179192 , 198–211.
Girardi G,Bulla R,Salmon JE,Tedesco F. The complement system in the pathophysiology of pregnancyMol Immunol.Year: 200643687716023727
Girling JE,Jones SM. In vitro progesterone production by maternal and embryonic tissues during gestation in the southern snow skink (Niveoscincus microlepidotus)Gen Comp Endocrinol.Year: 200313310010812899851
Gracheva EO,et al. Molecular basis of infrared detection by snakesNatureYear: 20104641006101120228791
Guarino FM,et al. Endocrine activity of the corpus luteum and placenta during pregnancy in Chalcides chalcides (Reptilia, Squamata)Gen Comp Endocrinol.Year: 19981112612709707472
Guillette LJ,Jones RE. Ovarian, oviductal and placental morphology of the reproductively bimodal lizard, Sceloporus aeneusJ Morphol.Year: 19851848598
Hagemann A,Nielson AH,Poulsen K. The uteroplacental renin-angiotensin system: a reviewExp Clin Endocrinol.Year: 19941022522617995347
Haggarty P. Placental regulation of fatty acid delivery and its effect on fetal growth—a reviewPlacentaYear: 200216S28S3811978057
Herbert JF,Lindsay LA,Murphy CR,Thompson MB. Calcium transport across the uterine epithelium of pregnant lizardsHerpetol Monogr.Year: 200620205211
Hsieh MY,Chen WY,Jiang MJ,Cheng BC,Huang TY,Chang MS. Interleukin-20 promotes angiogenesis in a direct and indirect mannerGenes Immun.Year: 2006723424216511554
Janssen BJC,et al. Structures of complement component C3 provide insights into the function and evolution of immunityNatureYear: 200543750551116177781
Jerez A,Ramírez-Pinilla MP. The allantoplacenta of Mabuya mabouya (Sauria: Scincidae)J Morphol.Year: 200124913214611466741
Jones SM,Swain R. Annual reproductive cycle and annual cycles of reproductive hormones in plasma of female Niveoscincus metallicusJ Herpetol.Year: 199630140146
Joyce J,et al. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesisCancer CellYear: 2004544345315144952
Joyce MM,et al. Uterine MHC class I molecules and β2-microglobulin are regulated by progesterone and conceptus interferons during pig pregnancyJ Immunol.Year: 20081812494250518684940
Kirschke H,Barrett AJ,Rawlings ND. Lysosomal cysteine proteasesYear: 19972nd edOxfordOxford University Press
Kwon H,Spencer TE,Bazer FW,Wu G. Developmental changes in amino acids in ovine fetal fluidBiol Reprod.Year: 2003681813182012606398
Langmead B,Trapnell C,Pop M,Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genomeGenome Biol.Year: 200910R2519261174
Li W,Godzik A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequencesBioinformaticsYear: 2006221658165916731699
Lindsay LA,Murphy CR. Aquaporins are upregulated in glandular epithelium at the time of implantation in the ratJ Mol Histol.Year: 200738879517342345
Loffing J,et al. Distribution of transcellular calcium and sodium transport pathwaysJ Comp Physiol B.Year: 2001177935943
Manso Filho HC,Costa HE,Wu G,McKeever KH,Watford M. Equine placenta expresses glutamine synthetaseVet Res Commun.Year: 20093317518218726164
Marioni JC,Mason CE,Mane SM,Stephens M,Gilad Y. RNA-Seq: an assessment of technical reproducibility and comparison with gene expression arraysGenome Res.Year: 2008181509151718550803
Masson GR,Guillette LJ. Changes in oviducal vascularity during the reproductive cycle of three oviparous lizards (Eumeces obsoletus, Sceloporus undulatus and Crotaphytus collaris)J Reprod Fertil.Year: 1987803613713656274
Moffett A,Loke C. Immunology of placentation in eutherian mammalsNat Rev Immunol.Year: 2006658459416868549
Moses EK,Freed KA,Brennecke SP,Rice GE. Distribution of the phospholipase A2 receptor messenger RNA in human gestational tissuesPlacentaYear: 19981935409481783
Murphy BF,Belov K,Thompson MB. Evolution of viviparity and uterine angiogenesis: vascular endothelial growth factor (VEGF) in oviparous and viviparous skinksJ Exp Zool B Mol Dev Evol.Year: 201031414815619676116
Murphy BF,Brandley MC,Murphy CR,Thompson MB. Morphology and development of the placentae in Eulamprus quoyii group skinks (Squamata: Scincidae)J Anat.Year: 2012 Advance Access published Mar 16, 2012, doi:10.1111/j.1469-7580.2012.01492.x.
Murphy BF,Parker SL,Murphy CR,Thompson MB. Angiogenesis of the uterus and the chorioallantois in the eastern water skink Eulamprus quoyiiJ Exp Biol.Year: 20102133340334720833927
Murphy BF,Thompson MB. A review of the evolution of viviparity in squamate reptiles: the past, present and future role of molecular biology and genomicsJ Comp Physiol B Biochem Syst Environ Physiol.Year: 2011181575594
Murphy BF,Thompson MB,Belov K. Evolution of viviparity and the maternal immune system: major histocompatibility complex (MHC) class I genes in skinksOrbitYear: 20091116
Murphy WJ,Pringle TH,Crider TA,Springer MS,Miller W. Using genomic data to unravel the root of the placental mammal phylogenyGenome Res.Year: 20071741342117322288
Myatt L,Cui X. Oxidative stress in the placentaHistochem Cell Biol.Year: 200412236938215248072
Nevalainen TJ,Haapamäki MM,Grönroos JM. Roles of secretory phospholipases A2 in inflammatory diseasesBiochim Biophys Acta.Year: 20001488839011080679
Nielsen AH,Schauser KH,Poulsen K. The uteroplacental renin-angiotensis systemPlacentaYear: 20002146847710940196
Parker SL,Manconi F,Murphy CR,Thompson MB. Uterine and placental angiogenesis in the Australian skinks, Ctenotus taeniolatus and Saiphos equalisAnat Rec.Year: 2010293829838
Parker SL,Murphy CR,Thompson MB. Uterine angiogenesis in squamate reptiles: implications for the evolution of viviparityHerpetol Conserv Biol.Year: 20105330334
Paulesu L. Cytokines in mammalian reproduction and speculation about their possible involvement in nonmammalian viviparityMicrosc Res Tech.Year: 1997381881949260849
Paulesu L,Romagnoli R,Bigliardi E. Materno-fetal immunotolerance: is interleukin-1 a fundamental mediator in placental viviparity?Dev Comp Immunol.Year: 20052940941515707662
Paulesu L,et al. Cytokines in the viviparous reproduction of squamate reptiles: interleukin-1a (IL-1a) and IL-1b in placental structures of a skinkPlacentaYear: 1995161932057792282
Paulesu L,et al. Evidence of Hβ58, a gene involved in mammalian placental development, in the three-toed skink, Chalcides chalcides (Squamata: Scincidae), a viviparous placentotrophic reptilePlacentaYear: 20012273574111597194
Paulesu L,et al. Cytokines in the oviparity/viviparity transition: evidence of the interleukin-1 system in a species with reproductive bimodality, the lizard Lacerta viviparaEvol Dev.Year: 2005728228815982365
Peng J,Zhang L,Drysdale L,Fong GH. The transcription factor EPAS-1/hypoxia-inducible factor 2alpha plays an important role in vascular remodelingProc Natl Acad Sci U S A.Year: 2000978386839110880563
Ramírez-Pinilla MP. Placental transfer of nutrients during gestation in an Andean population of the highly matrotrophic lizard genus Mabuya (Squamata: Scincidae)Herpetol Monogr.Year: 200620194204
Ramírez-Pinilla MP,De Pérez G,Carreño-Escobar JF. Allantoplacental ultrastructure of an Andean population of Mabuya (Squamata, Scincidae)J Morphol.Year: 20062671227124716850472
Robinson MD,McCarthy DJ,Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression dataBioinformaticsYear: 20102613914019910308
Robinson MD,Oshlack A. A scaling normalization method for differential expression analysis of RNA-Seq dataGenome Biol.Year: 201011R2520196867
Romagnoli R,Cateni C,Guarino FM,Bigliardi E,Paulesu LR. Potential role of interleukin-1 at the peri-ovulation stage in a species of placental viviparous reptile, the three-toed skink, Chalcides chalcides (Squamata: Scincidae)Reprod Biol Endocrinol.Year: 20031606514585105
Sahu A,Lambris JD. Structure and biology of complement protein C3, a connecting link between innate and acquired immunityImmunol Rev.Year: 2001180354811414361
Salamonsen LA,Nie G. Proteases at the endometrial-trophoblast interface: their role in implantationRev Endocr Metab Disord.Year: 2002313314312007290
Schwartz TS,et al. A garter snake transcriptome: pyrosequencing, de novo assembly, and sex-specific differencesBMC GenomicsYear: 20101169421138572
Self JT,et al. Glutamine synthesis in the developing porcine placentaBiol Reprod.Year: 2004701444145114736817
Song G,Bazer FW,Spencer TE. Differential expression of cathepsins and cystatin C in ovine uteroplacental tissuesPlacentaYear: 2007281091109817555811
Song G,Spencer TE,Bazer FW. Cathepsins in the ovine uterus: regulation by pregnancy, progesterone, and interferon tauEndocrinologyYear: 20061464825483316099855
Song G,et al. Cathepsin B, cathepsin L, and cystatin C in the procine uterus and placenta: potential roles in endometrial/placental remodeling and in fluid-phase transport of proteins secreted by uterine epithelia across placental areolaeBiol Reprod.Year: 20108285486420107207
Speake BK,Herbert JF,Thompson MB. Evidence for placental transfer of lipids during gestation in the viviparous lizard, Pseudemoia entrecasteauxiiComp Biochem Physiol A Mol Integr Physiol.Year: 200413921322015528170
Spencer TE,Bazer FW. Biology of progesterone action during pregnancy recognition and maintenance of pregnancyFront Biosci.Year: 2002718791898
Spencer TE,et al. Pregnancy recognition and conceptus implantation in domestic ruminants: roles of progesterone, interferons and endogenous retrovirusesReprod Fertil Dev.Year: 200719657817389136
Sterling D,Reithmeier RAF,Casey JR. A transport metabolon: functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangersJ Biol Chem.Year: 2001276478864789411606574
Stewart JR. Yolk sac placentation in reptiles: structural innovation in a fundamental vertebrate nutritional systemJ Exp Zool.Year: 1993266431449
Stewart JR,Ecay TW. Patterns of maternal provision and embryonic mobilization of calcium in oviparous and viviparous squamate reptilesHerpetol Conserv Biol.Year: 20105341359
Stewart JR,Thompson MB. Evolution of placentation among squamate reptiles: recent research and future directionsComp Biochem Physiol A Mol Integr Physiol.Year: 200012741143111154939
Surget-Groba Y,Montoya-Burgos JI. Optimization of de novo transcriptome assembly from next-generation sequencing dataGenome Res.Year: 2010201432144020693479
Takeda N,et al. Endothelial PAS domain protein 1 gene promotes angiogenesis through the transactivation of both vascular endothelial growth factor and its receptor, Flt-1Circ Res.Year: 20049514615315192019
Thompson MB,Speake BK. A review of the evolution of viviparity in lizards: structure, function and physiology of the placentaJ Comp Physiol B Biochem Syst Environ Physiol.Year: 2006176179189
Thompson MB,Speake BK,Russell KJ,McCartney RJ. Utilisation of lipids, protein, ions, and energy during embryonic development of Australian oviparous skinks in the genus LampropholisComp Biochem Physiol A Mol Integr Physiol.Year: 200112931332611423304
Thompson MB,Stewart JR,Speake BK. Comparison of nutrient transport across the placenta of lizards differing in placental complexityComp Biochem Physiol A Mol Integr Physiol.Year: 200012746947911154943
Varanou A,et al. The importance of cysteine cathepsin proteases for placental developmentJ Mol Med.Year: 20068430531716440214
Vieira S,De Pérez G,Ramírez-Pinilla MP. Invasive cells in the placentome of Andean populations of Mabuya: an endotheliochorial contribution to the placenta?Anat Rec.Year: 200729015081518
Vinson GP,Saridogan E,Puddefoot JR,Djahanbakhch O. Tissue renin-angiotensin systems and reproductionHum Reprod.Year: 1997126516529159419
Vitt LJ,Blackburn DG. Ecology and life history of the viviparous lizard Mabuya bistriata (Scincidae) in the Brazilian AmazonCopeiaYear: 19911991916927
Walker CG,et al. Modulation of the maternal immune system by the pre-implantation embryoBMC GenomicsYear: 20101147420707927
Wall CE,et al. Whole transcriptome analysis of the fasting and fed Burmese python heart: insights into extreme physiological cardiac adaptationPhysiol Genomics.Year: 201143697921045117
Wang L,Feng Z,Wang X,Wang X,Zhang X. DEGseq: an R package for identifying differentially expressed genes from RNA-Seq dataBioinformaticsYear: 20102613613819855105
Weekes HC. A review on placentation among reptiles with particular regard to function and evolution of placentaProc Linn Soc Lond.Year: 19352625645
Winchester B. Lysosomal metabolism of glycoproteinsGlycobiologyYear: 20051511515329357
Wooding FBP,Ramirez-Pinilla MP,Forhead AS. Functional studies of the placenta of the lizard Mabuya sp. (Scincidae) using immunocytochemistryPlacentaYear: 20103167568520557932
Wu G,Bazer FW,Tuo W. Developmental changes of free amino acid concentration in fetal fluids in the pigJ Nutr.Year: 1995125285928687472667
Wu Q,Thompson MB,Murphy CR. Changing distribution of cadherins during gestation in the uterine epithelium of lizardsJ Exp Zool B Mol Dev Evol.Year: 201131644045021612011
Zourbas S,Dubanchet S,Martal J,Chaouat G. Localisation of pro-inflammatory (IL-12, IL-15) and anti-inflammatory (IL-11, IL-13) cytokines at the foetal-maternal interface during murine pregnancyClin Exp Immunol.Year: 200112651952811737071

Figures

[Figure ID: fig1]
FIG. 1.— 

Aggregated GO biological processes for significantly regulated genes in the pregnant and nonpregnant uterine transcriptomes. For group descriptions, see text.



Tables
[TableWrap ID: tbl1] Table 1 

Characteristics of Contigs Assembled by ABySS Using the Subtractive Multiple-k Method (Surget-Groba and Montoya-Burgos 2010)


Number of Contigs 300,967
Maximum contig length 15,179
Mean contig length 530
Median contig length 165
N50 1,454

[TableWrap ID: tbl2] Table 2 

A Selection of Significantly Upregulated Genes in the Pregnant Chalcides ocellatus Uterus as Inferred by the MARS Differential Gene Expression Analysis of TMM Transformed Transcript Counts


Gene Symbol Gene Name Pregnant Transcript Count (Raw) Nonpregnant Transcript Count (Raw) Pregnant Transcript Count (Transformed) Nonpregnant Transcript Count (Transformed) Log2-Fold Change Z-Score Expression Rank
ACTB Actin, beta 95,506 39,786 133,776 55,728 1.3 154.2 16
AGTRAP Angiotensin II receptor–associated protein 1,177 657 840 284 1.6 16.9 1,058
ALDOA Aldolase A 10,459 9,017 7,467 3,900 0.9 33.7 384
ALDOB Aldolase B 7,194 291 5,136 126 5.3 74.4 94
ALDOC Aldolase C 275 123 196 53 1.9 9.3 1,991
ANG Angiogenin 14,189 4,337 10,130 1,876 2.4 78.7 80
ANGPT4 Angiopoietin 4 451 199 322 86 1.9 12.0 1,575
ANXA4 Annexin A4 12,674 3,524 9,048 1,524 2.6 76.8 87
APOA1 Apolipoprotein A-I 102 2 73 1 6.2 8.9 2,090
APOA2 Apolipoprotein A-II 1,123 0 802 0 10.6 23.4 720
APOA4 Apolipoprotein A-IV 331 109 236 47 2.3 11.7 1,617
APOE Apolipoprotein E 8,568 2,879 6,117 1,245 2.3 59.1 145
APOM Apolipoprotein M 34 2 24 1 4.6 5.0 3,188
AQPP11 Aquaporin 11 123 28 88 12 2.9 8.0 2,279
AQPP3 Aquaporin 3 859 837 613 362 0.8 8.1 2,266
AQPP5 Aquaporin 5 2,391 18 1,707 8 7.7 41.0 279
ARG2 Amino acid acetyltransferase 16,563 81 23,200 113 7.7 128.0 22
ATP1A1 ATPase, Na+/K+ transporting, alpha 1 54,438 10,240 38,865 4,429 3.1 176.0 11
BPI2 Bactericidal/permeability-increasing protein 2 17,327 97 12,370 42 8.2 107.9 40
BTG1 B-cell translocation gene 1 16,577 1,535 11,835 664 4.2 107.9 41
CA2 Carbonic anhydrase II 7,873 749 5,621 324 4.1 74.1 95
CAT Catalase 41,302 604 57,852 846 6.1 211.2 8
CDH1 Cadherin 1 12,711 3,753 9,075 1,623 2.5 75.4 91
CDH11 Cadherin 11 20,807 634 14,855 274 5.8 126.8 24
CDH15 Cadherin 15 1,735 1,214 1,239 525 1.2 17.2 1,037
CDH2 Cadherin 2 685 717 489 310 0.7 6.4 2,732
CDH24 Protocadherin 24 99 60 71 26 1.4 4.7 3,305
CDH4 Cadherin 4 4,170 21 2,977 9 8.4 52.5 185
CHY Chymosin 12,199 12 8,709 5 10.8 76.2 88
CKB Creatine kinase 20,847 3,963 14,883 1,714 3.1 108.6 38
CLDN1 Claudin 1 9,577 601 6,837 260 4.7 84.4 67
CLDN12 Claudin 12 214 222 153 96 0.7 3.6 3,732
CLDN23 Claudin 23 92 60 66 26 1.3 4.2 3,478
CLDN3 Claudin 3 2,806 2,719 2,003 1,176 0.8 14.8 1,245
CLDN4 Claudin 4 8,586 4,014 6,130 1,736 1.8 51.0 197
CLDN5 Claudin 5 557 587 398 254 0.6 5.7 2,963
CLDN6 Claudin 6 3,666 2,451 2,617 1,060 1.3 26.1 613
CLDN7 Claudin 7 3,596 1,716 2,567 742 1.8 32.6 417
CLDN8 Claudin 8 5,015 111 3,580 48 6.2 62.1 135
CO3 Cytochrome c oxidase subunit 3 37,325 14,477 52,281 20,278 1.4 102.2 48
COL6A3 Collagen, type VI, alpha 3 14,847 1,170 20,796 1,639 3.7 116.0 28
CPA2 Carboxypeptidase A2 18,052 4,020 25,286 5,631 2.2 98.1 50
CSTC Cystatin C 18,365 1,827 13,111 790 4.1 112.7 35
CSTM Cystatin 6/M/E 30,367 95 21,680 41 9.0 136.2 20
CTSA Cathepsin A 6,942 1,022 4,956 442 3.5 65.8 119
CTSB Cathepsin B 17,222 5,309 12,295 2,296 2.4 86.5 64
CTSD Cathepsin D 10,474 7,033 7,478 3,042 1.3 43.9 253
CTSE Cathepsin E 67 7 48 3 4.0 6.8 2,630
CTSF Cathepsin F 1,269 1,105 906 478 0.9 11.6 1,633
CTSL Cathepsin L1 497,141 918 354,922 397 9.8 523.9 1
CTSV Cathepsin V (cathepsin L2) 106,855 129 76,287 56 10.4 232.0 6
CTSZ Cathepsin Z 65,117 999 46,489 432 6.7 221.5 7
CYB Cytochrome b 39,111 14,696 54,783 20,585 1.4 107.3 44
DDIT4 DNA damage–inducible transcript 4 15,069 569 10,758 246 5.5 107.8 42
DSG2 Desmoglein 2 2,002 946 1,429 409 1.8 24.5 676
ENPP2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 13,373 60 9,547 26 8.5 93.2 56
EPAS1 Endothelial PAS domain protein 1 41,345 2,946 29,517 1,274 4.5 174.1 12
FABP1 Fatty acid–binding protein 1 14,898 0 10,636 0 14.4 60.2 140
FABP2 Fatty acid–binding protein 2 97 2 69 1 6.1 8.6 2,147
FABP3 Fatty acid–binding protein 3 2,247 395 1,604 171 3.2 36.2 340
FABP4 Fatty acid–binding protein 4 3,051 523 2,178 226 3.3 42.4 268
FABP5 Fatty acid–binding protein 5 1,198 388 855 168 2.3 22.4 765
FABP7 Fatty acid–binding protein 7 12,174 2 8,691 1 13.1 61.9 136
FN1 Fibronectin protein 15,382 2,046 21,546 2,866 2.9 106.9 45
FTL Ferritin, light polypeptide 36,692 10,208 51,395 14,298 1.8 126.0 25
FUCA1 Fucosidase, alpha-L1 145,697 220 104,017 95 10.1 277.5 5
FUCA2 Fucosidase, alpha-L2 129,789 765 92,660 331 8.1 296.6 4
GAPDH Glyceraldehyde-3-phosphate dehydrogenase 29,268 15,408 20,895 6,664 1.6 87.8 62
GGH Gamma-glutamyl hydrolase 12,615 85 17,670 119 7.2 113.8 33
GLA Galactosidase, alpha 16,970 486 12,115 210 5.9 114.5 32
GLB1 Galactosidase, beta 1 14,934 513 10,662 222 5.6 107.4 43
GLUL Glutamate–ammonia ligase (glutamine synthetase) 50,207 3,729 35,844 1613 4.5 191.3 10
GRN Granulin 10,060 1,385 7,182 599 3.6 80.0 74
GSN Gelsolin 19,199 5,489 13,707 2,374 2.5 93.7 54
GPX1 Glutathione peroxidase 1 3,871 1,307 5,422 1,831 1.6 36.4 337
GPX3 Glutathione peroxidase 3 2,969 23 4,159 32 7.0 55.6 161
GPX4 Glutathione peroxidase 4 2,996 957 4,197 1,340 1.6 33.2 396
GPX5 Glutathione peroxidase 5 1,484 22 2,079 31 6.1 40.0 287
HEXA Hexosaminidase A 196,426 183 140,234 79 10.8 305.3 3
HEXB Hexosaminidase B 15,870 192 11,330 83 7.1 108.3 39
HMGCS2 3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 2 9,362 55 6,684 24 8.1 79.7 76
HYAL3 Hyaluronoglucosaminidase 1 17,923 46 12,796 20 9.3 102.8 47
HYAL4 Hyaluronoglucosaminidase 3 12,479 0 8,909 0 14.1 56.5 157
IFI30 Interferon gamma–inducible protein 30/GILT 11,020 138 15,436 193 6.3 108.8 37
IL20RB Interleukin 20 receptor beta 8,497 261 6,066 113 5.7 81.0 69
KRT18 Keratin 18 19,527 3,222 27,352 4,513 2.6 113.5 34
KRT8 Keratin 8 20,650 3,619 28,925 5,069 2.5 114.5 31
LDHA Lactate dehydrogenase A 10,376 1,447 7,408 626 3.6 81.1 68
LGMN Legumain 57,922 430 41,352 186 7.8 201.3 9
MDH1 Malate dehydrogenase 1 8,071 3,725 5,762 1,611 1.8 49.7 207
MDH2 Malate dehydrogenase 2 5,076 3,831 3,624 1,657 1.1 27.4 572
NAGA Alpha-N-acetylgalactosaminidase 25,652 354 18,314 153 6.9 138.4 19
NDRG1 N-myc downstream–regulated 1 26,922 1,595 19,220 690 4.8 142.0 18
NEU1 Sialidase 1 29,234 76 20,871 33 9.3 131.4 21
NFAT5 Nuclear factor of activated T-cells 5 10,795 785 15,121 1,100 3.8 100.0 49
OVGP1 Oviductal glycoprotein 1, 120 kDa 29,231 217 40,944 304 7.1 174.0 13
PCK2 Phosphoenolpyruvate carboxykinase 2 8,634 511 6,164 221 4.8 80.4 73
PGAM1 Phosphoglycerate mutase 1 11,543 2,573 8,241 1,113 2.9 77.9 83
PKM2 Pyruvate kinase, isozymes M1/M2 25,858 6,134 18,461 2,653 2.8 114.8 30
PLA2G1B Phospholipase A2, group IB 22,787 187 16,268 81 7.6 127.0 23
PPIB Peptidyl-prolyl cis-trans-isomerase B 16,423 2,048 23,004 2,869 3.0 112.1 36
PRCP Prolylcarboxypeptidase (angiotensinase C) 2,944 173 2,102 75 4.8 47.0 229
PRSS1 Protease, serine, 1 2,681 0 1,914 0 11.9 32.4 420
PRSS12 Protease, serine, 12 1,290 49 921 21 5.5 31.5 443
PRSS16 Protease, serine, 16 90,516 9 64,622 4 14.0 154.5 15
PRSS2 Protease, serine, 2 148,046 2 105,694 1 16.7 147.5 17
PRSS27 Protease, serine, 27 19,852 1,096 14,173 474 4.9 122.3 26
PRSS3 Protease, serine, 3 161,271 5 115,136 2 15.8 169.8 14
PRSS33 Protease, serine, 33 994 60 710 26 4.8 27.3 578
PRSS36 Protease, serine, 36 466 12 333 5 6.1 19.0 935
PRSS8 Protease, serine, 8 11,814 400 8,434 173 5.6 95.5 52
REN Renin 15,355 32 10,962 14 9.6 93.3 55
SLC15A1 Solute carrier family 15, member 1 12,659 22 17,732 31 9.2 103.2 46
SPINT1 Serine peptidase inhibitor, Kunitz type 1 10,400 1,020 7,425 441 4.1 84.9 66
SPINT2 Serine protease inhibitor, Kunitz type 2 14,494 4,573 10,348 1,978 2.4 78.7 81
TFPI2 Tissue factor pathway inhibitor 2 17,908 393 12,785 170 6.2 117.3 27
TPP1 Polynucleotide 3′-phosphatase 162,913 2,062 116,308 892 7.0 347.8 2
VEGFA Vascular endothelial growth factor A 3,348 1,727 2,390 747 1.7 30.1 495
WSB1 WD repeat and SOCS box–containing 1 14,527 1,088 20,348 1,524 3.7 115.6 29

NOTE.—Only the top 50 most upregulated genes (as assessed by z-score), and those genes discussed in the text, are shown. The expression rank represents the order of expression in the entire upregulated transcriptome from highest to lowest (e.g., a rank of 1 is the highest change in expression). Complete results for the MARS analysis are provided in supplementary information 1 (Supplementary Material online).


[TableWrap ID: tbl3] Table 3 

A Selection of Significantly Downregulated Genes in the Pregnant Chalcides ocellatus Uterus as Inferred by the MARS Differential Gene Expression Analysis of TMM Transformed Transcript Counts


Gene Symbol Gene Name Pregnant Transcript Count (Raw) Nonpregnant Transcript Count (Raw) Pregnant Transcript Count (Transformed) Nonpregnant Transcript Count (Transformed) Log2-Fold Change Z-Score Expression Rank
ACTA2 Actin, alpha 2 1,340 7,007 1,877 16,201 −2.4 −64.8 19
AHNAK AHNAK nucleoprotein 6,465 15,345 9,056 35,479 −1.2 −61.0 23
BTF3 Basic transcription factor 3 3,393 9,969 4,753 23,049 −1.6 −58.1 28
C3 Complement component 3 2,256 10,334 3,160 23,893 −2.2 −74.8 9
CALD1 Caldesmon 1 9,97 5,162 1,397 11,935 −2.4 −55.4 31
CD74 CD74 molecule 503 4,806 705 11,112 −3.3 −62.9 21
CDH13 Cadherin 13 48 105 67 243 −1.1 −4.7 2164
CDH5 Cadherin 5 40 246 56 569 −2.6 −12.8 686
CNN1 Calponin 1 612 4,810 857 11,121 −3.0 −60.4 25
COL12A1 Collagen, type XII, alpha 1 306 6,613 429 15,290 −4.4 −82.0 6
CXCL14 Chemokine (C-X-C motif) ligand 14 56 3,551 78 8,210 −6.0 −62.0 22
DCN Decorin 361 3,191 506 7,378 −3.1 −50.5 41
DDX50 DEAD (Asp-Glu-Ala-Asp) box polypeptide 50 8,260 23,789 11,570 55,002 −1.5 −88.6 4
DES Desmin 1,883 9,420 2,638 21,780 −2.3 −73.9 11
EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 448 3,065 628 7,087 −2.8 −46.5 48
ESR1 Estrogen receptor 1 499 7,745 699 17,907 −4.0 −86.0 5
GAS1 1,3-Beta-glucanosyltransferase GAS1 1304 4,980 1,827 11,514 −1.9 −47.8 42
GPI Glucose-6-phosphate isomerase 6,118 14,803 8,570 34,226 −1.3 −61.0 24
GPR124 G protein–coupled receptor 124 228 4,267 319 9,866 −4.2 −65.1 18
HMGB1 High-mobility group 1 856 4,490 1,199 10,381 −2.4 −51.9 39
HSP90AB1 Heat shock protein 90 kDa alpha, class B member 1 6,564 15,181 9,194 35,100 −1.2 −59.3 27
IFITM3 Interferon-induced transmembrane protein 3 568 3,435 796 7,942 −2.6 −47.6 44
IGFBP5 Insulin-like growth factor–binding protein 5 289 5,002 405 11,565 −4.1 −69.9 12
LGR5 Leucine-rich repeat-containing G protein–coupled receptor 5 436 8,664 611 20,032 −4.3 −93.2 1
MEIS1 Meis homeobox 1 316 2,680 443 6,196 −3.1 −45.9 49
MHCIA MHC Class IA 1,651 11,834 2,313 27,361 −2.8 −92.6 2
MHCIIA MHC Class IIA 637 5,550 892 12,832 −3.1 −66.4 17
MHCIIB MHC Class IIB 835 4,703 1,170 10,874 −2.5 −54.4 34
MYH11 Myosin, heavy chain 11 1,940 9,598 2,717 22,191 −2.3 −74.3 10
PCLP1 Podocalyxin-like protein 1 965 6,741 1,352 15,586 −2.8 −69.4 13
PDLIM4 PDZ and LIM domain 4 364 6,474 510 14,968 −4.2 −79.8 8
PGR Progesterone receptor 283 2,345 396 5,422 −3.1 −42.7 61
PTGIS Prostaglandin I2 (prostacyclin) synthase 131 1,580 183 3,653 −3.6 −37.6 93
RPL10A 60S ribosomal protein L10A 5,856 17,979 8,203 41,569 −1.6 −80.4 7
RPL14 50S ribosomal protein L14 3,239 8,857 4,537 20,478 −1.5 −52.1 38
RPL17 50S ribosomal protein L17 6,268 13,974 8,780 32,309 −1.2 −54.9 32
RPL18A 50S ribosomal protein L18A 2,656 10,063 3,720 23,267 −1.9 −67.7 16
RPL19 50S ribosomal protein L19 4,645 10,270 6,506 23,745 −1.1 −46.6 47
RPL27A 50S ribosomal protein L27A 2,685 8,070 3,761 18,659 −1.6 −53.1 35
RPL32 60S ribosomal protein L32 3,513 8,440 4,921 19,514 −1.3 −45.7 50
RPL37 60S ribosomal protein L37A 2,163 8,028 3,030 18,561 −1.9 −59.9 26
RPL4 60S ribosomal protein L4 8,509 19,769 11,919 45,708 −1.2 −67.9 15
RPLP2 60S ribosomal protein LP2 2,023 6,650 2,834 15,375 −1.7 −51.0 40
RPS13 40S ribosomal protein S13 3,213 9,151 4,500 21,158 −1.5 −54.5 33
RPS2 40S ribosomal protein S2 12,945 22,719 18,132 52,528 −0.8 −52.1 37
RPS23 40S ribosomal protein S23 2,612 7,930 3,659 18,335 −1.6 −53.0 36
RPS7 40S ribosomal protein S7 2,504 13,326 3,507 30,811 −2.4 −89.9 3
RPS8 40S ribosomal protein S8 3,464 11,054 4,852 25,558 −1.7 −64.6 20
RPS9 40S ribosomal protein S9 5,272 11,266 7,385 26,048 −1.1 −47.2 45
SLC6A6 Solute carrier family 6, member A6 845 4,998 1,184 11,556 −2.6 −57.0 29
SLIT2 Slit homolog 2 276 2,727 387 6,305 −3.3 −47.7 43
SOX7 SRY (sex-determining region Y)-box 7 318 2,784 445 6,437 −3.1 −47.1 46
TNS3 Tensin 3 675 6,013 945 13,903 −3.2 −69.4 14
TPBG Trophoblast glycoprotein 271 3,464 380 8,009 −3.7 −56.1 30

NOTE.—Only the top 50 most downregulated genes (as assessed by z-score), and those genes discussed in the text, are shown. The expression rank represents the order of expression in the entire nonpregnant uterine transcriptome from highest to lowest (e.g., a rank of 1 is the highest change in expression). Complete results for the MARS analysis are provided in supplementary information 1 (Supplementary Material online).


[TableWrap ID: tbl4] Table 4 

Genes Whose Regulation Has Been Assessed Previously in Squamate Reptiles and Comparison with Chalcides ocellatus


Gene Species Regulatory Activity during Pregnancy Reference
Desmoglein 2 (DSG2) Chalcides ocellatus Increase This study
Lerista bougainvilliia No change Biazik et al. (2010)
Pseudemoia entrecasteauxii Decrease Biazik et al. (2010)
Pseudemoia spenceri Decrease Biazik et al. (2010)
Saiphos equalisa No change Biazik et al. (2010)
Occludin (OCLN) C. ocellatus No change This study
Eulamprus tympanum Absent Biazik et al. (2007)
P. entrecasteauxii Absent Biazik et al. (2007)
P. spenceri Increase Biazik et al. (2007)
S. equalisa Increase Biazik et al. (2007)
Claudin 5 (CLDN5) C. ocellatus Increase This study
E. tympanum Present Biazik et al. (2008)
L. bougainvilliia Present Biazik et al. (2008)
P. entrecasteauxii Present Biazik et al. (2008)
P. spenceri Present Biazik et al. (2008)
S. equalisa Present Biazik et al. (2008)
Cadherin (CDH)b C. ocellatus Varied, but mostly increase This study
L. bougainvilliia Decrease Wu et al. (2011)
Niveoscincus metallicus Decrease Wu et al. (2011)
Niveoscincus ocellatus Decrease Wu et al. (2011)
Vascular endothelial growth factor A (VEGFA) C. ocellatus Increase This study
E. tympanum Present Murphy, Belov, et al. (2010)
N. metallicus Present Murphy, Belov, et al. (2010)
P. entrecasteauxii Present Murphy, Belov, et al. (2010)
S. equalisa Increase Murphy, Belov, et al. (2010)
Interleukin 1A (IL1A) C. ocellatus Absent This study
Chalcides chalcides Present Paulesu et al. (1995)
Zootoca (Lacerta) viviparaa Present Paulesu et al. (2005)
Interleukin 1B (IL1B) C. ocellatus Absent This study
C. chalcides Present Paulesu et al. (1995)
Z. viviparab Present Paulesu et al. (2005)

NOTE.—Gene expression is identified as “present” if expression was inferred, but levels were not quantified between pregnant and nonpregnant individuals. Gene expression is identified as “no change” if expression was quantified, but there is no change in expression between pregnant and nonpregnant individuals.

aReproductively bimodal species. Only data for the viviparous individuals are presented.

bNonspecific “pan-cadherin” antibody used.



Article Categories:
  • Research Articles

Keywords: placenta, pregnancy, RNA-Seq, transcriptome, uterus, viviparity.

Previous Document:  Invitro controlled release of an anti-inflammatory from daily disposable therapeutic contact lenses ...
Next Document:  Purifying selection and molecular adaptation in the genome of Verminephrobacter, the heritable symbi...