Transcriptome analyses of bovine, porcine and equine endometrium during the pre-implantation phase

Animal Reproduction Science 134 (2012) 84–94

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Transcriptome analyses of bovine, porcine and equine endometrium during the pre-implantation phase夽 Stefan Bauersachs ∗ , Eckhard Wolf Chair for Molecular Animal Breeding & Biotechnology and Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377 Munich, Germany

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Article history: Available online 11 August 2012 Keywords: Bos taurus Sus scrofa Equus caballus Microarray RNA-Seq

a b s t r a c t Different reproductive strategies evolved in various mammalian groups to achieve recognition, establishment and maintenance of pregnancy. The complexity of these processes is reflected by a high incidence of embryonic loss during this critical period in many mammalian species. Besides studies in mice and humans a number of transcriptome studies of endometrial tissue samples and also of early embryos have been performed during the pre-implantation phase in cattle, swine and horse to identify genes associated with embryo–maternal interaction. Results of these studies are reviewed and compared between species. The comparison of data sets from different species indicated a general role of interferons for the establishment of pregnancy. In addition to many species-specific changes in gene expression, which may reflect different pregnancy recognition signals and mechanisms of embryo implantation, a number of transcriptome changes were found to be similar across species. These genes may have conserved roles during the establishment of pregnancy in mammals and reflect basic principles of mammalian reproduction. The relevance and strategies, but also the challenges of cross-species comparisons of gene expression data are discussed. © 2012 Elsevier B.V. All rights reserved.

Reproductive success depends on a number of biological processes, e.g. maturation and selection of gametes, fertilization, pre- and post-implantation embryonic development including endometrial support of embryo growth, placentation, fetal growth, and birth. A crucial process is probably embryo–maternal communication that facilitates establishment, recognition, and maintenance of pregnancy. Studies of early pregnancy in different mammalian species showed that the majority of embryo losses occur during the pre-implantation phase. For example in cattle, this

夽 This paper is part of the special issue entitled: 3rd Embryo Genomics, Guest Edited by D. Tesfaye and K. Schellander. ∗ Corresponding author at: Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany. Tel.: +49 89 2180 76701; fax: +49 89 2180 76849. E-mail addresses: [email protected] (S. Bauersachs), [email protected] (E. Wolf). 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.08.015

corresponds to the period before day 16 following breeding (Diskin and Morris, 2008). Furthermore, a reduction in fertilization and embryo survival rates has been suggested as the most important component for decreasing reproductive efficiency in dairy cattle (Santos et al., 2004). Likewise, the pre-implantation phase is most critical in the horse (Merkt and Gunzel, 1979; Ginther et al., 1985) and also in the pig (Wilson et al., 1999; Wessels et al., 2007). During the pre-implantation phase, the conceptus (embryo and associated extra-embryonic membranes) interacts with the uterine environment via paracrine signals, to coordinate attachment and implantation. In mammals with late implantation, the conceptus has also to signal its presence to prevent luteolysis for continuous progesterone (P4) production. Progesterone plays the most important role in uterine receptivity, i.e. the ability to support conceptus growth and development by the production of histiotroph (Spencer et al., 2007; Bazer et al., 2009). Thus,

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the analysis of gene expression changes during the cycle and the response of the endometrium to the conceptus at the gene expression level can be used to (i) find genes and pathways related to uterine receptivity; (ii) identify biological processes, which are stimulated or suppressed in the endometrium by the conceptus; and (iii) to evaluate the ability of embryos to elicit physiological responses in the endometrium, e.g. with regard to different types of embryos such as IVF-derived or cloned embryos. Different mechanisms of establishment of pregnancy have evolved in various mammalian orders and families as a result of adaptations to environmental conditions leading to substantial differences between mammalian species in many aspects of reproductive biology (Bazer et al., 2009). Therefore, a comparative analysis of endometrial changes during the sexual cycle and the pre-implantation phase for different mammalian species provides a unique opportunity for identification of conserved and distinct pathways. Since most physiological processes are associated with complex changes in RNA expression profiles, transcriptome analyses are a powerful strategy for a holistic description of cellular changes at the molecular level. A number of analytical approaches, such as DNA microarrays and RNA sequencing (RNA-Seq) have been developed for systematic analyses of mammalian transcriptomes (Stanton, 2001; Hoheisel, 2006; Bauersachs et al., 2008; Wang et al., 2009). In comparison to DNA microarrays, RNA-Seq can provide information on absolute transcript levels, transcript variants, and currently not annotated transcribed regions. In general, next-generation sequencing will have an increasing impact for solving the complex biological problems in agricultural sciences (Liu, 2011) and animal sciences, e.g. for identification of yet un-annotated genes (Derrien et al., 2011; Jager et al., 2011). 1. Identification of biological themes related to endometrial remodeling and receptivity in transcriptome studies of bovine endometrium Endometrial gene expression is mainly regulated by the complex interplay of the ovarian steroid hormones estradiol (E2) and progesterone (P4) during the estrous cycle and by progesterone during pregnancy (Goff, 2004; Spencer et al., 2004; Forde et al., 2011b). These steroid hormones act via classical nuclear steroid hormone receptors, but also via non-classical receptors such as progesterone receptor membrane component 1 and the novel family of membrane progestin receptors (Gellersen et al., 2009). P4 is the key hormone for preparation of the endometrium for embryo implantation and maintenance of pregnancy (Bazer et al., 2008) and genes with increased expression levels in the luteal phase are probably regulated by P4, directly or indirectly. The supportive role of P4 has been confirmed in a recent study in heifers where a positive influence of P4 on conceptus growth and development was found (Clemente et al., 2009; Forde et al., 2011a). In ruminants, conceptus implantation is late (in cattle after day 18 of gestation) after trophoblast elongation, which starts on day 14 of pregnancy in cattle. An epitheliochorial placenta is formed through a relatively non-invasive placentation process. Only limited fusion of

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endometrial epithelial and trophoblast cells occurs in the caruncular and also in the intercaruncular areas of bovine endometrium (Wathes and Wooding, 1980; King et al., 1981). Maternal recognition of pregnancy in cattle takes place around days 16 and 17 and is mediated by conceptus interferon-tau (IFNT) that prevents induction of luteolysis (Bazer et al., 1997). IFNT has been shown to suppress estrogen receptor-alpha (ESR1) and oxytocin receptor (OXTR) genes, which results in prevention of pulsatile release of luteolytic prostaglandin F2 alpha (PGF2␣) (Spencer and Bazer, 1996). In cattle, maximum secretion of IFNT was observed on day 17 (Bazer et al., 1997) coincident with the time of maternal recognition of pregnancy. To gain deeper insight into the highly complex molecular responses of the endometrium to the ovarian hormones during the estrous cycle, several transcriptome studies of bovine endometrium using microarrays have been performed (Bauersachs et al., 2005, 2008; Mitko et al., 2008; Forde et al., 2011a). These studies revealed several hundred differentially expressed genes (DEG) between different stages of the estrous cycle. In the study of Mitko et al. (2008), two major groups of genes could be distinguished according to their expression profiles, with highest mRNA levels during the estrus phase and highest levels during the luteal phase, respectively. Genes with highest mRNA levels at estrus were characterized by overrepresented functional terms such as ‘focal adhesion formation’, ‘cell motility’, ‘cytoskeleton’, ‘extracellular matrix’ (ECM), ‘ECM remodeling’, and ‘cell growth’. Furthermore, a number of genes showing lowest mRNA levels at diestrus were identified that have been described in the context of ‘positive regulation of invasive growth’. Thus, decreased levels of these mRNAs during the luteal phase may be characteristic for the non-invasive implantation process in cattle. Accordingly, a number of genes with higher mRNA levels during the luteal phase have been described in the context of ‘negative regulation of invasive growth’. Genes assigned to the functional categories ‘angiogenesis’, ‘vascular remodeling’, and ‘regulation of blood flow’ could also play an important role for endometrial remodeling during the estrous cycle. Several genes described to be associated with these processes were identified as differentially expressed during the estrous cycle, including members of the angiopoietin family, transcription factors controlling the expression of vascular endothelial growth factors (VEGF) and their receptors, and other genes involved, e.g. in endothelial differentiation and regulation of blood flow. Furthermore, elevated concentrations of mRNAs coding for a variety of proteins involved in metabolic and transport processes were found during the luteal phase, which could be related to increased secretion of nutrients and factors necessary for the development of the embryo (Allison Gray et al., 2000). This was also found in the study of Forde et al. (2011a) where endometrial tissue samples collected at different time points during the luteal phase were analyzed. In this study the authors found hardly any differences between days 5 and 7 but high numbers of DEG between days 7 and 13. This study also investigated the influence of exogenous supplementation of P4 and induction of low P4 concentrations in comparison to temporal changes in the expression of genes in the endometrium for normal serum

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P4 profiles. P4 supplementation markedly advanced and low P4 concentrations mainly delayed temporal changes in endometrial gene expression. The analysis of interaction networks derived from the DEG between days 7 and 13 of the estrous cycle revealed associated functional terms such as ‘lipid metabolism’, ‘molecular transport’, ‘cell cycle’, ‘carbohydrate metabolism’, ‘embryonic development’ and ‘organ development’, ‘cellular development’, ‘cellular growth’ and ‘proliferation’, and ‘connective tissue development and function’ (Forde et al., 2011a). Another study, conducted by Salilew-Wondim et al. (2010), compared similar stages of cyclic endometrium (day 7 and day 14) using the same Affymetrix bovine microarray. However, the comparison of differentially expressed genes revealed only a very small overlap. In consideration of the different sampling techniques, endometrial samples collected after slaughter in Forde et al. (2011a) and cytobrush samples in Salilew-Wondim et al. (2010), this small overlap may be due to cell type-specific gene expression changes in the bovine endometrium during the estrous cycle. The cytobrush technique mainly yields cells from the luminal surface, i.e. luminal epithelium (LE). Since the LE accounts only for a small proportion of the endometrium, specific changes may get lost when analyzing entire endometrial samples. The cell type-specific differences are probably associated with the specific down-regulation of the progesterone receptor gene (PGR) in LE and superficial glands during the luteal phase (Spencer and Bazer, 1995). In general, these results indicate that one of the future tasks for gene expression studies will be to unravel these cell typespecific gene expression changes. Furthermore, the effects of E2 and P4 on endometrial gene expression profiles were analyzed in ovariectomized cows in a microarray study by Shimizu et al. (2010). Cluster analysis of the DEG revealed independent and interdependent actions of E2 and P4. A set of genes was identified whose regulation by E2 depends on a P4 priming effect. The P4-primed E2 response genes comprised gene functions like ‘migration’, ‘cell differentiation’ and ‘cell adhesion’, and therefore may play a crucial role in tissue remodeling during estrus. One of the potential key regulators of this process, transforming growth factor, beta 2 (TGFB2), was found among the P4-primed E2 response genes. Functional annotation analysis of the P4 responsive gene clusters revealed very diverse molecular functions and processes, similar to the studies during the estrous cycle, but also some important fertility-related terms like ‘luteinization’, ‘oocyte maturation’ and ‘catecholamine metabolism’. A search for putative transcriptional regulators of the steroid hormone response in bovine endometrium identified the transcription factors SP1, NFYA, FOXA2, IRF2, ESR1 and NOBOX as candidate regulators of gene expression. In recent years, a number of studies of bovine endometrium during the peri-implantation period have been performed to get more detailed insights into the gene expression changes in response to the presence of a conceptus. The first transcriptome analyses were performed on endometrial samples recovered from day 18 pregnant animals and corresponding non-pregnant controls. In the first study, monozygotic twin cows were used, where one twin received two in vitro-produced embryos and the

corresponding twin a sham transfer (Klein et al., 2006). In the second experimental model, pregnancy was obtained by artificial insemination of heifers and control animals received a sham insemination (Bauersachs et al., 2006). Data analysis revealed that almost half of the obtained genes have been described as classical type I interferonstimulated genes (ISG) and could be directly assigned to the effects of IFNT on the endometrium. In addition, many of the identified DEG have been assigned to biological processes and/or molecular functions related to the preparation of the endometrium for embryo attachment and implantation, such as functional terms ‘ECM remodeling’, ‘vascular remodeling’, ‘cell adhesion’, and ‘immunomodulation’. Most of the genes involved in modulation of the immune system are probably regulated by the embryonic IFNT. In a recent study (Bauersachs et al., 2011), a microarray analysis of bovine endometrial tissue samples collected on days 12, 15 and 18 of pregnancy and on the corresponding days of the estrous cycle was performed. This analysis revealed differential gene expression on days 15 and 18 of pregnancy, but not on day 12, in comparison to the corresponding cyclic controls, which is in line with other recently published studies (Walker et al., 2010; Forde et al., 2011c). Most of the genes differentially expressed on day 15 were also found as differentially expressed on days 16 and 18 of pregnancy. A comparison of the data sets of this study (Bauersachs et al., 2011) with results of other studies that investigated day 16 (Forde et al., 2011c), day 17 (Walker et al., 2010), and day 20 (Mansouri-Attia et al., 2009a) of pregnancy revealed substantial overlaps, particularly for the genes up-regulated in pregnant samples, although different microarray platforms and methods for data analysis were used (Bauersachs et al., 2011). The bioinformatics analysis of the known and inferred functions of the genes up-regulated on days 15 and 18 of pregnancy in comparison to cyclic controls revealed mainly overrepresented functional categories related to the response to IFNT, similar to the results of earlier studies (Bauersachs et al., 2008). Although the results for days 15 and 18 were very similar, the gene contents of these IFN response-related categories showed some distinct differences indicating that there are early and late IFN response genes as suggested previously by Forde et al. (2011c). For example, genes of the SOCS family members 1, 3, 4 and 6 were found as up-regulated only on day 18 of pregnancy. SOCS genes have been described as negative regulators of IFNT signaling in ovine endometrium (Sandra et al., 2005). Likewise, genes of the cathepsin family were identified as up-regulated only on day 18 of pregnancy. The analysis of the genes found as down-regulated on day 18 of pregnancy (higher levels in cyclic controls) revealed functional categories related to ‘ECM remodeling’ and ‘protein secretion’ which is in line with previous results of a study of bovine endometrium during the estrous cycle (Mitko et al., 2008) and indicates the switch to a new estrous cycle. The observed massive induction of ISG during the preimplantation phase is possibly the main problem for the identification of more specific gene expression changes that may be hidden. To subtract the typical ISG, data from days 15 and 18 of pregnancy were compared to the effects

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of intrauterine application of human IFNA2 (Bauersachs et al., 2011). The comparison of the DEG after application of IFNA2 with the DEG of days 15 and 18 of pregnancy revealed a very similar induction of typical ISG in bovine endometrium after treatment with human IFNA2. Moreover, a number of differences in gene expression changes compared to days 15 and 18 of pregnancy were observed. These included genes that were more strongly up- or downregulated after IFNA2 administration compared to days 15 and 18 of pregnancy as well as genes that were not regulated after IFNA2 administration but were regulated on days 15 and/or 18 of pregnancy. The latter genes are probably specifically repressed or induced by the presence of an embryo and/or by IFNT. Most of the genes that showed stronger up-regulation in response to IFNA2 compared to early pregnancy were related to different immune functions indicating an attenuated response of the maternal immune system in pregnant endometrium due to specific effects of IFNT and/or other conceptus-derived factors. In contrast, the genes found as differentially expressed only during pregnancy were very diverse regarding biological functions, except for a group of genes assigned to transport functions. The strongest up-regulation on days 15 and 18 of pregnancy but no significant change after IFNA2 treatment was observed for the fatty acid binding protein 3 (FABP3) and placenta-expressed transcript 1 protein (PLET1) genes. Specific expression of FABP3 (Bauersachs et al., 2011) and PLET1 (Mansouri-Attia et al., 2009a) in the luminal epithelium suggests a regulatory role in these cells. FABP3 exhibits a very interesting expression profile. Messenger RNA expression decreases from day 7 to day 13 during the cycle and during pregnancy but then increases from day 13 to day 16 of pregnancy (Forde et al., 2011a,c). Furthermore, a number of genes coding for transcription factors of the HOXB family (B4, B5, B8) were found with lower expression levels in pregnant endometrium. Six members of the HOXB family (B2, B4–B8) were also contained in the most significantly overrepresented functional group of the genes down-regulated on day 18 of pregnancy. HOXB genes have been described in context of hematopoietic development and differentiation (Magli et al., 1997) and, so far, regulation of these genes was not described in the endometrium. In context of the genes involved in differentiation of immune cells induced by application of IFNA2, the lower levels of these transcription factors in pregnant vs. cyclic endometrium could be part of a mechanism to prevent unwanted effects of IFN. In an approach to find fertility-related genes using a pathological model, endometria from day 18 of pregnancy were compared after transfer of SCNT embryos and transfer of embryos derived by in vitro-fertilization (IVF) (Bauersachs et al., 2009). This microarray study revealed 58 differentially abundant transcripts between SCNT and IVF pregnancies. For many of these genes an important role in implantation and/or placentation has been shown or at least suggested. The gene serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 14 (SERPINA14, also known as uterine milk protein or UTMP) showed lower expression in endometrium from SCNT pregnancies compared to IVF pregnancies, whereas upregulation in bovine endometrium on day 18 of pregnancy

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compared to corresponding cyclic controls was shown (Ulbrich et al., 2009). Studies of SERPINA14 function indicated a role in mediating immunosuppressive effects of P4 on the endometrium (Arck et al., 2007). The most interesting candidate of the identified genes was nuclear receptor subfamily 2, group F, member 2 (NR2F2, alias COUP-TFII), a nuclear orphan receptor, which has been identified in the mouse as essential for P4 control of implantation and as a mediator of uterine epithelial–stromal crosstalk (Kurihara et al., 2007; Petit et al., 2007). During the peri-implantation period, NR2F2 regulates embryo attachment and decidualization through controlling ESR1 (Fig. 1) activity but is also required for placentation in the post-implantation period (Lee et al., 2010). Heterozygous Nr2f2-mutant mice show decreased fecundity (Takamoto et al., 2005). Up-regulation of NR2F2 was also found in bovine endometrium on day 18 (Bauersachs et al., 2006) and in equine endometrium on day 12 of pregnancy compared to non-pregnant controls (Merkl et al., 2010), suggesting a conserved function of NR2F2 during establishment of pregnancy in mammalian species. NR2F2 is a target gene of Indian hedgehog (IHH) (Fig. 1), which is induced by P4 via the progesterone receptor (PGR) (Simon et al., 2009). NR2F2 also down-regulates expression of the gene for oxytocin receptor (OXTR) in a complex with NR2F6 (Chu and Zingg, 1997) and interacts with a variety of other transcriptional regulators (Fig. 1). Abnormalities in the endometrial transcriptome profile on day 20 of gestation in SCNT pregnancies as compared to pregnancies derived by IVF were also described in an independent study (Mansouri-Attia et al., 2009b). In addition, MansouriAttia et al. compared endometrial samples derived from SCNT pregnancies to pregnancies after artificial insemination (AI) (Mansouri-Attia et al., 2009b). Gene expression differences between endometrium samples from IVF and AI pregnancies were less pronounced, whereas the comparison of SCNT with AI revealed many biological functions and canonical pathways related to metabolism and immune function. The basic findings of these two studies were similar, although a direct comparison of the results is not meaningful due to distinct differences in the experimental design and the different microarray platforms (for details see (Bauersachs et al., 2009). Both studies revealed differential gene expression between SCNT pregnancies and pregnancies initiated with fertilized embryos (after AI or IVF). In general, the results indicated an endometrial plasticity at the onset of implantation and that deregulation of the maternal environment during this time can lead to disturbed placental development with corresponding effects on the development of the embryo/fetus and the success of pregnancy. microarray studies of bovine Furthermore, endometrium were performed in the context of disturbed endometrial receptivity, e.g. to identify genes associated with delayed uterine recovery and compromised fertility due to postpartum negative energy balance in dairy cows (Wathes et al., 2009). Another study in the context of uterine receptivity analyzed endometrial gene expression profiles one estrous cycle before embryo transfer in correlation with pregnancy success and identified a number of expression differences between endometria derived from heifers with successful pregnancy and

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Fig. 1. Interaction network for the essential transcription factor nuclear receptor subfamily 2, group F, member 2 (NR2F2). The interaction network was generated with Cytoscape (v. 2.8.1) based on known interactions for NR2F2 and the interactions between the obtained NR2F2 interaction partners. Interactors are shown with their official gene symbol. pp, protein–protein interaction (green solid line); pd, protein–DNA interaction (promoter-binding, red dashed line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

non-pregnant endometria (Salilew-Wondim et al., 2010). Such genes could be useful for prediction of the receptivity of pre-transfer endometrium in cattle. In summary, the analysis of gene expression changes during the estrous cycle and during early pregnancy revealed a plethora of differentially expressed genes. In addition to bioinformatics approaches to figure out the most important genes and pathways, the analysis of pathological models can help to identify genes critical for fertility. Furthermore, the comparison of changes in response to pregnancy with changes in response to intrauterine application of human IFNA2 as a classical type I IFN uncovered specific effects of the presence of a conceptus in addition to the induction of ISG. This led to the identification of genes which are likely to play a role in fine-tuned modulation of endometrial functions during early pregnancy. 2. Analysis of gene expression in porcine endometrium during the pre-implantation phase In swine, implantation starts after conceptus elongation on day 14 of gestation and placentation is noninvasive

(epitheliochorial), similar to ruminants (Carter and Enders, 2004). The embryonic signal for maternal recognition of pregnancy in pigs is completely different to ruminants and has been identified as estrogens produced by the conceptus mainly on days 11 and 12 of pregnancy (Geisert et al., 1990; Ziecik, 2002). This is accomplished by regulation of synthesis and secretion of PGF2␣ and PGE2, which exert opposing actions on the CL critical either for the initiation of luteolysis or maintenance of pregnancy. One of the supportive mechanisms by which conceptuses inhibit luteolysis is changing PG synthesis in favor of luteoprotective PGE2. Results of recent studies indicate that the E2 signal from the conceptuses stimulates endometrial PGE2 synthesis. Combined with a positive PGE2 feedback loop in the endometrium, increased synthesis of PGE2 leads to a rise in the PGE2:PGF2␣ ratio, which helps to overcome the luteolytic effect of PGF2␣ (Waclawik, 2011). During apposition and beginning of implantation an extensive tissue remodeling in the endometrium can be observed (Cencic et al., 2003) and a pronounced vascularization is evident already from day 13 of gestation (Keys et al., 1986). In pigs, as in other species, implantation and

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establishment of pregnancy is associated with expression of proinflammatory factors, including cytokines, growth factors, and lipid mediators. The conceptus produces, e.g. IFN gamma (IFNG) and IFN delta (IFND), interleukins IL1B and IL6, and PG, which regulate inflammatory pathways in the endometrium. Together with P4 these embryonic signals further enhance uterine receptivity (Waclawik, 2011). To characterize the processes associated with the initiation of placentation at the level of gene expression, a microarray study of porcine endometrium at day 14 of pregnancy in comparison to corresponding nonpregnant controls was performed (Østrup et al., 2010). This study identified 263 DEG between pregnant and nonpregnant sows. Bioinformatics analysis revealed a number of overrepresented Gene Ontology (GO) terms, most of them containing more up-regulated than down-regulated genes. These GO terms included: ‘developmental process’, ‘transporter activity’, ‘calcium ion binding’, ‘apoptosis’, ‘cell motility’, ‘enzyme linked receptor protein signaling pathway’, ‘positive regulation of cell proliferation’, ‘ion homeostasis’, and ‘hormone activity’. Only three overrepresented GO terms contained more down-regulated genes, namely ‘oxidoreductase activity’, ‘lipid metabolic process’, and ‘organic acid metabolic process’. Many of the genes assigned to these terms are known to be involved in steroid hormone and prostaglandin metabolism. Based on the genes assigned to ‘developmental process’ an interaction network was built (Østrup et al., 2010) that revealed together with a literature search the genes coding for interleukin 6 receptor (IL6R), leukemia inhibitory factor receptor alpha (LIFR), interleukin 11 receptor, alpha (IL11RA), mucin 4 (MUC4), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) (ERBB3), fibroblast growth factor 9 (glia-activating factor) (FGF9), and fibroblast growth factor receptor 3 (FGFR3) likely to be involved in the process of placentation (Østrup et al., 2010). In our group, porcine endometrium from days 12 and 14 of pregnancy was also analyzed by the use of RNA sequencing (RNA-Seq) (Samborski, 2011, unpublished results). A comparison of the RNA-Seq data set for day 14 with the results of the microarray study showed very good agreement, further supporting validity of the microarray results. The comparison of the RNA-Seq results obtained for days 12 and 14 was performed by analysis of the overlap of DEG (pregnant vs. non-pregnant) and by a gene set enrichment analysis (GSEA) (Subramanian et al., 2005). This identified only a moderate overlap between these two data sets, reflecting the different functions of the endometrium during these stages, i.e. recognition of pregnancy on day 12 and preparation for conceptus implantation on day 14. Since secretion of IFNG and IFND by the porcine conceptus has been described (Waclawik, 2011), a GSEA was performed to compare the expression data derived from porcine endometrium at day 14 of pregnancy with a gene set of genes induced by IFNA in bovine endometrium (Bauersachs et al., 2011). This analysis revealed a substantial overlap of IFN-stimulated genes with the genes up-regulated in porcine endometrium at day 14 of pregnancy, further supporting the suggested role of IFNs in preparation for

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conceptus attachment and implantation in pigs (Johnson et al., 2009). 3. Analysis of gene expression in equine endometrium during the pre-implantation phase The mechanisms of embryo–maternal communication and maternal recognition of pregnancy in equids are still not completely understood. A number of features of equine pregnancy are unique compared to other mammals. The equine conceptus is completely enveloped by a tough glycoprotein capsule between days 7 and 21, which prevents elongation of the trophoblast and provides its typical spherical shape. The conceptus shows constant, self-induced mobility throughout the uterine lumen between days 6 and 16 after ovulation. After day 16, the conceptus becomes immobilized (“fixed”) at the base of one of the uterine horns. Placentation in horses is non-invasive, except for a short invasive period at days 35–37, when an ‘injection’ of specialized, gonadotropin-secreting trophoblast cells into the maternal endometrium takes place that form the endometrial cups. This short invasive period is followed by the establishment of a stable, microvillous contact of trophoblast cells with the luminal epithelium of the endometrium around days 40–42 – the beginning of placentation in the horse (Allen, 2001; Allen and Wilsher, 2009). So far, the nature of the equine embryonic signal of pregnancy recognition to prevent luteolysis is still unknown. However, the presence of a conceptus uncouples the oxytocin-induced release of luteolytic PGF2␣ (Goff, 1987). The equine conceptus produces a number of different secretory products during early pregnancy, including steroids, PG, different proteins and peptides (Betteridge, 2000), such as IFND, a member of the type I IFN family (Cochet et al., 2009). A form of mechanotransduction by the migrating conceptus could also play a role in preventing production and release of PGF2␣, since the application of intrauterine devices has been shown to prolong the luteal phase in the mare (Rivera Del Alamo et al., 2008). Very recently, intrauterine administration of plant oils has been shown to prolong luteal life span probably by modulation of prostaglandin metabolism (Wilsher and Allen, 2011). Recently, two microarray studies of equine endometrium during early pregnancy have been performed. In our study, days 8 and 12 of pregnancy in comparison to non-pregnant cyclic controls were analyzed (Merkl et al., 2010). In the second study (Klein et al., 2010) day 13.5 of pregnancy was investigated. Both studies identified several hundred DEG at day 12 and at day 13.5 of pregnancy, respectively. GSEA, DAVID functional annotation clustering and co-citation (CoPub) analyses were performed to identify related data sets, overrepresented functional terms and biological pathways for the DEG in day 12 pregnant endometrium (Merkl et al., 2010). Overall, GSEA, DAVID and CoPub identified biologically very different gene sets that could reflect (i) differential gene expression in different compartments of the endometrium and (ii) a response to different embryonic signals. This corresponds to the secretion of different molecules by the equine conceptus (Betteridge, 2000). Two

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major biological processes revealed by the bioinformatics analysis were ‘angiogenesis/vascular remodeling’ and genes described in context of steroid hormone and PG signaling. GSEA revealed many estrogen-induced genes and genes involved in regulation of estrogen signaling, but also genes known to be regulated by P4 and PGE2. Likewise at day 13.5 many genes with known or inferred functions are probably up-regulated by embryonic estrogen. Among the genes showing lower expression in pregnant mares on day 13.5, ESR1 was of particular interest because of its potential involvement in the initiation of luteolysis in cyclic mares (Klein et al., 2010). In addition to the response to different signaling molecules, a mechanical signaling induced by the migrating conceptus as suggested by a recent study (Rivera Del Alamo et al., 2008) could be indicated by some of the genes identified at day 12 of pregnancy which have been described in the context of mechanosensation in other physiological processes. A third microarray study of equine endometrial tissue samples, comparing day 16 of pregnancy and day 12 of the estrous cycle revealed more than 1300 DEG (Merkl et al., 2010, unpublished results) using the same microarray (Agilent Horse microarray) and the same sample collection procedure (Merkl et al., 2010). Comparison of this data with that from day 12 of pregnancy showed a very similar response to the presence of a conceptus on days 12 and 16 of pregnancy but with more pronounced differences to non-pregnant endometrium for day 16 of pregnancy. Furthermore, a number of genes were found as differentially expressed only for the comparison of samples from day 16 of pregnancy with day 12 cyclic controls, e.g. ESR1 that was down-regulated on days 13.5 (Klein et al., 2010) and 16 of pregnancy but not on day 12. In accordance with expression of IFND genes in the equine conceptus at day 16 of pregnancy (Cochet et al., 2009), a number of IFNstimulated genes were found as up-regulated in day 16 pregnant endometrium. Altogether, these microarray studies of equine endometrium during early pregnancy revealed potential target genes and pathways of conceptus-derived estrogens, P4, and PGE2 in the equine endometrium probably involved in the early events of establishment and maintenance of pregnancy in the mare. The data analysis of the microarray studies of bovine, porcine and equine endometrium during early pregnancy each revealed some major themes and pathways probably related to establishment and maintenance of pregnancy. A schematic summary of selected processes and functional groups of genes and their possible relationship to pregnancy recognition and endometrial receptivity is shown in Fig. 2. 4. Comparison of gene expression data sets from different mammalian species The comparison of endometrial gene expression data sets between species of different mammalian groups can be used for the identification of genes related to common and species-specific mechanisms for establishment and maintenance of pregnancy. For example, the data sets for gene expression analysis during the estrous cycle and

day 18 of pregnancy in bovine endometrium were compared to the results of related microarray studies in human, mouse, and Rhesus monkey (Bauersachs et al., 2008). This analysis revealed an overlap of 70 genes, which were also differentially expressed in at least one of the other studies. This reflects some common regulatory mechanisms between mammalian species but also specific differences between ruminant species and primates and rodents. Differences in endometrial gene expression changes between different mammals are probably related to specific histological changes in the endometrium during the cycle and the type of implantation. However, similar gene expression changes were found, e.g. for claudin 4 (CLDN4), a cell adhesion molecule in tight junctions involved in intercellular sealing in simple and stratified epithelia (Tsukita and Furuse, 2002). Likewise, dickkopf homolog 1 (DKK1) mRNA, coding for an inhibitor of WNT signaling (Glinka et al., 1998), has been found as up-regulated in four human studies during the receptive phase and in bovine endometrium during the luteal phase and at day 18 of pregnancy (Bauersachs et al., 2008). Dkk1 is secreted by decidual cells in murine endometrium, induces trophoblast cell invasion and has an essential role in embryo implantation (Li et al., 2008). Correlative expression differences were also found in two human studies for nuclear protein 1 (NUPR1, candidate of metastasis 1, P8), solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1 (SLC1A1) and decidual protein induced by progesterone (C10orf10) and the immune-related genes complement component 1, r subcomponent (C1R), serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 (SERPING1), and transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) (TAP1). In our recent study of equine endometrium at day 12 of pregnancy (Merkl et al., 2010) GSEA was used to compare the whole set of expressed genes to related gene sets (sets of DEG) derived from other mammals. The best enrichment towards the day 12 up-regulated genes was found for genes up-regulated in human endometrium during the receptive phase, indicating similarities in gene expression changes in equine und human endometrium. Overlap with the day 12 up-regulated genes was also obtained for genes induced during early pregnancy in porcine and bovine endometrium and for genes regulated during the estrous cycle in bovine endometrium. A comparison of the overlapping genes of gene sets derived from different species revealed a number of genes that probably have conserved functions across species such as crystallin, alpha B (CRYAB), ERBB receptor feedback inhibitor 1 (ERRFI1), fibroblast growth factor 9 (FGF9), insulin-like growth factor binding protein 2 (IGFBP2), NR2F2, stanniocalcin 1 (STC1), and tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10). Another approach to find genes or pathways associated with fertility represents the systematic analysis of endometrial transcriptomes across different mammalian species that goes far beyond simple comparisons of the results (DEG) of different microarray studies. For several reasons, e.g. hybridization kinetics, nucleotide compositions of microarray probes, location of microarray probes within the target transcript, incomplete microarray designs, etc.,

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Fig. 2. Overview of processes and signaling pathways inferred from transcriptome studies and existing knowledge, which are related to pregnancy recognition and endometrial receptivity in cattle, swine, and horse. IFNT, interferon tau; IFNG, interferon gamma; IFND, interferon delta; E2, estrogen; P4, progesterone and its derivatives; PGs, prostaglandins; PGE2, prostaglandin E2; D14, D16, days 14 or 16 of pregnancy.

the use of microarrays for direct across-species analyses is limited. In contrast, analysis by RNA sequencing (RNA-Seq) could represent a way to perform a more direct comparison between different species. RNA-Seq yields more quantitative expression data than microarrays (Wang et al., 2009) and, therefore, could be used to compare expression of orthologous genes. Although the technical basis already exists, the major hurdle is incomplete gene annotation, particularly for the pig. Furthermore, the knowledge of orthologous genes between mammalian species is also far from complete but is needed for a reliable cross-species analysis to get a unique gene identifier for comparison of gene expression data. Since official gene symbols are still not available for all bovine, porcine and equine genes (only locus IDs), gene symbols cannot be used as unique identifier. One possibility could be to assign putative human orthologous genes and use human Entrez Gene identifiers as a unique identifier. This could be accomplished by crosswise BLAST analyses of all known transcripts to obtain one-to-one orthologous genes. On the basis of these orthologous genes, gene expression data could be compared more directly. However, this task is complicated by duplicated genes, highly similar genes in closely related members of a

gene family, and incomplete genome sequences or assemblies and incomplete annotation. In a preliminary analysis of RNA-Seq data for bovine endometrium from day 18, porcine endometrium from day 14 and equine endometrium from day 16 of pregnancy were compared (Bauersachs, 2011, unpublished results) to identify genes with similar expression changes (Fig. 3). This analysis revealed a relatively low number of genes with similar changes for all three species (Fig. 3a and Table 1). For the interpretation of this result it has to be considered that in the mare implantation occurs after day 42 in contrast to cattle and pigs where implantation begins at day 19 and at day 14, respectively. Accordingly, the number of genes with similar changes in bovine and porcine endometrium was much higher. In addition, a comparison between the three species was performed for known ISG (Fig. 3b). A substantial overlap was found between bovine and porcine endometrium, but up-regulation of ISG was also found in equine endometrium, which is in line with a previous report showing expression of delta IFNs in the equine conceptus at day 16 (Cochet et al., 2009). Although this analysis was based on a very preliminary assignment of the genes to a unique identifier (human Entrez Gene ID)

Fig. 3. Venn diagrams for all differentially expressed genes (a) and for known interferon-stimulated genes (b) in bovine (day 18), porcine (day 14) and equine (day 16) endometrium in comparison of pregnancy with the corresponding cyclic stage. Endometrial tissue samples were collected from respective stages of confirmed pregnancy and from corresponding days of the estrous cycle (in mares from day 12 of estrous cycle) and analyzed by RNA sequencing. Bta: Bos taurus; Ssc: Sus scrofa; Eca: Equus caballus.

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Table 1 Genes with similar expression changes in bovine, porcine and equine endometrium. Hsa Entrez gene ID

Hsa gene symbol

Hsa gene name

Bta D18

Ssc D14

Eca D16

134548 1136 148327 60681 3200 4131 4188 4487 23467 54894 162515

ANKRD43 CHRNA3 CREB3L4 FKBP10 HOXA3 MAP1B MDFI MSX1 NPTXR RNF43 SLC16A11

−1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1

−1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1

−1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1

201232

SLC16A13

−1

−1

−1

25803 716 718 721 1159 1364 129607 441168 2633 2634 84941 439996 3437 24138 3965 23515 4599

SPDEF C1S C3 C4B CKMT1B CLDN4 CMPK2 FAM26F GBP1 GBP2 HSH2D IFIT1B IFIT3 IFIT5 LGALS9 MORC3 MX1

−1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

−1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

−1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

4600 4642 8013 4939 158158 710 6564 6775 8743 85363 54346 11274 8838

MX2 MYO1D NR4A3 OAS2 RASEF SERPING1 SLC15A1 STAT4 TNFSF10 TRIM5 UNC93A USP18 WISP3

Ankyrin repeat domain 43 Cholinergic receptor, nicotinic, alpha 3 cAMP responsive element binding protein 3-like 4 FK506 binding protein 10, 65 kDa Homeobox A3 Microtubule-associated protein 1B MyoD family inhibitor msh homeobox 1 Neuronal pentraxin receptor Ring finger protein 43 Solute carrier family 16, member 11 (monocarboxylic acid transporter 11) Solute carrier family 16, member 13 (monocarboxylic acid transporter 13) SAM pointed domain containing ets transcription factor Complement component 1, s subcomponent Complement component 3 Complement component 4B (Chido blood group) Creatine kinase, mitochondrial 1B Claudin 4 Cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial Family with sequence similarity 26, member F Guanylate binding protein 1, interferon-inducible, 67 kDa Guanylate binding protein 2, interferon-inducible Hematopoietic SH2 domain containing Interferon-induced protein with tetratricopeptide repeats 1B Interferon-induced protein with tetratricopeptide repeats 3 Interferon-induced protein with tetratricopeptide repeats 5 Lectin, galactoside-binding, soluble, 9 MORC family CW-type zinc finger 3 Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) Myxovirus (influenza virus) resistance 2 (mouse) Myosin ID Nuclear receptor subfamily 4, group A, member 3 2 -5 -oligoadenylate synthetase 2, 69/71 kDa RAS and EF-hand domain containing Serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 Solute carrier family 15 (oligopeptide transporter), member 1 Signal transducer and activator of transcription 4 Tumor necrosis factor (ligand) superfamily, member 10 Tripartite motif-containing 5 unc-93 homolog A (C. elegans) Ubiquitin specific peptidase 18 WNT1 inducible signaling pathway protein 3

1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1

−1 lower expression, +1 higher expression in pregnant compared to cyclic endometrium.

it revealed a number of interesting genes (Table 1). The known and inferred functions of these genes highlight the importance of immune-related processes for preparation for implantation. In summary, transcriptome studies of bovine, porcine and equine endometrium revealed complex changes in gene expression during the estrous cycle and during early pregnancy. These studies suggested many new pathways and genes with important roles in the context of uterine receptivity. The comparison of the results between different species showed many species-specific gene expression changes, which is in line with different pregnancy recognition signals and differences in the process of embryo implantation. In addition, a number of conserved gene expression regulations were found that could reflect the existence of basic principles in mammalian reproduction during this pregnancy phase. The application of RNASeq for analysis of endometrial transcriptome changes

during early pregnancy could be the basis for performing a deeper comparison between mammalian species to identify key fertility-related genes in mammalian reproduction.

Conflict of interest statement The authors declare no competing financial or other conflicts of interest.

Acknowledgements Our studies are supported by the Deutsche Forschungsgemeinschaft (FOR478, FOR1029) and the German Ministry for Education and Research (BMBF; programs FUGATO and FUGATO-plus; projects FERTILINK and COMPENDIUM).

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