Transcriptomic changes in the bovine conceptus between the blastocyst stage and initiation of implantation

Animal Reproduction Science 134 (2012) 56–63

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Transcriptomic changes in the bovine conceptus between the blastocyst stage and initiation of implantation夽 Solomon Mamo a , Dimitrios Rizos b , Patrick Lonergan a,∗ a

School of Agriculture and Food Science, University College Dublin, Dublin 4, Ireland Departamento de Reproducción Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de la Coruna, KM 5.9, 28040 Madrid, Spain b

a r t i c l e

i n f o

Article history: Available online 11 August 2012 Keywords: Transcript RNA-sequencing Conceptus Crosstalk Maternal recognition Progesterone

a b s t r a c t Conceptus-maternal communication is vital for the successful establishment and maintenance of pregnancy, yet relatively little information exists for many of the mechanisms and the nature of the conceptus signals responsible for this cross-talk. Sub-optimal communication, resulting from impairment of conceptus development and/or from abnormal uterine receptivity, contributes to a high incidence of embryonic mortality. Therefore, detailed examination of the mechanisms regulating both pre- and peri-implantation conceptus development are necessary to fully understand the factors regulating successful post-hatching development, pregnancy recognition and implantation signaling. Despite significant progress in understanding of the temporal changes in the transcriptome of the uterine endometrium, there is only a rudimentary knowledge of the genes and pathways governing growth and development of the cattle conceptus. Furthermore, although there are a large number of studies describing gene expression profiles in bovine embryos focused mainly during the earlier preimplantation stages (up to and including Day 7), very little information exists for the post-hatching embryo and elongating conceptus. This period of development is arguably more important as a significant proportion of all embryonic loss occurs between Days 8 and 16 of pregnancy in cattle, corresponding to the time of hatching of the blastocyst from the zona pellucida and its subsequent elongation coincident with the time of maternal recognition of pregnancy. Given that this is a critical period in development leading up to maternal recognition and establishment of pregnancy, the identification of key genes and pathways regulating these crucial developmental events is essential. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Declining reproductive efficiency is a worldwide problem affecting, in particular, the dairy industry (Lucy, 2001; Pryce et al., 2004), which has a major impact on profitability in commercial herds. There is substantial evidence of an association between greater milk production and

夽 This paper is part of the special issue entitled: 3rd Embryo Genomics, Guest Edited by D. Tesfaye and K. Schellander. ∗ Corresponding author. E-mail address: [email protected] (P. Lonergan). 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.08.011

decreased reproductive efficiency, although the evidence for a direct causal relationship is less clear (LeBlanc, 2010). The physiological adaptations to the rapid progress in genetics, nutrition and management in the dairy industry, associated with greater milk production may explain part of the decrease in reproduction (Lucy, 2001). Most embryonic loss in cattle occurs very early in pregnancy, prior to maternal recognition of pregnancy, which occurs around Day 16 post conception (Diskin and Morris, 2008). Although the potential causes are multifactorial, and may be linked to various checkpoints along the developmental axis (Lonergan, 2009; Rizos et al., 2010), early embryo mortality is the major contributing factor to

S. Mamo et al. / Animal Reproduction Science 134 (2012) 56–63

the decline in reproductive efficiency (Diskin and Morris, 2008). Normal conceptus development leading to successful pregnancy establishment involves reciprocal communication between the maternal uterus and the developing conceptus (embryo/fetus and associated extraembryonic tissues; Spencer et al., 2008). Evidence for this comes from the fact that post-hatching conceptus elongation does not occur in vitro (Brandao et al., 2004; Vejlsted et al., 2006) and fails to occur in vivo in the absence of uterine glands (Gray et al., 2002; Spencer and Gray, 2006). These studies confirm the importance of the maternal environment for normal embryo development. However, the developing embryo also plays a key role in determining its own fate by eliciting the correct response from the maternal endometrium (Bauersachs et al., 2009; Mansouri-Attia et al., 2009). Given this background, detailed examination of the mechanisms regulating both pre- and peri-implantation conceptus development and uterine receptivity are necessary to fully understand the mechanisms regulating successful post-hatching development, pregnancy recognition and implantation signaling. A large number of studies have described the expression of candidate genes and global gene analysis in blastocysts of cattle at around Day 7 post conception. This is, in part, a reflection of the fact that such embryos are relatively easy to obtain in vivo (following superovulation, artificial insemination and nonsurgical uterine flushing) and can be obtained in very large quantities following in vitro embryo production using oocytes derived from the ovaries of slaughtered animals. However, data on gene expression during the post-hatching stages in cattle are very sparse and, apart from a few exceptions, typically deal with candidate genes rather than being global in nature (Ushizawa et al., 2004; Hue et al., 2007; Degrelle et al., 2011). A comprehensive survey of transcript abundance can provide novel and detailed insights into the genomic biology of an organism (Harhay et al., 2010). Although some concerns related to depth of sequencing and data analyses have been raised (Oshlack and Wakefield, 2009; Tarazona et al., 2011), compared to the efficiency of various earlier types of analysis, the latest development in transcript analysis using RNA-sequencing is a major revolution in the field (Wang et al., 2009) that overcomes some of the drawbacks of other techniques and enables the examination of various aspects of the genome. Recent developments in high throughput sequencing technologies allow for the investigation of transcriptomes at unprecedented resolution. RNA sequencing is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies and provides a far more precise measure transcript and their isoform amounts than other methods. Such an approach has the advantage over microarray technology of being unbiased (no prior knowledge required), allowing almost complete coverage (i.e., very high sensitivity) and true genome-wide discovery (Werner, 2010). 2. Embryo development in cattle up to implantation Following fertilization of the oocyte in the oviduct, the resulting embryo moves toward the uterus as it undergoes

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the first mitotic cleavage divisions. The embryo of cattle enters the uterus at about the 16-cell stage on approximately Day 4 of pregnancy. It subsequently forms a morula, at which stage the first cell-to-cell tight junctions are formed. By Day 7 the embryo has formed a blastocyst consisting of an inner cell mass (ICM), which after further differentiation gives rise to the embryo/fetus, and the trophectoderm, which ultimately contributes to the placenta. According to (Guillomot, 1995) the phases of implantation comprise (i) hatching of the blastocyst from the zona pellucida, (ii) pre-contact and blastocyst orientation, (iii) apposition, (iv) adhesion, and (v) endometrial invasion, the latter being very limited in ruminants. After hatching on Days 9–10, the spherical blastocyst begins to grow and change in morphology from a spherical to ovoid shape during a transitory phase preceding the elongation or outgrowth of the trophectoderm to a filamentous form that usually begins between Days 13 and 15 (Maddox-Hyttel et al., 2003). The ovoid conceptus is about 2 mm in length on Day 13 and then reaches a length of about 60 mm by Day 16. Elongated conceptuses (∼20 cm) can be readily recovered by standard uterine flushing techniques up to Day 19. After Day 19, the fully elongated conceptus begins implantation with firm apposition and attachment of the trophectoderm to endometrial lumenal epithelium (LE) and projections of the trophectoderm that extend into the openings of the uterine glands (Guillomot et al., 1981). Until the blastocyst stage of development is attained, the embryo is somewhat autonomous (i.e., does not need contact with the maternal reproductive tract) as evidenced by the fact that blastocysts can be successfully developed in vitro in large numbers using IVF technology and transferred to reproductive cycle stage similar synchronized recipients. In contrast, development of the post-hatching and pre-implantation conceptus is dependent on substances in the uterine lumen, termed histotroph, that are derived from the endometrium, particularly the uterine glands, for growth and development. This is evidenced by the fact that: (i) post-hatching elongation does not occur in vitro (Brandao et al., 2004; Alexopoulos et al., 2005) but does occur after transfer of in vitro produced blastocysts to the uterus (Clemente et al., 2010); and (ii) the absence of uterine glands in vivo results in a failure of blastocysts to elongate (Gray et al., 2002; Spencer and Gray, 2006). With regard to maternal influence, preparation of the uterine luminal epithelium for attachment of trophectoderm and implantation in all mammals, including ruminants, involves carefully orchestrated spatio-temporal alterations in gene expression within the endometrium. In both oestrous cyclic and pregnant animals, similar changes occur in endometrial gene expression up to initiation of conceptus elongation (approximately Day 13), suggesting that the default mechanism in the uterus is to prepare for, and expect, pregnancy (Forde et al., 2010b, 2011). Indeed, it is possible to transfer an embryo to a synchronous uterus 7 days after estrus and establish a pregnancy, as is routine in commercial cattle embryo transfer. It is only in association with maternal recognition of pregnancy, which occurs on approximately Day 16 in cattle, that significant changes in the transcriptomic profile are detectable between oestrous

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cyclic and pregnant endometrial tissue (Forde et al., 2011), when the endometrium responds to increasing interferontau (IFNT) secreted by the filamentous conceptus and there is down-regulation of receptors for progesterone (PGR) in the endometrial epithelia (Okumu et al., 2010). 3. Role of progesterone Progesterone, from the ovarian corpus luteum, acts on the endometrium to regulate gene expression by the uterus required for elongation of the conceptus. Elongation of the blastocyst to form a conceptus is critical for developmentally regulated production of IFNT, the maternal recognition of pregnancy signal (Roberts, 2007). As indicated above, this process only occurs in the uterus, as evidenced by the fact that hatched blastocysts fail to elongate in vitro. IFNT is a Type I IFN and signals pregnancy recognition to the mother by functioning at the endometrium to inhibit development of the luteolytic mechanism and permit continued production of progesterone by the corpus luteum (Bazer et al., 1996). The key factor regulating IFNT production by the trophoblast is the increase in size of the rapidly elongating conceptus, which involves trophectoderm cell proliferation, migration and adhesion (Guillomot, 1995). Indeed, there is a strong positive correlation between conceptus length and IFNT production (Clemente et al., 2011). Inadequate development of the conceptus results in less IFNT production and an inability to maintain the corpus luteum, resulting in a decrease in progesterone and early pregnancy loss (Thatcher et al., 2001). Significant progress has occurred in recent years in clarifying the role of the maternal environment, in particular the role of progesterone, in the successful establishment of pregnancy in cattle. It has been demonstrated that: • Significant changes occur in the endometrial transcriptome during both the oestrous cycle and early pregnancy in cattle (Forde et al., 2009, 2010b, 2011). Indeed, the single factor having the greatest effect on the endometrial transcriptome is the day the tissues are collected; i.e., temporal changes occur irrespective of pregnancy status as the uterus prepares for pregnancy and it is only around the time of maternal recognition (∼Day 16) that significant changes occur between pregnant and oestrous cyclic animals due to the expression of IFNT-induced genes. • Elevated progesterone results in advancement in the normal changes that occur over time in the endometrial transcriptome (Forde et al., 2009) and localization of the progesterone receptor (Okumu et al., 2010), the consequence of which is advancement in conceptus elongation (Carter et al., 2008) that is associated with greater embryonic survival. • Using a combination of in vitro embryo production and in vivo embryo transfer techniques, we showed that the effect of progesterone on conceptus development is mediated exclusively via the endometrium (Clemente et al., 2009). Most convincingly, the embryo does not need to be present in the uterus during the period of progesterone elevation to benefit from it, suggesting that the

effect of progesterone is via the endometrium and altered histotroph composition (Clemente et al., 2009). • Reducing circulating concentrations of progesterone results in an alteration of the endometrial transcriptome and retarded embryonic development (Beltman et al., 2009; Forde et al., 2010a). • The ability of the oviduct/uterus of the postpartum lactating dairy cow to support early embryonic development is impaired compared to that of the non lactating heifer and this is likely due to low concentrations of progesterone in blood and an inadequate luminal environment (Rizos et al., 2010), presumably as a consequence of a variety of factors including high milk production, associated metabolic imbalances, and uterine infection. Collectively, these results highlight the importance of an optimal uterine environment to support successful development of the conceptus. However, the role of the developing conceptus itself in eliciting appropriate temporal and spatial changes in the endometrial functions should not be underestimated. For example, two recent key papers provide strong evidence that the endometrium of the cow reacts differently depending on the type of embryo present (Bauersachs et al., 2009; Mansouri-Attia et al., 2009). Embryos of different quality (i.e., with divergent developmental fates), therefore, signal differently to the endometrium and in turn elicit a different response in terms of the endometrial transcriptome. In this way, the endometrium can be considered as a biological sensor that is able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process (Mansouri-Attia et al., 2009). 4. Histotroph composition and conceptus elongation Although much information is known about prehatching blastocyst development in cattle from in vitro systems (Lonergan, 2007), very little is known about peri-implantation conceptus growth and development, particularly in cattle. Available evidence supports an unequivocal role for endometrial secretions as primary regulators of conceptus survival, growth and development during pregnancy (Gray et al., 2002; Spencer and Gray, 2006). During the peri-implantation period of pregnancy, the conceptus is bathed in and supported by uterine secretions (Spencer et al., 2004). The epithelial cells of the uterine lumen exhibit high secretory activity during the luteal phase of the oestrous cycle and at the beginning of implantation (Guillomot et al., 1981). The trophectoderm is a site of intense pinocytotic activity which increases as the conceptus develops. Both the onset and rate of conceptus elongation are variable and must depend on uterine secretions, because normal elongation has not been achieved in vitro despite marked expansion of cultured spherical blastocysts (Brandao et al., 2004; Alexopoulos et al., 2005). Uterine lumenal fluid is a rather undefined complex mixture of proteins, amino acids, sugars, lipids and ions that are derived from genes expressed in the endometrium as well as selective transport of components from maternal blood

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primarily via the uterine epithelia. Proteins in histotroph of the cattle uterus are not well defined, but include enzymes, growth factors, cytokines, adhesion proteins, transport proteins, and selective proteins from the serum (Bazer et al., 2011; Groebner et al., 2011). The role of endometrial glands in pre-implantation conceptus survival and growth is strongly supported by studies of the ovine uterine gland knockout (UGKO) ewe model. UGKO ewes are made by continuous administration of a synthetic, non-metabolizable progestin to neonatal ewes, which permanently ablate differentiation and development of uterine GE from LE (Gray et al., 2002). UGKO ewes exhibit recurrent early pregnancy loss, and transfer of blastocysts from normal fertile ewes into uteri of UGKO ewes failed to overcome this defect (Gray et al., 2002). Morphologically normal spherical blastocysts can be found in uterine flushes of bred UGKO ewes on Days 6 and 9, but not on Day 14 post-mating when uterine flushes of mated UGKO ewes contain either no conceptus or a severely growth-retarded ovoid conceptus (Gray et al., 2002). The absence of specific components of histotroph derived from the endometrial glands is proposed to be the primary cause of recurrent pregnancy loss in UGKO ewes. Surprisingly, the biochemical and molecular aspects of conceptus/endometrial interactions and blastocyst growth and conceptus development during early pregnancy are not well defined in cattle. A variety of studies in cattle, including studies from our own group (Forde et al., 2009, 2010b), evaluated genes expressed in the endometrium of the uterus during selected stages of the oestrous cycle and pregnancy (Bauersachs et al., 2005, 2006, 2008; Mitko et al., 2008; Mansouri-Attia et al., 2009; Walker et al., 2011). Further, a comprehensive review of the scientific literature found that few genes, proteins and pathways have been discovered in the endometrium or uterine lumen that potentially regulate peri-implantation blastocyst growth in cattle (see (Spencer et al., 2008) for review). Factors discovered within the post-hatching and/or preimplantation blastocysts include fibroblast growth factors 1, 2 and 10, epidermal growth factor receptor, retinol binding protein, and a few cell cycle regulatory genes (Kliem et al., 1998; Cooke et al., 2009). Most of the published studies are incomplete with respect to effects of day of pregnancy, cell type(s) expressing the genes, presence of secreted factors in histotroph from the uterine lumen, and whether or not receptors for gene products are present in the conceptus trophectoderm. Even fewer studies have used functional assays to determine the biological role(s) of secreted factors from the uterus or other components of histotroph in the uterine lumen on bovine conceptus or trophectoderm growth and development. In the majority of the studies mentioned thus far, the embryo has played a passive role, essentially being used as a bio-assay of the health or quality of the uterine environment. Despite significant progress in understanding of the temporal changes in the transcriptome of the uterine endometrium, there is only a rudimentary knowledge of the genes and pathways governing growth and development of the conceptus in cattle. Specifically, the functional mechanisms through which maternally derived

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molecules regulate embryonic development are not known. 5. Post hatching gene expression in bovine conceptuses Gene expression in elongating and gastrulating ruminant embryos was comprehensively reviewed by Hue et al. (2007) and therefore the following text focuses predominantly on more recent data. Using a bovine utero-placental specific cDNA microarray, Ushizawa et al. (2004) analyzed the changes in transcript abundance in cattle embryos on Days 7, 14 and 21, extra-embryonic membranes on Day 28 and fetuses on Days 28. The expression of 680 genes was up-regulated and 26 genes down-regulated from Day 7 to Day 14 including those encoding for enzymes, transcriptional regulators, oncogenes, tumor suppression, cell cycle control and apoptosis. A total of 452 genes were significantly up-regulated from Day 14 through Day 21 with only two being downregulated. Earlier work (Degrelle et al., 2005) compared gene expression in ovoid, tubular and filamentous conceptuses and presented data supporting an important role of the ovoid stage during blastocyst growth and differentiation. The persistent expression of epiblast- (Oct-4. Sox2, Nanog) and endoderm-specific (Gata-6) genes, the detection of markers of proliferation (Opn, Nap1L1) and the expression of trophoblast-specific genes (Cdx2, Hand1, Ets-2, IFNT) at the ovoid stage suggests that this stage represent an essential transition in polar and mural trophoblast development. We have (Clemente et al., 2011) examined the temporal changes in transcriptional profile of the cattle embryo as it develops from a spherical blastocyst on Day 7 to an ovoid conceptus at the initiation of elongation on Day 13 and to highlight differences in these temporal gene expression dynamics between in vivo- and in vitro-derived blastocysts which may be associated with embryonic survival/mortality. The main findings of this study were that: (1) major temporal changes occur in the embryo transcriptome between the blastocyst stage on Day 7 and the initiation of conceptus elongation on Day 13; (2) these changes are related to the environment to which the embryo is exposed up to the blastocyst stage (i.e., in vivo compared to in vitro), with marked differences appearing in the transcriptome of the Day 13 embryo depending on its origin which are reflective of its subsequent developmental fate and (3) there is a panel of differentially expressed transcripts between Day 7 and Day 13 which are common to both in vivo- and in vitro-derived embryos and which are likely essential for initiation of elongation. Similarly, groups of genes were uniquely associated with in vivo derived embryos and therefore potentially preferentially associated with increased embryo survival, while others were uniquely associated with in vitro-derived embryos and therefore potentially preferentially associated with an increased likelihood of embryo mortality (see Fig. 1). Using RNA sequencing (Mamo et al., 2011), described the temporal changes in transcriptional profiles of the bovine conceptus at five key stages of pre- and periimplantation development from a spherical blastocyst on

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MYL7 LDLR WBP5 APOA2 ADH6 TPI1 UCHL1

DNASE1L3 TKDP5 TKDP1 DNASE1L3 LOC781283 PAG8 PDZK1 IRX3 TDGF1 ARHGDIB

PAG11 GDPD1 FETUB DNAJB1 VDAC1 CHPT1 SLC40A1

SUSD3 APOA1 TSPAN2 APOBEC3B HMOX1 NEDD 9 PHLDA1

LAMA1 TUBB2A FOXA2 ANXA13 PAGE4 HABP2

CYP26A1 JAM2 NOV TKDP4 TPMT SSLP-1 HSPA5 SLC25A24 GJA1 REEP1

ADAMTS1 TMEM64 ZFP36L1 CFI UBA5 HSD17B1 CALR

SEC24D MCM6 ZNF503 MOGAT1 CYR61 TNFRSF21

LOC518469 ARRDC4 NCOA1 PNMA2 VRK3 AKAP12 PPFIBP2

Day 7 vs Day 13 In Vivo

225

444

219

180

465

285

673

1341

668

Day 7 vs Day 13 In Vitro

PWWP2A LHFP TMEM206 CDC42EP3 LOC532798 FCHSD2 AHCYL2

CD48 KRT5 LGALS4 GNMT ZAP70 GARNL3 AIF1L ENPEP STEAP3 DHRS7

TRIB2 KLF13 LOC786530 POU5F1 MTHFD2L CSTB PDLIM4

TBL2 MGC143285 IL6ST Bv1 TESK2 TRIB2

EFHD1 SLC5A11 MATN4 LOC785905 CLDN10 XDH CKB TIAM1 PRUNE2 KCTD1

CKMT1 TPPP PCOLCE TIMP2 LOC616498 TMEM51 COL1A2

FOXP1 HS6ST1 LOC512397 CCDC28B KIT TIMP1

Fig. 1. Venn diagram illustrations of differentially expressed genes between Day 7 and 13 embryos derived in vivo or in vitro showing the top 40 up- and down regulated genes on Day 13 unique to in vivo embryos, unique to in vitro embryos, and common to both. Taken from Clemente et al. (2011).

Fig. 2. Venn diagram showing the transcript overlaps between different developmental stages. More than 18,500 transcripts were shared among the five conceptus development stages, although the levels of each transcript vary between the stages. Taken from Mamo et al. (2011).

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Fig. 3. Some representative heat map images of the nine clusters generated from the cattle conceptus RNA sequencing transcript data after ANOVA analysis followed by self organizing map (SOM). Each block represents a single cluster and was formed from 25 separate sequencing results, generated from five replicates per stage and five developmental stages (as described on the top of each cluster). Green color represents down regulation, red color represents up regulation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Day 7 through Days 10, 13, 16 and 19, covering the critical period of the formation of an ovoid conceptus, initiation of elongation, maternal recognition of pregnancy to a filamentous conceptus at the initiation of implantation on Day 19. Compared to previous studies, a large number of transcripts were discovered in each of these key developmental stages, including novel transcripts. The majority of these transcripts were commonly detected across the five development stages (Fig. 2). However, significant differences were observed in the abundance of each particular gene across the five different developmental stages. Further analysis of these stage-specific transcriptional changes may explain the accompanied morphological changes and reveal the relative importance of a particular gene cluster during a particular developmental stage. Generally, based on the abundance during each developmental stage, the most prevalent 20 genes that appeared in most stages include, various trophoblast kunitz domain proteins, pregnancy associated glycoproteins, cytoskeletal transcripts, heat shock proteins, calcium binding proteins, APOA1, AHSG, BOP1, TMSB10, CALR, APOE, TPT1, BSG, FETUB, MYL6, GNB2L1, PRDX1, PRF1, IFNT, and FTH1 (Table 1). ANOVA followed by Self Organizing Map analysis revealed nine gene clusters with distinct stage-specific expression profiles containing a large number of known and novel transcripts that may play key physiological roles during the various stages of conceptus development (Mamo et al., 2011) (Fig. 3). Most of the detected genes (both novel and the known ones) have not been previously characterized in the bovine conceptus; therefore, establishing their functions, as well as horizontal and vertical relationship during various stages of conceptus growth and development will provide insight into their respective roles. For more details, see the recent publication of Mamo et al. (2011).

Table 1 Twenty most prevalent up regulated genes at each developmental stage based on expression intensity as measured by RPKMa values. Day 7

Day 10

Day 13

Day 16

Day 19

HSP70 TMSB10 KRT18 TKDP4 KRT8 EIF5A PAG11 DNAJB1 CLIC1 S100A14 TPT1 CFL1 CALR GNB2L1 EF2 MYL6 BSG APOA1 PRF1 HMGA1

TMSB10 TKDP4 HSP70 KRT18 KRT8 APOA1 KRT19 HSPB1 TPT1 MYL6 PAG11 GNB2L1 S100A14 PRF1 EF2 BSG CLIC1 CALR EIF5A PRDX1

TMSB10 KRT18 HSP70 FTH1 KRT19 HSPB1 KRT8 APOA1 TPT1 HMGA1 S100A14 EIF5A CALR EF2 PKLR SLCA3 PRDX1 MYL6 TKDP4 PRF1

TKDP4 TMSB10 APOA1 KRT19 TKDP3 KRT8 HSPB1 AHSG FETUB KRT18 APOE TKDP2 CALR RBP4 BSG PRF1 FTH1 GNB2L1 PAG2 SPARC

TKDP4 TMSB10 APOA1 KRT19 KRT8 TKDP3 AHSG BOP1 HSPB1 CALR APOE TPT1 BSG FETUB MYL6 GNB2L1 PRDX1 PRF1 IFNT2 FTH1

a RPKM (reads per kilobase of exon per million mapped sequence reads) (Mortazavi et al., 2008) is calculated as follows: RPKM = Total exon reads . Mapped reads (millions) × Exon length (KB)

6. Conclusions Survival and growth of the mammalian conceptus is dependent on a good quality embryo and an appropriate uterine environment. During early pregnancy, the endometrium synthesizes and secretes, as well as selectively transports, a variety of substances collectively termed histotroph into the uterine lumen (Bazer et al., 1979). Uterine secretions are of particular importance for survival and growth of conceptuses in ruminants because

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of the protracted peri-implantation period and the superficial nature of implantation. As important as the uterine milieu, the quality of the embryo has a significant effect on the signals elicited from the endometrium (Bauersachs et al., 2009; Mansouri-Attia et al., 2009) which may or may not be conducive to embryo survival. An appropriate uterine environment, therefore, may not be sufficient to ensure conceptus survival if the quality of the embryo is intrinsically compromised (e.g., in the case of IVF or cloned embryos). We have generated a large Affymetrix microarray dataset characterizing the transcriptome of the uterine endometrium of cattle at key stages of the oestrous cycle and early pregnancy (Days 5, 7, 13 and 16), corresponding to 16-cell/early morula-stage, blastocyst stage, initiation of conceptus elongation, and advanced conceptus elongation and secretion of IFNT for maternal recognition of pregnancy, respectively (Forde et al., 2009, 2010a,b). Using the same array platform, we have generated lists of differentially expressed genes in the conceptus as it develops from a spherical blastocyst on Day 7 to the initiation of conceptus elongation on Day 13 (Clemente et al., 2010). Using RNA Seq technology, we have generated transcriptomic profiles of bovine conceptuses across the entire preand peri-implantation periods (Day 7, 10, 13, 16 and 19) and identified clusters of genes associated with blastocyst formation, conceptus elongation, maternal recognition of pregnancy and initiation of implantation (Mamo et al., 2011). Combined, these data represent an unparalleled resource for evaluation of transcriptional changes occurring in both the endometrium and the conceptus during the critical stages of early development leading up to maternal recognition of pregnancy and initiation of implantation. Conflict of interest None of the authors have any conflict of interest to declare. Acknowledgements The authors’ work is supported by Science Foundation Ireland (SM and PL: 07/SRC/B1156) and the Spanish Ministry of Science and Innovation (DR: AGL2009-11810). References Alexopoulos, N.I., Vajta, G., Maddox-Hyttel, P., French, A.J., Trounson, A.O., 2005. Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture. Reprod. Fertil. Dev. 17, 799–808. Bauersachs, S., Mitko, K., Ulbrich, S.E., Blum, H., Wolf, E., 2008. Transcriptome studies of bovine endometrium reveal molecular profiles characteristic for specific stages of estrous cycle and early pregnancy. Exp. Clin. Endocrinol. Diabetes 116, 371–384. Bauersachs, S., Ulbrich, S.E., Gross, K., Schmidt, S.E., Meyer, H.H., Einspanier, R., Wenigerkind, H., Vermehren, M., Blum, H., Sinowatz, F., Wolf, E., 2005. Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes. J. Mol. Endocrinol. 34, 889–908. Bauersachs, S., Ulbrich, S.E., Gross, K., Schmidt, S.E., Meyer, H.H., Wenigerkind, H., Vermehren, M., Sinowatz, F., Blum, H., Wolf, E., 2006. Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity. Reproduction 132, 319–331.

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