RNA Sequencing Reveals Novel Gene Clusters in Bovine Conceptuses Associated with Maternal Recognition of Pregnancy and Implantation

Successful establishment and maintenance of pregnancy can be attained only through optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. In particular, very little information exists for the posthatching embryo and elongating conceptus. This period of development is arguably the most important, as approximately 40% of all embryonic loss occurs between Days 8 and 17 of pregnancy in cattle. Here, we describe the global transcriptome profile of the bovine conceptus at five key stages of its preand peri-implantation growth (Days 7, 10, 13, 16, and 19) using state-of-the-art RNA sequencing techniques. More than 287 million reads were generated at the five stages, and more than 22 700 unique transcripts were detected. Analysis of variance followed by self-organizing maps identified differentially regulated (P , 0.05) genes organized in nine gene clusters forming a sequential transcript dynamics across these developmental stages. Of particular interest, genes in clusters 3 (n 1⁄4 236) and 6 (n1⁄4 1409) were significantly up-regulated on Days 16 and 19, suggesting a role in maternal recognition and initiation of implantation. This transcriptome analysis of the bovine conceptus will provide a blueprint of the dynamic changes in gene expression occurring during maternal recognition and implantation and will complement existing knowledge of the temporal changes in the endometrial transcriptome, thus facilitating a better understanding of conceptus-maternal cross talk during the peri-implantation period of pregnancy. blastocyst, bovine, conceptus, embryo, implantation, maternal recognition of pregnancy, RNA sequencing INTRODUCTION Conceptus-maternal communication is vital for the successful establishment and maintenance of pregnancy [1, 2], yet relatively little information exists for many of the mechanisms and the nature of the conceptus signals responsible for this cross talk. Suboptimal communication, resulting from impairment of conceptus (embryo and associated extraembryonic membranes) development and/or from abnormal uterine receptivity [3], contributes to a high incidence of embryonic mortality, which is a major cause of infertility in domestic animal species and in humans. For example, despite fertilization rates of . 90% for most ruminant species [4], only about 35% of dairy cows produce live offspring in response to a single insemination [5]. Similarly, in humans, the success rate per cycle after each assisted reproductive technology procedure is only about 25% [6]. Therefore, detailed examination of the mechanisms regulating both preand peri-implantation conceptus development and uterine receptivity are necessary to fully understand the mechanisms regulating successful posthatching development, pregnancy recognition, and implantation signaling. The majority of studies examining the molecular mechanisms of conceptus-endometrial interactions carried out during the peri-implantation period of pregnancy have focused on the maternal side, describing changes in the transcriptome of the endometrium [7, 8]. Recent studies [8, 9] from our group indicate that the major factor affecting gene expression in the endometrium is day of the estrous cycle/early pregnancy. In other words, regardless of pregnancy status, the temporal changes in gene expression in the endometrium are similar in pregnant and cyclic animals up to the time of maternal recognition of pregnancy when conceptus-derived factors, such as interferon-tau (IFNT), induces or further amplifies expression of a large number of genes in pregnant animals. Furthermore, two recent key articles provide strong evidence that the endometrium of the cow reacts differently to different types of conceptuses around the time of initiation of implantation (Day 18 [10] and Day 20 [11]). These studies demonstrated that embryos of different quality (i.e., with divergent developmental fates) signal differently to the endometrium and in turn elicit a different response in terms of the transcriptome of the endometrium. In this way, it was proposed that the endometrium be considered as a biological sensor that is able to fine-tune its physiology in response to the presence of conceptuses whose development becomes altered much later after the implantation process [11]. Recent comprehensive reviews [3, 12] have acknowledged the deficiency in our knowledge of the molecular mechanisms Supported by Science Foundation Ireland under grant nos. 02/IN1/B78 and 07/SRC/B1156 (the opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the Science Foundation Ireland. Correspondence: FAX: 353 1 6288421; e-mail: pat.lonergan@ucd.ie Current address: Department of Animal Science, Washington State University, Clark Hall, Pullman, WA 99164-6351. Received: 31 March 2011. First decision: 12 May 2011. Accepted: 18 July 2011. 2011 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363 1143 D ow nladed rom w w w .biolrod.org. Downloaded from https://academic.oup.com/biolreprod/article-abstract/85/6/1143/2530571 by guest on 28 July 2018 regulating conceptus development. Despite a number of recent studies [13, 14] describing gene expression profiles in bovine embryos (up to and including Day 7), very little information exists for the posthatching embryos and elongating conceptus [15, 16]. This period of development is arguably more important as approximately 40% of all embryonic loss occurs between Days 8 and 17 of pregnancy in cattle [4], 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 in cattle. 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 critical developmental events is essential. Recent developments in high-throughput sequencing technologies allow for the investigation of transcriptomes at unprecedented resolution. RNA sequencing (RNA-Seq) is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies and provides a far more precise measure of levels of transcripts and their isoforms than other methods [17]. 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 genomewide discovery [18]. Using this state-of-the-art technique, the aim of this study was to examine the temporal changes in transcriptional profiles of the bovine conceptus at five key stages of preand peri-implantation development from a spherical blastocyst on Day 7 through the critical period of maternal recognition to a filamentous conceptus at the initiation of implantation on Day 19. Here, we report results of the first bovine conceptus global transcriptome profiling during its critical window of preand peri-implantation growth and development to provide a comprehensive data set that will drive our understanding of the regulation of maternal recognition and establishment of pregnancy and pave the way for detailed functional analysis of candidate genes. MATERIALS AND METHODS All experimental procedures involving animals were licensed by the Department of Health and Children, Ireland, in accordance with the Cruelty to Animals Act (Ireland 1897) and the European Community Directive 86/609/EC and were sanctioned by the Animal Research Ethics Committee of University