DOI: 10.21451/1984-3143-AR2018-0038 Proceedings of the 10th International Ruminant Reproduction Symposium (IRRS 2018); Foz do Iguaçu, PR, Brazil, September 16th to 20th, 2018. Applications of large-scale molecular profiling techniques to the study of the corpus luteum Joy L. Pate*, Camilla K. Hughes Penn State University, Center for Reproductive Biology and Health, Department of Animal Science, University Park, PA 16802 USA. Abstract Introduction The corpus luteum (CL) is vital for the In the 1600’s, Regnier de Graaf described his establishment and maintenance of pregnancy. observation of transient yellow globules that form from Throughout the history of luteal biology, cutting-edge emptied ovarian follicles after coitus, noting that the technologies have been used to develop a thorough number of globules was the same as the number of understanding of the functions of specific luteal cell fetuses (Jocelyn and Setchell, 1972), and Marcello types, the signaling pathways that result in luteal cell Malpighi first called this structure a corpus luteum, latin stimulation or demise, and the molecules that regulate for yellow body. The function of the corpus luteum specific functions of luteal cells. The advent of large- (CL) remained a mystery for 300 years, when scale profiling technologies such as transcriptomics, experimental evidence was obtained that the CL was proteomics, and metabolomics, has brought with it an necessary for the maintenance of pregnancy (Simmer interest in discovering novel regulatory molecules that 1971; Frobenius 1999). This was followed by the may provide targets for manipulation of luteal function discovery of the primary secretory product of the CL, or lifespan. Although the work to date is limited, progesterone, in the 1930’s. Despite a slow beginning to transcriptomics have been effectively used to provide a the understanding of the function of the CL, once it was global picture of changes in mRNA that relate to luteal identified that this small structure was absolutely development, steroidogenesis, luteolysis or luteal essential for the establishment and maintenance of rescue. Some studies have been reported that profile pregnancy in all mammals, it captured the attention of microRNA (miRNA) and proteins, and although not yet reproductive biologists, and none more so than those published, metabolomics analyses of the CL have been interested in reproduction of domestic ruminants. Thus, in undertaken. Thus far, these profiling studies seem to the last 50 years, great advances have been made in largely confirm earlier findings using targeted understanding luteal function, much of which came from approaches, although previously unstudied molecules studies in cows and sheep. While the goal of this research have also come to light as important luteal regulators. was to enhance fertility of these species, the knowledge These molecules can then be studied using traditional of the basic biology of the CL could be generally applied mechanistic techniques. Use of profiling technologies to nonruminants, and because of its ephemeral nature, the has presented physiologists with unique challenges CL has served as a model for many aspects of cellular associated with analyses of big data sets. An appropriate biology, including angiogenesis, tumor development, technique for balancing the risks associated with type I steroidogenesis, roles of tissue-resident immune cells, (false discoveries) and type II (overlooking a real and pathways of cellular death. change) statistical error has not yet been developed and During the mid- to late 20th century, the many big data studies may have potentially important hormonal regulators, second messenger molecules and differences that are overlooked. Also, it is imperative biochemical reactions in steroidogenesis were that attempts be made to integrate information from the elucidated. Sources of cholesterol as substrate for various -omics studies before drawing conclusions progesterone synthesis and intracellular signaling based on expression of only one class of molecule, to pathways were defined. Refined procedures to separate better reflect the interdependency of molecular networks cells based on size led to a race to determine the origins in cells. Currently, few analysis programs exist for such integrations. Despite challenges associated with these and distinct functions of the small and large techniques, they have already provided new information steroidogenic cells, and the discovery that oxytocin is about the biology of the CL, notably allowing produced in the CL prompted a flurry of research to identification of a key regulator of acquisition of determine if luteal oxytocin is necessary for uterine luteolytic capacity and providing a big-picture view of prostaglandin (PG)F2A release during luteolysis. The the subtle changes that occur in the CL during early cellular heterogeneity that characterizes the CL also pregnancy. As these technologies become more accurate intrigued researchers, whose work revealed the and less expensive, and as analysis becomes more user- contributions of endothelial cells, fibroblasts, pericytes friendly, their use will become much more widespread and immune cells to development, function, and and many new discoveries will be made. This review regression of the CL. The advent of technologies for will focus only on relevant studies in which these identifying and quantifying steady state concentrations technologies were used to study the CL of ruminants. of mRNA in cells and tissue, including northern blotting, PCR, and qPCR, brought about a revolution in Keywords: bovine, corpus luteum, molecular profiling. targeted-approach experimentation to elucidate how _________________________________________ *Corresponding author: [email protected] Received: February 26, 2018 Copyright © The Author(s). Published by CBRA. Accepted: May 15, 2018 This is an Open Access article under the Creative Commons Attribution License (CC BY 4.0 license) Pate and Hughes. Molecular profiling to understand luteal function. changes in luteal functions are driven by changes in metabolism. The reported purity of the separated cell gene expression. For more information about these populations was similar in these two studies, so the clear discoveries and the general biology of the CL, the discrepancy between them may be due to the cell-type reader is referred to a number of reviews on ruminant comparisons made. Baddela et al. used days 11-12 CL luteal function (Niswender et al., 2000; Pate et al., (n = 4) from timed estrous cycles. The stage of the cycle 2012; Wiltbank et al., 2012; Miyamoto et al., 2013; from which CL (n = 3) were collected in the study of Smith and Meidan, 2014). Romereim et al. was not described. Because differentiation of the small and large cells is a somewhat Transcriptomic profiling in the corpus luteum continuous process, it is possible that functions associated with small and large cells are stage- Using the technologies mentioned above, dependent. Although it remains to be determined which studies of mRNA concentrations in the CL have been steroidogenic cell type is responsible for recruiting hypothesis-driven, searching for the key changes in immune cells, other differentially abundant mRNA and mRNA relative to receptor activation, signal predicted functions of small and large cells were fairly transduction, steroidogenesis, cytokine production, and consistent between the two studies. cell death pathways. Much has been learned about Differentiation and maximal steroidogenic which pathways and genes were regulated during capacity of the ruminant CL is dependent on luteinizing development and regression of the CL using this type of hormone. As might be expected, gonadotropic approach. However, more recently, researchers have stimulation of the CL resulted in upregulation of genes used high throughput technologies to profile many related to lipid metabolism, cholesterol metabolism and (microarray) or all (sequencing) of the transcripts progesterone production (Fatima et al., 2012). The most present in the CL from selected times or physiological upregulated mRNA was fatty acid binding protein 5 states. This approach was at first criticized as being a (FABP5), which can transport lipids within cells to lipid fishing expedition, but identification of potentially droplets and mitochondria. To our knowledge, this important molecules that led to new hypotheses about potentially important regulator of steroidogenesis has luteal regulation has enhanced acceptance of these not been studied in the CL. Transcriptomic analysis of powerful approaches. Transcriptomic analyses have day 4 and day 11 bovine CL also indicated that largely confirmed our understanding of luteal functions steroidogenic and cholesterol biosynthetic genes are as determined by more targeted approaches, lending upregulated in the midcycle CL, along with genes further support to previously drawn conclusions. involved in immune response, whereas the day 4 CL is Perhaps more importantly, they have also shed light on characterized by genes related to cell cycle, DNA unexplored or potentially new cellular pathways and replication and metabolic processes (Kfir et al., 2018). functions. This analysis also revealed that the developing CL Development of the ruminant CL involves expresses angiogenesis-promoting genes, whereas the differentiation of follicular steroidogenic cells and it mature CL expressed genes related to cessation of blood was suggested from cell-labeling studies that the small vessel