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Nutrigenomics: Feeding the genes for improved fertility
Animal Reproduction Science 96 (2006) 312–322
Nutrigenomics: Feeding the genes for improved fertility夽 Karl A. Dawson ∗ Alltech North American Bioscience Center, Nicholasville, KY 40356, United States Available online 3 August 2006
Abstract The post genomic era will result in many new molecular tools for evaluating the factors influencing fertility and reproductive performance in domestic livestock and poultry. There is currently considerable interest and practical merit in examining the regulatory steps involved in the process of gene transcription. Currently, oligo-based and cDNA microarray techniques make it possible to understand many of the factors controlling the regulation of gene transcription and globally evaluate gene expression profiles by looking at the relative abundance of gene-specific mRNA in tissues. These techniques provide an unprecedented amount of information and are only now being used to examine key reproductive, developmental, and performance characteristics in cattle. They also promise to provide a tremendous amount of new information that can be used to understand and diagnose key issues that limit reproductive performance. The science of nutrigenomics has begun to use information obtained from basic studies of the genome to evaluate the effects of diet and nutrient management schemes on gene expression. Preliminary studies have shown the value of such techniques and suggest that it will be possible to use specific gene expression patterns to evaluate the effects of nutrition on key metabolic processes relating to reproductive performance. While the effects of nutrition on fertility are only partially understood, modern nutrigenomics will undoubtedly play a key role in developing strategies for addressing some of the limitations in reproductive performance. © 2006 Elsevier B.V. All rights reserved. Keywords: Fertility; Microarray; Nutrigenomics; Transcription; Oxidative stress
1. Introduction The completion of the human genome project in 2003 marked the dawn of a new era in biology. This new era based on functional genomics is the direct result of the massive accumulation of 夽 This paper is part of the special issue entitled Nutrition and Fertility in Dairy Cattle, Guest Edited by A. Evans and F.J. Mulligan. ∗ Tel.: +1 859 885 9613. E-mail address: [email protected].
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.08.009
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information that is resulting from studies of a variety of animal, plant, and microbial genomes. Applications of this new information and the concepts developed from genomic studies promises to revolutionize the way agriculturalists, animal scientists, and nutritionists think about animal production. This new information and technology is particularly well suited for studies of the complex metabolic interactions that dictate fertility and influence reproductive efficiency. Applications of molecular technology will provide new ways to evaluate reproductive potential and the basic physiological mechanisms that limit reproductive performance. These technologies will also provide new tools for managing and monitoring livestock fertility. In this paper, the use of basic gene expression to characterize the effects of nutrition and nutritional status on tissue function, the potential for using gene expression profiles to relate the effects of nutrition to reproductive performance, and the evaluation of the interactions between nutrition and fertility at the most basic molecular level will be examined. 2. Characteristics of the bovine genome The bovine genome is composed of 29 autosomes and two sex chromosomes, and contains approximately three billion nucleic base pairs (Lewin, 2003). In addition, genetic information is carried in a mitochondrial genome that contains roughly 16,000 base pairs. The bovine genome is roughly the same size as that of the human and is also similar in size to that from other mammals. Mapping strategies have resulted in many complimentary markers that have helped define the structural nature of the genome, and sequencing studies have identified and cataloged well over 300,000 expressed sequence tags (ESTs) in GenBank. The Bovine Genome Project was initiated in 2003 and, through an international effort, resulted in the release of the first draft of the complete bovine genome sequence in 2004 (NIH, 2004). While more detailed mapping of the genome will take place over the next decade, much of the basic information about the structural nature of the bovine genetic code is now available. 3. Using genomic information Studies of the basic biochemistry behind genetics, the genetic structure, and the basic flow of information in biological systems have fostered the development of a multitude of new genomic-associated disciplines. These are generally based on some basic molecular tools that were developed to increase our knowledge of the basic molecular structure of life. The sciences that make up functional genomics include transcriptomics, proteomics, and metabolomics, which study the quantitative relationships between the genome and gene expression, protein production, and metabolic processes, respectively. At its very basic level, biology can be defined by a central dogma that describes flow of biochemical information from DNA to RNA and then to protein. As a result, the information contained in the nucleotide base sequence in DNA determines the basic amino acid sequences in protein and ultimately determines the structural and functional nature of the encoded proteins. All biological processes, including those that are associated with reproductive performance, are dependent on the regulation or control of the information flow in this pathway. While this process is carefully controlled by the basic genetic determinants, many external factors can also influence its regulation. Such factors include disease challenge, exposure to environmental toxins, and nutrient supply. The basic understanding of these complex regulatory processes has changed considerably with the delineation of the various animal, plant, and microbial genomes. It is now possible to understand these regulatory processes in extremely fine detail. One step in this pathway, the
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transcription of a gene sequence into mRNA, is currently being described by examining factors that influence the expression of specific genes and the transcription of their corresponding mRNA. This particular step is one of the first points in the regulatory process, which controls the flow of information from the base genes. The science of transcriptomics is based on the examination of gene expression patterns resulting from quantitative examination of the abundance of mRNA copied from a basic nucleic acid blueprint contained in the genome. 4. Transcriptomics and the use of microarrays for evaluating gene expression In the last 5 years, our knowledge of nucleic acid sequences, nucleic acid hybridization, and cloning techniques has provided tools that can be used to get a clearer understanding of overall gene expression at the transcriptional level. While techniques to study the expression of individual genes have been available for many years, oligonucleotide and cDNA microarrays have provided powerful tools that will allow rapid evaluation of gene expression on an unprecedented scale. These techniques are based on a quantitative assay of the relative concentrations of specific RNA messengers (mRNA) in tissue samples. The relative amount of individual mRNA molecules present in a given tissue or cell directly reflects the level of gene regulation and can be used to quantitatively examine the factors that regulate the gene expression. The amount of the mRNA transcript present in tissues can be measured indirectly after it is extracted and then used to create a complimentary labeled strand of DNA. This labeled material can be hybridized with a complimentary strand on an array containing a known set of gene sequences that are attached to a solid glass slide or nylon substrate. The sequences are often organized as an array of small spots on the solid matrix and are generally referred to as probes. The intensity of the color that results during the hybridization process is directly related to the amount of target mRNA present and reflects the level of gene expression. In this way, it is possible to determine which gene is up-regulated or down-regulated as a result of specific biological manipulations or during normal tissue development. Comparison studies of gene regulation can be carried out using subtractive hybridization procedures that use contrasting color labels on complimentary DNA from two sets of messengers from different tissues (Moody, 2001). As a result, it is possible to quantitatively compare gene expression in two contrasting groups of tissue or animals. By using robotic techniques for producing arrays on a minute scale and laser techniques to discern the color of specific spots, it is possible to examine the expression of thousands of genes at one time. This is an extremely powerful tool that can be used to study metabolic processes at a very basic level and lends itself well to the complex understanding of interactions that regulate gene function. Since gene transcription is only one step in the regulatory pathway that leads to functional protein formation, it is not always possible to correlate the increased presence of mRNA in the tissue with phenotypic or protein changes in tissues (Moody, 2001; Muller and Kersten, 2003). While studies of gene transcription may have many drawbacks in this respect, the ability to globally evaluate the initial regulatory steps in gene expression provides many tools for elucidating the key processes in metabolic regulation. Powerful screening methods are now available to identify the key gene expression patterns that are influenced by environment, disease, and nutrition or simply during the process of tissue development. In the past, microarray studies have depended on specific arrays with relatively few nucleotides and limited amounts of information. These arrays were often generated to examine specific metabolic functions or immune responses. Recent work has reported the development of arrays that can be used to examine gene expression in a variety of species. These arrays range in size from a few hundred probes to systems that have over 40,000 elements. While the use of smaller,
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more defined arrays to examine regulation of specific tissue response have been useful, the development of standardized systems for examining the expression of large numbers of genes will greatly enhance our ability to understand basic metabolic and physiological functions. 5. Microarrays as a tool for studying biological processes in ruminants Transcriptional profiling of gene expression using microarrays promises to bring a new understanding to ruminant physiology. From a practical point of view, gene expression studies will allow for the identification of pathways and candidate genes responsible for economically important traits. Two basic approaches have been used to evaluate gene expression in ruminant tissues. The first uses arrays developed for studies in other animals (humans, rats, and mice). Such arrays are typically well characterized and annotated. As a result, it is easy to quickly relate the expression of specific genes with gene function. The approach is based on the assumption that there is sufficient cross-species hybridization to gain detailed insight into specific gene expression patterns (Moody et al., 2002). The second approach uses arrays that are specifically designed for bovine studies. At this point, many of the more complex arrays for ruminants are based on expressed sequence tags (ESTs) that have not been associated with specific genes or gene functions. While these arrays can be used to compare and differentiate gene expression patterns, the lack of well annotated probes limits their immediate usefulness in identifying specific functional changes or physiological processes. Numerous bovine-specific arrays have been developed and have been used to look at changes in gene expression in bovine tissue (Table 1). These have included the rather small, but very focused, 167 element arrays, like those used by Tao et al. (2004), to specifically examine the immuno-endrocrine interface to the larger 18,000 cDNA element high-density arrays produced by the Bovine Functional Genome Consortium (Suchyta et al., 2003b). In the last year, a more advanced high-density bovine microarray has been released (GeneChip® Bovine Genome Array, Affymetrix, Santa Clara, CA). This array is designed to monitor the expression of approximately 23,000 transcripts. This tool is expected to be extremely powerful and promises to help standardize gene expression studies in the future, but is still limited by a lack of understanding of gene annotations. Table 1 Some cDNA and oligo microarrays being used to examine gene expression in cattle Array
Source
Description
Reference
3300 Microarray
University of Illinois
Band et al. (2002)
NBFGC Microarray
National Bovine Functional Genomics Consortium Michigan State University
3800 genes from bovine spleen and placenta 18,263 transcripts from various bovine tissues 1056 transcripts from bovine leukocytes 167 specific sequences associated with immune and endocrine function 6887 elements largely from cDNA libraries of challenged epithelial and leukocyte cells 7872 select spleen and placenta cDNA sequences
BOTL-3 Microarray Immune-Endocrine Microarray
University of Guelph
Bovine Innate Immune Microarray
CSIRO Livestock Industries
cDNA Microarray
University of Illinois
Suchyta et al. (2003a) Madsen et al. (2004) Tao et al. (2004)
Donaldson et al. (2005)
Everts et al. (2005)
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Table 2 Some uses of oligo-based and cDNA microarrays to study gene expression in cattle Topic
Reference
Evaluating gene expression patterns associated with pathogenic microorganisms, disease challenge, and immune function
Burton et al. (2001), Coussens et al. (2003), Tao et al. (2004), Donaldson et al. (2005), Madsen et al. (2004), Jones et al. (2004), Davies et al. (2006), Klene et al. (2006), Marsh et al. (2006); Patel et al. (2006) Band et al. (2002)
Evaluating differential gene expression patterns in various body tissues Evaluating gene expression patterns and factors associated with milk performance, muscle development, and meat quality Examining effects of pregnancy on the expression of genes and animal physiology Examining expressed gene patterns that are important in follicular development and embryo development
Reverter et al. (2003), Lehnert et al. (2004), Byrne et al. (2005), Lehnert et al. (2006) Ishiwata et al. (2003), Madsen et al. (2004), Herath et al. (2004), Loor et al. (2005), Davies et al. (2006) Yao et al. (2004), Ushizawa et al. (2004), Corcoran et al. (2006), Klein et al. (2006)
There is still a limited amount of work using the global approaches in transcriptomics to evaluate gene expression and regulation in cattle (Table 2). Many of these studies have focused on evaluating the effects of immunological challenge and disease processes (Burton et al., 2001; Coussens et al., 2003; Tao et al., 2004; Donaldson et al., 2005; Chitko-McKown et al., 2004; Marsh et al., 2006; Patel et al., 2006). This work is still largely of a descriptive nature and has focused on the validation of gene expression and microarray techniques as tools for understanding the basic response of tissue to disease and toxin challenge. Several studies with microarrays in cattle have focused on characterization of gene expression during the development of embryos, during pregnancy, and during the periparturient periods (Ishiwata et al., 2003; Yao et al., 2004; Madsen et al., 2004; Ushizawa et al., 2004; Herath et al., 2004; Corcoran et al., 2006; Klein et al., 2006; Davies et al., 2006). These types of studies are important in our understanding of fertility, since it is during these critical reproductive periods that embryo survival and changes in physiological function are closely associated. It is still very early in these lines of investigation, as most of the recent work has been done only in a descriptive way. The basic approaches have simply begun to catalog the gene expression changes associated with embryo implantation and development. Ushizawa et al. (2004) have presented the results of some initial studies of gene expression during the development of the bovine embryo. This work is suggesting a number of candidate biomarkers that can be used to follow the key changes in the embryo during development. These gene expression studies have characterized some interesting specific gene expression changes that may provide clues about important physiological and developmental processes, but, to date, no universal gene markers that can be specifically associated with fertility or embryo survival have been elucidated. While a number of studies have examined the effects of pregnancy, embryo implantation, oocyte development, and follicular development on gene expression, most of these studies only emphasize the complexity of the gene expression patterns associated with these processes. There is a tremendous need for computational tools that can be used to extract the key information from the more complex gene expression profiles. These will be key to future interpretation of such complex processes and to development of the appropriate applications for this information.
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6. Nutritional genomics in ruminants Dietary manipulations and nutritional strategies are key tools for influencing ruminant production. It has long been known that reproductive performance and fertility in dairy cattle is influenced as much by nutrition as by genetic predisposition (Butler, 1998). This is particularly important during the transition period and early lactation, when the animal is particularly sensitive to nutritional imbalances. Nutrition has also become a greater concern as a consequence of animals being selected for greater milk production (Butler and Smith, 1989). Despite the importance of this issue in dairy production systems, methods for clearly elucidating the basic molecular mechanisms that explain these influences have been lacking. Nutrigenomics and nutritional genomics are sciences that examine the effects of nutrition on gene expression (Muller and Kersten, 2003; Swanson et al., 2003). These sciences are providing new tools that can be used to more clearly understand how nutritional management can be applied to address disease, performance, and fertility limitation in cattle. Gene expression studies are expected to revolutionize the methods for examining and elucidating the major limitations on fertility and help define nutritional strategies for addressing them. Nutrigenomic studies in beef and dairy cattle are still rare, but they are becoming important as we develop an understanding of the relationship between nutrition, genetics, fertility, and tissue growth (Table 2). In recent years, the goals of these studies have been to describe and catalog the effects of diet on changes in gene expression or regulatory processes that may be associated with normal biological processes and tissue development. Descriptive work by Reverter et al. (2003) has explored the basic statistical approaches that can be used to characterize and interpret the effects of diets on gene expression in muscles from steers fed diets of varying quality. Differential gene expression in these studies clearly show the value of using large microarrays to demonstrate the effects of diet on transcriptional regulation, even in the absence of information on specific annotated gene sequences. In studies of steers under nutritional restriction due to intake of poor quality feeds, expression of specific genes associated with protein turnover, cytoskeletal remodeling, and metabolic homeostasis was clearly influenced by diet (Byrne et al., 2005). Many of these changes in expression could be predicted from observed changes in animal growth and physiology during normal nutrient restriction. However, many changes seen at the gene expression levels are much more subtle. For example, the enhanced expression transcription activators and up-regulation of proteins associated with detoxification mechanisms in animals receiving nutrient restricted diets would not necessarily be predicted based on our current understanding of nutrition. Such work clearly suggests that nutrigenomic approaches will soon provide new and possibly more sensitive markers of nutritional status. Jones et al. (2004) have used microarray techniques and a commercially available rat microarray to examine the effects of endophyte-infected tall fescue on gene expression in luteal tissue of heifers. In these studies, specific differential expression of genes associated with neural functions, transport function, cell cycle regulation, and programmed cell death were all demonstrated in animals fed endophyte-free and endophyte-infected forages. This study clearly demonstrated that the expected toxin-induced changes in autonomic neuronal function were reflected in gene expression patterns at the transcriptional level and validated the use of expression profiling as a tool for demonstrating the effects of dietary management schemes at the molecular level. 7. Effects of selenium on gene expression At this point, there is little or no published information relating dietary-induced changes in gene expression to specific gene functions associated with reproductive performance in cattle or
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any other species. While it is still not possible to directly demonstrate the effects of nutritional strategies or diets on the expression of genes related to fertility in either male or female animals, it may be possible to begin to understand the importance of the relationship between individual nutrients and the regulation of gene expression. A good example is the recent work that has examined the effects of dietary selenium on gene expression in mice (Rao et al., 2001). In a study using a 12,000-gene mouse microarray, it has clearly been established that selenium deficiencies can influence the patterns of protein synthesis in mice by regulating the expression of specific genes at the transcriptional level. Genes that were up-regulated by selenium deficiencies in mice included those associated with stress responses, cell cycling and growth, and cell adherence (Rao et al., 2001). At the same time, genes that were down-regulated included those associated with detoxification mechanisms, selenoprotein production, oxidative stress protection, and lipid transport. It is important to note that these changes in gene expression can be used to account for many of the outward phenotypic characteristics of selenium deficiencies. The database used for studying selenium effects has been recently further enhanced by studies evaluating the effects of several selenium sources, including sodium selenite and selenium yeast (Sel-Plex® ), on gene expression in the intestinal tract of mice using a basic 23,000-element murine microarray (Table 3). This study showed dramatic changes in gene expression in intestinal tissues, with the expression of over 2500 genes being influenced by selenium supplementation, and at least 100 of these can be directly or indirectly associated with reproductive functions. While the Table 3 Examples of some fertility-associated gene expression patterns that are changed by supplementation of mouse diets with sodium selenite, selenomethionine, or selenium yeast (Sel-Plex® )a Gene
Protein
Relative increase in gene expression with dietary inclusion ofb Selenomethionine
Sodium selenite
Selenium yeast (Sel-Plex® )
Dio1
Iodothyronine deiodinase, type I
2.0-fold
2.8-fold
2.1-fold
Gpx1
Glutathione peroxidase 1
4.9-fold
4.1-fold
4.7-fold
Gpx3
Glutathione peroxidase 3 Thioredoxin 2
4.6-fold
3.5-fold
3.6-fold
NS
1.2-fold
1.4-fold
Thioredoxin reductase 1
1.8-fold
1.7-fold
1.8-fold
Txn2
Txnrd1
Functional role
Modulates metabolic activities during implantation and embryonic development Key enzymes in antioxidant systems protecting reproductive systems and embryos
Key electron carrier controlling implantation and embryo development Key enzyme supporting antioxidant activities, and controlling embryo development
a Data from an unpublished gene expression study of intestinal tissue from mice fed a low selenium (0.01 mgSe/kg) diet, a sodium selenite supplemented diet (1.0 mgSe/kg), a selenomethionine (1.0 mg/kg) or a selenium yeast (Sel-Plex® ) diet (1.0 mgSe/kg) (Weindruch et al., 2005, personal communication). b Changes in gene expression are provided as significant (p < 0.05) fold changes relative to the expression patterns observed in a selenium deficient control group. NS indicates a non-significant (p > 0.05) change in gene expression relative to the selenium-deficient control.
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direct effects of dietary selenium on gene expression in key reproductive tissue have yet to be examined, data from these studies can be used to identify candidate genes or biomarkers that are clearly regulated by various dietary forms of selenium in some tissues (Table 3). There are at least three examples that are worth examining. The thyroid hormone, triiodothyronine (T3 ) has many functions that can be related to reproductive performance. One of its major roles is to trigger gene transcription, which is important in the developing embryos during growth (Goodridge, 1986). As a result, it plays a critical role in embryo survival and the growth of young animals (Edens and Gowdy, 2004; Incerpi et al., 2005). The importance of this hormone in fertility has been established in studies of ovarian responses in cows with induced hypothyroidism (Bernal et al., 1999). In many animals, the formation of the active form of this hormone is accomplished through the action of the selenium-dependent enzyme, type 1 5 deiodinase, which deiodinates the relatively inactive thyroxin (T4 ) to deiodothyronine (T3 ), the most active form of thyroid hormone. The delay in the conversion of T4 to T3 has been associated with increased embryonic mortality in poultry (Christensen, 1985) and can be expected to have the same effects in other species where strict metabolic regulation of energy metabolism is needed for proper maintenance and development of embryos. While it would be expected that synthesis of this selenium-containing enzyme would be influenced by the presence of selenium, the recent gene expression studies with mice (Table 3) have established that changes in enzyme activities can be regulated at the level of gene expression, and are not simply the influenced by the amount of selenium needed to form a functional protein following transcription. Oxidative stress on reproductive tissue and during embryo development is believed to be a major determinant affecting reproductive efficiency (Bilodeau et al., 2000) and has been suggested to be a leading cause of male infertility (Shalini and Bansal, 2005). Many of the proteins associated with antioxidant systems are known to be influenced by both the level and form of selenium in the diet (Edens and Gowdy, 2004). The use of microarray technology in mice has allowed for identification of several oxidative stress-associated genes that are readily influenced by dietary supplementation with selenium (Table 3). The selenoproteins, glutathione peroxidase 1 and glutathione peroxidase 3, were both up-regulated by the use of selenium yeast and sodium selenite in the diets of mice. These are key proteins functioning as antioxidants in many tissues and would be expected to have a major impact on the reproductive tissue (Naziroglu and Gur, 2000), sperm quality (Bilodeau et al., 2000), and embryo development (Baek et al., 2005). The nutrigenomic approach has confirmed transcriptional regulation of these proteins in intestinal tissue and will provide the basis for designing studies to better understand the influence of dietary selenium in reproductive tissue. Another set of proteins that was readily influenced by selenium supplementation are those associated with the thioredoxin electron carrier systems (Table 3). Thioredoxin is an electron carrier protein involved in many aspects of cell cycling and in maintaining antioxidant systems. The thioredoxin system has been identified as a key player in cellular redox-mediated reactions (Miranda-Vizuete et al., 2004; Beckett and Arthur, 2005). The regulation of the thioredoxin system is believed to be key in differentiation and morphogenesis in embryonic tissue (Matsui et al., 1996) and can influence early embryo viability and maturation (Clarke, 1992). Thioredoxin mediates estradiol effects on antioxidant systems, which influences embryo survivability and implantation, as well as fertility in pigs (Deroo et al., 2004). While at least one component of the thioredxoin system, thioredoxin reductase, is a selenium-containing enzyme which would be regulated by selenium availability, thioredoxin itself is not a selenoprotein. The regulation of this component of this critical thioredoxin system by dietary selenium at the transcriptional level is unexpected
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(Table 3) and may suggest new approaches to understanding the role of this system in reproductive tissues and developing embryos. The search for other specific fertility-associated genes that may be readily influenced by nutrition is just beginning, but is currently limited by the lack of information about the effects of specific nutrients on transcription. From the selenium example in mice, it is clear that use of nutrigenomic tools in fertility studies can lead to the identification of key marker genes that will help predict and establish the effects of diet on fertility. Once identified, it is not hard to see how these approaches and the ever-increasing transcriptional information will be useful for formulating customized diets to specifically address fertility issues and improve reproductive performance. 8. Conclusions Discoveries made in the next decade using powerful molecular tools like microarray analysis will undoubtedly revolutionize our basic understanding of cattle physiology and help define new methods for managing animal nutrition and reproduction. In the long term, it is likely that transcriptional profiles will be able to provide a number of very basic tools for evaluating the nutritional and physiological status of individual animals or groups of animals. Such profiles could also be used to provide a detailed analysis of the reproductive status and help formulate nutritional strategies that could be implemented to address fertility problems. As a result, it will no longer be necessary to look at extreme nutritional treatments, induced deficiencies, or long-term production responses to understand the basic effects of diets. However, it can easily be seen from these types of gene expression studies that nutritional/gene interactions are extremely complex. When this is coupled with the differences in gene expression associated with the various critical tissues of the body and the potential for changes associated with age and developmental patterns, it is clear that the true challenges in the future will be in modeling and interpreting the massive amounts of information obtained from simple nutritional studies and dietary interventions. References Baek, I.J., Yon, J.M., Lee, B.J., Yun, Y.W., Yu, W.J., Hong, J.T., Ahn, B., Kim, Y.B., Kim, D.J., Kang, J.K., Nam, S.Y., 2005. Expression pattern of cytosolic glutathione peroxidase (cGPx) mRNA during mouse embryogenesis. Anat. Embryol. (Berl). 209, 315–321. Band, M.R., Olmstead, C., Everts, R.E., Liu, Z.L., Lewin, H.A., 2002. A 3800 gene microarray for cattle functional genomics comparison of gene expression from spleen, placenta and brain. Anim. Biotechnol. 13, 163–172. Beckett, G.J., Arthur, J.R., 2005. Selenium and endocrine systems. J. Endocrinol. 184, 455–465. Bernal, A., DeMoraes, G.V., Thrift, T.A., Willard, C.C., Randel, R.D., 1999. Effects of induced hypothyroidism on ovarian response to superovulation in Brahman (Bos indicus) cows. J. Anim. Sci. 77, 2749–2756. Bilodeau, J.F., Chatterjee, S., Sirard, M.A., Gagnon, C., 2000. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol. Reprod. Dev. 55, 282–288. Burton, J.L., Madsen, S.A., Yao, J., Sipkovsky, S.S., Coussens, P.M., 2001. An immunogenomics approach to understanding periparturient immunosuppression and mastitis susceptibility in dairy cattle. Acta Vet. Scand. 42, 407–424. Butler, W.R., 1998. Review: effect of protein nutrition on ovarian and uterine physiology in dairy cattle. J. Dairy Sci. 81, 2533–2539. Butler, W.R., Smith, R.D., 1989. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 72, 767–783. Byrne, K.A., Wang, Y.H., Lehnert, S.A., Harper, G.S., McWilliam, S.M., Bruce, H.L., Reverter, A., 2005. Gene expression profiling of muscle tissue in Brahman steers during nutritional restriction. J. Anim. Sci. 83, 1–12. Clarke, F.M., 1992. Identification of molecules and mechanisms involved in the ‘early pregnancy factor’ system. Reprod. Fertil. Dev. 4, 423–433.
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Development and application of a bovine cDNA microarray for expression profiling of muscle and adipose tissue. Aust. J. Exp. Agric. 44, 1127–1133. Lehnert, S.A., Wang, Y.H., Tan, S.H., Reverter, A., 2006. Gene expression-based approaches to beef quality research. Aust. J. Exp. Agric. 46, 165–172. Lewin, H.A., 2003. The future of cattle genomics: the beef is here. Cytogen. Gen. Res. 102, 10–15. Loor, J.J., Dann, H.M., Everts, R.E., Oliveira, R., Green, C.A., Janovick Gruretzky, N.A., Rodriguez-Zas, S.L., Lewin, H.A., Drackley, J.K., 2005. Temporal gene expression profiling of liver from perparturient dairy cows reveals complex adaptive mechanisms in hepatic function. Physiol. Genomics 23, 217–226. Madsen, S.A., Chang, L.C., Hickey, M.C., Rosa, G.J.M., Coussens, P.M., Burton, J.L., 2004. Microarray analysis of gene expression in blood neutrophils of parturient cows. Physiol. Genomics 16, 212–221. Matsui, M., Oshima, M., Oshima, H., Takaku, K., Maruyama, T., Yodoi, J., Taketo, M.M., 1996. Early embryonic lethality caused by targeted disruption of the mouse thioredoxin gene. Dev. Biol. 178, 179–185. Marsh, I.B., Bannantine, J.P., Paustian, M.L., Tizard, M.L., Kapur, V., Whittington, R.J., 2006. Genomic comparison of Mycobacterium avium subsp. Paratuberculosis sheep and cattle strains by microarray hybridization. J. Bacteriol. 188, 2290–2293. Miranda-Vizuete, A., Sadek, C.M., Jimenez, A., Krause, W.J., Sutovsky, P., Oko, R., 2004. The mammalian testis-specific thioredoxin system. Antioxid. Redox. Signal. 6, 25–40.
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Nutrigenomics: Feeding the genes for improved fertility夽 Karl A. Dawson ∗ Alltech North American Bioscience Center, Nicholasville, KY 40356, United States Available online 3 August 2006
Abstract The post genomic era will result in many new molecular tools for evaluating the factors influencing fertility and reproductive performance in domestic livestock and poultry. There is currently considerable interest and practical merit in examining the regulatory steps involved in the process of gene transcription. Currently, oligo-based and cDNA microarray techniques make it possible to understand many of the factors controlling the regulation of gene transcription and globally evaluate gene expression profiles by looking at the relative abundance of gene-specific mRNA in tissues. These techniques provide an unprecedented amount of information and are only now being used to examine key reproductive, developmental, and performance characteristics in cattle. They also promise to provide a tremendous amount of new information that can be used to understand and diagnose key issues that limit reproductive performance. The science of nutrigenomics has begun to use information obtained from basic studies of the genome to evaluate the effects of diet and nutrient management schemes on gene expression. Preliminary studies have shown the value of such techniques and suggest that it will be possible to use specific gene expression patterns to evaluate the effects of nutrition on key metabolic processes relating to reproductive performance. While the effects of nutrition on fertility are only partially understood, modern nutrigenomics will undoubtedly play a key role in developing strategies for addressing some of the limitations in reproductive performance. © 2006 Elsevier B.V. All rights reserved. Keywords: Fertility; Microarray; Nutrigenomics; Transcription; Oxidative stress
1. Introduction The completion of the human genome project in 2003 marked the dawn of a new era in biology. This new era based on functional genomics is the direct result of the massive accumulation of 夽 This paper is part of the special issue entitled Nutrition and Fertility in Dairy Cattle, Guest Edited by A. Evans and F.J. Mulligan. ∗ Tel.: +1 859 885 9613. E-mail address: [email protected].
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.08.009
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information that is resulting from studies of a variety of animal, plant, and microbial genomes. Applications of this new information and the concepts developed from genomic studies promises to revolutionize the way agriculturalists, animal scientists, and nutritionists think about animal production. This new information and technology is particularly well suited for studies of the complex metabolic interactions that dictate fertility and influence reproductive efficiency. Applications of molecular technology will provide new ways to evaluate reproductive potential and the basic physiological mechanisms that limit reproductive performance. These technologies will also provide new tools for managing and monitoring livestock fertility. In this paper, the use of basic gene expression to characterize the effects of nutrition and nutritional status on tissue function, the potential for using gene expression profiles to relate the effects of nutrition to reproductive performance, and the evaluation of the interactions between nutrition and fertility at the most basic molecular level will be examined. 2. Characteristics of the bovine genome The bovine genome is composed of 29 autosomes and two sex chromosomes, and contains approximately three billion nucleic base pairs (Lewin, 2003). In addition, genetic information is carried in a mitochondrial genome that contains roughly 16,000 base pairs. The bovine genome is roughly the same size as that of the human and is also similar in size to that from other mammals. Mapping strategies have resulted in many complimentary markers that have helped define the structural nature of the genome, and sequencing studies have identified and cataloged well over 300,000 expressed sequence tags (ESTs) in GenBank. The Bovine Genome Project was initiated in 2003 and, through an international effort, resulted in the release of the first draft of the complete bovine genome sequence in 2004 (NIH, 2004). While more detailed mapping of the genome will take place over the next decade, much of the basic information about the structural nature of the bovine genetic code is now available. 3. Using genomic information Studies of the basic biochemistry behind genetics, the genetic structure, and the basic flow of information in biological systems have fostered the development of a multitude of new genomic-associated disciplines. These are generally based on some basic molecular tools that were developed to increase our knowledge of the basic molecular structure of life. The sciences that make up functional genomics include transcriptomics, proteomics, and metabolomics, which study the quantitative relationships between the genome and gene expression, protein production, and metabolic processes, respectively. At its very basic level, biology can be defined by a central dogma that describes flow of biochemical information from DNA to RNA and then to protein. As a result, the information contained in the nucleotide base sequence in DNA determines the basic amino acid sequences in protein and ultimately determines the structural and functional nature of the encoded proteins. All biological processes, including those that are associated with reproductive performance, are dependent on the regulation or control of the information flow in this pathway. While this process is carefully controlled by the basic genetic determinants, many external factors can also influence its regulation. Such factors include disease challenge, exposure to environmental toxins, and nutrient supply. The basic understanding of these complex regulatory processes has changed considerably with the delineation of the various animal, plant, and microbial genomes. It is now possible to understand these regulatory processes in extremely fine detail. One step in this pathway, the
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transcription of a gene sequence into mRNA, is currently being described by examining factors that influence the expression of specific genes and the transcription of their corresponding mRNA. This particular step is one of the first points in the regulatory process, which controls the flow of information from the base genes. The science of transcriptomics is based on the examination of gene expression patterns resulting from quantitative examination of the abundance of mRNA copied from a basic nucleic acid blueprint contained in the genome. 4. Transcriptomics and the use of microarrays for evaluating gene expression In the last 5 years, our knowledge of nucleic acid sequences, nucleic acid hybridization, and cloning techniques has provided tools that can be used to get a clearer understanding of overall gene expression at the transcriptional level. While techniques to study the expression of individual genes have been available for many years, oligonucleotide and cDNA microarrays have provided powerful tools that will allow rapid evaluation of gene expression on an unprecedented scale. These techniques are based on a quantitative assay of the relative concentrations of specific RNA messengers (mRNA) in tissue samples. The relative amount of individual mRNA molecules present in a given tissue or cell directly reflects the level of gene regulation and can be used to quantitatively examine the factors that regulate the gene expression. The amount of the mRNA transcript present in tissues can be measured indirectly after it is extracted and then used to create a complimentary labeled strand of DNA. This labeled material can be hybridized with a complimentary strand on an array containing a known set of gene sequences that are attached to a solid glass slide or nylon substrate. The sequences are often organized as an array of small spots on the solid matrix and are generally referred to as probes. The intensity of the color that results during the hybridization process is directly related to the amount of target mRNA present and reflects the level of gene expression. In this way, it is possible to determine which gene is up-regulated or down-regulated as a result of specific biological manipulations or during normal tissue development. Comparison studies of gene regulation can be carried out using subtractive hybridization procedures that use contrasting color labels on complimentary DNA from two sets of messengers from different tissues (Moody, 2001). As a result, it is possible to quantitatively compare gene expression in two contrasting groups of tissue or animals. By using robotic techniques for producing arrays on a minute scale and laser techniques to discern the color of specific spots, it is possible to examine the expression of thousands of genes at one time. This is an extremely powerful tool that can be used to study metabolic processes at a very basic level and lends itself well to the complex understanding of interactions that regulate gene function. Since gene transcription is only one step in the regulatory pathway that leads to functional protein formation, it is not always possible to correlate the increased presence of mRNA in the tissue with phenotypic or protein changes in tissues (Moody, 2001; Muller and Kersten, 2003). While studies of gene transcription may have many drawbacks in this respect, the ability to globally evaluate the initial regulatory steps in gene expression provides many tools for elucidating the key processes in metabolic regulation. Powerful screening methods are now available to identify the key gene expression patterns that are influenced by environment, disease, and nutrition or simply during the process of tissue development. In the past, microarray studies have depended on specific arrays with relatively few nucleotides and limited amounts of information. These arrays were often generated to examine specific metabolic functions or immune responses. Recent work has reported the development of arrays that can be used to examine gene expression in a variety of species. These arrays range in size from a few hundred probes to systems that have over 40,000 elements. While the use of smaller,
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more defined arrays to examine regulation of specific tissue response have been useful, the development of standardized systems for examining the expression of large numbers of genes will greatly enhance our ability to understand basic metabolic and physiological functions. 5. Microarrays as a tool for studying biological processes in ruminants Transcriptional profiling of gene expression using microarrays promises to bring a new understanding to ruminant physiology. From a practical point of view, gene expression studies will allow for the identification of pathways and candidate genes responsible for economically important traits. Two basic approaches have been used to evaluate gene expression in ruminant tissues. The first uses arrays developed for studies in other animals (humans, rats, and mice). Such arrays are typically well characterized and annotated. As a result, it is easy to quickly relate the expression of specific genes with gene function. The approach is based on the assumption that there is sufficient cross-species hybridization to gain detailed insight into specific gene expression patterns (Moody et al., 2002). The second approach uses arrays that are specifically designed for bovine studies. At this point, many of the more complex arrays for ruminants are based on expressed sequence tags (ESTs) that have not been associated with specific genes or gene functions. While these arrays can be used to compare and differentiate gene expression patterns, the lack of well annotated probes limits their immediate usefulness in identifying specific functional changes or physiological processes. Numerous bovine-specific arrays have been developed and have been used to look at changes in gene expression in bovine tissue (Table 1). These have included the rather small, but very focused, 167 element arrays, like those used by Tao et al. (2004), to specifically examine the immuno-endrocrine interface to the larger 18,000 cDNA element high-density arrays produced by the Bovine Functional Genome Consortium (Suchyta et al., 2003b). In the last year, a more advanced high-density bovine microarray has been released (GeneChip® Bovine Genome Array, Affymetrix, Santa Clara, CA). This array is designed to monitor the expression of approximately 23,000 transcripts. This tool is expected to be extremely powerful and promises to help standardize gene expression studies in the future, but is still limited by a lack of understanding of gene annotations. Table 1 Some cDNA and oligo microarrays being used to examine gene expression in cattle Array
Source
Description
Reference
3300 Microarray
University of Illinois
Band et al. (2002)
NBFGC Microarray
National Bovine Functional Genomics Consortium Michigan State University
3800 genes from bovine spleen and placenta 18,263 transcripts from various bovine tissues 1056 transcripts from bovine leukocytes 167 specific sequences associated with immune and endocrine function 6887 elements largely from cDNA libraries of challenged epithelial and leukocyte cells 7872 select spleen and placenta cDNA sequences
BOTL-3 Microarray Immune-Endocrine Microarray
University of Guelph
Bovine Innate Immune Microarray
CSIRO Livestock Industries
cDNA Microarray
University of Illinois
Suchyta et al. (2003a) Madsen et al. (2004) Tao et al. (2004)
Donaldson et al. (2005)
Everts et al. (2005)
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Table 2 Some uses of oligo-based and cDNA microarrays to study gene expression in cattle Topic
Reference
Evaluating gene expression patterns associated with pathogenic microorganisms, disease challenge, and immune function
Burton et al. (2001), Coussens et al. (2003), Tao et al. (2004), Donaldson et al. (2005), Madsen et al. (2004), Jones et al. (2004), Davies et al. (2006), Klene et al. (2006), Marsh et al. (2006); Patel et al. (2006) Band et al. (2002)
Evaluating differential gene expression patterns in various body tissues Evaluating gene expression patterns and factors associated with milk performance, muscle development, and meat quality Examining effects of pregnancy on the expression of genes and animal physiology Examining expressed gene patterns that are important in follicular development and embryo development
Reverter et al. (2003), Lehnert et al. (2004), Byrne et al. (2005), Lehnert et al. (2006) Ishiwata et al. (2003), Madsen et al. (2004), Herath et al. (2004), Loor et al. (2005), Davies et al. (2006) Yao et al. (2004), Ushizawa et al. (2004), Corcoran et al. (2006), Klein et al. (2006)
There is still a limited amount of work using the global approaches in transcriptomics to evaluate gene expression and regulation in cattle (Table 2). Many of these studies have focused on evaluating the effects of immunological challenge and disease processes (Burton et al., 2001; Coussens et al., 2003; Tao et al., 2004; Donaldson et al., 2005; Chitko-McKown et al., 2004; Marsh et al., 2006; Patel et al., 2006). This work is still largely of a descriptive nature and has focused on the validation of gene expression and microarray techniques as tools for understanding the basic response of tissue to disease and toxin challenge. Several studies with microarrays in cattle have focused on characterization of gene expression during the development of embryos, during pregnancy, and during the periparturient periods (Ishiwata et al., 2003; Yao et al., 2004; Madsen et al., 2004; Ushizawa et al., 2004; Herath et al., 2004; Corcoran et al., 2006; Klein et al., 2006; Davies et al., 2006). These types of studies are important in our understanding of fertility, since it is during these critical reproductive periods that embryo survival and changes in physiological function are closely associated. It is still very early in these lines of investigation, as most of the recent work has been done only in a descriptive way. The basic approaches have simply begun to catalog the gene expression changes associated with embryo implantation and development. Ushizawa et al. (2004) have presented the results of some initial studies of gene expression during the development of the bovine embryo. This work is suggesting a number of candidate biomarkers that can be used to follow the key changes in the embryo during development. These gene expression studies have characterized some interesting specific gene expression changes that may provide clues about important physiological and developmental processes, but, to date, no universal gene markers that can be specifically associated with fertility or embryo survival have been elucidated. While a number of studies have examined the effects of pregnancy, embryo implantation, oocyte development, and follicular development on gene expression, most of these studies only emphasize the complexity of the gene expression patterns associated with these processes. There is a tremendous need for computational tools that can be used to extract the key information from the more complex gene expression profiles. These will be key to future interpretation of such complex processes and to development of the appropriate applications for this information.
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6. Nutritional genomics in ruminants Dietary manipulations and nutritional strategies are key tools for influencing ruminant production. It has long been known that reproductive performance and fertility in dairy cattle is influenced as much by nutrition as by genetic predisposition (Butler, 1998). This is particularly important during the transition period and early lactation, when the animal is particularly sensitive to nutritional imbalances. Nutrition has also become a greater concern as a consequence of animals being selected for greater milk production (Butler and Smith, 1989). Despite the importance of this issue in dairy production systems, methods for clearly elucidating the basic molecular mechanisms that explain these influences have been lacking. Nutrigenomics and nutritional genomics are sciences that examine the effects of nutrition on gene expression (Muller and Kersten, 2003; Swanson et al., 2003). These sciences are providing new tools that can be used to more clearly understand how nutritional management can be applied to address disease, performance, and fertility limitation in cattle. Gene expression studies are expected to revolutionize the methods for examining and elucidating the major limitations on fertility and help define nutritional strategies for addressing them. Nutrigenomic studies in beef and dairy cattle are still rare, but they are becoming important as we develop an understanding of the relationship between nutrition, genetics, fertility, and tissue growth (Table 2). In recent years, the goals of these studies have been to describe and catalog the effects of diet on changes in gene expression or regulatory processes that may be associated with normal biological processes and tissue development. Descriptive work by Reverter et al. (2003) has explored the basic statistical approaches that can be used to characterize and interpret the effects of diets on gene expression in muscles from steers fed diets of varying quality. Differential gene expression in these studies clearly show the value of using large microarrays to demonstrate the effects of diet on transcriptional regulation, even in the absence of information on specific annotated gene sequences. In studies of steers under nutritional restriction due to intake of poor quality feeds, expression of specific genes associated with protein turnover, cytoskeletal remodeling, and metabolic homeostasis was clearly influenced by diet (Byrne et al., 2005). Many of these changes in expression could be predicted from observed changes in animal growth and physiology during normal nutrient restriction. However, many changes seen at the gene expression levels are much more subtle. For example, the enhanced expression transcription activators and up-regulation of proteins associated with detoxification mechanisms in animals receiving nutrient restricted diets would not necessarily be predicted based on our current understanding of nutrition. Such work clearly suggests that nutrigenomic approaches will soon provide new and possibly more sensitive markers of nutritional status. Jones et al. (2004) have used microarray techniques and a commercially available rat microarray to examine the effects of endophyte-infected tall fescue on gene expression in luteal tissue of heifers. In these studies, specific differential expression of genes associated with neural functions, transport function, cell cycle regulation, and programmed cell death were all demonstrated in animals fed endophyte-free and endophyte-infected forages. This study clearly demonstrated that the expected toxin-induced changes in autonomic neuronal function were reflected in gene expression patterns at the transcriptional level and validated the use of expression profiling as a tool for demonstrating the effects of dietary management schemes at the molecular level. 7. Effects of selenium on gene expression At this point, there is little or no published information relating dietary-induced changes in gene expression to specific gene functions associated with reproductive performance in cattle or
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any other species. While it is still not possible to directly demonstrate the effects of nutritional strategies or diets on the expression of genes related to fertility in either male or female animals, it may be possible to begin to understand the importance of the relationship between individual nutrients and the regulation of gene expression. A good example is the recent work that has examined the effects of dietary selenium on gene expression in mice (Rao et al., 2001). In a study using a 12,000-gene mouse microarray, it has clearly been established that selenium deficiencies can influence the patterns of protein synthesis in mice by regulating the expression of specific genes at the transcriptional level. Genes that were up-regulated by selenium deficiencies in mice included those associated with stress responses, cell cycling and growth, and cell adherence (Rao et al., 2001). At the same time, genes that were down-regulated included those associated with detoxification mechanisms, selenoprotein production, oxidative stress protection, and lipid transport. It is important to note that these changes in gene expression can be used to account for many of the outward phenotypic characteristics of selenium deficiencies. The database used for studying selenium effects has been recently further enhanced by studies evaluating the effects of several selenium sources, including sodium selenite and selenium yeast (Sel-Plex® ), on gene expression in the intestinal tract of mice using a basic 23,000-element murine microarray (Table 3). This study showed dramatic changes in gene expression in intestinal tissues, with the expression of over 2500 genes being influenced by selenium supplementation, and at least 100 of these can be directly or indirectly associated with reproductive functions. While the Table 3 Examples of some fertility-associated gene expression patterns that are changed by supplementation of mouse diets with sodium selenite, selenomethionine, or selenium yeast (Sel-Plex® )a Gene
Protein
Relative increase in gene expression with dietary inclusion ofb Selenomethionine
Sodium selenite
Selenium yeast (Sel-Plex® )
Dio1
Iodothyronine deiodinase, type I
2.0-fold
2.8-fold
2.1-fold
Gpx1
Glutathione peroxidase 1
4.9-fold
4.1-fold
4.7-fold
Gpx3
Glutathione peroxidase 3 Thioredoxin 2
4.6-fold
3.5-fold
3.6-fold
NS
1.2-fold
1.4-fold
Thioredoxin reductase 1
1.8-fold
1.7-fold
1.8-fold
Txn2
Txnrd1
Functional role
Modulates metabolic activities during implantation and embryonic development Key enzymes in antioxidant systems protecting reproductive systems and embryos
Key electron carrier controlling implantation and embryo development Key enzyme supporting antioxidant activities, and controlling embryo development
a Data from an unpublished gene expression study of intestinal tissue from mice fed a low selenium (0.01 mgSe/kg) diet, a sodium selenite supplemented diet (1.0 mgSe/kg), a selenomethionine (1.0 mg/kg) or a selenium yeast (Sel-Plex® ) diet (1.0 mgSe/kg) (Weindruch et al., 2005, personal communication). b Changes in gene expression are provided as significant (p < 0.05) fold changes relative to the expression patterns observed in a selenium deficient control group. NS indicates a non-significant (p > 0.05) change in gene expression relative to the selenium-deficient control.
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direct effects of dietary selenium on gene expression in key reproductive tissue have yet to be examined, data from these studies can be used to identify candidate genes or biomarkers that are clearly regulated by various dietary forms of selenium in some tissues (Table 3). There are at least three examples that are worth examining. The thyroid hormone, triiodothyronine (T3 ) has many functions that can be related to reproductive performance. One of its major roles is to trigger gene transcription, which is important in the developing embryos during growth (Goodridge, 1986). As a result, it plays a critical role in embryo survival and the growth of young animals (Edens and Gowdy, 2004; Incerpi et al., 2005). The importance of this hormone in fertility has been established in studies of ovarian responses in cows with induced hypothyroidism (Bernal et al., 1999). In many animals, the formation of the active form of this hormone is accomplished through the action of the selenium-dependent enzyme, type 1 5 deiodinase, which deiodinates the relatively inactive thyroxin (T4 ) to deiodothyronine (T3 ), the most active form of thyroid hormone. The delay in the conversion of T4 to T3 has been associated with increased embryonic mortality in poultry (Christensen, 1985) and can be expected to have the same effects in other species where strict metabolic regulation of energy metabolism is needed for proper maintenance and development of embryos. While it would be expected that synthesis of this selenium-containing enzyme would be influenced by the presence of selenium, the recent gene expression studies with mice (Table 3) have established that changes in enzyme activities can be regulated at the level of gene expression, and are not simply the influenced by the amount of selenium needed to form a functional protein following transcription. Oxidative stress on reproductive tissue and during embryo development is believed to be a major determinant affecting reproductive efficiency (Bilodeau et al., 2000) and has been suggested to be a leading cause of male infertility (Shalini and Bansal, 2005). Many of the proteins associated with antioxidant systems are known to be influenced by both the level and form of selenium in the diet (Edens and Gowdy, 2004). The use of microarray technology in mice has allowed for identification of several oxidative stress-associated genes that are readily influenced by dietary supplementation with selenium (Table 3). The selenoproteins, glutathione peroxidase 1 and glutathione peroxidase 3, were both up-regulated by the use of selenium yeast and sodium selenite in the diets of mice. These are key proteins functioning as antioxidants in many tissues and would be expected to have a major impact on the reproductive tissue (Naziroglu and Gur, 2000), sperm quality (Bilodeau et al., 2000), and embryo development (Baek et al., 2005). The nutrigenomic approach has confirmed transcriptional regulation of these proteins in intestinal tissue and will provide the basis for designing studies to better understand the influence of dietary selenium in reproductive tissue. Another set of proteins that was readily influenced by selenium supplementation are those associated with the thioredoxin electron carrier systems (Table 3). Thioredoxin is an electron carrier protein involved in many aspects of cell cycling and in maintaining antioxidant systems. The thioredoxin system has been identified as a key player in cellular redox-mediated reactions (Miranda-Vizuete et al., 2004; Beckett and Arthur, 2005). The regulation of the thioredoxin system is believed to be key in differentiation and morphogenesis in embryonic tissue (Matsui et al., 1996) and can influence early embryo viability and maturation (Clarke, 1992). Thioredoxin mediates estradiol effects on antioxidant systems, which influences embryo survivability and implantation, as well as fertility in pigs (Deroo et al., 2004). While at least one component of the thioredxoin system, thioredoxin reductase, is a selenium-containing enzyme which would be regulated by selenium availability, thioredoxin itself is not a selenoprotein. The regulation of this component of this critical thioredoxin system by dietary selenium at the transcriptional level is unexpected
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(Table 3) and may suggest new approaches to understanding the role of this system in reproductive tissues and developing embryos. The search for other specific fertility-associated genes that may be readily influenced by nutrition is just beginning, but is currently limited by the lack of information about the effects of specific nutrients on transcription. From the selenium example in mice, it is clear that use of nutrigenomic tools in fertility studies can lead to the identification of key marker genes that will help predict and establish the effects of diet on fertility. Once identified, it is not hard to see how these approaches and the ever-increasing transcriptional information will be useful for formulating customized diets to specifically address fertility issues and improve reproductive performance. 8. Conclusions Discoveries made in the next decade using powerful molecular tools like microarray analysis will undoubtedly revolutionize our basic understanding of cattle physiology and help define new methods for managing animal nutrition and reproduction. In the long term, it is likely that transcriptional profiles will be able to provide a number of very basic tools for evaluating the nutritional and physiological status of individual animals or groups of animals. Such profiles could also be used to provide a detailed analysis of the reproductive status and help formulate nutritional strategies that could be implemented to address fertility problems. As a result, it will no longer be necessary to look at extreme nutritional treatments, induced deficiencies, or long-term production responses to understand the basic effects of diets. However, it can easily be seen from these types of gene expression studies that nutritional/gene interactions are extremely complex. When this is coupled with the differences in gene expression associated with the various critical tissues of the body and the potential for changes associated with age and developmental patterns, it is clear that the true challenges in the future will be in modeling and interpreting the massive amounts of information obtained from simple nutritional studies and dietary interventions. References Baek, I.J., Yon, J.M., Lee, B.J., Yun, Y.W., Yu, W.J., Hong, J.T., Ahn, B., Kim, Y.B., Kim, D.J., Kang, J.K., Nam, S.Y., 2005. Expression pattern of cytosolic glutathione peroxidase (cGPx) mRNA during mouse embryogenesis. Anat. Embryol. (Berl). 209, 315–321. Band, M.R., Olmstead, C., Everts, R.E., Liu, Z.L., Lewin, H.A., 2002. A 3800 gene microarray for cattle functional genomics comparison of gene expression from spleen, placenta and brain. Anim. Biotechnol. 13, 163–172. Beckett, G.J., Arthur, J.R., 2005. Selenium and endocrine systems. J. Endocrinol. 184, 455–465. Bernal, A., DeMoraes, G.V., Thrift, T.A., Willard, C.C., Randel, R.D., 1999. Effects of induced hypothyroidism on ovarian response to superovulation in Brahman (Bos indicus) cows. J. Anim. Sci. 77, 2749–2756. Bilodeau, J.F., Chatterjee, S., Sirard, M.A., Gagnon, C., 2000. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol. Reprod. Dev. 55, 282–288. Burton, J.L., Madsen, S.A., Yao, J., Sipkovsky, S.S., Coussens, P.M., 2001. An immunogenomics approach to understanding periparturient immunosuppression and mastitis susceptibility in dairy cattle. Acta Vet. Scand. 42, 407–424. Butler, W.R., 1998. Review: effect of protein nutrition on ovarian and uterine physiology in dairy cattle. J. Dairy Sci. 81, 2533–2539. Butler, W.R., Smith, R.D., 1989. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 72, 767–783. Byrne, K.A., Wang, Y.H., Lehnert, S.A., Harper, G.S., McWilliam, S.M., Bruce, H.L., Reverter, A., 2005. Gene expression profiling of muscle tissue in Brahman steers during nutritional restriction. J. Anim. Sci. 83, 1–12. Clarke, F.M., 1992. Identification of molecules and mechanisms involved in the ‘early pregnancy factor’ system. Reprod. Fertil. Dev. 4, 423–433.
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