New Frontiers in Contraceptive Research A Blueprint for Action (2004) / Chapter Skim
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2 Target Discovery and Validation
2 Target Discovery and Validation
Pages 27-77
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From page 27... ...
At least several hundred of these genes appear to be unique to reproductive cells and tissues, and more than 200 human genes or their counterparts in model organisms have been shown to play roles in reproduction in viva (Matzuk and Lamb, 2002~. Because genes that are highly or exclusively expressed in the reproductive tract continue to be discovered, it is likely that additional functional genomics studies will validate these gene products as putative contraceptive targets in men and women.
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From page 28... ...
This chapter provides a brief overview of several of the new methodologies used in basic biomedical research that could be brought to bear on the identification and validation of novel targets for contraception. Other methods or technologies might also make valuable contributions to the discovery and validation of new targets, but it was not possible to provide a comprehensive review of all possible scientific approaches to contraceptive research here.
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From page 29... ...
Once male germ cells complete meiosis to achieve a haploid chromosomal complement, they are called spermatids. Spermatids undergo a process of cellular differentiation known as spermiogenesis, progressing from round to elongated spermatids, and culminating in the development of spermatozoa.
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30 ; I tn m au o 11 IL _ _ ~ _ O I C,3 _ ~ , 11]
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Spermatogenesis requires a complex interaction of the various cellular compartments of the testis (seminiferous epithelium containing spermatogenic cells, Sertoli cells, and peritubular myoid cells; the interstitial cell compartment containing the steroidogenic Leydig cells, macrophages, and other interstitial cells; and the vasculature)
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From page 32... ...
Over the course of a woman's lifetime, there is a precipitous decline in the number of oocytes. The roughly 7 million germ cells in a 20-week-old fetus are reduced to 2 million oocytes at birth, and eventually to 300,000 oocytes at puberty.
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The sperm maturation process continues when sperm enter the female reproductive tract. It is here that they undergo a process known as capacitation, during which they acquire the ability to fertilize an egg.
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These limitations are well known in model organisms such as yeast and are likely to be amplified in the more complex human genome (reviewed by Snyder and Gerstein, 2003~. Thus, a variety of 2The definition of a "gene" has varied over time.
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EST sequencing projects have provided publicly available information on more than 100,000 sequences for vertebrate reproductive tract tissues and cell types (e.g., ovary, testis, pituitary gland, oviduct, uterus, cervix, vagina, epididymis, prostate, gametes, and early embryos)
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In silica approaches, in which sequences already available in EST and SAGE databases are compared by using special computer programs to identify those that may be unique to a given tissue or cell type, can also be very powerful analytical methods. For example, several genes that are expressed exclusively in germ cells (Rajkovic et al., 2001)
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From page 37... ...
Thus, projects to complete the determination of the novel reproductive tractspecific gene sequences of humans and model organisms, such as the mouse, are necessary and will need to include genomic and proteomic analysis of substitute nonhuman primate oocyte and early embryo cDNA libraries, similar to the exhaustive mouse sequence library analysis performed by Ko and colleagues (Ko et al., 2000~. Reverse Gene Discovery One of the ways to identify and study gene function is to look for evidence of expression that is, transcription of the gene into RNA.
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i°John J Eppig, The Jackson Laboratory, in a presentation at the International Symposium on New Frontiers in Contraceptive Research, Washington, DC, July 15-16, 2003.
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In theory, more extensive application of this approach to additional reproductive tract cells and tissues, including male germ cells or Sertoli cells or the epididymis, oviduct, cervix, or uterus, could lead to the identification of transmembrane and transport proteins that can be manipulated to produce a contraceptive effect. For example, tight junctions between the somatic Sertoli cells in the testis restrict the extracellular movement of molecules across the epithelium to the germinal cells on the luminal side.
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The proteomics community will need to work closely with scientists focused on biological problems to translate the broad but shallow proteomic data into deeper understanding of proteomics. One step in that direction has been taken by the Human Proteome Organization and the European Bioinformatics Institute, which together have started the Proteomics Standard Initiatives to exchange protein-protein interaction data and other proteomic data.
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From page 41... ...
Despite these current limitations, scientists have great hope for the potential benefits of proteomics research. Moreover, drug discovery and development could benefit greatly from the new information generated (Hanash, 2003; Pawson and Nash, 2003; Tyers and Mann, 2003; Vidal, 2001~.
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Herr, Professor of Cell Biology at the University of Virginia, in a presentation at the International Symposium on New Frontiers in Contraceptive Research, Washington, DC, July 15-16, 2003. i7Personal communication with John C
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The lipid components of membranes that are of particular interest include sterols, glycolipids, and ether lipids. Evidence from functional genomics experiments with mice has suggested important roles for lipid metabolism in male germ cell function.
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Efforts are also under way to identify and characterize the multitude of enzymes that synthesize and modulate lipids. Characterization of the lipid composition of reproductive cells, including any novel lipid structures and the biosynthetic pathways by which they are generated, could reveal opportunities for contraceptive targets, such as interference of sperm-egg membrane fusion or premature activation of sperm, which would result in a contraceptive effect.
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From page 45... ...
The potential role of glycobiology in contraceptive development is based on the evidence that specific glycan structures are involved in gamete maturation, function, sperm-reproductive tract interactions, and sperm-egg binding.2~ The evidence supporting a role for glycans in sperm-egg binding, as well as the interaction of gametes with the cells lining the male and female reproductive tracts, has been described in the literature for many decades; however, there is still debate about the exact components and mechanisms of the interaction (Primakoff and Myles, 2002; Talbot et al., 2003; reviewed by Wassarman, 2002~. It is well i8Anandamide, also known as arichidonylethanolamide, is derived from arachidonate an essential fatty acid that humans use to synthesize regulatory molecules such as prostaglandins and thromboxanes.
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From page 46... ...
Specific glycan structures are generated on sperm surfaces by selective processing in the testis, or are added during maturation as sperm pass through the epididymis, or during activation in the female reproductive tract (Srivastav, 2000~. One glycan in particular appears to be required for interaction between maturing sperm and cells of the testis (Akama et 22The addition of a chain of sugars to a protein in order to make a glycoprotein.
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In another study, inhibitors of glycoprocessing enzymes were used to induce reversible sterility in male mice with no other apparent phenotypic effects. Female mice treated with the inhibitors had normal fertility (van der Spoel et al., 2002~.
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From page 48... ...
Deciphering Cell Regulatory Networks Protein Networks To truly understand how cells work at the molecular level, one must know not only what proteins are present but also how they function and interact with each other in a cellular context. Conversely, identifying the interacting partners of proteins is very helpful in deducing protein function, as proteins that interact with one another or that are part of the same protein complex are generally involved in the same cellular processes.
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A variety of experimental methods are available for the identification and characterization of protein complexes, and each has different advantages and limitations (Box 2.2 and Table 2.1~. Two of the most commonly used techniques for identifying protein complexes are: the two-hybrid system to detect binary protein interactions in cells; and biochemical purification of protein complexes followed by protein identification by mass spectrometry.
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52 ._, o 5o u cry o o 5o H ._' o En V)
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The protein complexes of this network can now be tested by both biochemical and genetic means. The expectation is that understanding the network of cellular protein interactions will allow scientists to investigate proteins of interest in greater detail and in ways that are not currently possible.
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Currently, though, it is quite difficult to simplify complex genetic networks into a set of equations. Research on genetic networks has two major goals: first, to understand the dynamics and design principles of gene regulation and, second, to reverse engineer genetic networks from experimental measurements (Brazhnik et al., 2002~.
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Compounding these issues is a great need to integrate diverse data types and to construct tools that will assimilate the information into biological models (Banerjee and Zhang, 2002~. Defining genetic networks may be one of the most challenging tasks of functional genomics.
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56 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH TARGET VALIDATION STUDIES Model Organisms as a Necessary Step in the Development of Future Contraceptives Genetic screens can be conducted with a variety of model organisms, including mice, fish, frogs, worms, and flies.27 Both forward genetic screens (those that identify a gene on the basis of phenotype [e.g., infertil27Examples of screens in each of these model organisms were presented at the International Symposium on New Frontiers in Contraceptive Research, Washington, DC, July 15-16, 2003.
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From page 57... ...
Reverse Genetics The target discovery experiments described in the first part of this chapter will likely identify hundreds of candidate targets for follow-up studies. In most cases, the next step is to undertake reverse genetic studies with mammals.
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Use of this technology is ideal for the validation of contraceptive targets because the approach will more closely mimic the use of contraceptive targets by adults, and will also eliminate artifactual results due to developmental abnormalities. In addition, if a gene is expressed specifically in the reproductive tract, there would be less of a chance for unwanted phenotypic effects elsewhere.
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Wakimoto, professor of biology, University of Washington, Seattle, in a presentation at the International Symposium on New Frontiers in Contraceptive Research, Washington, DC, July 15-16, 2003.
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The Mouse Transposon Insertion Database30 already has sequence data on 5,000 insertional mutations. In a similar way, "genetrap" retroviral vectors have been invented to mutate genes in vitro in ES cells (Friedrich and Soriano, 1991; Hansen et 29John Schimenti, the Jackson Laboratory, in a presentation at the International Symposium on New Frontiers in Contraceptive Research, Washington, DC, July 15-16, 2003.
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. The epithelium of the female reproductive tract is a prime target for contraceptive research, since sperm entry, transport, maturation, and sperm-egg interactions occur in the environment of this epithelium.
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would be excellent candidates as targets for drug development efforts in the near future (Drews, 2000~. Thus, infertile knockout or knock-in mutant mouse models that lack or contain one of these types of proteins, respectively, would identify proteins with high priority as targets for drug development.
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From page 63... ...
The recent study demonstrates that the newly identified sperm OR causes the sperm to navigate in a specific direction when it detects a concentration of specific laboratory chemicals, while other chemicals can act to block this specific movement (Spehr et al., 2003~. However, the physiological chemical that attracts sperm in the female reproductive tract remains to be revealed.
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Animals from which 32Diana Myles, Ph.D., University of California, Davis, in a presentation at the International Symposium on New Frontiers in Contraceptive Research, Washington, DC, July 15-16, 2003.
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From page 65... ...
The germ cells of both male and female mice fail to progress through the first meiotic metaphase, thereby preventing maturation of the germ cells and the eventual ability of gametes to undergo fertilization. Agents that target CKS2 or the other meiotic proteins that interact with CKS2 could be considered contraceptive drugs.
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From page 66... ...
Recently, scientists discovered a novel family of proteins, known as CatSpers, that are uniquely expressed in male germ cells and that may form a multiprotein complex that regulates the influx of calcium into the sperm cell, thereby regulating motility (Lobley et al., 2003; Quill et al., 2001~. These proteins are localized to the flagellum, and sperm motility is markedly decreased in mice that lack CatSperl.
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From page 67... ...
The A form is generally a repressor of transcription, whereas the B form is the longer protein and is a transcriptional activator. Female mice lacking progesterone receptors are infertile because of a defect in the process of ovulation as well as a uterine abnormality because of the inability to develop a receptive state and an associated inflammatory infiltration (Lydon et al., 1995~.
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From page 68... ...
In addition, many more promising targets can be expected from continued work in target discovery using a variety of experimental approaches. Once these potential targets have been identified, they will need to be validated in model organisms through in depth phenotypic analysis using genomic and proteomic approaches.
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From page 69... ...
The Consortium for Functional Glycomics, funded by the National Institute of General Medical Sciences, could provide useful research tools for an initiative in reproductive glycomics. Recommendation 3: Validate existing and emerging contraceptive targets by using forward and reverse genetic approaches with model organisms.
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Proc Natl Acad Sci U S A 88~24~:1140811412. Boguski MS, McIntosh MW.
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Proc Natl Acad Sci U S A 99~7~:4495-4499. Dutton G
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Proc Natl Acad Sci U S A 100~17~:9918-9922. Harper MJ.1973.
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1995. Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.
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Proc Natl Acad Sci U S A 98~22~:12527-12531. Rajkovic A, Yan M S C, Klysik M, Matzuk M
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Proc Natl Acad Sci U S A 93~20~:10614-10619. Schlecht U
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Proc Natl Acad Sci U S A 99~26~:17173-17178. van Someren EP, Wessels LF, Backer E, Reinders MJ.
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1996. Leukaemia inhibitory factor in human endometrium throughout the menstrual cycle.
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