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New Frontiers in Contraceptive Research: A Blueprint for Action (2004)

Chapter: 2 Target Discovery and Validation

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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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Suggested Citation:"2 Target Discovery and Validation." Institute of Medicine. 2004. New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC: The National Academies Press. doi: 10.17226/10905.
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2 Target Discovery and Validation Major advances in biomedical research have occurred in the last decade of the 20th century, and these advances will be reflected in improvements in human health care as the 21st century proceeds. Technological advances in the area of genomics have permitted the complete or nearly complete sequencing of the genomes of several species, including humans as well as model organisms, such as the fruit fly, nematode worm, pufferfish, rat, and mouse. Advances in computational abilities and bioinformatics, along with the development of large-scale, high-throughput technologies such as DNA microarrays, have helped to define tissue- and cell-specific patterns of gene transcription, referred to as "transcriptomes." New tools for sequencing proteins have also provided information on the complete protein complements of cellular components, referred to as "proteomics." In parallel with these contributions, studies in "functional genomics" that use a variety of technologies in vitro and in viva have helped to iden- tify and characterize essential and novel functions of genes. In the process of performing these various analyses, thousands of genes have been found to be expressed in the cells and tissues that function in reproduction. 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 prod- ucts as putative contraceptive targets in men and women. 27

28 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH Mutations in a particular gene or genes that lead to an infertility phenotype in model organisms would suggest that specific modulation of the same gene product in humans could also, theoretically, result in a contraceptive effect. The more specific the expression and, more impor- tantly, the more specific the function of these genes in the reproductive tract, the less likely it is that a new reproductive tract-specific contra- ceptive would have unwanted side effects. Furthermore, genes whose protein products function as receptors, enzymes, and ion channels or transporter proteins, classes of molecules that have been targeted most commonly by pharmaceuticals (Figure 2.1), would likely be of greatest interest for developing new contraceptive drugs. This chapter provides a brief overview of several of the new method- ologies 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 contracep- tive research here. The chapter focuses primarily on early stage discovery approaches to research in reproductive biology, which should be viewed Receptors, 45% Enzymes, 28% DNA, 2% Hormones and factors, 1 1% Unknown, 7% channels, 5% receptors, 2% FIGURE 2.1 Molecular targets of drug therapy, with classification according to biochemical criteria. Based on modern standard work in pharmacology, the molecular targets of all known drugs that have been characterized as safe and effective have been collected and listed according to their biochemical nature. SOURCE: Drews, 2000.

TARGET DISCOVERY AND VALIDATION 29 as a long-term investment in the field, as it will likely take many years, perhaps even decades, to translate the new knowledge into clinically useful and acceptable contraceptives. The chapter also includes a few examples of promising targets that have already been identified and that could potentially be used to develop new contraceptives in a somewhat shorter time frame. Again, a comprehensive list of possible targets was not possible, but a general overview of potential contraceptive targets is provided in the following section. OVERVIEW OF REPRODUCTIVE BIOLOGY Reproduction is a complex process involving many different special- ized cells, tissues, and organs in both the male and the female (Institute of Medicine, 1996; reviewed in Matzuk and Lamb, 2002~. Figure 2.2 provides an overview of the male and female reproductive processes that could potentially be targeted by contraceptives. Recent advances in basic bio- logical research have provided a better understanding of these processes than ever before, and examples of the many genes that may play an im- portant role in reproduction at various stages are shown in Figures 2.3 and 2.4. A small number of germ cells are set aside from somatic cells early in embryogenesis, where they usually remain in an undifferentiated quies- cent state while somatic cells are dividing and forming tissues and organs. In order to form gametes, they must then begin to proliferate and enter meiosis, a process that is unique to germ cells and is required to halve the number of chromosomes in a gamete's nucleus so that it can combine with another haploid gamete at fertilization. Male spermatogenesis is initiated postnatally and is a continuous process characterized by three specific functional phases: proliferation, meiosis, and spermiogenesis. Proliferating cells known as spermatogonia undergo differentiation and enter meiosis as spermatocytes. Once male germ cells complete meiosis to achieve a haploid chromosomal comple- ment, they are called spermatids. Spermatids undergo a process of cellular differentiation known as spermiogenesis, progressing from round to elon- gated spermatids, and culminating in the development of spermatozoa. After spermatogenesis, spermatozoa are released from the Sertoli cells into the seminiferous tubule lumen. In the female, gametes develop in structures called follicles within the ovary. Oogenesis begins as follicles form during prenatal life in humans, and arrests at an early stage of meiosis. Recruitment of individual follicles leads to further growth and development of oocytes, with a resumption of meiosis, and culminates in ovulation, or release of the oocyte into the oviduct. At that point, the oocyte arrests again in a late stage of meiosis

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TARGET DISCOVERY AND VALIDATION Genes Cells Genes Adamts2 Apaf1 Bax Bmp8b Csf1 Dazl Gdnf Camk4 CsnkPa Crem Mtap7 Prm 1 Prm2 Ppptcc ~39562 Growth factors/ receptors Gonadotropins/ receptors Cell-cell adhesion ~ Sertoli cells Peritubular cells Interstitial cells Leydig cells _ Macrophanes _ Steroids and receptors Signal transduction Junctional complexes Proapoptotic/ survival Cell cycle Chromosome pairing/ synapsis _ Homologous recombination _ remodeling ~ .............. Cytoplasmic extrusion Spermiation _ Maturation in genital tract Capacitation Fertilization it, Growth factors Receptors Cytokines Melosis Genomic integrity DNA replication/ repair _ Spermatids ~ .................................................................. .. -............ Differentiation Chromatin packaging Nuclear condensation ~ Spermatozoa ........ - Ivlalurallon Motility Fertilization 31 Fas Gja1 Kit Pi3K Vasa Hyperactivated motility Sperm-zona- egg penetration Nuclear decon de nsati on Slc12a2 Styx Theg Tlp Tnp1 Tnp2 Ube2b 3~5~] P,m1 P~m2 [~752 FIGURE 2.3 Genes involved in the regulation of male reproduction in the mouse. Spermatogenesis requires a complex interaction of the various cellular compart- ments 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). Targeted mutation of the genes shown affects specific testicular cell types and reproductive function, resulting in male infertility or subfertility in the mouse. SOURCE: Matzuk and Lamb, 2002.

32 COO integrity GDF9, BMP1 5 PTGER2, PTX3, AMBP A. , Fertilization and pre-implantation development ZP1, ZP2, ZP3, MATER, DNMT10, PEASE, HSF1 Implantation COX2, HOXA10 HOXA11, LIF, IL11 R. NEW FRONTIERS IN CONTRACEPTIVE RESEARCH Pre~y Ovulatlon f(311~e LHR, COX2, PR, ... .. CEBPB NRIP1 , ~ INHA, INHBA, CX37 ERa ERp ~ , FSH, FSHR, IGF1, CCND2, L TAF4B Luteal differentiation/regression PTGFR, P27,CDK4 HMX3, PR, ERa PRL, PRLR F' GDF9, KITL r KITL, KIT, ASH FIGURE 2.4 Female fertility proteins. Knockout mouse models have defined key proteins that function at various stages of follicle formation, folliculogenesis, ovulation, and post-ovulatory events. Several proteins are needed for primordial follicle formation, oocyte and granulose cell (GC) growth and differentiation, ovulation, and the integrity of the cumulus oocyte complex (COC). SOURCE: Matzuk and Lamb, 2002. until fertilized by a sperm. In contrast to males, the formation of follicles in females results in a finite endowment of oocytes. 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. Only about 400 of those will ever undergo ovulation. Despite this sexual difference in meiosis, many regulators of the process are common to the germ cells of both males and females. Both are controlled from the brain through the production of gonadotropin- releasing hormone (GnRH) by the hypothalamus. GnRH in turn stimu- lates the anterior pituitary to secrete luteinizing hormone (LH) and follicle- stimulating hormone (FSH), both of which are required for normal gamete development as well as steroid hormone production (such as testosterone, estrogen, and progesterone). Chemical communication between develop-

TARGET DISCOVERY AND VALIDATION 33 ing gametes and their supportive somatic cells via growth factors and cytokines is also essential for egg and sperm production. When sperm leave the testis, they must undergo more morphologic and biochemical changes before they are capable of fertilization. These final developments take place in the epididymis, which also serves as a sperm storage reservoir. During maturation, much of the sperm surface appears to be remodeled. Products secreted by the epithelium of the epididymis mediate these changes. Although the precise components of this epididymal fluid have not been fully identified, it is known that its complex composition changes dramatically along the length of the epididymal tubule. The sperm maturation process continues when sperm enter the female reproductive tract. It is here that they undergo a process known as capaci- tation, during which they acquire the ability to fertilize an egg. For fertili- zation to take place, sperm must be motile and make their way to the oviduct, a step that may involve chemical signals from the egg or com- ponents of the female reproductive tract (Inaba, 2003~. Once a sperm comes in contact with the egg, several events must take place at the proper time in order for fertilization to take place. These include sperm penetra- tion of the egg's cumulus cell layer, binding to the zone pellucida (the egg's protective coat), the sperm acrosome reaction (release of enzymes), penetration through the zone pellucida, and fusion of the sperm and egg plasma membranes. The fertilized egg then progresses through the oviduct for another 72 hours and then enters the uterus. The egg undergoes several mitotic cycles during the first few days after fertilization, eventually forming a blasto- cyst. The blastocyst attaches to the uterine wall and then undergoes multiple steps to complete implantation. Human chorionic gonadotropin (hCG), a hormone released by the early embryo, is involved in events crucial to maintaining early pregnancy, including maintenance of the corpus luteum, which forms on the ovary after ovulation, progesterone production, and, perhaps implantation. STRATEGIES FOR TARGET IDENTIFICATION Recent advances in technology have provided new opportunities to use high-throughput methods to address biological questions on a larger scale than was previously possible. This new approach to biomedical research represents a shift from the traditional, reductionist approach of iAny approach using robotics, automated machines, and computers to process many samples at once.

34 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH studying one or a very small number of molecules at a time to a larger- scale discovery or systems approach to studying hundreds or even thou- sands of molecules at once (Institute of Medicine, 2003~. For example, with the recent sequencing of entire genomes of many different species, there is now great interest in studying the products of all the genes encoded by the sequences. However, defining the genes2 within a genome is not trivial. Most of an organism's DNA does not actu- ally encode functional genes for proteins or other cellular components. Several approaches are used to identify functional genes (Box 2.1), but each has limitations and is likely to identify a different set of potential genes (Figure 2.5~. 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. A current definition is "a complete chro- mosomal segment responsible for making a functional product" (Snyder and Gerstein, 2003~.

TARGET DISCOVERY AND VALIDATION / 711 Microarray 1 ,212 188 2,033 / / 106 1 163 Transposon \ / SAGE 35 FIGURE 2.5 Functional genomics is best used in a combined fashion. Illustrated is the number of open reading frames in the yeast genome that were found to be transcribed based on data from microarray hybridization, SAGE, and transposon tagging experiments. Areas of overlap indicate that some transcripts were identi- fied by more than one method. SOURCE: Snyder and Gerstein, 2003. approaches will be needed to identify and validate potential targets for contraceptive development. Some of these methods are described below. Forward Gene Discovery One method used to identify genes that are expressed in a particular cell or tissue type is the expressed sequence tag (EST) approach. This method entails sequencing many small nucleotide fragments, or tags, that have been derived from all the messenger RNAs (mRNAs) expressed in the given cell type. EST sequencing projects have provided publicly avail- able information on more than 100,000 sequences for vertebrate reproduc- tive tract tissues and cell types (e.g., ovary, testis, pituitary gland, oviduct, uterus, cervix, vagina, epididymis, prostate, gametes, and early embryos). These sequences are deposited in the public database at the National Cen- ter for Biotechnology Information3 and are also assembled into clusters at 3See htip://www.ncbi.nIm.nih.gov/.

36 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH UniGene.4 Complementary DNA (cDNA) subtraction hybridization meth- ods5 and serial analysis of gene expression (SAGE) (Neilson et al., 2000), another method for global gene expression profiling, have also been use- ful to identify genes uniquely expressed in reproductive tissues (Neilson et al., 2000; Wang et al., 2001~. In silica approaches, in which sequences already available in EST and SAGE databases are compared by using spe- cial computer programs to identify those that may be unique to a given tissue or cell type, can also be very powerful analytical methods. For ex- ample, several genes that are expressed exclusively in germ cells (Rajkovic et al., 2001) or epididymal tissues have been identified by this approach (Penttinen et al., 2003~. Because the ESTs are "tags," they are only a partial representation of the full-length mRNA. For transcripts that are long, short-lived, or in low abundance, the full-length mRNA and encoded proteins may be particu- larly difficult to identify. As a result, for a number of novel reproductive tract-specific transcripts, the full-length mRNA sequences and the protein products that they encode may still be unknown. This is problematic because many different mRNAs may be produced from a single gene via alternative splicing, transcription, or processing of the mRNA, and such alterations are known to result in gene transcripts that are unique to the testis (Kleene, 2001~. Hence, full-length transcripts must be determined by complete sequencing of full-length cDNAs. Thus, in certain situations, more exhaustive analysis may be necessary for complete determination of the reproductive tract transcriptome and proteome. The most extensive EST libraries of genes expressed in the male repro- ductive tract have been created by McCarrey and colleagues (McCarrey et al., 1999) and Ko and colleagues (Abe et al., 1998~. These analyses were undertaken with mouse tissues because of the powerful genetics tools that are available for the mouse and the readily available source of material. Thus far, McCarrey and colleagues have sequenced 16,833 cDNA se- quences. Additional sequencing is being done. In addition, the National Institutes of Health (NIH) Specialized Cooperative Centers Program in Reproduction Research has also sequenced alternative gene libraries and 4UniGene is an experimental system for automatically partitioning GenBank sequences into a nonredundant set of gene-oriented clusters. See http://www.ncbi.nlm.nih.gov/ entree / query. f cgi? db =unigene. 5A technique used to identify genes expressed differentially between two tissue samples. A large excess of mRNA from one sample is hybridized to cDNA from the other, and the double-stranded hybrids are removed by physical means. The remaining cDNAs are those that are not represented as RNA in the first sample and, thus, that are presumably expressed uniquely in the second sample. To improve specificity, the process is often repeated several times.

TARGET DISCOVERY AND VALIDATION 37 tissue sources.6 Although serial analysis of gene expression has been con- ducted with human oocytes (Neilson et al., 2000), full-length sequences for most of the novel sequences and libraries of full-length sequences of human oocytes and early embryos are unavailable at present. Thus, projects to complete the determination of the novel reproductive tract- specific 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 per- formed 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. A recently developed technology, DNA microarrays,7 provides a powerful, high-throughput method for studying transcription in specific cell types or tissues (Fodor et al., 1993; Schena et al., 1996~. Microarrays have already been used to generate the data used in thousands of peer-reviewed publi- cations and contributed to many public databases that are now used as research tools by the scientific community as a whole. This technology is widely thought to hold enormous potential for diverse applications, ranging from discovering new drug targets to identifying markers that could predict individual responses to drugs (reviewed by Chicurel and Dalma-Weiszhausz, 2002~. Microarrays that cover all known mouse genes as well as thousands of uncharacterized gene sequences are now being used to examine gene expression in numerous reproductive tissues, including the testis, ovary, and uterus of mice and humans (Borthwick et al., 2003; Burns et al., 2003; Carson et al., 2002; Giudice et al., 2002; Kao et al., 2002; Leo et al., 2001; McLean et al., 2002; reviewed by Schlecht and Primig, 2003; Schultz et al., 2003~. Schultz and colleagues (2003) used microarray analyses of testicu- lar cells to identify 351 mouse genes that appear to be sperm cell specific and that are expressed postmeiotically. The authors estimated that nearly 4 percent of the entire genome may be exclusively expressed in the male germ line. These genes thus represent a large number of germ cell-specific targets for contraceptive development. The databases containing the 6See http://www.nichd.nih.gov/about/cpr/rs/sccprr.htm (accessed August 2003~. 7A DNA microarray consists of a glass microscope slide or silicon chip onto whose surface thousands of specific DNA sequences are spotted. Incubation with a labeled sample of nucleic acid such as mRNA can reveal which of the genes represented on the array are expressed in the sample and their relative levels.

38 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH mouse testis results are publicly available and should be useful in the initial stages of developing a target for contraceptive development, although much work remains to characterize these genes and validate their potential as new targets for contraception. Since the majority of these genes appear to be homologous (similar in sequence and function) and expressed in the human testis, new targets for potential contraceptive development can now be investigated. However, the field could be greatly advanced by the availability of standardized microarrays that include all genes, as well as alternatively spliced tran- scripts that are expressed in the reproductive tissues of humans and model organisms (e.g., mice). This would make it easier to compare the results among multiple investigators and would reduce the cost of such research. For example, efforts are ongoing to develop a microarray called EndoChip that represents gene expression during the window of implantation.9 Selective Gene Parsing One can also discover putative genes of interest by identifying sequences that specify a particular function or location in the cell. For example, genes whose products function in specific cellular locations can be identified by searching for sequences that play an essential role in trans- porting proteins within the cell or in secreting proteins from a cell. Such proteins might facilitate communication between germ cells and their companion somatic cells or between germ cells or embryos and somatic cells of the reproductive tract. One such approach, called a signal sequence trap (SST), has recently been used to identify mouse proteins that are either secreted by oocytes or in the oocyte cell membrane (Taft et al., 2002~.~° The SST methodology is based on the ability of a cloned sequence to drive the secretion of the yeast invertase enzyme, which is essential for the growth and survival of genetically modified yeast that are incapable of secreting this critical enzyme (Jacobs et al., 1997, 1999~. When a piece of cDNA (derived from an oocyte, sperm cell, or other relevant reproductive tract cell) containing the appropriate signal peptide sequence is inserted in front of the yeast invertase gene, the ability of the enzyme-deficient yeast to secrete this critical enzyme is restored and the yeast can grow. The inserted cDNA See http://mrg.genetics.washington.edu/ (accessed July 2003~. 9This work is funded by the NIH Reproductive Sciences Branch and the NIH Office of Research on Women's Health (Linda Giudice, personal communication, July 2003~. 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.

TARGET DISCOVERY AND VALIDATION 39 can then be isolated from the growing yeast and the full-length cDNA containing this sequence can be identified. Because these cDNAs encode putative ligands and receptors ex- pressed by germ cells, embryos, or the reproductive tract, these regulators may serve as novel candidate targets for contraception. In theory, more extensive application of this approach to additional reproductive tract cells and tissues, including male germ cells or Sertoli cells or the epididy- mis, oviduct, cervix, or uterus, could lead to the identification of trans- membrane and transport proteins that can be manipulated to produce a contraceptive effect. For example, tight junctions between the somatic Ser- toli cells in the testis restrict the extracellular movement of molecules across the epithelium to the germinal cells on the luminal side. Little is known about the transport systems that deliver molecules to the protected compartment. Ion channels and transporter proteins may also be involved in sperm capacitation and fertilization. In addition to targeting the transport systems themselves, one can even imagine exploiting the properties of the transport systems to specifi- cally deliver contraceptives to testicular cells or other reproductive tract tissues, such as the uterus and prostate, that are also known to express many transporter proteins. Other approaches to selective gene parsing could potentially identify proteins with other functions that are amenable to modulation with drugs, including specific classes of enzymes such as kineses or phosphatases. For example, male germ-cell specific kineses have been identified (Nayak et al., 1998; Tanaka et al., 1999), but it is not certain that these are essential for spermatogenesis or sperm function, and at least some appear to be dispensable (Shinkai et al., 2002~. Proteomics Most genes encode protein products, but RNA-based methods such as DNA arrays cannot precisely predict protein levels or protein modifi- cations in a cell or tissue. Thus, many scientists are examining the expres- sion, structure, and function of proteins directly. Much effort has recently been devoted to the study of proteomics, which attempts to define and analyze the entire protein complement (the proteome) involved in a par- ticular biological process in a given cell, tissue, or organism. The chal- lenges associated with proteomics research are enormous (Phizicky et al., 2003; Sali et al., 2003; reviewed by Tyers and Mann, 2003~. Many different proteins can be produced from a single gene because of such regulatory events as alternate splicing of the mRNA as well as biochemical modifica- tions to the resultant protein, including glycosylation, acetylation, phos- phorylation, and proteolytic processing. An individual's proteome can change over time, depending on a whole host of variables, including stage

40 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH of development, exposure to external factors, and the state of health or disease. Furthermore, despite recent technological advances, current methods, instrumentation, and information technologies are still far from optimal; and many obstacles remain to the effective and efficient achievement of the goals of proteomics research (Boguski and McIntosh, 2003; Phizicky et al., 2003; Tyers and Mann, 2003; Vidal,2001~. For instance, additional high- throughput technologies and new computational methods to analyze the resultant large data sets are needed. The incorporation of medical informatics will also be critical, since clinical application of proteomics will increasingly require the integrated analysis of genetic, cellular, molecular, and clinical information and the expertise of pathologists, epi- demiologists, and biostatisticians. The plethora of information generated also poses challenges for data management. There is a great need for databases that use agreed-upon standards for protein data to facilitate comparisons and integration of complex and disparate kinds of protein information into a biological atlas. Because the current principal protein databases emphasize molecular and cellular features and annotation, they are not well suited to represent physiology, and there is currently no reliable way to retrieve groups of proteins based on well-known pathways or functional classifications (Boguski and McIntosh, 2003~. In addition, public access to the raw data is essential so that the numerous individuals working across the many fields of biomedical research can participate in the research. 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 In- stitute, which together have started the Proteomics Standard Initiatives to exchange protein-protein interaction data and other proteomic data. Access to the technologies needed to study proteomics may also be limited by their cost, so there is much interest in facilitating more wide- spread use of tools for proteomics research. At the same time, it is impor- tant to coordinate large-scale research efforts in proteomics both to avoid duplication and to provide strong rationale for funding agencies. One practical bottleneck to these approaches and in fact, to most large-scale, systematic research projects has been the limited availability of validated iiCollections of protein sequences date back to the 1960s, preceding GenBank by nearly 20 years. Since the early 1990s, important utilitarian goals of protein databases have included minimal redundancy, maximal annotation, and integration with other databases. These prin- ciples continue to be stressed today (see Boguski and McIntosh, 2003~. i2See http://psidev.sourceforge.net/ (accessed October 2003~.

TARGET DISCOVERY AND VALIDATION 41 genome-wide cDNA for use in the capture of protein complexes (Tyers and Mann, 2003~. The FlexGene consortiums between academic institu- tions and industry aims to develop complete cDNA collections for use by the biomedical community. A variety of other technical challenges also limit the quality and use- fulness of data generated by proteomics research. Meaningful interpreta- tion of results depends upon careful study design and high-quality~4 biological samples; but fundamental challenges involving biological vari- ability, preanalytic factors,~5 and analytical reproducibility remain to be resolved (reviewed by Boguski and McIntosh, 2003~. For instance, in the case of human proteomics, sample materials may be limited and of incon- sistent quality because of protein degradation or variability in collection, handling, and storage procedures. In addition, most gene expression and proteomic analyses involving human specimens will be observational studies out of necessity, leading to important questions about possible biases and confounding factors in the populations from which the samples are drawn. Indeed, in referring to functional genomics technologies and their relevance in clinical medicine, Margolin (2001) has admonished that "Scientists . . . need to avoid the tendency, often driven by the high price of some of the newer techniques, of running under-controlled experiments or experiments with fewer repeated conditions than would have been accepted with standard techniques" (p.234~. Boguski and McIntosh (2003) note that the same caveat applies to proteomics research, but perhaps even more so because a framework to estimate appropriate sample sizes has yet to be determined and the nature of the technology creates substantial challenges to progress in achieving this goal. 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~. Because most drugs target proteins, it is likely that proteomics will i3See http://www.hip.harvard.edu/ (accessed October 2003~. i4Two issues associated with specimens of almost any kind are sample quality and number. Quality involves both the preservation of molecular features (such as intact and representa- tive proteins) and the assurance of both inter- and intrasample homogeneity (Boguski and McIntosh, 2003~. i5These refer to those factors, both known and unknown, that may be present in a subject or that may arise in any of the steps before a laboratory test and data analysis. Examples include genotype; physiological attributes such as age, gender, reproductive status, lifestyle effects (for example, diet or smoking), and drugs taken; and specimen collection, handling, and processing protocols. Uncontrollable variables must be well understood to be able to separate their effects from the object or process under study. Most errors in clinical labora- tory tests are known to occur in the preanalytical phase (Boguski and McIntosh, 2003~.

42 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH play a major role in future drug discovery, development, and clinical application, provided that suitable platforms become available. The proteomics approach is already being used to identify potential new targets for contraceptives. John Herr and colleagues at the University of Virginia have been analyzing human sperm and mouse eggs using tandem mass spectrometry (a method for separating molecular and atomic particles by mass) to determine what proteins exist in these cells, the rela- tive abundance of individual proteins, and their unique properties. The objective of these analyses is to identify, clone, and characterize novel proteins that may be targets for drug-based contraceptives or that could potentially be used to make contraceptive vaccines (which would induce an immune response against proteins specific to the reproductive process and therefore block fertility). The databases created by these studies are referred to as the Human Sperm Proteome and the Mouse Egg Proteome. Similarly, the lists of proteins identified in each cell type are catalogued in the Sperm Protein Encyclopedia and the Egg Protein Encyclopedia. The former currently contains approximately 2000 proteins, of which 850 have been sequenced and 78 are novel or uncharacterized. About 280 pro- teins in the latter database have been sequenced and 32 are novel or uncharacterized.~7 To identify new candidates as targets for drug-based contraceptives, the group has examined key chemical modifications (phosphorylation) of regulatory proteins and the enzymes that catalyze those modifications (known as kineses). To optimize the identification of proteins that could potentially be useful for contraceptive vaccine production, the group has also focused on proteins that are found on the surfaces of sperm and eggs and on proteins that have already been found to induce antibody produc- tion in the serum of infertile men and women. Lipidomics The process of reproduction involves multiple events in which mem- branes of different cells come into intimate contact, including interactions of male germ cells with Sertoli cells, the binding of sperm to the cells lining the oviduct, the fusion of the sperm and the egg, the attachment of the embryo to the cells lining the uterus, and the formation of the placenta. While these interactions may involve proteins on the surface of the oppos- ing cells, the lipid structures of the cell membrane in which the proteins i6John C. 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. Herr, November 2003.

TARGET DISCOVERY AND VALIDATION 43 are embedded may also play key roles and are therefore potential targets for contraceptives. In addition, physiological processes occurring in germ cells may involve changes in membrane lipid composition that alter membrane structure or trigger signaling cascades. For example, sperm membranes have cholesterol-rich areas, and the removal of cholesterol from the sperm membranes is involved in triggering sperm capacitation (Davis, 1976, 1980, 1981; Travis and Kopf, 2002~. Changes in cholesterol occur during the natural process of sperm activation in the vagina or can be induced by incubation of sperm with cholesterol-binding substances (e.g., albumin and cyclodextrins). Signaling molecules, including a vari- ety of receptors, are localized in specialized membrane domains called rafts. Modulation of the lipid contents of these domains is known to alter protein function. A number of proteins are tethered to membranes by a phospholipid called phosphatidylinositol or as a result of posttranslational modifications with fatty acids, cholesterol, or intermediates in the choles- terol synthesis pathway. The integrity of cell membranes is critical to cell survival. Agents (e.g., detergents and surfactants) that disrupt the mem- brane lipid structure are well known to be spermicidal and are widely used as contraceptives (Bernstein, 1974), but they also have effects on non- germ cell membranes (e.g., the cells lining the reproductive tract). Finally, the entry of certain microorganisms into cells involves fusion with or up- take by the membrane, and therefore, entry into the cell is potentially sen- sitive to agents that affect lipid composition and membrane structure- function. 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. Spermatogenesis is severely impaired in mice lacking seminolipid, the major glycolipid of mature mammalian sperm (Fujimoto et al., 2000; Honke et al., 2002~. Mice that lack the genes for some of the enzymes required in the synthesis of specific types of lipids also have impaired fertility. For example, targeted deletion of the ganglio- side synthase gene in mice, which results in deficiencies in all ganglio- sides (a class of lipid), is associated with azoospermia and male infertility (Takamiya et al., 1998~. Mutation of another gene involved in the synthesis of ether lipids (also known as plasmalogens) is also associated with male infertility (Rodemer et al., 2003), as is inactivation of the hormone-sensitive lipase gene, which encodes an enzyme that hydrolyzes cholesterol esters and triglycerides (Chung et al., 2001; Osuga et al., 2000~. Data linking lipid function to female fertility are more limited, but at least one mouse model implicates such a link. Female mice lacking the scavenger receptor in- volved in the uptake of high-density lipoprotein cholesterol are infertile (Trigatti et al., 1999~.

44 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH Other types of experiments have also suggested a role for lipids in fertility. For instance, an inhibitor of the enzyme involved in the transfer of glucose to a type of lipid known as ceramide causes reversible infertil- ity in male mice without significant side effects (van der Spoel et al., 2002~. Sperm-immobilizing antibodies that react with seminolipid have been described as well (Tsuji et al., 1992~. The lipid content and composition of the testis and male reproductive tract and the cholesterol-rich domains of sperm have been the most studied, but very little effort has been made to examine the lipid content and composition of the oocyte or cells of the female reproductive tract. Human tissues may contain more than 1,000 major lipids, including classes such as glycolipids, sterols, and phospholipids, as well as an unknown number of less prevalent minor species (Wilson, 2003~. Quanti- fying and analyzing the lipid components of cells is thus a complex under- taking, but advances in technologies such as mass spectrometry and liquid chromatography are emerging that can increase the speed and scope of such research. 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, includ- ing 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 activa- tion of sperm, which would result in a contraceptive effect. A chemical genetics screen to discover agents that selectively act on and disrupt gamete membrane lipid domains is one possible exercise that could be fruitful for identification of new contraceptives. Lipids also play important signaling roles in reproduction. For example, platelet-activating factor, an acetylated glycerophospholipid, has been shown to be involved in several reproductive processes, including ovarian, uterine, and oviductal function (Harper, 1989; Ishii et al., 2002; Ishii and Shimizu, 2000; Logan and Roudebush, 2000; Toledo et al., 2003~. It causes activation of sperm and oocytes by inducing influx of extracellular cal- cium. Prostaglandin signaling plays a role in the implantation process. Mice deficient in the COX-2 cyclooxygenase (COX) display abnormalities in the implantation process, particularly the early decidual response (Lim et al., 1997~. Evidence suggestive of a role of prostaglandins in the human implantation process includes the presence of COX-1 and COX-2 in the human endometrium, mainly the glandular epithelium, during the pre- sumptive implantation period (Marions et al., 1999) and from an exami- nation of prenatal use of nonsteroidal anti-inflammatory drugs (NSAIDs), drugs that inhibit COX enzymes, that indicated an increased risk of mis- carriage in users of aspirin and other NSAIDs (Li et al., 2003~. One of the key prostaglandins involved in implantation is thought to be prostacyclin,

TARGET DISCOVERY AND VALIDATION 45 a ligand for peroxisome proliferator-activated receptor delta, a member of the nuclear receptor family expressed in subluminal stromal cells in the rodent uterus (Ding et al., 2003; Lim and Dey, 2002~. The transcription factor peroxisome proliferator-activated receptor delta is implicated in the implantation process (Lim and Dey, 2000; Matsumoto et al., 2001~. Another lipid implicated in implantation is the endocannabinoid anandamide,~8 a ligand for the cannabinoid receptors CB1-R and CB2-R (Habayeb et al., 2002; Paria and Dey, 2000; Paria et al., 2002), which are expressed in the preimplantation embryo and in the reproductive tract (CB1-R only). Uterine anandamide levels in the mouse are reduced at the time of implantation and are highest at interimplantation sites. Endo- cannabinoids at low levels accelerate trophoblast~9 differentiation, but at high levels they inhibit trophoblast differentiation and arrest embryo development. Natural and synthetic agonists of the cannabinoid receptors have similar effects. Thus, it has been postulated that endocannabinoids play an important role in controlling the synchrony of embryo develop- ment for implantation in rodents. Anandamide is present in human repro- ductive tract fluids (Schuel et al., 2002), and high anandamide levels are associated with failures of in vitro fertilization (Maccarrone et al., 2002~. Glycomics Scientists estimate that 50-80 percent of proteins contain glycan20 structures, and these structures can play an important role in cell inter- actions and signaling. 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 inter- actions, 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 prostaglan- dins and thromboxanes. It is also referred to as an endocannabinoid due to its ability to bind cannabinoid receptors. i9The part of the early embryo that attaches to the wall of the uterus. 20Any of many carbohydrates that are made up of chains of simple sugars. 2iThis section is based largely on written input provided by Diana Blithe, Ph.D., Contra- ception and Reproductive Health Branch NICHD, NIH, DHHS.

46 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH accepted that protein glycosylation22 is involved in these processes, and this assumption has been used as the basis for the theoretical develop- ment of contraceptive agents. As noted above, glycolipids play important roles in spermatogenesis as well. However, contraceptive product devel- opment has been slow because of difficulties in precisely analyzing the glycan structures involved and because gamete glycosylation is species specific, which likely contributes to the inhibition of cross-species fertili- zation. Advances in recombinant DNA technology have not simplified matters because the resultant recombinant proteins often do not contain native glycan structures. However, new technologies, such as carbohy- drate microarrays (Service, 2003), that could speed research progress and scope in this field are beginning to emerge. Similarly, technical advances that have improved the sensitivities of existing analytical techniques, such as mass spectrometry and nuclear magnetic resonance, have made it easier to characterize sugars from much smaller amounts of sample material. Moreover, the National Institute of General Medical Sciences at NIH has recently provided funding for the development of tools and resources for glycomics research through a "glue grant" a funding mechanism that supports large-scale collaborative efforts with the goal of addressing prob- lems beyond the reach of individual investigators. One of the many goals of the Consortium for Functional Glycomics is to determine glycan pro- files of mouse and human tissues, cells, and proteins, including the testis and ovary. Research tools such as arrays and mice with gene knockouts for enzymes that add or modify carbohydrate structures on proteins are being made available to scientists through core facilities.23 Potential glycan targets for contraception include immunogenic mol- ecules on the sperm surface that could be used to inhibit sperm motility in the reproductive tract, the enzymatic activity associated with sperm pen- etration of the cell layer surrounding the egg, or binding of the sperm to the egg. In addition, there is potential to interfere with cellular interactions during gamete maturation so that the appropriate glycans are not present on the mature gametes or the gametes fail to fully mature. In either case, function would be impaired. Specific glycan structures are generated on sperm surfaces by selec- tive processing in the testis, or are added during maturation as sperm pass through the epididymis, or during activation in the female reproduc- tive 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. 23See htip://web.mit.edu/glycomics/consortium/ (accessed November 2003~.

TARGET DISCOVERY AND VALIDATION 47 al., 2002; Muramatsu, 2002~. Production of a mouse in which the enzyme required for the formation of this glycan structure is knocked out leads to sterile male mice but fertile females (Akama et al., 2002~. 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~. The latter study was unable to determine whether the effects of the inhibitors were on glycoproteins or glycolipids, or both. Nonetheless, the ability to deliver an agent to the testis, as well as the apparent testis speci- ficity of the impact, makes these targets appealing for contraceptive development. A number of proteins are inserted into the sperm membrane as sperm progress through the testis and epididymis. One protein, an immunogenic structure called sperm agglutination antigen 1 (SAGA-1), is a membrane- anchored24 glycoprotein that is acquired in the epididymis and that is localized over the entire head of sperm. The protein core of this molecule is found on lymphocytes (a type of white blood cell) and sperm; however, the carbohydrate-specific portion of the molecule is found only on sperm, thus making it an attractive target for contraception (McCauley et al., 2002~. Glycan structures on a given protein are often tissue-specific. One such molecule is glycodelin. Glycodelin has two differently glycosylated forms, one secreted by uterine endometrium in the female reproductive tract (GdA) and the other secreted by seminal vesicle cells in the male reproductive tract (GdS). Unique glycosylation of GdA versus GdS deter- mines inhibitory activity in sperm-egg binding. GdA inhibits sperm-egg binding and is secreted into uterine fluid beginning about 4 days after ovulation. In contrast, GdS does not interfere with sperm-egg binding (reviewed by Halttunen et al., 2000~. Sperm-egg binding is a carbohydrate-mediated event. A zone pellu- cida protein known as ZP3 is believed to be a ligand for sperm binding to the zone pellucida of the egg (Primakoff and Myles, 2002; Talbot et al., 2003; reviewed by Wassarman, 2002~. Glycans present on ZP3 have been implicated in the binding of sperm. Early experiments suggested that an enzyme present on sperm could bind to oligosaccharide residues on ZP3 (Talbot et al., 2003~. Evidence also indicates that activated eggs lack the oligosaccharide structure that is the proposed binding target for sperm, thus protecting against fertilization by more than one sperm (Koyanagi and Honegger, 2003; Vo et al., 2003~. 24Via a glycosylphosphatidylinositol (GPI) membrane anchor. For more detail please see the section on sperm-egg interaction.

48 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH The species specificity of glycosylation remains an important issue in studies of protein function. Although ZP3 is widely accepted as the sperm receptor, the actual glycan structures involved have not been character- ized. Substitution of human ZP3 in a mouse ZP3 knockout resulted in restoration of fertility in the female, but specificity for mouse sperm rather than for human sperm was retained (Rankin et al., 1998~. The presump- tion is that human ZP3 was appropriately glycosylated by the mouse glycoprocessing machinery to provide species-specific binding. However, the potential functional impact of species-specificity or tissue specificity of glycosylation is often overlooked when recombinant technology is used to produce proteins (see Chapter 3 for more detail). For example, glyco- sylation of recombinant ZP3 is required for functional capability (Gahlay and Gupta, 2003~. Sperm incubated with recombinant glycosylated ZP3 could undergo the acrosomal reaction but sperm incubated with unglyco- sylated ZP3 made in E. cold could not undergo the reaction. In some cases, not only the presence of glycosylation but also the specific components of the glycan are critical. For instance, GdA synthesized in cultured human kidney cells retained the ability to inhibit sperm-egg binding, but this function was lost when GdA was produced in cultured hamster ovary cells (Van den Nieuwenhof et al., 2000~. Thus, despite the consensus that glycans are obligatory for sperm- egg interaction, the lack of knowledge about the specific structural com- ponents involved has hampered contraceptive development based on the egg as a target. Identification of the precise structure of the critical glycans on both the human egg and sperm will be necessary to plan an effective contraceptive strategy based on glycobiology. 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 func- tion, as proteins that interact with one another or that are part of the same protein complex are generally involved in the same cellular processes. Structured ensembles of proteins and other molecules perform many vital cellular functions. Frequently, these protein complexes comprise 10 or more components (Sali et al., 2003~. Small portions of proteins, known as interaction domains, mediate and regulate protein complex formation. Cells use a limited set of interaction domains, which are joined together in diverse combinations, to direct the actions of protein networks. Interaction

TARGET DISCOVERY AND VALIDATION 49 domains are remarkably versatile in their binding properties, as indi- vidual domains can engage several distinct targets, either simultaneously or at successive stages of signaling. Different members of the same domain type can also bind to quite different targets. Regulation of protein complex formation frequently depends on biochemical modifications of the inter- action domains, including phosphorylation, hydroxylation, acetylation, methylation, and ubiquitination (Pawson and Nash, 2003~. Interaction domains play a pervasive role in regulating the dynamic organization and function of cells. For example, these domains mediate the association of cell surface receptors with their targets; the formation of signaling protein complexes inside the cell; and protein movement, organization, function, and degradation within the cell. Moreover, protein interaction domains not only enable linear pathways but can also generate more complex net- works that facilitate cross talk between pathways and that integrate sig- nals from distinct sources (reviewed by Pawson and Nash, 2003~. The Protein Quaternary Structure (PQS)25 database currently contains ~10,000 structurally defined protein assemblies of presumed biological significance derived from a variety of organisms. It is very difficult to predict the potential number of different protein complexes with unique structures and biological functions, especially in complicated organisms such as humans. The calculation of such estimates is a formidable task because of the many possible components and the various life spans of the protein complexes (transient versus stable complexes). In addition, there is no obvious definition of a "protein complex" and no means to determine whether two protein complexes are of different types. How- ever, currently available data suggest that there are at least 30,000 protein- protein interactions in yeast, a simple model organism. This number corre- sponds to about nine partners per protein, but these are not all necessarily direct or occurring at the same time. The human proteome may have an order of magnitude more protein complexes than the yeast cell (reviewed by Sali et al., 2003~. By studying protein complexes, it is possible in principle to assemble an atlas of protein networks within a cell. A variety of experimental methods are available for the identification and characterization of pro- tein complexes, and each has different advantages and limitations (Box 2.2 and Table 2.1~. Two of the most commonly used techniques for identify- ing 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. Such approaches have been used to identify proteins that interact with an oocyte-specific 25See htip://pqs.ebi.ac.uk/pqs-doc.shtm! (accessed July 2003~.

50 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH

TARGET DISCOVERY AND VALIDATION 5

52 ._, o 5- o u cry o o 5- o H ._' o En V) o my U 5- o En ._ 5- 5- X U 5- ) At X ¢ O En ~ <:L) 5- CD ¢ CD :^ CD ¢ CD ~ ¢ CD 5- ·= O ou cn co :^ ~ 'O cn ·= .~ U cn co u .= , ~ Q1 cn ~ ~ 5 - En ~1 `1 `` ~:::~ 1 `\ `~` ~ ~ ~ 1 :^ g 'o co co 5 ~ ~_ 5 j O x ~ n ~ ~ u ¢ :^ g CD o 5- U CD :^ 5- ~ ~ ~0 ~ 8m ~ ~ o , b · ~ ~ o ~ 8 b~ ,~o => ~ ~ o o=~= ~ ~ o ° ~ ~ o ~ .-U ~ ~ .k ~ m~ ~ o ~ ~ ~ ~ ~U ~ o ,.o ~ 5—~ CD ~ ~ :^ ~ ~ ~ ~ ~ ~ ~ .= l ,~ U O ~L, m-~ ~ ~ ,~ ,= ~ ,= 'o ~ 'o -o ~ ·m o m~ ~ .Y ~ 3 ^~- ,= ~ ~ ~ ~ o ~ Co o ~ ~ ~ ~ ~ ~ U) ~ ~ ~ C ~ ~ ~ ~ ~, 5 U

TARGET DISCOVERY AND VALIDATION 53 enzyme that functions in a key protein degradation pathway (Suzumori et al., 2003~. With advances in automated technologies, protein interac- tions can now also be identified through a variety of high-throughput screening tests, but predictive data must still be validated by direct analy- sis of protein complexes from cells. Individually, each approach is prone to error and fails to capture all the relevant information about the dynamic activities of proteins within cells, but, taken together, they provide a higher level of information. For example, combining the results of several techniques has identified an interaction network in yeast (reviewed by Pawson and Nash, 2003~. 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. Moreover, the modulation of protein interactions is a promising approach to drug design because small molecules (e.g., kinase and phosphatase inhibitors) can be used to modify protein complexes in a variety of ways. The direct approach of inhibiting interactions is potentially of great value and has already yielded some potential drug compounds with in viva activity for thera- peutic indications (Cochran, 2000~. Genetic Networks Recent experiments using the techniques described in this chapter have generated data that can be used to ascertain the functions of many genes. A major challenge that remains is the development of a fundamen- tal description of cellular function at the DNA level by determining the complex dynamic interactions and regulation of all the genes of an organ- ism its genetic networks (Banerjee and Zhang, 2002; Hasty et al., 2001~. Genetic networks are models of how external signals affect gene expres- sion and how changing activity of one gene or a group of genes affects the activity of other genes (Brazhnik et al., 2002~. The goal is to link the genes and their products into functional pathways, circuits, and networks. Models of gene regulation commonly represent networks of genes as if they directly affect each other. Although genes do not interact directly with other genes, network models use this concept because gene induc- tion or repression occurs through the actions of specific proteins, which are, in turn, products of other genes. The theoretical concept of interacting genetic networks was first explored nearly 30 years ago, but progress in the field had to await technological advances that could facilitate the col- lection of data on gene expression and function on a larger scale (Hasty et al., 2001~.

54 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH Genes ~ >~ Gene6 t ~ \ Gene 2 | Gene 3 |~ \ /~// ~ Gent Gene 1 |1~/ FIGURE 2.6 An example of a hypothetical gene network. Lines show direct effects, with arrows indicating activation and bars indicating inhibition. Models of genetic networks are commonly represented by diagrams (Figure 2.6) that display causal relationships between gene activities (Brazhnik et al., 2002~. These schematics of genetic networks resemble cir- cuit diagrams, and in many ways this analogy highlights the motivation for a quantitative description of gene regulation. If a genetic network were a complex electrical circuit, there would be an accompanying set of equa- tions that would reliably describe its functionality. This description would be built from knowledge of the properties of the individual gene compo- nents (analogous to resistors, capacitors, inductors, and so on) and pro- vide a framework for predicting the behavior that would result from modifications in the circuit (Hasty et al., 2001~. 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 under- stand the dynamics and design principles of gene regulation and, second, to reverse engineer genetic networks from experimental measurements (Brazhnik et al., 2002~. In recent years, the majority of research in genetic networks has focused on methodologies for reconstructing genetic net- works from experimental observations of gene expression, presumably because of the abundance of microarray data. However, many interactions between genes have also been discovered through traditional molecular biology approaches. Genetic networks can be postulated by combining

TARGET DISCOVERY AND VALIDATION 55 data about these interactions from various sources. The GeNet database26 is an example of an electronic repository for such information. Experimental techniques alone are limited in their ability to deduce genetic networks. Similarly, although computational models can offer insight into basic mechanisms, modeling must ultimately be connected to experimental systems so that verifiable predictions can be made. The most useful methods will likely entail a combination of novel experimental approaches and new computational modeling techniques (Hasty et al., 2001~. Thus, it is necessary to develop sophisticated computational tools to extract relevant information from different data sources (van Someren et al., 2002~. Many different approaches for developing models of genetic networks have been proposed and tested, and each of the modeling tech- niques has its own merits and drawbacks (Brazhnik et al., 2002; Hasty et al., 2001~. Examples of methods used to decipher gene expression data are described in Box 2.3. So far, few computational modeling studies have involved tight coupling between modeling and experiments (Hasty et al., 2001~. Addi- tionally, current experimental data from which networks are inferred can be extremely difficult to analyze and interpret because of inconsistencies from one experiment to the next. Furthermore, in the foreseeable future, the numbers of samples and data points, even in the largest experiments, do not provide enough information to construct a full, detailed model with high statistical confidence. Thus, the results of many more experi- ments will be needed to develop genetic networks with high degrees of accuracy and predictability. 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. However, when successful, understanding the network of regulatory interactions could yield a wealth of useful informa- tion, such as the pathway to which a gene belongs, the function of the gene in terms of how it influences other genes, and the identification of genes that serve as pathway initiators and that are therefore potential drug targets (van Someren et al., 2002~. For example, genetic networks might allow genes to be ranked according to their importance in controlling and regulating cellular events. In this way, knowledge about genetic networks might provide valuable information for drug discovery and development by aiding the prioritization of targets (Brazhnik et al., 2002~. 26GeNet contains information on the functional organization of regulatory genes networks acting at embryogenesis. See http://www.csa.ru/Inst/gorb_dep/inbios/genet/genet.htm (accessed October 2003~.

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., infertil- 27Examples 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.

TARGET DISCOVERY AND VALIDATION 57 ity]) and reverse genetic screens (those that block a function of a repro- ductive tract-specific gene via gene knockout or knockdown approaches with the goal of defining the function of a gene in reproduction) are ex- tremely valuable and complementary. These in viva genetic screens are key steps in the validation process. Flies, fish, worms, and mice are the easiest in viva models to manipulate for contraceptive evaluation. The mouse has been an important mammalian model for understanding and studying reproduction in humans for many years because most of the reproductive hormones and growth factors are highly conserved between mice and humans. However, target validation in mice or lower organisms is not foolproof. For example, targeting a particular gene may inhibit fer- tility in mice but not humans, or vice versa, because of gene redundancy, a situation in which multiple genes within a genome can perform a simi- lar function in a given biological process or pathway. 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. Experimental approaches include "knockout" models, in which a specific gene is deleted or rendered nonfunctional in animals, and "knockdown" models, in which the level of expression of a particular gene is reduced. Knockdown approaches that use RNA interference tech- niques (described below) have been successful in the analysis of a few genes expressed by oocytes. Other knockdown approaches include RNA antisense methods and chimeric dominant negative protein inhibitors. Knockout Models The most exhaustive studies with mammals have used embryonic stem (ES) cell technology to create knockout mice. With this technology, a selected gene is mutated in mouse ES cells in vitro. These mutated ES cells are then transferred back into preimplantation embryos, and mutant mice with the selected mutation are produced. Mice that carry this mutation are called "knockout" mice. If the gene of interest is normally expressed only in reproductive organs, then mice lacking this gene are expected to be viable without any defects outside the reproductive tract. If a knockout mouse lacking a protein is fertile or subfertile, then that protein would be viewed as a less promising contraceptive target (i.e., humans in whom expression of that gene is blocked by drugs would also be fertile). However, if a knockout mouse lacking a protein is infertile, then that protein would be viewed as an excellent contraceptive target

58 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH (i.e., blockade of this protein target in humans would demonstrate a contra- ceptive effect). Technology is now available to conditionally knockout expression of a gene in a specific tissue in adult mice. 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 else- where. A few examples of these mutations are described later in this chap- ter. Despite the progress in this area, funding is limited for reverse genetic screens based only on analysis of the reproductive tract-specific expres- sion of a gene. The reason for this is that these gene-based screens are not viewed as hypothesis driven. However, these types of screens should be encouraged because extension of the screening database will provide more options for the identification of candidates with which to move forward for further validation. RNA Interference A new approach for studying the functions of specific genes is to reduce or eliminate gene expression through a process known as RNA interference (RNAi). RNAi is an innate cellular process that is initiated when a double-stranded RNA molecule enters the cell, causing the activa- tion of proteins that destroy both the invading RNA and the endogenous single-stranded RNAs with identical sequences inside the cell. First iden- tified in the nematode worm (Caenorhabditis elegans), this degradation pathway has since been found to operate in many organisms. The process most likely developed as a way to control pathogens and as a mechanism to control gene expression, but it has recently been harnessed by scientists as an efficient and powerful way to experimentally manipulate gene expression (reviewed by Hannon, 2002; Shuey et al., 2002~. By designing appropriate double-stranded RNA molecules to match the sequence of a specific target mRNA, RNAi provides a relatively simple and fast way to block gene expression in experimental organisms as varied as plants, worms, flies, and mammals. For example, the RNAi approach has already been shown to be a useful technique for studying the functions of oocyte- specific genes and other RNA transcripts that play critical roles in oocyte maturation in mice (Stein et al., 2003; Svoboda et al., 2000~. RNAi can also be used in high-throughput genetic screens to examine gene function on a whole-genome scale in organisms that range from worms to humans. Scientists are developing RNAi libraries that can be used to identify the relevant genes associated with a specific phenotype and also to subse- quently confirm the functions of genes that have been identified.

TARGET DISCOVERY AND VALIDATION 59 Although application of the technology is still in its infancy, there is great optimism for the potential of this new tool. Nobel laureate Phillip Sharp, professor and director of the McGovern Institute for Brain Research at the Massachusetts Institute of Technology, calls RNAi the most impor- tant breakthrough in the past decade (Dutton and Perkel,2003~. However, cautious optimism is warranted, given the as yet unfulfilled expectations of past advances such as antisense RNA and ribozymes, other genetic tools used to inhibit gene expression. Furthermore, a recent study showed that small double-stranded RNA molecules can sometimes change the expres- sion of nontarget genes, potentially confounding experimental results as well as therapeutic effects (Bridge et al., 2003; Jackson et al., 2003~. None- theless, dozens of companies are banking on the promise of RNAi tech- nology both as a research tool and for its potential in drug development (Dutton and Perkel, 2003~. Several collaborations between biotechnology and pharmaceutical companies are under way to design gene- and transcript-specific inhibitors for gene function evaluation and target validation. Efforts are also under way to develop RNAi interventions to target viruses, inflammatory diseases, and metabolic diseases. Scientists have already shown the efficacy of RNAi in blocking viral replication, including that of HIV (Martinez et al., 2002), as well as disease states like hepatitis and cancer in experimental models (Agami, 2002; reviewed by Dutton and Perkel, 2003; Shuey et al., 2002~. The first clinical trials are expected within the next few years. Forward Genetic Screens Another approach to the identification of important functional genes is the use of mutational screens. Known as forward genetics, this approach entails the induction of random mutations in an experimental cell or animal, identification of a relevant mutant phenotype, mapping and iso- lation of the gene that has been mutated, and finally, isolation and charac- terization of the protein encoded by the gene. Large-scale screens for mutations in the fruit fly (Drosophila melanogaster) recently led to the iden- tification and classification of nearly 2,400 mutations that result in sterile males.28 This collection of mutations has provided a wealth of material for defining the number and types of gene products that control the various aspects of male fertility, from germ cell formation and the development of sperm and eggs through fertilization and even into early embryonic 28Barbara T. Wakimoto, professor of biology, University of Washington, Seattle, in a pre- sentation at the International Symposium on New Frontiers in Contraceptive Research, Washington, DC, July 15-16, 2003.

60 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH development. Because of the striking conservation of many molecular pathways used in animal cells of different species, the lessons learned from studies with Drosophila have enormous potential to enable discovery of the proteins required for sperm function in humans and thus may pro- vide insight in the search for new contraceptives. Studies that used a similar approach were recently undertaken with mice as well. For example, large-scale production of chemically induced mouse mutations has led to the identification of a number of mutations that affect fertility (Kile et al., 2003~. In another study, a variety of muta- tions that affect essentially all stages of sperm and egg development have been identified, but more mutations were found to have an impact on male fertility than on female fertility.29 On the basis of the results of these preliminary studies demonstrating the effectiveness of forward genetic screens for infertility mutant genes in mice, NIH has provided funding to conduct a large-scale project to isolate scores of infertility mutant genes that represent a substantial fraction of all genes required for mammalian fertility. The results will be made available to the scientific community for further study. One limitation of forward genetics experiments is that they are very laborious and are thus quite expensive and time-consuming, even with current methods and technologies. In particular, the target of the gene in forward genetic mutagenesis screens is often difficult to identify. Hence, the development of alternative mutagenesis screens would be very helpful and should also be encouraged. One possible alternative is the transposon mutagenesis method developed by Largaespada and colleagues (Dupuy et al., 2002; Roberg-Perez et al., 2003~. A transposon is a short sequence of DNA that can change location in the genome and normally contains genes that code for proteins that enable it to change location. By inserting a trans- posable element into the mouse germ line, scientists can induce the move- ment of the transposon into a new (random) location. If the transposon inserts into a location containing a gene, then the transposon should mutate this gene, thereby creating a novel mutation. Mice can be bred to homozygosity to evaluate the phenotypic effects of the mutation. By DNA sequence analysis, the new site of insertion of the transposon can be accu- rately determined. 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 Sympo- sium on New Frontiers in Contraceptive Research, Washington, DC, July 15-16, 2003. 30See http: / /mouse.ccgb.umn.edu/transposon/ (accessed September 2003~.

TARGET DISCOVERY AND VALIDATION 61 al., 2003~. The site of the mutation (the trapped gene) can also be identified by DNA sequencing. The European ES Cell Consortiums has generated mutations in 11,000 genes in ES cells. These ES cells are publicly available. PROMISING NEW TARGETS There are many possible tissues and cells in the reproductive tract where inhibition or activation of gene product function could result in an infertility phenotype, and there are multiple options for either validation or rejection of target genes. Furthermore, after potential targets have been identified and validated, choosing the most promising targets for the next stages of drug discovery and development is enormously challenging. The drug development process is extremely costly and very risky, so wise selection of targets is essential to optimize the use of available funds and resources. A list of key criteria for target selection in contraception devel- opment is presented in Table 2.2. The committee considered the following areas to be particularly promising for the discovery of new targets: the male spermatogenesis pathway, sperm maturation (both sperm and epididymal proteins), sperm capacitation, motility and chemotaxis in the female reproductive tract, proteins and molecules in the female reproductive system (vagina, cervix, uterus, and oviduct), sperm-egg interactions (both sperm and egg pro- teins and molecules), and maturation and ovulation of the egg (both somatic cell- and egg-specific proteins and molecules). 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. The basic biologies of the epithelium of the vagina, the cervix, the endometrium, and the oviduct are fairly well understood with regard to the morphologic changes induced in these tissues by estrogen and progesterone. However, identification of targets for contraception by a genomics and subsequently proteomics approach is just beginning to be investigated. Several recent studies (Borthwick et al., 2003; Carson et al., 2002; Kao et al., 2002; Riesewijk et al., 2003) revealed potential targets in the human endometrium for inhibition of embryo implantation, including cell surface carbohydrate moieties, ion channels, essential element transporter proteins, and secretory proteins. Moreover, the role of the endometrial epithelium during sperm transport on its way to fertilize the egg is poorly understood and offers an impor- tant area of contraceptive target development. Likewise, identification of contraceptive targets in the epithelium of the vagina, cervix, and oviduct 3iSee htip://genetrap.de (accessed September 2003~.

62 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH TABLE 2.2 Defining the Key Criteria for Novel Contraceptive Target Selection Criteria Examples of Desirable Characteristics Expression Uniquely or selectively expressed in reproductive tract tissue or organs involved in reproduction Function Inhibiting function specifically and completely disrupts or alters a process unique to reproduction Timing of action Close to time of fertilization (e.g., postmeiotic events in gametogenesis) Potential for reversible Druggable protein classes such as enzymes, membrane modulation by a drug proteins, receptors, and ion channels and transporter proteins Potential route of Amenable to simple delivery systems ensuring ease of use and administration high rates of compliance Potential for product Inexpensive and easy to produce manufacture Potential for Dual protection against sexually transmitted infections and conception; protection against cancer or other diseases noncontraceptive benefits is needed and constitutes an important area for further investigation. This approach may offer the advantage of localized delivery through the vagina to minimize potential systemic effects of compounds that may impair epithelial function. It is clear from previous pharmaceutical development studies that specific types of proteins (e.g., receptors, ligands, enzymes, and ion chan- nels or transporters; see Figure 2.1) 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 pro- teins with high priority as targets for drug development. In addition to the general mouse models described above, specific examples of these types of proteins are discussed in the next section. This list is not meant to be exhaustive, and the committee recognizes that new targets will emerge even before the publication of this document.

TARGET DISCOVERY AND VALIDATION 63 Sperm Chemotaxis The sperm of marine invertebrates exhibits strong chemotaxis (attrac- tion by chemicals) in response to a variety of signals. It has long been hypothesized that mammalian sperm may use similar mechanisms to find and fertilize an egg. The existence of sperm chemotaxis was first shown experimentally in rabbits by Dickmann (1963~. Further experiments, also with rabbits, provided evidence that this phenomenon is triggered by the products of ovulation (Harper, 1973~; and this has been confirmed by Fabro et al. (2002), who concluded that rabbit sperm are chemotactically attracted to follicular fluid. Earlier studies by the latter group showed that sperm accumulation in follicular fluid was the result of both sperm chemo- taxis and chemokinesis, followed by hyperactivation of motility. They suggested that at least one chemotactic factor was a small (less than 10,000- dalton), nonhydrophobic molecule (Raft et al., 1994) and that there was a sequential acquisition of a chemotactic response by human sperm (Cohen- Dayag et al., 1994~. A recent study indicates that human sperm also have the ability to detect and respond to chemotactic signals through a novel receptor called hOR17-4 (Spehr et al., 2003~. This protein belongs to the olfactory receptor (OR) family, which includes 500 to 1,000 members that are expressed in the neural tissue of the nose. Despite the name, some ORs are also expressed in other tissues, but their role in these locations is unclear. About 50 ORs are known to be expressed in the testis, many predominantly or exclu- sively by cells involved in spermatogenesis. Although it has been known for some time that several OR proteins are present in sperm, their function has remained a mystery. The recent study demonstrates that the newly identified sperm OR causes the sperm to navigate in a specific direction when it detects a con- centration of specific laboratory chemicals, while other chemicals can act to block this specific movement (Spehr et al., 2003~. However, the physi- ological chemical that attracts sperm in the female reproductive tract remains to be revealed. It is also not clear whether the physiological chemoattractant must necessarily be secreted by the egg or whether production by other cells or tissues in the female reproductive tract might suffice (or perhaps be even more efficient) in properly directing the sperm toward the egg in the oviduct. In any case, the data suggest that sperm chemotaxis may be a critical component of the fertilization process, and thus inhibition of the hOR17-4 signaling system could potentially block fertilization and serve as a novel contraceptive (Spehr et al., 2003~. How- ever, this hypothesis is controversial and needs further examination. If validated, the goal is to find a compound that, when placed in the female reproductive tract, would confuse the directional signals that sperm need

64 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH to find and fertilize the egg. A better understanding of how the hOR17-4 signals the sperm to change direction or motility would be helpful in this regard, as the signaling pathways associated with OR proteins are gener- ally quite complex (Babcock, 2003~. Sperm-Egg Interaction Before fertilization, sperm must adhere to and fuse with the plasma membrane of the egg. Thus, molecules involved in these processes are candidate targets for new contraceptive development. With that goal in mind, a combination of experimental approaches has been used to deter- mine the protein molecules on the surface of the sperm or egg required for adherence and fusion. Several proteins have been shown to play a role in adhesion, and these adhesion-mediating proteins likely function within the context of multimeric complexes at the cell surface (Evans and Florman, 2002; Primakoff and Myles, 2002; reviewed by Talbot et al., 2003~. The molecular basis for the intercellular fusion, however, remains elusive. Some of the proteins involved in adherence were initially thought to play a role in fusion as well, but gene knockout studies have disproved that hypothesis. Research on other proteins on the egg surface has pointed primarily in two directions.32 Some proteins have a glycosylphosphatidylinositol (GPI) membrane anchor. GPI is a lipid that is attached to the protein and in- serted into the lipid bilayer of the plasma membrane to anchor the protein on the surface of the cell. Proteins with this membrane anchor have been implicated in fusion because treatment of eggs with an enzyme that dis- rupts this anchor and releases these proteins from the surface results in eggs that do not fuse with sperm (Alfieri et al., 2003~. Recent results show that female mice lacking the gene for an enzyme required to make the GPI membrane anchor (and that are therefore lacking GPI-anchored egg pro- teins on their surface) are infertile (Alfieri et al., 2003~. These animals pro- duce eggs that mature normally but are defective in sperm-egg fusion. Scientists are now searching for the particular GPI-anchored egg proteins that play a critical role in fusion and that could thus be suitable targets for contraceptives. The second direction for research on proteins of the egg surface involves CD9, a member of the protein family called tetraspanins (because they cross the plasma membrane four times) that binds to integrins and facilitates cell proliferation, motility, and adhesion. 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.

TARGET DISCOVERY AND VALIDATION 65 the gene for CD9 is deleted are infertile; their eggs cannot fuse with sperm. Ongoing efforts are aimed at determining what regions of the protein are directly involved in and essential for fusion and whether other proteins on the surface of the egg interact with CD9 to form a multiprotein struc- ture or complex in the process of fusion (Zhu et al., 2002~. Scientists are also searching for the protein on the surface of the sperm that interacts with CD9 on the egg. Significant insight into the membrane fusion process has also come from studies of virus-cell fusion (Evans and Florman, 2002~. Scientists have recently discovered the sites of fusion between HIV and other viruses in a host cell membrane. They have identified particular fusion proteins and have been gaining insights into how these proteins work. Importantly, precedent for therapeutic blocking of membrane fusion already exists in the literature. New drugs based on small peptides that can act as fusion inhibitors have been developed, and one of them, enfuvirtide (Kirby et al., 2002), was recently approved for use by the Food and Drug Administra- tion. Although less is known about cell-cell fusion events, new insights are emerging from studies with sperm from sea urchins and abalone (Evans and Florman, 2002~. -r Proteins That Function in Gamete Metabolism and Maturation Glyceraldehyde 3-phosphate dehydrogenase 2 (GAPDS or GAPD2) is an enzyme required for glycolysis (the breakdown of sugar to produce energy) that is expressed specifically in the testes of mice and humans (Welch et al., 2000~. GAPDS is associated with the sperm tail. Because of the important role of GAPDS in glycolysis, male knockout mice lacking GAPDS are infertile. Sperm from these knockout mice have a motility defect (i.e., they do not have the normal "swimming" motion). This sug- gests that contraceptive drugs in the male or female reproductive tract that block GAPDS activity also would prevent the sperm from reaching an egg (Qu et al., 2003~. CKS2 is one of the mammalian cyclin-dependent kinase 1 binding protein homologues. Although this protein is expressed in multiple tis- sues, knockout mice lacking the gene for CKS2 have a normal phenotype, except that they are infertile (Spruck et al., 2003~. 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 mei- otic proteins that interact with CKS2 could be considered contraceptive drugs. The egg protein known as zygote arrest 1 (ZAR1) is a novel oocyte- specific protein that was identified in sequence-based screens (Wu et al.,

66 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH 2003a, b). The protein is highly conserved and specific to the oocytes of fish, frogs, rats, mice, and humans (Wu et al., 2003a, b). The zarl gene is a member of a class of genes called maternal effect genes, which are ex- pressed in the maternal oocyte but which play a role after ovulation. The absence of the ZAR1 protein in the egg prevents the fusion of the male and female pronuclei, thereby arresting further development (Wu et al., 2003a). This results in infertility in females lacking the zarl gene. Blockade of the pathway in which the ZAR1 protein functions would be expected to cause a contraceptive effect. Sperm Motility Proteins Calcium ions play a major role in the regulation of sperm cell function. 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. The male knockout mice are sterile because their sperm cannot fertilize intact eggs, but the animals are otherwise normal and healthy (Ren et al., 2001~. Thus, molecules that target the CatSper proteins could potentially serve as an effective contra- ceptive. Another protein, known as kinase anchoring protein (AKAP) 4 (Miki et al., 2002), is the major component of the sperm flagellum. Akap4 is a male germ cell-specific gene that is transcribed only in the postmeiotic phase of spermatogenesis. AKAPs bind to the regulatory subunits of an enzyme called protein kinase A, as well as other proteins that control inter- cellular signal transduction pathways, such as kineses and phosphatases. Targeted deletion of the akap4 gene in mice results in a male infertility phenotype (Miki et al., 2002~. Sperm numbers were not reduced, but sperm failed to show progressive motility because of abnormal tail devel- opment. Consistent with the findings with the knockout mouse model, small peptides that block the protein kinase A regulatory subunit binding sites of AKAP cause sperm motility defects in bovine epididymal sperm (Vijayaraghavan et al., 1997), indicating that further efforts directed toward this protein as a potential contraceptive target should be pursued. Hormone and Cytokine Signaling The progesterone receptor mediates the genomic action of progester- one, a steroid hormone that is required for ovulation and preparation of the uterus for the process of implantation. The progesterone receptor gene

TARGET DISCOVERY AND VALIDATION 67 gives rise to multiple proteins as a result of alternative transcription and translation start sites. The best-characterized progesterone receptor isoforms are the A and B forms. The A form is generally a repressor of transcription, whereas the B form is the longer protein and is a transcrip- tional activator. Female mice lacking progesterone receptors are infertile because of a defect in the process of ovulation as well as a uterine abnor- mality because of the inability to develop a receptive state and an associ- ated inflammatory infiltration (Lydon et al., 1995~. In the periovulatory period, progesterone receptors are induced in ovarian granulosa cells. Drugs that block periovulatory progesterone synthesis as well as proges- terone receptor antagonists prevent ovulation (Shag et al., 2003~. Collec- tively, these findings demonstrate that events that are initiated by proges- terone and that act on the progesterone receptor are essential for the release of the oocyte from the mature follicle. Selective targeting experi- ments demonstrated that the progesterone receptor isoform required for ovulation is the A form (Mulac-lericevic et al., 2000~. The genes that are regulated by progesterone and that are essential for ovulation have yet to be elucidated, but analysis of differential gene expression through the study of transcript profiles of wild-type and progesterone receptor- deficient ovarian RNA suggests that degradative enzymes including cathepsin L and ADAMTS1 (a member of a family of proteins with a disintegrin and metalloprotease with thrombospondin type 1 repeat) are candidates for proteins that participate in the degradation of the follicle wall and thus release of the egg from the ovary (Richards et al., 2002; Robker et al., 2000~. Understanding the gene and protein networks associ- ated with progesterone action in the periovulatory period could yield new targets, including enzymes, for prevention of ovulation without disrup- tion of ovarian endocrine activity. A number of cytokines have also been identified as potential targets for development of anti-implantation strategies. For example, leukemia inhibitory factor (LIF) is essential for implantation in mice (Bhatt et al., 1991), and there is reason to believe that it plays a similar role in primates (Charnock-Iones et al., 1994; Chen et al., 1995; Cullinan et al., 1996; Vogiagis et al., 1996; Yue et al., 2000~. Interleukin-ll (IL-ll) is intimately involved in early decidualization in mice (Robb et al., 1998) and appears to play a similar role in humans (Dimitriadis et al., 2000, 2002~. Work is under way to develop peptide inhibitors that block activation of the uter- ine LIF and IL-ll signaling pathways (Fairlie et al., 2002~. RECOMMENDATIONS Major advances in identifying and verifying contraceptive targets in males and females have been made since publication of the 1996 Institute

68 NEW FRONTIERS IN CONTRACEPTIVE RESEARCH of Medicine report. These targets are, in some cases, uniquely and exclu- sively involved in reproduction. The committee recognizes the existence of several promising targets (i.e., proteins that have been shown to play key roles in gamete maturation, function, or interactions) and strongly recommends that these targets be pursued using high-throughput drug discovery approaches (see Chapter 3~. 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. Recommendation 1: Identify and characterize all genes and proteins uniquely or preferentially expressed in the testis, ovary, and repro- ductive tissues; and define the genetic and protein networks in cells relevant to reproduction, including construction of a protein inter- action map for the sperm and the egg. Emphasis should be placed on selective screening methods to iden- tify classes of molecules that have been traditionally targeted by pharma- ceutical compounds, including membrane proteins, enzymes, receptors, and ion channels or transporter proteins. An achievable short-term goal (less than 5 years) is the identification of all genes uniquely or preferen- tially expressed in all relevant reproductive cell and tissue types. This could be accomplished in part through the provision of continued and additional funding for a modest number of laboratories attempting to gen- erate the reproductive transcriptome. However, to complete this task more rapidly, the committee recommends that a broad group of reproductive biologists, bioinformaticists, biochemists, and physiologists convene a meeting with the sole purpose of verifying and annotating all gene expression data obtained through genomic methods. The information generated should then be stored in readily accessible World Wide Web- based databases (e.g., the Mammalian Reproductive Genetics database,33 the Mouse Genome Informatics database,34 or GermOnline35~. Equally im- portant, upkeep of the database will require vigilant review, annotation, and standardization if it is to be a useful and valuable tool for the research community (MacNeil, 2003~. In parallel with genomic approaches, the committee recognizes the need, and unique opportunity, to apply proteomic methods to contracep- tive research. One goal that is being pursued is the creation of sperm and 33See http://mrg.genetics.washington.edu/ (accessed September 2003~. 34See http: / /www.informatics.jax.org/ (accessed September 2003~. 35See http://www.germonline.unibas.ch/ (accessed January 2004~.

TARGET DISCOVERY AND VALIDATION 69 egg proteome databases. However, this is a large undertaking that will require additional allocation of human resources and financial capital. Recognizing that genes and proteins do not act autonomously, the com- mittee recommends that substantial new resources be devoted to identi- fying and constructing genetic and protein networks as well. This is a more long-term goal (greater than 5 years), but one that should be initi- ated immediately. Genomic and proteomic approaches can also identify metabolic path- ways and enzymes that are involved in the biosynthesis and catabolism of glycoproteins, glycolipids, and other lipids. Functional genomic approaches can reveal the importance of these pathways to reproductive processes. However, these techniques cannot establish the structures of unique carbohydrates on proteins and lipids or the content and organization of lipid domains within membranes. Consequently, different experimental methods are needed to characterize unique features of the glycomes and lipidomes of reproductive tract tissues and gametes. Recommendation 2: Generate a lipidome and glycome of the repro- ductive tract tissues and mature gametes. The glycomes and lipidomes of reproductive tract tissues and gametes have unique features not shared by other somatic tissues, and cell surface lipids and proteins represent logical targets for drugs, particularly small molecules that can interact with carbohydrate structures or insert in hy- drophobic domains. Thus, the committee recommends that the investiga- tion of the novel characteristics of the reproductive glycome and lipidome be pursued with the goal of identifying targets and small molecules that act to selectively disrupt membrane structure and function. The Consor- tium for Functional Glycomics, funded by the National Institute of Gen- eral 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. Some of the identified targets described here will be validated or rejected during the normal progress of typical NIH-sponsored research programs. However, in some circumstances, study sections may not fund studies for the genetic validation of targets with unknown biochemical or molecular functions. To address this bottleneck, the committee recom- mends that a small consortium of investigators (public or private) be funded for the sole purpose of completing the genetic validation of all potential targets. To accomplish this goal most efficiently, newly estab- lished genetic models should be rapidly distributed to the community of

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More than a quarter of pregnancies worldwide are unintended. Between 1995 and 2000, nearly 700,000 women died and many more experienced illness, injury, and disability as a result of unintended pregnancy. Children born from unplanned conception are at greater risk of low birth weight, of being abused, and of not receiving sufficient resources for healthy development. A wider range of contraceptive options is needed to address the changing needs of the populations of the world across the reproductive life cycle, but this unmet need has not been a major priority of the research community and pharmaceutical industry. New Frontiers in Contraceptive Research: A Blueprint for Action, a new report from the Institute of Medicine of the National Academies, identifies priority areas for research to develop new contraceptives. The report highlights new technologies and approaches to biomedical research, including genomics and proteomics, which hold particular promise for developing new products. It also identifies impediments to drug development that must be addressed. Research sponsors, both public and private, will find topics of interest among the recommendations, which are diverse but interconnected and important for improving the range of contraceptive products, their efficacy, and their acceptability.

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