B: Male Methods

David W. Hamilton, Ph.D.

Department of Cell Biology and Neuroanatomy,

University of Minnesota Medical School

Patricia M. Saling, Ph.D.

Department of Obstetrics and Gynecology and

Department of Cell Biology,

Duke University Medical Center

Introduction

The ideal male contraceptive should be safe, effective and reversible and should not have an effect on libido. In addition, it should be self-administered with little training and require neither elaborate surgical procedures nor prolonged periods of either abstinence or alternative contraceptive techniques to be effective. The biology of the male reproductive tract puts certain limitations on the development of novel contraceptive strategies yet, because of the unique features of cells in the male reproductive tract, new possibilities are presenting themselves that need to be carefully addressed.

Current Options

Contraceptive options for men are extremely limited. By far the most common contraceptive method in use today is the condom and, barring unforeseen complications such as puncture, this method can be highly effective both in contraception and in protection against sexually transmitted diseases for both men and women. A major advantage of the condom is that its effect is immediate and it does not require periods of abstinence or, indeed, use of other contraceptive approaches to be effective. Reversal is also immediate.

Vasectomy, on the other hand, which is the other most common and effective form of contraception used by men today (e.g., Wang et al. 1994), requires alternative contraceptive techniques for varying periods after the operation until there are no longer sperm in the ejaculate (azoospermia). Vasectomy also has the



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--> B: Male Methods David W. Hamilton, Ph.D. Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School Patricia M. Saling, Ph.D. Department of Obstetrics and Gynecology and Department of Cell Biology, Duke University Medical Center Introduction The ideal male contraceptive should be safe, effective and reversible and should not have an effect on libido. In addition, it should be self-administered with little training and require neither elaborate surgical procedures nor prolonged periods of either abstinence or alternative contraceptive techniques to be effective. The biology of the male reproductive tract puts certain limitations on the development of novel contraceptive strategies yet, because of the unique features of cells in the male reproductive tract, new possibilities are presenting themselves that need to be carefully addressed. Current Options Contraceptive options for men are extremely limited. By far the most common contraceptive method in use today is the condom and, barring unforeseen complications such as puncture, this method can be highly effective both in contraception and in protection against sexually transmitted diseases for both men and women. A major advantage of the condom is that its effect is immediate and it does not require periods of abstinence or, indeed, use of other contraceptive approaches to be effective. Reversal is also immediate. Vasectomy, on the other hand, which is the other most common and effective form of contraception used by men today (e.g., Wang et al. 1994), requires alternative contraceptive techniques for varying periods after the operation until there are no longer sperm in the ejaculate (azoospermia). Vasectomy also has the

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--> disadvantage of requiring a surgical procedure, although the degree of invasion is minimal (especially with nonscalpel vasectomy [NSV] with chemical or other methods of vas occlusion); however, reversal requires a more elaborate surgical procedure, with return of fertility dependent upon numerous unknown factors. Furthermore, vasectomy provides no protection against transmission of sexually transmitted infections. Variations on the theme of vasectomy have been suggested over the years, for example, valves that regulate vasal fluid flow (Kuckuck et al. 1975), and interruption of control of the vas musculature (Amobi and Smith 1995). A promising approach has been developed in China where percutaneous intravasal injection into the vas lumen of a quick-curing polymer results in a plug that effectively blocks seminal flow (Zhao et al. 1992). Still, this approach also requires a period of alternative contraceptive use until azoospermia is achieved (as much as 12 months [Chen et al. 1992]), although reversal is a simple and apparently effective procedure of merely removing the plug and does not require elaborate microsurgery. These mechanical methods rely on restricting movement of sperm into the female reproductive tract. An equally effective strategy could be to render sperm inactive prior to ejaculation or to produce azoospermia with drugs. In the following sections we provide an overview of research in male contraception and suggest strategies that might be employed to develop effective contraceptives for men. In order to provide some rationale for different strategies, we begin with a brief excursion into the structure and regulation of function in the male reproductive tract. Overview of the Male Reproductive Tract In developing contraceptives for men, it is obviously essential to take into account the dynamics of the male reproductive system, particularly spermatogenesis. Spermatogenesis is a precisely timed process whose regulation is a poorly understood, complex set of interactions among many cell types. Post-testicular sperm maturation is also highly complex, regulated as it is by circulating factors from the testis and from other organs, as well as by factors derived from intraluminal secretions of the testis itself. The intricacy of these processes is well recognized, but most of its details are unknown. The contraceptive strategy employed, therefore, will depend upon where in the male reproductive tract intervention occurs. Gross Structure Sperm are produced in the seminiferous tubule of the testis; are then released into the lumen of the tubule as immature cells, unable to fertilize eggs; and are carried by bulk flow through the rete testis and efferent ductule into the single,

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--> coiled tubule that comprises the epididymis. Movement of sperm into the epididymis from the testis is rapid, but as sperm traverse the efferent ductules, >90 percent of the water in the fluid that surrounds them is removed (Crabo 1965). This, in turn, produces a viscous, sperm-containing epididymal fluid that, over a time span of a week or more, is moved slowly through the epididymis by contractions of the smooth muscle surrounding the duct. During this transit, sperm acquire the ability to fertilize eggs through metabolic and other biochemical changes, and also develop the ability to move progressively; these modifications are collectively termed ''sperm maturation." The matured sperm are stored in the tail of the epididymis until ejaculation, at which time they move through the vas deferens and its ampulla; mix with secretions of the prostate, seminal vesicle, and bulbourethral glands; and exit through the penile urethra. From the time spermatogonia begin to differentiate into sperm until the time of ejaculation, the cellular and fluid environment, which is progressively modified, contributes to germ cell maturation, eventually assuring their ability to fertilize eggs. Contraceptive strategies can be developed that target events throughout this journey, that is, during the processes of sperm maturation. The Testis The seminiferous tubule is comprised of a structurally complex epithelium surrounded by muscle-like myoid cells, interstitial cells, and fluid. It is in this tubule that spermatogonia form into highly specialized sperm. The epithelium essentially comprises two cell types: Sertoli cells and germ cells. Three additional cell types—myoid cells, Leydig cells, and "immune cells"—surround the epithelium. Sertoli cells are tall, columnar somatic cells that extend from the base of the epithelium to the lumen of the duct. They surround and nurture the differentiating germ cells and, by means of structural specializations termed Sertoli-Sertoli junctions, segregate the germ cells undergoing meiosis (spermatocytes) and spermiogenesis (spermatids) from macromolecules in the blood and lymph vascular systems. Owing to this process of segregation—the so-called blood-testis barrier—the only germ cells accessible to macromolecules borne by blood or lymph are spermatogonia and early spermatocytes, although we do not know how or whether blood-borne factors affect spermatogonia. Germ cells undergo well-defined maturational events that comprise spermatogenesis, a process that begins at puberty with mitotic divisions of the primitive, Type A spermatogonia. This is followed by subsequent cell divisions—the number varies by species—of most of the daughter cells. Some daughter cells do not differentiate further; rather, these "resting" spermatogonia provide stem cells for future spermatogenetic events. The Type A spermatogonia that continue to divide eventually differentiate into Type B spermatogonia that are committed to enter meiosis. Once germ cells enter meiosis they are termed spermatocytes. The first meiotic division, carried out by primary spermatocytes, has a long prophase (see Dym 1983, or other histology texts for more detailed descriptions) and so these cells are frequently seen in histological sections of the seminiferous epithe-

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--> lium. The second meiotic division, carried out by secondary spermatocytes, has a short duration; as a consequence, these cells are more rare in histological sections than primary spermatocytes. Upon completion of meiosis, the cells enter spermiogenesis and begin the long process of differentiation from essentially round cells to highly polarized, highly differentiated sperm. In the human, the total process of spermatogenesis takes -64 days. In animals, the process takes less time but, interestingly, experimental studies to date have not been able to alter the timing of spermatogenesis. Germ cells comprise a histologically complex group of cells; the complexity derives in part from the fact that spermatogenesis, in humans, occurs in patches along the length of the seminiferous tubule and the patches are not all in synchrony. Within patches, however, there is synchronous differentiation. If one were able to sample one patch over the 64 days of spermatogenesis different cell associations, or stages (seven have been defined in the human), would be seen because of the timing of each phase (that is, mitosis, meiosis, and spermiogenesis). This lack of synchrony throughout the testis poses a major problem for the design of strategies for interrupting spermatogenesis, since it essentially determines a priori that any interruption of spermatogenesis would require at least a two-month delay before there was any noticeable decrease in sperm in the ejaculate. The myoid cells that surround the seminiferous epithelium have contractile properties. They release a glycoprotein, termed PmodS (Skinner and Fritz 1985) that affects Sertoli cells by activating the IP3 signaling pathway, although the function of PmodS is not known. Leydig cells produce testosterone, which supports spermatogenesis and also acts in the negative long-loop feedback system that regulates release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) (see below). There is considerable evidence that the "immune" cells (e.g., macrophages) affect Leydig cell functions (Hutson 1994). Interactions among these various major cell types in the testis have been amply documented. For instance, Sertoli cell products interact with germ cells to regulate their differentiation (see Sharpe 1993) and, recently, it has been shown that germ cells produce factors that affect Sertoli cell function (Onoda and Djakiew 1990, 1993). In addition, as described above, interstitial cells produce factors that affect Sertoli cells and possibly germ cells. In the aggregate, the observations on cell-cell interactions at the level of the testis ("short-loop feedback mechanisms") are extensive and could be targeted for contraceptive intervention. The Excurrent Ducts and Accessory Organs of Reproduction When sperm leave the testis and enter the first part of the epididymis, they are not capable of fertilizing eggs; it is the progression through the epididymis that results in their ability to fertilize. The maturation experienced by sperm in the epididymis involves both metabolic changes and interactions with the secretions of the epi-

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--> didymis and accessory organs of reproduction that lead to molecular modifications of the sperm surface (see below). Regulation of Function in the Male Reproductive Tract In addition to the regulation of function at the level of the seminiferous tubule through short-loop mechanisms mentioned above, there are endocrine feedback loops that support sperm development in the seminiferous tubule. These "long-loop interactions" (recently reviewed by McLachlan et al. 1995) are mediated by hormones produced in the brain and the gonads, the so-called hypothalamic-pituitary-gonadal axis. Secretion of the hypothalamic decapeptide GnRH (gonadotropin-releasing hormone) by hypothalamic neurons in the pituitary leads to release of the gonadotropins LH (luteinizing hormone) and FSH (folliclestimulating hormone) from gonadotroph cells in the anterior pituitary. LH and FSH are carried to the testis in blood, where LH stimulates production of testosterone by Leydig cells and FSH interacts with Sertoli cells to affect synthesis of proteins that are secreted both into the luminal compartment of the tubule and the blood vascular system (e.g., inhibin). Testosterone, carried by the blood and lymph, interacts with both the hypothalamus and pituitary to inhibit LH release, while inhibin inhibits FSH release. Regulation of function of the excurrent ducts occurs both by blood-borne factors, such as testosterone, and by factors not yet fully characterized that are secreted by the testis and carried through the tubule lumen where they can effect expression of proteins and glycoproteins secreted by the epididymis. Increasing evidence indicates that sperm, as well as innervation of the excurrent ducts, can directly affect epithelial cell function (Douglass et al. 1991). The long-loop feedback system has, in fact, already been targeted in a number of studies on male contraception (see Wang et al. 1994) by using such compounds as steroids and GnRH agonists and antagonists that have been previously used in studies on humans and for which toxicological information is available. Studies attempting interruption of the long-loop feedback mechanisms have involved injections of androgens to inhibit hypothalamic and pituitary functions related to gonadotropin release (e.g., Handelsman et al. 1992). Azoospermia was accomplished after variable periods of administration and, at the end of treatment, sperm returned to the ejaculate and the individuals were fertile. Similar strategies have been attempted using GnRH agonists and antagonists, but required ancillary injections of androgens to obviate loss of libido (Bagatell et al. 1993). (See Chapter 4 herein for further discussion of these approaches.) Potential Targets for Male Contraceptive Development In addition to the endocrine feedback loops referred to immediately above, there are many cellular and molecular events in the male reproductive tract that

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--> are unique to that tract. We would like to address potential areas of future research in two areas: the testis and the excurrent ducts. Inhibition of FSH Secretion and/or FSH Action Follicle-stimulating hormone (FSH) is a hormone produced by the anterior pituitary that regulates testicular function by binding to high-affinity receptors located in males exclusively on Sertoli cells. In mammals, the absolute requirement for FSH in the maintenance of adult spermatogenesis is controversial; however, it does appear to be generally agreed that in humans, as in nonhuman primates, qualitatively normal spermatogenesis requires FSH as well as testosterone (Zirkin et al. 1994). Therefore, contraceptive attack directed against either FSH or its receptor, if unique determinants are present, may offer a method to disrupt spermatogenesis without affecting steroid hormones. FSH is a heterodimeric 30 kD glycoprotein that contains about 30 percent carbohydrate. The hormone is composed of noncovalently associated subunits, an a chain 92 residues in length and a ß chain 111 residues in length. The FSH a subunit has a close structural relationship with the a subunits of LH, hCG and TSH (thyroid-stimulating hormone), whereas the ß subunit is unique and is considered to be responsible for the receptor specificity of FSH. The FSH receptor is an integral membrane protein containing the usual seven-pass transmembrane structure conserved for interaction with G-proteins at the cytoplasmic face of the plasma membrane. The extracellular domain of the FSH receptor is, however, unusually large for this class of receptor. The receptor exists in the membrane of the Sertoli cell as a homotetramer, and ligand-blot studies indicate that FSH binds only to the tetrameric form of the FSH receptor. Molecular details of the interaction of FSH with its receptor have been studied through the use of synthetic peptides corresponding to regions of the primary sequence of either FSH or its receptor, as well as by site-directed mutagenesis. These analyses reveal that the interaction between hormone and receptor is quite complicated. FSH appears to utilize different sites to effect receptor binding and signal transduction (Valove et al. 1994): FSH-ßArg35 is critical for efficient receptor binding but not for signal transduction whereas, in contrast, a specific oligosaccharide attached at FSH-aAsn52 has been reported to be critical for signal transduction but not receptor binding. Reciprocal studies dealing with the FSH receptor have mapped the linear nonhomologous sequence Arg265-Ser296 as important for FSH binding. These characteristics of the FSH/FSH receptor system suggest several possible and quite specific contraceptive tactics. Alteration of FSH Secretion Since the action of FSH is limited to the Sertoli cell alone, absence of FSH in the adult male would not be expected to lead to adverse side effects; indeed, trials that have been conducted thus far support this contention (see above). These studies utilized GnRH analogues or androgens to

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--> affect the long feedback pathway and produce infertility. The major stumbling block in this regard appears to be the metabolic clearance rate of either the androgens or the GnRH analogues. Some attention has been given to development of long-acting esters of testosterone (see Wang et al., 1994) but, to date, treatment still requires unacceptably frequent injections. Targeting of Unique Sequences of FSH for Immunologic Destruction Since FSH-ß is considered to be responsible for the receptor specificity of FSH, linear sequences—particularly those involved in binding to the receptor-—could be targeted by specific neutralizing antibodies. Thus, after defining the optimally immunogenic epitopes, individuals could be immunized with synthetic peptides corresponding to those epitopes. The desired consequence would be the elicitation of specific antibodies that would bind to FSH in the circulation and prevent the hormone's interaction with its receptor. Compared to the foregoing approach, this strategy has the advantage of greater selectivity; however, it has potentially adverse side effects relative to the anterior pituitary, which is not protected from immunological attack and could possibly be destroyed by cell-mediated immunity since it is the site of FSH synthesis. This would be analogous to the ovarian failure encountered in mammalian experiments with immunization with ZP3. One strategy that might be employed to circumvent this difficulty would be the identification of exclusive B-cell epitopes, if present, in the linear sequence of FSH-ß used as immunogen. Such exclusive B-cell epitopes would be predicted to elicit only specific antibodies without the cytotoxic arm of the immune response. Nevertheless, it may turn out that exclusive B-cell epitopes are not, in fact, present in the targeted sequence, or that the epitope is not sufficiently immunogenic on its own to generate blocking antibodies. Interference with FSH Binding to the FSH Receptor Because the biological effects of FSH are exerted through activation of its receptor on Sertoli cells, methods that prevent FSH receptor activation are predicted to disturb spermatogenesis. At least two different strategies can be considered as ways to produce this effect: (1) direct interference with hormone-receptor interaction by generation of specific anti-FSH receptor antibodies or mimetopes, or (2) indirect interference by preventing proper assembly of the functional tetrameric receptor. Direct Interference with Hormone-Receptor Interaction Current evidence suggests that the extracellular domain of the FSH receptor contains unique sequences that could be selectively targeted by antibodies or mimetopes so as to prevent FSH binding. This target is located at an accessible site since, although the sperm-producing portion of the testis is protected by the blood-testis barrier, the basal surface of Sertoli cells bears the FSH receptors and is available to the circulation. A potential disadvantage of this strategy is that the agent—antibody or mimetope—that binds to the FSH receptor may itself activate the receptor; this

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--> would obviate any contraceptive effect. Studies of receptor activation in a variety of other systems have revealed that both activating and nonactivating antibodies may be directed against epitopes in the extracellular domains of various receptors; in the case of the FSH receptor, exploration of this possibility would be required, using both in vitro and animal model assays. Indirect Interference with Hormone-Receptor Interaction As indicated above, FSH binding studies indicate that the hormone binds only to tetrameric FSH receptors. This means that any strategies that would prevent proper assembly of receptor subunits would effectively block receptor action. For most oligomeric receptors, assembly occurs intracellularly; improperly assembled receptors are retained within the cell and not inserted into the plasma membrane. Assuming that specific targets within the FSH receptor sequence can be identified—a reasonable assumption—the novel task is to determine how to prevent proper assembly. As far as we know, the information needed to achieve this objective is not available, but understanding of protein trafficking within cells is advancing at a remarkable rate and it can be anticipated that some strategies will be available in the future. Control of Meiosis Meiosis is the process by which the number of chromosomes in a diploid nucleus (2N) is reduced to the haploid state (1N) by cell division. There are two cell division cycles in meiosis; meiosis I and meiosis II. The former involves DNA synthesis and results in two daughter cells each of which are diploid. In the latter, there is no DNA synthesis, so that the four daughter cells that result from this division are haploid. Meiosis occurs only in germ cells and in no other cells in the body in both males and females and the basic mechanism underlying meiosis is the same in both sexes. However, there are dramatic differences between the two sexes in the way the process takes place. In males, meiosis is a continuous event, that is, once they have been initiated, the two cell divisions in meiosis are carried to completion. In the female, on the other hand, meiosis is arrested at the end of prophase in the first meiotic division and at metaphase in the second division. The first arrest can be years in duration and is relieved only when the oocyte is stimulated to grow in preparation for ovulation. The second arrest is relieved at fertilization. The basic question that arises, therefore, is: Why are there no meiotic arrests in the male similar to those in the female? The next question is: Can the mechanisms of arrest in the female be utilized in developing an approach to meiotic arrest in males that would be an effective contraceptive? The first meiotic arrest in females appears to be heavily dependent upon cAMP-dependent protein kinase A. In view of the general importance of this system in other cells

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--> in the body—in other words, it is very far from being specific—it does not seem likely that altering activity of this system would be a rewarding strategy. On the other hand, arrest of meiosis II appears to involve at least one unique germ cell protein, Mos, although there probably are other factors involved as well but it is not clear exactly when transcription occurs and studies to date are contradictory. In one study, c-mos was found to be transcribed in the male during meiosis, but the message was not translated until spermiogenesis began (Chapman and Wolgemuth 1994); in another study, c-mos was found expressed before meiosis (Van der Hoorn et al. 1991). This means that the protein is expressed either too late or too early to have an effect on meiosis in the male. One can speculate that if the protein was expressed during meiosis, then meiotic arrest would occur and would block further development of sperm. Targeting cells in the adluminal compartment of the blood/testis barrier is discussed below, but this is potentially such a powerful approach to fertility regulation that renewed investigation of the barrier is warranted. Genetic Manipulation of Sperm An ideal male contraceptive would interfere with the production or maturation of sperm without affecting testis function in any other way, including any reduction of hormone secretion or libido. An attack on developing germ cells within the testis would be a very direct approach to the disruption of sperm production. As spermatogonia differentiate into spermatocytes, they begin to advance toward the lumen of the seminiferous tubule. As noted above, primary spermatocytes engage in the first meiotic division to generate secondary spermatocytes; the latter cells undergo the second meiotic division to give rise to haploid spermatids. No further divisions occur: Spermatids differentiate into sperm (during spermiogenesis) and are liberated into the lumen of the seminiferous tubule. The progenitor spermatogonia are located at the base of the seminiferous tubule, are unprotected by the blood-testis barrier, and are therefore available to perturbation by agents distributed through the circulation. In contrast, the population of germ cells that initiates meiosis (primary spermatocytes) become protected by the blood/testis barrier during the first meiotic division. At that point, the germ cells are no longer directly accessible to the circulation. As an alternative to targeting spermatogonia, it may be possible to use Sertoli cells as a route of delivery to postmeiotic germ cells. This would be fairly complex since a two-stage delivery system would be necessary (targeting first to Sertoli cell, and then to germ cell), but it would make use of the extensive communicating junctions that exist between Sertoli and germ cells. Utilization of techniques developed for gene therapy should allow the targeting of accessible germ cells. In standard gene therapy, a stem cell population is targeted and new genes are inserted into these cells to overcome the effects of damaged genetic material. In a contraceptive context, these same techniques, for

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--> example, targeting surface components specific to spermatogonia, could be used but, instead of correcting a malfunction, a defect would be introduced. Sertoli cells could also (perhaps more easily?) be targeted in this way, and genes introduced that would disable normal function. In essence, this would be a "Sertoli cell knockout." Since the number of Sertoli cells limits the number of germ cells undergoing differentiation, the efficiency of this approach might not have to be near 100 percent to achieve a large impact on sperm numbers. The important task of identifying germ cell processes that are amenable to this strategy remains; several possible sites of contraceptive attack are described below. The creation of "conditional knockouts" in mice has been described wherein a specified gene was inactivated selectively in a particular cell type. These findings, which remain to be exploited for contraceptive development, pave the way for the selective inactivation of specific genes. In the context of the epididymis, for instance, one could envisage "knocking out" genes that encode epididymal secretory proteins that are important in maturing the sperm surface either by binding directly to the surface or by modifying it enzymatically. In this way, critical determinants of the sperm surface would not appear properly, leading to compromised function during fertilization. Disturbance of Normally Functioning Transcription Factors An important approach to disrupting sperm production would be to interfere with differentiation itself. Spermatogenesis is under elaborate, yet strict, control both spatially and temporally. This control is exerted in part by transcription factors, proteins that bind to DNA with the effect of switching genes on or off. Although this field is relatively unexplored in terms of germ cells, it can be imagined that the schedule of transcription factor expression is extremely important. Inappropriate expression of a transcription factor, either absence of expression when needed or overexpression, is likely to have profound effects on differentiation. Transcription factors specific to germ cells have already been identified (Chen et al. 1994), making this topic a very attractive one to pursue. Since spermatogonia are not protected by the blood-testis barrier, events that occur in this population should be emphasized with this approach until it can be established that the delivered agent can operate postmeiotically. As a specific example, an attractive process to target with this approach would be the replacement of nuclear histones with protamines. This replacement process is exclusive to developing sperm cells and occurs in spermatids. Spermatocytes, however, synthesize protamines, highly charged basic proteins that, when complexed with DNA, are thought to facilitate the dense chromatin packing found in the sperm head. Failure to package the chromatin in this way could have important implications for chromatin structure or stability. Among other processes that might be targeted productively are any that are unique to sperm development, for instance, acrosome formation (see below). Like histone/protamine replacement, acrosome formation normally occurs in

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--> postmeiotic germ cells. Nevertheless, several of the acrosomal contents are synthesized much earlier, some as early as in late spermatogonia/early spermatocytes. Thus, thorough understanding of these fundamental processes of spermatogenesis will assist in identifying possible targets. Mapping of Genes Responsible for Fertility and/or Infertility Classic genetic techniques offer an alternative approach to trying to identify unique transcription factors or processes involved in sperm production. Many of the processes of meiosis and gamete production are highly conserved across all animal phyla, and important information can be gained from the study of well-established model systems, with the aim of then relating that information back to the human situation. For instance, the genetics of Drosophila have been studied extensively. Of the -4,000 genes in the total genome, mutation of -400 of them causes sterility in females; of these, -150 are without any obvious effects elsewhere in the organism (see Chapter 5). Parallel studies for male fertility might identify a similar subset of mutations. While all of these mutations are unlikely to have human correlates, some of them may serve as a previously unrecognized site for contraceptive focus. Inhibition of Acrosome and Tail Formation Two important events in sperm development are, first, formation of the acrosome and, second, the establishment of the longitudinal polarity of the cell by attachment of centrioles to the nucleus leading to formation of the sperm tail. These events occur in no other cells in the male and are therefore potentially susceptible to unique intervention for contraception. As far as we know, these processes are not regulated by hormones. Interference with Acrosome Formation The acrosome is bound tightly to the outer surface of the nuclear envelope, just deep to the plasma membrane overlying the sperm head. It is a product of the Golgi apparatus (Thoren-Tjomsland et al. 1988) and first appears morphologically in spermatids; however, it has been shown that the mRNA for acrosomal contents can be synthesized as early in pachytene spermatocytes. During spermiogenesis, selected vesicles in the trans-Golgi network begin to accumulate a granular content and fuse into a large acrosomic body that moves from the Golgi to attach to the nucleus. The remaining Golgi is partially lost in residual bodies and phagocytosed by Sertoli cells, although portions of Golgi membranes remain in the cytoplasmic droplet found on the sperm tail. The trans-Golgi network in most somatic secretory cells faces away from the nucleus toward the apical surface of the cells. In somatic cells, secretory vesicles move from the Golgi toward the apical plasma membrane to eventually fuse with it and release their contents. In contrast, the trans-Golgi network in spermatids faces toward the nucleus so that the forming acrosomic

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--> body is ideally placed to move to the nucleus. In essence, the acrosome is a secretory vesicle that stores its products during the later phases of spermiogenesis and post-testicular maturation until a signal is given by the binding of sperm to the zona pellucida to release the contents (see below). Since formation of the acrosome is absolutely essential for fertility, it seems reasonable that interfering with the process could result in sperm that could not fertilize an egg. There are several questions that arise about formation of the acrosome that may provide a strategy for interrupting either its formation or its function: What mechanisms are involved in orientation of the Golgi apparatus that are different from those found in most somatic cells? What signals target acrosomic contents to only a subset of vesicles in the trans-Golgi network? What structural features participate in coalescence of the acrosomic vesicles? What are the mechanisms for tight interactions between the membrane of the acrosomic body and the nuclear membrane? Interference with Centriole Attachment and Sperm Tail Formation Like the acrosome, the sperm tail is essential for fertility. The tail derives from centrioles but, in sperm, the flagellum that forms is highly modified compared to those in other cells. Centrioles and the Golgi apparatus in most somatic cells are structurally closely related to form the cytocentrum. In all cells, centrioles have the potential to germinate cilia or flagella, but only in spermatids do the centrioles migrate around the periphery of the cell to bind tightly to the nuclear envelope 180 degrees away from the attachment of the acrosome. The first indication of tail formation is movement of the centrioles to lie just deep to the plasma membrane. They then appear to migrate around the cell and, at a certain point, move toward the nucleus, pulling with them the plasma membrane in the region. The centrioles bind to the outer nuclear membrane, becoming highly modified themselves; the longitudinal centriole germinates the axoneme that comprises the sperm tail. All of these modifications, as well as the formation of the mitochondrial sheath, fibrous sheath, the annulus, and outer dense fibers, are unique events that occur only in spermatids. Presumably these unique events require unique processes to bring them about, which can be targeted for contraceptive intervention. Alteration of Sperm Surface Proteins During Spermatogenesis and Post-Testicular Development During spermatogenesis, sperm acquire a highly polarized morphology, with a quadripartite structure consisting of a head, which contains the haploid genome, a midpiece, a principal piece, and an end piece. The extreme state of compart-

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--> mentalization observed in this cell at maturity extends as well to the organization of the sperm plasma membrane, where both lipids and proteins are organized into highly regionalized domains. Many questions about the formation and maintenance of the membrane domains remain, but at least some domains are thought to be important functionally. During spermiogenesis, when the haploid cell is remodeled, many of these domains make their first appearance, although several are refashioned during post-testicular maturation in the epididymis. Several sperm proteins thought to be important in fertilization have been identified and their appearance in the membrane of the developing sperm is being charted (Bartles 1995). Although few studies dealing with the development of membrane domains have been conducted directly with human sperm, human homologues for some of the nonhuman tracked proteins have been identified and are presumed to behave similarly. Many of these fertilization-related proteins are inserted into the plasma membrane during spermatogenesis and display a restricted distribution from the start, whereas others are more uniformly distributed in testicular sperm but are directed into domains during epididymal passage. Both fertilin and PH-20 represent sperm proteins important for fertilization that fall into the latter category (Myles and Primakoff 1991). In testicular sperm, while both these proteins are distributed over the entire sperm surface, they are restricted during epididymal transit to specialized regions of the sperm head. In vitro studies of guinea pig sperm indicate that this redistribution can be affected by trypsin treatment of immature sperm, giving rise to the notion that redistribution is proteolysis-dependent in vivo. This sort of alteration is presumably caused by secretory products of the epididymal epithelium; disruption of the elaboration of such components will be considered in the following section. In terms of intrinsic sperm structure and its relationship to fertilizing capacity, however, these concepts give rise to possible contraceptive strategies. Disruption of Membrane Domains A conserved feature of all mammalian sperm analyzed to date is the extreme regionalization of membrane components. While membrane domains are also a common feature of somatic cells, they are rarely built to such an extent. Although at present the specific function(s) of this polarized organization in any cell type is not clear, germ cells invest heavily in elaborating and maintaining this organization. As more becomes known about the cellular components involved in generating and maintaining membrane domains, it may be possible to target these pathways so that domain formation is disrupted. The absence of a high local concentration of a particular sperm protein in a defined region, or the inability of specific components to couple to appropriate intracellular constituents, are likely to lead to dysfunctional sperm. Disruption of Delivery of Fertilization-related Proteins to the Membrane As mentioned earlier, fertilin is delivered to the membrane of the developing sperm cell during spermatogenesis; in fact, initial delivery to the surface occurs in

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--> spermatids. Eddy and O'Brien (1994) have hypothesized that the temporal regulation of surface expression may be responsible for directing proteins to the correct surface domain. If this concept is accurate, then alteration of the timing of protein expression could subtly disorder the intricate organization of the sperm membrane, with infertile sperm as the result. Of course, fertilin need not be the sole sperm protein that could be targeted with this strategy: Any fertilization-related protein that displays a restricted surface distribution in the mature sperm would be an equally attractive candidate. Indeed, it may be that emphasis on fertilization-related proteins is unnecessary, since the inappropriate timing of any surface protein may so disrupt the positioning of other functional components that detrimental effects would ensue. Interruption of Epididymal Function In addition to serving as a sperm reservoir, the epididymis provides an environment for morphological and biochemical alterations in the sperm that are necessary for fertility, that is, the ''sperm maturation" that takes place during the one- to two-week period that sperm require for passage through the single convoluted tubule that comprises this organ. Whereas the morphologic changes that occur with maturation are fairly subtle, the functional changes effected are essential for the sperm's fertilizing ability. These include the capacity for (1) vigorous motility; (2) productive interaction, both binding and acrosomal exocytosis, with the egg's extracellular matrix, the zona pellucida; and (3) productive interaction with the egg's plasma membrane. These functions of the epididymis, and indeed the maintenance of the organ itself, are androgen-dependent; in the absence of androgen, the epididymis decreases dramatically in size and sperm maturation does not occur. Thus, transit through this post-testicular site confers on sperm the ability to fertilize an egg; as such, it constitutes what may be among the best locations for contraceptive attack for several reasons. First, the epididymis is an end-organ for hormone action but apparently is not itself involved in hormone synthesis in vivo. Second, the only known functions of the epididymis, that is, transit, maturation, and storage, involve sperm exclusively. Third, sperm are fully formed genetically in the testis and are fairly inert biosynthetically upon arrival in the epididymis. Consequently, compared to manipulations of the testis, contraceptive strategies directed toward the epididymis are considerably less likely to produce adverse hormonal or genetic side effects. Much of the sperm surface appears to be remodeled during maturation and modifications have been detected in all types of plasma membrane components, including lipids, proteins, and carbohydrates. Several theories have been advanced with regard to the mechanisms involved in these changes, including (1) modification (glycosylation, phosphorylation, and so forth) or unmasking (hydrolysis, either limited or complete) of preexisting components; (2) redistribution

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--> of preexisting components; or (3) binding of new components to preorganized domains at the sperm surface following synthesis and secretion by the epididymal epithelium. Sperm surface changes are mediated by products of the epididymal epithelium secreted into the lumen of this tubule. Although the specific components of the epididymal fluid that are responsible for maturing sperm have not been fully identified, it is clear that the composition of the fluid is complex and changes dramatically along the length of the tubule, suggesting that different components of the sperm surface may be modified at different locations along the length of the epididymis. Although the molecular details of sperm maturation are not well known, it is clear nevertheless that this is an ideal organ to target for contraception. Several possible approaches are outlined below but they are just a beginning. Further research into the molecular mechanisms underlying sperm maturation are very likely to be richly rewarding in their potential to produce novel strategies of a sort that we may not even have imagined. Modification of Expression of Epididymal Proteins Important in Sperm Maturation Several proteins that are secreted into epididymal fluid and subsequently adsorbed onto the sperm surface have been identified in humans and in other mammalian species. We do not yet know whether any of these proteins are essential for sperm maturation, since it is only recently that techniques have become available that suggest the feasibility of this kind of approach (Gu et al. 1994). If further research supports feasibility, then the modified expression of such critical proteins would constitute an excellent strategic avenue for male contraceptives. Application of Epididymis-specific Antiandrogens As indicated earlier, the functioning of the epididymis depends upon androgen. Impaired delivery of androgen to this site markedly disrupts a large number of parameters, including the secretion of glycosidases known to modify the sperm surface. Recent progress in the administration of organ-specific antiestrogens makes it possible to consider a parallel route for the delivery of antiandrogens to the epididymis. If such an agent were to become available, it might be one of the best ideas for reversible contraception in the male. Immunoneutralization of Epididymal-specific Proteins If epididymis-specific proteins that are important in human epididymal and/or sperm function were to be identified, a highly likely possibility, then it should be possible to target these for neutralization by specific antibodies using an immunocontraceptive approach. Just as might be the case for females, males would be immunized with a peptide corresponding to an epididymal-specific portion of the protein of interest. The probability is that the antibodies generated would have considerably better access

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--> to epididymal cells than they would to testicular cells, since the blood-testis barrier is not present in the epidiymis. Regulation of the Inhibition of Immune Reactions by Sperm A little recognized aspect of sperm development is the fact that, in many species, proteins secreted by the seminal vesicle are immunosuppressive and attach covalently to the sperm surface by a transglutaminase-catalyzed reaction. This phase of sperm maturation may be as important to the viability of sperm in the female tract as any of the events that occur in the epididymis, since the sperm would be essentially immunologically silent and would evade the female immune system (see review by Hamilton 1994). Whether similar events occur in human males and females are unknown. Induction of Premature Acrosome Reaction During the gamete interactions that lead to fertilization, one of the regulators of success is the appropriate occurrence of exocytosis in the sperm cell. Normally, this event occurs after the sperm has bound to the egg's protective coat, the zona pellucida, and the enzymes released from the sperm's acrosome permit penetration through this egg coat. Inappropriate release of the acrosomal enzymes when the sperm is some distance from the egg results in infertility. Recently, progress has been made in defining some of the sperm proteins important in triggering exocytotic release of the acrosome. One of these is termed ZRK (zona receptor kinase), a ZP3 receptor that has the structure of a receptor tyrosine kinase (Burks et al. 1995). It is thought that sperm binding to ZP3 in the zona promotes the oligomerization of ZRK and thereby activates the receptor by autophosphorylation, by analogy with the mechanism of activation for other well-characterized receptor tyrosine kinases. Thus, strategies of the type described just below that would influence the premature occurrence of any of these events-—either directly or indirectly—is likely to have profound consequences for fertility. Delivery of a ZP3-mimic to Sperm in the Male Theoretically, a reagent that binds ZRK and is multivalent or minimally bivalent could aggregate these ZP3 receptors and activate them. Activation would lead to exocytosis and premature release of the acrosomal enzymes. Either peptide or nonpeptide reagents could be considered as ZP3 mimics; it should be possible to identify these with relative straightforwardness since the primary structure of ZRK is known and there is at least some initial information about ligand binding sites. Delivery of such a mimic could occur in the epididymis and thus potentially be present on sperm for a relatively long period of time. Alternatively, the ZP3 mimic could be targeted to any of the accessory organs of the male reproductive tract—seminal vesicles,

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--> prostate, Cowper' s glands, or bulbourethral glands—with the choice made on the basis of targeting potential, stability of mimic, etc., and therefore be applied to the sperm surface at ejaculation. There are, at the outset, at least two possible problems with this approach. One is that sperm binding to the natural ligand ZP3 normally occurs after sperm capacitation, which is a poorly defined final maturational phase accomplished within the female reproductive tract during which both the intrinsic (e.g., increased membrane fluidity) and the extrinsic (e.g., glycosylation) structure of the sperm membrane are modified. Therefore, the binding of a multivalent ZP3 mimic to sperm prior to capacitation, as proposed here, may not be sufficient to stimulate exocytosis. The other is that sperm are present at high concentration in the epididymis, particularly the cauda epididymidis; the accessibility of such a reagent to sperm in the male reproductive tract would have to be extremely high for this approach to be effective. Identification of the Acrosome Reaction-Eliciting Substance Produced by t-Bearing Sperm Although the strategy outlined above is attended by caveats, it turns out that there is actually a natural method of producing precisely this effect. It has been known for several decades that mouse sperm heterozygous for a mutation in the T-locus will make equivalent amounts of t-bearing and wild-type sperm. However, when mated, there is a transmission distortion for the t-bearing sperm such that 95 percent of the offspring are also heterozygous for the T-locus mutation. This phenomenon has been studied extensively and found to depend on the ability of the t-bearing sperm to provoke premature acrosome reactions in the wild-type population (Brown et al. 1989). The mechanism(s) underlying this effect are completely unknown, and further study of this system could reveal the component(s) that evoke this effect. Inhibition of Gamete (Sperm-Egg) Interaction Sperm-egg interaction can be broken down into at least the following six steps (Saling 1995): Cumulus layer penetration Primary binding to the zona (ZP3-mediated) Triggering of acrosomal exocytosis Secondary binding to the zona (probably ZP2-mediated) Penetration through the zona pellucida Fusion between the sperm and egg plasma membranes. Since each of these events is necessary for fertilization under normal circumstances, potential blockade of any step would impair, if not prevent, fertility. Sperm proteins associated with individual steps of gamete interaction have also

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--> been identified recently, which permits selective focus on particular steps and/or proteins. To develop a male-directed contraceptive aimed at eventually blocking these events requires detailed knowledge about the proteins that will be attacked, both in terms of their mechanisms of action as well as potential modification during epididymal transit. An appropriate location for attack appears to be the epididymis, principally for the reasons outlined above; as such, we have already dealt with general strategies that might be employed for this purposes earlier in this paper. There are also in this category strategies using specific sperm proteins. •   PH-20: It may turn out that the role of PH-20 in secondary binding depends upon glycosylation. Delivery of antiandrogens to the epididymis might alter expression of the glycosidase required for this effect and therefore result in a PH-20 protein on the sperm surface that is 90 percent less effective in secondary binding to the zona. •   Fertilin: We have already referred to the fact that proteolytic processing of fertilin in the epididymis is required for redistribution of the protein to the appropriate subcellular domain in mature sperm. Were the protease responsible for this effect to be identified, it might be possible to design a "conditional knockout" of the gene encoding this enzyme in the epididymis, thereby preventing fertilin redistribution and functioning. •   ZRK: This ZP3 receptor appears to be sperm-specific, which may make it attractive for an immunocontraceptive strategy. If antibodies directed against the extracellular domain of ZRK were able to gain access to the epididymal lumen and bind to ZRK on sperm, either or both of two events might occur: (1) steric blockade of sperm-ZP3 interaction or (2) premature release of the acrosome. Both of these events are predicted to lead to infertility. Because of the extremely large number of cells that would require neutralization, optimized delivery of any reagent would be essential. Concluding Remarks This paper has followed the general discussion at the symposium on New Frontiers in Contraceptive Technology held at the Institute of Medicine in December 19941 and expands on some of the more general concepts that emerged during that meeting. The fundamental message is that there are many plausible targets for development of contraceptives for males. The largest obstacle to targeting the male reproductive tract is its biology. Approaches to inhibition of spermatogenesis (for example, work with the long-loop feedback system) and vasectomy show that infertility is not produced immediately and that lag times can be long. Behavior in men does not conform well to such delays or, in fact, to regimen in general. Still, this does not mean that all approaches will entail comparable problems. Further developments in this field can come only through

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--> cooperative interactions among scientists in academia and in industry, and by prioritizing allocation of resources sufficient to achieve the goals. References Amobi NI, IC Smith. Differential inhibition in the human vas deferens by phenoxybenzamine: A possible mechanism for its contraceptive action. Journal of Reproduction and Fertility 103:215-221, 1995. Bagatell CJ, AM Matsumoto, RB Christensen, et al. Comparison of a gonadotropin releasing hormone antagonist plus testosterone (T) versus T alone as potential male contraceptive regimens. Journal of Clinical Endocrinology and Metabolism 77(2):427-432, 1993. Bartles JR. The spermatid plasma membrane comes of age. Trends in Cell Biology 5:400-404, 1995. Brown J, JA Cebra-Thomas, JD Bleil, et al. A premature acrosome reaction is programmed by mouse t haplotypes during sperm differentiation and could play a role in transmission ratio distortion. Development 106:769-773, 1989. Burks DJ, R Carballada, HDM Moore, et al. Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization. Science 269:83-86, 1995. Chapman DL, D Wolgemuth. Regulation of M-phase promoting factor activity during development of mouse male germ cells. Developmental Biology 165(2):500-506, 1994. Chen ZW, YQ Gu, XW Liang, et al. Safety and efficacy of percutaneous injection of polyurethane elastomer (MPU) plugs for vas occlusion in man. International Journal of Andrology 15(6):468-472, 1992. Chen F, AJ Cooney, Y Wang, et al. Cloning of a novel orphan receptor (GCNF) expressed during germ cell development. Molecular Endocrinology 8:1434-1444, 1994. Crabo BG. Studies on the composition of epididymal content in bulls and boars. Acta Veterinaria Scandinavica 6 (Suppl.5):1-94, 1965. Douglass J, SH Garrett, JE Garrett. Differential patterns of regulated gene expression in the adult rat epididymis. Annals of the New York Academy of Sciences 637:384-398, 1991. Dym M. The male reproductive system. IN Histology. L Weiss, ed. New York: Elsevier Biomedical. 1983. Eddy EM, DA O'Brien. The Spermatozoon. IN The Physiology of Reproduction. E Knobil, JD Neill, eds. New York: Raven Press. 1994. Gu H, JD Marth, PC Orban, et al. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science 265:103-106, 1994. Hamilton DW. Local immunity and sperm processing in the male reproductive tract. IN Local Immunity in Reproductive Tract Tissues. PD Griffin, PM Johnson, eds. New York: Oxford University Press. 1994. Handelsman DJ, AJ Conway, LM Boylan. Suppression of human spermatogenesis by testosterone implants. Journal of Clinical Endocrinology and Metabolism 75(5): 1326-1332, 1992. Hutson JC. Testicular macrophages. International Review of Cytology 149:99-143, 1994. Kuckuck L, GS Chhina, SK Manchanda. Development and initial evaluation of a vas deferens valve. Indian Journal of Physiology and Pharmacology 19(1):20-27, 1975. McLachlan RI, NG Wreford, DM Robertson, et al. Hormonal control of spermatogenesis. Trends in Endocrinology and Metabolism 6:95-101, 1995. Myles DG, P Primakoff. Sperm proteins that serve as receptors for the zona pellucida and their posttesticular modification. Annals of the New York Academy of Sciences 637:486-493, 1991. Onoda M, D Djakiew. Modulation of Sertoli cell secretory function by rat round spermatid protein(s). Molecular and Cellular Endocrinology 73:35-44, 1990.

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--> Onoda M, D Djakiew. A 29,000 Mr protein derived from round spermatids regulates Sertoli cell secretion. Molecular and Cellular Endocrinology 93:53-61, 1993. Saling PM. Gamete interactions leading to fertilization in mammals: Principles, paradigms, and paradoxes. IN Reproductive Endocrinology, Surgery, and Technology. EY Adashi, JA Rock, Z Rosenwaks, eds. New York: Raven Press. 1995. Sharpe R. Experimental evidence for Sertoli-germ cell and Sertoli-Leydig cell interactions. IN The Sertoli Cell. L Russell, M Griswold, eds. Clearwater, FL: Cache River Press. 1993. Skinner MK, IB Fritz. Testicular cells secrete a protein under androgen control that modulates Sertoli cell function. Proceedings of the National Academy of Sciences, USA 82:114-118, 1985. Thoren-Tjomsland G, Y Clermont, L Hermo. Contribution of the Golgi apparatus components to the formation of the acrosomic system and chromatoid body in rat spermatids. Anatomical Record 221:591-598, 1988. Valove FM, C Finch, JN Anasti, et al. Receptor binding and signal transduction are dissociable functions requiring different sites on follicle-stimulating hormone. Endocrinology 135:2657-2661, 1994. Van der Hoorn FA, JE Spiegel, MF Maylie-Pfenninger, et al. A 43 kD c-mos protein is only expressed before meiosis during rat spermatogenesis. Oncogene 6(6):929-932, 1991. Wang C, R Swerdloff, GMH Waites. Male contraception: 1993 and beyond. IN Contraceptive Research and Development 1984 to 1994: The Road from Mexico City to Cairo and Beyond. PFA Van Look, G Pèrez-Palacios, eds. Delhi: Oxford University Press. 1994. Zhao SC, SP Zhang, RC Yu. Intravasal injection of formed-in-place silicone rubber as a method of vas occlusion. International Journal of Andrology 15(6):460-464, 1992. Zirkin BR, C Awoniyi, MD Griswold, et al. Is FSH required for adult spermatogenesis? Journal of Andrology 15:273-276, 1994. Note 1.   The proceedings from this meeting, including a participants list, are included in this report as Appendix E.