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5 Biologic Markers of Epididymal Structure and Function This chapter focuses on markers of epididymal function. Very few direct markers of human epididymal function are available. However, there is a useful array of indirect markers. The chapter discusses markers of tissue structure: markers of structural, biochemical, membrane compositional, and functional changes of the maturing spermatozoa; biochemical markers of the luminal fluid; and histologic and biochemical markers of the epithelium. Many of the assessments must be performed on biopsied or autopsied tissue or in animals. These studies are essential to the understanding of basic mechanisms of action of toxicants. The epididymis is a single highly con- voluted duct whose length varies from 3-4 meters in man to 80 meters in horses (Maneely, 1959~. The duct begins where the efferent ductsthe ductuli effer- entes, numbering from 4 to 20, depending on species (Nistal Martin de Serrano and Paniagua Gomez-Alvarez, 1984; Hemeida et al., 1978-come together. The epididy- mal duct continues as a straight tube, the vas deferens, that is surrounded by a thick muscular layer. The vas deferens connects with the urethra, which empties outside the body. The epididymis is usually divid- ed into three gross anatomic segments: head (caput), body (corpus), and tail (cauda). An initial segment lies between the efferent ducts and the remainder of 63 the caput epididymis, has a characteristic histologic appearance and function and is also often identified (Benoit, 1926; Robaire and Hermo,1987~. Interest in the epididymis has grown over the past 2 decades, since the demon- stration that it was in this tissue that spermatozoa mature enough to become able to fertilize eggs (Bedford, 1967; Orgebin- Crist, 1967a). On leaving the testis, spermatozoa have a light microscopic appearance similar to that of spermatozoa in semen, but are incapable of fertilizing eggs. Several reviews on various facets of epididymal structure and function have appeared in the past 15 years (Hamilton, 1972; Bedford, 1975; Hamilton, 1975; Neaves, 1975; Orgebin-Crist et al., 1975; Turner, 1979; Hinton, 1980; Courot, 1981; Orgebin-Crist, 1981; Brooks, 1982; Glo- ver, 1982; Howards, 1983; Orgebin-Crist, 1984; Cooper, 1986; Amann, 1987; Robaire and Hermo,1987~. To monitor whether a toxicant has com- promised male fertility, it is essential to determine not only whether the normal physiologic functions of the epididymis are still being performed, but also whether the toxicant has so altered the spermatozoa while they were in the epididymis that they cannot fertilize eggs or can fertilize eggs but produce only nonviable or abnormal offspring. A discussion of potentially useful mark-

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64 ers of epididymal function that are avail- able, are under development, or ought to be developed is presented below. We begin by discussing markers of the tissue as a whole and then focus on markers that reflect changes in maturing spermatozoa, the implications of the makeup of the fluid in the luminal compartment that bathes maturing spermatozoa, and the activities of the epithelial compartment of the epididymis. MARKERS OF EPIDIDYMAL TISSUE Few noninvasive direct determinations are used to assess whether a substance has deleteriously affected the epididy- mis. The tissue can be palpated and the size, consistency, and shape assessed. Such simple markers are sometimes of value in diagnosing tubal obstruction or epidid- ymitis, but they are highly subjective and do not lend themselves to assessment of variability, limits of detection, and so forth. The more recently developed imaging techniques are only now being test- ed as tools in assessing the epididymis; these probably will yield a reliable index of such characteristics as size and shape. If the tissues are available, the weight of the epididymis is, in fact, a simple and useful biologic marker. The epididymis is a tissue with two comnart- MALE REPRODUCTIVE TOXICOLOGY the biologic regulation of epididymal function. The epididymis contains recep- tors for a variety of hormones, e.g., tes- tosterone, estradiol, prolactin, and vitamin D; but the value, with respect to epididymal function, of measuring the serum concentrations of the hormones as markers has not been established. The topography of the blood vessels entering and leaving the testis is complex and spe- cies-specific (Chubb and Desjardins, 1982~. It is evident from numerous castra- tion and hormone-replacement studies that blood vessels are a major route by which hormonal signals are received by the epi- didymis. Our knowledge of capillary flow and its impact on tissue function is sparse, but it is likely that we will soon be able to monitor blood flow in epididymal capillaries. Whether such measurement will become an important marker of epididy- mal function will depend on the ability of the capillary system to adapt to dele- terious actions of toxicants. Very little is known about epididymal lymphatic input and drainage. As the na- ture of the blood/epididymis barrier be- comes better understood and the use of immunosuppressive agents (which alter the immune system and lymph content) grows, we will increase our understanding of this system, although no lymphatic markers regarding epididymal function are likely meets: epithelium and lumen. The lumen to tee developed soon. is filled with fluid and spermatozoa and, Neuronal input is responsible for the under normal conditions in many mammals, basal epididymal contractility, which makes up approximately 50% of the weight is at least partially responsible for of the tissue (e.g., Robaire et al., 1977~; the transport of sperm and fluid down the small decreases in epididymal weight are duct system from the testis. Assessment usually associated with proportionately of whether drugs that affect the autonomic larger decreases in epididymal sperm re- nervous system can modulate epididymal serves. Accessory sex tissues become mark- contractility and hence the rate of sperm edly hypertrophic in the presence of large transit and the acquisition of fertilizing excesses of androgen, even in a castrated animal. However, the epididymis does not become hypertrophic. Thus, changes in epididymal weight can be a valuable index of the status of the tissue. As with other hormone-dependent tis- sues, blood is the major source of regula- tory substances that reach the epididymal epithelium. Measurement of serum concen- trations of hormones, especially testos- terone, is useful and routine in monitoring ability in viva has not been attempted. It would be surprising if they could not; but such drugs affect an array of other systems, so their effects on the epididymis might be masked. Although the exact epi- didymal effects of modulating neuronal activity would be of interest, measure- ments of neuronally mediated epididymal contractility and of the factors that regu- late it will probably not become selective markers.

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EPIDIDYAfAL STRUCTURE AND FUNCTION Some epididymal functions apparently are regulated in a paracrine manner, i.e., by factors from the testis that enter the epididymal lumen directly (Ro- baire and Hermo, 1987~. Factors secreted by the testis into the efferent ducts that directly modulate epididymal function are probably of Sertoli cell origin (Scheer and Robaire, 1980; Robaire and Zirkin, 1981~. Their identities are not resolved, but androgen-binding protein has been proposed as a likely candidate. Determin- ing the concentrations of various factors in rete testis fluid is difficult for many species, because it is technically dif- ficult to obtain the fluid, only small volumes can be obtained, and the procedure cannot be repeated in most small animals and cannot be done in humans. However, it is apparent that some key regulators of epididymal function, which are also prob- ably useful epididymal markers, are to be found in this fluid. The presence and concentrations of such compounds in semen might reflect not only testicular but also epididymal function, and research to identify them and to develop means of monitoring them should be strongly encour- aged, although it will certainly take several years for such markers to become readily available. CHANGES IN MATURING SPERMATOZOA A number of morphologic and biochem- ical changes reported to occur in sper- matozoa during transit through the epidid- ymis and vas deferens have been reviewed extensively (Bedford, 1975; Bedford, 1979; Olson and Orgebin-Crist, 1982; Eddy et al., 1985; Cooper, 1986; Robaire and Hermo, 1987~. Structural Changes The most consistent morphologic change that takes place in spermatozoa during ductal transit is the migration of the cytoplasmic droplet from the neck region of the flagellum to the end of the midpiece of the sperm (mitochondrial sheath). The change has been noted in snakes and birds (Bedford, 1979), in many mammals (Branton 65 and Salisbury, 1947; Phillips, 1975; Kap- lan et al., 1984), and in humans (Hafez and Prasad, 1976~. In rats, lysosomal and other degradative enzymes are present in the droplet (Dots and Dingle, 1968; M.L. Roberts et al., 1976~; however, the func- tional importance of the enzymes and of the droplet remains to be resolved. The percentage of spermatozoa that re- tain cytoplasmic droplets or the number of cytoplasmic droplets per spermatozoon might well be a useful indication of the state of maturation of spermatozoa and might also reflect the activity of clear cells in some species. It might be a sensi- tive measure, but there will probably be large variability in it. This marker has not yet been validated as a good correlate of epididymal function, but it should be fairly straightforward to do so. Such a measure could be particularly useful in selected cases in which clear cell function or droplet shedding is impaired. Spermatozoa undergo a change in acro- somal size, shape, and internal structure as they pass through the duct system. This has been demonstrated in many species (Fawcett and Hollenberg, 1963; Fawcett and Phillips, 1969; Bedford and Nicander, 1971; Jones et al., 1974; Bedford and Mil- lar, 1978~. In animals in which acrosomal shape clearly changes during epididymal transit, observation of spermatozoa should reveal whether exposure to a given chemical has altered this marker of sper- matozoal maturation. Further studies are needed with primates to resolve whether such changes are correlated with spermato- zoal quality, i.e., fertilizing ability. Biochemical Changes An array of biochemical alterations in spermatozoa as they traverse the ducts has been reported (Bedford, 1975; Bedford and Cooper, 1978; Olson and Orgebin-Crist, 1982~. Only the characteristics with the greatest potential for use as markers are discussed here. In spite of numerous de- tailed studies on how sperm change during epididymal transit, it is still not possi- ble to dissociate factors that cause the maturation of spermatozoa from factors that result from maturation or are inciden-

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66 tat to it. Hence, any of the markers sug- gested above might correlate well with changes in spermatozoa that alter their fertilizing potential or their ability to produce normal offspring under a given set of conditions without having the desired predictive value. An increase in the relative number of disulfide bonds in the nuclei of spermato- zoa as they reach the cauda epididymis has been observed in many species (Calvin and Bedford, 1971; Bedford et al., 1973; Bed- ford, 1975; Johnson et al., 1980b). The increase is associated with a more con- densed (cross-linked) spermatozoa! nu- cleus-spermatozoa taken from the cauda epididymis are harder to Recondense than those taken from the initial segment (Zirkin et al., 1985b). The state of chro- matin condensation can be assessed by de- termining how long it takes for detergent- treated nuclei to dissolve in the presence of a given concentration of a disulfide reducing agent. If chemicals can affect spermatozoa by preventing the proper chro- matin condensation that takes place while spermatozoa are in the epididymis, then measuring such rates might provide a simple and useful biologic marker. The anionic charge on spermatozoa in- creases as they reach the cauda epididymis (Bedford, 1975; Toowicharanont and Chula- vatnatol, 1983~; the increase is probably acquired during passage through the corpus epididymis (Fain-Maurel et al., 1983~. The exact cause of the change in charge has not been resolved, but it is most likely due to changes in glycoprotein composition of the sperm plasma membrane that take place during epididymal transit. The membrane proteins of spermatozoa obtained from different segments of the epididymis present a markedly different pattern after separation on polyacryla- mide gel electrophoresis (Jones et al., 1981; Chulavatnatol et al., 1982; Brown et al., 1983; Dacheux and Voglmayr, 1983; Jones et al., 1983; Brooks and Tiver, 1984~. During epididymal transit, the number of concanavalin A binding sites on the sperm membrane decreases (Lewin et al., 1979; Nicolson and Yanagimachi, 1979; Olson and Orgebin-Crist, 1982), the ability of spermatozoa to activate comple- MCALE REPRODUCTIVE TOXICOLOGY ment decreases (Witkin et al., 1983), and the activity of protein methyltransferase in spermatozoa decreases dramatically (Gagnon et al., 1984~. Gonzalez Echeverria et al. (1984) have noted that the addition of a protein extract from hamster epididy- mis could increase the fertilizing ability of hamster spermatozoa. Blaquier et al. (1987) found that polyclonal antibodies to human epididymal sperm proteins bound selectively in the acrosomal cap region in fertile men, but that a large percentage of sperm bound these antibodies in a non- specific manner in infertile men. Such data lead to the proposal that several highly specific sperm-surface protein markers of epididymal origin might be al- tered, not only in cases of infertility, but also in response to various drugs. Antigenic determinants on the surface of spermatozoa change markedly as sperm traverse the epididymis (Eddy et al., 1985~. Monoclonal antibodies to mouse "sperm-maturation antigens" that arise in mice during epididymal passage have been characterized, and at least one (SM4) has been shown to appear on sperm only while they are in the corpus epididymis (Eddy et al., 1985; Vernon et al., 1985~. The appearance of such determinants might be due not only to the addition of a protein to the surface of spermatozoa, but also to the removal of proteins or to the enzyme- mediated exposure of pre-existing sites. This new approach to the characterization of changes that take place in spermatozoa during epididymal transit might provide some useful biologic markers, if a clear linkage between the state of maturation of a spermatozoon and the presence or absence of a given epitope can be established. The lipid composition of spermatozoa (Dacheux, 1977; Evans and Setchell, 1979), particularly that of their plasma mem- branes (Nikolopoulou et al., 1985), changes dramatically during epididymal transit: the amount of cholesterol de- creases, and the amounts of desmosterol and cholesterol sulfate increase (Legault et al., 1979; Inskeep and Hammerstedt, 1982; Nikolopoulou et al., 1985~; the rela- tive distribution of the different polar lipids also changes, some increasing while

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EPIDIDYMAL STRUCTURE AND FUNCTION others decrease (Nikolopoulou et al., 1985~. The change in lipid composition has been proposed as the cause of the in- creased sensitivity of caudal spermatozoa to cold (Nikolopoulou et al., 1985~. The relationship between the state of matura- tion of spermatozoa and the lipid makeup of their cell membrane has not yet been de- termined, but there might well be a sig- nificant relationship, because of the constraints put on membrane fluidity in sperm-egg interaction. If such a rela- tionship were established, measurements of lipid composition or membrane fluidity of spermatozoa might become useful markers. The changes in metabolic activity of spermatozoa during epididymal transit are numerous (Voglmayr, 1975; Brooks, 1981~. Increases in the glycolytic and respiratory activity of spermatozoa dur- ing epididymal transit have been reported (Dacheux et al., 1979; Voglmayr and White, 1979~. The increase in cAMP associated with spermatozoa as they mature (Del Rio and Raisman, 1978; Amann et al., 1982) has been attributed to both an increase in the synthesis of this second messenger and a decrease in its hydrolysis (Purvis et al., 1982~. There are so many indices of metabolic activity that it is not clear which, if any, would be useful markers of sperm maturation (Cooper et al., 1988~. Research in this subject could yield impor- tant results, but for the present it must be viewed as Fishing expeditions Functional Changes Content of Epididymal Spermatozoa One of the most powerful available bio- logic markers of epididymal function is the number of spermatozoa in different regions of the epididymis (Amann, 1981~. Because the heads of spermatozoa are very tightly condensed and resistant to homog- enization, it is possible to homogenize a segment of epididymis, filter the ho- mogenate, and count the number of condensed sperm heads with a hemacytometer (an in- strument usually used for counting blood cells). The method requires the destruc- tion of the tissue, but it is sensitive and 67 has the same degree of variability as any other method that depends on hemacyto- metric determinations; because of inter- ference by debris, it has not been possible to adapt electronic cell-counting methods to this application. Results from hemacy- tometric determinations not only give an accurate index of sperm content in dif- ferent epididymal regions where various functions take place (Robaire et al., 1977; Trasler et al., 1988), but also can be used to indicate the sperm reserve within a tissue (Amann, 1981~. Transport of Spermatozoa In spite of the very large range in sperm production rate in different spe- cies, there is great consistency in how long it takes for spermatozoa to traverse the epididymis: approximately 10 days (see Table4- l ~ (Robaire end Hermo, 1987~. How- ever, the transit time for spermatozoa in the human epididymis is variable (Rowley et al., 1970; Amann, 1981; Orgebin-Crist and Olson, 1984~. The time spent by sper- matozoa in the cauda epididymis is longer and more variable than that spent in any other segment. That is presumably because sperm transit in the caput and corpus epi- didymis is independent of ejaculatory fre- quency, whereas that in the cauda epididy- mis depends on this frequency (Amann and Almquist, 1962; Swierstra, 1971; Kirton et al., 1967~. The rate of luminal flow in different segments of the rat epididymis decreases from 210 mm/in in the initial seg- ment to 32 mm/in in the distal caput and 12 mm/in in the cauda epididymis and vas defer- ens(Jaakkola, 1983~. The mechanisms responsible for driving the luminal contents through the efferent ducts and epididymis include hydrostatic pressure (Johnson and Howards, 1976; Pholpramool et al., 1984), muscular con- tractions (Jaakkola and Talo, 1083), and ciliary action (Markkula-Viitanen et al., 1979~. Norepinephrine (Pholpramool and Triph- rom, 1984), acetylcholine (Pholpramool and Triphrom, 1984), and vasopressin (Jaakkola and Talo, 1981) have been pro- posed as regulators of the muscular con- tractions and ciliary action. In addition,

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68 epididymal tubular contraction can be regulated by prostaglandins and by drugs that affect their synthesis (Cosentino et al., 1984~. The relative contributions of hydrostatic pressure, electric activ- ity, and ciliary action in driving luminal flow are still unresolved; it is unlikely that any factor accounts fully for luminal flow in all mammals. Assessment of transit time of spermato- zoa through the epididymis and the rate of luminal flow are unlikely to become practical markers for studies in humans. However, numerous chemicals can probably affect transit time through the epididymis by altering neuronal activity or affecting one of the other regulatory mechanisms. Studies are needed to establish which drugs can modulate such transport and whether the effects will alter the ability of sper- matozoa to fertilize eggs or to produce normal, viable offspring. Acquisition of Fertilizing Ability In mammals, spermatozoa leaving the testis do not have the ability to fertilize eggs, whereas those in the cauda epididymis have acquired this function. It took several decades to resolve whether the role of the epididymis in that function was passive or active, but several elegant studies by Orgebin-Crist and her col- leagues (Orgebin-Crist et al.; 1975) and by Bedford (1966) clearly established that the epididymis was actively involved in converting immature to mature (i.e., fer- tile) spermatozoa. The acquisition of the potential to fertilize eggs and, separ- ately, produce viable offspring appears not to be a simple on-off situation (Nishi- kawa and Waida, 1952; Orgebin-Crist, Af4LEREPRODUCTII~E TOXICOLOGY et al., 1975; Bedford, 1979; Turner, 1979; Courot, 1981; and Orgebin-Crist and Olson, 1984.) Whether, and if so where, within the epididymis spermatozoa acquire not only the ability to fertilize eggs, but also to produce normal, viable offspring is the most important marker of epididymal function. To use such a marker, spermato- zoa from different regions of the epididy- mis need to be removed and inseminated into females of known fertility, and the result- ing progeny outcome must be analyzed. Such studies are expensive and tedious and require the use of large numbers of animals; however, they are the only proven means of determining whether a substance will selectively affect the site of matura- tion of spermatozoa, if there is any matur- ation at all. These tests need to be used more extensively to determine whether there are substances that alter the ability of the epididymis to mature sperm or the site where such maturation is acquired. The development of new simple approaches with great predictive value that will clearly establish whether failure of spermatozoa to mature has been caused by an epididymal defect has high priority. Associated with the acquisition by sper- matozoa of the ability to fertilize eggs is a gain in potential for motility. In studies in which different regions of the epididymis were ligated and spermatozoa were removed, it became apparent that sper- matozoa could acquire the potential for motility without acquiring the ability to fertilize eggs (Bedford, 1967; Orgebin- Crist, 1967a; Cummins,1976~. Except possibly in rabbits, the cauda fluid composition prevents movement of spermatozoa. The underlying mechanism 1967b; Orgebin-Crist, 1968; Blandau and of the acquisition of potential for motili- Rumery, 1964; Dyson and Orgebin-Crist, ty is unknown. A number of factors have 1973; Frenkel et al., 1978; Fournier-Del- been proposed as regulators or mediators, nech et al 1979. 1981). There is some including forward-motility protein (Hoskins et al., 1978; Acott et al., 1979), acidic epididymal glycoprotein (Pholpra- mool et al.. 1983). albumin (Pholoramool species variation with respect to the site at which spermatozoa gain their fertiliz- ing potential, but it is evident that pas- sage through some part of the caput is es- sential and that no species relies on the entire length of the epididymis for acquir- ing fertilizing potential. (For reviews, see Orgebin-Crist, 1969; Orgebin-Crist et al.3 1983)3 carnitine (Hinton et ale 1981; Inskeep and Hammerstedt31982)3 cAMP (Hoskins and Casillas3 1975; Amann et al.3 1982)3 sperm-motility inhibiting factor (Turner and Giles3 1982)3 sperm-motility

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EPIDIDYMAL STRUCTURE AND FUNCTION quiescence factor (Carr and Acott, 1984), and immobilin (Usselman and Cone, 1983~. Only in the past few years has quantita- tive objective assessment of spermatozoa! motility become available. The difficulty in monitoring sperm motility lies in the variability in the degree and type of mo- tion of a large number of spermatozoa. The method routinely used in most laboratories was the subjective determination of the percent of spermatozoa that were motile and the type of motility on a 0-4 or 0-10 scale, with 0 being immotile and with in- creasing numbers reflecting more progres- sive motility (shaking, circular, for- ward, forward progressive). The advent of high-speed videomicrography made it possible to record spermatozoa! motility accurately. Such recordings can be ana- lyzed with computer imaging techniques to provide accurate, objective assessment of the distribution of spermatozoa! speed, extent and angle of circular movement, lateral head displacement, beat angle of the flagellum, and so on. These methods have been developed for human semen analy- sis (Ginsburg et al., 1988; Mahony et al., 1988) and are only beginning to be used to characterize the acquisition of sperm motility during epididymal transit (Work- ingandHurtt,1987~. Whether and how sperm motility can be altered by chemicals that affect the epididymis has yet to be deter- mined. Various measures of sperm motility have the potential of being powerful mark- ers of one of the major functions of the epididymis, but fulfilling the potential will require much more research, which is now technically feasible. Storage of Spermatozoa The major site for storage of spermato- zoa in the mammalian duct system is the cauda epididymis. Although normal transit time of spermatozoa through the cauda is some 3-10 days (Robaire and Hermo, 1987), they can be stored in this tissue for peri- ods of over 30 days (Orgebin-Crist et al., 1975~. On storage in the cauda, a loss in fertilizing ability was found to occur before a loss in motility (Martin-Deleon etal., 1973;Cummins,1976~. An important difference between humans 69 and a number of other mammalian species is that other mammals have a high sperm pro- duction rate so that the number of stored spermatozoa available for ejaculation Is 3-, times greater than the daily sperm production rate and 2-3 times greater than that found in a"typical" ejaculate (Amann, 1981). In contrast, humans have a sperm production rate well below that of most other mammals and a sperm reserve available for ejaculation that is only about equal to the number of sperm in an ejaculate, whether the person has been at sexual rest or not. To assess the ability of the cauda epi- didymis to store spermatozoa, one either would have to count the sperm in the tissue after homogenization (a method clearly limited to animal experimentation) or would have to obtain serial ejaculates (minutes to hours apart) to assess the size of the sperm reserve in the cauda epididy- mis. This marker of epididymal function might provide some useful information about the ability of the cauda epididymis to store spermatozoa, but it is not very practical and has not yet been used to monitor damage to the epididymis other than that caused by heat (Bedford, 1977; Bedford, 1978a,b). EPIDIDYMAL LUMINAL FLUID The epididymal lumen contains water, ions, small organic molecules, proteins and glycoproteins, spermatozoa, and other particulate matter of undefined origin. From the efferent ducts all the way through to the vas deferens, numerous changes take place in the makeup of this complex of substances. The only available means of monitoring changes in the composition of the luminal fluid, without removing or irreversibly damaging the tissue, is analysis of the epididymal contribution to semen. Such analysis has been used extensively to monitor several organic molecules and proteins as markers of epididymal function, but is of no value in assessing changes in ionic makeup along the epididymis. Micropuncture of epididy- mal tubules has provided valuable informa- tion, but does not allow for serial studies and cannot be usect in humans (Hinton and

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70 Howards, 1982~. We therefore focus here on luminal markers that either can easily be measured in semen or are particularly closely related to the epididymis but need to be measured in isolated tissues. There is a precipitous decrease (by nearly 100 mM) in the concentration of chloride between the efferent ducts and the caput epididymis, whereas a similar decrease in sodium is observed between the caput and cauda epididymis (Crabo, 1965; Levine and Marsh, 1971; Jenkins et al., 1980~. The increase in potassium ion (more than 30 mat) does not account for the change in osmolarity, and it has therefore been proposed that the epididymis is involved in the secretion of organic ions (Levine and Marsh, 1971~. Phosphorus, whose serum concentration is usually around 2 mM, becomes higher than 90 mM in the corpus epididymis; this high con- centration is accounted for, in part, by the incorporation of phosphorus into glycerylphosphorylcholine and phospho- choline, which make up approximately 50 mM and 20 mM, respectively, leaving an inorganic phosphorus concentration of more than 10 mM in the cauda epididymis and vas deferens (Hinton and Setchell, 1980a). It would be of interest to deter- mine whether the very high inorganic phosphorus concentration is maintained by the action of parathyroid hormone or vitamin D in this tissue. AL4LE REPRODUCTIVE TOXICOLOGY inositol and no other sugars is most in- triguing, in that no component of inter- mediary metabolism of either spermatozoa or epididymal epithelium seems to have a preference for inQsito1 as a source of energy; such high concentrations are also unlikely to be required to mediate hormone action via the phosphatidyl-inositol phosphate system. Testosterone is the most abundant androgen entering the epi- didymis, and dibydrotestosterone becomes the major androgen in the caput epididymis, increasing by more than 200-fold between the rate testis and the caput epididymis in bulls and rats (Ganjam and Amann, 1976; Turner et al., 1984~. All the above chemicals have been meas- ured in semen as potential indicators of epididymal function; the large collec- tion of studies has not provided a clear answer as to their value as markers (Mann and Lutwak-Mann, 1981; Cooper et al., 1988~. That stems in part from the fact that many of the clinical studies were poorly controlled and in part from the lack of basic physiologic knowledge about the epididymis, which would permit rational interpretation of the results. The pos- sibility that chemicals or enzymes se- creted by sex accessory tissues can alter the concentrations of substances secreted by the epididymis should also be kept in mind. Amp specific nrntein.s senarahle hv Carnitine (Hinton and Setchell, 1980b), glycerylphosphorylcholine (Hinton and Setchell, 1980a), phosphocholine (Hinton, 1980), inositol (Hinton, et al., 1980), sialic acid (Arora et al., 1975), glycerol (Cooper and Brooks, 1981), and steroids (Ganjam and Amann, 1976; Turner et al., 1984) are dramatically concentrated in the lumen or change radically in concentra- tion as one moves down the testicular duct system. The physiologic role of high concentrations of carnitine has not yet been resolved, but one of the proposed functions of carnitine is as a precursor to acetylcarnitine, which is used as a source of energy by spermatozoa or is used to promote the maturation of spermatozoa during epididymal transit (Casillas and Chaipayungpan, 1979~. Why the epididymal lumen should accumulate specifically ~ ~ , electrophores~s, are present in the lumen of the epididymal duct system of mammals. Using micropuncture methods, Koskimies and Kormano (1975) and Turner (1979) demonstrated not only that the disk gel electrophoretic patterns of proteins of different segments of the epididymis and vas deferens differed from those of serum or rete testis fluid, but also that they differed from each other-i.e., there were gradual changes in electrophoretic pattern from the caput to the corpus epi- didymis and vas deferent. The identities of most of the proteins have not yet been established. However, in the past few years, a few molecules found in the epididymal lumen with specific physicochemical characteristics or identifiable biological activity have been described. Albumin, a2-macroglobu-

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EPIDIDYMAL STRUCTURE AND FUNCTION fin, transferrin, and androgen-binding protein have been found in epididymal luminal fluid (Amann et al., 1973; Skinner and Griswold, 1982; Turner et al., 1984~. Other proteins-such as acidic epididymal glycoprotein (Lea and French, 1981), dimeric acidic glycoprotein (Sylvester et al., 1984), forward-motility protein (Brands et al., 1978), angiotensin- 1 - converting enzyme (Vanha-Perttula et al., 1985), and ~x-lactalbumin-like protein (Hamilton, 1981)-are also likely to be in the mammalian epididymal lumen, but evidence of their presence in the luminal compartment is indirect. Many of the proteins are beginning to be used as mark- ers of epididymal function; no clear picture has yet emerged regarding their ability to reflect epididymal functions, but such results are likely to become available soon. It is important to note that 90-95% of the fluid coming from the testes is re- sorbed in the efferent ducts and the ini- tial segment of the epididymis (see, e.g., Robaire and Hermo, 1987~. This water resorption will be a factor in the con- centration measured for all the chemicals mentioned above. EPIDIDYMAL EPITHELIAL FUNCTION Histology Presence and Relative Distribution of Various Cell Types Detailed descriptions of the appear- ance, at the light and electron microscopic levels, of the epididymis in a number of animals and humans are available (Benoit, 1926; Hamilton, 1975; Connell and Don- jacour, 1985), and have been reviewed by Robaire and Hermo (1988~. The regional changes in epididymal histology have been described (e.g., for rat, see Reid and Cleland,1957~. The epididymal epithelium is pseudo - stratified and is made up of principal, basal, clear, and halo cells. The major cell type is the tall, columnar principal cell, which makes up about 80% of all epi- didymal epithelial cells (Robaire and 71 Hermo, 1987~. These cells are involved in resorption of fluid and organic material and in secretion of small organic mole- cules, as well as proteins and glycopro- teins. The next most important cell type (about 15%) is the basal cell, flat elon- gated cells that are found throughout the epididymis in contact with the basement membrane; their function has not yet been ascertained. Clear and halo cells are more sparsely distributed along the epididy- mis. In the species in which they are found, clear cells might be involved in resorbing the elements of cytoplasmic droplets that are shed by spermatozoa as they mature. These cells are not found in all mammals, e.g., the ram epididymis does not have any. Halo cells are believed to be part of the immune system and have been described as either monocytes or lymphocytes (Robaire end Hermo, 1987~. The quantitative distribution of these cell types as one moves from one segment of the epididymis to the next has recently been described not only in intact rats, but also in animals that had been treated chronically with the antitumor and im- munosuppressive agent cyclophosphamide (Robaire and Hermo, 1987; Trasler et al., 1987b). There were marked changes in the proportion of various cell types from the initial segment of the caput to the cauda epididymis and changes in the relative cell surface area of a particular cell type in selected segments of the epididymis at specified times after initiation of treatment. For several reasons, epididymal biop- sies have not become routine. Because the epididymis is a single long convoluted tubule, a biopsy would result in a disrup- tion of the tubule and hence prevent normal transport of spermatozoa through it. Kelatively little is known about the functional histology of the tissue, so the practical consequences of an altered histologic appearance would not be evi- dent. Thus, although it is clear that biopsies of epididymal tissues should not be routine, it is also evident that de- tailed analyses of the relative distribu- tion of various cell types and of histolog- ic appearance can provide valuable infor- mation on the actions of some drugs on this

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72 tissue and accordingly can act as useful markers. Blood-Epididymis Barrier The anatomic and functional existence of a blood-testis barrier is well estab- lished (Setchell and Waites, 1975), but only since the late 1970s has there been substantial evidence of a blood-epididy- mis barrier (Friend and Gilula. 1972: Howards et al., 1976; Suzuki and Nagano, 1978: Cavicchia. 1979: Greenberg and , , , Forssmann, 1983; Hoffer and Hinton, 1984), as reviewed by Robaire and Hermo (1988~. Given the presence in spermatozoa of proteins that are recognized by the body as foreign, it stands to reason that there should be a continuation of a functional barrier beyond the testis. There is extensive functional evidence of such a barrier: the large differences in the concentrations of inorganic and organic compounds between the luminal fluid and the blood (Crabo and Gustafsson, 1964; Jenkins et al., 1980; Turner et al., 1984; Hinton, 1985~. The barrier is repor- tedly resistant to various treatments, such as high-dose administration of estra- diol (Turner et al., 1981), the application of gossypol (Hoffer and Hinton, 1984), vasectomy (Turner and Howards, 1985), or the presence of varicocele (Turner and Howards, 1985), but little is known about its role in protecting spermatozoa from immunoglobulins and toxicants. Indeed, the ability of the barrier to maintain its tightness under conditions of stress might be pivotal in allowing the epididymis to sustain its functions. There is no evi- dence that environmental toxicants or drugs can disrupt the blood-epididymis barrier. However, if it were disrupted, the potential consequences could include immobilization of spermatozoa by antibod- ies and alteration of the sperm genome by chemicals. It is therefore important to develop simple markers that will allow for the monitoring of the integrity of the blood-epididymis barrier without disrupt- ing epididymal function. Spermiophagy The fate of spermatozoa that are not A[4LE REPRODUCTION TOMCOL~ ejaculated has been and remains controver- sial. There is now morphologic evidence, especially for the vas deferens, that spermiophagy does occur in a number of species under normal conditions or after mechanical or chemical manipulation. Such a process might involve the epithelial cells that line the duct system or the presence of luminal macrophages. In either case, spermatozoa in various stages of degeneration have been reported in epithe- lial cells and in luminal macrophages (Cooper and Hamilton, 1977; Holstein, 1978; Murakami et al., 1982a,b; Murakami et al., 1984), and it has been suggested that the spermatozoa are undergoing lysis. Most observers have reached the conclusion that the epithelial cells of the epididymis and the vas deferens are not involved in such activity under normal conditions (Bedford, 1975; Orgebin-Crist, 1984~. However, mechanical or chemical manipula- tion of the epididymis and vas deferens has been shown to cause epithelial cells of the different segments of the duct system to become phagocytic and to engulf and digest spermatozoa (Grover, 1969; Flickinger, 1972; Alexander, 1973; Hoffer et al., 1973; Hoffer and Hamilton, 1974; Hoffer et al., 1975; Neaves, 1975~. Also, spermiophagy by luminal macrophages has been shown to occur under abnormal condi- tions, (Neaves, 1975; Phadke, 1975; Bed- ford, 1976; Flickinger, 1982~. The ability of the epithelial lining of the duct system to become active in spermiophagy apparently depends on either the presence of excess spermatozoa or the presence of ~abnormal" spermatozoa in the lumen; the mechanism is not clear. How- ever, histologic observation and measure- ment of the extent of spermiophagy by the epididymal or vas deferens epithelium might provide a good indication of the extent of sperm damage. This potential marker has not been tested systematically for any family of drugs. Biochemical Markers Hormone Receplors and Regulation of Epididymal Function Androgensespecially 5 OCR for page 63
EPIDIDYMAL STRUCTURE AND FUNCTION tosterone (DHT), the 5c'-reduced metabo- lite of testosteroneare the primary modulators of epididymal function. How- ever, it has become apparent that many other regulatory molecules play special- ized roles in maintaining normal epididy- mal function. The factors that regulate epididymal function-i.e., those for which there are specific binding proteins-do not reach the epididymis only through the circulation (endocrine), but also through direct input from the testis (paracrine). Androgens. The dependence of the epidid- ymis on androgens has been established for over 60 years (Benoit, 1926~. Many studies during the past 3 decades have tested the effects of castration and testosterone replacement on a large array of characteristics (Orgebin-Crist et al., 1975; Brooks, 1979a), and several general conclusions can be drawn. The epididymis atrophies and treatment with physiologic concentrations of androgen only partially maintains epididymal weight; the remaind- er attributable to spermatozoa and luminal fluid (Karkun et al., 1974; Robaire et al., 1977; Brooks, 1979b). In that respect, changes in epididymal weight could be a marker of changes in testosterone. Androgens regulate intermediary metab- olism (Brooks, 1979a), the transport of ions across the epididymal epithelium (Won" and Young, 1977), the transport of inositol and carnitine across the mem- branes of epididymal epithelial cells (Bohmer et al., 1977; Brooks, 1980; Phol- pramool et al., 1982), and the synthesis and secretion of a number of epididymal glycoproteins as well as the activity of several enzymes, e.g., glutathione S- transferase (Rastogi et al., 1979; Jones et al., 1980; Moore, 1981; Mayorga and Bertini, 1982; Robaire and Hales, 1982; Brooks, 1983~. Whether any of these ef- fects are directly mediated by androgens or require the synthesis of new mRNA and therefore the synthesis of new proteins is being investigated. Acquisition of fertilizing ability and storage of spermatozoa both depend directly on androgens (Benoit, 1926; Bedford, 1975; Cohen et al., 1981~. The presence of an inhibitor of 5~-reductase caused decreases in the number of motile 73 spermatozoa, in the percentage of oocytes fertilized, and in the number of blas- tocysts foundbut only when the inhibitor was given with testosterone. There is no debate that androgens regu- late epididymal function, but the mechan- ism of their action is far less clear. Two molecules in the epididymis have high affinity for DHT; these have been named ~androgen receptor~ and "androgen- binding protein." It must be stressed, however, that the classical conditions necessary to demonstrate that a given molecule is the androgen receptor in the epididymis have not been fulfilled. Exogenously administered testosterone is transformed to DHT and is found bound to a cytosolic protein in the epididymis of a number of animals (Blaquier, 1971; Ritzen et al., 1971; Danzo et al., 1973; Younes and Pierrepoint, 1981; Carreau et al., 1984~. Epididymal cytoplasmic recep- tor seems to have many features in common with prostatic androgen receptor (Tindall et al., 1975; Younes and Pierrepoint, 1981). The binding of DHT to the cytoplas- mic receptor is blocked by antiandrogens, such as cyproterone acetate and flutamide (Tindall et al., 1975; Danzo and Eller, 1975~. Androgen-binding protein (ABP) has been found in many animals (Danzo et al., 1977; Fabre et al., 1979; Carreau et al., 1980) and humans (Hsu and Troen, 1978~. It has a number of physicochemical proper- ties that differ markedly from those of the cytoplasmic androgen receptor (Hans - son et al., 1975~. ABP is synthesized by Sertoli cells and enters the epididymis via the efferent ducts (Attramadal et al., 1981; Musto et al., 1982~. Its functions are still unresolved, but it has been proposed that it acts as an androgen sink in seminiferous tubules (Hansson et al., 1975), transports androgens to the epidid- ymis (Ritzen et al., 1971), and acts as a regulator of epididymal 5~-reductase (Robaire et al., 1981). In a rat model, a correlation between the amount of epidid- ymal ABP and the fertilizing ability of spermatozoa has been established (Anthony etal.,1984~. Several biologic markers have been used to assess androgenic action OI1 the

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74 AL4LE REPRODUCTIVE TOXICOLOGY epididymis. The first is the serum con- tritiated DHT and estradiol in the adult centration of testosterone. Although mouse epididymis revealed differences of limited value (it does not specifically in distribution of grains in different reflect epididymal activity), it is a cell types, as well as in different seg- useful gross indicator. The concentra- ments of the epididymis (Schleicher et lions of ABP in semen and in epididymal al., 1984~. That finding brings up the tissue fractions or luminal fluid have intriguing possibility that endogenous ' ^^ circulating, or potentially locally synthesized, estradiol serves a specific function in the epididymis, e.g., modula- tion of clear cell function. The difficul- ty in identifying estradiol receptors and aromatase activity in adult mammalian epididymis might be due to the localization of activity to cells (e.g., clear cells) that are not abundant in this tissue. The inherent obstacles in establishing the role of estradiol and of its receptor as indicators of specific epididymal func- tions suggest that the development of these markers should be given a low priority. Aldosterone. The concentrations of ions change along the duct system, but the mechanism responsible for controlling these ion fluxes has not yet been eluci- dated. Because of the similarities in ion fluxes between the epididymis and the kidney, Wong and Lee (1982) and Jenkins et al. (1983) have proposed similar regula- tory mechanisms. Indeed, specific binding sites for aldosterone, the adrenal mineralocorticosteroid normally con- sidered to have the kidney as its target site, have been found autoradiographical- ly to be selectively disposed over clear cells (Hinton and Keefer, 1985) and have been shown to be involved in regulating the concentration of spermatozoa in the epididymis (Turner and Cesarini, 1983~. More studies are required to elucidate the role of aldosterone in epididymal function and to determine whether specific markers can be developed to reflect its epididymal activity. Prolactin. In light of the possible coreg- ulation of gonadotropin and prolactin release, the apparent presence of prolac- tin "receptors" in the epididymis (Aragona and Freisen, 1975; Orgebin-Crist and Djiane, 1979), and the proposal that prolactin regulates ion transport (Shiu and Friesen, 1980), it is plausible that this hormone plays a major role in regulat- ing some facets of epididymal function. been used as a combined indicator of Ser- toli cell and epididymal functions. Androgen-receptor and 5~-reductase activities have been measured as markers of the androgenic status of the epididymis; although these two markers are highly specific and are probably among the most useful of the markers discussed in this section, they require removal of tissue, which is possible only under experimental conditions. If noninvasive techniques could be devised to monitor these aspects of androgen-related epididymal activity, they would be extremely useful. Estrogens. The administration of estro- gens can affect the male reproductive system in general (Gay and Dever, 1971; Swerdloff and Walsh, 1973; Karr et al., 1974; Verjans et al., 1974; Ewing et al., 1977) and the epididymis in particular (Meistrich et al., 1975; Orgebin-Crist et al., 1983~. Although it has been gener- ally accepted that mediation of estrogen action takes place via the hypothalamo- pituitary-gonadal axis (Swerdloff and Walsh, 1973; Verjans et al., 1974; Robaire et al., 1979), substantial evidence of the direct action of estrogens on androgen target tissues has accumulated over the past 2 decades. Specific, well-regulated, high-affinity cytosolic and nuclear estrogen-binding proteins, presumably receptors, have been identified in the epididymis of mice (Schleicher et al., 1984), rats (Van Beurden-Lamers et al. 1974), rabbits (Danzo and Eller, 1979), dogs (Younes et al., 1979), and humans (Murphy et al., 1980~. It is proposed that stromal (as opposed to epithelial) cells (Cunha et al., 1980) act as primary target sites because estrogen adm~n~stration to young dogs (Cornell and Donjacour, 1985) or rabbits (Orgebin-Crist et al., 1983) leads to increases in epididymal weight that were due almost entirely to stromal hyperplasia. Autoradiographic localization of

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EPIDIDYMAL STRUCTURE AND FUNCTION Prolactin and its binding protein might eventually become useful markers of epi- didymal function, but the available data suggest that this subject be given low priority for research. Vitamin D. The known primary function of vitamin D (a sterol hormone) is the homeostasis of calcium and phosphorus; its major sites of action are bone, intes- tine, and kidney (De Luca,1978~. A number of complementary observations suggest that the epididymis can also be a target for its action. First, specific binding proteins for 1,25-(OH)2 vitamin D have been found in the rat epididymis (Walters et al., 1982~. Second, the epididymis was found to take up 25-(OH) vitamin Do and metabolize it to both 1,25-(OH)2 vitamin D3 and 24,25- OCR for page 63
76 system is situated on the basolateral membrane, cannot transport other hexoses, and can be inhibited by 3-O-methylglucose, a nonmetabolizable analogue that is recog- nized by the transport system. Oxidative metabolism is high right after birth in the rat epididymis, decreases for the first 2 weeks of life, and then increases in parallel with the increase in circulating androgens (Delongeas et al., 1984~. A number of other enzymatic processes, such as prostaglandin biosynthesis and metabolism, have been described for the epididymis (Robaire and Hermo, 1987~. We limit our discussion here to glutathi- one, because of its potential usefulness as a marker of epididymal function. Sever- al enzymes involved in the biosynthesis, metabolism, and conjugation of glutathi- one are present in the epididymis. They are particularly important, because free glutathione is viewed by many toxicolo- gists as a mechanism of "mopping up" elec- trophiles that might damage cellular components. The family of epididymal enzymes involved in the conjugation of glutathione with electrophilic chemicals, the glutathione S-transferases, can be resolved into six peaks, each with a char- acteristic isoelectric point and sub- strate specificity (Hales et al., 1980~; the most acidic peak is much higher in the epididymis than in other tissues and might be specific for it. Results of studies on the longitudinal distribution of these enzymes in the epididymis indicate a general trend toward decreasing activity for each of the substrates from the caput to the cauda epididymis (Hales et al., 1980~. Thiol oxidase, an enzyme that converts 2 R-SH + O2 into R-S-S-R and H2O, has been reported in rat and hamster epi- didymis (Chang and Zirkin, 1978~; enzyme activity was higher in the cauda than in the caput epididymis. It was proposed that such an enzyme could protect spermatozoa from endogenous free sulfhydryls. Excep- tionally high concentrations of q-gluta- myl-transpeptidase have also been report- MALE REPRODUCTIVE TOXICOLOGY ed in the rat epididymis (DeLap et al., 1977~. That enzyme is involYed in the transport of q-glutamyl amino acids and can act as a glutathione oxidase, convert- ing reduced to oxidized glutathione (fate end Orlando, 1979~. EPIDIDYMALLY MEDIATED TOXIC DRUG EFFECTS A growing number of compounds have been shown to affect the epididymis, e.g., a- chlorohydrin, 6-chloro-6-deoxyglucose, and possibly gossypol (Robaire and Hermo, 1987~. The epididymis, however, has not usually been studied as one of the target tissues for the toxic effects of drugs or environmental toxicants, so little is known about the importance of this tissue as a target for toxic drug effects. For some compounds, such as dibromo- chloropropane (DBCP), it has clearly been shown that there is an effect on the epidid- ymis, e.g., on epididymal weight or sperm reserves (Amann and Berndtson, 1986~; but it is still not clear whether the deleter- ious effects of this drug, and others like it, on reproductive outcome are directly linked to their action on the epididymis or epididymal spermatozoa. Clear evidence has emerged from some recent studies that-after a week of treat- ment with such drugs as cyclophosphamide, an anticancer and immunosuppressive drug (Trasler et al., 1985, 1986~; methylnitro- sourea, an alkylating agent (Nagao, 1987~; or methyl chloride, an industrial gas (Chellman et al., 1986~-a sharp increase in preimplantation and postimplantation loss (dominant lethal mutations) could be found. Such male-mediated adverse effects on reproductive outcome were associated, in the case of cyclophospha- mide, with specific changes in the dis- tribution of cell types in the epididymal epithelium and an increase in the number of spermatozoa with abnormal flagellae in the rat epididymis (Trasler et al., 1988~.