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6 Biologic Markers of Accessory Sex Organ Structure and Function This brief review on markers of accessory sex organ function shows that few non- invasive or minimally invasive markers exist for the assessment of accessory sex organ function in human males exposed to toxicants. That is probably because little is known about the function of these glands, because they are inacces- sible, and because they vary between mam- malian species in their presence, size, and structure. Those difficulties are exacerbated by the mixing of the accessory sex organ secretions at ejaculation, by the variation in the volume and chemical makeup of the secretions between ejacu- lates even of the same individual, and by the presence in semen of enzymes that alter the chemical composition during clotting, liquefaction, and storage. However, it has been demonstrated that many exogenous chemicals are trapped in accessory sex organ secretions. There- fore, research in appropriate animal mod- els will be important. The male accessory sex organs charac- teristic of mammals are the prostate, semi- nal vesicles, ampullae of the vas deferens, bulbourethral (Cowper's) glands, urethral (Littre's) glands, and preputial glands (Fig. 6-1~. The prostate is purported to be the only accessory sex organ present in all mammals (Coffey and Isaacs, 1981~. Species vary in the presence, size, and structure of the male accessory sex organs, 77 but the organs share some important charac- teristics: they all contain secretory epithelium that is magnified greatly by villous infoldings or a compound tubulo- alveolar structure, and they all depend on androgen for differentiation, growth, and secretory function (Coffey, 1986~. Little is known about the function of these organs beyond their obvious secre- tion of fluids that mix with sperm at ejacu- lation. It has been suggested that the accessory sex organ secretions are bac- teriostatic and can protect the male geni- tal tract from bacterial infection. (Refer to reviews Coffey (1986) and Mann and Lutwak-Mann (1981) for detailed de- scriptions of the structures and functions of the male accessory sex organs.) The growth and structure of accessory sex organs and their secretion of specific chemicals into seminal plasma constitute biologic markers of exposure to or effects of toxicants. This chapter discusses pri- marily the prostate and to a lesser extent the seminal vesicles. It does not discuss the ampullae, the bulbourethral glands, or the preputial glands. There are several reasons for the exclusion: the prostate is probably the only accessory sex organ that occurs in all mammals; the human pros- tate has been studied extensively, not because of its response to toxicants, but because prostatitis, benign prostatic hyperplasia, and prostatic cancer are

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78 PROSTATE GLAND - TUBULES COILED IN PLACE TEST In _ v~c nFFFRF FO SSA NAVICULARIS major human diseases; and general concepts established for the prostate probably will have application to other accessory sex organs. It is impossible to restrict our comments to the human, because data on potential biologic markers are fragmen- tary. Therefore, we include animal data- frequently obtained from the dog, whose prostate has many similarities with the human prostate. The dog is a particularly valuable animal model, because it has no seminal vesicles or bulbourethral glands, and prostatic secretion therefore con- stitutes more than 95% of its seminal plasma. PHYSICAL MARKERS Size is an important consideration in the clinical evaluation of the prostate in humans (Coffey, 1986) and dogs (Blum et al., 1985~. Accessory sex organ weight in general and prostate weight in particu- lar are used widely as androgen bioassays, because androgen is required for growth and secretion. Although useful in animal experiments, prostatic weight is not use- ful in humans as a marker, because changes in weight are nonspecific (growth does ABLE REPRODUCTIVE TOXICOL{X;Y FIGURE ~1 Diagrammatic representation of testis, showing duct system and relation of ducts to accessory sex glands and penis. Source: Rev printed with permission of Macmillan Publish- ing Co. from The Human Body: Its Stmcture atld Physiology, Ed ea., Grollman, 1987. Copy- ~ht 19871~r Sigmund Grollman. not necessarily reflect secretion) and imprecise and its measurement requires autopsy. Estimates of prostatic volume obtained by measuring length, width, and height in situ with calipers at laparotomy are highly correlated with prostatic weight in the dog (Walsh and Wilson, 1976; Deklerk et al., 1979~. That relationship is not as applicable to irregularly shaped struc- tures, such as the seminal vesicles or multilobular prostates. Even with the dog prostate, there are practical limits to the frequency and number of such meas- urements, because of the long recovery time associated with laparotomy, the for- mation of prostatic adhesions, the risk of infections, and the tedious, time-con- suming effort associated with repeated surgery. Berry et al. (1985) recently developed a method of generating a three-dimensional representation of the dog prostate from a pair of orthogonal radiographs of the prostate in situ to which six metal markers are surgically attached. The externally estimated prostate weight is highly corre- lated with actual prostate weight (r 0.90~. The technique affords precise es-

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AL4RKERS OF ACCESSORY SEX ORGANS timates of changes in weight within a pros- tate over time. Although it allows con- tinuous monitoring of a dog's prostate during the course of an experiment, it requires extensive surgery and access to sophisticated x-ray equipment. A simple, noninvasive technique of imaging the pros- tate is still needed. Transrectal and transabdominal ultra- sound have been used to assess prostatic volume in the human (Henneberry et al., 1979; Bartsch et al., 1982~. There is a statistically significant relationship between the ultrasound estimate and the size of the prostate (Henneberry et al., 1979; Bartsch et al., 1982~. Blum et al. (1985) recently compared prostatic size measured by in viva ultrasound with actual volumetric measurement of the dog prostate at autopsy; they also reported a statisti- cally significant correlation between the two measures. The advantages of using ultrasound to determine prostatic size are the ease and rapidity of the technique, the involvement of only minimal trauma, and the avoidance of prostatic scarring. However, greater precision and accuracy are required. STRUCTURAL MARKERS The human prostate, a complex organ, is divided into five zones: anterior fibro- muscular stroma, peripheral zone, central zone, preprostatic tissue, and transition zone (Coffey, 1986), containing stromal and epithelial cells. Efforts are under way to identify these various zones with magnetic resonance imaging in situ (Iverson al., 1988; Sommer et al., 1988; Allen et al., 1989~. The complex and highly differentiated cytoarchitecture of the prostate has been used at the light microscopic level to diagnose a variety of clinical prostatic disorders. But it has not been used widely to study the effect of toxicants on pros- tatic function in humans or experimental animals, because it requires invasive biopsy techniques or autopsy specimens; because diagnostic histopathology re- quires judgment by a highly trained spe- cialist and is qualitative, rather than quantitative; and because it is difficult 79 to determine whether a toxicant acts di- rectly on a specific cell type in the epi- thelium, indirectly via an effect on a stromal element, or even more indirectly via the hypothalamo-hypophyseal-gonadal axis. Stereologic techniques have been used to quantify the volume of prostatic lumen, epithelial cell, and stromal ele- ments in the human (Bartsch and Rohr, 1977; Bartsch et al., 1979) and dog (Zirkin and Strandbergh, 1984) at the light micro- scopic level. The same approach has been extended to the ultrastructural level to quantify the volume of cytoplasmic or- ganelles in specific cell types in dog prostate (Zirkin and Strandbergh, 1984~. Although invasive and tedious, these are sensitive and precise biologic markers of prostatic structure and will be particu- larly useful in understanding mechanisms of toxicity. FUNCTIONAL MARKERS Accessory sex organs secrete fluids that are emitted into the urethra and later ejaculated or otherwise excreted. The average human ejaculate contains ap- proximately 3 ml; 1% of the volume is sper- matozoa and 99% is seminal fluid derived from accessory sex organ secretions (Coffey, 1986). In the human, about 1.5- 2.0 ml of the seminal plasma in an ejaculate is derived from the seminal vesicles and about 0.5 ml from the prostate. The volume of seminal plasma and the chemical consti- tuents in seminal plasma can be used as markers to monitor the effect of toxicants on accessory sex organ secretion. Human seminal plasma can be obtained noninvasively and repetitively by mastur- bation. The volume of the human ejaculate is only a crude measure of the secretory capacity of the accessory sex organs, be- cause it is influenced by frequency. Also, the volume is nonspecific, in that it re- flects the accumulation of secretions not only of the prostate and seminal vesicles, but also of the epididymis, ampullae of the vas deferens, bulbourethral glands, and urethral glands. In contrast, the dog ejaculate reflects prostatic secretion more directly, because the dog lacks semi-

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80 nal vesicles and bulbourethral glands. Efforts have been made to obtain more spe- cific information in humans by mechanical- ly splitting ejaculates obtained through masturbation into temporal fractions, the first of which are rich in sperm and prostatic secretions and the latter in seminal vesicular secretions (Coffey, 1986~. That approach has met with only limited success, because of individual variability and the difficulty in collect- ing split ejaculates. Inaccuracies and imprecision in the volumetric measurement of human seminal fluid are also caused by interindividual variation and by intra- individual variation over time. More specific information regarding accessory sex organ secretory function can be derived by measuring the concentra- tions of specific endogenous chemicals in seminal fluid. It is beyond the scope of this report to review exhaustively the 100 or more endogenously produced chemi- cals that appear in mammalian seminal plas- ma, and the reader is referred to the re- views by Coffey ( 1986) and Mann and Lutwak- Mann (1981~. We limit our discussion here to a few well-known chemical markers of individual human accessory sex organ func- tion and later discuss some potential mark- ers discovered in experimental animals. Individual human accessory sex organs have been shown to be the principal sources of specific chemicals in seminal fluid. For example, fructose is derived from human seminal vesicles (Mann and Lutwak-Mann, 1981), and zinc, acid phosphatase, and spermine from the human prostate (Coffey, 1986~. Measurement of those chemicals in seminal fluid should reflect the expo- sure and the effects of exposure of the prostate or seminal vesicles to toxicants (Bygdeman and Eliasson, 1969~. However, such data will not prove that a toxic chemi- cal acted directly on a specific accessory sex organ. Repetitive samples must be collected from the same person over time to offset the variability inherent in the volume and chemical makeup of a single sample of seminal fluid. And the collec- tion and preservation of semen must be controlled rigorously, because clotting and liquefaction can take place, because enzymatic activities in seminal fluid ~1I-E REPRODUCTIVE TOXICOLOGY alter its chemical composition, because the concentration of enzyme inhibitors is variable, and because such seminal fluid constituents as fructose are metabolized by spermatozoa (Rui et al., 1986~. Perhaps the existence of those characteristics explains why the technique has not been used widely as a biologic marker. Research and development might make it more useful. Numerous drugs and toxic chemicals have been identified in human semen (Reeves et al., 1973; Stamey et al., 1973; Malmborg, 1978; Cohn et al., 1982~. Ex- amples of exogenous chemicals transported into and accumulating in semen of various mammals are carcinogens, antibiotics (including basic macrolides and sulfona- mides), and methadone. In general, these chemicals are assumed to cross cell mem- branes of the accessory sex organs by non- ionic diffusion. Several factors probably are important in trapping these chemicals in accessory sex organ secretions, includ- ing lipid solubility, pK of the chemical, binding of the chemical to proteins, and pH of sex organ secretions. Chemicals are absorbed intravaginally in the human fe- male (Benziger and Edelson, 1983~. It has been shown that thalidomide in the rabbit (Lutwak-Mann, 1964) and cyclophosphamide in the rat (Hales et al., 1986; Trasker et al., 1987) cause paternally mediated ad- verse effects on the progeny. Therefore, it is possible that toxic agents in human semen absorbed by the mother can affect the conceptus. Alternatively, the same chemicals might affect the quantity or viability of or the genomic information in spermatozoa. Clearly, the subject war- rants high priority for research. Human seminal plasma contains a variety of enzymes, metalloproteins, flavopro- teins, and mucoproteins (Coffey, 1986; Mann and Lutwak-Mann, 1981), many of which occur in other bodily compartments and therefore are not specific markers. How- ever, some proteins exhibit organ specifi- city; for example, serum acid phosphatase concentration has been used as a marker of prostatic carcinoma (Coffey, 1986; Mann and Lutwak-Mann, 1981~. It is surprising that macromolecules in semen have not been exploited by toxicologists as markers of individual accessory sex organ function.

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MARKERS OFACCESSORY SEX ORGANS More specific and therefore more promis- ing biologic markers have been identified. Kistler and colleagues (Ostrowski et al., 1979, 1982) purified an androgen-depen- dent rat seminal vesicular protein IV. Recently, they isolated and characterized a genomic clone for rat seminal vesicular protein IV (Kandala et al., 1983~. French and coworkers (Lea et al., 1979) purified a major secretory protein of rat ventral prostate (prostatein). French and col- leagues also have purified two androgen- dependent secretory proteins of rat dorsal prostate and coagulating gland that, al- though anatomically distinct, synthesize similar proteins in response to androgens (Wilson and French, 1980; Wilson et al., 1981). A single (unnamed) protein accounts for over 90% of the total protein in dog seminal plasma (Isaacs and Shaper, 1983~. Clearly, a primary function of the dog prostate is to synthesize and secrete that protein in response to androgens (Isaacs and Shaper, 1985~. Therefore, its measure- ment in seminal fluid is a sensitive and specific marker of androgen-dependent prostate function in the dog. The protein has been shown to have many characteristics of a glandular kallikrein (Isaacs and Cof- fey, 1984~. Kallikreins are proteolytic enzymes that have the capacity to process precursor molecules into biologically active peptides. It was recently discov- ered that the human prostate secretes a 81 kallikrein into seminal fluid (Fink et al., 1985~. Those findings suggest that macromole- cules secreted into seminal plasma have characteristics that make them desirable biologic markers of exposure to or effect of toxic chemicals. First, individual accessory sex organs synthesize and se- crete specific macromolecules into semi- nal fluid. Second, the process is regu- lated by androgen in some instances and therefore reflects the adequacy of the production of androgen. Third, many small molecules in seminal fluid might result from diffusion into accessory sex organ secretions from the blood; that would limit their usefulness as biologic markers, but this is unlikely to be a problem with macro- molecules synthesized in accessory sex organs under the influence of androgens. Fourth, specific antibodies can be gener- ated against a purified protein and thus permit the study of their cellular origin, the development of immunoassay measure- ment techniques, and the development of a cDNA clone to ensure production of ade- quate amounts of the protein for molecular biologic analysis. Much research remains to be done to identify specific proteins in appropriate animal models and in humans. Once that is achieved, it will be im- perative to test the effects of a series of toxic agents on specific markers in a standardized protocol.

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