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4 Biologic Markers of Testicular Function This chapter focuses on physical and chemical markers of the testes; stere- ologic and biochemical assessments of Leydig cells, Sertoli cells, and germ cells; and molecular biologic analyses of DNA and RNA in germ cells. Some markers other than semen analysis (discussed in Chapter 7) are noninvasive or minimally invasive and can be used to assess testicu- lar function in human males exposed to toxicants. In addition, several promising noninvasive or minimally invasive markers using new imaging techniques, molecular biologic assays, and biochemical assays of saliva, serum, and urine are identified. The testis has two compartments: the interstitium and the seminiferous tug bules. The interstitium contains Leydig Figure 4-1 shows the human germ cell cells that produce the male hormone tes- types, the life span of each, and the time tosterone. Testosterone causes the dif- required for each to reach the ejaculate. ferentiation and development of the The figure illustrates three important fetal reproductive tract, the neonatal points. First, the human testis has 14 organization of what will become androgen- recognizable types of germ cells. Second, dependent target tissues in puberty and toxic effects on a specific germ cell might adulthood, the masculinization of the male not be manifested in the semen for alive car at puberty, and the maintenance of growth and function of androgen-dependent organs in the adult. The seminiferous tubules contain ger- minal epithelium and supporting cells. The supporting cells include Sertoli cells; in adults, Sertoli cells are static nonproliferating cells that are inti- mately associated with and support ger- minal cells involved in spermatogene- sis, the production of spermatozoa. The germinal epithelium is populated by cells that give rise to spermatozoa. Spermato- genesis encompasses a phase during which primitive spermatogonia divide either to replace their number (stem cell renewal) or produce new spermatozoa that are com- mitted after additional mitotic divi- sions to become spermatocytes; a meiotic phase during which spermatocytes undergo the first and second meiotic divisions that result in haploid spermatids; and a spermiogenic phase during which sper- matids undergo a dramatic metamorphosis in size and shade to form snermat~zon weeks, because more mature unaffected cells will continue to develop and appear in the ejaculate. Third, the time of ap- pearance of defective, immotile, or re- duced numbers of spermatozoa in the ejacu- late provides important information about the germ cell type affected by a toxi- cant. Figure 4-2 presents an example to 47

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48 ll MALE REPRODUCTIVE TOXICOLOGY go o o CL CD CD lo o :~ - ~ LL c 11_' CC ~ ~ a) Duration of Stage (days) en =0 Z ~ ~ ~ O`r Em lL ~ llJ m~ On Oh oh ~ Oh ~ Oh <0 Z O ~ LL oh an an N IL cat LL ~ LL Ant fir O co Z a,,) _ Oh ~ _ IL ~ C) C) Cal ~ > Ad Ap B PL L Z P II Sa Sb1 Sb2 SO Sd1 Sd2 us 1 1 ~ _ 18.5 1 8.8 1.0 3.7 ~ 2.9 15.6 0.8 8.0 1.0 1.2 5.8 4.8 T 1.6 1 -12 r c' LU r I , , , ' ~ 1 1 ' - ' ~ 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 1 , 1 1 1 1 1 1 ~ 1 1 ~ 1 1 1 1 1 1 1 1 Time to reach ~ I I I I ~ ~ I I Ejaculate I I I I I I I I I (days) 85.7 67.2 58.4 57.4 53.7 50.8 35.2 34.4 26.4 25.4 - 1 1 1 1 1 1 1 1 1 1 1 1 24.2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 18.4 13.6 12 1 1 l o FIGURE ~1 Spermatogenesis in man, showing life span of each cell and time necessary to reach ejaculate. Redrawn from da Cunha et al., 1982. illustrate those points. Spermatozoal concentration and motility were monitored at various times after 12 courses of treat- ment of a melanoma patient with AMSA [4'- (9 -acridinylamino Methane - sulfon - m - anisididel. The slow decline in the num- ber of motile spermatozoa in the ejaculate was interpreted by the investigators as suggesting that there was no immediate damage to spermatozoa transport or the epididymis. They believed that primarily postspermatogonial germ cells were killed by the AMSA therapy. (Initially, the cells that were killed were primary spermato- cytes; later, type B spermatogonia were also killed.) They interpreted the rapid recovery of sperm concentration and motil- ity in the semen 13 weeks after the first nine courses of AMSA treatment as showing that type A (stem) spermatogonia were not irreversibly affected. PHYSICAL AND CHEMICAL MARKERS OF TESTICULAR FUNCTION Markers in Use Testicular consistency and size have been considered important in the clinical evaluation of human male fertility. With tonometers, one can measure testicular consistency through the stretched scrotal skin. Tonometer readings have been corre- lated with clinical impressions of tes- ticular consistency in human males (Lewis et al., 1985) and with sperm morphology in bulls (Hahn et al., 1969~. The advan- tages of testicular consistency as a bio- logic marker are that it is non~nvas~ve and simple to measure. The disadvantages are that it is unrelated to a specific de- fect in testicular function and that large changes are required to detect ef- fects of treatment. Testicular weight is directly corre- lated with testicular volume, which can be estimated from testicular size in many vertebrates (Bailey, 1950; Kenagy, 1979; Handelsman and Staraj, 1985~. In mammals, the bulk of testicular volume and therefore

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L4RKERS OF TESTICULAR FUNCTION Courses of AMSA 1 2 3 4 5 6 7 ~ 9 10 11 12 ,,, 10' a) _ ._ O a) ~ ~ ~ 1 o6 I- O 105 1o2 \60% ~40% \10% \5% \ 7 ospermia ~/////////////~///////~///////////////~ 0 20 40 60 Weeks in Observation weight is accounted for by germ cells (Amann, 1 970a). Estimated testicular volume based on measured size is a simple and noninvasive marker of sperm production (Foote, 1969; Chubb and Nolan, 1985~. However, it is imprecise, because errors are inherent in the measurement process itself and in the calculation of volume of nonspherical objects. The use of weight measurements, although precise, has the disadvantage of being invasive, in that the testes must be removed. Markers Requiring Research and Development Organ chemiluminescence can give read- ily detectable, continuously monitorable, noninvasive signals of oxidative metabol- ism (Boveris et al., 1980~. It is possible that chemiluminescence can be used to moni- tor the effect of toxicants on the radical reactions of lipid peroxidation in testes in situ or in testes perfused in vitro. Kopp et al. (1986) used phosphorus-31 magnetic resonance imaging (MRI) to deter- mine functional metabolic correlates, temporal relationships, and intracellular actions of cardiotoxic chemicals nonde- structively in isolated intact perfused rat hearts. They obtained useful informa- tion on biochemical mechanisms respon- 49 FIGURE ~2 Sperm concentration and motility during chemotherapy with AMSA [~4'-9-acndinyl-aniino) methan~sulfon~ -ariisidide] . Source: da Cunha et al., 1982. sible for the cardiotoxic actions of xeno- biotics. This approach can probably be applied profitably to an in vitro perfused rat testis model immediately and perhaps to the human testis in situ eventually. One potential problem is that MRI might alter testicular temperature. Other in- direct measures of testicular size and function are promising but have received little attention, including ultrasound, positron-emission tomography, and com- puted axial tomography. LEYDIG CELLS Leydig cells are the principal source of testosterone in the mammalian male (Ew- ing and Zirkin, 1983~. Leydig cell growth and differentiated function depend on anterior pituitary production of lutein- izing hormone (LH) (Ewing and Zirkin, 1983~. Toxicants can interfere with tes- tosterone production indirectly by in- terfering with gonadotropin-releasing hormone (GnRH) stimulation of pituitary gonadotropes, by interfering with LH pro- duction by pituitary gonadotropes, or by interfering with receptor-mediated LH stimulation of testosterone secretion by Leydig cells. Toxicants might also inhibit the Leydig cell steroidogenic apparatus directly.

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so Markers in Use Stereologic techniques allow determina- tion of Leydig cell numbers per testis at the light microscopic level and of Leydig cell cytoplasmic organelle volume and membrane surface area (e.g., area of inner mitochondrial membrane and smooth endo- plasmic reticulum) at the electron micro- scopic level in both humans (Mori et al., 1982) and experimental animals (Mori and Christensen, 1980~. Although they are invasive and tedious to carry out, these biologic markers of Leydig cell structure have been shown to be highly correlated with Leydig cell steroidogenic function under a variety of experimental and physio- logic conditions (Christensen and Pea- cock, 1980; Zirkin et al., 1980; Ewing et al., 1981~. For example, prolonged expo- sure to lead causes a diminution in tes- tosterone production and in the surface area of smooth endoplasmic reticulum in rat Leydig cells (Zirkin et al., 1985a). In addition, Leydig cell numbers diminish with advancing age in humans (Kaler and Neaves, 1978~. These morphologic markers are particularly helpful in studies aimed at understanding mechanisms of toxicity. Testosterone is commonly secreted epi- sodically by Leydig cells in the mammalian testis (Ewing et al., 1980~. There are annual and diurnal rhythms in testosterone production, and a diurnal rhythm and fre- quent sporadic bursts of testosterone in some species. The episodic nature of tes- tosterone secretion in man is blunted, compared with that in many species. The variation complicates the assessment of Leydig cell steroidogenic activity by measurement of peripheral blood testos- terone concentration, because a regimen of frequent sampling must be followed, especially in experimental animals (Ismail et al., 1986~. Measurement of testosterone concentration in blood serum at 10-minute intervals for 8 hours will usually indicate whether a toxicant has altered the entire hypothalamo-adeno- hypophysial-testicular axis in experimen- tal animals. But toxic effects in humans might be diagnosable with less frequent or even a single serum testosterone meas- urement, because testosterone production MALE REPRODUCT~ TOMCOL~ is not so episodic. The function of the hypothalamo-adenohypophysial link can also be assessed by measuring serum LH concentrations every 3-10 minutes for 24 hours. Measurement of LH and testosterone concentrations in peripheral blood for less than 24 hours but at the same frequency will impart the same information regarding the hypothalamo-hypophysial function. Measurement of serum LH concentration might be more useful than testosterone pulses for diagnosing defects in the hypo- thalamo - adenohypophysial- testicular axis in humans, because of the attenuation of testosterone pulses. In both men and experimental animals, the capacity of pituitary gonadotropes to respond to GnRH can be assessed by in- jecting a bolus of GnRH either intravenous- ly or subcutaneously and then measuring LH in peripheral blood. Similarly, the capacity of Leydig cells to respond to LH can be assessed by injecting a bolus of human chorionic gonadotropin (hCG) intravenously and measuring testosterone in peripheral blood. Again, measurement of LH and testosterone concentrations in peripheral blood for a shorter period but at the same frequency will impart the same information; with less frequent sam- pling, there is a loss of sensitivity and precision in detecting an effect of expo- sure to a toxicant. The advantages of meas- uring the concentration of LH and testos- terone in peripheral blood serum of humans or experimental animals are that it direct- ly monitors the gonadotrope and Leydig cell function, respectively, and that repetitive measurements can be obtained for assessing temporal effects of a treat- ment. Disadvantages of measuring LH and testosterone in peripheral blood are that only small amounts of blood can be collect- ed from small rodents, unless red blood cells are replaced and that, because LH and testosterone production are episodic, several measurements are required. Most testosterone (over 90%) in periph- eral blood is bound to albumin and tes- tosterone estradiol-binding globulin and therefore is biologically inert. If it is important, free biologically active testosterone can be measured by separating free and protein-bound testosterone

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MARKERS OF TESTICUI~4R FUNCTION (Vermeulen et al., 1971~. This issue be- comes important when increased sex ster- oid-binding globulin results in a decrease in free testosterone and causes hypogonad- ism. An example is the effect of chronic alcoholism in human males (Van Thiel et al., 1974~. Inhibition of Leydig cell steroidogene- sis in viva causes a diminution in acces- sory sex organ weight, discussed in Chapter 6 as a bioassay for peripheral blood tes- tosterone concentration in experimental animals (Dorfman and Shipley, 1956; Chubb and Nolan, 1985; Coffey, 1986~. Finally, Leydig cell steroidogenesis can be monitored by removing testes from experimental animals and measuring their capacity to produce testosterone in vitro. For example, testes of some species (e.g., mice and rats) can be dispersed with col- lagenase and the production of testoster- one by testicular cells measured (Bordy et al., 1984~. Alternatively, testes of numerous species have been perfused in vitro (Ewing et al., 1981~. These tech- niques are invasive and limited to a few hours duration in vitro. But they can be particularly helpful in studies aimed at understanding mechanisms of toxicity. Clearly, this approach is impractical in humans. Markers Requiring Research and Development It has been suggested that measurement of testosterone in saliva is an excellent biologic marker for Leydig cell testoster- one production, because saliva can be col- lected from humans repetitively in a non- stressful manner and because salivary testosterone concentration is correlated closely with the biologically active free testosterone in blood (Riad-Fahmy et al., 1982~. Studies have borne this idea out: human salivary testosterone concentration has been shown to exhibit a circadian rhy- thm (Magrini et al., 1986), to increase after hCG stimulation (Nahoul et al., 1986), and to be highly correlated with pathophysiologic conditions that result from modifications in serum testosterone concentrations (Riad-Fahmy et al., 1982~. To our knowledge, this technique has not 51 been used to monitor the effect of xeno- biotics on Leydig cell function in humans. It should be added to the armamentarium of reproductive toxicologists and epide- miologists, because it provides a nonin- vasive, specific, accurate, and sensitive marker to monitor exposure to or effects of a toxic agent on Leydig cell steroido- genic function in human males. Measurement of serum or salivary tes- tosterone concentration does not repre- sent the integrated 24-hour rate of tes- tosterone production. That can still be achieved only by sophisticated study of metabolic clearance rate, which is tedi- ous, time-consuming, expensive, and im- practical for application to humans. How- ever, considerable evidence shows that it is possible to monitor ovarian function and pregnancy status by measuring gonado- tropin and gonadal steroids and/or steroid metabolites in morning or random urine specimens from females of numerous species (Lasley et al., 1980; Czekala et al., 1981~. The advantage of this approach is that the marker of interest accumulates in urine, thus obscuring the episodic na- ture of hormone production and making it possible to estimate integrated hormone production over time with fewer samples. There are shortcomings (Edwards et al., 1969; Curtis and Fogel, 1970), and a care- ful evaluation will determine whether specific hormones can be measured in urine samples as biologic markers of the functional status of the hypothalamo- adenohypophysial-Leydig cell axis in experimental animals and humans. Leydig cells probably have functions other than testosterone production. Therefore, considerable research is re- quired to uncover potentially new and useful Leydig cell markers. The testis contains peptides that are also formed elsewhere in the body. There is evidence of a GnRH-like factor, thyrotropin- releaslng hormone, arglnlne vasopressin (AVP), somatomedins, oxytocin, mitogens, epidermal growth factor, and several pro - opiomelanocortin (POMC) - derived peptides (Hsueh and Schaeffer, 1985; Boi- tani et al., 1986; Kasson et al., 1986) in mammalian testes. The most experimental detail is available on the GnRH-like factor

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52 and AVP, which have been shown to alter Leydig cell steroidogenesis, and on POMC- derived peptides, which apparently are produced in Leydig cells. It is beyond the scope of this report to discuss each of these testicular peptides in detail. We focus our attention here on the POMC- derived peptides, for three reasons: con- cepts established for the use of one pep- tide as a biologic marker might have ap- plication to others; the GnRH-like peptide seems to be restricted only to the rat tes- tis, whereas POMC-derived peptides seem to be more widely distributed; and more information is available on the physicochemical characteristics of POMC- derived peptides than on those of the other testicular peptides. The reader is re- ferred to comprehensive reviews for more information on testicular GnRH-like fac- tor (Hsueh and Schaeffer, 1985) and AVP (Cooke and Sullivan, 1985; Kasson et al., 1986). POMC-derived peptides have been local- ized by immunocytochemical procedures in Leydig cells, but not in myoid and Ser- toli cells of rat, mouse, hamster, guinea pig, and rabbit testes (Tsong et al., 1982a,b). mRNA for POMC-like proteins was localized in Leydig cells of mouse testes by in situ hybridization (Gizang- Ginsberg and Wolgemuth, 1985~. It was later shown that genes for POMC-like pro- teins and the concentration of Leydig cell POMC-derived peptides are regulated by LH (Shaha et al., 1984; Boitani et al., 1986; Valenca and Negro-Vilar, 1986~. Together, those results suggest that Ley- dig cells might produce POMC-like pro- teins. The function of such molecules is unknown. Nevertheless, it is possible that a Leydig cell-specific POMC-derived peptide can be secreted by testes and that its measurement might constitute a biolog- ic marker for some as yet poorly understood Leydig cell function. SEMINIFEROUS TUBULES The seminiferous tubules in the adult mammalian testis contain germ cells in various developmental phases and nonpro- liferating Sertoli cells. The reader is referred to several comprehensive reviews AL4LE REPRODUCTIVE TOXICOLO(;Y (Roosen-Runge, 1969; Clermont, 1972; Ewing et al., 1980; Griswold, 1988) for detailed descriptions of spermatogenesis and the structure and function of Sertoli cells. Briefly, the primary function of the seminiferous tubules is the produc- tion of spermatozoa. A major difficulty in elucidating the site or mechanism of action of a toxicant on spermatogenesis in mammals is that, as germ cells differentiate, they physi- cally interact with and are affected by each other, somatic cells of the seminifer- ous tubules (e.g., Sertoli cells), and indirectly through chemical signals (e.g., from follicle-stimulating hormone (FSH), LH, testosterone). It is extremely difficult to ascertain whether a toxicant acts directly on a specific cell type in the germinal epithelium (e.g., a spermato- gonium or spermatid) or indirectly via the Sertoli cells, Leydig cells, or (even more indirectly) cells in the hypothalamus or adenohypophysis. Toxicologic elucidation is further complicated by the presence of Sertoli- Sertoli junctional complexes that sub- divide the seminiferous epithelium into a basal compartment and an adluminal com- partment in many species, including hu- mans. It is believed that these special- ized junctional complexes constitute the principal site of the blood- testis barrier that restricts the free movement of specific chemicals between the blood and seminiferous tubular fluid. Apparent- ly, spermatogonia and young spermatocytes are outside the permeability barrier, in the basal compartment next to the base- ment membrane of the seminiferous tubule, and presumably exposed to xenobiotics in blood and lymph. In contrast, mature sper- matocytes and spermatids are sequestered within the permeability barrier, in the adluminal compartment. A practical con- sideration is the possibility of differen- tial drug access to the cells sequestered behind the barrier. However, studies with labeled alkylating agents in mice have indicated that spermatocytes and sper- matids can be exposed and that exposure can result in cell-killing and induced mutations. Despite these complex interac- tions, we have divided the following dis-

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MARKERS OF TESTICULAR FUNCTION cussion of biologic markers of the semi- niferous tubules into a section on Sertoli cells and a section on the germinal epithe- lium to simplify the presentation. Sertoli Cells The structure and function of Sertoli cells have been the subject of several comprehensive reviews (Fawcett, 1975; Dym et al., 1977, Ewing et al., 1980; Gris- wold, 1988~. Briefly, Sertoli cells in the adult mammal are nondividing or slowly dividing cells that rest against the base- ment membrane with projections into the lumen of the seminiferous tubule. Their shape is complex and constantly changing, depending on the stage of the cycle of the seminiferous epithelium. Generally, however, Sertoli cell shape is character- ized by an irregular nucleus, prominent nucleolus, and filamentous cytoplasm. Although the structure of the Sertoli cell has been described for numerous spe- cies, its functions remain enigmatic, because of its intimate association with a population of germ cells that change over time and space (Ewing et al., 1980~. Sertoli cells must be involved at least in germ cell division and differentiation, in view of their direct and specialized membrane contact with germ cells, their formation and presumed control of the mi- lieu of the adluminal compartment of the seminiferous tubule via the tight junc- tions between adjacent Sertoli cells, and their apparent transduction of hormonal signals (e.g., from FSH and testosterone) that are known to regulate spermatogene- sis. Although Sertoli cells can be count- ed, few biologic markers of Sertoli cell function have been developed, and their used is complicated by differential and variable secretion from Sertoli cells into the blood/lymph or seminiferous tu- bule fluid draining into the rete testes and epididymal and seminal fluids. Markers in Use Numerous stereologic procedures have been used to learn the number of Sertoli cells per testis (Wing and Christensen, 1982; Johnson et al., 1984a; Johnson, 1986) 53 and the numbers of several specific germ cell types associated with an average Ser- toli cell (Wing and Christensen, 1982; Johnson et al., 1984a). Knowing the former allows an investigator to test the effect of a toxicant on the viability of Sertoli cells; knowing the latter allows one to test the effect on the functional capacity of Sertoli cells to support each germ cell type. To our knowledge, however, these markers have not been used to test the effect of a xenobiotic chemical on Sertoli cell number or structure, probably because they require biopsy or autopsy specimens, are tedious to use, and are subject to artifacts of tissue prepara- tion. These morphologic markers should be particularly helpful in studies of mech- anism of toxicity in experimental animals, because they can provide information about germ-Sertoli cell interaction. Sertoli cells cultured in vitro secrete at least 60 proteins, as measured by the incorporation of radioactive amino acids into spots on two-dimensional gels (Wright et al., 1981~. Sertoli cells secrete both serum proteins and testis-specific pro- teins. Serum proteins identified include transferrin, ceruloplasmin, somatomedin C, and sulfated glycoproteins 1 and 2; testis-specific proteins include andro- gen-binding protein (ABP), inhibin, Mul- lerian-inhibiting substance (MuIS), Ser- toli-derived growth factors, and cyclic proteins-2 (Griswold, 1988~. With the exception of MuIS, and perhaps transferrin and ABP, these proteins have poorly under- stood functions. However, each is a poten- tially useful marker of Sertoli cell func- tion in vitro. It is important to note that these data are derived largely from the culture of Sertoli cells from immature, rather than mature, rats. Therefore, ex- trapolation of the results from immature rat Sertoli cells in vitro to the human in viva situation must be made cautiously. Markers Requiring Research and Development Transferrin, ABP, MuIS, and inhibin are the most extensively characterized Sertoli cell products and therefore the best candidates for in viva markers of the

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54 pathophysiologic state of Sertoli cells. Transferrints usefulness is limited, because it also is synthesized in the liver (Skinner et al., 1984~. The other three hold considerable promise as biologic markers, because they are specific prod- ucts of the Sertoli cell, because they probably are secreted into the peripheral blood, and because considerable progress has been made in cloning genes for them. The discovery of ABP (Ritzer. et al., 1971; Hansson and Djoseland, 1972) and its identification in peripheral blood of rats (Gunsalus et al., 1980) suggest that peripheral blood concentrations of ABP might serve as a marker of the patho- physiology of Sertoli cells in viva. It was recently shown (Orth and Gunsalus, 1987) that ABP concentration in the peri- pheral blood of rats is highly correlated with Sertoli cell number. That finding was possible because the rat has no tes- tosterone-estradiol binding globulin (TEBG), which is probably identical with ABP in other species. It will require con- siderable research and development, how- ever, to validate a radioimmunoassay that differentiates between ABP and TEBG in species other than the rat, to elucidate the relationship between serum ABP derived directly from Sertoli cells and ABP derived indirectly from the epididymis, and final- ly to describe the correlation between Sertoli cell function and serum or seminal ABP concentrations. A number of studies (McCullagh, 1932; Rich and De Kretser, 1977; de Jong, 1979) have shown selective increases in serum FSH after destruction of the germinal epi- thelium. Sertoli cells secrete inhibin, a factor that diminishes the release of FSH from cultured pituitary cells (Stein- berger and Steinberger, 1976~. Further research and development are required. however, to purify inhibin from testes, to raise a specific antibody against sub- units of inhibin, to validate a radio- immunoassay for inhibin, and finally to elucidate the relationship between Ser- toli cell pathophysiology and serum in- hibin concentration. These studies are under way as this report is written (see, e.g., de Jong,1987~. MuIS is a glycoprotein that causes re- AL4LE REPRODUCTIVE TOXICOLOGY gression of the Mullerian duct (Picard et al., 1986~. The bovine and human genes for MuIS were recently isolated, and the human gene can be expressed in animal cells (Cate et al., 1986~. Its C-terminal domain shows a marked homology with human trans- forming growth factor ~ and the ,8 chain of porcine inhibin (Mason et al., 1985~. Considerable research must be completed to determine whether MuIS is secreted by adult testes, whether it has any function in adults, and whether it can serve as a biologic marker of Sertoli cell function. Germ Cells Spermatogonia are the most undifferen- tiated germ cells in the seminiferous epi- thelium. Spermatogenesis is the process by which undifferentiated spermatogonia divide and differentiate into spermato- zoa. The spermatogonia undergo a number of mitotic divisions, enter the meiotic phase of spermatogenesis as diploid sper- matocytes, and undergo the first and second meiotic divisions to produce haploid sper- matids, which differentiate into sper- matozoa. Spermatogonia are of two major types: one is a stem cell that divides oc- casionally to replenish itself, or two, produces a committed spermatogonium that undergoes several mitotic divisions and then forms spermatocytes, and eventually gives rise to spermatozoa. As spermato- gonia divide and differentiate, they are also replenished by a process termed stem cell renewal. Spermatogenesis consists of a series of events that takes a different amount of time in different species (Clermont, 1972~. Table 4-1 compares several charac- teristics of spermatogenesis and sperm production in mice, hamsters, rats, rab- bits, beagles, rhesus monkeys, and humans. Species differ substantially in charac- teristics of spermatogenesis and sperm production; they differ less in epididymal transit time. Amann (1986) concluded that no species is identical with the human in these respects, so care must be taken when extrapolating from animals to humans. The potential for a toxic effect on sper- matogenesis might be greater in humans because sperm production per gram of testis

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?~9RKERS OF TESTICULAR FUNCTION 55 TABLE ~1 Species Differences In Spermatogenes~s, Daily Spenn Production, and Epididymal Transit Thea Dog Monkey Mouse Hamster Rat Rabbit ~eagle) Jesus! Man Duration of spermatm genesis, days Duration of cycle of seminiferous epithelium, days Life span, days B ppermatogonia IA~ptotene Pachytene spermatopytes Golgi spermatids Cap spermatids Testicular weight (total) Daily sperm production, millions Per gram of testis Per male Sperm reserves In caudae epidi~mes (at sexual rest), millions Epididymal transit tune (at sexual rest), days 3~35 35-36 8.9 8.7 1.5 1.6 2.0 0.8 8.0 8.1 1.7 23 3.6 3.5 0.2 3.0 28 24 5.6 72 49 1,020 14.8 48 4~51 12.9 10.7 2.0 13 1.7 2.2 11.9 10.7 2.9 2.1 5.0 5.2 3.7 6.4 24 25 89 160 440 1,600 8.1 12.7 62 70 13.6 9.5 4.0 2.9 3.8 2.1 12.4 9.5 6.9 1.8 3.0 3.7 12.0 49.0 20 23 240 1,127 2,100 5,700 113 10.5 7~74 16.0 63 3.8 12.6 7.9 1.6 34.0 4.4 150 420 5.5-12 aData derived largely from a table constructed by Amann (1986~. in humans is approximately one-fourth to one-sixth that in the other species. Daily sperm production in all species probably is in excess of that required for fertili- ty. For example, a 90% reduction in fertile sperm available for ejaculation did not suppress fertility in rats (Amann, 1986~. Thus, especially in animal species, but even in humans, fertility is unlikely to be a sensitive indicator of toxic insult of spermatogenesis. Therefore, our task is to elucidate bio- logic markers that reflect the exposure to or effect of toxicants on the number and function of germ cells, from primitive spermatogonia to fully formed spermato- zoa. The complexity and species variation of spermatogenesis suggest that a battery of markers will be required to detect toxic effects on spermatogenesis. For example, one toxic chemical might act on cells un- dergoing rapid mitosis, and another pri- marily on meiotic cells in which complex genetic rearrangements occur. Slowly dividing stem spermatogonia might be re- sistant to cytotoxic chemicals, but vul- nerable to DNA alterations. The nondivid- ing condensed spermatids are resistant to direct effects of toxic chemicals on development and function, but sensitive to point mutations and chromosomal break- age. Detection of each alteration might require a different biologic marker. Markers in Use In any given region of a seminiferous tubule, germ cells are differentiating. That combination of phenomena creates a complex histologic appearance at the light microscopic level, where adjacent cross sections through seminiferous tubules generally appear quite different. Cler- mont and coworkers (Leblond and Clermont, 1952; Helter and Clermont, 1964) showed that distinct cellular associations or stages exist and that their number depends on the species-e."., 6 in humans and 14 in rats. The complex histologic cell as- sociation pattern allows trained obser- vers to evaluate subjectively whether external factors have specific effects

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56 on germ cells in different steps of dif- ferentiation. This constitutes a rela- tively simple marker, which is particular- ly applicable to studies with experimental animals, whose testicular cytoarchitec- ture can be well preserved with large tis- sue samples and in which enough spermato- genesis remains to allow staging of spe- cies-specific cellular associations. Studies with human biopsy specimens are complicated by the small amount of tissue available, by artifacts induced by the biopsy procedure, and by the fact that a cross section of a human seminiferous tu- bule usually contains more than one stage of the cycle of the germinal epithelium (Heller and Clermont, 1964~. A simple quantitative approach is to determine the percentage of seminiferous tubules with mature spermatids lining the lumen versus the percentage of tubules without spermatids lining the lumen. That approach assumes that a chemical simply does or does not have a toxic effect on a tubule and, therefore, a clear judgment can be made as to which category a particu- lar tubule belongs to. Alternatively, it is possible to measure the minor diame- ter of 15 seminiferous tubules, which di- minish with the loss of germ cells (Courot, 1964~; the advantage of this quantifica- tion is that it is sensitive, easy to meas- ure, and provides a spectrum of values from a maximal to a minimal diameter of semi- niferous tubules. The disadvantages of both approaches are that they are nonspe- cific and relatively insensitive and re- quire autopsy or biopsy specimens. Again, great care must be taken to prevent arti- facts associated with fixing, embedding, and sectioning the testicular tissue (Amann, 1981). It is possible to count stem cells in histologic sections of rodent testes (Oakberg, 1978~. But the technique is subjective and laborious, because stem cells are difficult to identify and are few. Alternatively, a functional test for stem cell renewal can be used. Methods to measure the effect of toxi- cants on germ cells in tine testis have been described and reviewed (Amann, 1970a; Berndtson,1977,Amann,1981~. Thesemeth- ~ . OdS are applicable to humans and experlmen- Af 4LE REPRODUCTIVE TOXICOLOGY tat animals, provided that the testicular ~n~nim~n~ non be fixed appropriately and biopsy artifacts can be prevented. One widely used method involves counting germ cells in a fixed number of tubule cross sections of one cellular association (Amann,1970b; Berndtson,1977~. The germ cell numbers are generally but not always expressed per Sertoli cell nucleolus, to correct for seminiferous tubule shrink- age caused by histologic processing and experimental treatment. The method has proved sensitive in assessing the effect of antimitotic agents on spermatogenesis (Amann,1981). However, the numbers gener- ated are relative, rather than absolute; and the results might be nonspecific, be- cause numerous agents can cause a morpho- logically identical pattern of response (Russell et al., 1981~. In a simplified version of the procedure, the mean number of spermatids per tubule from a testicular biopsy has been used to predict sperm count in humans (Silber and Rodriguez-Rigau, 1981~. Another method applies stereologic principles (Van Dop et al., 1980a,b; Wing and Christensen, 1982; Jones and Berndt- son, 1986) to obtain quantitative informa- tion on the diameter and volume of the semi- niferous epithelium and lumen and to learn the absolute, rather than relative, num- bers of Sertoli and germ cells (from pre- leptotene primary spermatocytes to step 10 spermatids). The method allows an ex- perimenter to determine precisely the effect of a treatment on the number of cell types in the germinal epithelium. It has disadvantages: it is invasive, labor- intensive, and subject to numerous arti- facts of tissue preparation. To our knowl- edge, the latter method has not been used to test the effect of xenobiotic chemicals on the seminiferous tubular epithelium in animals or humans. An alternative to direct counting of stem cells is to count the stem-dependent cells after a toxic insult. One way to do that is to count the number of repopulating and nonrepopulating seminiferous tubular cross sections in a series of experimental animals killed at different times after treatment with a toxic chemical (Meis- trich, 1986~. It is assumed that each re-

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MARKERS OF TESTICULAR FUNCTION populating cross section of seminiferous tubules results from the presence of at least one surviving stem cell and that nonrepopulating cross sections result from the absence of stem cells. In a second assay used in mice, sperm heads in the testis 50 days after exposure to a toxic insult are counted (Meistrich, 1986~. Both methods are simple and rapid. But both are invasive and therefore not readily applicable to humans; and repopu- lating tubules cannot be counted in low- dose situations, because extensive kill- ing of stem cells is required for empty tubular cross sections to be produced. One method of counting spermatid nuclei in homogenates of testicular parenchyma is based on the fact that late spermatid nucleoprotein becomes highly condensed and therefore resistant to homogenization (Amann and Lambiase, 1969~. It is simple to use and its results are highly corre- lated with daily sperm output in rabbits (Amann, 1970b; Amann. 1981: Berndtson. 1977~. It is also applicable to humans (Amann and Howards, 1980; Johnson et al., 1980a, 1984b). The disadvantage of the technique is that it is invasive (or re- quires autopsy specimens) and therefore has only limited human application as a biologic marker. The amount of testicular LDHC4, a germ cell-specific isozyme of lactate dehydro- genase, is proportional to the numbers of meiotic and postmeiotic germ cells in testes of mice and can be used to estimate the survival of spermatogonial stem cells after treatment with toxicants (Meis- trich, 1982~. The method is indirect, more difficult than measuring spermatid num- bers in testicular homogenates, and in- vasive. But, it holds promise, because the LDHC4:sperm ratio in seminal plasma of human males might serve as an indicator of the function of the seminiferous epithe- lium (Eliasson and Virji, 1985; Virji, 1985. The approach should be investigated thoroughly, not only with LDHC4, but with other proteins specific for meiotic and postmeiotic germ cells. Severe damage to the germinal epithelium in the testes of many species results in increased serum FSH concentrations (Mc- Cullagh, 1932; Rich and De Kretser, 1977; 57 de Jong, 1979), in part because a Sertoli cell product (inhibin), which regulates FSH secretion, is produced in low amounts in azoospermic animals and in high amounts in normospermic animals. Major insults to spermatogenesis can be monitored in- directly by measuring FSH concentration in peripheral blood. The principal advan- tage of this biologic marker is that it can be measured in peripheral blood samples, which are easy to collect repetitively. The disadvantages are that the inverse relationship between FSH concentration in serum and germinal epithelium damage are not tightly coupled; FSH production is episodic, so the results are variable. That renders the marker relatively insen- sitive to changes in spermatogenesis, despite the sensitivity of the radio- immunoassay. In addition, very low sperm counts are required for FSH increase to become evident. Finally, gonadal steroids (testosterone and estradiol) can account for selective FSH increase when testoster- one production is low (Sherins et al., 1982~. Consequently, FSH concentration might not always reflect only inhibin pro- duction. The marker is nonspecific, in that any toxic insult that depletes germ cells causes an increase in FSH production and testosterone and estradiol also par- tially control FSH production. Markers Requiring Research and Development The application of molecular biology to the study of mammalian testicular dif- ferentiation is providing investigators with insights into many of the molecular mechanisms that regulate male germ cell formation. Gene expression during sper- matogenesis is temporally and spatially regulated with precision; many macromole- cules and organelles are synthesized in specific cell types during the continu- um of testicular cell differentiation (Hecht, 1987~. Although variants of ubi- quitous enzymes and structural proteins are expressed in many organs, the testis appears to be an especially rich source of isozymes (Goldberg, 1977~. Presumably because of the specialized requirements for producing a spermatozoon, unique tes-

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58 ticular isozymes that code for proteins such as lactate dehydrogenase, phospho- glycerate kinase, and cytochrome c-have evolved (Fig. 4-3~. In addition to the testicular isozymes, many structural proteins of the maturing spermatid and the spermatozoon have been identified. Results of biochemical analyses of the differentiating haploid germ cells that transform into the highly polarized spermatozoon suggest that the definition of all the possible sperm-specific molecu- A~E REPRODUCTIVE TOXICOLOGY tar markers has only begun. As a result of such studies and the well-characterized sequence of events leading to the formation of spermatozoa, efforts to monitor the effects of toxicants on male germ cells can be based on substantial knowledge and use the numerous DNA probes already avail- able for the mammalian testis to study the mechanism of toxic action of individual chemicals on spermatogenesis. Most DNA cloning efforts during sper- matogenesis have been directed toward two MOUSE SPERMATOGENESIS CELL TYPE ~ SPERMATOGOb IA | SPERMATOCyTES I | SPE STATICS SPERM (Pachytene) 11 (Rounds Elongating) Ploidy 2N | 4N | 2N N | N Rate of Differen- tiation 6 11 1 9 20 34 | DNA Synthesis I ~ | _ l l mRNA Synthesis Cyto. RNA (pg/cell) 9.1 2.2 0.9 Poly (A) RNA (ng/~1 9) 6 1 1 3 Protein Synthesis Histones _ _ PGK-2 _ _ _ _ _ _ __ Protamines Acrosin pActin __ (2~1 kB) Y Actin (2~1 kB) Actin (?) (1 5 kB) (6.2 and 8.0 kB) c-ab L (4.7 kB) l l I I ~ FIGURE ~3 Periods of active synthesis of DNA, RNA, and proteins diagrammed for various cell types. Source: Hecht, 1987b.

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MARKERS OF TESTICULAR FUNCTION intervals of spermatogenesis: meiosis and spermiogenesis (Kleene et al., 1983; Dudley et al., 1984; Fujimoto et al., 1984~. That is because the meiotic pachy- tene spermatocytes and the haploid round spermatids represent two critical periods for gene expression during male germ cell development. Moreover, highly enriched populations of pachytene spermatocytes and round spermatids can be obtained readi- ly with cell separation techniques, be- cause of their marked size differences and abundance in the sexually mature testis (Romrell et al., 1976; Meistrich, 1977; Hecht, 1987b). Almost all DNA probes for genes ex pressed in testis cells have been isolated from animals, such as mice, rats, and bulls. The origin of these probes will not pose a problem for application to human DNA. Because DNA probes for homologous genes in humans and other vertebrates share substantial sequence homology, the animal probes can be used to isolate equivalent human sequences from available human cDNA or genomic DNA libraries, and investiga- tors committed to human toxicology studies should seriously consider the direct iso- lation and characterization of human DNA probes. For cases in which both rodent and human probes are available, the animal studies can be used to validate the DNA probe marker for human use and, more impor- tant, to reveal the mechanisms by which specific chemicals interact with the ge- nome. MeioticDNAProbes. Lactatedehydrogen- ase C4 (LDHC4) is one of the best-charac- terized proteins in the testis (Goldberg, 1977~. It appears to be testis-specific and is synthesized initially during meio- sis and in decreasing amounts during early spermiogenesis (Meistrich, 1977~. LDHC4 mRNA makes up as much as 0.18% of total func- tional mRNA in mouse pachytene spermato- cytes (Wieben, 1981~. LDHC4 is also present on the surface of mature spermatozoa, so it has been used extensively in immunocon- traceptive studies (Wheat et al., 1985~. The protein sequence of rodent LDHC4 has been known for some time (Pan et al., 1983), but only recently has a cDNA probe for human LDHC4 been isolated (Millan et al., 1987~. Two distinct forms of phosphoglycerate 59 kinase (PGK) have been characterized in mammals. PGK-1 is an X-linked gene that is expressed in somatic cells, whereas the autosomally derived isozyme, PGK- 2, is specific to the testis (Kramer and Erickson, 1981~. Although PGK-2 is syn- thesized during spermiogenesis, the gene appears to be initially transcribed during meiosis, with increased synthesis of PGK- 2 mRNA in spermatids (Gold et al., 1983~. Recent elegant studies of the human PGK multigene family have produced detailed sequence knowledge and DNA probes for these important enzymes (Michelson et al., 1985). Cytochrome c, the electron-transport protein from the mitochondrial respira- tory chain, exists in two forms in the tes- tis (Goldberg et al., 1977~. One variant, cytochrome c~, is restricted to the testis; the other variant, cytochrome c~, is presumably found in all tissues. Indirectimmunofluorescence with monospe- cific antibodies first detects cytochrome c~ in the mitochondria of pachytene sper- matocytes and in later stages of spermato- genesis, whereas cytochrome c,, is found in the mitochondria of interstitial cells, Leydig cells, and spermatogonia. Sequence analysis of the two mouse cytochrome c molecules has revealed that cytochrome cl; differs from cytochrome c~ in 13 amino acid residues (Hennig, 1975~. Although DNA probes exist only for the c~ variant of cytochrome c, knowledge of the sequence of the testicular form of cytochrome c would allow appropriate oligonucleotide DNA probes to be prepared. Such oligonu- cleotides would facilitate the isolation of cDNA or genomic DNA probes for human cytochrome cl;. During spermatogenesis, a dramatic reorganization of the germ cell nucleus occurs. The transformation ultimately produces a sperm nucleus with highly com- pacted DNA and accompanied by the replace - ment of histones with a group of transient- ly associated nuclear proteins and finally with protamines (Hecht, in press). In addition to the standard com- plement of histone molecules found in mam- malian cells, the meiotic pachytene sper- matocyte contains several additional histone variants believed to be peculiar .

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60 to the testis. DNA sequence analysis of one testis-specific histone variant, Hit, has revealed it to be a unique gene product, and not a posttranslational modification of an existing histone (Cole et al., 1986~. The availability of a specific DNA probe for one of these meiotic histones of the rat provides a means to obtain the equiva- lent probe for the human male meiotic his- tone gene. Probes for other testis-specif- ic histones can also be isolated. Postmelotic DNA Probes. The predominant proteins in mammalian spermatozoa are the protamines, a group of small arginine- rich DNA-binding proteins that aid in nuclear DNA compaction during spermio- genesis (Hecht, 1987c). With the exception of a few species, most mammalian spermato- zoa have been reported to contain one type of protamine. In the mouse, two protamine variants, MP1 and MP2, have been identified by DNA sequence analysis of isolated cDNA clones (Kleene et al., 1985; Yelick et al., 1987~. Protein sequence studies have iden- tified similar P1 and P2 human protamine variants (McKay et al., 1985; Ammer et al., 1986; McKay et al., 1986~. Although the P1 and P2 protamines differ substantially in size and sequence, in the mouse they are closely linked on chromosome 16 and are temporally and translationally regu- lated during spermiogenesis (Hecht et al., 1986a). The human protamine genes probably are also chromosomally linked. The P1 human protamine was recently shown to be on human chromosome 16 (R.H. Reeves, Johns Hopkins University, and N.B. Hecht, Tufts University, unpublished observa- tion, 1987~. Northern blots of RNA from prepubertal testes and from isolated meiotic and post- meiotic testicular cell types have re- vealed that the protamines are expressed solely during the haploid interval of sper- matogenesis (Kleene et al., 1983. 1984: Hecht et al., 1986a,b). Moreover, changes in length of protamine mRNAs during sper- matogenesis allow the protamine probes to be used as molecular markers to evaluate the extent of spermiogenesis in wild-type, mutant, or chemically induced sterile animals (Kleene et al., 1984~. For in- stance, if the MPl-cDNA probes are used, no MPl-mRNA is detected in testicular ex- M>llLE REPRODUCTIVE TOXICOLOGY tracts of prepubertal mice up to 20 days old (a time when spermatogenesis has ad- vanced to meiosis), whereas a 580-nucleo- tide form of MPl-mRNA is present in the testes of 22-day-old mice (early sperma- tids are present by day 22) and a heterogen- eous population of 580- and 450-nucleotide MP1 -mRNAs is present in the testes of sexu- ally mature animals (Hecht et al., 1986b). Results of cell-separation studies con- firm that no MP1 -mRNA is presentin pachy- tene spermatocytes, a 580-nucleotide MP1- mRNA is found in round spermatids, and elongating spermatids contain an addi- tional 450-nucleotide mRNA (Kleene et al., 1984~. These mRNA length changes result from a partial deadenylation of the prota- mine mRNAs that takes place when they move from the ribonucleoprotein particle frac- tion of the cytoplasm (in round spermatids) to polysomes (in elongating spermatids). Similar size changes occur in the MP2 mouse protamine and in P1 and P2 rat and hamster protamine mRNAs (Bower et al., in press). The protamine genes appear to be excel- lent candidates to serve as probes to moni- tor genomic defects induced during sper- miogenesis. They are two single-copy genes that express abundant postmeiotic tes- ticular mRNAs essential for sperm func- tion. Moreover, the cDNA probes show much homology to the DNA and RNA of many other vertebrates, including humans. Several laboratories are seeking to isolate human probes for these male-specific DNA-bind- ing proteins. In mammals, histones are not directly replaced by protamines, but by a presumably heterogeneous group of basic proteins called testis-specific proteins (lP) (Hecht, 1987c). TPs are associated with the spermatid nucleus during its transition from its nucleosome-like structure to the smooth branching fibril _7 ~ _ ~ of the spermatozoan nucleus. TPs are re- placed by protamine during spermiogene- sis. Recently, cDNA probes for the mouse and rat TPs have been identified (Hecht et al., 1986b; M.A. Heidaran and W.S. Kist- ler, University of South Carolina, per- sonal communication). Phylogenetic stud- ies have indicated a strong sequence con- servation of TPs in rodents and humans. The identical pattern of expression of

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MARKERS OF TESTICuL~4R FUNCTION mouse TP and MPl and MP2 suggests that these three genes are coordinately and temporal- ly regulated in the postmeiotic testicular cells and could be used together to monitor postmeiotic gene expression. In mammals, actins are encoded by a mul- tigene family that expresses at least six distinct but closely related forms of ac- tin. In addition to the general role that actin plays in cell motility and division, secretion, organelle movement, and main- tenance of cellular cytoarchitecture, testicular actins are likely to be involved in chromosomal movement during meiosis, in shaping specific nuclear structures during spermiogenesis, and in spermatozoa! function (Hecht et al., 1984~. Although the mRNAs coding for the cytoplasmic ,6 and ~ actin isotypes have been detected in all testicular cell types throughout spermatogenesis, mRNA that encodes an additional actin variant is first detected during spermiogenesis (Waters et al., 1985~. It should be pos- sible to obtain several distinct DNA probes for actin isotypes that are expressed con- stitutively during spermatogenesis and of other actin isotypes that are expressed temporally in specific stages or cell types. Microtubules consist of heterodimers of a and ~ tubulin. The ~ and ,6 tubulin sub- units are distinct sequences, each encoded by multigene families. In the testis, the tubulins are involved in mitotic and mei- otic divisions, in changes in cell shape and structure, in the species-specific shaping of the sperm nucleus, and in the synthesis of the axoneme of sperm tails. Results of protein gel electrophoresis and DNA cloning studies have suggested that multiple isoforms of c' and,B tubulin are expressed during spermatogenesis (Hecht et al., 1984~. The availability of cDNAs for a number of mouse testicular c' tubulins and a detailed study of the ex- pression of the ~ tubulin multigene family will provide a set of useful probes to moni- tor the differential expression of several cytoplasmic structural genes during tes- ticular germ cell development. Results of in vivo and in vitro postmei- otic protein synthesis studies and the many morphologic changes in cell shape . . . 61 and structure that occur during spermio- genesis have indicated that many addition- al unique macromolecules are synthesized during spermiogenesis. Continuing stud- ies in many laboratories suggest that such proteins as acrosin, hyaluronidase, a sperm-specific enolase, and sperm tail proteins (e.g., the dyneins and outer dense-fiber proteins) will provide addi- tional sources of stage-specific DNA probes for this critical interval of sper- matogenesis. DNA probes are also available for some proto-oncogenes, such as c-abl and c-myc, that are differentially ex- pressed during spermatogenesis. Although c-abl mRNA is present in premeiotic, mei- otic, and postmeiotic cell types, a novel c-abl mRNA of distinct size is first de- tected in postmeiotic cells (Ponzetto and Wolgemuth, 1985~. Because of its unique size, a probe specific to this shortened c-abl transcript could be prepared. In contrast, the proto-oncogene c-myc ap- pears not to be expressed in testicular germ cells (Stewart et al., 1984~. Stem Cell DNA Probes. Spermatogonia make up only a few percent of the cells found in the sexually mature mammalian testis. It has therefore been difficult to work biochemically with this cell type, and no DNA probes peculiar to spermatogonia have yet been isolated for this critical stage of spermatogenesis. Because genetic alterations in stem cell DNA will produce persistent heritable defects, a major effort needs to be commenced to obtain an armamentarium of DNA probes specific to animal and human testicular stem cells. Recent improvements in testicular cell separation methods make this possible, in that highly enriched populations of several types of spermatogonia can be ob- tained from the prepubertal testis. With poly(A)+ RNA isolated from enriched popu- lations of spermatogonia, radiolabeled cDNAs can be prepared and a differential- hybridization approach similar to that used previously to obtain postmeiotic cDNAs can be conducted to isolate stem cell-specific cDNAs (Kleene et al.. 1983). In brief, a total testicular cDNA library or a cDNA library enriched with spermato- gonial cDNAs would be differentially hy- bridized with radiolabeled cDNAs prepared

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62 from spermatogonial, meiotic, or postmei- otic cell types. The cDNAs that appear to be preferentially expressed in spermato- gonia would be isolated and their temporal appearance confirmed by the early appear- ance of RNA in the testes of prepubertal- staged mice (Kleene et al., 1983~. DNA sequence analysis could be used to help to identify the proteins coded for by the stem cell cDNAs. One possible candidate DNA probe for a protein expressed in sper- matogonia would be the DNA that codes for the H2A histone stem cell variant found in mouse embryonic spermatogenic cells (Gizang-Ginsberg and Wolgemuth, 1985~. Clearly, these macromolecular probes at several cell stages represent an excel- lent collection of biologic markers for the dynamic process of spermatogenesis. They can be used to assess the effect of a toxicant on the genome that directs spe- cific biochemical events in spermatogene- sis. The approach would be particularly useful in studies of the mechanism of ac- tion of toxicants in spermatogenesis. Disadvantages include the invasiveness AL4LE REPRODUCTIVE TOXICOLOaY of the present techniques and their re- quirement for autopsy or biopsy specimens. Development of these probes holds out the possibility of analyzing potential toxic effects at the DNA level in ejacu- lated spermatozoa. However, that is un- likely to occur soon. The primary limita- tions on the application of DNA probe tech- nology to evaluate genomic DNA alterations in spermatozoa are three: DNA probes are available for only a very small portion of the genome; current procedures severely limit the number of probes that can be as- sayed at one time, thereby restricting the percentage of the genome examined in each analysis; and the DNA from single cells, such as spermatozoa, cannot be ana- lyzed. Theoretically, the limitations can be overcome. Procedures can be devel- oped to monitor large fragments of genomic DNA with a battery of DNA probes that cover vast regions of the human genome. Advances in fluorescence detection of DNA combined with computer imaging of samples will aid in the analysis of DNA from single cells.