Histopathology of chemically induced testicular atrophy in rats

Review

Exp Toxic Pathol 1995; 47: 267-286 Gustav Fischer Verlag Jena

Institute of Toxicology, ASTA Medica AG, HallelWestf., Germany

Histopathology of chemically induced testicular atrophy in rats T. NOLTE, J. H. HARLEMAN and W. JAHN With 12 figures and 3 tables Received: April 24. 1994, Revised: October 4. 1994, Accepted: October 12. 1994 Address for correspondence: T. NOLTE, Institute of Toxicology, ASTA Medica AG, KantstraBe 2, D-33790 HallelWestf., Germany. Key words: Testis, atrophy; Atrophy, testis; Chemically induced testicular atrophy; Male genital tract, rat; Spermatogonial toxicants.

Introduction The male genital tract is one of the main organ systems examined in the safety evaluation of chemical and pharmaceutical compounds. Increasingly questions are being asked on the possible effect of those compounds on the male reproductive system. One of the reasons for the interest in the effects of chemical and pharmaceutical materials on the male genital tract is the observation of a decrease in mean sperm counts in man over the past 50 years (26). Until now no clear cause for this phenomenon has been determined. However, examples have also been published of induced infertility and testicular abnormalities in workers after exposure to industrial chemicals. In the United States, male factory workers became sterile after exposure to 1,2-dibromo-3-chloropropane. Other instances include: factory workers in battery plants in Bulgaria, lead mine workers in the State of Missouri and workers in Sweden who handled organic solvents. All of them suffer from low sperm counts, abnormal sperm and varying degrees of infertility. It has also been reported that diethylstilbestrol, lead, kepone, methylmercury and many cancer chemotherapeutis are toxic both to the male and female genital system (24). Many of the above mentioned effects are reproducible in animal experiments. In toxicity studies a wide variety of industrial chemicals and drugs have been found to cause testicular damage and to inhibit spermatogenesis. Examples of such compounds include phenacetin and paracetamol (7), a number of esters of o-phtalic acid (34) or the metal ion cadmium (79). Although there has been extensive literature published on testicular atrophy there is still a lack of detailed morphologic and photographic evidence. The aim of this article is to present various histological observations from standard toxicity studies on chemical and pharmaceutical

compounds. This morphological description of H & E or PAS stained paraffin sections may be of more practical use in the day to day setting than the excellent morphology that can be obtained after application of special methods, i. e. elaborative and cost extensive plastic embedding and sectioning or electron microscopy. Such extended methods should be used in studies with substances which in routine studies have been shown to damage the male genital system.

Tissue preparation and histological examination The testis is extremely sensitive to fixation, and for any critical evaluation of morphology additional specialised fixation methods may be required. An overview on the different effects of various fixatives on the histology of the testis has been presented by LILLE and FULLENER (77). The testis should be fixed as the whole organ, due to the loss of tubular or interstitial tissue organisation when ist is trimmed prior to fixation. All of the immersion fixation procedures can be improved by pricking the capsule of the testis 15-20 times to increase the penetration of the fixative (13). Conventional immersion fixation in buffered formalin gives relatively poor penetration of fixative, resulting in not optimal cellular definition. Immersion fixation in Bouin's or Zenker's fluid gives more rapid penetration and improves cellular contrasts, but gives rise to severe shrinkage artefacts. If Helly's fixative is used, these artefacts are less pronounced, however, the fixative has to be prepared freshly each day. The most successful but time consuming fixation technique, is perfusion with buffered formalin or glutaraldehyde via the heart or locally via the testicular or iliac artery. Optimal histological examination is possible if perfusion fixed tissue samples are embedded in either methacrylate or epon resin. Exp Toxic Patho147 (1995) 4

267

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Fig.t. Series of drawing illustrating the 14 stages (roman numerals I-XIV) ofthe cycle of the rat seminiferous epithelium according to the classification of Leblond and Clermont (56) as seen in PAS-hematoxylin stained sections. Each drawing represents a portion of seminiferous epithelium and gives cellular composition of the stages as well as arrangement of various generations of germ cells within the epithelium; drawings represent complete series of successive cellular associations appearing in anyone area of a seminiferous tubule (after stage XIV, stage I reappears, and so on). A, In, and B: type A, intermediate-type and type B spermatogonia; InM, BM: intermediate and type B in mitosis; PI: preleptotene primary spermatocytes; L: leptotene spermatocytes; Z: zygotene spermatocytes; P: pachytene spermatocytes; Di: diplotene spermatocytes; II secondary spermatocytes; 1M, lIM: primary and secondary meiotic divisions; 1-19: spermatids at various steps of spermatogenesis; RB: residual bodies. PAS-positive acrosomic system of spermatids closely associated to nucleus is shown in black or deep grey. First 14 steps of spermiogenesis were utilized to identify the 14 stages of cycle. Reprint with permission from Clermont Y (1972). The histological examination of the testis must include evaluation of seminiferous tubules, interstitial cells and stromal vasculature to establish normal morphology. The seminiferous tubules should show normal spermatogenesis and spermiation. The composition of all cell types should be normal. For the exact identification of the target cell population knowledge of the various stages of the spermatogenic cycle of the species under study is required. In a routine section through the rodent testis it is obvious that not all seminiferous tubules are in the same stage of development. An excellent description of the ge268

Exp Toxic Pathol47 (1995) 4

nerally used classification of the stages of the seminiferous epithelium of the rat has been published by LEBLOND and CLERMONT (56) and by CLERMONT (15) (fig. 1 giving a detailed scheme of the staging). This scheme is based on PAS-staining, however, some but not all stages can be identified using routine H & E stained sections. In H & E stained sections it can be difficult to identify stages I-VII. These stages can be roughly assessed by the migration position of elongate maturing spermatids and the estimation of the number of round spermatids containing a Golgi zone. A more precise differentiation is

Fig. 2. The five main stages of rat seminiferous epithelium as seen in H & E stained sections. Top left: stage I-VII tubule; round spermatids in golgi phase. Middle left: stage VIII tubule; elongate spermatids exclusively in luminal position, residual bodies (arrows). Bottom left: stage IX tubule; mature spermatids are released, numerous residual bodies. Top right: stage X-XIII tubule; only one generation of spermatids present. Middle right: stage XIV tubule; spermatocytes in metaphase of meiosis. bar = 20 11m. possible with PAS stained sections. The PAS methods stains the developing acrosome and thus facilitates differentiation of stages I-VII more precisely, although the demarcation between the end of one stage and the beginning of the next is often imprecise (15). The differentiation of stages VIII-XIV is possible on both H & E and

PAS-stained sections. Stage VIII is characterized by mature spermatids which are exclusively in luminal position and residual bodies. Stage IX can be identified by residual bodies, absence of round spermatids and the presence of elongate spermatids in the acrosomic phase. The differentiation between stages X-XIII is only possible Exp Toxic Pathol47 (1995) 4

269

according to the position of elongate spermatids. Stage XIV tubules can readily be identified by the presence of numerous spermatocytes in the metaphase of meiosis (61). Figure 2 gives the morphology of these five main stages (I-VII, VIII, IX, X-XIII, XIV) of the spermatogenic cycle in routine H & E stained sections. The information obtained from the above described investigation procedures is in most cases sufficient for the purposes of a routine toxicity study. If mechanistic explanations are sought then the complex submicroscopic structure of the testis must be examined. This requires a lengthly sampling procedure, especially if stage specific lesions are the subject of investigation.

Target sites and mechanisms of injury Testicular atrophy caused by a wide variety of compounds results in histologically comparable end points, however, the primary target sites and mechanisms of injury may be entirely different. Primary target sites may be intratesticular or extratesticular. Intratesticular target sites are the vasculature, Sertoli cells, Leydig cells and germ cells. The complex interactions between the various cells ofthe testis make it sometimes extremely difficult to determine cell-specific toxicity. This can only be done on the basis of a thorough examination and understanding of the histological pattern of damage. Dose-response and time-course studies are needed to identify the lowest dose and pathomechanism of a lesion. Ultrastructural examination may provide additional information on the pathogenesis of the lesion. The clear identification of the primary target site as precise as possible is of great importance when appreciating the end point and reversibility of a given lesion and thereby assisting in the evaluation of the safety assessment and extrapolation to man. Table I shows examples of xenobiotics and their respective testicular target cells. The vast majority of compounds which primarily act on an extratesticular target site affect the hormonal axis of hypothalamus-pituitary-gonads. Primary toxic effects to a number of other organs, e.g. adrenals and thymus, are associated with secondary testicular atrophy. Tab. 1. Testicular target sites of xenobiotics. 6-Chlolodesoxy-glucose Nitroimidazole

I

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Exp Toxic Pathol47 (1995) 4

Only examples of the known or suspected testicular toxicants (Tab. 2) will be described in this article. The agents discussed will illustrate the potential sites and mechanisms of injury.

1. Intratesticular target sites 1.1. Vasculature: The seminiferous tubules are devoid of blood vessels. The germ cells are supplied with oxygen by diffusion from the interstitium to the lumen of the tubules. The low oxygen tension of the seminiferous tubules makes the germinal epithelium highly susceptible for a reduced blood supply. Any agent damaging the vascular structures is liable to rapidly produce anoxic conditions in the organ leading to widespread ischemic cell death. The most cited example ofaxenobiotic acting on the testicular vasculature is the heavy metal cadmium. The ischemic necrosis of seminiferous tubules caused by this agent is usually associated with vascular thrombosis, capillary stasis, edema and hemorrhage (25, 29, 52, 55, 79, 80, 81, 97). Necrotic tubules tend to calcify within 2-3 months after administration. The dose levels producing testicular damage are below those causing extratesticular effects in most species. This high susceptibility of the testes is caused by testicular accumulation of cadmium. Accumulation is due to stimulation of metallothionein synthesis and binding of cadmium to metallothionein (74). A second testicular target site of cadmium ist the Leydig cell which may undergo neoplastic transformation, although this effect may be secondary to the atrophy of the seminiferous epithelium. Other substances suspected of causing testicular atrophy by primarily disturbing the vascular supply are carbon disulfide (2) and trivalent chromiun (3). Ischemic changes after reduction of the testicular blood flow by physical means reflect the existence of two testicular compartments with a different oxygen tension. After temporary or permanent occlusion of the testicular artery hypoxic damage occurs first at the germinal epithelium and eventually affects the Sertoli cells. Ischemia lasting 90-105 min selctively affects type A spermatogonia in stage XII and XIV tubules (15), intermediate spermatogonia in stages I-IV and spermatocytes in stage VII and VIII tubules. The loss of precursor cells in affected tubules leads to a maturation depletion of subsequent stages (11). The Leydig cells in the interstitial compartment are more resistant but are also destroyed by occlusion of the artery for a period of 6 hr or more (76). In contrast to the high susceptibility of spermatogonia to ischemic changes we have observed selective damage on elongate spermatids after treatment of dogs with a vasoactive compound which resulted in a marked transitient lowering of blood pressure. Tubules with round spermatids in Golgi phase and cap phase lacked elongate spermatids (fig. 3). It is not clear whether this change resulted from a toxic effect on elongate spermatids in late stage tubules or from a Sertoli cell damage which affected the

a

b

Fig. 3. Testis of a Beagle dog treated intravenously with a vasoactive compound over a period of 13 weeks. a) Absence of elongate spermatids in early stage tubules (stages I-VI); H & E, bar = 50 11m. b) Longitudinal section of a seminiferous

tubule with round spermatids in golgi-phase; note prominent perinuclear golgi-zone (arrows); only few elongate spermatids present; H & E, bar = 10 11m.

supportive function of these cells, possibly by poor perfusion. The effect was not clearly dose related but occurred most pronounced in those animals which showed a marked decrease in blood pressure. 1.2. Sertoli cells: Sertoli cell morphology and function are pivotal to the integrity of the seminiferous epithelium. The Sertoli cells rest with their basal aspect on the basement membrane and extend with their apical parts to the tubular lumen, giving out sheet-like cell processes that separate and surround the germ cells. These processes with their specialized junctions are named apical or lateral processes. They supply structural support for the elongate spermatids and mediate spermiation (30,87,89, 90, 92, 94, 95). Basal occluding junctions between adjacent Sertoli cells form the main part of the blood-tubule barrier (19, 73, 86). Sertoli cells support metabolically the germ cells by the FSH-stimulated secretion of pyruvate and lactate, which are the exclusive energy source for spermatocytes and spermatids (45,47). Sertoli cells secrete tubular fluids (99) which are essential for sperm

transport, and are responsible for the phagocytosis of degenerating germ cells and residues of germ cell cytoplasm occurring during the spermatogenesis (residual bodies). The Sertoli cells are also thought to secrete factors involved in gonadotropin feedback (inhibin) and in paracrine control of the Leydig cells (66). Disturbances of any of these functions and their structural basis are likely to disrupt spermatozoal production. Such effects on germ cells may be very marked and may result in "Sertoli only" atrophy, which may histologically be misinterpreted as a direct germ cell toxicity. The Sertoli cell itself is generally considered to be extremely resistant to lethal injury or necrosis and to show rapid recovery on cessation of treatment (23). Sertoli cell damage is histologically visible as cytoplasmic rarification and vacuolization. Ultrastructrually, this change is associated with dilatation of the ER (48) and mitochondrial swelling (14). Much larger vacuoles may also be seen, but are often phagocytotic vacuoles remaining after the digestion of necrotic germ cells (23). A common effect is the retraction of the apical processes or Exp Toxic Patho147 (1995) 4

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the alteration of their ectoplasmic specializations and tubulobulbar complexes. These changes subsequently lead to contact loss to germ cells, resulting in premature exfoliation or sloughing of germ cells. The displaced germ cells may be present in the tubular lumen or the epididymal duct. In the subsequent section a description is given for the various pathogeneses of Sertoli cell toxicity. Many of these effects have also been observed in man. The potential Sertoli cell target sites are summarized in tab. 3. One of the best investigated group of chemicals primarily affecting the Sertoli cell are certain phthalate esters such as di-(2-ethylhexyl)phthalate (DEHP). Phthalate esters are extensively used as plasticizers in polyvinyl chloride plastics. With the most active esters (di-n-pentyl phthalate, di-n-butyl phthalate) histologic Sertoli cell damage occurs after 6 hours of dosing. Basal Sertoli cell cytoplasm rarefication and closure of the tubular lumen progresses to germ cell degeneration and sloughing of cells into the lumen. The first ultrastructural changes occur 3 h after the administration of a single oral dose of 2.2 gram di-n-pentyl phthalate/kg body weight, consisting of a vacuolation of the perinuclear smooth endoplasmic reticulum which extends to the apical cytoplasm 6 h after dosing (20). The lesion progresses to disruption of the ectoplasmic specializations and enlargement of mitochondria. These degenerative changes occur only in tubules of stages I, II and XI to XIV of the spermatogenic cycle. This stage specificity of the lesion may coincide with a proposed functional cycle of the Sertoli cells (82) with plasminogen activators as the most important substances with cyclic variations in their production. A retraction of the Sertoli cell apical processes and consequent loss of contact with germ cells results in sloughing of spermatocytes and spermatids which show early degenerative changes (20, 22). This indirect germ cell toxicity is restricted to tubules with Sertoli cell damage and therefore affects only pachytene spermatocytes and step 1 spermatids in stage I-II and XI-XIV tubules. Daily phthalate treatment for a period of up to 4 days results in a gradual depletion of germinal cells from all tubules, leaving a Sertoil cell matrix with only occasional normal spermatogonia (20) (fig. 4). This end point lesion may histologically be misinterpreted as a "Sertoli only" change caused by a germ cell toxicant. The mechanism of phthalate toxicity on Sertoli cells is mediated by its endogenous metabolite mono(2-ethylhexyl)phthalate (MEHP), acting at the level of the FSHreceptor by competetive inhibition (23). Additionally, MEHP and di-n-octyl phthalate may alter the Sertoli cell metabolism by a direct inhibition of the mitochondrial respiratory functions (78). Two hypothetical mechanisms have been proposed for germ cell loss after Sertoli cell damage with 2,5-hexanedione. The first suggests that the compound alters Sertoli cell tubulin by forming tubulin dimers via covalent binding. The subsequently altered microtubule assembly may result in compromised sustentacular functions. Loss of 272

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postspermatogonial germ cells is the consequence of these non supportive Sertoli cells (5, 6). This mechanism of 2,5-hexanedione toxicity shows the critical role of microtubules in supporting and translocating germ cells (93, 108). The second hypothesis is based on biochemical changes observed in Sertoli cells after 2,5-hexanedione treatment prior to histological changes (10). A reduction in the Sertoli cell-specific enzymes beta-glucuronidase and gamma-glutamyl transpeptidase is interpreted as a sign of inhibited glycolysis. This results in depletion of lactate and pyruvate which are the only source of energy for spermatocytes and spermatids (46, 71,83). The main component of the blood-tubule barrier is formed by the basal occluding cell junctions between adjacent Sertoli cells, situated at the level above the spermatogonia and below the spermatocytes (109). 2,3,7,8tetrachlorodibenzo-p-dioxin would appear to disturb the blood-tubule barrier by affecting these organelles. In doses of 3 and 5 fJg/kg body weight the substance caused a reduction in the number of Sertoli cell tight junctions. The acrosomes of spermatids when stacked in the Sertoli cell cytoplasm were commonly surrounded by a vacuole instead of usual ectoplasmic specializations. Additionally, altered germ cells in all developmental stages were observed (9). A stage specific Sertoli cell attack is described for 1,3dinitrobenzene which affects Sertoli cells in stage IX and X tubules (4). The high metabolic rate of Sertoli cells in stage IX and X tubules may be a cause of their particular vulnerability to toxic damage (4, 82). Another effect observed in the testicular toxicity of several chemicals and which is presumably Sertoli cell-mediated is the failure of sperm release from the seminiferous epithelium. Under normal conditions mature spermatids are released from the seminiferous epithelium in stage VIII and very rarely occur in later stages. In cases of a failure in sperm release, e.g. after treatment with low doses of cadmium which do not induce vascular damage, step 19 spermatids occur in stage IX through stage XIV tubules. Groups of step 19 spermatids are still attached to the luminal margin of Sertoli cells. A few sperm heads are visible at the tubule periphery, probably being phagocytosed by Sertoli cells (39). 1.3. Germ cells: Degeneration of germ cells is a physiologic event during spermatogenesis (50). It is most frequently observed in stage VIII-XIV and stage I tubules where pyknotic step 19 spermatids and basally situated pyknotic germ cells occur (50). These apoptotic processes have to be carefully distinguished from an increased germ cell death caused by a noxious agent. Necrosis and loss of germ cells are the most frequent manifestation of testicular injury. They are produced directly or by an effect secondary to Sertoli cell damage as shown above for the example of the phthalate esters. Because of the close communication between Sertoli cells and germ cells, an initial effect on germ cells may result

5 4 Fig. 4. Induced "Sertoli only" change in a 5 month old rat; tubules lined by Sertoli cells (arrow) with their cytoplasmic extensions and few degenerating germ cells; note thickened basement membrane; PAS, bar = 10 11m.

Fig. 5. Low magnification of the same lesion as in fig. 4; completely atrophic tubules adjacent to less severely and intract tubules; hypertrophy and hyperplasia of Leydig cells surrounding atrophic tubules; PAS, bar =50 11m. in later changes of Sertoli cells (23). Germ cell effects which indicate a primary Sertoli cell damage are the loss of spermatogenic synchrony with the appearance of abnormal giant cells undergoing meiosis, delays in spermiation and the disorganization of the germinal epithelium. The susceptibility of seminiferous epithelium to toxic damage varies with the tubular stage. This explains the sometimes noted effect of some approximately normal tubules besides severely affected tubules in short term toxicity studies. Many components specifically affect certain spermatogenic stages. Nevertheless, this specificity is in most cases dose dependent. Bis-(2-methoxyethyl)ether for example selectively damages pachytene spermatocytes at an inhalative dose of 110 ppm. At 370 ppm also round spermatids and at 1100 ppm all spermatogenic stages were affected (59). The most vulnerable stages of germ cells are type A spermatogonia (stages XI-XIV), mid pachytene spermatocytes (stage VII), spermatocytes undergoing meiotic division (stage XIV) and step 7 and step 19 spermatids (round and elon-

gate spermatids in stage VII tubules). A problem for histologic evaluation of germ cell effects is the rapid sloughing of degenerated cells into the lumen or their phagocytosis by Sertoli cells (23). After a short treatment period (shorter than duration of spermatogenesis) tubules with germ cell depletion are often irregularly distributed throughout the cut surface of the testis (fig. 5), interspaced between histologically normal tubules. This may be the result of the stage specificity of the lesion. The reason for such a patchy distribution after a treatment lasting longer than the duration of the spermatogenesis is still not known. Atrophic tubules in testes affected in this way are preferentially located in the subcapsular region and sometimes around the rete testis. This uneven distribution has been associated with the kinetics of the blood supply (23). 1.3.1. Spermatogonia The spermatogonia are the major proliferative cells of the testes. According to functional aspects they are diviExp Toxic Pathol47 (1995) 4

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6 7 Fig. 6. Seminiferous tubule of a rat treated with an antineoplastic agent for 26 weeks; large vacuoles in all layers of the germinal epithelium resulting in hypocellularity; H & E, bar = 10 /lm. Fig. 7. Acute degeneration of spermatocytes and round spermatids in an early stage tubule induced by four weeks administration of a chemical compound; degenerating spermatocytes and spermatids appear as shadow cells (arrows); PAS, bar = 20 /Jm. ded into two main cell populations, the slowly dividing stem cells and the cells that differentiate into spermatocytes (16, 17,27). Morphologically the stem cells represent undifferentiated type A spermatogonia (42, 75). The differentiating spermatogonia undergo six mitotic divisions, the last giving rise to the spermatocyte. Because of this high mitotic activity the population of differentiating spermatogonia is the primary target for anti tumor agents. Cells past the DNA-synthesizing stages, including spermatocytes, spermatids and spermatozoa, are generally resistant to such substances. The slow dividing stem cell spermatogonia show an intermediate sensitivity (27, 70). Adriamycin and cyclophosphamide are examples of these substances. The inhibition of mitotic divisions is histologically visible as a reduction of spermatogonia and subsequent maturation depletion and hypocellularity (fig. 6). The end stage of this lesion is the "Sertoli only" change (fig. 4), characterized by tubules which are lined only by Sertoli cells. The "Sertoli only" change occurs if stem 274

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cell spermatogonia are destroyed and results in long term and possibly permanent sterility (70). Dystrophic mineralization occasionally occurs in tubules with such marked degenerative changes. 1.3.2. Spermatocytes The spermatocytes are the cells that undergo meiosis, i.e. the two successive divisions leading to the haploid spermatids. The primary spermatocytes emerge from type B spermatogonia. Preleptotene, leptotene, zygotene, pachytene and diplotene spermatocytes are successive stages of the prophase of the meiotic division. With completion of the meiotic division the secondary spermatocytes appear (15). Compounds that affect spermatocytes commonly act simultaneously on spermatids (fig. 7). Examples are certainnitroimidazoles. Degenerative changes of spermatocytes appear as nuclear chromatin clumping, cytoplasmic vesiculation (58) and as an increase in cytoplasmic

8 9 Fig. 8. Progression ofthe lesion in fig. 7: Absence of most spermatocytes and round spermatids in an early stage tubule; spermatogonia directly oppositing to elongate spermatids; H & E, bar = 10 11m. Fig. 9. Multinucleated giant cells after orat treatment with a pharmaceutical compound over a period of 26 weeks; H & E, bar = 10 11m. PAS-staining which is more easily detectable after GMA embedding than in routine paraffin material (13). Additionally, the uniform staining normally found in the cytoplasm is replaced by an uneven, patchy staining distribution (11). In our own material we additionally observed degenerating spermatocytes and round spermatids as shadow cells (fig. 7). Spermatids, followed by spermatocytes, are the cells most sensitive to the compound hexafluoroacetone-sesquihydrate. In vitro studies have shown that the testicular atrophy induced by this substance was associated with a decreased sterol synthesis (36). This may indicate a Leydig cell damage, manifested as a reduced androgen synthesis. Interestingly, spermatocytes and spermatids, especially mid pachytene primary spermatocytes and step 7 and step 19 spermatids are the germ cells most susceptible to degeneration after hypophysectomy. Other stages remain unaffected. This selective degeneration could be largely prevented by supplementation with LH and completely brought to normal low levels after supplementa-

tion with both LH and FSH, FSH alone had no effect (91). Therefore, the simultaneous degeneration of certain stages of spermatocytes and spermatids may in many cases be a result of their similar and high dependence on high concentrations of gonadotropic hormones and androgens. It may be possible that many compounds which produce degerneration of spermatocytes and spermatids primarily act on higher centres of the hormonal regulation, i.e. pituitary and/or Leydig cells. The best known examples of compounds specifically acting on spermatocytes are certain ethylene glycol alkyl ethers. Ethylene glycol monomethyl ether (EGME) has been extensively used in the industry as a solvent in paint, dyes, ink, varnish or hydraulic fluid (98, 112). Inhalation exposure to 300 ppm EGME for 3 days produced degenerative changes in pachytene spermatocytes of stage XIV tubules in rats (58). The stage specificity of this lesion is to some extent dose dependent; Twenty-four hours after oral treatment with a relative high dose (500 mg/kg body weight), pachytene spermatocytes in all stages of Exp Toxic Pathol47 (1995) 4

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a b Fig. 10. Examples of stage specific spermatid toxicity: a) normal appearing stage J-Vll tubule (top) next to a tubule in comparable cycle stage with a reduced number of round spermatids in golgi-phase after treatment with the same chemical compound as in fig. 6; H & E, bar = 10 11m. b) Stage XIV tubule of a rat treated for 26 weeks with a pharmaceutical compound; several degenerating spermatocytes in metaphase of meiotic division; H & E, bar = lO 11m. the cycle were affected (21). The specific toxic effect to spermatocytes resulted in seminiferous tubules lined with a single layer of stem cells and directly oppositing spermatids or mature spermatocytes. Such a pattern of lesion is a common finding in testicular atrophy induced by a variety of chemical toxicants and drugs (12, 57, 62) (fig. 8). EGME additionally acts on Sertoli cells, resulting in cytoplasmic vacuolation, contact loss to germ cells and fragmentation of cytoplasmic processes. It is not clear wether the spermatid degeneration is a primary effect of EGME or occurs as an effect apparently secondary to disrupted Sertoli-germ cell associations (31, 58). Another sensitive stage of spermatocyte development is the metaphase of meiotic division in stage XIV tubules which may preferentially be attacked by spermatocyte toxicants (fig. 10, own unpublished data). Other xenobiotics induce stage specific and morphologically characteristic spermatocyte lesions. An example is that mycotoxin alpha-amanatin selectively inhibits RNA polymerase II, leading to fragmentation of nucleoli in the pachytene spermatocytes (10). 276

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1.3.3. Spermatids Spermatids undergo the extremely complex series of changes giving rise to the spermatozoa. The main steps in spermatid development are described as seen with the light microscope. Young spermatids, characterized by their prominent PAS-positive Golgi-zone are in Golgiphase. With the appearance and extension of the PAS-positive cap in the region of the golgi zone they reach the cap phase. This stage is followed by the acrosomic phase with its species specific changes in nuclear shape. In the maturation phase spermiogenesis is completed by further condensation of nuclear chromatin. For detailed descriptions see CLERMONT (15), LEBLOND and CLERMONT (55). Degeneration of spermatids is histologically visible as the formation of ring-like nuclei, caused by chromatin margination (13). Multicellular aggregates with fused nuclear acrosomes are commonly formed by degenerating spermatids. These aggregates are often called multinucleated giant cells (fig. 9). Degenerating spermatids can also form eosinophilic globules which are located at

the luminal surface of the germinal epithelium. Ultrastructurally they contain myelin bodies, lipid droplets, clear vesicles, mitochondria, flagellae and a condensed nucleus (61). Lesions produced by spermatid toxicants are characterized by degenerative changes of spermatids, while spermatocytes are not or less severely affected. The susceptibility of the various stages of spermatids upon toxic agents seems to differ. Hence, selective effects on spermatids in Golgi-phase and cap-phase on spermatids in the metaphase of meiotic division (Fig. 10, own unpublished data) and elongate spermatids in stage I-VIII tubules (fig. 3) have been observed. Spontaneous testicular atrophy of young male rats with cryptorchid testes initially affects spermatids in maturation phase. Degenerating spermatids form eosinophilic globules. A failure in sperm release, present as retention of eosinophilic globules and mature spermatids in stage IX-XIV tubules (61) may indicate an involvement of Sertoli cells. With more advanced degenerative changes elongate spermatids almost disappear and the tubular lumen may be closed by exfoliated round spermatids. Multinucleated giant cells and Sertoli cell vacuolation may also occur. The end stage lesion is the "Sertoli only" change (61). Spontaneous testicular atrophy is reported to occur in an incidence of 2.5 % in control animals of short term oral studies and 9.4 % in control animals of short term nose only inhalation studies (combined incidence of bilateral and unilateral atrophy). The high incidence of testicular atrophy in inhalation studies was interpreted as the result of stress associated with the immobilization during the exposure period (61). In respect to the exteme temperature sensitivity of spermatogenesis it could also be an effect of elevated body surface temperature during the nose only inhalation. The incidence of spontaneous unilateral atrophy seems to differ only minimally between oral and inhalation studies. Ethylene oxide is a highly reactive and infiltrative alkylating agent, which is widely used as a precursor of industrial chemicals and as a sterilant for heat-sensitive medical and hospital supplies (40). Inhalative exposure of rats to ethylene oxide results in degenerative changes of spermatids. Early and mid mature elongate spermatids were more susceptible than round spermatids or mature elongate spermatids. Elongate spermatids which showed no morphological alterations at the light microscopic level, showed ultrastructural deformation in both head and tail portion. These stage specific germ cell effects we~e presumably secondary to simultaneously occurring Sertoli cell damage, predominantly consisting of deformation of the ectoplasmic specializations, tubulobulbar complexes and the smooth endoplasmic reticulum (48). Since these structures play an important role in the suspension of elongate spermatids (30, 87, 89,90,92,95), it is likely that the alterations in the apical processes induced an irregular arrangement, degeneration and sloughing of the elongate spermatids (48).

1.3.4. Spermatozoa Spermatozoa are highly motile cells with an intensive catabolic metabolism. The vast majority of compounds affecting these cells interfere with their energy requirements. Examples of substances that reduce ATP-Ievels are the chlorosugars (e.g. 6-chlorodesoxyglucose, alphachlorhydrin) which block glycolysis. The thiosugar 5thio-D-glucose causes testicular toxicity by the same mechanism, i.e. competetive inhibiting of glucose utilization (41). Nevertheless, in mice the primary target cells of this compound are spermatocytes and spermatids (44). Toluene, which has been identified as a contaminant of municipal tap water (28), caused decreased sperm motility in mice when administered in a concentration of 8.67 Ilg/ml drinking water (114). The mechanism of the action of toluene on the sperm is still controversially discussed (106, 114). 1.4. Leydig cells: The usual mechanism of toxicity on Leydig cells is the inhibition of its primary function, the production of testosterone. This inhibition is caused by a wide variety of mechanisms.

Fig. 11. Seminiferous tubules of a rat treated with a GnRHantagonist over a period of 26 weeks; maturation arrest of the germinal epithelium, inactive Sertoli cells and small Leydig cells; H & E, bar = 10 11m. Exp Toxic Pathol47 (1995) 4

277

.

a A very specific Leydig cell effect is induced by the inhibition of the secretion of luteinizing hormone (LH) by gonadotropin-releasing hormone (GnRH)-agonists and -antagonists. The absence of the hormonal stimulation causes atrophic changes in Leydig cells, which are microscopically visible as scant cytoplasm and a small condensed nucleus (fig. 11) (own data, 104, 110). The results of a morphometric study on rat Leydig cells posthypophysectomy suggest that on the ultrastructural level the atrophic changes presumably include a reduction of all organelles (96). All structural parameters related to volume and surface area of the Leydig cell and its organelles were lowered 6 and 28 days after hypophysectomy . In cases of Leydig cell atrophy thorough histological examination of the pituitary is necessary to identify the primary target site of the agent and its mechanism of action. The interruption of the axis hypothalamus-pituitary-gonads is indicated by a reduced number and size of FSH- and LH-producing basophils in the adenohypophysis (fig. 12), as it is caused by LHRH-agonists and -antagonists. These compounds also inhibit the Leydig cell steroidogenesis directly ans reduce the number of Leydig cell-LH-receptors (101). Perhaps the most widely studied Leydig cell toxicant is ethylene dimethane sulfonate, a sulfonic acid ester. Its specific cytotoxicity causes almost complete loss of Ley278

Exp Toxic Pathol47 (1995) 4

'

b

Fig. 12. a) Pituitary gland of a control rat; immunohistochemical detection ofLH-producing basophils with a monoclonal antibody; bar = 10 /lm. b) Pituitary gland of a rat treated with a GnRH-antagonist for 26 weeks; reduction of LH-producing basophils in number and size; bar = 10 /lm.

dig cells in vivo, particularly in mature rats. The testosterone ablation caused by Leydig cell toxicants leads to changes of the germinal epithelium, characterized as maturation arrest. The affected tubules lack spermatozoa, spermatids and late stages of spermatocytes (fig. 11). The number of spermatogonia appears normal, the number of spermatocytes is reduced (103). In the acute phase necrosis of mid pachytene spermatocytes as well as step 7 and step 19 spermatids is seen in testes with disrupted hormonal balance (23, 82). The selective necrosis of these cell types is caused by their high susceptibility to androgen ablation. Additionally, there is evidence of an androgen receptor in the nuceli of spermatogenic cells (54), which makes a direct effect of testosterone ablation on spermatogenic cells possible. Other authors assume that germ cell loss after hormonal ablation is mediated via the Sertoli cells (8, 82, 107). This again shows the complex cellular interactions in the testis. An indirect toxic effect which is more common in Leydig cells than atrophy is hypertrophy. Many testicular toxicants produce atrophy of the germinal epithelium only in a portion of tubules (fig. 5). Leydig cells adjacent to the atrophic tubules show morphological evidence of stimulation (fig. 4), whereas in the same testis unaffected seminiferous tubules were flanked by normal Leydig cells. A paracrine mechanism is proposed for this inter-

action between Leydig cells and seminiferous tubules with "GnRH-like substances" and a high molecular weight factor (37) as endogenous peptides secreted from the seminiferous tubules.

2. Extra-testicular sites Most compounds which induce testicular atrophy by a primary extratesticular effect act on the hypothalamuspituitary axis. 2.1. Hypothalamus-pituitary: A wide variety of factors are capable of modifying the function of the hypothalamus-pituitary axis and thereby leading to testicular effects via an influenced production of gonadotrophic hormones. The classic example of GnRH-agonists and -antagonists was given above. In animals treated with these substances the pituitary is histologically characterized by an atrophy of FSH- and LH-producing basophilic cells. Comparable effects can also be achieved by sex steroids. Natural and synthetic estrogens are proven to be potent inhibitors of spermatogenesis (72, 83). They act on two levels of the hormonal regulation: (1) by pituitary inhibition of LH-secretion and (2) by an inhibitory effect on Leydig cell testosterone production. 17beta-hydrolase and 17,20 desmolase have been identified as the target enzymes of the estrogen effects on Leydig cells (111). Androgens act on the hypothalamus-pituitary axis as part of the physiologic negative feedback mechanism. In unphysiologic high concentration they suppress gonadotrophic activity (1, 32). Testicular atrophy induced by high doses of testosterone-propionate and testosteroneenanthate is characterized by degenerative changes in Sertoli cells, hypocellularity and disorganization of the germinal epithelium, which may in the case of testosterone-propionate result in "Sertoli only" change (44). This pattern of changes may indicate a predominating role of reduced FSH-Ievels on Sertoli cells in the testicular toxicity of exogenous administered testosterones. Drugs that alter nervous function can inhibit the secretion of reproductive hormones. Such CNS-active drugs include marijuana, barbiturates, transquillizers and reserpine (64, 85, 102). In most cases of pronounced testicular atrophy caused by agents which directly act on the testes the adenohypophysis shows morphologic characteristics of a stimulated FSH- and LH-secretion. Basophilic cells of the lateral lobes are hypertrophic and hyperplastic and resemble the so-called castration cells. Mild hypertrophy and hyperplasia of basophils can easier be evaluated on sections stained with Perjod Acid Schiff-Orange G method than on routinely stained H & E sections. Environmental conditions associated with stress are reported to cause a reduction in the number of seminiferous tubules and disorganisation of the seminiferous epithelium (35) which is likely to be mediated via the in-

hibitory effect of corticosterone on LH (49). These observations should be kept in mind when evaluating a treatment related testicular atrophy. In female animals also a partial caloric food deprivation is associated with reduced fertility, nevertheless, it has only little or no effect on spermatogenesis in male rats (65). Well documented nutritional deficiencies leading to testicular degeneration include zinc (84), essential fatty acids, certain amino acids and some vitamins (100). 2.2. Epididymis: Occlusion of the efferent ductules or of the epididymidal duct primarily leads to an increase of testicular weight and tubular diameter by inhibition in the discharge of tubular fluids secreted by the Sertoli cells. If this inhibition persists for a longer period complete tubular atrophy may result. Occlusion of efferent ductules has been described as an effect of the fungicide bleomyl (methyl l-[butykarbamoyl] 2-benzimidazole carbamate) which also causes a closure of the lumen of the epididymal duct by the formation of granulomas (38). 2.3. Other sites: A number of organs have been implicated in testicular dysfunction, e.g. adrenals and thymus. Thymectomy and chemically induced thymic involution have been shown to be associated with testicular atrophy. Theobromine treatment was reported to produce thymic injury and testicular atrophy in the rat (l05). The mechanism involved is not clear so far.

Reversibility of testicular atrophy Reversibility of testicular atrophy is a critical point in estimating the testicular effects of chemical and pharmaceutical compounds under regulatory aspects. In the testes, recovering from damage of the germinal cells occurs in two processes: 1. regeneration, which is the increase in numbers of stem cells and 2. repopulation, which is the differentiation of stem cells and the refilling of tubules by their progeny (70). The spermatogonial stem cells are most important for both processes. Therefore, the presence of stem cells is crucial in estimating the reversibility of testicular atrophy. However, stem cell identification is difficult and time consuming, and the presence of a stem cell does not guarantee its subsequent viability and function. Alternatively, two functional assays have been developed to measure stem cell survival. The one of Withers et al. (113) involves counts of repopulating tubuli cross sections, usually at 5 wk. after cessation of treatment. Cross sections showing repopulation with spermatogenic cells indicate the presence of at least one surviving stem cell in that region. The second assay involves counts of sperm heads in the testis at 56 days after treatment (63). The sperm heads visible after that recovery period must have been arisen from cells that were stem spermatogonia at the time of treatment (63). In most if not all species including man, regeneration of testicular atrophy starts after a certain period of delay. Exp Toxic Patho147 (1995) 4

279

Mice do not show regeneration during the first month after cessation of exposure to radiation or treatment with alkylating agents (69). Regeneration and repopulation start int the second month (43, 51) and result in the production of an appreciable number of sperm within 8 weeks after exposure (63). We have made comparable observations of stem cell regeneration in rats in our laboratory. Six weeks after cessation of treatment with an antineoplastic compound which induced testicular atrophy with a pronounced reduction in the number of spermatogonia, only a distinct regeneration and an incomplete repopulation was observed. In extreme cases with no surviving stem cell spermatogonia, as it is the case in cryptorchidism, there is no regeneration and irreversible azoospermia (53). A much more pronounced delay in stem cell regeneration is proposed for the human testis, where treatment with intermediate doses of anticancer drugs often results in infertility for many years (18, 88). These data would suggest that testicular atrophy caused by spermatogonial toxicants is more or less irreversible and that atrophy caused by substances which act on spermatocytes, spermatids and spermatozoa is completely reversible. Nevertheless, there are a number of experiments which indicate incomplete reversibility of spermatocyte and spermatid effects even after a post exposure period of more than 80 days (59, 60). E.g., exposure of rats to the spermatocyte and spermatid toxicant hexafluoroacetone for 90 days results in severe testicular atrophy. This atrophy was only partially reversible within a 84 days post exposure period (60). Since we have shown in our experiments with a GnRH antagonist that ablation of exclusively spermatocytes and spermatids is fully reversible, it is most likely that in the case of hexafluoroacetone stem cells and/or Sertoli cells are additionally affected. This may also be true for the majority of spermatocyte/spermatid toxicants which induce irreversible or partially irreversible testicular atrophy. Instructive information concerning the repopulation was gained from experiments with a GnRH-antagonist. The maturation arrest induced by this compound was mentioned above and is shown in fig. 11. The exclusive absence of spermatocytes, spermatids and spermatozoa and the presence of obviously unaffected spermatogonia offers the possibility to study repopulation without overlapping stem cell regeneration. Complete repopulation was observed in an experiment with a 120 days post exposure period after a treatment duration of 26 weeks. Even in a 13 weeks toxicity study with a 42 days post exposure period a strong tendency towards repopulation was present, but it was still incomplete in a number of animals. In the rat, spermatogenesis takes 48-53 days (23). Sertoli cell damage appears to be of limited reversibility, since testicular atrophy after DEHP administration was irreversible within 45 days post exposure period (77). Atrophy of the seminiferous epithelium induced by 2,5-hexandione was still irreversible in most animals 280

Exp Toxic Patho! 47 (1995) 4

after a post exposure period of 119 days (6). If Sertoli cells are destroyed or permanently functionally compromised, regeneration is not possible since these cells do not divide and are necessary for the coordination of spermatogenesis (23). Leydig cells are able to undergo complete recovery after chemical ablation with compounds such as ethylene dimethane sulfonate. The precise derivation of this population of cells is still controversly debated (23).

Extrapolation to man The qualitative response of the male reproductive system is analogous across the species including man (67). Therefore, animal models are clearly useful for identifying testicular toxicants. A practical method to compare effects produced in test animals with those observed in man is the introduction of the interspecies extrapolation factor (IEF). It is defined as the relationship between the dose necessary to produce a given toxic effect in a test animal to the dose which produces the same effect in man. The IEF is a ratio between administered doses. Nevertheless, a testicular effect depends not only on the dose but also on the agent's pharmacokinetics, the rate of metabolism and the susceptibility of the target cells. Interspecies differences in these factors may cause IEF's higher than 1 (man more susceptible than the species under study) or below 1 (man less susceptible than the species under study). In determining the IEF's of a given agent it is of great importance to choose the appropriate end point. Fertility measurement is not appropriate for interspecies extrapolation of reproductive risks, primarily because laboratory and domestic animals are highly selected for their reproductive abilities and tend to produce more spermatozoa than are required for efficient fertilization (33). The end point of fertility testing in these species may therefore be very insensitive for detecting reductions in sperm numbers by toxins. Qualitative and semiquantitative histology are much better parameters with the only disadvantage that histologic material of damaged human testes is often limited. Another useful end point is the number of sperms produced, obtained by counting sperms in the ejaculate. An important source for differences between laboratory animals and man is the kinetic in the regeneration of stem cells. If a stem cell damage took place, the delay in stem cell regeneration, i.e. the delay in increasing the number of stem cells, is much longer in man than in laboratory animals. This makes obvious that the time of measurement is of importance in determining the IEF: It is higher than I if the effects of a spermatogonial toxicant are measured during chronic exposure or short time after chronic exposure, but 1 or below 1 a long time after exposure. The IEF of radiation is for example between 11 and 21 in the mouse (67). For cyclophosphamide the IEF is greater than 2.6 if examined during chronic exposure and

Table 2. Agents which cause testicular atrophy in rats

Fortsetzung table 2

Leydig ceU toxicants - acetaldehyde - cannabinols - ethanol - ethylene dimethane sulfonate - hexachlorocyclohexane - ketoconazole - LHRH, LHRH-analogues - LHRH-antagonists

-

Sertoli cell toxicants - 1,3-dinitrobenzene - 2,4-dinitrotoluene - 2,5-hexanedione - 2-ethyl-hexahydro-7 -methyl-5-p-tolyl-2-hindeno(1 ,2 C)pyridine hydrochloride - chlorobenzyl-I-indazole-3-carboxylic acid - cyproterone acetate - DL-6-(N-2-picolinomethyl)-5-hydroxy-indane - ethylene glycol monomethyl ether - m-nitrosobenzene - methylchloride - pepicolino-methyl-hydroxyindane - phtalate esters (di-2-ehtylhexyl-phthalate, mono-2-ethylhexyl-phthalate, di-n-pentyl-phthalate, di-n-butyl-phthalate) Spermatogonial toxicants - antitumor antibiotics; adriamycin, actinomycin D, bleomycin, daunomycin, mitomycin C - alkylating agents: sulfonic acid esters (busulfan), ethylenimines, hydrazines (procarbazine), nitrogen mustards (chlorambucil, cyclophosphamide), nitrosoureas - alkaloids (vinblastine, vincristine) - antimetabolites: amino acid analogues (azaserine), folic acid antagonists (methotrexate), nucleic acid analogues - borax - bendamustin - doxorubicin Spermatocyte toxicants - hexafluoroacetone-sesquihydrate - hexafluoroacetone - bis (2-methoxyethyl)ether - 2-methoxyethanol - 5-thio-D-glucose - heterocyclic compounds (nitrofurans, thiopens) - dinitropyrrols - bis-(dichloroacetyl)-diamines - alpha-amantin Spermatid toxicants - ethylene acid - ethylene oxide - hexafluoroacetone-sesquihydrate Spermatozoa toxicants - 6-chlorodesoxyglucose - alpha-chlorhydrin - toluene Failure in sperm release - fungizone - procarbazine - dibutyryl-c-AMP

vitamin A-deficiency dimethyl-methylphosphonate methylchloride 1,3-dinitrobenzene ethanol boric acid vitamin B6 5-fluorouracil cis-platinum doxorubicine ametopterin cadmium methylxanthines (caffeine, theobromine)

Extratesticular sites A. Hypothalamus-pituitary - GnRH-antagonists - GnRH-agonists - growth hormone -prolacin - natural and synthetic androgens - estrogens - progestins - DDT-analogues - marijuana - barbiturates - transquillizers (reserpine) - ethanol - clomiphene citrate B. Central nervous system - anesthetics: enflurane, halothane, methoxyflurane, nitrous oxide - opioidsd - anti parkinson drugs (levodopa) - appetite suppressants - neuroleptics (phenothiazines, imipramine, amitriptyline) C. Others - hypoglycemia induced by hyperinsulinemia Blood supply - cadmium - 5-hydroxytryptamin - epinephrin - mepivacaine hydrochloride - carbondisulfid - trivalent chromium Others (testicular target cell not specified) - antispermatogenic drugs: derivatives of I-beinzylindazole-3-carboxylic acid, I-p-chlorobenzyl-IH-indazol-3carboxylic acid, chlorohydrins, dichloracetyldiamine derivatives, dihydronaphtha1enes, dinitropyrrols, monothioglycerol - consumer products: tris(2,3-dibromopropyl)-phosphate (TRIS), - cyclic and linear organosilane polymeric fluids - food additives and contaminants: aflatoxins, cyclamat, dimethylnitrosamine, gossypol, metanil yellow, monosodium glutamate, nitrofuran derivatives - fungicides, sterilants: dibromochloropropane, ethylene dibromide, thiocarbamates, triphenyltin Exp Toxic Pathol47 (1995) 4

281

Fortsetzung table 2 - herbicides: chlorinated phenoxyacetic acids, diquat, paraquat - industrial chemicals: chlorinated hydrocarbons (hexafluoracetone, polybrominated biphenyles, polychlorinated biphenyls, TCDD, vinylchloride, chloroprene, 1,2dibromo-3-chloropropane), polycyclic aromatic hydrocarbons (dimethylbenzanthracene, benzo(a)pyrene), solvents (benzene, glycolethers, hexane, thiopene, xylene), diethyl adipate, ethylene oxide cyclic tetramer, trifluoroethanol, deuterium oxide - insecticides: lindane, carbaryl, aldrin, chlordane, dieldrin, dichlorvos, hexamethylphosphoramide, chlordec one - metals: aluminum, arsenic, cobalt, lead, mercury, methylmercury, molybdenum, nickel, silver, uranum - Mg-ions - nutrient deficiencies in: - certain amino acids - essential fatty acids - some vitamins -zinc - rodenticides: fluoroacetate - spontaneous in the spontaneous diabetic BB rat - therapeutic agents: aldactone, amphotericin B, chlorycyline, chloroquine, chlorpropamide, cimetidine, colchicine, diphenylhydantoin, hexachlorophene, hycantone, niridazole, nitrofuran derivatives, phenacetin, quinacerine, quinine, sulfasalazine, tetraethylthiuram disulfide, thiazides - miscellaneous: personal habits (alcohol consumption, tobacco smoking), physical factors (heat, light, hypoxia), radiation.

Table 3. Sertoli cell target sites. Sertoli cell

MEHP

FSH-receptor

2,5-hexanedione

microtubules

2,5-hexanedione

glycolysis

MEHP

mitochondrial respiratory functions

2,3,7,8-TCDD, ethylene oxide

tubulo-bulbar complexes, ectoplasmic speCializations

2,3,7,8-TeDD

ethylene oxide

.. basal occluding cell junctions smooth endoplasmic reticulum

greater than 0.6 after a long post exposure period (doses in mg/m2 body surface area). The IEF should be closely to I if the target cell of an agent is post-spermatogonial, the Sertoli cell or the Leydig cell. The IEF between rat and man for suppression of sperm production with testosterone is for example 1.3 (68). In general, a factor of lOis applied to animal data to ac282

Exp Toxic Pathol 47 (1995) 4

count for possible interspecies differences in sensitivity. With the determination of a reliable IEF it is possible to estimate the interspecies differences more precisely.

Conclusion Atrophy is the most common non-neoplastic lesion in the rat testis. A great variety of chemicals and environmental conditions can cause testicular atrophy (tab. 2). End stage lesions of these processes may be (1.) hypocellularity, (2.) maturation arrest, (3.) "Sertoli only" change or (4.) complete tubular necrosis. The complex intratesticular intercellular interactions are responsible for the relative uniformity of end stage lesions caused by so may agents. Examination of the tubular end stage lesions is not sufficient in elucidating the mechanism of action of a testicular toxicant. Associated changes of Leydig cells and adenohypophysis many provide further information and it may be possible to recognize a reaction pattern which is characteristic for a certain group of compounds. For some common groups of substances the reaction patterns are as follows:

1. Antiproliferative agents - "Sertoli only" change of the germinal epithelium because of their spermatogonial toxicity; - Sertoli cells not affected or active; - Leyding cells diffuse hyperplastic; gonadotropin-producing basophils increased in number and size; - plasma testosterone levels are normal or increased; - accessory sex glands normal. The pituitary effect is mediated via a reduced secretion of inhibin which is physiologically part of the negative feedback mechanism for the regulation of FSH-secretion. Leydig cell hyperplasia is caused by an increased plasma LH-Ievel and an increased secretion of a GnRH-like peptide by Sertoli cells. This pattern of lesions is caused by sufficient concentrations of most spermatogonial, spermatocyte and spermatid toxicants as well as by bilateral cryptorchidism.

2. Germ cell toxicants with hormonal mechanism of action maturation arrest of the germinal epithelium with only spermatogonia and eventually spermatocytes present; - Sertoli cells small and inactive; - Leydig cells small and inactive; - gonadotropin-producing basophils reduced in number and size; - plasma testosterone levels decreased; - accessory sex glands atrophic. The hypothalamus-pituitary-gonads-axis is interrupted by a suppression of gonadotropin secretion.

3. Leydig cell toxicants - maturation arrest of germinal epithelium; - Leydig cells degenerated, or in later stages, ablation of Leydig cells; - Sertoli cells small and inactive; - gonadotropin producing basophils increased in number and size; - plasma testosterone level decreased; - accessory sex glands atrophic. The germinal cells most sensitive to the testosterone ablation are the step 7 and 19 spermatids which explains the maturation arrest of the germinal epithelium.

4. Disturbances of the testicular blood supply - maturation depletion (hypocellularity); in more severe cases complete tubular necrosis with subsequent mineralization; - Leydig cell effects depend on severity of circulatory disturbances: in slight cases Leydig cell hyperplasia, in severe cases Leydig cell degeneration; - reaction of gonadotropin producing basophils, plasma testosterone levels and accessory sex glands depend on Leydig cell reaction. The maturation depletion results from the toxicity of lowered oxygen tension primarily on type A spermatogonia. The above examples of patterns of reaction may help to suggest a certain mechanism of action for substances which affect the testes. Identification of the primary target cell, however, is only possible if early changes are detected. A thorough histopathological examination of early testicular changes including the examination of all spermatogenic stages and the cellular composition of the tubules is extremely useful in the identification of the primary target cell and may in some cases elucidate the mechanism of action of the xenobiotic agent (e.g. LHRH-antagonists). The identification of the primary target cell is predictive in assessing the reversibility of the change in the species under investigation. It is necessary to emphasize the importance of the dose chosen as this can influence the spectrum of primary target cells. Several substances affect certain spermatogenic stages only at relatively low doses. At higher doses all germ cells, including the resistant stem cell spermatogonia, are damaged. Such substances could therefore cause reversible testicular damage at low doses with irreversible atrophy at higher doses. An intensive histopathology may be sufficient to identify the primary target cell, to determine the reversibility and to estimate the dose response relationship under qualitative and quantitative aspects. Conclusive results relating to the mechanisms of action can not always be expected. For this purpose additional studies are necessary which include time course and hormone assays.

Acknowledgements: The authors thank Mrs. LYNDA NOLAN for the kind assistance in language and Mrs. ANNEDORE ROSENKRANZ for the preparation of the list of references.

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36. 37.

38.

39. 40.

41. 42. 43.

44.

45.

46.

47.

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