J. Biosci., Vol. 2, Number 4, December 1980. pp. 361-367 © Printed in India Abnormalities in the fine structure of the spermatids of ratsinjected with cadmium

N. H. GOPAL DUTT and KAN KOBAYASHI*Department of Post-Graduate Studies and Research in Zoology, University of Mysore, Manasagangotri,Mysore 570 006, * Department of Anatomy, School of Medicine, Gunama University, Maebashi, Japan MS received 14 December 1978; revised 12 September 1980 Abstract. The degenerative changes in the spermatids as measured by changes in fine structureabnormalities increased with time following injection of Cd2+ into rat testis. The spermatids inthe twelve hours group appear as peculiarly club shaped and elongated structures with one or twosmall but perceptible vacuoles. The subacrosomal area and the space between the nucleus andthe middle piece are seen abnormally dilated. In the 30 day group, the central filaments are themost susceptible unit of 9+2 axoneme complex. The plasma membrane, the cytoplasmic matrix,the mitochondria of the middle piece and the fibrous sheath appear shrunken, discontinuous anddegenerative. Keywords. Spermatids; fine structure; rats; Cd2+ injection; testis.

Introduction

The fine structure of the testis of rat after a single injection of CD2+ has beendescribed by Chiquone (1964). He drew attention to the site of action of Cd2+ andpointed out that the testicular necrosis is due to the effects of this metal upon theendothelium of the vascular bed. Dutt et al. (1979) have reported ultrastructuralchanges in the interstitial cells of Leydig after a single injection of Cd2+ . Thepresent study provides a description of spermatids exhibiting various abnormalitiesand degenerative changes in the rat testis as a result of Cd2+ treatment. Materials and methods

Adult male rats of Wistar strain (300 g fed stock diet ad lib were maintained on a 12h:12 h, light:dark schedule. Cd2+ (0.04 M) was dissolved in distilled water. (Cd2 +

(9.1 mg/kg body weight) was injected subcutaneously as recommended by Parizek(1957). The animals were sacrificed by decapitation at intervals of 6, 12, 48 h and30 days. Distilled water injected rats served as controls. Small pieces of testis ofexperimental as well as the control rats were fixed, stained with uranyl acetatefollowed by lead citrate and examined with JEM-7 A, JEM-110v and JEM-100Celectron microscopes (Dutt et al., 1979).

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362 Dutt and Kobayashi Results In the following description, the spermatids, which were identified in the testis ofCd2+-injected rats are grouped into the following stages: a) early spermatids andb) late spermatids. Only these stages were used for comparison with the controls.In some cases the cytotoxic damage was so extensive that it was difficult to assignthe spermatids to any particular stage. For these reasons abnormalities were describedmainly with reference to the acrosomal system, the nucleus, the mitochondria of themiddle piece and the axoneme complex of the tail. Early and late spermatids of the controls

These cells are situated in the area between the primary spermatocytes and thelumen. Their nucleus is spherical and contains uniformly distributed chromatin.The periphery of the nucleus is conspicuous by the presence of a distinct electrondense acrosomal granule enclosed in a vesicle (figure 1). The nucleus of the latespermatid is elongated and flattened, and the mitochondrial sheath surrounding themiddle piece is distinct (figures 2 and 3). Various parts of the head such as the outerplasma membrane, the outer and the inner acrosomal membranes and a well developednucleus with its condensed chromatin are clearly seen. The axial filament complexconsists of nine pairs of circumferentially arranged peripheral filaments and a singlepair of centrally placed tubular filaments. Some electron dense material is alwaysseen between the peripheral and the central filaments. The flagella contain additionalstructures at different levels. Thus, the spermatids possess mitochondria, coarsefibres surrounding the middle piece and a dense fibrous sheath (figure 4). The axialfilaments are surrounded by a distinct plasma membrane all along their length. Six hour group A rather common abnormality of this group is the shrinkage of the nucleus of earlyspermatids into a flattened structure with a decrease in the stainability of the hemi-spherical acrosomal granule and the transformation of chromatin into loosely scatteredmasses within the nucleus (figure 5). Often a number of darkly stained bodies,probably the degenerating nuclei of spermatids, are also observed. They appear toundergo fragmentation into small bodies representing a sign of further disintegration.

Acrosomal deformation as evidenced by an abnormal growth is observed in certain late spermatids. The acrosome of these cells is seen thick and hypertrophiedwith thin regions at some points. The oval nucleus undergoes a reduction in size andits posterior region is differentiated into a thread like structure. A perforatorium isabsent in many spermatids and where it persists it looks almost empty. No changesare encountered in the sperm tail. The cytoplasmic matrix shows uneven texture.The mitochondria are often blown. Twelve hour group The late spermatids are very much elongated. The acrosomal system appears wavyand is seen parallel to the thread like structure of the nucleus (figure 6). Thespermatids appear peculiarly club shaped. The perforatorium is completely lost.A prominent gap is usually noticed in the acrosome (figure 7). The nucleus containsone or two breaks near its flat base. The apical region of the nucleus assumes aconical shape. A few spermatids show a considerably large space beneath theirnuclei (figure 8). The spermatid flagellum retains, however, its normal structure.

Fine structure of spermatids in cadmium injected rats 363

Figure 1-4. 1. A portion of spermatid from the testis of a control rat showing uniformly distri-buted chromatin and dense acrosomal granule ×16,000. 2. Late-spermatid from the testis of acontrol rat with its intact head, middle piece, mitochondrial sheath and axial filaments (M-mito-chondrial sheath)×8,400. 3. Enlarged view of an intact spermatid head to show the plasmamembrane, acrosomal membranes and the nucleus (PM-plasma membrane, ΟAM-outer acrosomalmembrane, IAM-inner acrosomal membrane, N-nucleus)×20,000. 4. A longitudinal sectionthrough the principal piece of the spermatid tail from a control rat with intact fibrous sheath andcentral filaments. ×20,000.

364 Dutt and Kobayashi

Figures 5-8. 5. A shrunken spermatid head from the testis of rat 6 h after Cd2+ injectionshowing loosely scattered chromatin masses in the nucleus N-nucleus, Ag-aerosomal granule.×16,000. 6. 12 h after Cd2+ injection. Notice the wavy and dense acrosomal margin of anabnormally elongated spermatid. ×13,000. 7. Group same as above showing a prominent gapin the dense acrosomal system around the degenerating nucleus. ×8,900. 8. Spermatid showinga large space below its nucleus. × 13,000.

Fine structure of spermatids in cadmium injected rats 365

Figures 9-12. 9. Cross section of the middle piece of the flagellum 48 h after Cd2+ injectionshowing loss of cristae in the mitochondria. ×13,400. 10. Group same as above. Illustratingthe empty mitochondria in the spermatid of the same group as above. 11. 30 days after Cd2+

injection to show loss of central filaments in the axoneme. ×18,900. 12. Spermatid tailrevealing discontinuity in its outer fibres. ×18,900.

366 Dutt and Kobayashi Forty eight hour group The deformativei changes in the acrosomal system and in the nucleus are much morepronounced than those in the 12 h group. The longitudinal row of mitochondriaalong the outer dense fibres shows degenerative changes like reduction in the stainabilityand complete loss of cristae (figure 9). The plasma membrane surrounding themiddle piece is also greatly disrupted and the mitochondria appear empty (figure 10). Thirty day group

The spermatid heads are not commonly observed in this group. When seen theyshow profiles similar to those of the 12 h group in the process of degeneration. Thecentral filaments of the axoneme complex were not seen in most of the spermatid-rats (figure 11). The fibrous sheath was often discontinuous (figure 12), and appearto decrease in size and in staining capacity. There are other signs of disruptionincluding shrinkage and dissolution of the various components of axonene complex. Discussion Recently Chowdhury and Steinberger (1975) have shown that the administration oftestosterone propionate, dehydrotestosterone or 5 ∝ -androstenediol to the ratresults in the formation of spermatids upto the stage of spermiogensis. In the testisof Cd2+ treated rat, necrosis of interstitial cells was reported earlier by Dutt et al.,(1978, 1979). These authors have also reported a marked decrease in the level ofserum androgens in the Cd2+ treated rats. In view of the dynamic role of androgensin the intiation and maintenance of spermatids, it is believed that the degenerativechanges observed in the present studies in the spermatids may be due to deprivationof androgen under the influence of Cd2+ Whether this effect is due to direct actionof Cd2+ on the interstitial cells and if so the question of such mechanism is not clear.

In the testis of Cd2+ injected rats abnormality usually manifests itself in theuncontrolled growth of the acrosome and reductipn in the nuclear material. Thecontact between the nucleus and the middle piece is also destroyed. These changesresult in the development of an unusually large space beneath the nucleus. Pleon(1973) also believed that during experimental cryptorchidism the formation of subacro-somal space is the result of a decreased firmness of contact between the acrosomeand the nucleus.

Many workers have interpreted the abnormalities of axoneme complex as factorsor signal marks contributing to defective motility, thus leading to infertility of spermsdue to experimental cryptorchidism (Nagano, 1963), antispermatogenic agent, Win18446 (Reddy and Svaboda, 1967), neutron irradiations (Hunt and Johnson, 1971)and sterility in the natural populations (Cooper and Hamilton, 1977). The presentwork has shown that the flagellum of the sperm of rat is resistant to the influence ofCd2+ upto 12 h after the injection. At 48 h, however, the mitochondria of themiddle piece appear as empty membrane bound spaces without cristae or any stainablematerial in them. The plasma membrane surrounding the fibres is also not seen. Inthe 30 day group the fibrous sheath reveals discontinuity in structure and loss ofuniformity in staining. According to Fawcett (1975) the mitochondria provide thenecessary energy for the maintenance of the function of the outer fibres. In view ofthis it may be interpreted that the mitochondrial degenerative changes probably play

Fine structure of spermatids in cadmium injected rats 367 an important role in the process of necrosis of the outer fibres in the Cd2+ treatedrat. Further, in the 30 day group, the central filaments of the tails frequentlydisappear from view. Such an obscurity of central filaments has been describedearlier m the experimentally induced cryptorchid rat (Nagano, 1963). It seems likely,as in the cryptorchid rat, the central filaments are far more sensitive to Cd2+ thanthe other components of the axoneme-complex in rat. It is further noteworthy thatthe longer the duration after Cd2+ injection the greater is the impairment of axialfilaments. References Chiquone, A. D. (1964) Anat. Rec., 149, 23. Chowdhury, A. K. and Steinberger, E. (1975) Biol. Reprod., 12, 609. Cooper, T. G. and Hamilton, D. W. (1977) Am. J. Anat., 149, 93. Dutt, N. H. G., Wakabayashi, K. and Inoue, S. (1978) Proc. Indian Acad. Sci., B87, 161. Dutt, N. H. G., Inoue, S. and Wakabayashi, K. (1979) Curr. Sci., 48, 1049. Fawcett, D. W. (1975) Dev. Biol., 44, 394. Hunt, D. M. and Johnson, D. R. (1971) J.Embryol. exp. Morph., 26, 111. Nagano, T. (1963) J.Cell. Biol., 18, 337. Parizek, J. (1957) J. Endocrinol., 15, 56. Ploen, L. (1973) Virchows Arch. Abt. Β. Zellpath., 14, 159. Reddy, K. J. and Svaboda, D. J. (1967) Arch. Pathol., 84, 376.