741
J. Anat. (1977), 124, 3, pp. 741-755 With 14 figures Printed in Great Britain
Ultrastructural studies on the differentiation of spermatids in the domestic fowl V. K. GUNAWARDANA* AND M. G. A. D. SCOTT Department of Anatomy, Royal Veterinary College, London
(Accepted 10 November 1976) INTRODUCTION
Studies on the ultrastructure of spermatids and spermatozoa of the domestic fowl are relatively few when compared to similar studies in mammals. The available reports include those of Nagano (1962), McIntosh & Porter (1967), Lake, Smith & Young (1968) and Tingari (1973). While Nagano in his studies focused attention mainly oni the development of the acrosome and the arrangement of the centrioles in spermatids, the studies of McIntosh & Porter were confined to the microtubular systems and their functional significance. The other two reports deal with the ultrastructure of spermatozoa. Lake, Smith & Young investigated the ultrastructure of ejaculated fowl spermatozoa. Tingari reported the changes in fine structure during the passage of spermatozoa through the excurrent ducts. Thus it is apparent that a comprehensive study of spermiogenesis in this species at an ultrastructural level is lacking. The present investigation was undertaken to repair this deficiency. The observations recorded here establish a sequence of changes which occurs in a spermatid differentiating into a spermatozoon. MATERIALS AND METHODS
Sexually mature White Leghorns were used. The birds were killed with an overdose of pentobarbitone sodium (Nembutal - Abbott Laboratories). Pieces of testis were removed soon after death and immersed in fixative. Two methods of fixation were employed. In the first, tissues were fixed directly in buffered osmium tetroxide according to the method of Zetterqvist (1956). In the second, tissues were fixed overnight in 3 %0 glutaraldehyde in phosphate buffer at pH 7 4, and subsequently postfixed in 1 %0 osmium tetroxide, also in phosphate buffer, for 2 hours. In all instances fixation was carried out at 4 'C. After fixation by either method the tissues were dehydrated in a graded series of methyl alcohol, cleared in propylene oxide and embedded in Araldite. Sections were cut on a Porter Blum ultratome using glass knives. Sections for light microscopy were stained with 1 % methylene blue in 1 0 borax. Ultrathin sections of selected areas were stained with lead citrate (Reynolds, 1963) and examined using a RCA EMU-3F electron microscope operating at 50 kW. * Present address: Department of Veterinary Pre-Clinical Studies, School of Veterinary Science University of Sri Lanka, Peradeniya Sri Lanka.
742
V. K. GUNAWARDANA AND M. G. A. D. SCOTT RESULTS
Based on their nuclear structure, spermatids could be assigned to one of the following four phases: (I) Spermatids with round nuclei. (II) Spermatids with irregular nuclei. (III) Spermatids with long nuclei containing coarse chromatin granules. (IV) Spermatids with long homogeneously dense nuclei. Phase I spermatids possess one or more membrane-bound proacrosome granules in the region of the Golgi complex (Fig. 1). The latter appears in the form of a stack of cisternae, interspersed with vesicles (Figs. 1, 2). Figure 2 illustrates the formation of the tail in this phase. The proximal and distal centrioles comprising the centriolar complex are seen in transverse and sagittal section respectively. The proximal centriole is located about half way between the cell membrane and the nuclear membrane, while the distal centriole extends from this point to the cell membrane, at which point the tail filament originates. The cell membrane is reflected on to and encloses the developing tail. A dense transverse slightly convex plate, with the convexity on the centriolar aspect, separates the centriole from the tail. The peripheral fibres of the latter structure are continuous with the wall of the distal centriole, but the central pair of fibrils seem to commence at the transverse plate. Dense deposits and clusters of vesicles are seen close to the proximal centriole. The nuclei of phase IL spermatids show varying shapes corresponding to different degrees of nuclear elongation (Figs. 3-7). The chromatin is finely granular and of uniform density. The nucleoli are either compact (Figs. 3, 4), or they are diffuse and apparently disappearing (Figs. 5, 6). The proacrosome is lodged in a concavity of the nuclear membrane which is thickened over the area of contact (Figs. 3, 4). Closely associated with the developing acrosome is a dense droplet lying within an invagination of the nuclear membrane (Fig. 6). Later, at a time when the nucleus is more elongated, the acrosome is flattened on either side on the end of the nucleus forming a crescentic structure (Fig. 7). The cavity formed by the invagination of the nuclear membrane and the dense droplet within it are enlarged. The proximal centriole is also lodged in a concavity of the nucleus, while the junction of the distal centriole with the tail filament is in a position at the base of an invagination of the cell membrane (Figs. 4, 5, 9). Dense material is deposited in this region, which is referred to as the annulus. Microtubules occur in the cytoplasm of spermatids in both phases. In phase I and early phase II these occur as short lengths near the proximal centriole (Fig. 2). Tangential sections of the more elongated nuclei show microtubules disposed in an oblique direction, seemingly parallel to one another (Fig. 7). The mitochondria have rounded profiles and show pronounced cristae. The elongated nuclei of phase III spermatids contain coarse granules. At the acrosomal end of the nucleus is a U-shaped cavity; lying within this cavity is a rodlike structure, the perforatorium (Fig. 8). A salient feature of the spermatids in this group is the presence of microtubules arranged in a regular pattern. Some spermatids cut in a longitudinal plane show cross sections of microtubules arranged in two rows on either side of the nucleus. They are seen to begin near the cell membrane caudal to the acrosome and to continue beyond the centrioles towards the tail (Figs. 8, 9). On transverse section these microtubules have the appearance of being concentrically arranged (Fig. 10). In others, where the coarse granular material of the nucleus is
Spermatid differentiationi in the domestic fowl X.?U).
A
,
t
743
a
Fig. 1. Phase I spermatid showing a proacrosome granule (PA) near the Golgi complex (GC). MVB, multivesicular bodies. Phase III spermatids are seen below.
2 Fig. 2. Phase I spermatids showing the beginning of tail formation. An abundance of vesicles is seen near the proximal centriole (PC). Note the proximity of the microtubules (MT1) to these vesicles (arrows). AL, annulate lamellae.
744
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
Fig. 3. Bi-nucleate spermatid in early phase II. A peripheral vacuole of a membrane body (MB) is continuous with the annulate lamellae (AL). Note the compact nucleolus (NU).
4
"
Fig. 4. Phase II spermatid. The nuclear membrane is thickened near the acrosome (A). Some vacuoles of the membrane body (MB) contain dense material. AL, annulate lamellae; CC, cytoplasmic canal; NU, nucleolus.
Spermatid differentiation in the domestic fowl
745
Fig. 5. Phase II spermatid. The proximal centriole (PC) is within a nuclear concavity. Arrow indicates the beginning of the cytoplasmic canal near the annulus (AN). A granular satellite (S) is seen adjacent to the membrane bodv (MB). DC, distal centriole; NU, nucleolus.
6 ~ ' ,0. x ~ Fig. 6. Phase II spermatid. The dense droplet (P) appears to originate from the nuclear membrane. MB, membrane body; NU, nucleolus.
746
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
Ti~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Fig. 7. Phase II spermatid which is older than that of Fig. 6. Note the crescentic acrosome (A) and enlarged dense droplet (P), which would give rise to the perforatorium. MT1, microtubules; AL, annulate lamellae.
'UM Vj .t #
.*
-i w-4 aq
'k
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Fig. 8. Phase III spermatid with rod shaped perforatorium (P). Note the regular arrangement of the microtubules (MT1). A, acrosome.
Spermatid differentiation in the domestic fowl J
747
.i;
'0.. U..,...
)
*.jk6
_
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Fig. 9. Phase III spermatid. Coarse granules are abundant in the nucleus. Microtubules (MT1) extend beyond the distal centriole. Arrow indicates dense material near the annulus (AN). CC, cytoplasmic canal.
P
,.
. ..
l'' Fig. 10. Phase III spermatids sectioned transversely. The microtubules (MT1) appear concentrically arranged. The spermatid at top left is sectioned at its anterior end and shows the perforatorium (P) centrally, acrosome (A) peripherally and the nucleus (N) in between.
748
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
4;.p. -
_
.-
A
.
Fig. 11. Phase III spermatids sectioned transversely. Note the distinctive pattern of the microtubules (MT2) forming the manchette. Arrows indicate dense ring-like structures. The spermatid at bottom left is in phase I. GC, Golgi complex; MVB, multivesicular body; PA, proacrosome.
more in evidence, the arrangement of the microtubules is completely different. They are distributed as longitudinal straight tubules extending from the anterior extremity of the nucleus to the end piece of the tail, forming a complete wall which encircles these structures (Fig. 11). At lower levels in the tail, gaps are noticeable in this wall. In rare instances where both sets are present, the longitudinal microtubules are placed internal to those which are concentrically arranged (Fig. 12). The mitochondria in most instances are oval in outline and are seen external to the microtubules. In the main piece of the tail a dense layer surrounds the nine doublets. Dense ring-like structures, and vacuoles of varying shape and size, are present in the cytoplasm. The nuclei and axial filament complexes in some of the spermatids in phase IV are seen to be surrounded by the wall of longitudinally arranged microtubules (Figs. 12, 13). These however do not exhibit the distinctive scalloped pattern seen previously. The tubules are crowded together, their walls are thicker, and they seem to be coalescing (Fig. 12). In other spermatids of this phase the microtubules are absent (Fig. 14). The distal centriole appears to be of a shorter length when compared to the earlier phases. The annulus is at the junction of the mid piece with the main piece, and the dense material of the annulus is continuous with the dense material surrounding the main piece. Mitochondria with longitudinal cristae are located at the posterior pole of the cell and in some spermatids they are in a position around the mid piece. The entire spermatid is slightly curved. During spermiogenesis there is a gradual reduction in cytoplasm, the acrosome region being devoid of cytoplasm by phase II. Clusters of multivesicular bodies are
Spermatid differentiation in the domestic fowl
MT2 ~~
~
~
~
749
~~T
Fig. 12. Phase IV spermatids sectioned transversely. The microtubules (MT2) appear to be coalescing. The spermatid at top right is sectioned through the centriole and shows both sets of microtubules (MT1 and MT2). IB, intercellular bridge; MB, membrane body.
always seen in all phases of spermiogenesis. Two or more narrow cisternae lying parallel to one another, and showing intermittent constrictions, are interpreted as annulate lamellae (Figs. 2, 3, 4, 7). Some such cisternae are observed in close apposition to the nuclear membrane (Fig. 2). A unique structure, referred to as a membrane body, is seen consistently in phase II and less frequently in other phases. It is located in the caudal half of the cell and consists of a central spherical vacuole surrounded by radially arranged peripheral vacuoles leaf-like in shape (Figs. 3-6, 12). In some micrographs one of the peripheral vacuoles is seen to be in continuity with the cisternae of the annulate lamellae (Fig. 3). Varying amounts of dense material are seen within the vacuoles. An aggregate of granular material is at times observed in close proximity to the membrane body (Fig. 5). Multinuclear spermatids, and spermatids connected by intercellular bridges, occur frequently in the testes of the domestic fowl. DISCUSSION
The observations presented here elucidate the fine structural changes undergone by spermatids during their differentiation. The classification of spermatids into four phases was based on nuclear changes. When these changes are considered along with the degree of development of the acrosome and tail filament, it seems evident that these are successive phases of development. Thus the formation of the acrosome and tail, which began in phase I, had progressed further in phase II. 48
ANA I24
750
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
13 Fig. 13. Phase IV spermatid3 sectioned longitudinally. Microtubules (MT2) acrosom.; P, perforatorium; RB, residual body.
are
present. A,
Fig. 14. Phase IV spermatid showing mitochondria (M) in position around the mid piece. Note the length of the distal centriole (DC) and the location of the annulus (AN). Note also the curvature of the spermatid.
Spermatid differentiation in the domestic fowl
751 In many mammalian species a proacrosome granule arises within a vesicle which subsequently gives rise to the head cap (Burgos & Fawcett, 1955; Gardner, 1966). In the toad a large vesicle is present, but a proacrosome granule is not formed (Burgos & Fawcett, 1956). Acrosome formation in the fowl is distinct from both these. The proacrosome granule closely invested by its membrane attaches itself to the nuclear membrane by phase II and spreads over the anterior pole of the nucleus to form a crescentic structure which is termed the acrosome from then onwards (see Fig. 7). There is no acrosome vesicle, and hence a head cap comparable to that of mammalian spermatids is not formed. Grigg & Hodge (1949) described the acrosome of the spermatozoon as consisting of an apical cap and an apical spine. Lake et al. (1968) and Tingari (1973) referred to these structures as the acrosome cap and acrosome spine respectively. Tingari states that the acrosome cap loosely fits the nucleus during spermiogenesis, but is more closely applied to the nucleus in spermatozoa lying free in the lumina of the seminiferous tubules. In the present study the acrosome (which is the equivalent of the acrosome cap in Tingari's study) was seen to be closely applied to the nucleus throughout the process of spermiogenesis. It is thought likely that the looseness of the acrosome encountered in Tingari's investigation is a consequence of the fixation procedure adopted. Lake et al. (1968) considered the acrosome spine to be the equivalent of the perforatorium in the rat. Brokelmann (1963) regarded the perforatorium and the acrosome as identical structures, and compared a ventral thickening of the acrosome cap in the rat to a dense droplet described by Nagano (1962) in the rooster. Bedford (1967) thought that the subacrosome space in mammals was synonymous with the perforatorium. The rod-shaped perforatorium in this study lies within the space bounded by the inner membrane of the acrosome cap and the nuclear surface, and extends caudally between these structures. Thus the perforatorium in the fowl is located in a position identical to that described for mammals by Bedford (1967). The perforatorium is believed to arise from the dense droplet referred to earlier, thereby confirming the results of Nagano (1962). Clermont et al. (1955) described the perforatorium as an extension of the nuclear membrane in the rat. The present findings reveal that the perforatorium is attached to the nuclear membrane of spermatids. This is contrary to the observations of Tingari (1973) in spermatozoa. It is likely that this attachment is loosened during the final phase of spermiogenesis. Again, the possibility of fixation artifact cannot be totally disregarded when comparing and interpreting these observations. The arrangement of the centrioles and the axial filament complex in general agrees with that of Nagano (1962). However, he reported that the distal centriole increases in length with increasing age of spermatids. Lake et al. (1968), in their study of fowl spermatozoa, described a short neck region containing a complex centriolar structure. The results of this investigation reveal that during the final phase of spermiogenesis there is a reduction in the length of the distal centriole. Nagano, in his study, did not record any spermatid stage showing a homogeneous dense nucleus, as was seen in the final phase in the present study. The point of reflexion of the plasma membrane on to the tail filament lies at the distal extremity of the distal centriole. This point, which is referred to as the annulus, is drawn into the cytoplasm with the movement of the centriolar complex towards the nucleus, thereby forming a cytoplasmic canal. In rat spermatids a cytoplasmic canal arises in an identical manner (Sotelo & Trujillo-Cenoz, 1958). The cytoplasmic canal is obliterated late in spermiogenesis when the annulus, together with the dense 48-2
V. K. GUNAWARDANA AND M. G. A. D. SCOTT 752 material associated with it, migrates down the axial filament complex. A similar process is reported in other species, and Sapsford, Rae & Cleland (1967) excluded the annulus from the neck region of the bandicoot spermatids for this reason. Microtubules form very early in spermiogenesis, and they appear to originate from the cluster of vesicles seen in the vicinity of the proximal centriole. During the course of spermiogenesis two sets of microtubules are formed. The first set has been shown by McIntosh & Porter (1967) to be helically arranged. This arrangement became evident in late phase II and persisted during the early part of phase III. In late phase III the helix had been replaced by the longitudinal array of microtubules which corresponds to the manchette or caudal sheath of light microscopy. The presence of microtubules showing two distinctive patterns of distribution has been reported in reptiles (Clark, 1967). This author proposed that the helix unwinds as the nucleus elongates, resulting in the longitudinally orientated microtubules. It is thought unlikely that such a phenomenon occurs in the fowl since spermatids possessing both sets of microtubules at the same time were encountered, although
rarely. Nagano (1962), in his study, does not refer to the presence of helically arranged microtubules. McIntosh & Porter (1967) attribute this to the method of fixation employed by Nagano, and argue that temperature is a factor which determines the stability of microtubules. However, in the present investigation the helix was preserved when material was fixed in cold osmium tetroxide alone, which is one method used by Nagano. The manchette or caudal sheath has been described by this author, but he states that it was not always present in late spermatids. According to his interpretation these are cells with coarse granular nuclei. On the contrary, from the present results, it is clear that the manchette is visible even in spermatids with homogeneously dense nuclei. Nagano associated the manchette with elongation of spermatids. In a study of spermatids with the light microscope, Gunawardana (1973, 1976) reported that maximum length was attained at stage 8, which is prior to the formation of the manchette. This stage of spermatid, when examined under the electron microscope, falls into the category of early phase III, where the helical microtubules are clearly developed (Gunawardana, 1973). These observations tend to support the view of McIntosh & Porter (1967) that the helix is concerned with nuclear elongation. The same workers suggested that the manchette determines the final curvature of the nucleus. While agreeing with this suggestion, it should be pointed out that the curvature involves not only the nucleus but the axial filament complex as well (see Fig. 14). This could be explained by the relatively long extent of the manchette as far caudal as the end piece of the spermatids. In contrast, most species show the manchette ending just caudal to the distal centriole (Clark, 1967; Sapsford et al. 1967). A structure referred to as a chromatoid body has been recognized in many species (Sud, 1961 a). In the domestic fowl this structure does not seem to have received much attention, except for a reference to an accessory body by Zlotnik (1947). Burgos & Fawcett (1955) described the chromatoid body in the cat as an irregular mass of closely aggregated osmiophilic granules. Sapsford et al. (1967) thought it possible that dense ring-like bodies seen in bandicoot spermatids were derived from an aggregate of granular material which bears some resemblance to the chromatoid body. More recently, Susi & Clermont (1970), in a detailed study of this structure in the rat, described it as being composed of dense osmiophilic material and vacuoles. They stated, further, that during later stages the chromatoid body appeared as a
Spermatid differentiation in the domestic fowl
753
dense sphere surrounded by radially arranged vesicles. No single structure conforming to any of the above descriptions was seen regularly enough to warrant its identification as the chromatoid body in the domestic fowl. The one structure which could be equated with the chromatoid body, and which appeared regularly during the course of spermiogenesis, was the structure referred to in this study as the 'membrane body'. This structure, in comparison to the chromatoid body of the rat (Susi & Clermont, 1970), consists of a central spherical vacuole containing dense material instead of a dense sphere. Radially arranged vesicles are similar to those of the rat. Sud (1961 b) observed a granular satellite accompanying the chromatoid body in the rat. In guinea-pig and chinchilla germ cells a satellite is shown to be intimately related to the chromatoid body. It is possible that the granular aggregate seen in phase II spermatids represents a satellite. Dense rings seen in later stages (Fig. 11) are comparable to those described in bandicoot spermatids (Sapsford et al. 1967). Fawcett & Phillips (1967) suggested a nuclear origin for the chromatoid body in certain mammalian species. In the species investigated here (the fowl) the membrane body appears to arise from the annulate lamellae. In the oocytes of Necturus, annulate lamellae are said to be formed by the budding of vesicles from the nuclear membrane (Kessel, 1963). The proximity of some annulate lamellae to the nuclear membrane of spermatids in the fowl suggests a similar mode of origin in this species. Swift (1956) postulated that annulate lamellae may function in the transfer of specific material from the nucleus to the cytoplasm. Taking the above views into consideration, it is proposed that during the stages ofnuclear condensation, when considerable shrinkage of nuclear volume occurs, there is a loss of excessive nuclear material which is transferred to the chromatoid body (membrane body) via the annulate lamellae. Vaughn (1966), using autoradiographic and microspectrophotometric methods, demonstrated that basic protein lost from the nucleus was present in the 'sphere chromatophil' of the rat spermatid. Histochemical techniques at an ultrastructural level, together with the above techniques, may prove useful in confirming the validity of the suggested proposal. With regard to the ultimate fate of the chromatoid body (membrane body), if its presence is merely to serve as a reservoir for excessive nuclear material, it is very likely that it is lost in the residual cytoplasm. The presence of multinuclear spermatids may be related to the intercellular bridges frequently encountered. Leblond, Steinberger & Roosen-Runge (1963) noted that, in pathological conditions, cells connected by intercellular bridges tended to fuse to form multinuclear cells. Bryan (1971) is of the opinion that cells produced as a result of coalescence of conjoined cells are normal, and lead to the production of normal spermatozoa. The occurrence of binuclear cells with an acrosome differentiating in relation to each nucleus is comparable to the situation in the cat (Burgos & Fawcett, 1955). The frequency with which such cells were seen in apparently normal testes suggests that they might very well eventually produce normal spermatozoa. Lake (1956), who was of similar mind, considered multinuclear spermatids to be produced as a result of post-meiotic multiplication which would eventually produce clusters of spermatozoa. SUMMARY
Four phases of spermatid maturation are recognized on the basis of nuclear morphology. The formation of the acrosome, perforatorium and axial filament complex are described in relation to these phases. The functional significance of the microtubular systems in nuclear elongation and spermatid curvature are discussed.
754
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
A membranous structure, hitherto unidentified, and referred to in this study as the 'membrane body' is compared with the chromatoid body. Morphological evidence suggests that this structure, together with the annulate lamellae, removes excessive nuclear material. ADDENDUM
The development of the tail and the transformation of the shape of the nucleus of the domestic fowl has been investigated recently (Okamura & Nishiyama, 1976). With reference to the development of the tail the authors suggest that the plasma membrane of the flagellum is derived from the Golgi vesicles. The present findings favour the view that the membrane of the flagellum is a reflexion of the cell membrane. Our results are in agreement with those of Okamura & Nishiyama regarding the presence of two sets of microtubules. However, a few points of difference were noted. It has been reported that the formation of the first set (circular manchette) begins rostrally and proceeds caudally. From our observations the reverse seems to be happening (see Fig. 7). Microtubules are first seen in the region of the centrioles. Therefore it is even more reasonable to suppose that the circular manchette starts to form at the caudal end of the nucleus. The appearance of the longitudinal manchette of Okamura & Nishiyama was said to be coincident with the beginning of nuclear condensation. In the present work nuclear condensation preceded the formation of the manchette. With respect to the abberant spermatid, the conclusions of Okamura & Nishiyama are based on the assumption that a longitudinal manchette was not formed in this spermatid. However, it is also possible that a longitudinal manchette formed but disappeared with advancing spermiogenesis. Furthermore, one cannot be certain whether or not this particular spermatid produced a circular manchette in the first instance. In which case, the abnormality in shape may just as well be attributed to the absence of a circular manchette. In fact, the results of McIntosh & Porter (1967) show abnormalities in nuclear shape which are due to faulty orientation of the first set of microtubules or circular manchette. Finally, the spermatid under discussion could have resulted from the absence of both sets of microtubules. REFERENCES BEDFORD, J. M. (1967). Observations on the fine structure of spermatozoa of the Bush Baby (Galago senegalensis), the AfricanGreen Monkey (Cercopithecus aethiops) and man. American JournalofAnatomy 121, 443-460. BROKELMANN, J. (1963). Fine structure of germ cells and Sertoli cells during the cycle of the seminiferous epithelium in the rat. Zeitschriftfiir Zellforschung und mikroskopische Anatomie 59, 820-850. BRYAN, 1. H. D. (1971). On the presence of multinuclear spermatogenic cells in the seminiferous epithelium of the mouse. Zeitschrift fir Zellforschung und mikroskopische Anatomie 112, 333-349. BuRGos, M. H. & FAWCETr, D. W. (1955). Studies on the fine structure of the mammalian testis. 1. Differentiation of the spermatids in the cat (Felis domestica). Journal of Biophysical and Biochemical Cytology 1, 287-300. BuRGos, M. H. & FAWCETT, D. W. (1956). An electron microscope study of spermatid differentiation in the toad Bufo aerenarum, Hensel. Journal of Biophysical and Biochemical Cytology 2, 223-240. CLARK, A. W. (1967). Some aspects of spermiogenesis in a lizard. American Journal of Anatomy 121, 369-400. CLERMONT, Y., EINBERG, E., LEBLOND, C. P. & WAGNER,S. (1955). The perforatorium. An extension of the nuclear membrane in the rat spermatozoon. Anatomical Record 121, 1-12. FAwcETr, D. W. & PHILLIPS, D. M. (1967). Further observations on mammalian spermiogenesis. Journal of Cell Biology 35, 152A. GARDNER, P. J. (1966). Fine structure of the seminiferous tubule of the Swiss mouse. The spermatid. Anatomical Record 155, 235-249.
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GRIGG, G. W. & HODGE, A. J. (1949). Electron microscopic studies of spermatozoa. 1. The morphology of the spermatozoon of the common domestic fowl (Gallus domesticus). Australian Journal ofScientific Research Series B 2, 271-279. GUNAWARDANA, V. K. (1973). The microscopic structure of the reproductive system of the male domestic fowl, with some observations on the effects of sex hormones and vitamins A and E. Ph.D. thesis, University of London. GUNAWARDANA, V. K. (1976). Stages of spermatids in the domestic fowl - a light microscope study using Araldite sections. Journal ofAnatomy 123, 351-360. KESSEL, R. G. (1963). Electron microscope studies on the origin of annulate lamellae in oocytes of Necturus. Journal of Cell Biology 19, 391-414. LAKE, P. E. (1956). The structure of the germinal epithelium of the fowl testis with special reference to the presence of multinuclear cells. Quarterly Journal of Microscopical Science 97, 487-497. LAKE, P. E., SMITH, W. & YOUNG, D. (1968). The ultrastructure of the ejaculated fowl spermatozoon. Quarterly Journal of Experim?ntal Physiology 53, 356-366. LEBLOND, C. P., STEINBERGER, E. & ROOSEN-RUNGE, E. C. (1963). Spermatogenesis. In Mechanisms Concerned with Conception. (ed. C. G. Hartmann). Oxford: Pergamon Press. McINTOsH, J. R. & PORTER, K. R. (1967). Microtubules in the spermatids of the domestic fowl. Journal of Cell Biology 35, 153-1 75. NAGANO, T. (1962). Observations on the fine structure of the developing spermatids in the domestic chicken. Journal of Cell Biology 14, 193-205. OKAMURA, F. & NISHIYAMA, H. (1976). The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl, Gallus gallus. Cell Tissue Research 169, 345-359. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208-212. SAPSFORD, C. S., RAE, C. A. & CLELAND, K. W. (1967). Ultrastructural studies on spermatids and Sertoli cells during early spermiogenesis in the bandicoot Perameles nasuta Geoffroy (Marsupialia). Australian Journal ofZoology 15, 881-909. SOTELO, J. R. & TRUJILLO-CENOZ, 0. (1958). Electron microscope study of the kinetic apparatus in animal sperm cells. Zeitschriftfiir Zellforschung und mikroskopische Anatomie 48, 565-601. SUD, B. N. (1961 a). The chromatoid body in spermiogenesis. Quarterly Journal of Microscopical Science 102, 273-292. SUD, B. N. (1961 b). Morphological and histochemical studies of the chromatoid body and related elements in the spermatogenesis of the rat. Quarterly Journal ofMicroscopical Science 102, 495-505. Susi, F. R. & CLERMONT, Y. (1970). Fine structural modifications of the rat chromatoid body during spermiogenesis. American Journal of Anatomy 129, 177-191. SwIFr, A. (1956). The fine structure of annulate lamellae. Journal of Biophysical and Biochemical Cytology 2, II Supplement, 415-418. TINGARI, M. D. (1973). Observations on the fine structure of spermatozoa in the testis and excurrent ducts of the male fowl, Gallus domesticus. Journal of Reproduction and Fertility 34, 255-265. VAUGHN, J. C. (1966). The relationship of the 'sphere chromatophile' to the fate of displaced histones following histone transition in rat spermiogenesis. Journal of Cell Biology 31, 257-278. ZETTERQVIST, H. (1956). Cited in Techniques for Electron Microscopy. (1961) (ed. D. Kay). Oxford: Blackwell. ZLOTNIK, I. (1947). The cytoplasmic components of germ cells during spermatogenesis in the domestic fowl. Quarterly Journal of Microscopical Science 88, 353-365.
J. Anat. (1977), 124, 3, pp. 741-755 With 14 figures Printed in Great Britain
Ultrastructural studies on the differentiation of spermatids in the domestic fowl V. K. GUNAWARDANA* AND M. G. A. D. SCOTT Department of Anatomy, Royal Veterinary College, London
(Accepted 10 November 1976) INTRODUCTION
Studies on the ultrastructure of spermatids and spermatozoa of the domestic fowl are relatively few when compared to similar studies in mammals. The available reports include those of Nagano (1962), McIntosh & Porter (1967), Lake, Smith & Young (1968) and Tingari (1973). While Nagano in his studies focused attention mainly oni the development of the acrosome and the arrangement of the centrioles in spermatids, the studies of McIntosh & Porter were confined to the microtubular systems and their functional significance. The other two reports deal with the ultrastructure of spermatozoa. Lake, Smith & Young investigated the ultrastructure of ejaculated fowl spermatozoa. Tingari reported the changes in fine structure during the passage of spermatozoa through the excurrent ducts. Thus it is apparent that a comprehensive study of spermiogenesis in this species at an ultrastructural level is lacking. The present investigation was undertaken to repair this deficiency. The observations recorded here establish a sequence of changes which occurs in a spermatid differentiating into a spermatozoon. MATERIALS AND METHODS
Sexually mature White Leghorns were used. The birds were killed with an overdose of pentobarbitone sodium (Nembutal - Abbott Laboratories). Pieces of testis were removed soon after death and immersed in fixative. Two methods of fixation were employed. In the first, tissues were fixed directly in buffered osmium tetroxide according to the method of Zetterqvist (1956). In the second, tissues were fixed overnight in 3 %0 glutaraldehyde in phosphate buffer at pH 7 4, and subsequently postfixed in 1 %0 osmium tetroxide, also in phosphate buffer, for 2 hours. In all instances fixation was carried out at 4 'C. After fixation by either method the tissues were dehydrated in a graded series of methyl alcohol, cleared in propylene oxide and embedded in Araldite. Sections were cut on a Porter Blum ultratome using glass knives. Sections for light microscopy were stained with 1 % methylene blue in 1 0 borax. Ultrathin sections of selected areas were stained with lead citrate (Reynolds, 1963) and examined using a RCA EMU-3F electron microscope operating at 50 kW. * Present address: Department of Veterinary Pre-Clinical Studies, School of Veterinary Science University of Sri Lanka, Peradeniya Sri Lanka.
742
V. K. GUNAWARDANA AND M. G. A. D. SCOTT RESULTS
Based on their nuclear structure, spermatids could be assigned to one of the following four phases: (I) Spermatids with round nuclei. (II) Spermatids with irregular nuclei. (III) Spermatids with long nuclei containing coarse chromatin granules. (IV) Spermatids with long homogeneously dense nuclei. Phase I spermatids possess one or more membrane-bound proacrosome granules in the region of the Golgi complex (Fig. 1). The latter appears in the form of a stack of cisternae, interspersed with vesicles (Figs. 1, 2). Figure 2 illustrates the formation of the tail in this phase. The proximal and distal centrioles comprising the centriolar complex are seen in transverse and sagittal section respectively. The proximal centriole is located about half way between the cell membrane and the nuclear membrane, while the distal centriole extends from this point to the cell membrane, at which point the tail filament originates. The cell membrane is reflected on to and encloses the developing tail. A dense transverse slightly convex plate, with the convexity on the centriolar aspect, separates the centriole from the tail. The peripheral fibres of the latter structure are continuous with the wall of the distal centriole, but the central pair of fibrils seem to commence at the transverse plate. Dense deposits and clusters of vesicles are seen close to the proximal centriole. The nuclei of phase IL spermatids show varying shapes corresponding to different degrees of nuclear elongation (Figs. 3-7). The chromatin is finely granular and of uniform density. The nucleoli are either compact (Figs. 3, 4), or they are diffuse and apparently disappearing (Figs. 5, 6). The proacrosome is lodged in a concavity of the nuclear membrane which is thickened over the area of contact (Figs. 3, 4). Closely associated with the developing acrosome is a dense droplet lying within an invagination of the nuclear membrane (Fig. 6). Later, at a time when the nucleus is more elongated, the acrosome is flattened on either side on the end of the nucleus forming a crescentic structure (Fig. 7). The cavity formed by the invagination of the nuclear membrane and the dense droplet within it are enlarged. The proximal centriole is also lodged in a concavity of the nucleus, while the junction of the distal centriole with the tail filament is in a position at the base of an invagination of the cell membrane (Figs. 4, 5, 9). Dense material is deposited in this region, which is referred to as the annulus. Microtubules occur in the cytoplasm of spermatids in both phases. In phase I and early phase II these occur as short lengths near the proximal centriole (Fig. 2). Tangential sections of the more elongated nuclei show microtubules disposed in an oblique direction, seemingly parallel to one another (Fig. 7). The mitochondria have rounded profiles and show pronounced cristae. The elongated nuclei of phase III spermatids contain coarse granules. At the acrosomal end of the nucleus is a U-shaped cavity; lying within this cavity is a rodlike structure, the perforatorium (Fig. 8). A salient feature of the spermatids in this group is the presence of microtubules arranged in a regular pattern. Some spermatids cut in a longitudinal plane show cross sections of microtubules arranged in two rows on either side of the nucleus. They are seen to begin near the cell membrane caudal to the acrosome and to continue beyond the centrioles towards the tail (Figs. 8, 9). On transverse section these microtubules have the appearance of being concentrically arranged (Fig. 10). In others, where the coarse granular material of the nucleus is
Spermatid differentiationi in the domestic fowl X.?U).
A
,
t
743
a
Fig. 1. Phase I spermatid showing a proacrosome granule (PA) near the Golgi complex (GC). MVB, multivesicular bodies. Phase III spermatids are seen below.
2 Fig. 2. Phase I spermatids showing the beginning of tail formation. An abundance of vesicles is seen near the proximal centriole (PC). Note the proximity of the microtubules (MT1) to these vesicles (arrows). AL, annulate lamellae.
744
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
Fig. 3. Bi-nucleate spermatid in early phase II. A peripheral vacuole of a membrane body (MB) is continuous with the annulate lamellae (AL). Note the compact nucleolus (NU).
4
"
Fig. 4. Phase II spermatid. The nuclear membrane is thickened near the acrosome (A). Some vacuoles of the membrane body (MB) contain dense material. AL, annulate lamellae; CC, cytoplasmic canal; NU, nucleolus.
Spermatid differentiation in the domestic fowl
745
Fig. 5. Phase II spermatid. The proximal centriole (PC) is within a nuclear concavity. Arrow indicates the beginning of the cytoplasmic canal near the annulus (AN). A granular satellite (S) is seen adjacent to the membrane bodv (MB). DC, distal centriole; NU, nucleolus.
6 ~ ' ,0. x ~ Fig. 6. Phase II spermatid. The dense droplet (P) appears to originate from the nuclear membrane. MB, membrane body; NU, nucleolus.
746
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
Ti~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Fig. 7. Phase II spermatid which is older than that of Fig. 6. Note the crescentic acrosome (A) and enlarged dense droplet (P), which would give rise to the perforatorium. MT1, microtubules; AL, annulate lamellae.
'UM Vj .t #
.*
-i w-4 aq
'k
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Fig. 8. Phase III spermatid with rod shaped perforatorium (P). Note the regular arrangement of the microtubules (MT1). A, acrosome.
Spermatid differentiation in the domestic fowl J
747
.i;
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)
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_
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Fig. 9. Phase III spermatid. Coarse granules are abundant in the nucleus. Microtubules (MT1) extend beyond the distal centriole. Arrow indicates dense material near the annulus (AN). CC, cytoplasmic canal.
P
,.
. ..
l'' Fig. 10. Phase III spermatids sectioned transversely. The microtubules (MT1) appear concentrically arranged. The spermatid at top left is sectioned at its anterior end and shows the perforatorium (P) centrally, acrosome (A) peripherally and the nucleus (N) in between.
748
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
4;.p. -
_
.-
A
.
Fig. 11. Phase III spermatids sectioned transversely. Note the distinctive pattern of the microtubules (MT2) forming the manchette. Arrows indicate dense ring-like structures. The spermatid at bottom left is in phase I. GC, Golgi complex; MVB, multivesicular body; PA, proacrosome.
more in evidence, the arrangement of the microtubules is completely different. They are distributed as longitudinal straight tubules extending from the anterior extremity of the nucleus to the end piece of the tail, forming a complete wall which encircles these structures (Fig. 11). At lower levels in the tail, gaps are noticeable in this wall. In rare instances where both sets are present, the longitudinal microtubules are placed internal to those which are concentrically arranged (Fig. 12). The mitochondria in most instances are oval in outline and are seen external to the microtubules. In the main piece of the tail a dense layer surrounds the nine doublets. Dense ring-like structures, and vacuoles of varying shape and size, are present in the cytoplasm. The nuclei and axial filament complexes in some of the spermatids in phase IV are seen to be surrounded by the wall of longitudinally arranged microtubules (Figs. 12, 13). These however do not exhibit the distinctive scalloped pattern seen previously. The tubules are crowded together, their walls are thicker, and they seem to be coalescing (Fig. 12). In other spermatids of this phase the microtubules are absent (Fig. 14). The distal centriole appears to be of a shorter length when compared to the earlier phases. The annulus is at the junction of the mid piece with the main piece, and the dense material of the annulus is continuous with the dense material surrounding the main piece. Mitochondria with longitudinal cristae are located at the posterior pole of the cell and in some spermatids they are in a position around the mid piece. The entire spermatid is slightly curved. During spermiogenesis there is a gradual reduction in cytoplasm, the acrosome region being devoid of cytoplasm by phase II. Clusters of multivesicular bodies are
Spermatid differentiation in the domestic fowl
MT2 ~~
~
~
~
749
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Fig. 12. Phase IV spermatids sectioned transversely. The microtubules (MT2) appear to be coalescing. The spermatid at top right is sectioned through the centriole and shows both sets of microtubules (MT1 and MT2). IB, intercellular bridge; MB, membrane body.
always seen in all phases of spermiogenesis. Two or more narrow cisternae lying parallel to one another, and showing intermittent constrictions, are interpreted as annulate lamellae (Figs. 2, 3, 4, 7). Some such cisternae are observed in close apposition to the nuclear membrane (Fig. 2). A unique structure, referred to as a membrane body, is seen consistently in phase II and less frequently in other phases. It is located in the caudal half of the cell and consists of a central spherical vacuole surrounded by radially arranged peripheral vacuoles leaf-like in shape (Figs. 3-6, 12). In some micrographs one of the peripheral vacuoles is seen to be in continuity with the cisternae of the annulate lamellae (Fig. 3). Varying amounts of dense material are seen within the vacuoles. An aggregate of granular material is at times observed in close proximity to the membrane body (Fig. 5). Multinuclear spermatids, and spermatids connected by intercellular bridges, occur frequently in the testes of the domestic fowl. DISCUSSION
The observations presented here elucidate the fine structural changes undergone by spermatids during their differentiation. The classification of spermatids into four phases was based on nuclear changes. When these changes are considered along with the degree of development of the acrosome and tail filament, it seems evident that these are successive phases of development. Thus the formation of the acrosome and tail, which began in phase I, had progressed further in phase II. 48
ANA I24
750
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
13 Fig. 13. Phase IV spermatid3 sectioned longitudinally. Microtubules (MT2) acrosom.; P, perforatorium; RB, residual body.
are
present. A,
Fig. 14. Phase IV spermatid showing mitochondria (M) in position around the mid piece. Note the length of the distal centriole (DC) and the location of the annulus (AN). Note also the curvature of the spermatid.
Spermatid differentiation in the domestic fowl
751 In many mammalian species a proacrosome granule arises within a vesicle which subsequently gives rise to the head cap (Burgos & Fawcett, 1955; Gardner, 1966). In the toad a large vesicle is present, but a proacrosome granule is not formed (Burgos & Fawcett, 1956). Acrosome formation in the fowl is distinct from both these. The proacrosome granule closely invested by its membrane attaches itself to the nuclear membrane by phase II and spreads over the anterior pole of the nucleus to form a crescentic structure which is termed the acrosome from then onwards (see Fig. 7). There is no acrosome vesicle, and hence a head cap comparable to that of mammalian spermatids is not formed. Grigg & Hodge (1949) described the acrosome of the spermatozoon as consisting of an apical cap and an apical spine. Lake et al. (1968) and Tingari (1973) referred to these structures as the acrosome cap and acrosome spine respectively. Tingari states that the acrosome cap loosely fits the nucleus during spermiogenesis, but is more closely applied to the nucleus in spermatozoa lying free in the lumina of the seminiferous tubules. In the present study the acrosome (which is the equivalent of the acrosome cap in Tingari's study) was seen to be closely applied to the nucleus throughout the process of spermiogenesis. It is thought likely that the looseness of the acrosome encountered in Tingari's investigation is a consequence of the fixation procedure adopted. Lake et al. (1968) considered the acrosome spine to be the equivalent of the perforatorium in the rat. Brokelmann (1963) regarded the perforatorium and the acrosome as identical structures, and compared a ventral thickening of the acrosome cap in the rat to a dense droplet described by Nagano (1962) in the rooster. Bedford (1967) thought that the subacrosome space in mammals was synonymous with the perforatorium. The rod-shaped perforatorium in this study lies within the space bounded by the inner membrane of the acrosome cap and the nuclear surface, and extends caudally between these structures. Thus the perforatorium in the fowl is located in a position identical to that described for mammals by Bedford (1967). The perforatorium is believed to arise from the dense droplet referred to earlier, thereby confirming the results of Nagano (1962). Clermont et al. (1955) described the perforatorium as an extension of the nuclear membrane in the rat. The present findings reveal that the perforatorium is attached to the nuclear membrane of spermatids. This is contrary to the observations of Tingari (1973) in spermatozoa. It is likely that this attachment is loosened during the final phase of spermiogenesis. Again, the possibility of fixation artifact cannot be totally disregarded when comparing and interpreting these observations. The arrangement of the centrioles and the axial filament complex in general agrees with that of Nagano (1962). However, he reported that the distal centriole increases in length with increasing age of spermatids. Lake et al. (1968), in their study of fowl spermatozoa, described a short neck region containing a complex centriolar structure. The results of this investigation reveal that during the final phase of spermiogenesis there is a reduction in the length of the distal centriole. Nagano, in his study, did not record any spermatid stage showing a homogeneous dense nucleus, as was seen in the final phase in the present study. The point of reflexion of the plasma membrane on to the tail filament lies at the distal extremity of the distal centriole. This point, which is referred to as the annulus, is drawn into the cytoplasm with the movement of the centriolar complex towards the nucleus, thereby forming a cytoplasmic canal. In rat spermatids a cytoplasmic canal arises in an identical manner (Sotelo & Trujillo-Cenoz, 1958). The cytoplasmic canal is obliterated late in spermiogenesis when the annulus, together with the dense 48-2
V. K. GUNAWARDANA AND M. G. A. D. SCOTT 752 material associated with it, migrates down the axial filament complex. A similar process is reported in other species, and Sapsford, Rae & Cleland (1967) excluded the annulus from the neck region of the bandicoot spermatids for this reason. Microtubules form very early in spermiogenesis, and they appear to originate from the cluster of vesicles seen in the vicinity of the proximal centriole. During the course of spermiogenesis two sets of microtubules are formed. The first set has been shown by McIntosh & Porter (1967) to be helically arranged. This arrangement became evident in late phase II and persisted during the early part of phase III. In late phase III the helix had been replaced by the longitudinal array of microtubules which corresponds to the manchette or caudal sheath of light microscopy. The presence of microtubules showing two distinctive patterns of distribution has been reported in reptiles (Clark, 1967). This author proposed that the helix unwinds as the nucleus elongates, resulting in the longitudinally orientated microtubules. It is thought unlikely that such a phenomenon occurs in the fowl since spermatids possessing both sets of microtubules at the same time were encountered, although
rarely. Nagano (1962), in his study, does not refer to the presence of helically arranged microtubules. McIntosh & Porter (1967) attribute this to the method of fixation employed by Nagano, and argue that temperature is a factor which determines the stability of microtubules. However, in the present investigation the helix was preserved when material was fixed in cold osmium tetroxide alone, which is one method used by Nagano. The manchette or caudal sheath has been described by this author, but he states that it was not always present in late spermatids. According to his interpretation these are cells with coarse granular nuclei. On the contrary, from the present results, it is clear that the manchette is visible even in spermatids with homogeneously dense nuclei. Nagano associated the manchette with elongation of spermatids. In a study of spermatids with the light microscope, Gunawardana (1973, 1976) reported that maximum length was attained at stage 8, which is prior to the formation of the manchette. This stage of spermatid, when examined under the electron microscope, falls into the category of early phase III, where the helical microtubules are clearly developed (Gunawardana, 1973). These observations tend to support the view of McIntosh & Porter (1967) that the helix is concerned with nuclear elongation. The same workers suggested that the manchette determines the final curvature of the nucleus. While agreeing with this suggestion, it should be pointed out that the curvature involves not only the nucleus but the axial filament complex as well (see Fig. 14). This could be explained by the relatively long extent of the manchette as far caudal as the end piece of the spermatids. In contrast, most species show the manchette ending just caudal to the distal centriole (Clark, 1967; Sapsford et al. 1967). A structure referred to as a chromatoid body has been recognized in many species (Sud, 1961 a). In the domestic fowl this structure does not seem to have received much attention, except for a reference to an accessory body by Zlotnik (1947). Burgos & Fawcett (1955) described the chromatoid body in the cat as an irregular mass of closely aggregated osmiophilic granules. Sapsford et al. (1967) thought it possible that dense ring-like bodies seen in bandicoot spermatids were derived from an aggregate of granular material which bears some resemblance to the chromatoid body. More recently, Susi & Clermont (1970), in a detailed study of this structure in the rat, described it as being composed of dense osmiophilic material and vacuoles. They stated, further, that during later stages the chromatoid body appeared as a
Spermatid differentiation in the domestic fowl
753
dense sphere surrounded by radially arranged vesicles. No single structure conforming to any of the above descriptions was seen regularly enough to warrant its identification as the chromatoid body in the domestic fowl. The one structure which could be equated with the chromatoid body, and which appeared regularly during the course of spermiogenesis, was the structure referred to in this study as the 'membrane body'. This structure, in comparison to the chromatoid body of the rat (Susi & Clermont, 1970), consists of a central spherical vacuole containing dense material instead of a dense sphere. Radially arranged vesicles are similar to those of the rat. Sud (1961 b) observed a granular satellite accompanying the chromatoid body in the rat. In guinea-pig and chinchilla germ cells a satellite is shown to be intimately related to the chromatoid body. It is possible that the granular aggregate seen in phase II spermatids represents a satellite. Dense rings seen in later stages (Fig. 11) are comparable to those described in bandicoot spermatids (Sapsford et al. 1967). Fawcett & Phillips (1967) suggested a nuclear origin for the chromatoid body in certain mammalian species. In the species investigated here (the fowl) the membrane body appears to arise from the annulate lamellae. In the oocytes of Necturus, annulate lamellae are said to be formed by the budding of vesicles from the nuclear membrane (Kessel, 1963). The proximity of some annulate lamellae to the nuclear membrane of spermatids in the fowl suggests a similar mode of origin in this species. Swift (1956) postulated that annulate lamellae may function in the transfer of specific material from the nucleus to the cytoplasm. Taking the above views into consideration, it is proposed that during the stages ofnuclear condensation, when considerable shrinkage of nuclear volume occurs, there is a loss of excessive nuclear material which is transferred to the chromatoid body (membrane body) via the annulate lamellae. Vaughn (1966), using autoradiographic and microspectrophotometric methods, demonstrated that basic protein lost from the nucleus was present in the 'sphere chromatophil' of the rat spermatid. Histochemical techniques at an ultrastructural level, together with the above techniques, may prove useful in confirming the validity of the suggested proposal. With regard to the ultimate fate of the chromatoid body (membrane body), if its presence is merely to serve as a reservoir for excessive nuclear material, it is very likely that it is lost in the residual cytoplasm. The presence of multinuclear spermatids may be related to the intercellular bridges frequently encountered. Leblond, Steinberger & Roosen-Runge (1963) noted that, in pathological conditions, cells connected by intercellular bridges tended to fuse to form multinuclear cells. Bryan (1971) is of the opinion that cells produced as a result of coalescence of conjoined cells are normal, and lead to the production of normal spermatozoa. The occurrence of binuclear cells with an acrosome differentiating in relation to each nucleus is comparable to the situation in the cat (Burgos & Fawcett, 1955). The frequency with which such cells were seen in apparently normal testes suggests that they might very well eventually produce normal spermatozoa. Lake (1956), who was of similar mind, considered multinuclear spermatids to be produced as a result of post-meiotic multiplication which would eventually produce clusters of spermatozoa. SUMMARY
Four phases of spermatid maturation are recognized on the basis of nuclear morphology. The formation of the acrosome, perforatorium and axial filament complex are described in relation to these phases. The functional significance of the microtubular systems in nuclear elongation and spermatid curvature are discussed.
754
V. K. GUNAWARDANA AND M. G. A. D. SCOTT
A membranous structure, hitherto unidentified, and referred to in this study as the 'membrane body' is compared with the chromatoid body. Morphological evidence suggests that this structure, together with the annulate lamellae, removes excessive nuclear material. ADDENDUM
The development of the tail and the transformation of the shape of the nucleus of the domestic fowl has been investigated recently (Okamura & Nishiyama, 1976). With reference to the development of the tail the authors suggest that the plasma membrane of the flagellum is derived from the Golgi vesicles. The present findings favour the view that the membrane of the flagellum is a reflexion of the cell membrane. Our results are in agreement with those of Okamura & Nishiyama regarding the presence of two sets of microtubules. However, a few points of difference were noted. It has been reported that the formation of the first set (circular manchette) begins rostrally and proceeds caudally. From our observations the reverse seems to be happening (see Fig. 7). Microtubules are first seen in the region of the centrioles. Therefore it is even more reasonable to suppose that the circular manchette starts to form at the caudal end of the nucleus. The appearance of the longitudinal manchette of Okamura & Nishiyama was said to be coincident with the beginning of nuclear condensation. In the present work nuclear condensation preceded the formation of the manchette. With respect to the abberant spermatid, the conclusions of Okamura & Nishiyama are based on the assumption that a longitudinal manchette was not formed in this spermatid. However, it is also possible that a longitudinal manchette formed but disappeared with advancing spermiogenesis. Furthermore, one cannot be certain whether or not this particular spermatid produced a circular manchette in the first instance. In which case, the abnormality in shape may just as well be attributed to the absence of a circular manchette. In fact, the results of McIntosh & Porter (1967) show abnormalities in nuclear shape which are due to faulty orientation of the first set of microtubules or circular manchette. Finally, the spermatid under discussion could have resulted from the absence of both sets of microtubules. REFERENCES BEDFORD, J. M. (1967). Observations on the fine structure of spermatozoa of the Bush Baby (Galago senegalensis), the AfricanGreen Monkey (Cercopithecus aethiops) and man. American JournalofAnatomy 121, 443-460. BROKELMANN, J. (1963). Fine structure of germ cells and Sertoli cells during the cycle of the seminiferous epithelium in the rat. Zeitschriftfiir Zellforschung und mikroskopische Anatomie 59, 820-850. BRYAN, 1. H. D. (1971). On the presence of multinuclear spermatogenic cells in the seminiferous epithelium of the mouse. Zeitschrift fir Zellforschung und mikroskopische Anatomie 112, 333-349. BuRGos, M. H. & FAWCETr, D. W. (1955). Studies on the fine structure of the mammalian testis. 1. Differentiation of the spermatids in the cat (Felis domestica). Journal of Biophysical and Biochemical Cytology 1, 287-300. BuRGos, M. H. & FAWCETT, D. W. (1956). An electron microscope study of spermatid differentiation in the toad Bufo aerenarum, Hensel. Journal of Biophysical and Biochemical Cytology 2, 223-240. CLARK, A. W. (1967). Some aspects of spermiogenesis in a lizard. American Journal of Anatomy 121, 369-400. CLERMONT, Y., EINBERG, E., LEBLOND, C. P. & WAGNER,S. (1955). The perforatorium. An extension of the nuclear membrane in the rat spermatozoon. Anatomical Record 121, 1-12. FAwcETr, D. W. & PHILLIPS, D. M. (1967). Further observations on mammalian spermiogenesis. Journal of Cell Biology 35, 152A. GARDNER, P. J. (1966). Fine structure of the seminiferous tubule of the Swiss mouse. The spermatid. Anatomical Record 155, 235-249.
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GRIGG, G. W. & HODGE, A. J. (1949). Electron microscopic studies of spermatozoa. 1. The morphology of the spermatozoon of the common domestic fowl (Gallus domesticus). Australian Journal ofScientific Research Series B 2, 271-279. GUNAWARDANA, V. K. (1973). The microscopic structure of the reproductive system of the male domestic fowl, with some observations on the effects of sex hormones and vitamins A and E. Ph.D. thesis, University of London. GUNAWARDANA, V. K. (1976). Stages of spermatids in the domestic fowl - a light microscope study using Araldite sections. Journal ofAnatomy 123, 351-360. KESSEL, R. G. (1963). Electron microscope studies on the origin of annulate lamellae in oocytes of Necturus. Journal of Cell Biology 19, 391-414. LAKE, P. E. (1956). The structure of the germinal epithelium of the fowl testis with special reference to the presence of multinuclear cells. Quarterly Journal of Microscopical Science 97, 487-497. LAKE, P. E., SMITH, W. & YOUNG, D. (1968). The ultrastructure of the ejaculated fowl spermatozoon. Quarterly Journal of Experim?ntal Physiology 53, 356-366. LEBLOND, C. P., STEINBERGER, E. & ROOSEN-RUNGE, E. C. (1963). Spermatogenesis. In Mechanisms Concerned with Conception. (ed. C. G. Hartmann). Oxford: Pergamon Press. McINTOsH, J. R. & PORTER, K. R. (1967). Microtubules in the spermatids of the domestic fowl. Journal of Cell Biology 35, 153-1 75. NAGANO, T. (1962). Observations on the fine structure of the developing spermatids in the domestic chicken. Journal of Cell Biology 14, 193-205. OKAMURA, F. & NISHIYAMA, H. (1976). The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl, Gallus gallus. Cell Tissue Research 169, 345-359. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208-212. SAPSFORD, C. S., RAE, C. A. & CLELAND, K. W. (1967). Ultrastructural studies on spermatids and Sertoli cells during early spermiogenesis in the bandicoot Perameles nasuta Geoffroy (Marsupialia). Australian Journal ofZoology 15, 881-909. SOTELO, J. R. & TRUJILLO-CENOZ, 0. (1958). Electron microscope study of the kinetic apparatus in animal sperm cells. Zeitschriftfiir Zellforschung und mikroskopische Anatomie 48, 565-601. SUD, B. N. (1961 a). The chromatoid body in spermiogenesis. Quarterly Journal of Microscopical Science 102, 273-292. SUD, B. N. (1961 b). Morphological and histochemical studies of the chromatoid body and related elements in the spermatogenesis of the rat. Quarterly Journal ofMicroscopical Science 102, 495-505. Susi, F. R. & CLERMONT, Y. (1970). Fine structural modifications of the rat chromatoid body during spermiogenesis. American Journal of Anatomy 129, 177-191. SwIFr, A. (1956). The fine structure of annulate lamellae. Journal of Biophysical and Biochemical Cytology 2, II Supplement, 415-418. TINGARI, M. D. (1973). Observations on the fine structure of spermatozoa in the testis and excurrent ducts of the male fowl, Gallus domesticus. Journal of Reproduction and Fertility 34, 255-265. VAUGHN, J. C. (1966). The relationship of the 'sphere chromatophile' to the fate of displaced histones following histone transition in rat spermiogenesis. Journal of Cell Biology 31, 257-278. ZETTERQVIST, H. (1956). Cited in Techniques for Electron Microscopy. (1961) (ed. D. Kay). Oxford: Blackwell. ZLOTNIK, I. (1947). The cytoplasmic components of germ cells during spermatogenesis in the domestic fowl. Quarterly Journal of Microscopical Science 88, 353-365.
