Observations on the Fine Structure of Spermatozoa in the Testis and Excurrent Ducts of the Male Fowl, Gallus Domesticus

OBSERVATIONS ON THE FINE STRUCTURE OF SPERMATOZOA IN THE TESTIS AND EXCURRENT DUCTS OF THE MALE FOWL, GALLUS DOMESTICUS

M. D. TINGARI A.R.C. Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh EH9 3JS (Received 30th June 1972)

Summary. Ultrastructural observations were made on fowl spermatids and free spermatozoa, fixed in situ, in the lumina of the seminiferous tubules, the rete testis, the ductuli efferentes, the connecting ductules, the ductus epididymidis and different levels of the ductus deferens. Changes occurred in the acrosome during the differentiation of the spermatid. The early ellipsoidal form of the acrosome attains a mature, elongated shape at the late spermatid stage. During this stage, remodelling and condensation of chromatin of the nuclear region also occurs. The acrosomal cap changes from a loosely fitting structure before spermiation to become relatively closely apposed to the sperm nucleus in the excurrent ducts. The plasma membrane overlying the acrosomal cap of the free spermatozoon is closely applied whilst that over the head is slightly loose and swollen, especially in the ductus epididymidis. The inner mitochondrial membrane becomes thicker when the spermatozoon reaches the rete testis. A cytoplasmic droplet is not seen in the middle piece of the fowl spermatozoon. It is concluded that the structural differentiation of the fowl spermatozoon is almost complete directly after spermiation, and is earlier than the achievement of the fertilizing capacity which is reported to occur in the ductus epididymidis. There is no obvious correlation at an ultrastructural level between the morphological and the functional maturation of the fowl spermatozoon.

INTRODUCTION A brief account of some aspects of the fine structure of partially disrupted ejaculated fowl spermatozoa was given by Grigg & Hodge (1949). Attention was focused chiefly on the kinetic apparatus of the tail (mid-piece and main- piece). Later, Nagano (1962) studied some features of the development of the spermatid with only a brief reference to the acrosome and nucleus. Mclntosh & * Present address: Department of Anatomy, Faculty of Veterinary Science, University of Khartoum, P.O. Box 32, Khartoum North, Sudan. 255 Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access 256 M. D. Tingan Porter (1967) discussed developmental changes in the shape of the head of the spermatozoon in relation to the presence of microtubules in the spermatid and Nicander & Hellström (1967) reported an increase in the thickness of the inner membrane of the mitochondria during the maturation of the spermatozoon. A full account of the normal ultrastructure of ejaculated fowl spermatozoa was given by Lake, Smith & Young (1968). Changes in the fine structure of the fowl spermatozoon during passage through the excurrent ducts have not hitherto been studied and this was the purpose of the present work. Similar extensive studies on mammalian sperma¬ tozoa (Bedford, 1963, 1965; Fawcett & Hollenberg, 1963; Dickey, 1966; Fawcett & Phillips, 1969a; Bedford & Nicander, 1971) have indicated that cer¬ tain changes occur in the shape and size of the acrosome and in the closeness of fit of the overlying plasma membrane during passage through the epididymis. A well-known observation in the mammalian spermatozoon is the movement of the cytoplasmic (kinoplasmic) droplet from the neck region to the caudal part of the mid-piece as the sperm-cell progresses from the caput to the cauda epi¬ didymidis. These changes are completed in the epididymis and are considered to be associated with a progressive increase in the fertilizing capacity of the spermatozoa. Lake & El Jack (1966) did not see a cytoplasmic droplet in the fowl spermatozoa when they were examined as whole mounts with the electron microscope.

MATERIALS AND METHODS Fifteen Brown and White Leghorn cocks of proven fertility were killed on separate occasions by dislocation of the neck vertebrae or by the administration of an overdose of Nembutal (Abbott Laboratories Ltd). The abdominal cavity was immediately opened and most of the organs were removed, leaving the reproductive tract exposed in situ. The cavity was filled with an ice-cold fixative solution containing 4% formaldehyde and 0-8% glutaraldehyde in phosphate buffer (Millonig, 1962) at pH 7-4. The reproductive tract was dissected out after a few minutes and small pieces of the testis, epididymal region and upper, middle and lower parts of the ductus deferens (Tingari, 1971) were transferred to fresh fixative for about 30 min. The tissues were then osmicated for 1 hr in 1% osmium tetroxide in phosphate buffer. Occasionally, the reproductive tract was dissected out immediately after killing, and small samples of tissue were transferred to Millonig's phosphate-buffered osmium tetroxide fixative (Glauert, 1967) for 2 hr. After fixation, the blocks were dehydrated in ethanol, cleared in propylene oxide and embedded in Araldite according to the schedule described by Maxwell & Trejo (1970). Thick sections were cut with glass knives and stained with toluidine blue. Such sections from the epididymal region were used to identify the various types of tubules in this region, namely, rete testis, ductuli efferentes, connecting ductules and ductus epididymidis (Tingari, 1971). Those tubules which contained an appreciable number of spermatozoa, and blocks from the testis and ductus deferens, were cut with a diamond knife. Thin sec¬ tions were mounted on uncoated copper grids, stained with alcoholic uranyl

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access Ultrastructural changes andfowl sperm maturation 257 acetate (Stempak & Ward, 1964) and lead citrate (Reynolds, 1963) and examined with an EM 6B or Philips EM 300 electron microscope.

RESULTS Acrosome The acrosomal cap is elliptical in shape (PL 1, Fig. 1) at the beginning of its formation within the cisternae of the spermatid Golgi complex. It is composed of electron-lucent homogeneous material. It becomes attached to the nuclear membrane of the early spermatid and during subsequent elongation of the nucleus in spermateliosis, the acrosome gradually becomes crescent shaped. With further development, it attains a U-shape (PL 1, Fig. 2) and elongates rostrally in late spermatids (PL 1, Fig. 3). At this stage, the acrosome protrudes from the spermatid and its rostral tip is enclosed by Sertoli-cell processes (PL 1, Fig. 3) which permeate between the cells of the germinal epithelium. There is no further elongation of the acrosome following the release of the spermatozoon into the lumen of the seminiferous tubule or when in the excurrent ducts. The acrosomal cap appears loosely fitting to the nucleus during spermateliosis (PL 2, Fig. 4) but, in the free spermatozoon in the lumen of the seminiferous tubule, it is more closely applied (PL 2, Fig. 5). This change is accentuated when the spermatozoon is in the lumina of the excurrent ducts, particularly in the connecting ductules and ductus epididymidis (PL 2, Figs 6 and 7). The acrosomal spine arises as a small dense granule in a rostral nuclear invagination of early spermatids (Nagano, 1962) and lies close to the acrosomal cap (PL 3, Fig. 8). The spine is embedded in finely granular homogeneous material which appears to be confluent with the spermatid cytoplasm at the postacrosomal region (PL 1, Fig. 3; PL 3, Fig. 8). The spine itself does not show a uniform density, as lighter, longitudinally oriented areas are encountered along its axis (PL 2, Fig. 4). The spine shows no attachment to either the acro¬ somal cap or the head of the spermatozoon. However, granular material is present between the cap and spine and the nucleus and spine at an early stage of spermateliosis. The acrosomal cap is covered by two closely adherent membranes ; the outer one is the investing plasma membrane. The close apposition of these mem¬ branes to the underlying acrosomal cap is established early in spermateliosis (PL 1, Fig. 3) and is maintained throughout the subsequent stages of develop¬ ment.

Head The texture of the chromatin of the head changes during the formation of the spermatozoon. The chromatin of the early-stage nucleus is finely granular and evenly distributed except for a distinct nucleolus (PL 3, Fig. 8). The chromatin does not abut onto the nuclear membrane as some areas of clear nucleoplasm separate it from the wavy nuclear membrane (PL 3, Fig. 8). At this stage, the head is irregularly elongated with the attached acrosome marking its rostral end (PL 3, Fig. 8). When the slender shape characteristic of the more advanced form of the spermatozoan head is achieved, the nucleoplasm becomes packed

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access 258 M. D. Tingari EXPLANATION OF PLATES Electron micrographs of spermatids and spermatozoa from domestic fowls. PLATE 1 Fig. 1. The anläge of the acrosomal cap (C) arising within the cisternae of the Golgi complex of a spermatid. Fig. 2. The U-shaped acrosome cap (C) applied to the rostral end of the elongated nucleus in a late spermatid stage. The chromatin is clumped into small dense granules. Note the cytoplasmic microtubules (arrows) running parallel to the long axis of the nucleus. Fig. 3. Late spermatid showing rostral elongation of the acrosome cap (C) which lies ad¬ jacent to the Sertoli-cell cytoplasm (SG). The membranes overlying the cap are closely applied to it. The subacrosomal region is confluent with the postacrosomal cytoplasm (arrows). S, acrosome spine; N, nucleus. PLATE 2 Fig. 4. Acrosomal cap (C) loosely fitting over the rostral part of the nucleus (N) during the late spermatid stage. Parts of the acrosomal spine (S) are less dense than others. Fig. 5. Free spermatozoon in the lumen of a seminiferous tubule. Acrosomal cap (G) is closely applied to the nucleus (N) which displays the mature form. The plasma mem¬ brane over the acrosome shows no distortion. Figs 6 and 7. A close apposition of the acrosomal cap (C) to the nucleus (N) is noted in spermatozoa in the connecting ductules (Fig. 6) and ductus epididymidis (Fig. 7). Note the loosely fitting plasma membrane over the nuclear region especially in Fig. 7 (double arrows) ; the nuclear membrane (NM) is closely applied to the chromatin. PLATE 3 Fig. 8. Acrosomal spine (single arrow) arising in a rostral nuclear invagination close to the acrosomal cap (G). The nuclear chromatin of the early spermatid is finely granular. Note the loose nuclear membrane and the confluence between the sub- and postacroso¬ mal regions (double arrows). N, nucleus. Osmium fixation. Fig. 9. Later stage from that shown in Fig. 2. The spermatid nucleus is packed with granules. Microtubules run close to the nucleus and parallel to its long axis (arrows). Fig. 10. Later stage of spermatid development from that shown in Fig. 9 showing further condensation of the head chromatin. Note the presence of microtubules (arrows). Fig. 11. Proximal centriole (P) of a spermatid occupying a concavity in the caudal end of the nucleus (N). The distal centriole (D) lies perpendicular to it. The central pair of microtubules (M) terminates at the middle of the distal centriole. The alignment of mitochondria to constitute the mid-piece is almost complete. PLATE 4 Fig. 12. Late spermatid showing the relationship of the central pair of microtubules (M) to the distal centriole (D). Note the cylindrical form of the latter and the alignment of mitochondria to constitute the mid-piece. The plasma membrane in the tail region is wavy. Inset shows dense matrix rostral to the proximal centriole (P) being confluent with that in which the centriolar microtubules are embedded (arrows). Figs 13 and 14. Spermatozoa in the rete testis showing some coalescence between the dense masses ofthe proximal centriole. Figure 14 also illustrates the difference in the thick¬ ness of the outer and inner mitochondrial membranes (arrows). P, proximal centriole ; D, distal centriole; N, nucleus. PLATE 5 Fig. 15. Complete coalescence of the dense matrix of the proximal centriole is achieved in the ductus epididymidis. P, proximal centriole; N, nucleus. Fig. 16. Larger masses of dense matrix (DM) are deposited around the distal centriole of the spermatid stage. The plasma membrane is reflected at the caudal end of mitochon¬ drial sheath. Note the electron-dense material deposited on the inner surface of the membrane at this site (arrows). Fig. 17. Transverse section at the level of the distal centriole showing cavitation of the masses (DM) illustrated in Fig. 16. D, distal centriole; M, microtubules. Fig. 18. Cytoplasmic protrusion (CP) displayed by a spermatozoon in a ductulus eflerens. Note the absence of organdíes and inclusions. Osmium fixation.

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access PLATE 1

(Facing p. 258)

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access PLATE 2

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access PLATE 3

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access PLATE 4

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access PLATE 5

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access Ultrastructural changes andfowl sperm maturation 259 with small dense granules except for some parts which appear electron-lucent (PL 1, Fig. 2). Gradual coalescence of the granules occurs during further development (PL 3, Figs 9 and 10). Just before spermiation, this condensation of the head chromatin is almost complete. Zlotnik (1947) and Lake & Smiles (1952) report that the nuclear material contracts and shrinks during the very late stages of spermateliosis. During these stages of the formation of the head of the spermatozoon, a highly ordered system of cytoplasmic microtubules, or manchette, is observed around the developing nucleus (PL 1, Fig. 2; PL 3, Figs 9 and 10). A detailed description of these tubules is given by Mclntosh & Porter (1967) and they consider that they aid nuclear elongation and determine the curvature which is typical of the head of the mature spermatozoon. Most of the heads of the free spermatozoa found in the lumen of the seminiferous tubules (PL 2, Fig. 5) are similar to those of the mature, ejaculated cells described by Lake et al. (1968). In this respect, they resemble the spermatozoa of the rabbit and the monkey which remain morphologically unchanged during and after the process of spermiation (Bedford & Nicander, 1971). The plasma membrane covering the head of a testicular spermatozoon is little distorted, but that of spermatozoa from any part of the excurrent ducts tends to become tortuous (PL 2, Figs 6 and 7). This is more marked in epididy¬ mal spermatozoa; the nuclear membrane, on the other hand, remains straight and closely applied to the condensed chromatin of the head. Tail During early spermateliosis, the centrioles take up a position immediately caudal to the developing head. The juxtanuclear, or proximal, centriole is transversely orientated and occupies a concavity in the base of the head from which it is separated by the nuclear membrane. The distal centriole is orientated perpendicularly to the long axis of the proximal centriole. The rostral side of the proximal centriole is applied closely to the nuclear membrane (PL 3, Fig. 11). Its structure is unmodified, being composed of nine triplet microtubules embedded in an electron-dense material, with an irregu¬ larly shaped lumen (PL 3, Fig. 11; PL 4, Figs 12 and 14). Separate masses of dense material arise in the space between this centriole and the nuclear mem¬ brane and when the condensation of the head chromatin is complete, they fuse with the dense material of the centriole and with each other (PL 4, Figs 12, 13 and 14). In epididymal spermatozoa, these fused masses form the articular facet of the future capitulum, as described in mammals (Fawcett & Phillips, 1969b) ; the articular surface of the capitulum is irregular in conformity with the head invagination, the articular fossa, to which it is applied (PL 5, Fig. 15). In the excurrent ducts, the reduction in size of the central cavity of the proximal centriole may be due to increasing deposition of dense matrix. This appearance of the centriole in the neck region persists in the spermatozoa in the remaining parts of the excurrent duct system. In longitudinal sections, the distal centriole is cylindrical, similar to that of a basal body, and gives rise to the flagellum in the early spermatid stage. Its parallel walls are composed of the nine doublets of the axial filament complex

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access 260 M. D. Tingari embedded in a dense matrix (PL 4, Fig. 12). The central pair of microtubules, unlike that of the mammalian spermatozoon, appear to terminate at the middle of the distal centriole (PL 3, Fig. 11 ; PL 4, Fig. 12). No connection between the central pair of microtubules and the proximal centriole was seen at any stage of development, although, in the ejaculated spermatozoa, Lake et al. (1968) occasionally found evidence of ill-defined central structures ascending to the proximal centriole. Irregular dense masses were seen around the distal centriole in the spermatid stage but not thereafter (PL 5, Fig. 16); transverse sections revealed that they were not solid, but showed some cavitation (PL 5, Fig. 17). A cytoplasmic droplet, characteristic of mammalian spermatozoa, was never observed in connection with fowl spermatozoa but a cytoplasmic protrusion from a corresponding location to that of the droplet was observed once in a spermatozoon from a ductulus efferens (PL 5, Fig. 18). Structurally, it was bounded by the plasma membrane enclosing cytoplasm devoid ofany organdíes. Before the formation of the spermatid, the mitochondria are elongated, oval or circular with irregular or transverse internal cristae. In the developing spermatid, the mitochondria assume a slender shape with little matrix, and the cristae show a regular longitudinal orientation. When the head chromatin has condensed, the mitochondria are aligned end-to-end in the cytoplasm around the axial filaments, thus forming part of the mid-piece which begins at about the level of the proximal centriole (PL 4, Fig. 12). The mitochondria are elongated in both transverse and longitudinal sections of the mid-piece of the ejaculated fowl spermatozoon and hence Lake et al. (1968) conclude that they are in the form of rectangular plates curved along the longitudinal axes. Such a con¬ figuration of mitochondria is established by the late spermatid stage and main¬ tained in subsequent stages of the development. The inner and outer mito¬ chondrial membranes in the spermatid stage are of equal thickness. However, in spermatozoa from the rete testis and remaining parts of the duct system, there is an increase in the thickness ofthe inner membrane of the mitochondrion (PL 4, Fig. 14). The plasma membrane is reflected at the caudal end of the mitochondrial sheath indicating the distal end of the mid-piece and the beginning of the main- piece of the tail (PL 5, Fig. 16). This has been noted in the developing spermatid (Nagano, 1962) as well as in the ejaculated spermatozoon (Lake et al., 1968) and is regarded as the annulus region. Electron-dense material is deposited on the inner surface of the plasma membrane at the site of its reflection. This is quite visible in the spermatid stage (PL 5, Fig. 16) but not thereafter. This region has no ultrastructural characteristics of a centriole and, since it appears in sperma¬ teliosis at the point where the axial fibres project from the spermatid, Lake et al. (1968) suggested that it represents merely a thickened portion of the membrane around the point of exit. The axial filament complex, which constitutes the main-piece of the tail, is formed of nine doublets and a central pair surrounded by an amorphous sheath of moderate electron opacity. In no stage of development did this sheath show any structural detail; this observation agrees with Lake et al. (1968) who report the absence of transverse segmentation. The disposition of the covering mem¬ brane of the spermatid tail is rather loose and wavy (PL 4, Fig. 12), otherwise

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access Ultrastructural changes andfowl sperm maturation 261 the structural features of the contained filaments of all stages examined in the excurrent ducts are the same as those described in the ejaculated spermatozoon (Lake et al., 1968). DISCUSSION The acrosome of the fowl spermatozoon shows the characteristic shape of that in the ejaculated spermatozoon as early as the late spermatid stage of develop¬ ment, when the nucleus elongates and condenses. This is different from the situation in the mammal ; for example, the shape assumed by the mature acro¬ some in the guinea-pig and chinchilla (Fawcett & Hollenberg, 1963; Fawcett & Phillips, 1969a) is not shown by spermatozoa in the ductuli efferentes or in the first part of the epididymis but is gradually acquired during passage through the epididymis. Less striking modifications of the acrosome by continuous remodel¬ ling occur in the rabbit and monkey (Bedford, 1965; Bedford & Nicander, 1971). In the latter species, a more discrete acrosomal outline, typical ofthat seen in the mature spermatozoon, is displayed by spermatozoa only in the distal parts of the caput epididymidis. It is commonly observed that, in mammalian spermatozoa, a reduction in the dimensions of the acrosome occurs during passage through the epididymis and this has been attributed to an absolute decrease in the width of that part of the acrosome which extends both rostrally and laterally beyond the edge of the nucleus (Fawcett & Hollenberg, 1963; Bedford, 1965; Fawcett & Phillips, 1969a; Jones, 1971). Jones (1971) observed an overall reduction in the size of the acrosome of the boar spermatozoon, but pointed out that it was not pos¬ sible to determine whether there was an actual reduction in volume or just a redistribution of the acrosomal contents. The reduction in acrosomal width may be associated with the movement of the cytoplasmic droplet away from the neck region in mammalian spermatozoa (Bedford, 1963). If there is a strict correlation, then the absence of a cytoplasmic droplet in the fowl spermatozoon may account for the absence of an apparent reduction in the size of the acro¬ some seen in the present work. The remodelling of the acrosome after spermiation in the mammal may re¬ present one aspect of the process of the maturation of the spermatozoon in the excurrent ducts. However, many spermatozoa in the proximal part of the rabbit epididymis, which are infertile at that stage, possess morphologically mature acrosomes (Bedford, 1966; Bedford & Nicander, 1971). These authors consider it unlikely that there is an absolute correlation between the maturation of the acrosome and the fertilizing ability of the epididymal spermatozoon. The inner acrosomal spine in the fowl spermatozoon arises as a small dense granule in the early spermatid (Nagano, 1962) and develops rapidly before the time of spermiation to attain the characteristic shape of that in the mature spermatozoon described by Lake et al. (1968). The acrosomal spine may be homologous to a similar structure termed the 'perforatorium' in some mammals (Fawcett & Phillips, 1969a).The perforatorium develops during the maturation of spermatozoa in the epididymis, whereas the acrosomal spine of the fowl spermatozoon is fully developed before spermiation. Different views exist as to the function of the subacrosomal space in the

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access 262 M. D. Tingari mature spermatozoon. Buongiorno-Nardelli & Bertolini (1967), after studying some lysosomal enzymes in spermatozoa of Triturus cristatus, find that they are exclusively localized in the subacrosomal space. They suggest, therefore, an analogous function between this region and lysosomes; thus, the acrosome causes lysis when penetrating the egg membrane and lysosomal contents can lyse exogenous bodies. Allison & Hartree (1970) demonstrated, biochemically, lysosomal enzymes in the detached acrosome of ram spermatozoa and suggested this rôle for them during fertilization. Fawcett & Phillips (1969a) suggested that the material in the subacrosomal space maintained the cohesion between the acrosomal cap and nucleus rather than being directly involved in perforation or lysis of the egg membrane. The plasma membrane over the acrosomal cap of the fowl spermatozoon is smooth and closely applied but a looseness is observed over the nuclear region, especially in epididymal spermatozoa. This appears to differ from mammalian spermatozoa where a change in the closeness of fit of the plasma membrane occurs over the acrosomal cap and not the nucleus during the passage ofsperma¬ tozoa through the epididymis (Bedford, 1965; Fawcett & Phillips, 1969a; Bed¬ ford & Nicander, 1971). These changes in the covering membranes are taken to indicate a progressive change in the volume of the underlying structures, or an increase in the amount of membranes. Recently, however, Jones (1971) con¬ sidered the possibility that loosening of the plasma membrane over the acro¬ some might be due to the action of fixatives. The structure and fate of the two centrioles of the fowl spermatozoon are similar to those of mammals (Fawcett & Phillips, 1969b). Spermatozoa of cer¬ tain species of insects possess neither a proximal nor a distal centriole (Phillips, 1969). The structure of the centriole in the early spermatid in both the fowl and the mammal is like that of the centrioles in somatic cells except for the ac¬ cumulation of dense matrix between the microtubules. More matrix is deposited rostral to the juxtanuclear centriole during development of the spermatozoa in mammals and birds ; in mammalian spermatozoa, this region is called the neck (Fawcett & Phillips, 1969b). The proximal centriole persists in the mature spermatozoon of both fowl and mammals while the distal centriole changes during differentiation and ultimately disappears as a distinct entity. The axial filament complex of the tail of mammal or fowl spermatozoa does not arise from a basal body as seen with cilia and flagella. The proximal centriole of the early mammalian spermatid increases in length by the addition of material to its free distal end (Fawcett & Phillips, 1969b). This added material has been called the 'centriolar adjunct' and it disappears in late spermiogenesis. Such a structure is not seen in the spermatids of the fowl. The characteristic orientation and shape of mitochondria in the mid-piece of the ejaculated spermatozoon (Lake et al., 1968) is already achieved before spermiation in the fowl, but certain structural changes in the mitochondrial membranes take place during passage of the spermatozoa along the excurrent ducts. The outer and inner mitochondrial membranes are equally thick during the spermatid stage and, following the release of spermatozoa from the semini¬ ferous epithelium but later, there is an increase in the thickness of the inner mitochondrial membrane in spermatozoa in the rete testis and in subsequent

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access Ultrastructural changes andfowl sperm maturation 263 parts of the duct system. This confirms the observations of Nicander & Hell¬ ström (1967), who report thicker inner mitochondrial membranes in fowl spermatozoa taken from the epididymal region and ductus deferens. The fowl 'epididymal region' is formed of a tubular complex (Tingari, 1971) and the present study indicates more precisely where the increase in thickness of the membrane first occurs, namely, the rete testis. Nicander & Hellström (1967) suggest that the thickening of the membrane is a means of concentrating respira¬ tory enzymes within a reduced volume of mitochondria. As mammalian spermatozoa pass through the epididymis, the cytoplasmic droplet moves from the neck region ('proximal bead') to the caudal end of the mid-piece ('distal bead'). This droplet is bounded by the plasma membrane and consists of numerous cisternae and vesicles of various sizes, reminiscent of the Golgi complex, in an admixture with endoplasmic reticulum and small amounts of cytoplasm (Bloom & Nicander, 1961 ; Dott & Dingle, 1968; Garbers, Waka- bayashi & Reed, 1970). The only comparable avian structure to the mam¬ malian droplet is that illustrated in PL 5, Fig. 18. However, it is rarely seen and is merely a mass of cytoplasm bounded by a plasma membrane and no or¬ gandíes are present. It is concluded therefore, that a droplet homologous to that of mammals does not exist in fowl spermatozoa, which confirms the ob¬ servations made by Lake & El Jack (1966) on whole mounts with the electron microscope. The mechanism of release of spermatozoa from the germinal epithelium in the fowl remains to be explained. In mammals (Fawcett & Phillips, 1969a), there is a slender cytoplasmic stalk between the residual bodies and the necks of the spermatids. This stalk breaks at the time of spermiation, and the retraction and rounding up of the proximal end of the stalk forms the cytoplasmic droplet in the neck region of the free spermatozoon. The remainder of the spermatid cytoplasm is engulfed by Sertoli cells. Functional changes in spermatozoa during passage through the excurrent ducts of the cock are reported by Munro (1938). Motility is minimal in the testis, noticeably increased in the epididymal region and maximal in the ductus deferens. Fertilizing capacity is achieved by the spermatozoa during their pas¬ sage through the epididymal region or in the initial part of the ductus deferens. Considering the complex tubular nature of the epididymal region (Tingari, 1971), the sample of semen examined by Munro must have been obtained at random; the precise location could not have been known as it is impossible to identify the different kinds of tubules except in histological sections. The present study indicates that the morphological differentiation of spermatozoa is vir¬ tually complete when they are released from the seminiferous epithelium of the fowl. In the light of Munro's findings, this cannot mean that they are then functionally mature. It is concluded that there is no structural development which can be directly correlated with achievement of fertilizing capacity, though this property may be attained earlier in the excurrent ducts of the bird than in those of the mammal. ACKNOWLEDGMENTS Grateful thanks are due to Dr P. E. Lake and Professor A. R. Muir for reading

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access 264 M. D. Tingari and criticizing the manuscript. The work has been carried out while on leave of absence from the University of Khartoum, Sudan, during a tenure of a Popula¬ tion Council fellowship.

REFERENCES

Allison, A. C. & Hartree, E. F. (1970) Lysosomal enzymes in the acrosome and their possible rôle in fertilization. J. Reprod. Fert. 21, 501. Bedford, J. M. (1963) Morphological changes in rabbit spermatozoa during passage through the epididymis. J. Reprod. Fert. 5, 169. Bedford, J. M. (1965) Changes in fine structure of the rabbit sperm head during passage through the epididymis. J. Anat. 99, 891. Bedford, J. M. (1966) Development of the fertilizing ability of spermatozoa in the epididymis of the rabbit. J. exp. Zool. 163, 319. Bedford, J. M. & Nicander, L. (1971) Ultrastructural changes in the acrosome and sperm membranes during maturation of spermatozoa in the testis and epididymis of the rabbit and monkey. J. Anat. 108, 527. Bloom, G. & Nicander, L. (1961) On the structure and development of the protoplasmic droplet of spermatozoa. Z· ZeWmsck. mikrosk. Anat. 55, 833. Buongiorno-Nardelli, M. & Bertolini, B. (1967) Subcellular localization of some acid hydrolases in Triturus cristatus spermatozoa. Histochemie, 8, 34. Dickey, J. F. (1966) An electron microscope study of bovine spermatozoa undergoing maturation in the male reproductive system. Diss. Abstr. 24, 6274. Dott, H. M. & Dingle, J. T. (1968) Distribution of lysosomal enzymes in the spermatozoa and cyto¬ plasmic droplets of bull and ram. Expl Cell Res. 52, 523. Fawcett, D. W. & Hollenberg, R. D. (1963) Changes in the acrosome of guinea-pig spermatozoa during passage through the epididymis. Z- ZeWmsc^· mikrosk. Anat. 60, 276. Fawcett, D. W. & Phillips, D. M. (1969a) Observations on the release ofspermatozoa and on changes in the head during passage through the epididymis. J. Reprod. Fert. Suppl. 6, 405. Fawcett, D. W. & Phillips, D. M. (1969b) The fine structure and development of the neck region of mammalian spermatozoa. Anat. Ree. 165, 153. Garbers, D. L., Wakabayashi, T. & Reed, P. W. (1970) Enzyme profile of the cytoplasmic droplet from bovine epididymal spermatozoa. Biol. Reprod. 3, 327. Glauert, A. M. (1967) Fixation and embedding of biological specimens. In : Techniques for Electron Micro¬ scopy, p. 166. Ed. D. Kay. Blackwell Scientific Publications, Oxford. Grigg, G. W. & Hodge, A. J. (1949) Electron microscopic studies ofspermatozoa. I. The morphology ofthe spermatozoon of the common domestic fowl (Gallus domesticus). Aust.J. scient. Res. Series B, 2,271. Jones, R. G. (1971) Studies of the structure of the head of boar spermatozoa from the epididymis. J. Reprod. Fert. Suppl. 13, 51. Lake, P. E. & El Jack, M. H. (1966) The origin and composition offowl semen. In: Physiology of the Domestic Fowl, p. 44. Eds. C. Horton-Smith and E. C. Amoroso. Oliver & Boyd, Edinburgh. Lake, P. E. & Smiles, J. (1952) Some observations on the morphology of fowl spermatozoa. Proc. Soc. Study Fert. 4, 18. Lake, P. E., Smith, W. & Young, D. (1968) The ultrastructure of the ejaculated fowl spermatozoon. Q_.Jl exp. Physiol. 53, 356. McIntosh, J. R. & Porter, K. R. (1967) Microtubules of the spermatids of the domestic fowl. J. Cell Biol. 35, 153. Maxwell, M. H. & Trejo, F. (1970) The ultrastructure of white blood cells and thrombocytes of the domestic fowl. Br. vet. J. 126, 583. Millonig, G. (1962) Further observations on a phosphate buffer for osmium solutions in fixation. In: Proc. 5th Int. Gonf. Electron Microsc. Vol. II, p. p-8. Ed. S. S. Bréese. Academic Press, New York and London. Munro, S. S. (1938) Functional changes in fowl spermatozoa during their passage through the excurrent ducts of the male. J. exp. Z°°l· 79, 71. Nagano, T. (1962) Observations on the fine structure of the developing spermatid in the domestic chicken. J. Cell Biol. 14, 193. Nicander, L. & Hellström, . (1967) Increased thickness ofthe inner mitochondrial membrane during sperm maturation in the domestic rooster. Expl Cell Res. 48, 622. Phillips, D. M. (1969) Exceptions to the prevailing pattern of tubules (9+9+2) in the sperm flagella of certain insect species. J. Cell Biol. 40, 28.

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access Ultrastructural changes andfowl sperm maturation 265 Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208. Stempak, J. G. & Ward, R. T. (1964) An improved staining method for electron microscopy. J. Cell Biol. 22, 697. Tingari, M. D. (1971) On the structure of the epididymal region and ductus deferens of the domestic fowl (Gallus domesticus). J. Anat. 109, 423. Zlotnik, I. (1947) The cytoplasmic components of germ-cells during spermiogenesis in the domestic fowl. Q_.Jl microsc. Sci. 88, 353.

Downloaded from Bioscientifica.com at 10/10/2021 01:57:59AM via free access