Observations on the Fine Structure of Spermatozoa in the Testis and Excurrent Ducts of the Male Fowl, Gallus Domesticus
Total Page：16
File Type：pdf, Size：1020Kb
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, remodel- ling 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 spermato- zoon 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 spermato- zoon 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 ultrastruc- tural 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.