J. Anat. (1993) 183, pp. 525-535, with 14 figures Printed in Great Britain 525 Spermiogenesis and spermiation in the Japanese quail (Coturnix coturnix japonica)
M. LIN AND R. C. JONES Department of Biological Sciences, University of Newcastle, New South Wales, Australia
(Accepted 11 May 1993)
ABSTRACT The ultrastructure of 12 steps of spermatid development and the process of spermiation are described for the Japanese quail in order to clarify the classification proposed for determining the stages of the cycle of the seminiferous epithelium (Lin et al. 1990) and to assess disagreements in the literature about sperm development in birds. It was concluded that acrosomal development involves the formation of proacrosomal granules which do not contain dense granules like the mammalian acrosome. Material which forms the perforatorium initially accumulates as a nuclear granule before appearing in the subacrosomal space. A circular and longitudinal manchette develop sequentially during nuclear elongation. Microtubules of the circular manchette initially form around several parts of the spherical nucleus of step 4 spermatids and subsequently occur most frequently around the narrowest regions of the elongating nucleus. Fibrous sheath development starts in step 2 spermatids indicating that it forms much earlier in the quail than in mammals. Spermiation differs from the process described in mammals in that the residual body is released from near the rostral end of the sperm nucleus leaving no cytoplasmic droplet in quail spermatozoa.
Okamura & Nishiyama, 1976; Gunawardana & Scott, INTRODUCTION 1977; Guraya, 1987). There is also a lack of detail The differentiation of mammalian spermatids has about the process and occurrence of spermiation, and attracted considerable attention because it provides a ultrastructural studies have not been related to a good model of cellular differentiation and because classification of the stages of spermatogenesis. Conse- classifying the steps (or phases) of spermiogenesis quently, the study described in this report was carried provides a basis for classifying the stages of the cycle out to clarify the processes of spermiogenesis and of the seminiferous epithelium. However, there have spermiation in the Japanese quail and to provide a been few ultrastructural studies of spermiogenesis in basis for more detailed studies of spermiogenesis in birds. Most work has been on the domestic fowl birds. (Nagano, 1962; McIntosh & Porter, 1967; Tingari, 1973; Okamura & Nishiyama, 1976; Gunawardana & Scott, 1977). Some aspects of development of the MATERIALS AND METHODS sperm head of the pigeon and sperm tail of the finch Adult male Japanese quails (Coturnix coturnix ja- have also been reported (Fawcett et al. 1971). ponica) more than 48 d old (mass approximately Spermiogenesis in other birds has been studied by 250 g) were used. They were maintained for at least light microscopy including work on the drake (Cler- 4 wk before use on a regime with a constant 16L: 8D mont, 1958), Japanese quail (Yamamoto et al. 1967; light schedule and a temperature of 21 'C. Lin et al. 1990) and guinea fowl (Aire et al. 1980). Four quails were killed with an overdose of sodium However, there are some disagreements among these pentobarbitone (30 mg/kg, intraperitoneally). Their reports such as in the occurrence of the proacrosomal testes were fixed by vascular perfusion (Forssmann et granule (Yamamoto et al. 1967; Lin et al. 1990) and al. 1977) with 3 % (v/v) glutaraldehyde in phosphate the nature of the manchette (Fawcett et al. 1971; buffer (pH 7.4) and then immersion-fixed overnight in
Correspondence to Dr M. Lin, Department of Biological Sciences, The University of Newcastle, New South Wales 2308, Australia. 526 M. Lin and R. C. Jones
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Fig. 1. Electron micrograph of a step 1 spermatid. The round nucleus contains 2, or more, centrally located chromatin bodies and a narrow band of chromatin lines the nuclear membrane. Mitochondria with widened spaces between the cristae (M) are evenly dispersed in the cytoplasm. Centrioles (C), multivesicular bodies (MV) and smooth endoplasmic reticulum (SR) are present. Fig. 2. Electron micrograph of a step 2 spermatid. Chromatin within the nucleus has started to condense to form a centrally located mass. A membrane-bound proacrosome granule (AG) appears in a juxtanuclear position. In the same area, the flagellum (F) develops from the centriole complex (CC) and a fibrous sheath (arrow) has developed in the flagellum. Spermiogenesis and spermiation in the Japanese quail 527
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Fig. 3. Electron micrograph of a step 3 spermatid. Chromatin has condensed into a large mass in the centre of the nucleus. The acrosomic granule (AG) attaches to the nuclear membrane. The flagellum (F) is moving away from the acrosomal region. Fig. 4. Electron micrograph of a step 4 spermatid. The nuclear chromatin is finely granular and almost uniform in density. The proacrosome (A) is settled in a concavity of the nuclear membrane. A few scattered microtubules (MT, see inset), the precursor of the manchette, are present just outside the nucleus. the same fixative. The samples were postfixed in 1 % an Ultracut E ultramicrotome (Reichert-Jung, osmium tetroxide and embedded in Spurr's resin Austria) using a diamond knife (Diatome Ltd, Bienne, (Agar Scientific Ltd, Essex, UK). Sections were cut on Switzerland). Sections (70-100 nm) were stained with 528 M. Lin and R. C. Jones
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Fig. 5. Electron micrograph of a step 5 spermatid. The nucleus is roughly spherical and contains fine homogeneous chromatin and a few small accumulations of chromatin. A dense droplet (P), a precursor of the perforatorium, is present in the nucleoplasm. The proacrosome (A) is attached to the nucleus over a wider area than in step 4 spermatids (see Fig. 4). Fig. 6. Electron micrograph of a step 6 spermatid. It has an elongated irregular nucleus containing fine homogenous chromatin granules. A dense droplet (P) destined to be the perforatorium is obvious in the nucleoplasm. The acrosome (A) is ellipsoid. Its rostral end is in contact with the plasma membrane and it is embedded in the Sertoli cell (SC). Microtubules (MT) destined to form the manchette occur around the nucleus. Spermiogenesis and spermiation in the Japanese quail 529
Fig. 7. Electron micrograph of step 7 spermatids sectioned longitudinally. The roughly cylindrical nucleus is surrounded by considerable cytoplasm. The acrosome (A) is crescent shaped and covers most of the rostral surface of the nucleus. A small cavity (C) occurs at the end of the nucleus beneath the acrosome. Microtubules of the circular manchette (CM, also see inset) run in bands around the nucleus (N). M, mitochondria. Fig. 8. Electron micrograph ofstep 8 spermatids which are grouping together with their acrosomal ends embedded in the Sertoli cell cytoplasm (SC). The perforatorium (P) lies within a cavity of the nucleoplasm under the acrosome. Microtubules of the circular manchette (CM) surround the entire surface of the nucleus (N). A, acrosome. 530 M. Lin and R. C. Jones
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Fig. 10. Electron micrographs of longitudinal sections of step 10spermatids. (a) The nuclear chromatin is condensed itocorsarldesegranules.
The acrosome (A) has become densely electron opaque. (b) The mitochondrial sheath (MS) of the middle piece is forming. CM, the circular manchette; N, nucleus. Fig. 11. (a) Electron micrograph of a longitudinal section through the tip of the head of a step 11 spermatid. The chromatin has condensed into large coarse granules. The acrosome (A) is cylindrically shaped. Microtubules of the longitudinal manchette (LM) lie parallel to the Spermiogenesis and spermiation in the Japanese quail 531
1% uranyl acetate in 30% (v/v) ethanol (Watson, Step 3 spermatids (Fig. 3). The nucleus remains 1958), then lead citrate (Reynolds, 1963) and exam- spherical and has a large accumulation of chromatin ined in a JEOL-IOOCX or a JEOL-1200EX trans- in the centre. It is distinguished from step 2 spermatids mission electron microscope (JEOL, Tokyo, Japan) by the attachment of the proacrosomal granule to the operating at 80 kV. nuclear membrane, and by the partial migration ofthe flagellum towards the pole of the nucleus opposite the acrosome. RESULTS Step 4 spermatids (Fig. 4). The nucleus remains Spermatid development was classified into the 12 spherical and contains finely granular chromatin of steps recognised by Lin et al. (1990). The classification uniform density and some centrally located chromatin is mainly based on the morphological development of particles. The acrosome is lodged in a concavity ofthe the acrosome, nucleus and flagellum. nuclear membrane and the nucleoplasm stains densely Step I spermatids (Fig. 1.). These are products of below the area of contact. The formative flagellum the 2nd meiotic division and show little differentiation. continues to migrate to the side of the nucleus They are usually associated with the old generation of opposite the acrosome. A few microtubules pre- step 11 spermatids located close to the lumen of the sumably destined to form the circular manchette are seminiferous tubule. They are characterised by a in close apposition to the nuclear membrane (see spherical nucleus containing a narrow band of insert in Fig. 4). flocculent chromatin under the nuclear membrane Step 5 spermatids (Fig. 5). The nucleus is roughly and 2 or more, centrally located, dense chromatin spherical and similar in content to step 4 spermatids. bodies. Mitochondria are evenly distributed through- The acrosome embeds deeper into the nucleus and out the cytoplasm. They are spherical with large increases its area of contact with the nucleolemma. A intercristae spaces (see Fig. 1). The Golgi apparatus is precursor of the perforatorium begins to form within inconspicuous and rare in ultrathin sections. A pair of the nucleoplasm under the acrosome. The formative centrioles are located on the luminal side of the flagellum has completed its migration and its base is nucleus close to the plasmalemma. Multivesicular embedded into a recess of the nuclear membrane bodies, smooth endoplasmic reticulum and some lipid opposite the acrosome. droplets are present, but ribosomes and rough Step 6 spermatids (Fig. 6). The nucleus contains endoplasmic reticulum are rare. finer homogenous chromatin granules than step 5 Step 2 spermatids (Fig. 2). The nucleus is spherical spermatids. It begins to undergo 2 processes, nuclear and contains chromatin which is beginning to con- elongation (steps 6 to 12) and rotation (steps 6 and 7). dense. The Golgi apparatus is located on the luminal It elongates along an axis perpendicular to the widest side of the nucleus and forms one or more membrane- diameter of the acrosome, initially forming a pear- bound, proacrosomal granules which move towards shaped structure in step 6 spermatids. The nucleus the nucleus. The proximal centriole is surrounded by rotates (with the acrosome and flagellum attached) to Golgi lamellae and is located about half way between reverse the polarity of the cell along the radial axis of the nucleus and the distal centriole. The latter lies the seminiferous tubule. The acrosome becomes under the plasmalemma with its formative flagellum ellipsoidal and spreads over the nuclear surface, and protruding from the cell surface towards the lumen of its rostral surface contacts the plasma membrane as the seminiferous tubule. The formative annulus the nucleus elongates. The precursor of the perfora- appears as an electron dense ring of amorphous torium enlarges and becomes a dense droplet located material attached to the base of the invagination of in the nucleoplasm beneath the acrosome. Micro- the plasmalemma around the axoneme. The fibrous tubules destined to form the circular manchette scatter sheath is forming around the tail distal to the annulus around the nuclear membrane (arranged in a plane as a series of circumferentially oriented loops. The perpendicular to the longitudinal axis of the nucleus; other cytoplasmic organelles are the same as in step 1 see Fig. 6), particularly accumulating in the narrow spermatids. part of the nucleus.
longitudinal axis of the nucleus (N). (b) A portion of neck region of a step 11 spermatid showing the longitudinal manchette (LM) and microtubules (MT) extend to middle piece region. F, flagellum. Fig. 12. (a) Electron micrograph of the anterior heads of step 12 spermatids. The nuclear chromatin is densely opaque. The acrosome (A) is fully elongated and electron opaque. The perforatorium (P) is fully extended. (b) The microtubules of the longitudinal manchette (LM) are present in the early step 12 spermatids. L, lumen of the seminiferous tubule; N, nucleus. 532 M. Lin and R. C. Jones
Step 7 spermatids (Fig. 7). The spermatids have Step 11 spermatids (Fig. 11 a, b). The spermatids are moved towards the lumen of the seminiferous tubule. clustered in groups and continue to ascend to the The nuclei continue to rotate so that some are lumen ofthe seminiferous tubule. They possess longer, oriented randomly whilst others have started to group more cylindrical nuclei than step 10 spermatids (Fig. together with their acrosomes directed towards the 11 a) and the chromatin continues to condense basement membrane of the seminiferous epithelium. increasing the size of the chromatin granules. The The acrosome becomes crescent-shaped and almost acrosome continues to elongate. The circular man- covers the rostral end of the nucleus. A small cavity chette has disappeared and is replaced by the occurs within the nucleoplasm at the end of nucleus longitudinal manchette, an array of almost straight beneath the acrosome. Mitochondria begin to elon- microtubules lying parallel to the longitudinal axis of gate. Microtubules of the circular manchette continue the nucleus (Fig. 11 a, b). Cytoplasmic bridges between to accumulate around the nucleus. The cytoplasm adjacent spermatids are maintained. surrounding the rostral end of the nucleus begins to Step 12 spermatids (Fig. 12a, b). The nucleus is very migrate caudally. slim and highly condensed and there is little cytoplasm Step 8 spermatids (Fig. 8). The spermatids have around the head. The acrosome is more elongated and completed the rotation process and group together condensed than step 11 spermatids (Fig. 12a). The with their rostral ends further embedded in the perforatorium also appears to have condensed further. cytoplasm of Sertoli cells directed towards the The longitudinal manchette is present early, but basement membrane of the seminiferous epithelium. disappears towards the end of the step 12 (Fig. 12b). The nuclear chromatin remains in the form of fine The cytoplasm not destined to form part of the homogenous granules, but the nuclei continue to spermatozoon is displaced rostrally to form a putative elongate and become more cylindrical than step 7 residual body just caudal to the acrosome and leaving spermatids. The acrosome elongates rostrally. The little cytoplasm around the spermatid neck. The putative perforatorium is contained within a cavity residual body contains numerous large vesicles, lipid formed by caudal extensions ofthe lateral edges of the droplets, mitochondria and remnants of other organ- acrosome. Microtubules of the circular manchette elles. surround the entire surface of the nucleus. The Spermiation (Fig. 13, 14). When step 12 spermatids cytoplasm surrounding the nucleus continues to are mature only the rostral part of the acrosome is in migrate caudally. contact with a Sertoli cell (Fig. 13) and most of the Step 9 spermatids (Fig. 9a, b). The nucleus con- spermatid projects into the lumen of the seminiferous tinues to elongate and its chromatin condense (Fig. tubule. The putative residual body is almost com- 9a). The acrosome is more slender than in step 8 pletely surrounded by Sertoli cell cytoplasm and it is spermatids. The circular manchette persists. The only separated from the spermatid by a short narrow cytoplasm continues to migrate caudally reducing the bridge of cytoplasm (Fig. 13). The bridge breaks near amount around the rostral end of the nucleus. the spermatid when the spermatozoon is released and Mitochondria elongate, form longitudinal cristae and the residual body is phagocytosed by the Sertoli cell migrate to the caudal cytoplasm (Fig. 9b). (Fig. 14). Step 10 spermatids (Fig. IOa, b). The spermatids ascend towards the lumen of the seminiferous tubule leaving only the acrosome and anterior part of the DISCUSSION nucleus embedded in Sertoli cell cytoplasm. The nucleus continues to elongate and condense, forming Spermiogenesis coarse chromatin granules. The acrosome becomes This report confirms the observation by Lin et al. denser and its rostral tip continues to elongate. The (1990) that quail spermatids develop proacrosomal perforatorium condenses to form a dense rod-shaped granules. Yamamoto et al. (1967) failed to identify the structure within the subacrosomal cavity (Fig. 10a). granules in paraffin sections, presumably because Most ofthe spermatid cytoplasm is displaced caudally paraffin sections lack the resolution of epoxy em- from the anterior third of the nucleus. The circular bedded material. It is also suggested that acrosomal manchette is present early, but disappears towards the development in the quail (this study), drake (Cler- end of step 10. Mitochondria accumulate around the mont, 1958) and domestic fowl (Nagano, 1962; de axoneme between the nucleus and annulus forming Reviers, 1971; Okamura & Nishiyama, 1976; Guna- the mitochondrial sheath of the middle piece (Fig. wardana, 1977; Gunawardana & Scott, 1977) differs lOb). from the process described in some mammals in that Spermiogenesis and spermiation in the Japanese quail 533
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Fig. 13. Electron micrograph ofthe residual body (RB) which remains connected to a step 12 spermatid through a narrow cytoplasmic bridge (CB) extending from the head close to the acrosome (A). Large vesicles (V), lipid droplets (LD), and mitochondria (M) are present in the residual body. SC, Sertoli cell. Fig. 14. Electron micrograph ofthe head ofa mature spermatid separated from the seminiferous epithelium. The rostral part of the acrosome (A) is the last area of contact between the spermatid and Sertoli cell (SC). RB, residual body. the contents of the developing avian acrosome are of Burgos & Fawcett, 1955; Clermont & Leblond, 1955; homogeneous density and lack the dense granule of Gardner, 1966; Plben & Courtens, 1986). mammalian acrosomes (Leblond & Clermont, 1952; The perforatorium in birds is much larger than in 534 M. Lin and R. C. Jones mammals (Bedford, 1967; Fawcett & Phillips, 1969; radiography, Irons and Clermont (1982a, b) found Jones, 1971). The development of the perforatorium that the fibrous sheath formation in rats begins at the described in this report differs from the process distal end of the principal piece in step 8 spermatids described in the fowl. The early researches (Nagano, and ends at the proximal end of the principal piece in 1962; Okamura & Nishiyama, 1976; Gunawardana & step 19 spermatids. They also found that from step 8 Scott, 1977) considered that the perforatorial material to 14 spermatids ofthe rat only the anlage ofthe outer accumulates from the nucleus in a nuclear invagina- dense fibres was formed (Irons & Clermont, 1982a). tion under the acrosome offowl spermatids whereas it Using a monoclonal antibody, Sakai et al. (1986) was initially recognised as a nuclear granule in this detected the appearance of components of the fibrous study on the quail. sheath in step 14 spermatids of mice. However, the Okamura & Nishiyama (1976) reported that the study described in this report showed that the fibrous formation of the circular manchette in fowls starts sheath was present in step 2 spermatids in the quail rostrally and proceeds caudally along the nucleus, (see Fig. 2) indicating that the fibrous sheath develops while Gunawardana & Scott (1977) supposed that the much earlier in the quail than that in mammals. circular manchette begins to form at the caudal end of However, further studies are required to determine the nucleus. Our findings in the quail disagree with the actual timing and pattern of formation of the both these suggestions. In quails, microtubules of the fibrous sheath in the quail. circular manchette are first seen around the spherical nucleus of step 4 spermatids which have not yet started to elongate (Fig. 4). It is suggested that the Spermiation formation of the circular manchette in quail sperm- To our knowledge this is the first report ofspermiation atids may commence in a number of regions around in a bird. Compared with spermiation in mammals, the nucleus during the nuclear condensation and some significant differences have been identified. elongation as the distribution of microtubules of the Firstly, there is a difference in the elimination of circular manchette may vary over the surface of early spermatid cytoplasm destined to form the residual elongating spermatids (see Figs 6-8). body. In mammals the excess cytoplasm accumulates The study described in this report found that the around the neck of spermatids, and during spermi- circular and longitudinal manchette develops sequen- ation a long slender stalk joins the spermatid to the tially around elongating spermatid nuclei of the quail putative residual body held within the Sertoli cell as has been described in the domestic fowl (McIntosh cytoplasm (see Fawcett & Phillips, 1969). When the & Porter, 1967; Okamura & Nishiyama, 1976; connecting stalk is broken, the proximal end retracts Gunawardana & Scott, 1977) and the mammals which to form the cytoplasmic droplet around the neck of have been studied (see review by Fawcett et al. 1971). the spermatozoon (Fawcett & Phillips, 1969; Russell Although the significance of the manchette during & Clermont, 1976; Holstein & Roosen-Runge, 1981). nuclear elongation of mammalian spermatids is still In the quail, the excess spermatid cytoplasm accumu- debatable (see reviews by Fawcett et al. 1971 and lates just behind the acrosome and it is only separated Guraya, 1987), work on the fowl (Nagano, 1962; from the putative residual body by a short bridge of McIntosh & Porter, 1967; Gunawardana, 1977; cytoplasm (Fig. 13). Consequently, as in the domestic Okamura & Nishiyama, 1976; Gunawardana & Scott, fowl (Lake & El Jack, 1966; Tingari, 1973), virtually 1977; Lin et al. 1987) and quail (this report) suggests no cytoplasmic droplet remains on the spermatozoon that the microtubules of the manchette play an when the bridge is broken at spermiation. It is important role in nuclear morphogenesis since the suggested that the very long head of quail and other development of microtubules closely coincides with avian spermatozoa may explain why the connecting the process. Furthermore, in early elongating quail stalk originates far from the neck of the spermatid. spermatids, the circular manchette always accumu- The 2nd difference in spermiation between the quail lated around the narrowest part of the nucleus (see and mammals is the absence of the tubulobulbar Fig. 6). However, we could not identify any cross- complex in the quail, a structure which is considered bridges connecting the successive turns of the helix of to be involved in the elimination of spermatid the circular manchette as has been described in the cytoplasm (Russell, 1979, 1980). domestic fowl (McIntosh & Porter, 1967). However, further studies of the quail are required The present study shows that the time course of to confirm this finding as the tubulobulbar complex formation ofthe fibrous sheath in the quail is different would be present for a shorter period than in the rat from that in mammals. Using high resolution auto- (due to the shorter duration of spermatogenesis in the Spermiogenesis and spermiation in the Japanese quail 535 quail; Lin et al. 1990), and the small area of cellular JONES RC (1971) Studies of the structure of the head of boar associations in the quail (Lin & Jones, 1990, 1992) spermatozoa from the epididymis. 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