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 2K fl.% 10pIm A <.ez.,<\* ,; F, ,,a.,;s^., S7 'w ,' 44,} s ,, S t 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 r 4 -r.,,,;~~'.s*'!w;k ,, "0..r.. (4e,,, - _ ~ N+ 4 C;,.-', v* 44 VA-4 . * 0 * ; - *dI i4t^I ~ ' 3 ''* -| ;, ,0 2',AGm'e 41.*'~.*iP ':7.. L.Opm '.*,] ^ - ( t;4 ~~* $ / ** *-2 P>.. 0.,'% t' V44- V~Xsfs;ArC'.4^> ¢'u V'[Y W * $4 4k iX, - tt7 Fig. 3. Electron micrograph of a step 3 spermatid. Chromatin has condensed into a large mass in the centre of the nucleus. The acrosomic granule (AG) attaches to the nuclear membrane. The flagellum (F) is moving away from the acrosomal region. Fig. 4. Electron micrograph of a step 4 spermatid. The nuclear chromatin is finely granular and almost uniform in density. The proacrosome (A) is settled in a concavity of the nuclear membrane. A few scattered microtubules (MT, see inset), the precursor of the manchette, are present just outside the nucleus. the same fixative. The samples were postfixed in 1 % an Ultracut E ultramicrotome (Reichert-Jung, osmium tetroxide and embedded in Spurr's resin Austria) using a diamond knife (Diatome Ltd, Bienne, (Agar Scientific Ltd, Essex, UK). Sections were cut on Switzerland). Sections (70-100 nm) were stained with 528 M. Lin and R. C. Jones "0* 7w..Yj(i ~~~~~~~~~~~~P t:, A /"S 0~~~~~~~~~~~~~. *~~~~~~~~~~~~~~~~~~~~14 ~~~~~O ( * s~~~~~~~~~~~~~~~~~~4 ~~~~~~~~~~~~~A ~AtJ`~ ~ ~ ~ ~ ~ * t f jC~~~~~~~~~~~~~~~~~~~~~1 rt.~ p ~¾, ."-~~. - -~~~~~~~~~~~~~~ A -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~'t 4stri -$ -rr ~~~~~~ ~~44.4kF 4~~~~~~~t" W&''5 rwJ'V. C,.tA OP aKfrr~~~~~¶( ~ ~~~zS4c~~~~r~t ux"1b".&'*"~~~~~~~" -~~~ 4 ~~~~'ji. 4'~~~z2-,! .4 4~~~~~Z - *I-e AC"~~~~~~~~~~~~~~tt64;sC ~~~~~~~~~~~~~~a L 4 *SZCAI4. -~~~~~~~W"4- *,. 4J ____ 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 )4 R'DI j.:,.- MF 12~~~~~~~~~~~~~~~~~~1 A A~~~~~~~~~~~~~~~~~~~~~ *~1. tM j84 F ,'I , ;I6 ' 4!q ~ AP * C M.....-- -. Fi.9 Eetonmcogah o ogiuialscioso se sentis a)Tenula crmti scodnedt or mlldne ass The crosme A) i slederand -shaed n logitdinlscinkhwefrtru P a ecm o-hpd h irtblso h circular m~~~~~~ancet C)aefrelrudtences b iohnra(M ihlniuia rsa r ctee ntectpama 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.