Ultrastructural Study of Spermatogenesis in Phoronopsis Harmeri (Lophophorata, Phoronida)
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Helgol Mar Res (2004) 58:1–10 DOI 10.1007/s10152-003-0153-3 ORIGINAL ARTICLE Arkadiy Reunov · Waltraud Klepal Ultrastructural study of spermatogenesis in Phoronopsis harmeri (Lophophorata, Phoronida) Received: 10 October 2002 / Revised: 2 June 2003 / Accepted: 2 June 2003 / Published online: 8 July 2003 Springer-Verlag and AWI 2003 Abstract The process of sperm development in Phoronop- Introduction sis harmeri was studied by electron microscopy. Develop- ing spermatogenical cells are aggregated around the Present ultrastructural investigations in Lophophorata capillaries of the haemal plexus. The spermatogonia, which (Tentaculata) allow a general view of mature sperm are situated around the capillary walls of the caeca, are structure in representatives of all five sub-groups: remarkable for the presence of germ-line vesicles and Phoronida, Brachiopoda, Bryozoa, Entoprocta and Cy- contain their centrioles near the cell membrane. The cliophora (see Herrmann 1997; James 1997; Mukai et al. spermatocytes and spermatids are flagellated cells arranged 1997; Nielsen and Jespersen 1997). However, the data on in clusters. During spermiogenesis the basal body/flagellum spermatogenesis are insufficient, as the complete process complex migrates to the apical pole of the spermatid. The has so far been studied only in two species of brachiopods acrosome-like structure arises from material produced by (Hodgson and Reunov 1994). the Golgi complex. It lacks a surrounding membrane and Spermatozoa of Phoronida are typically ‘V’-shaped has a fibrillar content. The nucleus elongates and the (see Ikeda 1903; Cori 1939; Silen 1952; Franzn 1956; condensation of chromatin is caused by an activation of Zimmer 1967; Franzn and Ahlfors 1980; Herrmann ‘initiation centres’. The late spermatid and the spermato- 1997). In accordance with ultrastructural findings (Fran- zoon appear as two-armed ‘V’-shaped cells in which one zn and Ahlfors 1980) one arm of the V contains the arm contains the nucleus and posteriorly located mitochon- flagellum, and the other the nucleus and two elongated dria, and the other one is the axoneme. Spermatogenesis of mitochondria. In Phoronis muelleri Selys-Longchamps, P. harmeri is an interesting example of gamete differen- 1903 the spermatozoon has a needle-like acrosome, a tiation where advanced sperm structure is combined with a short, almost round nucleus and a flagellum (Herrmann plesiomorphic pattern of sperm development characterized 1997). An ultrastructural study of phoronid sperm devel- as ‘flagellate spermatogenesis’. opment has been carried out on Phoronis pallida Schnei- der, 1862 with a consideration of spermatid development Keywords Phoronopsis harmeri · Flagellate only (Franzn and Ahlfors 1980). The early stages of spermatogenesis · Spermatozoon sperm development (spermatogonia and spermatocytes) have never been investigated in Phoronida. To fill this gap, spermatogenesis in Phoronopsis harmeri Pixell, 1912 has now been studied through all stages of sperm differentiation. Communicated by H.-D. Franke Methods A. Reunov ()) Samples of Phoronopsis harmeri were collected in August 1996 in Institute of Marine Biology, the intertidal zone of ‘Vostok’ Biological Station (Japan Sea, Far East Branch of the Russian Academy of Sciences, Russia). The species is gonochoristic (Zimmer 1967; Herrmann 690041 Vladivostok, Russia 1997). The males were identified by dissection and the gametes e-mail: [email protected] were observed using light microscopy. For the ultrastructural study the metasoma of three males were W. Klepal fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer Institute for Zoology, (pH 7.4, fixative osmolarity=1100 mosmol) and in 2% osmium University of Vienna, tetroxide buffered by filtered sea water. Following dehydration in a Biozentrum, 14 Althanstrasse, 1090 Vienna, Austria graded series of ethanol and acetone, the material was embedded in 2 Epon-Araldite (Sigma-Aldrich, St Louis). Sections were cut on an during the pachytene period. Each primary spermatocyte Ultracut-E ultramicrotome (Reichert-Jung, Austria) using glass and has a single flagellum (Fig. 8). diamond knives, stained with uranyl acetate and lead citrate, and examined with an EM 9S-2 transmission electron microscope The clusters of the secondary spermatocytes can be (Zeiss, Germany). For scanning electron microscopical investiga- recognized in the vasoperitoneal tissues by the cell size tions, samples of P. harmeri were fixed as above, critical-point intermediate between that of primary spermatocytes and dried, coated with gold and photographed with a 35CF scanning early spermatids (Fig. 9). The secondary spermatocytes electron microscope (Jeol, Japan). The examination of the speci- mens with the electron microscopes was carried out in the have one cilium and a single daughter centriole (Fig. 10). Zoological Institute at the University of Vienna. Spermiogenesis Results Early stage Male gamete forming tissue Early spermatids are organized in clusters of cells (Fig. 9) Developing spermatogenical cells are aggregated around connected by intercellular bridges (Fig. 11). The cells are the capillaries of the haemal plexus. The diameter of these monociliated and have an almost spherical nucleus with vessels is quite small and corresponds to the size of a chromatin at the initial stage of condensation. From this blood cell (Fig. 1). The wall of the capillaries consists of stage, there is normally no other centriole in sperm cells vasoperitoneal cells lined by an inner layer of extracel- except the basal body. lular matrix (Fig. 2). In some areas the projections of these cells do not join up and spermatogenical cells adhere to the matrix layer (Fig. 2). The vasoperitoneal Middle stage tissue cells are spread among the forming gametes. The highly vacuolized cytoplasm of these cells has character- During this stage of spermiogenesis, the condensation of istic phagosome-shaped inclusions (Fig. 3). chromatin proceeds (Fig. 12) and the nucleus then becomes trapeziform (Fig. 13). The basal body/flagellum complex migrates to the apical pole of the spermatid Spermatogonia (Figs. 12, 13). This displacement involves the formation of an invagination of the plasma membrane which forms a Groups of elongate spermatogonia are usually arranged cytoplasmic channel through which the flagellum is around the capillary walls of the caeca (Fig. 1). These passed (Fig. 14). The mitochondria tend to fuse at the cells have an ovoid nucleus with small patches of lateral side of the nucleus and form two elongated ovoid chromatin. One prominent nucleolus is near the periphery mitochondria (Fig. 15). Golgi vesicles supply a substance of the nucleus. The cytoplasm of the spermatogonia for the formation of the acrosome-like structure (Figs. 16, contains electron-dense vesicles which supposedly re- 17). Finally, this organelle consists of fibrillar condensed present the germ-line substance (Figs. 4, 5). Two material and is not surrounded by a membrane (Fig. 18). perpendicularly oriented centrioles are near the cell After the acrosome-like structure is formed, the Golgi membrane (Fig. 5). complex is eliminated, along with residual cytoplasm (Fig. 19). Spermatocytes Groups of spermatocytes usually cover the spermatogonia distally (Fig. 1), but sometimes they are connected to the capillary walls (Fig. 2). Developing spermatocytes are Fig. 1 Scanning electron micrograph (SEM) of fracture of va- organized in multicellular clusters inside which the cells soperitoneal tissue. Spermatogonia (Sg) and primary spermatocytes are connected by intercellular bridges (Fig. 6). The (Sc I) situated around the capillary blood vessel. Star shows a blood spermatocytes develop synchronously, i.e. the cells in cell in the lumen of the vessel each cluster are always at the same stage. The cytoplasm Fig. 2 Transmission electron migrograph (TEM) of the blood of the spermatocytes contains mitochondria, Golgi com- vessel wall. Asterisks show the vasoperitoneal tissue cells. Arrows indicate the inner matrix of the blood vessel. Triangles demonstrate plexes, endoplasmic reticulum and vacuoles. The elec- an area of direct contact between spermatogenic cells and the tron-dense vesicles, characteristic of spermatogonia, are matrix. Sc spermatogenical cells, B blood cell no longer seen. Fig. 3 TEM of vasoperitoneal tissue cells (V) surrounded by Primary spermatocytes are almost round (Fig. 1). One spermatogenical cells (Sc). P phagosome character of the primary spermatocytes is the presence of Fig. 4 TEM of spermatogonia (Sg) and spermatocytes (Sc). Arrows synaptonemal complexes in the zygotene-pachytene stage show vesicles of germ plasm of meiosis (Fig. 7). The centriolar complexes consist of Fig. 5 TEM of the centriolar complex in the spermatogonium. two maternal and two developing daughter centrioles Arrow indicates the vesicles of germ plasm. Dc distal centriole, Pc proximal centriole, G Golgi complex, N nucleus 3 4 5 Late stage P. harmeri is an example of ‘flagellate spermatogenesis’ (see Reunov 2001) which is typical of Metazoa with During the late stage of spermiogenesis, the nucleus external or external/internal fertilization and ‘primitive’ elongates rapidly and the chromatin condensation, char- (sensu Franzn 1956) spermatozoa. This type of sperm acterized by many ‘initiation centres’ proceeds (Figs. 20, development has been described for representatives of 21). These ‘initiation centres’ gradually disappear during Porifera (Paulus 1989), Cnidaria (Larkman 1984), Pria- chromatin condensation.