Ultrastructural Study of Spermatogenesis in Eisenia foetida (Annelida, Oligochaeta)
Rolando, A.; Romanini, M. C. Mugnaini, M. T. Bozzo, A. Pastorino, I. & Soñez, C. A.
Int J. MorphoL, 25(2)-.277-284, 2007. Open Access Article.
SUMMARY: Spermatogenesis and the spermatozoon ultrastructure follow a common pattern in terrestrial oligochaetes. However, many of their characteristics are distinctive of each species and they are of great importance for phylogenetic and taxonomic studies.
Germinal cells were studied through conventional electronic microscope techniques in order to go deep on spermatogenesis, especially on the mature spermatozoon ultrastructure of Eisenia foetida.
Spermatogenesis occurs in seminal vesicles and follows a pattern comparable to that of some other oligachaetes species. Spermatozoon ultrastructural details revealed that it is a filiform cell, with the acrosome placed in anterior position, followed by the nucleus, the midpiece and the tail which flagellum has a 9+2 typical microtubular configuration. We may conclude that Eisenia foetida spermatozoon is of a apomorphic type since it presents several evolved characteristics such as: a very large acrosome; the primary acrosomic vesicle is held within the acrosomal tube, the axial rod is very elongated and ended in a developed capitulum and a number of mitochondria of the midpiece equal to six.
KEY WORDS: Oligochaeta; Spermatogenesis, Ultrastructure; Spermatozoa; Eisenia.
In earthworms spermatogenesis takes place in seminal vesicles where germinal cells arrive after the beginning of their development in testis. In the interior of seminal vesicles the germinal cells appear in groups called morulae. These morulae have different number of cells that may grow in geometric progression according they belong to morulae of spermatogonia, spermatocytes or spermatids. In all cases the germinal cells are attached through cytoplasmatic bridges to a central anucleate mass of cytoplasm and rich in organelles: the cytophore. The only independent cells are the spermatozoa since they detached from cytophore once the spermiogenesis processes is over.
Spermatogenesis including spermiogenesis and the mature spermatozoon ultraestructure have been studied in several orders and oligochaete families (Jamieson et al, 1981; Anderson et al, 1967; Gluzman, 1999; Ferraguti, 1984). These studies have demonstrated that spermatogenesis follows a basic patter common for all oligochaete (Ferraguti & Jamieson, 1984). It has also been demonstrated that mature spermatozoon, significantly differs in its ultrastructure, from others clitellate spermatozoa (Branchiobdelliday Hirudinea) and even more from Polychaetes. It can be assured that each family has its own distintictive form of basic oligochaetes spermatozoon (Jamieson et al, 1982).
Spermatozoa are extremely specialized cells which morphology would be determined by the interaction between the phylogenetic history of the group and the local adaptations to different reproductive modalities (Ferraguti etal, 1996; Baldo et al, 2005; Purschke et al, 2000). The knowledge of spermatozoon ultrastructure details of a certain species allows to use the spermatozoon as a tool for phylogenetic and taxonomic studies. The spermatozoon ultraestructure can be used even to distinguish species within a genre (Ers鵳 et al, 1995). Several authors have considered the spermatozoon ultrastructure study very useful for the phylogenetic studies of oligochaetes as well as of many other invertebrates and vertebrates groups, such as cephalopods (Ribes et al, 2002), insects (Jamieson et al, 1999; Jamieson, 2000) and fish (Jamieson, 1991).
According to Franz鮧s observations (1974 and 1977) annelides spermatozoa are of the primitive type en Polychaetes and Archiannelides and of the modified type in Oligochaetes and Hirudinea, which is correlated with fertilization modalities. When there is an internal fertilization or the spermatozoa are liberated near the female genital pore, the spermatozoon morphology varies from its primitive form to the one of the modified type (Franz鮬 1974) or introsperm (Jamieson & Rouse, 1989). In these last ones the nuclei and acrosome are strongly elongated. From this classification, it is observed that oligochaetes have a modified type of spermatozoon or introsperm. Thus, the study of the acrosome being especially informative is very important. The acrosome is one of the most complex structures of animal kingdom and in the clitellate has variations within each group (Westheide & Purschke,1996).
Several authors have studied Eisenia foetida spermatogenesis (Herlant-Meewis, 1959, Martinucci& Felluga, 1975; Martinucci et al., 1977) but there are still ultrastrectural details of the mature spermatozoon to be known.
The aim of this work is the spermatogenesis ultrastructural study and, particularly, Eisenia foetida s spermatozoon to contribute to get new knowledge for phylogenetic and taxonomic studies.
Clitellate specimens of Eisenia foetida were obtained from static piles of stabilized aviar waste, settled for worm cultures and placed in the campus of the Universidad Nacional de RCuarto, Argentina.
Mature worms were dissected and the seminal vesicle, seminal funnels and spermathecae removed for processing. After 2h in fixer Karnovsky (2% glutaraldehyde and 2% parafhormaldehyde in 0,1M cacodylate buffer at pH 7,4) the tissue pieces were washed in 0,1 M cacodylate buffer (pH 7,4) and post-fixed in 1% osmium tetroxide, similarly buffered, for lh. Following dehydration in an acetone series, the tissues were embedded in resin Epon 812 and polymerized by maintaining in a stove for 72 hours at 60°C.
Ultrathin sections were stained with uranyl acetate followed by lead citrate, examined and photographed with a Siemens Elmiskop 101 and JEM 1200 EXII electron microscope.
Spermatogenesis. Spermatogenesis begins in testis where spermatogonia divide mitotically and with an incomplete cytokinesis originating a common cytoplasmatic mass, the cytophore which connects, polarizes and synchronizes the surrounding cells through cytoplasmatic bridges. From there, spermatogonia move to the seminal vesicles where spermatogenesis is completed. Spermatogonia are irregular cells with a very big nucleus with disperse chromatin that allows to visualize the nucleolus. The cytoplasm is scarce; Golgi complexes, free ribosomes and cisterns of the rugose endoplasmic reticulum are observed in this cytoplasm. Spermatogonia ma are composed of a few cells attached to a cytophore in formation. On the contrary, spermatocyte ma are composed of numerous cells attached to the cytophore (Fig.1a). These cells nucleus is round with the chromatin partially condensed and it is surrounded by a double membrane with a well-defined perinuclear cistern (Fig.1b). In the cytoplasm the organelles are generally placed at a distal position of the cytoplasmatic bridges.
Immature spermatids are isodiametric cells with a nucleus more or less round which occupies the majority of the cell. The chromatin, in the interior of the nucleus, is uniformly distributed in small cords and it only occasionally adheres to the internal nuclear membrane (Fig.2). Perinuclear cistern is normally dilated abundant free ribosomes, rugose endoplasmic reticulum, polyribosomes and a few mitochondria can be observed in the small cytoplasm while microtubules are not. Cytophore reaches it maximum expansion, it has an irregular form and multivesicular bodies and numerous mitochondria. It also presents well developed cisterns of the rugose endoplasmic reticule and abundant free ribosomes (Fig. 3). At this stage the nucleus sends finger like projections towards the cytophore through cytoplasmatic bridges (Fig. 2).
Spermiogenesis. The beginning of this stage is characterized by the formation of manchette. This is a row of microtubular elements around the spermatids external nuclear membrane. After the apparition of the manchette a round nucleus with the chromatine forming dense filaments and the perinuclear cistern collapsed is observe (fig. 3). Later, the nucleus begins to elongated and its size is reduced. Chromatin appears condensed and forming a peripheric ring attached to the internal nuclear membrane (Fig. 4). Manchette's microtubules and free microtubules in the cytoplasm are observed around the nucleous (Fig. 4).
A great chromatin condensation can be observed in the final spermiogenesis stages. The nucleus reduces it size and shows an elonged and cilindric shape (Fig. 5). Manchette's microtubules decrease in number until they disappear in the mature spermatozoon. When these microtubules disappear the perinuclear cistern visualizes again (Fig. 5).
During spermiogenesis the primary acrosomal vesicle appears in spermatide cytoplasm. It is small and originate in close proximity to the very developed Golgi apparatus (Fig. 4). This vesicle is covered by the plasmatic membrane that protrudes in this region. The early second rudiment of the acrosome is a dense granular material that appears under the primary acrosomal vesicle which is called dense granule and it is considered as the precursor of the axial rod or acrosomal rod (Fig. 6). The acrosomal tube, which is first a short cylinder to elongated later, appears under the dense granule (Fig. 6). The primary acrosomal vesicle grows becoming more concave and giving place to the secondary acrosomal vesicle or subvesicular space. This vesicle withdraws into within the primary acrosomal vesicle and strangles itself lightly (Fig. 6). The axial rod is formed from the dense granule. This axial rod is a medium electrodense structure which develops into the secondary acrosomal vesicle or subvesicular space causing its elongation. The rod distal extreme is widened and is called capitulum. From the dense granule it also appears, a short tube called periaxial sheath. Finally, the acrosomal tube grows very much and covers the primary and secondary acrosomal vesicles, the axial rod and the periaxial sheath. Thus, the acrosomal complex of the mature spermatozoon is formed. In its morphogenesis, this acrosomal complex changes its position until it definitely places on the tip of the nucleus (Fig.7).
During spermiogenesis the mitochondria migrate towards the nucleus distal extreme where they place in a six mitochondria annular configuration; thus they constitute the midpiece (Figs. 8 a y b). The distal centriole, which will originate spermatozoon flagellum, places following this midpiece.
At the beginning of spermiogenesis, the cytophore is as big as it was in the previous stage. Globular mitochondria frequently clustered, abundant multivesicular bodies and rugose endoplasmic reticulum are found in the cytophore in this stage (Fig. 3). The cytophore shrinks and its components undergo condensation at the end of spermatogenesis.
Mature spermatozoa. Mature spermatozoa were observed in seminal funnels and in spermateca. They are free cells independent of the cytophore. They are filiform, very elongated are surrounded by a cellular waved membrane. Acrosomal complex, nucleus, midpiece and flagellum can be distinguished in spermatozoa.
Acrosomal Complex. In the mature spermatozoon, the acrosomal complex is a very elongated structure and surrounded by the plasmatic membrane, which shows electrodense granules arranged with an unimterrupted sequence. It is placed in the cellular nucleus apix and presents several parts: primary acrosomal vesicle, secondary acrosomal vesicle, axial rod and capitullum, acrosomal tube and periaxial sheath (Fig. 7).
Nucleus. Mature spermatozoon nucleus is elongated, cylindrical and erected. It is truncated in its apical part, in the joint with the acrosome. Its chromatin is very condensed and electrodense. A complete residual cytoplasm reduction and the disappearance of the "manchette" microtubules can be observed (Fig. 7).
Midpiece. The midpiece of the mature spermatozoon is a cylindrical and straight structure placed between the nucleus and distal centriole from which the flagellum axonema will originate (Fig. 8a). In cross section the midpiece has a circular shape and is formed by six mitochondria surrounded by the plasmatic membrane. The six mitochondria are placed around a central axis which has electrodense material. The membranes of the adjacent mitochondria are very close to each other and tight forming septum that irradiate from the central axis (Fig. 8b). A few crests of an irregular form and projected inside the mitochondrial matrix are observed.
4. Flagellum. The flagellum follows the pattern described for some other species. The spermatozoon flagellar axonema has the conventional configuration 9 + 2: nine pairs of peripheric microtubules and two central microtubules (Fig. 9). Two dense fibers appear next to the central microtubules and form with them a tetragonal configuration, typical for oligachaetes. Big dense granules corresponding to glycogen appear around the peripherical microtubules (Fig. 9). These granules decrease in quantity towards the tail distal extreme. The plasmatic membrane covers the axonema and glycogen granules.
Spermatogenesis in Eiseniafoetida follows a pattern similar to the other earth oligochaetes but increasing research is clarifying our knowledge on this process. Germinal cells are spheric during the first spermatogenesis stages (Anderson et ah; Felluga & Martinucci, 1975; Martinucci & Felluga, 1972). Then, the spermatids undergo a rapid elongation during the spermiogenesis; these changes would be associated to the apparition of the "manchette" (Anderson et ah; Jamieson, 1981; Ferraguti, 1984). A nuclear morphogenesis inducting role has been assigned to the manchette; besides, it would produce perinuclear cistern collapse and chromatine condensation (Ferraguti & Lanzavecchia, 1971; Felluga & Martinucci; Jamieson, 1981; Jamieson et ah). Lanzavecchia and Lora Lamia Donin (1972) demonstrated this for Tubiflcidae and Lumbricus. They observed that in the area where microtubulescontact the perinuclear cistern, this collapses and the first chromatin condensations appear. In agreement, this work results show that at the time the manchette microtubules are observed, the perinuclear cistern disappears and chromatin begins to condense. It was also possible to observe mature spermatids with condensed chromatin nuclei corresponding to stages D, E and F described by Ferraguti & Lanzavecchia for Tubiflcidae. According to Jamieson (1981), the nuclear condensation is accompanied by the nucleoplasm elimination within the vesicles. Particularly, in Eisenia foetida, the transfer of nucleoplasm towards the cytophore would produce the nucleoplasm elimination. This phenomenon, indicated by the presence of nuclear projections, was observed in this work and by Martinucci et al.
Another point concerning spermatide morphogenesis to be remarked is the acrosomal complex formation. Anderson et al. demonstrated for the first time, in Lumbricus terrestris the fusion of the vesicles of the Golgi apparatus to form the acrosome. The acrosome originates on the spermatide base, the first part to be formed is the primary acrosomal vesicle, which grows and projects, pushing the spermatide plasmatic membrane (Anderson et al.; Ferraguti & Lanzavecchia). Then, this structure migrates to the tip of the nucleus which is now highly condensed and elongated.
Thus, the origin of the axial rod from a dense electron granule is demonstrated. One or two granules starting this origin are common for the oligochaetes (Jamieson, 1981).
On the other hand, mitochondria in spermatids undergo a modification in its form and intracellular distribution. At spermiogenesis beginning there numerous mitochondria distributed in the cytoplasm but only six are moved to the region of the midpiece. During the spermiogenesis process the spherical mitochondria transform into six pyramidal sub-unities constituting the midpiece. This mitochondria have very irregular crests. This is not a common detail and it is not frequently described in literature for other oligochaetes species. The microtubules surrounding the midpiece in early spermiogenesis stages here observed were also observed by Anderson et al. in Lumbricus. These microtubules probably, provide the force for lateral fusion and mitochondria reorganization. The midpiece with its mitochondria provides an energetic resource for locomotion and fertilization.
The dense granules observed in the spermatozoon tail, placed between the axonema microtubules and the plasmatic membrane, were described by Anderson et al. also for Lumbricus terrestris. These granules correspondiycogen deposits that would be used as substrate for the ATP production by the mitochondria of the midpiece. They are also observed in Tubiflcidae (Ferraguti & Lanzavecchia; Jamieson & Daddow, 1979) and in Megascolecidae and Phreodrilidae (Jamieson, 1981).
Jamieson (1981) refers that glycogen would also be an important energetic reservoir for spermatozoon anaerobic respiration when it remains stored for long periods in the spermateca.
We may conclude that Eisenia foetida spermatozoon ultrastructure corresponds, in a general aspect, with the descriptions done for other oligochaetes spermatozoons. They are apomorphic type evolved spermatozoons as they present many of the characteristics described by Jamieson et al for this classification. These characteristics are: even nuclear extreme in the joint with the acrosome; a very large acrosome; acrosomic vesicle held and deeply withdrew within the acrosomal tube; the axial rod is very elongated and ended in a developed capitulum and a number of mitochondria of the midpiece equal to six, which is considered another evolved characteristic.
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Fig. 2. Immature spermatids. Nucleus (N) with chromatin distributed in small cords. The nucleus sends finger like projections (*) towards the cytophore. X 14,000
Fig. 3. spermatids morulae. Nuclei (N) with perinuclear cistern collapsed. The cytophore (C) is big. Mitochondria (M), abundant multivesicular bodies (Mb) and rugose endoplasmatic reticulum (ER) are found in the cytophore in this stage. X 8,000
Fig. 4. Cross section of the spermatids nuclei. It its surrounded by a single circlet (arrow) of microtubules that form the manchette. Chromatin appears condensed and forming a peripheric ring joined to the nuclear envelope. Early primary acrosome vesicle (pav) near the Golgi apparatus is observed. X 20,000.
Fig. 5. Longitudinal and cross sections of the immature spermatozoon. Elongated nucleus (N) with the condensed chromatin and surrounded by the perinuclear cistern (arrow). X 30,000.
Fig. 6. In this elongate spermatid the formation of the acrosomic complex is observed, The enlarged primary acrosome vesicle (PAV) is causing protrusion of the plasmatic membranes and is invaginated to form the secondary acrosome vesicle (SAV). The acrosome tube (ST) and the dense granule (DS) are also observed. X 30,000.
Fig. 7. Mature spermatozoon. Detail of the acrosome: primary acrosome vesicle (pav); secondary acrosome vesicle (sav); axial rod (ar); capitulum (ca); acrosome tube (at); secondary tube (st); and nucleus. The nuclear tip is plane. X 40,000.
Fig. 8 a) The elongating mitochondria of the midpiece (M)have aggregated around the longitudinal axis between the nucleus (N) and the distal centriole (dc), wich forms the basal body of the axoneme. X 40,000. b) Cross sections of the midpiece (M), in wich the six mitochondria are around a central axis. X 80,000
Fig. 9. Longitudinal sections of the spermatozoon tail. Central and peripheral microtubules (m) are observed. Dense glycogen granules (gg) are arranged in a linear.