were sectioned at a thickness of 4 gm and stained with erythrosine aurantia-orange G and toluidine blue. For cytological study, pieces of gonads were fixed in ...
Cell Tissue Res (1994) 276:123-132
Cell&Tissue Research 9 Springer-Verlag 1994
Resorption of unemitted gametes in Lithognathus mormyrus (Sparidae, Teleostei): a possible synergic action of somatic and immune cells Laurence Besseau*, Elisabeth Faliex Laboratoire de Biologie Marine, Universit6 de Perpignan, 52, Avenue de Villeneuve, F-66860 Perpignan Cedex, France Received: 21 May 1993/Accepted: 5 August 1993
Abstract. The resorption of unemitted gametes during the post-spawning period of the male and female reproductive cycles in Lithognathus mormyrus was studied by histochemical, histological and cytological methods. The resorption of residual spermatozoa involved the phagocytotic activity of Sertoli cells bounding the seminiferous cysts of spermatozoa, and those associated with spermatogonia lining the lobular lumen. Spermatozoa remaining in the sperm duet were phagocytozed by the lining epithelial cells. Eosinophilic granulocytes and macrophages were identified in the vicinity of residual spermatozoa. The remnants of oocytes underwent an atretic phenomenon in which follicle cells were firstly involved, inducing a progressive fragmentation of the oocyte cytoplasm. Subsequently, eosinophilic granulocytes invaded oocyte degenerative areas and clung to the remaining vitelline inclusions ensuring their biotransformation into waste products (brown bodies). The analogy of the resorption processes of both male and female unemitted gametes during the post-spawning period of natural reproductive cycle, involving first the enveloping somatic cells and then immune cells, is emphasized.
During the post-spawning period, the resorption occurs of mature genital products that have not been emitted, and the gonial recolonization of the gonad takes place. Although studies of the recovery processes are numerous, few data have been reported concerning the cellular mechanisms involved in the resorption of unemitted gametes (follicle cells: Lang 1981; Sertoli cells: Billard et al. 1972). Nevertheless, in sparids, these unemitted gametes, especially female gametes, have frequently been detected by authors (Mich61e 1972; Pollock 1984). A histocytological study concerning the sexuality of the stripped bream, Lithognathus rnorrnyrus (Besseau and Faliex 1990; Besseau 1991), has shown that the residual gametes of both sexes are particularly numerous. L. mormyrus is a c o m m o n mediterranean sparid, in which protandric hermaphroditism has been reported (D'Ancona 1949). Therefore, we have analysed by histo-cytological criteria, the cellular processes involved in the resorption of unemitted gametes during the post-spawning period of male and female reproductive cycles in L. mormyrus.
Key words: Unemitted gametes - Resorption - Somatic cells - Immune cells - Post-spawning period Lithognathus mormyrus (Teleostei)
Materials and methods
Introduction Teleosts from temperate areas exhibit reproductive cycles with an annual rythm, discernible by the chronology of structural modifications of the gonad (Billard and Breton 1981; Brusl6 1982). The cycle can be divided into four main gonadal stages: the sexual resting, gametogenic activity, spawning, and post-spawning phases. * Present address and address for correspondence: Observatoire Oc~anologique de Banyuls, Histo-Cytophysique EPHE, Laboratoire Arago CNRS URA 117, F-66650 Banyuls sur mer, France
A total of 397 Lithognathus mormyrus was caught monthly over 3 consecutive years (1986-1988), using a beach seine, professional trawls and nets, on the north-western coast of the Mediterranean Sea (Gulf of Lion, Roussillon, France). Fishes were measured (furcal length), weighed and killed by decapitation. Gonads were removed and fixed in Bouin-Hollande's solution and embedded in paraffin for histological study. Samples were sectioned at a thickness of 4 gm and stained with erythrosine aurantia-orange G and toluidine blue. For cytological study, pieces of gonads were fixed in 3% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.4, at 4~ C for 4 h. Samples were post-fixed in 2% OsO4 in the same buffer, dehydrated in a graded acetone series and finally embedded in Epon (Brusl+ 1983). Semi-thin sections (1 gm thick) were stained according to Richardson et al. (1960) and ultra-thin sections (50-60 nm), according to Reynolds (1963). Immune-cell recognition was performed, on histological sections, by Giemsa-L staining. Natural pigments were characterized by the following histochemical tests: PAS, Hueck, Ziehl-Neelsen,
124 Schmorl and Perls (Martoja and Martoja-Pierson 1967; Gabe 1968; Ganter and Jo!les 1970).
Results
Sparids are characterized by the peculiar morphology of their heterosexual gonad, the ovotestis, which consists of a medio-dorsal ovarian area and a latero-ventral testicular territory, separated by a connective wall (Fig. 1). Each gonadal zone, successively functional according to the protandric pattern (male ~ female), shows a similar structure to that known in gonochoristic teleosts (seminiferous lobules: Billard 1986; ovarian lamellae: Nagahama 1983).
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Male post-spawning period
The recovery phase occurs shortly after spermiation and consists of the colonization of seminiferous lobules, by increasing numbers of spermatogonia, in a centripetal direction. During, and particularly at the beginning of this recovery period, voluminous residual bodies can be recognized in the cytoplasm not only of Sertoli cells bounding cysts of spermatozoa (Fig. 2), but also of Sertoli cells associated with spermatogonia lining the lobular lumen (Fig. 3). The presence of membranous digitations located at the periphery of the Sertoli cells surrounding spermatogonia and directed towards residual spermatozoa, emphasized by the presence of concomitant intracytoplasmic heterophagous vacuoles, suggests that these cells phagocytose unemitted male gametes. In addition, during this post-spawning phase, numerous immune cells, such as granulocytes, can be observed in the central part of the testicular territory at the histological level. Giemsa-L tests on paraffin sections of such areas have revealed the eosinophilic affinity of these cells, allowing us to diagnose them as eosinophiiic granulocytes. At the ultrastructural level, these immune cells have been also observed among degenerative spermatozoa, in the lobular lumen (Fig. 4). However, no phagocytotic activity has been detected in these granulocytes. A few scattered macrophages can also be observed in the lobular lumen; they exhibit phagocytotic activity against residual spermatozoa. Numerous heterophagic vacuoles are found in the cytoplasm of the lining epithelial cells, along the sperm duct (Fig. 5). In addition, these cells show many membranous digitations directed towards the remaining spermatozoa, leading to a possible exfoliating-like process (Fig. 6). The male sexual resting phase begins at the end of residual sperm elimination and of spermatogonial recoIonization of seminiferous lobules. Female post-spawning period
In L. morrnyrus, in accordance with previous descriptions in teleosts, ovarian lamellae after spawning show many lacunae that correspond to the location of emitted
semi Iobulneisferou~
Fig. 1. Morphology of the sparid heterosexual gonad
oocytes. These lacunae, still limited by the remaining follicle and thecal envelopes, become filled up in a centripetal direction by both follicle and thecal cells. Finally, they are resorbed inducing an important decrease in ovarian lamellae depth and consequently in the volume of the ovary. At this stage of the ovarian cycle, the ovarian lamellae still contain vitellogenic oocytes that have not been emitted during the spawning period. In L. mormyrus, the number of unemitted oocytes varies among individuals and is, in many cases, high. These variably sized unemitted oocytes correspond to different vitellogenic stages and are devoid of a pellucid zone, so that follicle cells are contiguous with the oocyte cytoplasm (Fig. 7). The residual oocytes become degenerative according to an atretic process in which follicle cells are first involved. The latter cells develop membranous digitations that extend towards the oocyte cytoplasm, which they finally penetrate, losing contact with the basal laminae (Fig. 8). The oocyte cytoplasmic membrane disrupts as soon as the atretic process begins. Follicle envelopment continues by the sliding of follicle cells around the oocyte (Fig. 9). Membranous digitations extended by these follicle cells contribute to a progressive fragmentation of the oocyte cytoplasm, residual elements of which appear within voluminous phagosomes located in the cytoplasm of the follicle cell (Fig. 9). Concomitantly, numerous membranous organelles, such as dictyosomes, rough endoplasmic reticulum and lysosomal vesicles, can be observed in the cytoplasm of the
125
Figs. 2-6. Male post-spawning period in L. mormyrus Fig. 2. Residual spermatozoa (rSZ) in heterophagic vacuoles contained in the cytoplasm of a Sertoli cell (SC) bounding a cyst. z9950. Bar: 1 Ixm Fig. 3. Residual spermatozoa (rSZ) in the lobular lumen. Residual bodies in the cytoplasm of Sertoli cells (SC) surrounding spermato-
gonia (SG). Note the membranous digitations (rod) extending from the Sertoli cells, x 8200. Bar: 1 gm Fig. 4. Eosinophilic granulocytes (eG) among residual spermatozoa (rSZ) in the lobular lumen. Spermatogonium (SG). • 6000. Bar: 2 gm
follicle cells (Fig. 10). During the atretic process, the content of heterophagic vacuoles increases in electron density, thereby revealing its progressive degradation and leading to remnants in the shape of myelin figures and lipofuscins (Fig. 11). This atretic process occurs in a centripetal direction so that lacunae appear in the oocyte;
the follicle cells also become degenerative (disruption of cellular limits, loosening of cytoplasmic cohesion) (Fig. 12). G r a n u l a r leucocytes, identified by the Giemsa-L test as eosinophilic granulocytes, are secondarily involved in the atretic process. They invade degenerative oocyte
126
Figs. 5, 6. Male post-spawning period in L. mormyrus
Fig. 5. In the sperm duct, epithelial cells (EC) show heterophagic vacuoles (by) and membranous digitations (rod) extend towards residual spermatozoa (rSZ). x 3000. Bar: 3 gm
areas through the basal laminae which show, at this stage of the atretic process, weakened zones revealed by high electron density and vague limits. Eosinophilic granulocytes cling to the remaining vitellin inclusions and most often release their granule contents, which probably contain lytic enzymes (Figs. 13, 14), thereby ensuring their progressive biotransformation into waste products. At the end of the atretic process, granulocytes, now devoid of their granule contents, become degenerative within these areas of follicle and oocyte waste, which appear as deposits of yellow-brownish pigments (Figs. 15, 16). These clusters are preferentially located in the median area of the ovarian lamellae because of their active recolonization by germ cells. Histochemical tests have been carried out on paraffin sections of such ovaries in order to characterize these waste products, usually n a m e d " brown bodies". The results of this analysis, summarized in Table 1, have led to their identification as chromolipoids, and more precisely lipofuscins. Moreover, the presence of polyunsaturated fatty acids and carboxyl residues has been suggested by the PASpositive reaction. These clusters of waste products progressively disappear during further reproductive cycles, probably via the bloodstream, in view of their close proximity to blood vessels. During resorption of lacunae and oocyte atresia, numerous oogonia, meiotic oocytes and small previtellogenic oocytes have been observed, testifying to the reconstitution of a new stock of female germ cells. The ovarian territory subsequently enters the resting phase.
Fig. 6. Epithelial cells show an exfoliating-Iike process in the most distal part of the spermduct. Membranous digitations (mar),heterophagic vacuoles (hv), residual spermatozoa (rSZ). x 3000. Bar: 3 gm
Discussion
Cellular processes involved in the resorption of unemitted gametes have been investigated in a sparid species described as a protandric hermaphrodite, L. morrnyrus; this species possesses a heterosexual gonad, viz. the ovotestis, as do all the species of this family. These processes have been followed during the post-spawning period of both male and female reproductive cycles.
Male post-spawning period
In L. mormyrus, during the male post-spawning period, special attention has been paid to the resorption of unemitted spermatozoa. These unemitted gametes are firstly submitted to phagocytotic activity, which involves not only the Sertoli cells lining the spermatozoa cysts and those surrounding spermatogonia located in the vicinity of the lobular lumina, but also the epithelial cells from the sperm duct. Subsequently, macrophages exhibiting phagocytotic activity and eosinophilic granulocytes participate in the elimination of residual spermatozoa. Sertoli cells surrounding spermatozoa-containing cysts have often been recognized as being responsible for the elimination of male gametes remaining in the lobular lumen after spawning, before themselves entering into a degenerative process (Billard et al. 1972; Gresik et al. 1973; Grier and Linton 1977; Van Den Hurk et al. 1978; Grier 1981; Billard 1970; 1986; Grier et al. 1989; Lahnsteiner and Patzner 1990). Our observations are in agreement with these previous studies. However, the phagocytotic activity of the spermatogonia-sur-
127
Figs. 7-9. Female post-spawning period in L. mormyrus Fig. 7. Unemitted vitellogenic oocyte (aO), devoid of the pellucid zone, at the beginning of the atretic process. Follicle cells (FC) developing membranous digitations (rod) towards the oocyte cytoplasm, x 4900. Bar: 2 gm
Fig. 8. Follicle cell (FC) within the atretic oocyte cytoplasm (aO). x 7900. Bar: 1 gm Fig. 9. Follicle cell (FC) showing voluminous phagosomes (ph). x 8000. Bar: 1 Ixm
128
Figs. 10-12. Female post-spawning period in L. mormyrus Fig. 10. Numerous dictyosomes (d), rough endoplasmic reticulum (rer) and lysosomal vesicles (lv) within the follicle cell cytoplasm (FC). The heterophagic vacuoles (by) show an increasing electron density, testifying to the progressive biodegradation of their content. x 10100. Bar: 1 pm
Fig. 11. Lipofuscin residual bodies (/J) of high electron density within the cytoplasm of the follicle cell (FC). x 4700. Bar: 2 gm Fig. 12. Appearance of lacunae (la) in the atretic oocyte. Degenerating follicle cells (dFC) containing voluminous lipofuscin residual bodies (/.1). Basal lamina (b/), previtellogenetic oocyte (pO). x 2800. Bar: 3 gm
129
Figs. 13, 14. Female post-spawning period in L. mormyrus Fig. 13. Eosinophilic granulocytes (eG) invading the oocyte degenerative area; yolk inclusions (yi). x 5000. Bar: 2 gm
Fig. 14. Degranulating process in the vicinity of the remaining yolk inclusions (yi). Eosinophilic granulocyte (eG), granule (gr). x 20200. Bar: 0.5 gm
rounding Sertoli cells against residual spermatozoa has never been recorded in the literature. In L. mormyrus, this phagocytosis is clearely revealed by the presence both of membranous digitations developing towards residual spermatozoa and heterophagic vacuoles within cytoplasm of the Sertoli cells. The subsequent development of these Sertoli cells remains to be elucidated: either they degenerate after their phagocytotic activity and are eliminated by an exfoliating-like process in the lobular lumen (in this case, spermatogonial envelopment would be ensured by the division and/or migration of the remaining Sertoli cells that have not intervened in the phagocytotic process), or they would eliminate the residual bodies by exocytosis during the following resting phase (in this case, they should be capable of recovery). Thus, the functional duality of the Sertoli cells surrounding spermatogonia during the post-spawning period in L. mormyrus is worth emphasizing. With regard to the possible activity of immune cells within post-spawning testes, although the presence of phagocytes of undefined cellular identity has been previously described (for a review, see Grief 1981), that of macrophages has rarely been mentioned (Billard et al. 1971 ; Billard 1986). The presence of eosinophilic granu-
locytes in such testes is described here for the first time. In L. morrnyrus, more eosinophilic granulocytes are present in the ovotestis (Besseau and Faliex 1990; Besseau 1991) than is usually reported in teleosts, in contrast to the situation in cases of bacteriosis, parasitosis or inflammation (Lester and Desser 1975; Lester and Daniels 1976; Powell et al. 1990). Although no phagocytotic activity or degranulating process of eosinophilic cells, as previously described (Page and Rowley 1983; Hine and Wain 1988), has been observed in the testes of L. rnormyrus, the presence of these cells is probably related to the resorption of unemitted spermatozoa. However, mechanisms involving immune cells and their functioning in the resorption of residual sperm remain to be elucidated. Finally, in L. mormyrus, phagocytotic activity against residual spermatozoa had also been detected in the epithelial cells lining the lumen of the sperm duct. This observation has also been previously mentioned in teleosts, particularly in Oncorhynchus mykiss, kept in captivity and for which the quantity of residual sperm was considerable (50%-80%) (Billard and Takashima 1983).
130
Figs. 15, 16. Female post-spawning period in L. mormyrus Fig. 15. Eosinophilic granulocytes (eG) in the vicinity of the oocyte and follicle waste, giving deposits of yellow-brownish pigments (brown-bodies, BB). Follicle cell (FC) x 4300. Bar: 2 gm
Fig. 16. Brown-body (BB): lipofuscin (/J) and myelin figures (ma0. x 5700. Bar: 2 p~m
1?able 1. Histochemical characterization of the waste products h~the ovarian part of the ovotestis of L mormyrus Products
Methods
According to Martoja and Martoja-Pierson (1967) Cbomolipoid Ceroid
Chromolipoid Ceroid Lipofuscin
HUECK ZIEHL Fluorescence (excitation at 360 ,ma) SCli~O~
Melanin Polyunsaturated fatty acids
PAS
Our results Melanin
Lipofuscin
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