Ultrastructural studies of oogenesis in Bolinus brandari s ( Gastropoda : Muricidae ) *

Muricidae comprise more than 2000 Neogastropod species distributed worldwide. Some of these species are commercially exploited in, for instance, Thailand (Nugranad et al. 1994) and Chile (Gutiérrez and Gallardo, 1999). Bolinus brandaris (Linnaeus, 1758) is common in the Mediterranean and constitutes a locally important resource in Spain, Italy and Turkey (Martín et al. 1995). On the other hand, B. brandaris, like many other prosobranchia species, is affected by the imposex phenomenon (i.e. penis and spermduct are superimposed onto the female gonochoristic ducts; Smith, 1971), in response to tributyltin (TBT) pollution in sea water, mainly caused by anti-fouling paints. This phenomenon is caused by the alteration of the steroid metabolism (Oehlmann et al. 1993) and has been shown to cause alterations to the genital tract (Oehlmann et al., 1991). One of the first reports about prosobranch reproduction is by Schitz (1920), who examined gametogenesis with light microscopy in Hexaplex trunculus. Further details on the ultrastructure of members of the family Muricidae are provided by Bottke (1972) in SCI. MAR., 68 (3): 343-353 SCIENTIA MARINA 2004


INTRODUCTION
Muricidae comprise more than 2000 Neogastropod species distributed worldwide.Some of these species are commercially exploited in, for instance, Thailand (Nugranad et al. 1994) and Chile (Gutiérrez and Gallardo, 1999).Bolinus brandaris (Linnaeus, 1758) is common in the Mediterranean and constitutes a locally important resource in Spain, Italy and Turkey (Martín et al. 1995).On the other hand, B. brandaris, like many other prosobranchia species, is affected by the imposex phenomenon (i.e.penis and spermduct are superimposed onto the female gonochoristic ducts; Smith, 1971), in response to tributyltin (TBT) pollution in sea water, mainly caused by anti-fouling paints.This phenomenon is caused by the alteration of the steroid metabolism (Oehlmann et al. 1993) and has been shown to cause alterations to the genital tract (Oehlmann et al., 1991).

MATERIALS AND METHODS
Thirty female individuals of Bolinus brandaris were collected in April 1999 from a coastal Mediterranean site (Sant Carles de la Ràpita, Spain) at depths of 15 to 25 m using an artisanal dragged gear (Martín et al. 1995).Imposex in this site was monitored in a previous study (Ramón and Amor, 2001) and reached 99.7% of the females examined (N=301).The shell was cracked with a vice and the gonads of imposex females were carefully removed.Thin sections were fixed in 10% formalin and stained with hematoxylin-eosin and the PAS technique (i.e.cytochemical stain with periodic acid and Schiff reagent) for study by light microscopy.Thinner sections were processed for transmission electron microscopy following routine double fixation, i.e. glutaraldehyde and 2.5% OsO 4 , both buffered using Sörensen's phosphate buffer.Samples were embedded in Spurr's resin after progressive dehydration.About 1 mm-thick sections were obtained and stained with methylene-blue borax to select the areas most suitable for the ultrathin sections.These were about 30 nm thick and were cut using a Reichert-Omu ultramicrotome with a diamond knife.Sections were picked up on copper grids and stained with uranyl acetate and lead citrate.Thiery's technique (Thiery 1967) was sometimes used, and then the sections were picked up on gold grids.We used a 301 Philips transmission electron microscope at the Serveis Científico-Tècnics of the University of Barcelona.

RESULTS
Oogenesis of Bolinus brandaris followed four main stages: premeiosis, meiosis, vitellogenesis and the formation of the mature oocyte.Premeiosis and meiosis are characterised by the presence of a round nucleus 6 µm in diameter, in which drops of heterochromatin are spread throughout the nucleoplasm.The cytoplasm is nearly empty, except for some mitochondria and dictyosomes (Fig. 1A).The most developed apparatus is the centriole, shown as a microtubule organising centre (Fig. 2A).
During meiosis, the most outstanding phase is prophase I, in which the nucleus enlarges and chromosomes and synaptonemal complexes appear.As is usual at this stage, the cells are undifferentiated and the nucleus/cytoplasm ratio is high.
Vitellogenesis in B. brandaris can be divided into three main stages: In the first stage, the cell is 30 µm in diameter and the nucleus (22 µm) is round or oval.It shows one or two nucleoli (1.6 µm), in which we can often distinguish granular and fibrillate phases (Figs.1B(a) and 2B).The nuclear envelope shows abundant well-developed pores (1 pore per µm 2 of about 90 nm in diameter).Slight invaginations increase the envelope area and favour the passage of nuclear precursors for vitellogenesis (Fig. 1B(a)).The cytoplasm increases in volume and its organelles increase in both number and in volume.Thus, we can distinguish vacuoles of various sizes, PAS positive ß-glycogen granules and vesicles (4-5 µm) full of material of varying electrondensity.Mitochondria are well-developed (1.5 µm long) and can be rodshaped, curved or elongate.The cristae and matrix are well formed.Both are near the nucleus and their number increases progressively, leading to the formation of mitochondria clusters.Mitochondria divide by bipartition or gemmation (Fig. 2C).At the end of this stage, several mitochondria loose their cristae and become vesicles, and some are invaded by pre-vitelline material (Figs.1B(a) and 2C).
In the plasma membrane, intercellular bridges and desmosomes can be seen among young oocytes, as well as microvilli (Fig. 1B).Desmosomes and intercellular spaces showing accumulations of previtellogenic material are also shown among oocytes and follicle cells caused by electrondense vesicles possibly from follicle cells origin (Figs.3A(a,b) and 3B).Sometimes, the plasma membrane invaginates to form small vacuoles, which aggregate to create a large, round reticule of annulate lamellae (Figs.3A,B).The Golgi body is well developed and is formed by several dyctiosomes, with abundant cisternae.These produce vesicles of varying electrondensity, multivesicular bodies and also annulate lamellae (Fig. 3C(b)).Clusters of annulate lamellae similar to the nuclear envelope are also often found (Fig. 3C(a)).
The rough endoplasmic reticulum (RER) can appear in different shapes and is highly developed.It usually surrounds the nucleus, although it can be found elsewhere.In the latter case, it can be arched, round or cupulate (Figs.2D,E,F).The rough ER can be found next to lipidic vesicles (Fig. 2E), as well as surrounding mitochondria (Fig. 2F).
The advanced stage of vitellogenesis is characterised by the formation of vitelline platelets.The nucleus is still active, although the number of nuclear pores decreases.On the other hand, the amount of cytoplasm and the number of organelles both increase.Thus, during this phase, mitochondria, endoplasmic reticulum and the Golgi body are all well developed.Also, various granules can also be seen entering empty vesicles (Fig. 4A(a) inset, 4A(b)).Glycogen granules, lipidic vesicles and granulated vesicles with striated formations are also detected (Fig. 4A(a) inset, 4A(c,d)).The mature vitelline platelets (Figs.5B(c,e) and 6(h)) typically show a central, electrondense core surrounded by a clear envelope.The core is formed by dark vitelline material surrounded by a membrane; the external envelope of the vitelline platelet is also membranous.
The most important feature of this phase is the vitelline platelet formation.Previtellin material enters empty vesicles, becoming the core (Fig. 4A(a Although all the females examined were affected by the imposex syndrome in degree 4 (Ramón and Amor, 2001), oogenesis alterations at ultrastructural level were not found.

DISCUSSION
The most remarkable features of the oogenesis process in Bolinus brandaris were observed during vitellogenesis.The first stages were characterised by a large nucleus, non-condensed chromatin and developed nucleolus and the existence of nuclear pores which allowed transport of ribonucleoproteins to the cytoplasm, as reported in Mollusca by Davenport and Davenport (1965), Durfort (1973a), Popham (1975), Jong-Brink et al. (1983), and Swenson et al. (1987), as well as in other invertebrates by Coimbra and Azevedo (1984), Larkman (1984), Ribes (1986), and Sciscioli et al. (1991).Although in some Muricidae species a nucleolus vacuolisation was described (Bolognari et al. (1981) in Hexaplex trunculus), this phenomenon has been not detected in B. brandaris.Some cytoplasmic organelles were very developed, and associated with high synthetic activity.Mitochondria were also conspicuous, and various descriptions can be found in Bruslé (1972), Hill and Bowen (1976), Pfanestiel and Grünig (1982), Larkmann (1984), Sciscioli et al. (1991), andSukhomlinova et al. (1998) for several invertebrate groups and in Weakley (1976) for vertebrates.However, no "mitochondria cloud" was detected, as described for Actinia sp. by Larkman (1984).Mitochondria can divided in several ways, as shown for other molluscs in Taylor andAnderson (1969), andin Jong Brink et al. (1976).Mitochondria were often associated to the rough ER, as reported for Mytilus edulis by Durfort (1976a,b).The progressive degeneration of their cristae means that they became empty vesicles, which later appeared to be invaded by vitellogenic material, as has already been described in other molluscs (e.g.Durfort, Durfort 1973a,b;Amor and Ribes, 1995) and invertebrates (Pfanestiel and Grünig, 1982;Larkman, 1984).Despite the similarities, we did not observe the transference of mitochondria from nurse cells to oocytes, as described in some insects (e.g.Tourmente et al., 1990;Stebbings, 1997).
As is common in this phase, annulate lamellae were also observed (Dhainaut and Richard, 1976;Durfort 1973a, b;Durfort, 1976;Hill and Bowen, 1976;Pfanestiel and Grünig, 1982;Kessel et al., 1986).The morphology and localisation of annulate lamellae suggest three possible origins.One is the nuclear envelope, as proposed by Pfanestiel and Grünig (1982), Kessel et al. (1986), andRibes (1986).A second possible origin is the Golgi body, suggested by Jong-Brink et al. (1976).A third possibility derives from the observation of invaginations of the plasma membrane to form large reticulelike annulate lamellae.Similar features were reported by Kessel et al. (1986), leading him to propose a plasma membrane origin to annulate lamellae.Our observation of vesicles that later fill in with electrondense material agrees with this hypothesis.We believe that an endoplasmic reticulum origin for annulate lamellae, as in Mytilus edulis and Trachidermon cinereus (Durfort, 1973a,b;Durfort, 1976a,b), is less probable.The annulate lamellae could represent a reservoir for membranes needed by the cell to coat the previtelline material.
The presence of intercellular open bridges among oocytes in the first stages of oogenesis could indicate a synchronisation role as in the eupyrene spermatogenesis (Amor and Durfort, 1990a).The observation of microvilli-like interdigitations among oocytes connected to the plasma membrane of the follicle cells could indicate an enhanced capture of vitelline material.This material is often accumulated in the intermembranous spaces between oocytes and follicle cells.Similar features have also been described by Durfort (1973a), Popham (1975), andJong-Brink et al. (1976).Griffond and Gomot (1979) also reported this irregular distribution of the oocyte plasma membrane in the prosobranchia Viviparus viviparus.
On the other hand, vitelline material has two possible origins: exogenous, (i.e.produced by follicular cells), and endogenous, (i.e.produced by the oocyte).The exogenous origin means stocks of material origined in follicle cells and accumulated in the spaces between both plasma membranes (i.e.those of follicle cells and of oocytes).The presence of microvilli in the oocyte plasma membrane suggested that this material enters the cytoplasm of the oocyte by endocytosis, as described by Pfannestiel and Grünig (1982) in a polychaete.However, this entrance was apparently not by direct contact, as reported in other species (Harrison and Huebner 1997).The endogenous origin of vitellin material means that it is synthesised by the endoplasmic reticulum and later processed by the Golgi body.
Both organelles were highly developed, which supports this hypothesis.Similar observations have been reported from the mollusc Mytilus edulis (Durfort Durfort 1976a,b).These two possible mechanisms for vitelline material formation were also described for Aplysia depilans (Bolognari and Licata 1981), Planorbis corneus and Lymnaea stagnalis (Bottke, 1972) and Helix aspersa (Barre et al. 1991 andBride et al. 1992).In the advanced stages of vitellogenesis, nuclear activity decreases, as is shown by the reduction in the number of nuclear pores, the higher density of chromatin and the decreasing nucleolus activity.Mitochondria and dyctiosomes were spread throughout the cytoplasm, together with vesicles of different sizes and electrondensity, glycogen granules, lipidic droplets and vitelline platelets in formation.
A central electrondense core surrounded by a clear envelope, as described by Durfort (1973a), forms the characteristic vitelline platelet.The core is formed by vitelline material surrounded by a membrane.There are three possible origins for the core membrane.The first is that it is a degenerate mitochondrium as reported Ribes (1986) and Amor and Ribes (1995).The second is that it origins directly from the Golgi body.Hill and Bowen (1976) described this fact, and the high development of this organelle in the oocytes of B. brandaris agrees with this hypothesis.A further hypothesis suggests that the core membrane comes from the annulate lamellae.The latter idea is in agreement with our hypothesis about the role of the annulate lamellae as a membrane reservoir.A group of Thiery positive membranes surrounded the previtellogenic material as it entered the vesicles.These membranes twisted around the vesicles, thus becoming curly-shaped.This twisting progressively increased, giving the membranes a resemblance to myelin structures.As the twist increased, gaps between membranes closed and it began to fuse, in a way similar to chromatin condensation in male spermatogenesis (Amor and Durfort, 1990a).Vitelline platelets showed different behaviours in response to two cytochemic carbohydrate reactions.There were highly PAS positive (under light microscopy) but non-positive for Thiery's reaction (under electron microscopy).This could have been due to the presence of carbohydrates mixed with other non-positive material, such as the proteins often found in vitellin platelets.We also observed oval striated bodies and vesicles containing striated formations, similarly to what has been observed in the gastropod Murex elenensis (Durfort, 1973a) and in the sponge Stelletta grubii (Sciscioli et al., 1991).Moreover, other vesicles, slightly dyed using Thiery's technique, were also present.Lipidic vesicles were abundant in the cytoplasm, as reported by Popham (1975), Durfort (1976a), and Hill and Bowen (1976) for molluscs, and Pfannestiel and Grünig (1982) for a polychaete.Two origins have been described for the formation of lipid vesicles, i.e. the synthesis by the endoplasmic reticulum (Durfort, 1976b) and the capture from the blood by endocytosis (Ritcher, 1976;Durfort et al. 1982 andJong-Brink et al. 1983).However, in B. brandaris we only found ER vesicles associated with the formation of lipid vesicles.Therefore, the ER seems to be the only way of forming lipid vesicles.
Glycogen granules from the Golgi body were also detected by Thiery's technique.The oocyte plasma membrane increased in thickness.This, and the presence of abundant dyctiosomes, lead us to think that the latter originated in the Golgi body.

FIG. 1
FIG. 1. -A: Ultrastructural panoramic view of the ovary.Young oocytes can be seen (star).The nucleus (n) showing the chromatin dispersed in small drops, the nucleolus (arrowhead) and the mitochondria (m) are apparent in the cytoplasm.Note the non-germinal cells, apparently connective cells (asterisk), too.B: Detail of an oocyte in early stages of oogenesis.(a): The nucleus (black N) shows a compact and large nucleolus (white N).The chromatin is granulated, although some condensed droplets appear in the caryoplasm (arrowheads).The nucleus envelope shows 'undulations' and nuclear pores (black asterisk).The cytoplasm is limited by the plasma membrane (pm) showing microvilli (white arrowhead).In the cytoplasm, note the clusters of mitochondria (mt).Vesicles of variable electrondensity (star) are detected, together with lysosomes (L).The white asterisk shows electrondense material embedding degenerate mitochondria, which still remain cristae (black arrow).Other electrondense vesicles with mitochondria morphology are also observed (black arrows).Outside the oocyte, a follicular cell(F) with glycogen granules (g) are shown and below, connective tissue (C) is detected; (b): microvilli (asterisk), present between two oocytes (O1 and O2), are shown.
FIG. 5. -A:Longitudinal section of mature oocytes limited by the young vitelline membranes (P).A decreasing nucleus (N)/cytoplasm ratio is apparent, caused by the numerous organelles incorporated into the cytoplasm.The most remarkable are the characteristic vitelline platelets (arrow).There are also other vesicles, different in both size and electrondensity (asterisks).B: Different performances of the evolution of the vitellin platelets using the Thiery's technique.(a): shows non-twisted membranes around the core; (b): membranes around the core begin to twist.See the positive reaction of their membranes to Thiery's technique; mature vitellin platelets are present (asterisks in c and e).The homogeneous envelope around the core is shown.On the other hand, note the contrast between the reaction to Thiery's of glycogen granules (white asterisk in c and e) and the poor reaction shown by the core.Lipidic plateles are also seen in (c) (square); (d): highly Thiery-positive vesicles are seen.
FIG. 6. -Sequence of the progressive organisation of vitelline platelets.(a): membranes are still non-twisted; (b): membranes begin to curve; (c): the twisting of membranes continues; (d): membranes become curly-shaped; (e): curling increases; (f): Membranes fuse as curling process and the vitelline platelet periphery seems homogeneous; (g): the vitelline platelet is almost formed; (h): mature vitelline platelet.The core and the periphery are homogeneous.