Histochemical characterisation of oocytes of the swordfish Xiphias gladius

SUMMARY: This paper reports a histological, histochemical and immunohistochemical characterisation of growing oocytes of the swordfish Xiphias gladius . The presence and distribution of carbohydrates, proteins, lipids, calcium, iron, vitellogenin/ Vg, zona radiata protein/Zrp, metallothionein/Mt, and thyroid hormones/T 3 -T 4 were studied during oogenesis (cortical al- veoli, globules, yolk-granules, cytoplasm, follicular and radiata envelopes). During the initial vitellogenic phase, the oocytes showed cortical alveoli and oil globules containing neutral lipids exclusively. During this phase, small yolk granules ap- peared around the peripheral cytoplasm, and they increased through exogenous vitellogenesis. Yolk granules were composed of glycoproteins, calcium, iron, and proteins rich in lysine, arginine, tyrosine, tryptophan, cysteine and cystine. Vg and Mt were immunohistochemically detected in yolk. The follicular envelope contained proteins rich in amino acids. Moreover, calcium and thyroid hormones (triiodotyronine and thyroxine/T 3 , T 4 ) were detected in this cell envelope. Cortical alveoli, which contained carboxylated and neutral glycoconjugates, were especially rich in N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, galactose and sialic acid. Finally, the zona radiata was mainly proteinaceous in nature and was composed of calcium and neutral glycoproteins. The egg envelope or chorion and the liver showed specific immunoreactivities by using anti-salmon Zrp as the primary antiserum. cisteina y cistina, y contienen Vg y Mt. La capa folicular de los ovocitos del pez espada está compuesta por glicoproteínas y proteínas ricas en diferentes aminoácidos. Esta envuelta contiene calcio y hormonas tiroideas/ T 3 y T 4 . Los alveolos corticales están constituidos por glicoconjugados carboxilados y neutros, y son especialmente ricos en N-acetil-D-galactosamina, N-acetil- D-glucosamina, galactosa y ácido siálico. Finalmente, la capa radiada, fundamentalmente proteica, contiene calcio y glicoproteínas neutras. Utilizando anti-salmon Zrp, como anticuerpo primario, el corion o capa radiada y el hígado muestran una immunoreactividad específica. Palabras clave : histoquímica, carbohidratos, lípidos, glicoconjugados, vitelogenina, proteína radiada, metalotioneinas, hor- monas tiroideas, cationes, ovocitos, Xiphias gladius .

Cytological analysis has revealed that fish oogenesis is characterised by the appearance of membrane-limited round structures known as the cortical alveoli. During vitellogenesis, the alveoli are gradually displaced to the oocyte cortex, due to the centripetal storage of yolk (Gutiérrez et al., 1985;Motta et al., 2005). At fertilisation, alveoli polysialoglycoprotein content is proteolytically cleaved, leading to the formation of specific glycopeptides (L-hyosophorin) which are released into the perivitelline space (Inoue and Inoue, 1986;Seko et al., 1989), and this contributes to the transformation of the vitelline envelope into the fertilisation membrane (Kudo and Teshima, 1998). The presence of high molecular mass polysialoglycoproteins or H-hyosophorins has been reported in Fundulus heteroclitus , in medaka, Oryzias latipes  and in several salmonids (Inoue et al., 1987;Kitajima et al., 1988). Glycoconjugates may be involved in binding of hormones; in bacteria agglutination; in the transport of metabolites and ions across the plasmalema; in sperm-egg binding; and in polyspermic inhibition (Prokop et al., 1974;Miller and Ax, 1990;Skutelsky et al., 1994;Motta et al., 2005).
In teleost fish species, oocyte growth and yolk protein formation is mainly due to the hormonal regulation of hepatic synthesis and ovarian uptake of exogenous proteins, such as vitellogenin (Vg) and zona radiata proteins (Zrp) (Wallace, 1985;Hamazaki et al., 1987;Hyllner and Haux, 1992;Celius and Walter, 1998). Vg is a complex glycolipophosphoprotein (300-640 kDa) thought to be a heterodimer or to have multiple forms (Mommsen and Walsh, 1988;Walther, 1993;Tyler and Sumpter, 1996;Hiramatsu et al., 2006). In the egg, Vg undergoes proteolytic cleavage into yolk proteins, the lipid-rich lipovitellins, highly phosphorylated phosvitins and a β′-component (Hiramatsu et al., 2001 andRomano et al., 2004), which are the major source of nutrients for eggs and developing embryos. In plasma, Vg binds Ca 2+ , Mg 2+ and K + to provide minerals to the developing fish (Palumbo et al., 2007). Additionally, the primary degradation products of Vg have also been shown to play a role in regulating oocyte hydration and buoyancy of teleostean eggs (Matsubara et al., 1999).
In vertebrates, the egg envelope, referred to as the zona pellucida in mammalian eggs, is a fibrous and noncollagenous extracellular matrix surrounding the eggs and composed of three to four homologous glycoproteins with a common ZP domain. While ZP1 and ZP3 are present in all vertebrate species, ZPX is not found in mammals and ZP2 is not found in zona radiata of fish eggs (Waclawek et al., 1988;Hamazaki et al., 1989;Yamaguchi et al., 1989;Hyllner et al., 1994;Epifano et al., 1995;Modig et al., 2006). In fish species, proteins of the zona radiata are synthesised in either the ovary or the liver (Hyllner et al., 2001;Hamazaki et al., 1987). The precursors of these proteins have been identified as choriogenin H (high molecular weight) and choriogenin L (low molecular weight), respectively (Lee et al., 2002). During oogenesis, choriogenins are produced in the liver as a response to oestrogens and released into the bloodstream to be incorporated into the egg envelope (Lee et al., 2002;Arukwe and Goksøyr, 2003). In the Japanese medaka, Oryzias latipes, the respective precursors of choriogenin subunits contain a domain characterised by aligned cysteine residues, which is frequently found in glycoproteins of the fish egg envelope (Murata et al., 1994;Grau et al., 1996;Sarasquete et al., 2002a,b).
The swordfish is an important commercial species with an extensive seasonal migration and a circumglobal distribution. It is a gonochoristic species, with females attaining larger sizes than males, and it lives to at least 9 years of age (Rey, 1988). Moreover, in the Mediterranean areas this species often suffers stress due to lipophilic contaminants (Fossi et al., 2001), which could be analysed and characterised by using specific cell biomarkers. The present study investigates the presence and cellular distribution of carbohydrates, glycoconjugates, metal binding proteins, aminoacids, lipids, thyroid hormones and cations on oocytes of the swordfish Xiphias gladius by using a battery of histochemical and immunohistochemical techniques.

Histological procedures
Adult specimens of the swordfish Xiphius gladius were caught by harpoon from traditional Sicilian "passerella" fishing boats during the spawning period (June-August) in the Strait of Messina (Sicily, Italy).
Fragments of the liver and ovary were dissected and fixed in Bouin (picric acid, formaldehyde, acetic acid, 15/5/1, v/v/v) for 24 hours and conserved in 70% ethanol, dehydrated through graded alcohols, cleared in xylene and embedded in paraffin. Serial sections (6-7 µm thick) were cut and mounted in gelatinised slides. To identify lipid inclusions, some samples were preserved in 70% ethanol, treated with 1% osmium tetraoxide and 2.5% potassium dichromate for 8 h, washed in running water and dehydrated before the wax embedding procedure (Luna, 1968). Haematoxylin-Eosin and Haematoxylin-VOF polychromic stains were performed according to Gutiérrez (1990) and Sarasquete and Gutiérrez (2005).

Lectin histochemistry
To analyse glycoconjugates, sections were treated with 0.3% hydrogen peroxide for 15 minutes (to in-hibit endogenous peroxidase) in Tris buffered saline (TBS) at pH= 7.2. The sections were then incubated for 1 to 4 hours at room temperature with different horseradish peroxidase-conjugated lectins (HRPlectin conjugated) dissolved in TBS at the concentrations indicated in Table 1. After three washes in TBS, peroxidase activity was visualised with TBS containing 0.05% 3,3' diaminobenzidine tetrahydrochloride (DAB) and 0.015% hydrogen peroxide. Sections were washed in running tap water for 10 minutes, dehydrated, cleared and mounted in Eukitt. Controls were: omission of the respective lectin; substitution of lectin-HRP conjugated by TBS; and treatments with different enzymes (neuraminidase Type V, β-galactosidase Grade VI, α-mannosidase Type III, β-N-acetylglucosamine, β-N-acetylgalactosamine and L-fucosidase). Controls for lectin specificity included: 1) substitution of the substrate medium with buffer without lectin; and 2) incubation with each lectin in the presence of its hapten sugar (0.2-0.5 M in Tris buffer). Lectins and enzymes were purchased from Sigma Chemical Co. St Louis, MO, USA.

Immunohystochemical procedure
Endogenous peroxidase activity was blocked in the dark with 3% hydrogen peroxide in Coons buffer with Triton X-100 (CBT-0.01M veronal, 0.15M NaCl, 0.1% Triton X-100) for 15 min. Non-specific protein binding to sections was blocked with 0.5% (wt/vol) bovine serum albumin (BSA) in CBT for 30 min. T 4 (thyroxine) and T 3 (triiodothyronine) primary monoclonal antibodies (Fitzgerald Industries) diluted in CBT/0.5% BSA (T 4 antiserum diluted 1:50 and T 3 antiserum diluted 1:25) and with anti-seabream Vg, anti-salmon Zrp and anti-cod Mt primary polyclonal antibodies (Biosense laboratories, Bergen, Norway) were used. Antibodies were diluted 1:250 to 1:500 and incubations were performed overnight in a humidified chamber at room temperature. Sections were washed in CBT and incubated for 1 h at room temperature with goat antimouse IgG peroxidase conjugated for monoclonal primary antibodies and with goat anti-rabbit IgGperoxidase conjugated for polyclonal primary antisera (Sigma, St Louis, MO, USA), with a 1:1500 dilution. Sections were washed again in CBT and in Tris-HCl (0.05 M, pH 7.4). Peroxidase activity was visualised in the dark with 0.025% 3-3'-diamino benzidine tetrahydrochloride/DAB (Sigma) in Tris-HCl 0.05 M, pH 7.6 containing 0.5% hydrogen peroxide. The stained sections were mounted in an aqueous mounting medium (Aquatex, Merck). To confirm the specificity of immunostainings, negative controls were performed by replacing the primary antibody with pre-immune serum (from the same animals from which the antiserum was obtained) or BSA, and by omission of primary and secondary antibodies. Results were visualised using a Diaplan Leitz light microscope equipped with an Insight Spot digital camera.

Histochemical characteristics of oocytes of the swordfish Xiphius gladius
Oocytes at different stages of development (oogonia, previtellogenic, vitellogenic, maturating, atretic and post-ovulatory follicles) were detected in most X. gladius ovaries studied ( Fig. 1A-J). A high percentage of vitellogenic and maturation oocytes, and the presence of atretic oocytes and especially of post-ovulatory follicles, were indicative of advanced vitellogenesis, maturation and spawning phases.
The histochemical results are summarised in Table 2. Yolk granules showed orange G (Haematoxylin-VOF) or eosin affinities (Haematoxylin-Eosin) and lipid globules appeared unstained with both morphological dyes. Neutral lipids (globules or vacuoles in paraffin sections) showed a strong affinity towards osmium tetraoxide. Lipid globules increased in number and size and gradually fused and coalesced during the maturation phase (Figs. 1, 2A-C).
Yolk granules reacted weakly with PAS and diastase-PAS (presence of neutral glycoproteins/ glycolipids and absence of glycogen) ( Fig 2D) and they were strongly stained with protein techniques, especially those rich in -SH-and -SS-groups (i.e. cysteine and cystine) (Fig. 2E-H) and unstained with Alcian Blue techniques (AB, pH: 0.5, 1 and 2.5). Intergranular cytoplasm contained proteins rich in different amino acids, especially those rich in tyrosine, tryptophan, cysteine and cystine . Intergranular cytoplasm also contained iron (Fe 3+ ) and calcium. Cortical alveoli were composed of carbohydrates (carboxylated and neutral glycoproteins) and proteins rich in -SH and -SS-groups exclusively In X. gladius, the zona radiata was mainly proteinaceous in nature. This egg envelope showed a strong acidophilia/eosinophilia and was composed of an internal part (ZrI) with an affinity for acid fuchsin and an external part (ZrE) with a tinctorial affinity for light green (H-VOF stain). Proteins in general and proteins rich in amino acids (i.e. arginine, cystine, cysteine and tryptophan) were detected in the zona radiata. The ZrE showed a stronger PAS-positive reaction (neutral glycoproteins) than the ZrI, which contained proteins rich in disulphide (-S-S-) groups (Figs. 2D-H, 3A-C)

Immunohistochemical distribution of Mt, Vg and Zrp, T 3 and T 4
Immunohistochemical localisation of Mt, T 3 , T 4 , Vg and Zrp in oocytes of the swordfish X. gladius is summarised in Table 4. Cortical alveoli and follicular envelopes showed strong Mt immunostaining and weak or moderate immunoreactivities were detected in cytoplasm and yolk granules (Fig. 5A-C) (Table  4). Calcium was strongly positive in yolk granules and zona radiata and a weak alizarin red staining was detected in both oocyte cytoplasm and the follicular envelope (Fig 5d). Mt was also detected in the liver, within the hepatocytes and vascular system of both males and females.
Cortical alveoli showed a moderate anti-T 3 immunoreactivity, whereas the follicular envelope and intergranular cytoplasm showed variable T 3 and T 4 immunostaining. However, no immunoreactivity was detected in oil globules, yolk granules and zona radiata using both thyroid primary antisera (Table 4; Fig 5 E, F).
Intensity of reaction: +/-: weak; +: moderate; ++: strong.      showing numerous acidophilic yolk granules (g) (orange G affinity) and lipid globules (lg) (empty vacuoles). The zona radiata (zr) shows a bipartite structure constituted by a thinner external zone (zrE/II) and a less acidophilic internal radiata zone (zrI/I). The follicular envelope was divided into two differentiated layers: the granulosa (grl) and a thin external cell layer or theca (t). D, anti-Zrp immunoreactivity detected in the external radiata zone (zrE/II) and less intense immunostaining in the internal zone (I). E, late stage vitellogenic oocyte showing a striated zona radiata (zr). F, strong anti-Zrp immunoreactivity in the internal homogeneous zone (I) and moderate Zrp staining in the longitudinally-striated external zone (II). No Zrp immunostaining was detected in the follicular envelope. n : hepatocyte nucleus; s: sinusoids; grl: granulosa layer; t: theca layer. Scale bars represent 100 µm.
oocytes displayed a strong Vg staining in numerous yolk granules located within the ooplasm. Moderate Vg immunostaining was detected in intergranular cytoplasm of the swordfish vitellogenic oocytes. The liver also showed positive Vg immunostaining, especially evident in the plasma content of the vascular system, in the cytoplasm of hepatocytes, and surrounding the canalicular border of hepatic cells. However, no Vg immunostaining was detected in the sinusoidal endothelium ( Fig. 6A-F).
Both the ZrE and ZrI layers of the zona radiata showed a strong Zrp immunoreactivity, with higher staining in the internal layer than in the ZrE. The liver also showed Zrp immunoreactivity, which was located as condensed granules within the cytoplasm and along the canalicular border of parenchymal cells (Fig. 7A-F; Table 4).

DISCUSSION
The histological characteristics of the oogenesis of the swordfish Xiphias gladius were similar to those previously described by Corriero et al. (2004). The ovarian maturation consisted of oocytes at different developmental stages, and especially a high percentage of vitellogenic, maturating, atretic and post-ovulatory follicles, which is typical of a multiple spawner species during the freezing period.
In swordfish oocytes, as in those of other fish species Sarasquete et al., 2002 a,b;Motta et al., 2005), cortical alveoli contain a considerable cellular heterogeneity of glycoconjugate sugar residues (i.e. N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, galactose and sialic acid), suggesting that they may exert a primary intracellular role by acting, for example, as carriers or crosslinking agents for the glycosylated alveolar components (Olden et al., 1982). In medaka eggs, Shibata et al. (2000) reported the existence of a specific metalloproteinase, the alveolin, also responsible for chorion hardening following fertilisation. The fact that alveoli lectins and glycoconjugates are extruded into the perivitelline space at fertilisation could indicate that they are not an important source of nutrients to the embryos (Yamamoto, 1962), but different glycoconjugates may be involved in sperm and bacteria agglutination (Prokop et al., 1974) or in binding of sperm to the egg surface, as suggested by Kothbauer and Schenkel-Bruner (1974).
It is well known that thyroid hormones play an important role during embryogenesis and organogenesis, and most vertebrates are unable to grow and reach their normal adult form without them (Liu et al., 2000;Power et al., 2001). The presence of thyroid hormones in fish eggs prior to hatching has been reported in several fish species and these are presumably of maternal origin (De Jesus et al., 1991;Lam, 1995;Yamano, 2005). Recently, in Solea senegalensis larvae development, Ortiz-Delgado et al. (2006) detected T 3 and T 4 immunostaining in the yolk sac matrix at hatching. Interestingly, a weak presence of thyroid hormones/T 3 and metallothionein/Mt was detected in swordfish cortical alveoli. Effects of the ovarian fluids as hormones, which act through the hypothalamic-pituitary axis to stimulate the activation of the thyroid gland, were pointed out (Finn, 2007). Moreover, according to Finn, ovarian fluids can act as an important trap for cations, being also the major barrier to diffusion of gases and solutes.
The zona radiata of swordfish oocytes, also known as the chorion, is immunostained with antisalmon Zrp. This acellular protein egg envelope contains calcium and is composed of neutral mucopolysaccharides and especially of proteins rich in cysteine and cystine, which suggests that most of the proteinaceous material of this multilamellar layer is determined by the formation of disulphide bonds (Hagenmainer, 1973;Gutiérrez et al., 1985;Sarasquete et al., 1993Sarasquete et al., , 2002Grau et al., 1996;Mayer et al., 1988). In Salmo salar radiata envelopes, Hamor and Garside (1973) detected proteins, mucopolysaccharides, phospholipids, cholesterol, nucleic acids and oxidative enzymes. Studies in other fish species (Tesoriero, 1977) have shown that the external layer of the zona radiata was rich in polysaccharides, whereas the inner layer was rich in proteins. In Carassius auratus (Khoo, 1979) and Dicentrarchus labrax (Mayer et al., 1988), polysaccharides adhere to the outer layer which might contribute to the adhesion of the eggs. However, in Siganus rivulatus eggs, the chorion consists exclusively of proteins (Lahnsteiner and Patzner, 1999).
The liver of swordfish females showed moderate to strong Zrp, Vg and Mt immunoreactivies. In X. gladias oocytes, Vg was detected in yolk granules and intergranular cytoplasm but not in the follicular envelope. According to Abraham et al. (1984) the follicular cells are not involved directly in the transfer of exogenous material into the oocytes. However, in other fish species Vg was present in the follicular envelope (Susca et al., 2001;Sarasquete et al., 2002 a, b). Vg immunostaining was also strongly detected in hepatocytes, especially in the canalicular border, and it was weakly detected in the hepatic vascular system. Hepatocytes have previously been identified as the site of exogenous vitellogenesis protein synthesis, and the vascular system has been identified as the transferral route between the liver and ovary (Wallace, 1978). Vg inmunoreactivity was detected in the liver of brown trout, Salmo trutta females, and no staining was observed in the liver of males (Wahli et al., 1998). Interestingly, as indicated previously in Mediterranean swordfish specimens (Fossi et al., 2001, 2007, Desantis et al., 2005, we observed positive Vg immunoreactivity in the liver of some male specimens (data not shown), which also presented high plasma oestradiol, Vg and Zrp concentrations, as well as high endocrine disrupting chemical levels (DDTs, PCBs, PAHs, etc) in the liver, gonads and plasma (Mori et al., 2005). The capacity of the liver of fish males to synthesise Zrp and Vg after exposure to natural estrogens or man-made chemicals has been widely reported (Oppen-Berntsen et al., 1992, Fossi et al., 2001, 2007Desantis et al., 2005).
In the reproductive cycle of vertebrate and invertebrate species, Mt-like proteins play an important physiological role during vitellogenesis, because these proteins participate in homeostasis of zinc and copper (Webb, 1987;De Prisco et al., 1991;Banks et al., 1999), and Mt could also participate in the regulation of Vg synthesis. In X. gladius oocytes, Mt was strongly detected in the follicular layer (granulosa cells), cortical alveoli and intergranular cytoplasm, and was weakly detected in yolk granules of the vitellogenic oocytes, as well as in the liver of both male and female swordfish specimens. In fish species, Olsson et al. (1989) and Carpene et al. (1994) suggested that zinc is an essential macronutrient for oocyte development. Banks et al. (1999) in channel catfish indicated the importance of Mt-like proteins in zinc regulation during exogenous vitellogenesis and fast oocyte growth.
In conclusion, the histochemical composition of oocytes of the swordfish Xiphias gladius (cytoplasm, cortical alveoli, yolk and egg envelopes) can indicate specific physiological reproductive functions. Sugar residue of gycoconjugate cortical alveoli contents (β-D-GlcNAc, sialic acids, α-D-GalNAc, α or β-D-GalNAc, β-D-Gal(1-3)-GalNAc), which are discharged into the perivitelline space at fertilisation, may be involved in chorion hardening, binding of sperm to the egg surface, and polyspermy block after gamete fusion, among other functions. As in most fish species, globules consisting of neutral lipids exclusively and glycophospholipoprotein yolk granules are the major energetic and nutritional resources respectively of eggs, embryos and larvae. During swordfish oogenesis, from a histochemical point of view, possible interactions of Mt and Vg synthesis could be suggested. Both complex proteins are localised within the yolk granules and intergranular cytoplasm during the vitellogenesis and maturation phases. Finally, immunohistochemical localisation of thyroid hormones in the cortical alveoli, intergranular ooplasm and follicular envelope could indicate their maternal origin, as well as an essential role of T 3 /T 4 during swordfish oogenesis.
Studies are currently being performed to analyse inorganic and organic contaminants, xenobiotic induction of molecular, biochemical and cell biomarkers (Vg, Zrp, Mt, oestrogens, thyroid hormones, etc.), and presence of molecular and histopathological disorders in swordfish males and females from several Mediterranean areas.