Introduction
The periblast or yolk syncytial layer (YSL) is a continued cytoplasmic layer located between the blastoderm disc and the yolk, and resulting from the incomplete division of the blastomeres. Initially, it is a ring-shaped layer around the yolk that it spreads eventually under the entire blastoderm (Kimmel et al., Reference Kimmel, Ballard, Kimmel and Ullmann1995). This layer is greatly important for the embryonic development of teleost eggs. Due to its location, all the nutrients from the yolk pass through this layer to reach the blastoderm (Trinkaus, Reference Trinkaus1951, Reference Trinkaus1993; Devillers, Reference Devillers1961). In addition, the YSL works as a force for the epiboly movement of the blastoderm (Trinkaus, Reference Trinkaus1984a, Reference Trinkausb, Reference Trinkaus1993).
Recent research on the epiboly and somite stages, suggests that the major problem for cryopreservation of zebrafish embryos is the difficulty in gaining sufficient cryoprotectant penetration, particularly in the yolk compartment (Rawson et al., Reference Rawson, Zhang, Kalicharan and Jongebloed2000). Hagedorn et al. (Reference Hagedorn, Hsu, Pilatus, Wildt, Rall and Blackband1996, Reference Hagedorn, Kleinhans, Wildt and Rall1997) showed cryoprotectant penetration in the blastoderm, but hardly any in the yolk, suggesting that the YSL is one of the main permeability barriers to water and cryoprotectants, hindering the chilling of fish embryos. Therefore, there is a considerable interest in the study of this syncytial layer, which envelopes the yolk, in order to identify its structural, functional and physiological features (Rawson et al., Reference Rawson, Zhang, Kalicharan and Jongebloed2000).
The knowledge of YSL characteristics has great importance for the development of cryopreservation techniques for fish embryos. Inasmuch as there is no detailed study about this subject in Neotropical teleosts, the present study has aimed at anylysing the structure and ultrastructure of YSL, using as a model Prochilodus lineatus, a Neotropical fish that belongs to the Prochilodontidae family, with a wide distribution in southeastern Brazil, and found in the entire Paraná-Paraguay and Paraíba river basins (Corrêa e Castro, Reference Corrêa e Castro1990).
Materials and Methods
Embryos were obtained through artificial fertilization of mature adult individuals of Prochilodus lineatus from the broodstock kept at Aquaculture Section, in the Animal Production Department of the Faculdade de Medicina Veterinária e Zootecnia–UNESP, Botucatu. The material was further processed and analyzed at the Biology and Fish Genetics Laboratory at the Morphology Department of the Biosciences Institute–UNESP, Botucatu, Brazil.
Embryos were collected at different developmental stages up to hatching and fixed with 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3). To carry out the light microscopic (LM) analyses, representative embryo samples from each embryonal stage were dehydrated in a series of increasing alcohol concentrations. They were sequentially and individually put into glycol-methacrylate for further microtomy, in order to obtain transverse and sagittal serial sections of 3 to 5 μm. Subsequently, the sections were stained with Harris eosin hematoxylin and toluidine blue, analyzed and then photographed under a photomicroscope (Axiophot, Zeiss).
To perform the transmission electron microscopy (TEM), the samples were post-fixed in 1% osmium tetroxide for 2 h, contrasted with an aqueous solution of 0.5% uranyl acetate, dehydrated in acetone and placed in epoxy resin. For contrast, ultra-fine sections were counter-stained with uranyl acetate (Watson, Reference Watson1958), washed in 50% alcohol and then stained with lead citrate (Reynolds, Reference Reynolds1963). The material was analyzed and photographed using transmission electron microscopy (Phillips, CM100).
In the scanning electron microscopy (SEM) process, pre-fixed embryos were previously cryofractured, after placing the samples in a 30% sucrose solution until they settled, washing in 1% phosphate buffer, cryofractured in liquid nitrogen and submerging in a 2.5% glutaraldehyde solution. After 24 h, these embryos were transferred to a 13 mm cover slide coated with 1% poly-l-lysine, post-fixed in 0.5% osmium tetroxide, dehydrated in alcohol, dried in Balzer's critical point dryer CPD-20, coated with 10 nm of gold in Balzer's MED-010 sputtering unit, and then observed and photographed under a scanning electron microscope (Phillips SEM 515).
Results
YSL formation in P. lineatus started at the end of the cleavage stage (morula) and at the beginning of blastula stage, on the margin of blastoderm, giving rise to a surrounding ridge (Fig. 1a, c). It was characterized as a cytoplasmic layer bearing several nuclei with no separating cytoplasmic membranes and possibly showed fractionated yolk globules within its cytoplasm (Fig. 1c, d, f, g). The first nuclei were derived from peripheral blastomeres of incomplete cytokinesis that, when in contact with the cytoplasmic layer surrounding the yolk (YCL), released their contents into this layer (Fig. 1f).
Photomicrographies of P. lineatus embryo cuts: (a, b) transversal sections of embryo at the initial and late stage, respectively (toluidine blue); (c) lateral transversal section showing the yolk syncytial layer (YSL) covering the entire surface under the blastoderm (toluidine blue); (d) gastrula stage (Toluidine blue; 50% epiboly), pointing out the euchromatic nuclei of the yolk syncytial layer (YSL); (e) yolk plug made up by YSL (90% epiboly) (HE); (f) blastomere releasing its content into YSL (toluidine blue); (g) yolk globules within the YSL (HE); (h) view of YSL enveloping the yolk completely (segmentation stage) (HE); (i) longitudinal cut of the embryo at the larval stage showing the YSL enveloping the primitive intestine (HE). b, blastomere; bl, blastoderm; n, nucleus; no, notochord; s, somite; ycl, yolk cytoplasmic layer; yg, yolk globules; ysl, yolk syncytial layer. Open blastomere to the ycl; pi, primitive intestine. Bars: (a, b) 50 μm; (c) 50 μm; (d) 50 μm; (e) 25 μm; (f) 6.25 μm; (g) 25 μm; (h) 100 μm; (i) 333.33 μm.
Initially, the peripheral YSL presented a thicker aspect (Fig. 1a). However, as long as the nuclei underwent divisions and migrated to the YCL portion located below the blastoderm, its peripheral portion became thinner (Fig. 1b). At the end of blastula stage, a distinctive multinucleated intermediate layer was observed, arranged between the yolk vacuoles and blastoderm cells (Fig. 1c).
The nuclei under LM were presented as basophilic and uncondensed structures, showing a high metabolic activity (Fig. 1a, d, f). During epiboly, the YSL spread along the embryo until it encompassed the yolk mass entirely, forming the yolk plug (late gastrula) (Fig. 1e). The YSL was present as a thin layer involving the yolk completely at the segmentation stage (Fig. 1g, h) and, at the larval stage, it reached the primitive intestine (Fig. 1i).
The YSL analysis under TEM at several embryonal development phases revealed that this layer contained many ribosomes, a large net of endomembranes, several mitochondria, vacuoles and some multivacuole bodies (Fig. 2e, f). Individualized Golgi complexes were not found. The nuclei were elongated and euchromatic, presenting uneven contours and a high amount of pores, which indicated high metabolic activity (Fig. 2c–e). The cytoplasm of this cell type was more electron dense than that found from adjacent cells. The posterior face (PF) of YSL was delimited by endoderm embryo cells, while the anterior face (AF) was delimited by yolk globules, and evaginations from this membrane into these structures could be seen (Fig. 2a, c). Microvilli, mostly from the YSL, were detected in the PF (Fig. 2a, b). In addition, yolk globules were observed throughout the cytoplasm and their size decreased towards PF (Fig. 2c, d).
Ultrastructure (TEM) of the yolk syncytial layer: (a) view of arrangement between the endoderm, the yolk syncytial layer (YSL) and the yolk; (b) detail of microvilli present between the YSL and the endoderm; (c, d) show that the nuclei of the YSL are cylindrical and uneven; (e, f) point out the large quantity of mitochondrias and extensive net of endomembranes found in the cytoplasm of the YSL as well as fibres, multivacuole bodies and yolk vacuoles. ed, endomembranes; en, endoderm; f, fibres; m, mitochondria; mv, microvilli; n, nucleus; vb, vacuole body; yg, yolk globules; ysl, yolk syncytial layer. Bars: (a) 2.3 μm; (b) 0.59 μm; (c, d) 3.1 μm; (e) 0.75 μm; (f) 0.59 μm.
SEM observations showed that the YSL was located just below the endoderm, in direct contact with the yolk, confirming previous LM and TEM observations (Fig. 3a, b). Its cytoplasm presented a granular aspect, probably related to the large number of ribosomes, mitochondria and vacuoles found within the cytoplasm (Fig. 3c). The microvilli became more evident, revealing that they are present in high number and spread over the entire outer surface of this layer (Fig. 3b, d). However, in some PF parts of the YSL (stains), the microvilli were present either in a reduced size or absent (Fig. 3e, f).
Eletronmicrographies (SEM) of cryofractured P. lineatus embryos (4–6 somite phase): (a, b) show the overlapping of ectoderm layer, endoderm, yolk syncytial layer and the yolk; (c, d) show the cytoplasm of the YSL and its arrangement with the yolk globules; (e, f) show the microvilli of the YSL and the stains on its surface. ec, ectoderm; en, endoderm; mv, microvilli; n, nucleus; st, stains; yg, yolk globules; ysl, yolk syncytial layer. Bars: (a) 30.77 μm; (b) 13.89 μm; (c) 2.35 μm; (d) 3.97 μm; (e) 4.5 μm; (f) 0.87 μm.
Discussion
Observations made on the start of YSL formation in P. lineatus were supported by previous analyses in Fundulus heteroclitus and Brachydanio rerio (Lentz & Trinkaus, Reference Lentz and Trinkaus1967; Kimmel et al., Reference Kimmel and Law1985; Trinkaus, Reference Trinkaus1993) and confirmed that YSL nucleation is observed at the end of the cleavage (morula) stage, through the releasing of the nucleus and the cytoplasmic content of some peripheral blastomeres within the yolk cytoplasmic layer (YCL). The YSL undergoes the epiboly process regardless of the blastoderm (Trinkaus, Reference Trinkaus1993; Devillers, Reference Devillers1961) and it acts as a primary force for blastoderm epiboly (Betchaku & Trinkaus, Reference Betchaku and Trinkaus1986; Trinkaus, Reference Trinkaus1993). The peripheral YSL undergoes contraction, resulting in several events such as facilitating the migration of the nuclei to its inner part and completes its formation with thinning towards the vegetal pole along the blastoderm, to which it is firmly adhered, leading to the epiboly movement (Trinkaus, Reference Trinkaus1993). Analyses in P. lineatus corroborate the observations of the authors mentioned above, pointing out that the YSL appears clearer from nucleation onwards, as was also reported by Lentz & Trinkaus (Reference Lentz and Trinkaus1967) for F. heteroclitus, which suggests that besides the volume increase, there is a change in membrane characteristics, making it more visible.
Regarding the importance of YSL in the yolk incorporation by P. lineatus embryos, the presence of microvilli in its upper part and the membrane projections between the yolk globules, as well as the lack of yolk globules close to the blastoderm, suggest that the vitelline material is degraded by hydrolytic enzymes, as reported by Lentz & Trinkaus (Reference Lentz and Trinkaus1967), and then transferred to the blastoderm (Walzer & Schönenberger, Reference Walzer and Schönenberger1979). Nevertheless, as pinocytic or phagocytic vacuoles were not observed close to the microvilli, we could infer that the nutritive material passes through the plasmatic membrane as small molecules, as reported by Lentz & Trinkaus (Reference Lentz and Trinkaus1967).
TEM analysis showed some different external structure characteristics in the YSL of the plasma membrane in P. lineatus in relation to those found by Rawson et al. (Reference Rawson, Zhang, Kalicharan and Jongebloed2000) in B. rerio. For instance, the membrane in P. lineatus is wrinkled and covered by microvilli and, at ultrastructural level (TEM, SEM), presented neither pores nor similar structures. On the other hand, our observations are in agreement with those reported by Rawson et al. (Reference Rawson, Zhang, Kalicharan and Jongebloed2000) on the granular aspect of the cytoplasm of the YSL.
Inversely to the impermeability of the YSL to cryoprotectants proposed by Hagedorn et al. (Reference Hagedorn, Kleinhans, Artemov and Pilatus1998), the present analysis of YSL ultrastructure showed no morphological evidence that it could act as a barrier to cryoprotectant substances, as was reported by Rawson et al. (Reference Rawson, Zhang, Kalicharan and Jongebloed2000). However, there is evidence that the YSL, besides connecting, sustaining and separating the yolk and the embryo cells, makes the yolk content available to the embryo. Furthermore, this finding also suggests that the YSL has some control over the entrance and exit of substances to the vitelline vacuole.
Acknowledgements
This work was supported by Aquaculture Division of the Faculdade de Medicina Veterinária e Zootecnia/ UNESP/Botucatu/São Paulo/Brazil, which provided the fish and the facilities used in this study, and also by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Pesquisa).
