**7. The egg case: structure and function during embryo development**

After the spawning period, the genitors die and leave their eggs in the marine environment without any parental protection. Thus, the sustainability of the species depends on the reproduction success and more precisely on the ability of the eggs to complete their development.

Egg-laying regulation in cuttlefish is a complex mechanism that involves peptide and protein regulatory factors of different nature produced by the central nervous system, the ovary, and

**Figure 10.** Protein precursor of a retinol-binding protein able to release the tetra-peptide ILME. The predicted signal peptide is highlighted in yellow and the convertase cleavage sites in red. ILME is highlighted in gray, and the stop codon

The neuropeptides trigger egg-laying by integrating environmental stimuli across a neurosensory network. The ovarian regulatory peptides synchronize oocyte transport and egg capsule secretion, and their concentration is correlated to the number of smooth oocytes stored in the genital coelom. As they are short and unprotected peptides, they have a short life time

The waterborne sex pheromones cleaved from three protein precursors overexpressed in the oviduct gland stimulate and facilitate mating and reproduction behaviors by aggregating mates in egg-laying areas. Short pheromones participate to the release of oocytes in the mantle cavity, and large pheromones are suspected to modulate reproduction behaviors by

These multiple regulatory layers can be correlated with the complexity of the successive steps of the egg-laying mechanism that involves the ovary and ASGs and is performed thanks to a

the ASGs.

18 Biological Resources of Water

after secretion, hence a very dynamic regulation.

at the end of the coding sequence is indicated by an asterisk.

**Table 2.** Primary sequences of ovarian regulatory peptides.

aggregating mates in egg-laying areas.

**Figure 11.** (A) Photograph of female reproductive glands during secretion of the egg case (red arrow). ANG, accessory nidamental gland; IB, ink bag; MNG, main nidamental gland; OG, oviduct gland. (B) Schematic representation of a mature female cuttlefish in ventral view showing the localization of the MNG and ANG. (C) Longitudinal section of the MNG and ANG stained in Prenant-Gabe triple staining. Longitudinal section of the MNG lamellae stained in alcian blue and periodic acid of Schiff highlighting the secretion of acid mucopolysaccharides (D), and neutral mucopolysaccharides and glycoproteins (E). (F) Longitudinal section of the ANG stained in Prenant-Gabe triple staining showing that tubules are composed of a single layer of ciliated epithelium and filled with bacteria in the lumen. (G) Thin section of the lumenal surface of accessory nidamental gland tubules showing a single layer of ciliated epithelium with microvilli and a few lumenal bacteria in TEM (x 12,000). (Photo credits: V. Cornet. D. Goux).

Cuttlefish eggs are large oocytes containing all the nutrient reserves required for embryo development. To withstand physical and microbial threats, mature oocytes are enclosed within a protective egg case produced by secretions of the female genital apparatus [53, 54]. This egg case is composed of two distinct envelopes. The inner layer is in direct contact with the chorion surrounding the oocyte; it is formed by secretions added, while the egg passes through the oviduct gland [53]. The oviduct gland secretes proteins and polypeptides. The main proteins secreted by this gland correspond to sex pheromones. Afterward, the oocyte is released inside the mantle cavity and embedded with an outer layer secreted by the two nidamental glands and stained with ink (**Figure 11A**).

The conserved innate immune Toll/NF-κB pathway was described for the first time in *Sepia officinalis* ANG [59]. The transcriptomic analysis of ANG led to the identification of different constitutive elements of the Toll/NF-κB pathway. Five related Toll receptors (TLRs) have been characterized. Among them, TLRα shares 89% sequence identity with the unique TLR found in the light organ of *E. scolopes*. In addition, eight phosphorylation cascade elements have been demonstrated such as IRAK, TRAF6, and Rel/NF-κB. These immune pathway proteins (α2-macroglobulin-like protein, CD-63 antigen, transferrin) are probably involved in the establishment and maintenance of the bacterial symbionts like those in the light organ of *E. scolopes* [60]. Although several studies have been carried out about the subject, the real function of this ANG and its symbionts remains unknown. Several studies in squid suggest protection of the egg via the secretion of antimicrobial or antifouling compounds by ANG or

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 21

The function of the main nidamental gland (MNG) in egg case formation is clearer (**Figure 11A**). This white gland secretes the mucopolysaccharides and glycoproteins that form the egg case.

A recent unpublished analysis of the MNG proteome reveals the occurrence of proteins involved in glycolysis/gluconeogenesis (6-phosphofructo-2-kinase, type-2 Hexokinase, Pyruvate kinase, Glyceraldehyde 3-phosphate dehydrogenase, Fructose-1,6-bisphosphatase, fructosebisphosphate aldolase) and in glycogenolysis/glycogenesis (Glycogen phosphorylase, Glycogen synthase). These results indicate a large amount of energy production and consumption by the MNG due to an intense production and secretion of egg case components. Some of the identified proteins are also involved in the metabolism of polysaccharides or glycoproteins, like glycosyltransferases, which catalyze the transfer of oligosaccharide moieties from activated nucleotide sugars to nucleophilic glycosyl acceptor molecules or GDP-mannose pyrophosphorylase, involved in the production of N-linked oligosaccharides. Finally, the MNG secretes the main capsular components, the Egg Case Proteins, involved in the formation of a

narrow mesh that provides elasticity and resistance properties to the egg case [63].

At the time of fertilization in the oral cavity of the female cuttlefish, the oocytes are already wrapped in the thick and complex egg case. The female's arms form a chamber to keep the freshly embedded oocytes near the oral copulatory pouch where spermatophores have been deposited by the male during mating (**Figure 1**). Fertilization of the oocytes by spermatozoa is facilitated by a diffusible chemoattractant factor: SepSAP (Sepia Attracting Sperm Peptide). This hexapeptide is expressed in the vitellogenic follicles and released by embedded oocytes through the various capsular envelopes to facilitate fertilization by increasing chances of gamete collision. SepSAP has an attractant effect on sperm from low concentrations around

During fertilization, the eggs are also in contact with saliva. As early as 1934, Jecklin suggested that salivary secretions could protect the eggs during spawning [54]. A recent study of the transcriptome and proteome of *Sepia officinalis* posterior salivary gland seems to confirm this

**9. The oral cavity: completion of the eggs**

10−17 M [64].

its symbionts [61, 62], but no molecule has been characterized yet.
