**3. Egg-laying: regulation**

The first work on the contractile structures of the female reproductive system led to the identification of numerous myotropic or myosuppressor regulatory peptides. The first of them, a neuropeptide belonging to APGWamide family, was identified from a sample of optic lobes purified by rpHPLC on the basis of its myosuppressive effect on the contraction of the distal oviduct [2].

Then, new myotropic bioassays on the contractile organs of the female genital apparatus were performed, and the bases of the functional control of spawning and the related activities were laid, for example, blocking or inducing oocyte transport in the oviduct and the secretion of internal and external oocyte capsules before fertilization.

From the papers published on this topic between 1997 and 2006, it appears that the successive steps of egg-laying are mainly governed by two classes of regulatory peptides: neuropeptides involved in the integration of environmental cues and ovarian regulatory peptides that modulate the activity of the genital tract [2–8]. The recent development of "-omics" approaches based on *de novo* RNAseq and mass spectrometry led to the identification of transcripts and mature cleavage products.

Using a transcriptomic approach, Enault and collaborators [9] discovered a third category of regulatory peptides, namely the sex pheromones expressed and released by the oviduct gland, and cleaved from three protein precursors into bioactive peptides ranging between 1.3 and 8 kDa.

Thanks to the sequencing of the neurotranscriptome, several neuropeptide families involved in the regulation of egg-laying were identified on the basis of expression pattern and tissue localization out of the 38 families composing the cuttlefish neuropeptidome [10]. Finally, the RNA sequencing of ovary tissue revealed that most of the ovarian regulatory peptides involved in oocyte release were cleaved from a single yolk protein (unpublished results).

The identification of the cuttlefish neuropeptidome by *de novo* RNAseq and mass spectrometry screening was the next step that provided an overview of the neuropeptidome *via* a deep

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 7

Based on the filtering criteria applied to the 38 identified neuropeptide families—expression level, neuropeptide tissue mapping, and mRNA localization—seven neuropeptide families were finally selected: allatostatins, APGWamide, crustacean cardioactive peptides (CCAPs),

Several neuropeptides cleaved from the protein precursor of **allatostatin A1** and **A2** issued from alternative splicing were detected by nanoliquid chromatography tandem mass spectrometry (nLC-MS/MS) in the oviduct gland and the main nidamental glands, suggesting a role in egg capsule secretion. In insects, **FGLamide allatostatins** (also called **allatostatins A** or **buccalins**) are involved in reproduction [15] and feeding decisions interacting with the adipokinetic hormone (AKH) and insulin-like peptides [16]. In cuttlefish, **allatostatins A1** and **A2** were also detected in the hemolymph, in accordance with the status of neurohormones that

The **APGWamide** was detected by nLC-MS/MS in the CNS, and the **GWamide** was characterized from the CNS [2]. Moreover, large amounts of mRNAs were found in the OGs, MNGs, and ANGs of egg-laying females. Similar observations were reported in the pond snail *Lymnaea stagnalis* by van Minnen and Bergman [17]. High amounts of mRNAs encoding the egg-laying hormone were detected in the nerve terminals after a stimulus, as well as polyribosomes, supporting that the translation of egg-laying hormone transcripts could occur in the axonal compartment. These data are supported by recent papers revealing the occurrence of rough endoplasmic reticulum, smooth reticulum, and Golgi apparatus in the axonal compartment [18]. Otherwise, Martin and Kim [19] used Aplysia as a model to show that netrin-1, already known to promote translation in axonal growth cones [20], increased translation of subcellular mRNAs localized at the level of dendrites or axons by binding the cytoplasmic domain of the netrin-1 receptor called DCC (for deleted colorectal cancer). The rapid reaction of female

cuttlefish can be related to the state of readiness of the axons that innervate the ASGs.

peptide family is strongly suspected to regulate egg capsule secretion.

The three neuropeptides predicted from the protein precursor of **CCAPs** were detected by nLC-MS/MS in the CNS, the oviduct gland and the main nidamental glands [10]. **CCAPs** are also overexpressed in the sub-esophageal mass of egg-laying females (as opposed to mature males), which is the only part of the CNS that innervates the genital apparatus. This neuro-

**CCAPs** were initially described in crustaceans. They are usually C-terminally amidated neuropeptides in arthropods [21–24], as well as in mollusks [14, 25–27], whereas the cuttlefish preprohormone predicted three nonamidated peptides confirmed by nLC-MS/MS analysis. The four neuropeptides predicted from the protein precursor of **FaRPs** were detected by nLC-MS/MS in the CNS, and the decapeptide ALSGDAFLRFamide was the only one detected in the neurohemal area connected to the sub-esophageal mass and in the oviduct gland and main nidamental glands, confirming the immunostaining results obtained by Henry and collaborators [3]

FaRPs, FLGamide, myomodulins, and small cardioactive peptide (SCP).

could regulate the biosynthesis of egg capsule products during vitellogenesis.

structural and functional investigation [10].
