**5. Sex pheromones**

regulation of the synthesis of yolk proteins and/or egg capsule proteins. In addition to the neuropeptides directly involved in the regulation of egg-laying, RNAseq revealed a substantial overexpression of neuropeptides Y (NPY), also called neuropeptides F (NPF) in protostomes because of a C-terminal tyrosine amide substituted by a phenylalanine amide (**Figure 3**). The

The expression level of NPF 1, the most overexpressed of them, reaches 45-fold the expression level found in mature males in the same part of the CNS. NPFs are probably overexpressed in females to stimulate feeding in order to support gametogenesis and egg capsule synthesis between each spawning step. In this species, asynchronous gametogenesis allows females to resume egg-laying by replenishing their batch of mature oocytes and biosynthesizing egg

More generally, the overexpression of many neuropeptides found in the sub-esophageal mass of egg-laying females (as compared to males) could be due to the regulation associated to the production of several batches of oocytes that contain a large quantity of vitellus for embryonic development and to the mobilization of the energy needed to carry out gametogenesis. By contrast, in English Channel males, gametogenesis ends 6months before reproduction and produces a much smaller volume of gametes than in females. The energy required for male gametogenesis is probably very low compared with the energy required for female gametogenesis.

Finally, a 36-amino-acid neuropeptide called Egg-Laying Hormone (ELH) can induce egglaying following a single intramuscular injection in the foot of Gastropods. In cuttlefish, ELH

ANG: accessory nidamental gland; MNG: main nidamental gland; OG: oviduct gland; OS: ovarian stroma; SubEM:

**Table 1.** Tissue mapping of neuropeptides and mRNAs in the female cuttlefish.

five transcripts of cuttlefish NPF are unequally overexpressed.

capsule products until they die.

10 Biological Resources of Water

sub-esophageal mass.

During a short life cycle of about 22 months, English Channel cuttlefish can perform four horizontal migrations from the Normandy coasts to the western part of the English Channel [35]. After a last migration to reach specific mating and spawning coastal areas, cuttlefish aggregate for mating and egg-laying between April and June on the Normandy coasts. This behavior suggests the occurrence of some kind of chemical communication *via* waterborne molecules that induce the aggregation of mates. Chemical communication in cuttlefish was first demonstrated by Boal and collaborators [36, 37] using y-mazes. They showed that recently laid eggs, ovary extracts and nidamental glands, induced an increase in ventilation rate and the attraction of sexually mature cuttlefish in the arm of the y-maze containing purified extract *versus* artificial sea water. In the same way, Cummins and collaborators [38] identified a 10 kDa protein in Loligo termed Loligo β-microsemino-protein (Loligo β-MSP) that immediately changes the behavior of male squid from calm swimming and schooling to extreme fighting. Loligo β-MSP is synthesized in the accessory sex gland of females—the oviduct gland, the main and accessory nidamental glands—and is secreted with the proteins of the outer tunic of egg capsules. When a male is attracted to the eggs visually, upon touching them and contacting Loligo β-MSP, it immediately escalates into intense physical fighting with any nearby males.

Loligo β-MSP was originally discovered in human seminal plasma and prostatic fluids [39]. It is only described in other vertebrates [40–44] and in the basal chordate amphioxus [45]. It is a highly variable 91-amino-acid protein, with 10 spatially conserved cysteine residues than can potentially form five intramolecular disulfide bonds, giving resistance to proteolytic cleavage to prolong its activity on the egg surface.

In *Sepia officinalis*, Enault and collaborators [9] identified three major related transcripts encoding secreted peptides and expressed in the oviduct gland. RT-PCR and mass spectrometry analyses revealed that transcripts and expression products were co-localized in the oviduct gland. The two very similar protein precursors termed SPα and SPα′ (**Figure 4**) diverge by only four amino acids in the α3 and α3′ peptides. They yield seven putative expression products ranging from 1.3 kDa (α5) to 7 kDa (α3 and α3′).

All peptides except α1 contained at least one cysteine, and two of them, α3 and α3′, are C-terminally amidated like many bioactive peptides (**Figure 5A**).

The third protein precursor, termed SPβ, shares 56.7% similarity with SPα and SPα′ (**Figure 4**) and yields five putative expression products ranging from 1.1 kDa (peptide β1) to 8.3 kDa


of cleavage of SP precursors coexist and generate low-molecular-weight peptides (prohormone convertase cleavages) and also 22–26 kDa polypeptides/proteins released by the

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 13

The mechanism that leads to the release of both low- and high-molecular-weight pheromones processed from a same protein precursor has to be elucidated. The occurrence of C-terminal amidation for peptides β2 and α3 demonstrates that two distinct processings are performed in the Golgi apparatus, which means that low-molecular-weight pheromones (LMWPs) are not degradation products of high-molecular-weight pheromones

The present functional hypothesis could be that LMWPs induce mating and the release of oocytes into the mantle cavity, and that HMWPs, as described in *Aplysia*, facilitate the

**Figure 5.** Schematic diagrams showing the organization of *Sepia officinalis* pheromone precursors (A) SP α and α′; (B) SPβ. Precursors encode a complex cocktail of peptides and polypeptides resulting from dibasic cleavages. Black box, signal peptide; vertical black line, potential dibasic residue cleavage site; asterisk, predicted N-linked glycosylation site; S, Cys

eggs into the surrounding medium.

(HMWPs).

residue [9].

**Figure 4.** Amino acid alignments of the three protein precursors SPα-α′ and SP β. Red asterisks indicate conserved cysteines. Predicted signal sequences are highlighted in yellow, the conserved sequence between SPs in green, differences between Spα and SPα′ in blue, and potential basic residue cleavage sites in red [9].

(peptide β3), with C-terminal amidation (peptide β2), disulfide bonds (peptides β3, β4, β5), or N-glycosylation (peptide β3) (**Figure 5B**).

For most of the expression products derived from SPα-α′ and SPβ, predicted post-translational modifications such as C-terminal amidation and disulfide bonds have been confirmed by nLC-MS/MS analysis. These modifications can provide a strong protection against protease and peptidase activity and can be expected to confer the peptides a long life in marine environments. As most of the predicted peptides were recovered by nLC-MS/MS analysis, the processing of SPα-α′ and SPβ should lead to the release of a cocktail of waterborne pheromones. Peptides α3 and α2 strongly stimulate the contraction of the penis and the gills when they are applied on these parts (**Figure 6**).

Therefore, peptides expressed and secreted by a female's accessory sex gland can modulate the activity of a male's genital apparatus. Recent unpublished data show that the protein precursors SPα and SPβ are also able to release a second batch of high-molecularweight (22–26 kDa) pheromones secreted with the egg capsule proteins and integrated to the inner layer of the egg capsule. Finally, they are detected in the sea water around egg masses once they have crossed the outer layer of the egg capsule. The presence of these high-molecular-weight pheromones identified by proteomic analysis of the oviduct gland and egg capsule also implies the presence of high-molecular-weight polypeptides/ proteins derived from SP precursors. These analytical results demonstrate that two modes of cleavage of SP precursors coexist and generate low-molecular-weight peptides (prohormone convertase cleavages) and also 22–26 kDa polypeptides/proteins released by the eggs into the surrounding medium.

The mechanism that leads to the release of both low- and high-molecular-weight pheromones processed from a same protein precursor has to be elucidated. The occurrence of C-terminal amidation for peptides β2 and α3 demonstrates that two distinct processings are performed in the Golgi apparatus, which means that low-molecular-weight pheromones (LMWPs) are not degradation products of high-molecular-weight pheromones (HMWPs).

The present functional hypothesis could be that LMWPs induce mating and the release of oocytes into the mantle cavity, and that HMWPs, as described in *Aplysia*, facilitate the

(peptide β3), with C-terminal amidation (peptide β2), disulfide bonds (peptides β3, β4, β5), or

**Figure 4.** Amino acid alignments of the three protein precursors SPα-α′ and SP β. Red asterisks indicate conserved cysteines. Predicted signal sequences are highlighted in yellow, the conserved sequence between SPs in green, differences

For most of the expression products derived from SPα-α′ and SPβ, predicted post-translational modifications such as C-terminal amidation and disulfide bonds have been confirmed by nLC-MS/MS analysis. These modifications can provide a strong protection against protease and peptidase activity and can be expected to confer the peptides a long life in marine environments. As most of the predicted peptides were recovered by nLC-MS/MS analysis, the processing of SPα-α′ and SPβ should lead to the release of a cocktail of waterborne pheromones. Peptides α3 and α2 strongly stimulate the contraction of the penis and the gills when they are

Therefore, peptides expressed and secreted by a female's accessory sex gland can modulate the activity of a male's genital apparatus. Recent unpublished data show that the protein precursors SPα and SPβ are also able to release a second batch of high-molecularweight (22–26 kDa) pheromones secreted with the egg capsule proteins and integrated to the inner layer of the egg capsule. Finally, they are detected in the sea water around egg masses once they have crossed the outer layer of the egg capsule. The presence of these high-molecular-weight pheromones identified by proteomic analysis of the oviduct gland and egg capsule also implies the presence of high-molecular-weight polypeptides/ proteins derived from SP precursors. These analytical results demonstrate that two modes

N-glycosylation (peptide β3) (**Figure 5B**).

12 Biological Resources of Water

between Spα and SPα′ in blue, and potential basic residue cleavage sites in red [9].

applied on these parts (**Figure 6**).

**Figure 5.** Schematic diagrams showing the organization of *Sepia officinalis* pheromone precursors (A) SP α and α′; (B) SPβ. Precursors encode a complex cocktail of peptides and polypeptides resulting from dibasic cleavages. Black box, signal peptide; vertical black line, potential dibasic residue cleavage site; asterisk, predicted N-linked glycosylation site; S, Cys residue [9].

aggregation of mature cuttlefish in the coastal egg-laying areas. All these data confirm that cuttlefish eggs are a source of pheromones, as described in other mollusks such as marine gastropods of the genus *Aplysia* [46–48]. Behavioral tests now have to be performed to clarify the mechanism of action of LMWPs and HMWPs in sexually mature cuttlefish.

**6. Ovarian regulatory peptides**

primocultures of glandular cells from main nidamental glands.

ates according to the successive spawning events.

peptide ILME and (C) synthetic SepOvotropin [4, 5].

lated the incorporation of 3

The role of the ovary in the regulation of the synthesis of capsular products secreted by MNGs was highlighted for the first time by Henry and Boucaud-Camou [49]. Ovary extract stimu-

Seawater used for incubating oocytes also modified the contractile activity when applied on perfused oviduct (**Figure 7A**). The first ovarian regulatory peptide ever characterized was the tetrapeptide ILME [4], followed by SepOvotropin [5], SepCRPs (Sepia Capsule Releasing Peptides) [6, 8], and OJPs (Ovarian Jelly Peptides) [7]. All these peptides modulate the contraction of the distal oviduct, and some of them also regulate the contraction of the main nidamental glands (**Figures 7B**, **C**, and **8A**–**E**). They are expressed in vitellogenic follicles and smooth oocytes and secreted into the lumen of the oviduct during egg-laying to regulate the contractions that permit oocyte transport to the mantle cavity. They are suspected to be keyplayers in the synchronization of the accessory sex glands and oviduct. This regulation takes into account the number of oocytes stored in the genital coelom, which substantially fluctu-

A recent transcriptomic approach showed that SepOvotropin, SepCRPs, and OJPs are cleaved from a single large protein precursor of 1634 amino acids expressed in the ovarian follicle and

The occurrence of a signal peptide reveals that the expression products released by this protein precursor are secreted. The spatial and temporal expression patterns of the transcripts show that it is probably a yolk protein (unpublished results: **Figure 9B**) involved in embryo development. This implies that yolk proteins could be submitted to successive processes, leading to the release of regulatory peptides. A comparison of the protein precursors with the primary sequences obtained from MS/MS analysis, and Edman degradation revealed some mistakes probably due to the tool used to determine molecular weights (ionic trap) and to analyze MS/MS spectra by a *de novo* strategy. In SepCRPs, there was a mistake about the

**Figure 7.** Perfusion of distal oviduct with (A) seawater used for incubating mature oocytes (SWO), (B) the synthetic

smooth oocytes and as yet never described in the animal kingdom (**Figure 9A**).

HLeucine and 14CGlucose into the proteins and polysaccharides of

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 15

**Figure 6.** Bio-activity of synthetic β2 and α3 peptides. β2-induced contractions on (A) female gill and (B) penis from a threshold of 10−8 M. No activity on (C) rectum. α3-induced contractions on (D) penis from a threshold of 10−9 M [25].
