**6. Ovarian regulatory peptides**

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

**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].

the mechanism of action of LMWPs and HMWPs in sexually mature cuttlefish.

14 Biological Resources of Water

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 stimulated the incorporation of 3 HLeucine and 14CGlucose into the proteins and polysaccharides of primocultures of glandular cells from main nidamental glands.

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 fluctuates according to the successive spawning events.

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

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 The spatial and temporal expression patterns of the transcripts show that it is probably a yolk protein (unpublished results: **Figure 9B**) involved in embryo development.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 peptide ILME and (C) synthetic SepOvotropin [4, 5].

**Figure 8.** Effects of increasing concentrations of SepCRP on (A) the main nidamental gland and the whole female genital tract, (B) before the laying of a first batch of eggs, and (C) after the laying of a first batch of eggs. Effects of increasing concentrations of DQVKIVL on the whole female genital tract (D) and on the main nidamental gland (E) [6–8].

amino acid in position 5: aspartate (D) should be replaced by asparagine (N) (EISLNKDEVK instead of EISLNKD). In OJPs, the same amino acids should be swapped in the C-terminal moiety (EISLNKDEVK instead of EISLNKD), and in position 2, glutamate (E) should be replaced by glutamine (Q). In each case, the amino acids involved have very similar molecular masses that only diverge by 1 Da.

As for tetrapeptide ILME, it could be cleaved from the protein precursor of a retinol-binding protein (**Figure 10**) expressed in the ovarian follicles and the oocytes. *In silico,* data mining showed that it was the only protein precursor expressed in the ovary and containing the sequence ILME. The specificity of ovarian regulatory peptides lies in the fact that they come from the secondary cleavage of functional proteins. As they are cleaved at atypical cleavage sites, this makes

**Figure 9.** (A) Protein precursor of yolk-protein-releasing SepOvotropin, SepCRPs, and OJPs. The predicted signal peptide is highlighted in yellow and the convertase cleavage sites in red. Ovarian regulatory peptides are highlighted in gray, and the stop codon at the end of the coding sequence is indicated by an asterisk. (B) Expression pattern of the yolk protein. PF: previtellogenic follicles; VF: vitellogenic follicles; SO: smooth oocytes; ANG: accessory nidamental gland; MNG: main nidamental gland; OG: oviduct gland; PSG: posterior salivary gland; CNS: central nervous system, FPKM:

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 17

Similar regulatory peptides have been described in insects, such as TMOFs for "Trypsin-Modulating-Oostatic Factors." Bioactive peptides cleaved from vitellin membrane proteins

it difficult to predict their primary sequence on the basis of protein precursor structure.

[50] control egg development [51] and inhibit ecdysone biosynthesis [52].

fragments per kilobase of exon per million fragments mapped x 10−3.

Finally, SepCRPs and OJPs are smaller families initially described in [6–8].

A similar mistake was made at the level of SepOvotropin because the sequence PKDSML LLQVPVYamide has the same molecular weight as PKDSMoxLLLQVPVMox. The primary sequence of SepOvotropin released by the protein precursor is PKDSMLLLQVPVM. The corrected sequences of SepOvotropin, SepCRPs and OJPs are summarized in **Table 2**. They reveal the occurrence of a conserved domain suggesting that they could bind the same receptor.

**Figure 9.** (A) Protein precursor of yolk-protein-releasing SepOvotropin, SepCRPs, and OJPs. The predicted signal peptide is highlighted in yellow and the convertase cleavage sites in red. Ovarian regulatory peptides are highlighted in gray, and the stop codon at the end of the coding sequence is indicated by an asterisk. (B) Expression pattern of the yolk protein. PF: previtellogenic follicles; VF: vitellogenic follicles; SO: smooth oocytes; ANG: accessory nidamental gland; MNG: main nidamental gland; OG: oviduct gland; PSG: posterior salivary gland; CNS: central nervous system, FPKM: fragments per kilobase of exon per million fragments mapped x 10−3.

amino acid in position 5: aspartate (D) should be replaced by asparagine (N) (EISLNKDEVK instead of EISLNKD). In OJPs, the same amino acids should be swapped in the C-terminal moiety (EISLNKDEVK instead of EISLNKD), and in position 2, glutamate (E) should be replaced by glutamine (Q). In each case, the amino acids involved have very similar molecular

**Figure 8.** Effects of increasing concentrations of SepCRP on (A) the main nidamental gland and the whole female genital tract, (B) before the laying of a first batch of eggs, and (C) after the laying of a first batch of eggs. Effects of increasing concentrations of DQVKIVL on the whole female genital tract (D) and on the main nidamental gland (E) [6–8].

A similar mistake was made at the level of SepOvotropin because the sequence PKDSML LLQVPVYamide has the same molecular weight as PKDSMoxLLLQVPVMox. The primary sequence of SepOvotropin released by the protein precursor is PKDSMLLLQVPVM. The corrected sequences of SepOvotropin, SepCRPs and OJPs are summarized in **Table 2**. They reveal the occurrence of a conserved domain suggesting that they could bind the same receptor.

Finally, SepCRPs and OJPs are smaller families initially described in [6–8].

masses that only diverge by 1 Da.

16 Biological Resources of Water

As for tetrapeptide ILME, it could be cleaved from the protein precursor of a retinol-binding protein (**Figure 10**) expressed in the ovarian follicles and the oocytes. *In silico,* data mining showed that it was the only protein precursor expressed in the ovary and containing the sequence ILME. The specificity of ovarian regulatory peptides lies in the fact that they come from the secondary cleavage of functional proteins. As they are cleaved at atypical cleavage sites, this makes it difficult to predict their primary sequence on the basis of protein precursor structure.

Similar regulatory peptides have been described in insects, such as TMOFs for "Trypsin-Modulating-Oostatic Factors." Bioactive peptides cleaved from vitellin membrane proteins [50] control egg development [51] and inhibit ecdysone biosynthesis [52].


stereotyped behavior: (1) ovulation, with the release of mature oocytes in the genital coelom, (2) oocyte transport by the oviduct, (3) secretion of the inner egg capsule by the OG, (4) secretion of the outer egg capsule by the MNGs, (5) black pigmentation of the egg capsule by the ink bag, (6) fertilization of oocytes by the sperm stored in the female's copulatory pouch, and

Egg-Laying in the Cuttlefish *Sepia officinalis* http://dx.doi.org/10.5772/intechopen.71915 19

**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.

**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).

(7) attachment of eggs to the sea bottom to form an egg mass.

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

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

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 the ASGs.

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 after secretion, hence a very dynamic regulation.

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 aggregating mates in egg-laying areas.

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 stereotyped behavior: (1) ovulation, with the release of mature oocytes in the genital coelom, (2) oocyte transport by the oviduct, (3) secretion of the inner egg capsule by the OG, (4) secretion of the outer egg capsule by the MNGs, (5) black pigmentation of the egg capsule by the ink bag, (6) fertilization of oocytes by the sperm stored in the female's copulatory pouch, and (7) attachment of eggs to the sea bottom to form an egg mass.
