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. 2015 Jul 13;10(7):e0132836.
doi: 10.1371/journal.pone.0132836. eCollection 2015.

How Egg Case Proteins Can Protect Cuttlefish Offspring?

Valérie Cornet  1 , Joël Henry  2 , Didier Goux  3 , Emilie Duval  4 , Benoit Bernay  5 , Gildas Le Corguillé  6 , Erwan Corre  6 , Céline Zatylny-Gaudin  1
Affiliations

How Egg Case Proteins Can Protect Cuttlefish Offspring?

Valérie Cornet et al. PLoS One. .

Abstract

Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Egg Case and SepECP structure analysis by histological techniques.
(A) Longitudinal section of a whole 15-day-old egg. Black box, magnificence zone of Fig 1.B. (B) Longitudinal section of a 15-day-old egg stained in Prenant-Gabe trichromic. 1: outer layer of the extrachorion, 2: inner layer of the extrachorion, 3: chorion, 4: vitelline membrane, 5: oocyte. (C) Longitudinal section of a 24-hour-old egg focused on the outer layer of the extrachorion stained in Periodic Acid of Schiff. Glycoproteins polymerize and are assembled in narrow meshes (black arrows) that are organized in layers. (D) Section of the outer layer of the extrachorion of an egg in TEM (b, bacteria; mg, melanin granules).
Fig 2
Fig 2. Egg case and Main Nidamental Gland protein and glycoprotein profiles.
M: Molecular weight standard, 1: Egg Case protein extract, 2: Main Nidamental Gland protein extract, 3: fetuin, 4: β-lactoglobulin. (A) SDS-PAGE profiles of Egg Case and MNG protein extracts stained in Coomassie blue. a: 135kDa band containing SepECP 1 and SepECP 2, b: 75kDa band containing SepECP 2, c: 60kDa band containing SepECP 1. Black lines show bands submitted to tryptic digestion and MS/MS identification. (B) SDS-PAGE stained in PAS. a: 135kDa band containing SepECP 1 and SepECP 2, b: 75kDa band containing SepECP 2, c: 60kDa band containing SepECP 1, f: fetuin.
Fig 3
Fig 3. Identification of Egg Case proteins in the Main Nidamental Gland (MNG) and egg case (EC).
(A) MS spectrum of the tryptic peptide from the 135kDa band. In blue, m/z corresponding to SepECP1, in red, m/z corresponding to SepECP2. (B) MS/MS spectra of the two tryptic peptides AYVYGIGVGNAIR (m/z = 1352.52) and VHMAAFAFNDHISK (m/z = 1586.47) recovered from SepECP 1 and SepECP 2 proteins, respectively. (C) SepECP sequence alignment. In red, conserved amino acids; black frame, signal peptide; N-glycosylation predicted sites are underscored; blue box, cysteine-rich conserved domain.
Fig 4
Fig 4. SepECPs forming a mesh observed in TEM
(A) SepECP protein extract precipitated and solubilized in sterile seawater observed in TEM. Black arrow, protein network. (B) SepECP protein extract precipitated, solubilized and boiled in sterile water observed in TEM. Red arrow, protein alone.
Fig 5
Fig 5. Tissue-specific expression patterns for SepECPs by RT-PCR.
Expression patterns for SepECP 1, SepECP 2, elongation factor EFγ and actin transcripts in Ovary; OG, Oviductal Gland; MNG, Main Nidamental Gland; ANG, Accessory Nidamental Gland; CNS, Central Nervous System; SG, seminal Gland; AG, Accessory Gland. Ctrl−, negative control (no template added to the PCR mix).
Fig 6
Fig 6. SepECP transcript coverage obtained from Main Nidamental Gland transcriptome.
(1) In SepECP 1 and SepECP 2, respectively: Bases with coverage: 76.119% and 90.158%; Average coverage rate: 9.198 and 10.959; Maximum coverage depth: 22 and 34 reads. (2) N-terminal SepECP protein sequence, red frame: signal peptide. (3) 5’-end transcript sequence. (4) 5’-end read coverage.
Fig 7
Fig 7. SepECP/Vibrio aestuarianus complex in SEM.
(A) Vibrio aestuarianus with SepECP agglomerates (white arrows). (B) Vibrio aestuarianus without SepECPs (red arrow).
See this image and copyright information in PMC

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