Characterization of structure and protein of vitelline membranes of precocial (ring-necked pheasant, gray partridge) and superaltricial (cockatiel parrot, domestic pigeon) birds.

Of all the known oviparous taxa, female birds lay the most diverse types of eggs that differ in terms of shape, shell pigmentation, and shell structure. The pigmentation of the shell, the weight of the egg, and the composition of the yolk correlate with environmental conditions and the needs of the developing embryos. In this study, we analyzed the structure and protein composition of the vitelline membrane (VM) of ring-necked pheasant, gray partridge, cockatiel parrot, and domestic pigeon eggs. We found that the VM structure is characteristic of each species and varies depending on whether the species is precocial (ring-necked pheasant and gray partridge) or superaltrical (cockatiel parrot and domestic pigeon). We hypothesize that a multilayer structure of VM is necessary to counteract the aging process of the egg. The multilayer structure of VM is only found in species with a large number of eggs in one clutch and is characterized by a long incubation period. An interesting discovery of this study is the three-layered VM of pheasant and partridge eggs. This shows that the formation of individual layers of VM in specific sections of the hen's reproductive system is not confirmed in other species. The number of protein fractions varied between 19 and 23, with a molecular weight ranging from 15 to 250 kDa, depending on the species. The number of proteins identified in the VM of the study birds' eggs is as follows: chicken-14, ring-necked pheasant-7, gray partridge-10, cockatiel parrot-6, and domestic pigeon-23. The highest number of species-specific proteins (21) was detected in the VM of domestic pigeon. This study is the first to present the structure and protein composition in the VM of ring-necked pheasant, gray partridge, cockatiel parrot, and domestic pigeon eggs. In addition, we analyzed the relationship between the hatching specification of birds and the structure of the VM.

Introduction

The vitelline membrane (VM) is a multilayered structure that protects and gives shape to the egg yolk and separates it from the egg white. Together with the chalaza, VM keeps the egg yolk in the central part of the egg, thereby preventing its integration with the shell membranes. In addition, it acts as a diffusion barrier by transporting water and nutrients between the egg yolk and the egg white. It protects the embryo during the first 96 h of incubation against the strongly alkaline nature of the egg white [1, 2].

The specific structure of the VM helps it performs the aforementioned functions. In general, it consists of an inner layer (IL; lamina perivitellina), which is formed before ovulation from the follicular epithelium, and an outer layer (OL; lamina extravitellina), which is formed after ovulation from the mucinous secretion of infundibulum glands (the first segment of the oviduct) [3-6]. The components of IL are expressed by the hepatic cells, as well as granulosa cells, of the female birds. Between the IL and OL lies a granular “continuous membrane” (CM; lamina continua), the composition of which is not known [1]. Electron microscopic results have shown that IL is a single-layered structure and is formed of a network of cylindrical fibers. However, fibrous OL consists of a different number of sublayers [7, 8]. The IL primarily consists of glycoproteins of the zona pellucida, five of which have been identified and described previously [9]. The OL contains numerous proteins analogous to those known as the components of the egg white (ovalbumin, lysozyme C, and ovomucin) and yolk (serum albumin, immunoglobulins, lipovitellin, and apolipoprotein B) [2]. Mann conducted a proteomic analysis and has expanded the number of known VM proteins from 13 [10-12] to 137 [2]. Many of these proteins are VM-specific (ovocalyxin-36, apolipoprotein A-I, ovocleidin-116, semaphorin C3, actin, filamin, clusterin), but their functions remain to be elucidated.

So far, only the data on the structure and protein composition of VM of the hens’ eggs are available [1-6]. Much less attention has been paid to the VM of quail eggs [9, 13], and only a few studies have focused on the VM of the eggs of other bird species [7-9]. Chung et al. [7] compared the structure of the VM of hen and duck eggs, and Damaziak et al. [8] compared the structure of the VM of ostrich, emu, and rhea eggs. These authors [7-8] have demonstrated that pattern and thickness of the fibers, as well as the presence of additional structures serving, inter alia, toward the consistency of IL and OL, differ among different species. The differences observed in the strength of VM in different poultry species also suggest different structures that form the membrane [14]. All the previously studied species belong to the precocial group, in which the offsprings are partially independent at clutching, that is, they are covered with down and move and feed themselves but are dependent on parental care. According to our knowledge, there is no data regarding the effect of structure and composition of protein of VM of birds belonging to the other categories on the degree of chick development at the time of hatching. Birds are distinguished into four basic categories: precocial—covered with fluff, seeing, self-feeding, but remaining under the care of parents (e.g. chicken, turkey, duck, and quail); superprecocial—completely independent (e.g. malleefowl and brushturkey); altricial—covered with down and sighted but fed by parents (e.g. seagull); and superaltricial—blind, partially naked, unable to walk in the first few days of life, and completely dependent on parents (e.g. parrot, pigeon, and passerine) [15]. Although this classification was made on the basis of the morphological features and behavior of chicks at clutching, the differences in the structure of the egg and the course of embryogenesis [16-18] themselves suggest differences in the structure of the VM. It is known that eggs from superaltricial birds are characterized by a very small proportion of egg yolk (~20%) to white, unlike eggs from precocial birds, in which case, egg yolks constitute about 45% [19]. Moreover, the eggs of superaltricial birds are incubated immediately after laying, and the entire clutching is characterized by asynchronization, unlike the eggs of precocial birds, in which case, the incubation begins when the last egg in the clutch is laid. Consequently, most eggs of precocial birds remain in the nest for a few days before the process of incubation begins. It should be mentioned that delayed incubation time has a negative effect on the structure of VM [20].

Therefore, in this study, we hypothesize that the structure of the VM of the eggs of precocial and superaltricial birds is different due to the evolutionary adjustment to clutching behavior. We also identified the proteins present in VM using the NanoAcquity Ultra Performance LC (Waters) system, since the current knowledge on VM proteins is limited. Proteomic analysis might throw some light on the proteins involved in the protection of embryos before and during incubation. The results of this study might be useful to identify birds on par with genetic analysis.

Materials and methods

Egg collection

Eggs from three species of precocial birds (ring-necked pheasant (Phasianus colchicus), gray partridge (Perdix perdix), and laying-type ISA Brown chicken (Gallus gallus; only used for proteomic analyses) belonging to Galliformes) and two species of superaltricial birds (cockatiel parrot (Nymphicus hollandicus) representing the Psittaciformes and domestic pigeon (Columba livia) representing the Columbiformes) were studied. The eggs of pheasant and partridge were obtained from game bird breeding centers (64–061 Kamieniec, PL and 26–070 Łopuszno, PL, respectively), and the eggs of cockatiel parrot and pigeon were obtained from private farms. All birds were kept in breeding flocks, and all eggs were assumed to be fertilized because the breeding flocks included fertile males in them. Each egg was obtained from a different female on the day of laying. A total of 10 eggs from each species of the bird were stored in a refrigerator at 4°C for 24 h. The egg weight was determined (±0.1 g) after storage, and the samples were taken for analysis. VM was obtained from each egg at 48 h after laying.

Preparation of the vitelline membrane

For scanning electron microscopy (SEM), VM samples were prepared by following the methodology described by Kirund and McKee [20]. Briefly, six eggs from each of the four species were analyzed yielding a total of 24 samples. The shells of the eggs were broken, and the contents were poured onto a separator to separate the egg yolk from the white. After determining the weight (±0.1 g), the yolk was placed on a glass pan such that the germ disc was visible on the surface. VM was cut with a scalpel around the egg yolk about halfway up. The collected VM was rinsed in deionized water (~4°C) until all residues of the yolk visible to the naked eye were removed. The weight of the whole VM (±0.1 mg) was determined, and the area of the embryonic disc (not the analyzed area) was separated.

Scanning electron microscopy

Round fragments of VM (about 2–3 mm in diameter) were cut out for analysis, ensuring that they did not contain any chalaza. The fragments were placed in glass vials containing fixing agent (6 mL of 3% glutaraldehyde solution + 100 mL of 1% paraformaldehyde in 0.1 M potassium phosphate buffer, pH 7.2). Prefixation was performed for 24 h at a storage temperature of 4°C. Following this, the fragments were fixed in 1% osmium tetroxide (OsO4) prepared in phosphate buffer at room temperature for 1 h. The fixed samples were washed with distilled water and dehydrated by placing it in a series of ethanolic solutions (25, 50, 75, and 95% solutions for one time and 100% solution for three times). The samples were dried with carbon dioxide and mounted on a stub and coated with 200 Å gold. VM was observed with an FEI QUANTA 200 scanning electron microscope (Hillsboro, OR, USA), operated at 25 kV, at various magnifications.

Transmission electron microscopy (TEM)

Six eggs from each of the four species were analyzed yielding a total of 24 samples. VM samples were fixed in 2.5% glutaraldehyde for 2 h at 4°C. The fixed samples were washed with phosphate buffer (pH 7.2) for about 2 h at 4°C. Then, the samples were fixed in 2.5% glutaraldehyde for 2 h at 4°C. The fixed samples were washed with phosphate buffer (pH 7.2) for about 2 h at 4°C. Then, the samples were fixed in 1% OsO4 at 4°C for 1 h and dehydrated in an increasing gradient of ethanol and saturated with acetone. Then, the samples were immersed in Epon 812. Following polymerization of the Epon, the samples were cut with a diamond knife on an ultramicrotome (LKB, Sweden) and transferred to copper nets, which were then contrasted in uranyl acetate and lead citrate. The prepared material was examined under TEM (JEM 1220 TEM, JEOL, Japan). From the TEM image, the thickness of the samples was measured and the number of layers of individual bird species was counted using the Nikon optical microscope (type 104c, Japan) equipped with Nis Elements, version 5.10.

Protein extraction and gel electrophoresis

The obtained VMs were dried in SpeedVac. Eight eggs from each of the 5 species were analyzed yielding a total of 35 samples. Proteins were extracted from the samples by using a buffer consisting of 50 mM Tris–HCl (pH 8.0), 10% glycerol, 2% sodium dodecyl sulfate (SDS), 25 mM ethylenediaminetetraacetic acid, and protease inhibitor cocktail (Sigma-Aldrich, Poland). Samples were incubated overnight under constant stirring and at room temperature. After this, the samples were centrifuged (12000 g, 30 min, 4°C), the supernatant was collected, and the concentration of proteins was determined by using the Lowry method.

Electrophoresis in SDS-polyacrylamide gel (SDS-PAGE) was conducted under denaturing conditions in 4% thickening and 14% separating gels. The samples for analysis were prepared by mixing 15 μL of proteins with 5 μL of reducing buffer and then incubating at 95°C for 5 min under shaking (Eppendorf Thermomixer Comfort, Germany). Electrophoresis was performed in Mini Protean® 3 (Bio-Rad) at a constant current of 20 mA in 1× Tris–glycine buffer (pH 8.3). To visualize protein bands, the gels were stained with Coomassie Brilliant Blue R-250. The electrophoretically separated proteins were documented with the GelDoc 2000 gels registration system (Bio-Rad, France) and analyzed using Quantity One computer program [21, 22].

Protein identification

Followed by the precipitation of proteins by using acetone, the samples were dissolved in 0.1% RapidGest™ surfactant (Waters) in 50 mM ammonium hydroxide. After the reduction and alkylation of the cysteine residues, the samples were digested at 30°C for 12 h using trypsin (Sigma-Aldrich, Poland). The reaction was stopped by adding trichloroacetic acid at a final concentration of 1% (v/v). Low molecular weight proteins were digested with chymotrypsin (Sigma-Aldrich, Poland) in addition to trypsin. The digested peptides were analyzed using the NanoAcquity Ultra Performance LC (Waters) system combined with a mass spectrometer. Peptides were added to the Symmetry® C18 column (5 μm × 180 μm × 20 mm) (Waters) at a flow rate of 10 μL/min in 99% buffer A (0.1% formic acid in water) and 1% buffer B (0.1% formic acid in acetonitrile) for 3 min. The trapped peptides were separated on the BEH 130 C18 analytical column (1.7 μm × 75 μm × 200 mm) balanced in 97% buffer A and 3% buffer B. The column was eluted with a linear gradient of buffer B at a constant flow rate of 300 nL/min at 35°C. Online MSE analyses were performed in positive ionization mode using the Synapt G2 HDMS mass spectrometer (Waters). The fragmentation spectra were recorded in the range of 50–2000 Da, and the energy of transfer collision was increased in the range of 15–35 V. The accuracy of raw data of molecular weights was corrected with leucine enkephalin (flow rate 2 ng/μL, 1 μL/min, 556.2771 Da/e [M+H]+). Each sample was analyzed at least thrice and mixed with bovine albumin (60 fmol) as an internal standard during the tryptic digestion process. To identify the proteins, peak lists were created from raw datasets and used to search for proteins in a database using Protein Lynx Global Server, version 2.4 (Waters) [23].

Statistical analysis

All the analyzed traits were compared between the species using Duncan’s test at a significance level of P ≤ 0.05. Calculations were performed using Statistica 12 software [24].

Results

Characteristics of egg and VM

Table 1 (S1 Data) presents a comparison of the weight of the eggs between the examined species of precocial and superaltricial birds. The weight of the egg and yolk, the proportion of yolk weight to the egg weight, and the weight of the VM were found to be significantly higher in precocial birds than that of superaltricial birds (P < 0.05). Among the precocial birds, egg weight and yolk weight were higher in ring-necked pheasant eggs than that of the gray partridge eggs (P < 0.05). However, gray partridge eggs had a much higher content of egg yolk than that of ring-necked pheasant eggs (P = 0.025). Among the superaltricial birds, egg weight, yolk weight, and VM weight were found to be higher in pigeon eggs than that of cockatiel parrot eggs (P < 0.05).

Table 1

Results (mean ± SD) of the comparative analysis of the egg and yolk weights and VM characteristics of eggs from some precocial and superaltricial birds.

Items		Species
		Ring-necked pheasant	Gray partridge	Cockatiel parrot	Domestic pigeon
Egg weight	(g)	31.82 ± 0.59d	19.53 ± 1.67c	4.70 ± 0.26a	16.70 ± 0.81b
Yolk weight	(g)	10.30 ± 0.20d	7.46 ± 0.47c	0.97 ± 0.06a	3.33 ± 0.13b
Yolk ratio1	(%)	32.37 ± 0.56b	38.26 ± 1.21c	20.61 ± 0.64a	19.98 ± 1.10a
VM weight	(g)	6.70 ± 0.46c	6.50 ± 0.42c	3.89 ± 0.26b	4.61 ± 0.33a
VM ratio2	(%)	20.69 ± 1.45c	17.03 ± 1.48a	18.92 ± 1.52b	23.09 ± 1.62d
VM thickness	(μm)	37.68 ± 0.45d	33.59 ± 0.77c	7.33 ± 0.56a	21.75 ± 1.14b

a–dMeans within a row without a common superscript differ significantly, P < 0.05

VM = vitelline membrane

SD = standard deviation

1Yolk weight ratio to egg weight

2VM weight ratio to yolk weight

The highest proportion of the weight of VM in the weight of egg yolk was found to be in pigeon eggs, followed by the ring-necked pheasant and cockatiel parrot eggs, and the lowest proportion was found in gray partridge eggs (P < 0.05). The VM in the egg yolk of precocial birds was significantly thicker than that of egg yolk of superaltricial birds (P < 0.05). A thicker VM was observed in the egg yolks of ring-necked pheasant eggs than that of egg yolks of gray partridge (P = 0.016), and the VM in the egg yolk of pigeon eggs was thicker than that of egg yolk of cockatiel parrot eggs (P = 0.001).

VM structure

Figs 1 and 2 show the SEM images of the structure of the VM of egg yolks of the studied bird species. The structure of the OL (Fig 1) of ring-necked pheasant and gray partridge eggs was found to be uniformly formed by thin and thick fibers of protein that were densely arranged. The course of the fibers formed a three-dimensional network along the lines of a truss. A similar structure was observed for the OL of cockatiel parrot eggs, but the fibers showed a uniform thickness (Fig 1). A completely different structure of OL was observed in the case of pigeon eggs, as the OL in this species did not have a fibrous structure and was entirely formed from strongly branched sheets. The branches of the sheets were not regular and had a few pores of a much larger diameter than that of the pores in the networks of OL fiber of other examined bird species. However, when observed from the inside, IL did not show a typical fibrous structure in any of the examined species, even at a magnification of up to ×10000 under the SEM (Fig 2). In the case of ring-necked pheasant, gray partridge, and pigeon eggs, the IL was similar and appeared like a homogeneous layer of the membrane. In contrast, the IL of the cockatiel parrot eggs was made up of densely arranged protein grains with an irregular structure (Fig 2).

Fig 1

Scanning electron micrograph.

Outer layers of the vitelline membrane in the egg yolk of precocial (ring-necked pheasant and gray partridge) and superaltricial (pigeon and cockatiel parrot) birds.

Fig 2

Scanning electron micrograph.

Inner layers of the vitelline membrane in the egg yolk of precocial (ring-necked pheasant and gray partridge) and superaltricial (pigeon and cockatiel parrot) birds.

In the TEM image, the structure of the VM of ring-necked pheasant and gray partridge eggs showed an analogous three-layered structure (Fig 3). In both species, it was possible to distinguish the three primary layers of VM formed by IL (IL1–3) and OL (OL1–3). It was also possible to distinguish a few sublayers of different thicknesses in the cross-section of the main VM layers. The difference in the VM structure between ring-necked pheasant and gray partridge eggs was visible during the course and continuity of IL and OL. In the VM of ring-necked pheasant eggs, both IL1–3 and OL1–3 ran strictly parallel, whereas in the VM of gray partridge eggs, numerous branches of individual layers and blindly ended deviations giving an impression of internal connectors were observed in the cross-section (Fig 4). The cross-section of the whole width of the VM of cockatiel parrot eggs formed a single layer as observed in the case of ring-necked pheasant and gray partridge eggs. The TEM image of the cross-section of the VM of pigeon eggs indicated a completely different structure, in which case, the OL and IL were conventionally distinguished, but their cross-section differed significantly from the cross-section of the VM of other discussed species (Fig 3). In general, the VM of the pigeon eggs had 15–18 sublayers forming the OL and a similar number of sublayers, but with a twofold greater thickness, forming the IL. However, all the layers were much loose than that of the closely adjacent layers observed in the VM of the other examined bird species. Since the OL of pigeon VM was formed solely from sheets and not from longitudinal fibers, as in the case of ring-necked pheasant, gray partridge, and cockatiel parrot eggs, it appeared as a more homogeneous structure with less porosity.

Fig 3

Transmission electron micrograph.

The vitelline membrane of the egg yolk of precocial (ring-necked pheasant and gray partridge) and superaltricial (pigeon and cockatiel parrot) birds. OL = outer layer; CM = continuous membrane; IL = inner layer.

Fig 4

Transmission electron micrograph.

Cross-section of the vitelline membrane of the egg yolk of gray partridge eggs.

VM proteome

Fig 5 shows the electrophoretic separation of the proteins isolated from VM. The selected protein bands (red arrows) were marked, for the additional identification of the proteins. Significant differences were found between the protein bands after electrophoretic separation. In the first lane that had electrophoretically separated VM of pigeon egg yolks, a total of about 23 protein bands was observed. In the case of the VM of cockatiel parrot, gray partridge, and ring-necked pheasant eggs, the number of protein bands observed was 20, 19, and 22, respectively. The highest variability between the proteins of the VM of the birds was found for the fractions with molecular weights ranging from 37 to 100 kDa. Moreover, the protein bands (37–15 kDa) obtained from the VM of parrot and partridge eggs were found to be of less intensity compared to the VM of other bird species. A similar intensity of about 46 kDa was observed for the protein bands of pigeon and cockatiel parrot eggs. The VM protein bands of partridge and pheasant eggs in the range of high molecular weight were very similar, and the differences were observed only in the case of low molecular weight proteins. In pheasant eggs, bands of 17, 20, and 23 kDa were observed, which were not found in partridge. It should be noted that the pheasant VM resulted in a protein band of 11 kDa, which was not found in other birds (excluding hen). Similar protein profiles were found for the VM of hen and pheasant eggs in the low molecular weight range (20–10 kDa), which may indicate the similarity between the two species. Thus, the obtained results showed that the number of protein bands obtained from VM on electropherograms depended on the species of the birds.

Fig 5

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole vitelline membrane.

Analysis of proteins of the vitelline membrane in the egg yolk of superaltricial (1: pigeon and 2: cockatiel parrot) and precocial (3: gray partridge, 4: ring-necked pheasant, and 5: chicken) birds. The red arrows indicate the protein bands selected for detailed analysis.

Tables 2 and 3 and (S2 Data and S3 Data) show the results of the proteomic identification of VM protein fractions of the studied birds.

Table 2

The proteomic analysis of water-washed vitelline membrane (VM) of selected bird species.

All proteins in the whole VMs were identified by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Swiss-Prot/Trembl accession	Protein	mW (Da)	pI (pH)	PLGS score	Peptides	Theoretical peptides	Coverage (%)	Products	Digest peptides	Protein ID
PRECOCIAL
Chicken (layer hen); Gallus gallus
P00698	LYSC CHICK Lysozyme C OS Gallus gallus OX 9031 GN LYZ PE 1 SV 1	16228	9.2	18802.8	7	12	36.1	252	7	5495
tr	A0A2P4SB66 BAMTH Uncharacterized protein OS Bambusicola thoracicus OX 9083 GN CIB84 01490	20279	8.5	12399.5	14	16	47.0	250	11	789063
tr	A0A140JXP0 CHICK Zona pellucida sperm-binding protein 1 OS Gallus gallus OX 9031 GN ZP1 PE	102171	8.3	9989.5	12	27	11.0	226	9	290653
tr	A0A2H4Y814 CHICK OVA Fragment OS Gallus gallus OX 9031 GN OVA PE 2 SV 1	42838	4.9	9123.7	9	27	38.6	185	9	282735
P01012	OVAL CHICK Ovalbumin OS Gallus gallus OX 9031 GN SERPINB14 PE 1 SV 2	42853	5.0	8592.4	9	27	38.6	186	9	3671
P79762	ZP3 CHICK Zona pellucida sperm-binding protein 3 OS Gallus gallus OX 9031 GN ZP3 PE 1 SV 4	46736	5.9	7070.4	18	22	23.1	225	12	9092
tr	A0A1D5P1X2 CHICK Clusterin OS Gallus gallus OX 9031 GN CLU PE 3 SV 1	53785	5.4	3729.2	15	38	30.5	139	14	319290
tr	A0A146J2U8 CHICK Protein TENP OS Gallus gallus OX 9031 GN TENP PE 2 SV 1	47387	5.6	2215.3	4	24	11.2	60	4	400552
P53478	ACT5 CHICK Actin cytoplasmic type 5 OS Gallus gallus OX 9031 PE 3 SV 1	41808	5.1	1481.4	4	34	15.4	31	4	7156
tr	A0A0K0PUH6 CHICK Chemerin OS Gallus gallus OX 9031 GN RARRES2 PE 2 SV 1	18219	9.0	832.9	5	17	27. 8	35	5	344118
Q25C36	OLFL3 CHICK Olfactomedin like protein 3 OS Gallus gallus OX 9031 GN OLFML3 PE 2 SV 1	44817	5.7	404.5	5	31	11.2	34	5	3280
P01875	IGHM CHICK Ig mu chain C region OS Gallus gallus OX 9031 PE 2 SV 2	48142	6.0	290.5	3	26	11.2	15	3	5166
tr	A0A140T8F5 CHICK Polymeric immunoglobulin receptor OS Gallus gallus OX 9031 GN PIGR PE 4	70526	4.8	260.9	3	42	7.9	25	3	145853
P02845	VIT2 CHICK Vitellogenin 2 OS Gallus gallus OX 9031 GN VTG2 PE 1 SV 1	204677	9.3	174.8	8	131	5.8	42	8	1087
tr	A0A087VPD3 BALRE Vitellogenin 2 Fragment OS Balearica regulorum gibbericeps OX 100784 G	201826	9.2	138.2	5	123	3.6	30	5	306649
P02789	TRFE CHICK Ovotransferrin OS Gallus gallus OX 9031 PE 1 SV 2	77726	6.8	133.3	2	74	4.1	13	2	1940
Q98UI9	MUC5B CHICK Mucin 5B OS Gallus gallus OX 9031 GN MUC5B PE 1 SV 1	233393	5.2	56.8	2	139	1.3	22	2	2906
tr	A0A1D5P2X2 CHICK Alpha-2-macroglobulin-like 1 OS Gallus gallus OX 9031 PE 4 SV 1	163357	8.0	36.1	2	99	1.6	16	2	145034
Ring-necked pheasant; Phasianus colchicus
P00702	LYSC PHACO Lysozyme C OS Phasianus colchicus colchicus OX 9057 GN LYZ PE 1 SV 2	16154	9.2	6282.7	7	13	40.8	121	7	5343
tr	Q4VTT5 PHACC Zona pellucida c OS Phasianus colchicus OX 9054 PE 4 SV 1	47813	5.2	5835.7	11	24	21.1	142	9	357975
tr	A0A140JXP0 CHICK Zona pellucida sperm-binding protein 1 OS Gallus gallus OX 9031 GN ZP1 PE	102171	8.3	5648.6	9	27	9.1	149	7	290653
P79762	ZP3 CHICK Zona pellucida sperm-binding protein 3 OS Gallus gallus OX 9031 GN ZP3 PE 1 SV 4	46736	5.9	5294.3	10	22	18.1	126	8	9092
tr	A0A091UKP2 PHORB Vitelline membrane outer layer protein 1 OS Phoenicopterus ruber ruber O	19988	6.8	4644.9	9	12	30.1	116	6	608332
P41366	VMO1 CHICK Vitelline membrane outer layer protein 1 OS Gallus gallus OX 9031 GN VMO1 PE 1 SV 1	20221	8.5	4493.6	13	16	41.0	169	10	9423
tr	G1NME9 MELGA Alpha-2-macroglobulin-like 1 OS Meleagris gallopavo OX 9103 GN A2ML1 PE 4 SV 2	163475	8.9	2235.6	19	95	12.1	234	17	508606
tr	A0A2H4Y7W8 CHICK OVA Fragment OS Gallus gallus OX 9031 GN OVA PE 2 SV 1	42680	5.4	2178.5	1	27	5.2	37	1	250032
tr	A0A226MZF6 CALSU Clusterin OS Callipepla squamata OX 9009 GN ASZ78 001440 PE 3 SV 1	51670	5.4	2127.0	9	40	21.8	83	9	489139
P53478	ACT5 CHICK Actin cytoplasmic type 5 OS Gallus gallus OX 9031 PE 3 SV 1	41808	5.1	1991.00	7	34	20.2	55	6	7156
tr	A0A226N891 CALSU Uncharacterized protein OS Callipepla squamata OX 9009 GN ASZ78 008997 P	84169	5.4	1449.8	8	66	8.0	96	7	478906
tr	G1MYK6 MELGA Ovalbumin OS Meleagris gallopavo OX 9103 GN SERPINB14 PE 3 SV 2	42988	5.0	1060.2	2	26	8.5	43	2	502906
tr	G1MVV5 MELGA Ovotransferrin OS Meleagris gallopavo OX 9103 GN TF PE 3 SV 2	77727	6.6	723.6	6	72	11.5	47	6	498065
Q98UI9	MUC5B CHICK Mucin 5B OS Gallus gallus OX 9031 GN MUC5B PE 1 SV 1	233393	5.2	310.7	3	139	2.2	41	3	2906
tr	A0A1V4JWT9 PATFA Ovalbumin-related protein X OS Patagioenas fasciata monilis OX 372326 GN	44274	7.8	271.7	5	30	3.1	27	1	130111
tr	A0A091G4Q7 9AVES Alpha-2-macroglobulin-like 1 Fragment OS Cuculus canorus OX 55661 GN N	107871	6.5	267.4	2	61	2.5	26	2	173711
tr	A0A2I0TKM1 LIMLA Type II cytoskeletal 5-like OS Limosa lapponica baueri OX 1758121 GN lla	63071	5.0	231.8	2	55	2. 5	50	2	528417
P01013	OVALX CHICK Ovalbumin-related protein X Fragment OS Gallus gallus OX 9031 GN SERPINB14C PE 3 SV 1	26274	4.9	185.6	1	15	5.2	11	1	3669
Gray partridge; Pedrix pedrix
tr	G1MYK6 MELGA Ovalbumin OS Meleagris gallopavo OX 9103 GN SERPINB14 PE 3 SV 2	42988	5.0	7342.1	7	26	31.3	122	7	502906
tr	G1NMV6 MELGA Clusterin OS Meleagris gallopavo OX 9103 GN CLU PE 3 SV 2	35883	6.6	6536.1	11	28	29.6	143	9	552529
tr	A0A2P4SV45 BAMTH Uncharacterized protein Fragment OS Bambusicola thoracicus OX 9083 GN	46710	5.4	6413.0	2	27	8.6	59	2	747230
tr	P84496 ALOAE Lysozyme OS Alopochen aegyptiaca OX 30382 PE 3 SV 1	14408	9.5	5976.3	8	11	25.6	117	4	689231
tr	A5HTY5 COTCO ZP1 protein Fragment OS Coturnix coturnix OX 9091 GN ZP1 PE 2 SV 1	101001	8.0	5353.4	9	30	8.2	150	6	33861
tr	Q4VTT5 PHACC Zona pellucida c OS Phasianus colchicus OX 9054 PE 4 SV 1	47813	5.2	5352.3	12	24	21.1	169	9	357975
tr	Q4VTT4 9GALL Zona pellucida c OS Lyrurus tetrix OX 1233216 PE 4 SV 1	47747	5.7	5170.9	12	24	22.6	155	10	201628
tr	G1MVV5 MELGA Ovotransferrin OS Meleagris gallopavo OX 9103 GN TF PE 3 SV 2	77727	6.6	3204.9	15	72	20.4	155	14	498065
O42273	TENP CHICK Protein TENP OS Gallus gallus OX 9031 GN TENP PE 2 SV 1	47404	5.5	3146.7	1	25	4.8	39	1	9683
tr	G1NME9 MELGA Alpha-2-macroglobulin-like 1 OS Meleagris gallopavo OX 9103 GN A2ML1 PE 4 SV 2	163475	8.9	2869.8	21	95	16.6	263	20	508606
tr	A0A2P4TFF1 BAMTH Uncharacterized protein OS Bambusicola thoracicus OX 9083 GN CIB84 00115	185200	8.0	1146.6	7	111	4.5	82	6	740122
tr	A0A226MNG6 CALSU Uncharacterized protein OS Callipepla squamata OX 9009 GN ASZ78 006884 P	49818	4.9	962.9	2	41	5.9	22	2	510342
tr	A0A087R1Y4 APTFO Uncharacterized protein Fragment OS Aptenodytes forsteri OX 9233 GN AS	26013	4.7	790.6	4	14	5.2	20	1	341341
tr	A0A2I0LY02 COLLI Alpha-2-macroglobulin-like protein 1 OS Columba livia OX 8932 GN A306 00	165215	9.2	743.6	4	95	3.0	52	4	59852
P53478	ACT5 CHICK Actin cytoplasmic type 5 OS Gallus gallus OX 9031 PE 3 SV 1	41808	5.1	464.7	3	34	12.0	21	3	7156
Q98UI9	MUC5B CHICK Mucin 5B OS Gallus gallus OX 9031 GN MUC5B PE 1 SV 1	233393	5.2	126.7	3	139	1.9	30	3	2906
tr	A0A091G4Q7 9AVES Alpha-2-macroglobulin-like 1 Fragment OS Cuculus canorus OX 55661 GN N	107871	6.5	104.2	2	61	2.5	21	2	173711
tr	G3UU60 MELGA Uncharacterized protein OS Meleagris gallopavo OX 9103 PE 4 SV 1	75040	8.7	62.9	3	59	6.0	15	3	846630
SUPERALTRICIAL
Domestic pigeon; Columba liva
tr	A0A2I0MW20 COLLI Ovalbumin-related protein X OS Columba livia OX 8932 GN A306 00001787 PE	43832	7.9	15955.0	39	31	61.3	546	25	59432
tr	A0A2I0LY02 COLLI Alpha-2-macroglobulin-like protein 1 OS Columba livia OX 8932 GN A306 00	165215	9.2	12830.3	59	95	31.9	1050	48	59852
tr	A0A2I0M6I1 COLLI Zona pellucida sperm-binding protein 3 OS Columba livia OX 8932 GN A306 00	47079	6.5	8856.7	12	29	15.2	168	7	51054
tr	A0A1V4JAY6 PATFA Uncharacterized protein OS Patagioenas fasciata monilis OX 372326 GN AV5	119578	9.0	7324.3	28	60	20.6	464	22	631533
tr	A0A1V4JYZ2 PATFA Vitellogenin 2-like OS Patagioenas fasciata monilis OX 372326 GN AV530 0	175956	8.2	5428.3	21	106	15.7	254	20	288584
tr	A0A2I0LGQ9 COLLI Uncharacterized protein OS Columba livia OX 8932 GN A306 00000120 PE 4 S	34862	5.5	5357.3	10	18	21.0	153	7	59471
tr	R7VU42 COLLI Ig gamma chain C region Fragment OS Columba livia OX 8932 GN A306 11075 PE 4 S	10819	4.5	4089.1	4	8	34.0	61	3	56564
tr	A0A094KPL4 ANTCR Alpha-2-macroglobulin-like 1 Fragment OS Antrostomus carolinensis OX 2	80882	9.6	3225.3	8	49	7.7	134	5	737869
tr	A0A2I0LIU5 COLLI Lysozyme OS Columba livia OX 8932 GN LYZ PE 3 SV 1	16950	9.7	1297.8	5	15	23.3	45	4	50139
tr	A0A2I0MDV9 COLLI Vitellogenin 1 OS Columba livia OX 8932 GN A306 00005842 PE 4 SV 1	155410	9.9	1280.7	25	104	24.8	271	24	60028
tr	U3JPW5 FICAL Uncharacterized protein OS Ficedula albicollis OX 59894 PE 3 SV 1	59786	5.6	1149.3	5	41	10.3	51	5	88292
tr	A0A1V4JZ59 PATFA Vitellogenin 1 OS Patagioenas fasciata monilis OX 372326 GN AV530 007742	188213	8.6	1140.6	26	132	18.0	237	25	130590
tr	H0Z0C5 TAEGU Uncharacterized protein OS Taeniopygia guttata OX 59729 PE 3 SV 1	55310	4.8	1081.6	3	37	5.4	36	3	456330
P63256	ACTG ANSAN Actin cytoplasmic 2 OS Anser anser anser OX 8844 GN ACTG1 PE 2 SV 1	41850	5.2	852.9	9	34	34.7	49	8	7055
tr	A0A2I0MWA2 COLLI Ovalbumin-like OS Columba livia OX 8932 GN A306 00001789 PE 3 SV 1	42895	4.6	832.3	9	25	38.6	64	8	62894
tr	A0A2I0LJ29 COLLI Uncharacterized protein Fragment OS Columba livia OX 8932 GN A306 0000	24662	6.6	504.8	2	19	11.4	16	2	687600
tr	A0A091VIN1 NIPNI Alpha tectorin Fragment OS Nipponia nippon OX 128390 GN Y956 09212 PE	46906	7.1	461.7	5	27	7.9	29	4	182954
tr	A0A1V4JQ70 PATFA Uncharacterized protein OS Patagioenas fasciata monilis OX 372326 GN AV5	18384	8.4	451.0	2	15	18.1	15	2	636963
tr	A0A2I0LN01 COLLI BPI fold-containing family B member 2 OS Columba livia OX 8932 GN BPIFB	54428	4.6	352.7	4	23	11.2	30	4	686748
tr	A0A2I0LY07 COLLI Alpha-2-macroglobulin-like protein 1 OS Columba livia OX 8932 GN A306 00	145725	8.0	340.1	9	86	9.8	67	9	692380
tr	A0A2I0TKM1 LIMLA Type II cytoskeletal 5-like OS Limosa lapponica baueri OX 1758121 GN lla	63071	5.0	299.9	3	55	3.7	36	3	528417
tr	R7VRC4 COLLI Complement C3 OS Columba livia OX 8932 GN A306 14901 PE 4 SV 1	181394	6.6	271.1	5	134	4.5	36	4	55053
tr	A0A2I0LIU4 COLLI Uncharacterized protein Fragment OS Columba livia OX 8932 GN A306 0000	51191	5.9	267.9	4	26	12.9	27	4	62521
tr	A0A1V4KQV2 PATFA Ovotransferrin OS Patagioenas fasciata monilis OX 372326 GN TF PE 3 SV 1	77253	6.9	194.4	6	68	12.5	42	6	629419
tr	A0A2I0ME42 COLLI Vitellogenin 2-like OS Columba livia OX 8932 GN A306 00005830 PE 4 SV 1	189533	8.8	151.3	10	127	7.7	73	10	54877
Cockatiel parrot; Nymphicus hollandicus
tr	A0A0Q3PS33 AMAAE Ovalbumin-related protein Y-like protein OS Amazona aestiva OX 12930 GN	45758	8.1	3217.7	16	30	14.8	192	8	115683
tr	A0A091GPI0 BUCRH Zona pellucida sperm-binding protein 3 Fragment OS Buceros rhinoceros si	32669	5.5	2184.3	2	20	9.0	29	2	516948
tr	U3JXV8 FICAL Uncharacterized protein OS Ficedula albicollis OX 59894 GN LOC101815176 PE 4 SV	160510	9.5	1178.0	5	108	4.6	91	5	82838
tr	A0A091IIQ9 CALAN Zona pellucida sperm-binding protein 1 Fragment OS Calypte anna OX 9244	95792	7.1	579.4	2	34	3.6	16	2	593785
tr	A0A2I0TKM1 LIMLA Type II cytoskeletal 5-like OS Limosa lapponica baueri OX 1758121 GN lla	63071	5.0	237.4	2	55	2.5	18	2	528417
tr	A0A2I0THL3 LIMLA Uncharacterized protein OS Limosa lapponica baueri OX 1758121 GN llap 16	79366	5.6	129.6	2	64	3.5	14	2	524668
P87498	VIT1 CHICK Vitellogenin 1 OS Gallus gallus OX 9031 GN VTG1 PE 1 SV 1	210497	9.2	43.2	2	144	1.4	9	2	1086

PLGS = ProteinLynx Global SERVER

Table 3

The proteomic analysis of water-washed vitelline membrane (VM) of selected bird species.

Proteins were identified from the selected bands (according to Fig 5).

Swiss-Prot/Trembl accession	Protein	mW (Da)	pI (pH)	PLGS score	Peptides	Theoretical peptides	Coverage (%)	Products	Digest peptides	Protein ID
PRECOCIAL
Ring-necked pheasant; Phasianus colchicus
Not determined
Gray partridge; Pedrix pedrix
Tr	G1MVV5 MELGA Ovotransferrin OS Meleagris gallopavo OX 9103 GN TF PE 3 SV 2	77727	6.6	1902.1	14	72	19.3	135	14	498065
O42273	TENP CHICK Protein TENP OS Gallus gallus OX 9031 GN TENP PE 2 SV 1	47404	5.5	734.1	1	25	4.8	18	1	9683
Tr	G1MW54 MELGA Zona pellucida glycoprotein 1 OS Meleagris gallopavo OX 9103 GN ZP1 PE 4 SV 1	95510	7.6	623.5	4	29	6.0	43	4	499970
SUPERALTRICIAL
Domestic pigeon; Columba livia
Tr	H0Z0C5 TAEGU Uncharacterized protein OS Taeniopygia guttata OX 59729 PE 3 SV 1	55310	4.8	471.5	2	37	3.8	21	2	456330
Cockatiel parrot; Nymphicus hollandicus
Tr	A0A087R4H2 APTFO Alpha-2-macroglobulin-like 1 Fragment OS Aptenodytes forsteri OX 9233	106930	8.0	208.5	2	61	3.2	19	2	336274
Tr	U3JXV8 FICAL Uncharacterized protein OS Ficedula albicollis OX 59894 GN LOC101815176 PE 4 SV	160510	9.5	207.7	4	108	3.5	38	4	82838
Tr	A0A091FSD8 9AVES Mucin 5B OS Cuculus canorus OX 55661 GN N303 00192 PE 4 SV 1	233620	5.4	200.9	2	140	1.4	26	2	652317

PLGS = ProteinLynx Global SERVER

The analysis of the characteristics of the proteins in the VM of hen eggs using a Venn diagram (Fig 6) revealed 14 proteins, which were not identified in the VM of other birds. In the VM of ring-necked pheasant eggs, seven proteins were identified, which were absent in the eggs of other birds. It should be emphasized that the protein structure of the VM pf pheasant eggs was more closely related to other birds in terms of the proteins present in the VM as well. The most pronounced similarity with the VM of pheasant eggs was found for gray partridge (five proteins) and hen eggs (two proteins). Among the studied avian species, the lowest number of proteins (six) was found in the VM of cockatiel parrot eggs. It should also be emphasized that one protein (A0A2IOTKM1) was found commonly in cockatiel parrot, ring-necked pheasant, and pigeon eggs. The VM of pigeon eggs demonstrated the highest number of protein bands (23). Proteomic analysis of the protein fractions of about 170 and 15 kDa obtained from the VM of pigeon eggs showed the presence of zona pellucida sperm-binding protein 1 (ZP1) and keratin protein (type II cytoskeletal 5-like) in the keratin structure of the VM, respectively.

Fig 6

A representative list of proteins of the vitelline membrane (VM) extracted from the Venn diagrams (a full list is given in Table 2 and S2 Data).

In addition, individual protein bands obtained through electrophoretic separation (Fig 5, marked with red arrows) were selected and subjected to an in-depth analysis to detect the presence of specific proteins (Fig 7). The analysis confirmed the presence of four proteins with a weight of >250 kDa and three proteins weighing approximately 35 kDa in the protein band of VM isolated from the cockatiel parrot eggs. The proteomic analysis confirmed that the 250-kDa protein found in the VM of all the birds analyzed was alpha-2-macroglobulin-like 1 protein, which is an endopeptidase inhibitor or mucin 5B. In the case of partridge and pheasant eggs, this protein was found to be in the lowest quantity. According to the results of proteomic data obtained from ProteinLynx Global SERVER (PLGS), the protein weighing 35 kDa was found as ZP3, which is present in the transparent casing. It is a thick, glycoprotein coat surrounding the oocyte. Such proteins are characterized by variability in their structure. The most conservative glycoprotein is ZP3, which consists of about 400 amino acids. The protein fraction obtained in the case of pheasant and hen eggs weighing 20 and 12 kDa, respectively, showed the presence of type II cytoskeletal keratin proteins present in the protein cytoskeleton. It should be noted that the proteins of the complement system (R7VRC4) were found in the VM of pigeon eggs (Fig 5, S2 Data) but not in the VM of other birds’ eggs.

Fig 7

Species-specific list of proteins of the vitelline membrane (VM) extracted from the Venn diagrams (a full list is given in Table 3 and S3 Data).

In the case of gray partridge and cockatiel parrot eggs, only three and seven proteins, respectively, were observed (Fig 7). The 15-kDa protein band obtained after the separation of the VM of domestic pigeon demonstrated the presence of H0Z0C5 protein, the function of which has not yet been identified.

Discussion

According to our results, the structure of the VM of superaltricial birds’ eggs was much more complex than that of precocial birds’ eggs. First, the VM of both pheasant and partridge eggs was composed of three analogous layers: IL, CM, and OL. This finding is interesting because previous studies on the structure of VM of hen eggs have shown the presence of one fibrous IL and one OL separated by a thin continuous layer of CM [1, 25–27]. All three species of birds, namely, hen, pheasant, and partridge belong to the category of precocial birds. This result indicates however the species differences between them. Using the example of VM of hen eggs, Waclawek et al. [28] and Takeuchi et al. [6] previously demonstrated that the IL components are secreted by granulosa cells in the ovarian follicle. According to Bausek et al. [29], at least one of the major IL components—chkZP1—is synthesized in the liver and is transported via the bloodstream to the ovarian follicle. These authors showed that the protein components constituting CM and OL are formed after ovulation in the infundibulum or other parts of the oviduct during the shift of the yolk sac. However, this cannot be possible in the case of the VM structure of pheasant and partridge because the individual layers are overlapped. Consequently, only IL3 can be formed from the products of the granulosa cells of the ovarian follicle. Both IL1 and IL2 must be produced after ovulation, similar to the three layers of CM and OL. The specification of granulosa cells in the ovarian follicle, including both oocytes and oviduct walls, for glycoproteins producing IL and other proteins such as ovomucin, lysozyme, and VMO-II, which have been characterized as typical for OL, is not excluded [12, 30].

According to the TEM images, the VM of the eggs of superaltricial birds has a much less diversified structure compared to the VM of precocial ones, including that of the hen eggs known from the literature. It is noteworthy that the structure of the VM of pigeon eggs is completely different. By convention, a single layer of IL and OL was marked in the TEM image, but the differences between them are so subtle that the whole structure of VM can be considered as IL formed from multiple thin sublayers. This may be partly due to the fact that the whole VM is made up of flat sheets and not cylindrical fibers as observed in the other species. In the literature, only Chung et al. [7] described the similarity in the structure of the VM of duck and hen eggs. Nevertheless, the structure of the VM of both these species was formed from cylindrical fibers of different thicknesses and less number of sheets and thus appearing similar to the structure observed for pheasant and partridge eggs. Based on these observations, it can be concluded that the different structure of the VM of the pigeon egg is not, however, a characteristic feature of the precocial species, as evidenced by the structure of the VM of parrot eggs, in which case, the VM consisted of three clearly separated layers (as per TEM images)—IL, CM, and OL—and the OL was fibrous and sheet-free structure (as per SEM images), and hence was similar to the VM structure of hen eggs described in the literature. However, compared to the structure of the VM of the hen egg described earlier by Kido and Doi [25], it was completely thin with a much less developed OL.

According to Kido and Doi, structural differences in the VM of the birds of the studied superaltricial and precocial species, and other altricial species for which such a characterization had been performed previously, maybe due to the differences in clutching specificity. First, the functions of the VM can be divided into those that take place during the inhibition of embryonic development before incubation and those that occur during incubation. The embryonic development is inhibited when the egg is laid as a result of temperature reduction from 41°C (body temperature of bird) to ambient temperature. This period differs between the bird species and depends on the number of eggs in a single clutch and whether the species clutching is synchronized or asynchronized. In the case of species with multiple clutches, in which a strong synchronization is the key hatching strategy, most of the eggs go through a period during which the embryo lives but does not develop while waiting for the incubation to begin. However, the mechanisms of egg “aging” in which the VM plays a significant role constantly occur during this period. The basic mechanism of aging involves the penetration of water through the VM from the egg white to the yolk due to the loss of CO2 in the shell pores and the increase in the pH of the egg white (from 7.6 to 9.7). As a result, the ovomucin–lysozyme complex is disintegrated, and the VM is loosened and becomes more permeable to pathogens [20, 31]. Among the studied species, this threat is incomparably greater for pheasants and partridge eggs. A single clutch consists of 8–12 eggs in the case of pheasant [32] and even up to 20 eggs in the case of partridge [33]. Both these species are characterized by a strong synchronization of clutching, and hence, incubation starts only when the last egg is laid. As a result, the duration between the laying of the first egg and hatching spans even several weeks. On the contrary, pigeons lay only two eggs in a clutch and start brooding as soon as the first one is laid [34]. Parrots use a similar strategy; they lay 4–6 eggs but incubation them after laying the first or second egg and the whole clutch is characterized by asynchronization [35]. Thus, a highly developed VM seen in the eggs of pheasant and partridge may help them in counteracting the negative effects of the long waiting time for the eggs to start incubation. However, it is interesting to explore as to why the structure of the VM of pheasant and partridge eggs is so different from that of the hen eggs presented by other authors, even though they are all precocial birds [1, 25–27]. Analyzing the natural clutching strategies of the wild ancestor of the domestic hen, Red Jungle Fowl, it was observed that its clutch consisted of a small number of eggs (4–6) and the period from the laying of the first egg to the start of brooding did not last longer than 8 days [36]. There is no information available in the literature on the effect of domestication and hen selection on the structure of the VM. Therefore, the structure of the VM of the hen eggs presented by Kido and Doi [25], Tan et al. [26], and Li et al. [27] is probably the same as in their ancestor. Kirunda and McKee [20] also demonstrated that the structure of the VM of a hen egg loosens as early as 7 days after laying, becoming more susceptible to interruption. Seven days is also considered an optimal storage period for the hatching of hen eggs, followed by a significant decline in their biological value [37]. Therefore, it can be assumed that due to a more abundant clutch and a consequently longer period of inhibition of embryo development compared to hens and superaltricial birds, the eggs of pheasant and partridge have a strongly expanded VM. It is assumed to slow down the negative effects of the natural “aging” of eggs.

Apart from a long period of residence in the state of inhibition of embryogenesis, pheasant and partridge eggs are also characterized by a relatively long incubation time, which lasts for about 23–24 days [32, 33]. Despite their high egg weight, the incubation time of hen eggs is short (21 days) [37], whereas the incubation time of the eggs of superaltricial birds is even shorter—18 days in the case of a parrot [35] and only 14 days in the case of a pigeon [34]. During incubation, VM performs both antioxidant and antibacterial functions due to the presence of specific proteins, as well as the mechanical functions favored by its structure. It is also primarily responsible for the transport of nutrients between the yolk and the walls of the blood vessels of the embryo, takes part in the formation of fetal membranes, and enables the sac to be drawn into the body cavity before the clutch [38]. The supply of the substance of the yolk sac in the precocial species is much higher than in the altricial ones, since the chicks of the precocial birds are not fed by the parents, and it must be sufficient for them until they are able to feed themselves. However, the chicks of the superaltricial birds stay in the nest for several weeks and are fed by the parents from the first day, and therefore, they do not need a large supply of yolk material. Thus, maintaining the continuity of the walls of a heavy yolk sac and pulling it into the body cavity requires a much stronger structure of the VM in the case of precocial birds than that of the superaltricial ones.

The use of the NanoAcquity Ultra Performance LC (Waters) system combined with a mass spectrometer for proteomic analysis enabled us to conduct a minimalist approach for the processing of the concentrated extract of VM. As a result, it was possible to identify a large number of protein components in the VM of as many as five bird species (Tables 2 and 3 and S2 Data and S3 Data). This is the first comprehensive report on the comparison of the VM proteome of superaltricial and precocial species, which provides a starting point for the determination of the function and the molecular basis of the properties of VM. Most of the components of protein of the VM were previously determined from the other parts of the egg structure (e.g. ovalbumin and ovomucin) [2, 39]. However, the biochemical function of ovalbumin is still undefined. It is most abundant in egg white and is a nonfunctional member of the SERPIN family and has no antimicrobial activity [40, 41]. It probably functions only as a reserve material for a developing embryo. However, ovomucin is responsible for the viscosity of egg white and maintaining its proper structure [42, 43].

In this study, however, proteins with antibacterial activity were mainly identified in the VM of the eggs: ovotransferrin and lysozyme C. Ovotransferrin is an acidic glycoprotein (pI 6.0), which constitutes about 12–13% of the egg white in birds [44]. It inhibits the development of various microorganisms possibly through binding with iron ions, which is an essential growth factor and is responsible for the proper maintenance of the cellular redox status [45]. Lysozyme C is a bacteriolytic enzyme occurring in the form of a monomer and belongs to the group of glycosidic hydrolases. This enzyme may cause damage to the bacterial cell wall membrane system by hydrolyzing the polypeptide bonds [46]. Among the lesser-known proteins, clusterin, alpha-macroglobulin, and olfactomedin were found in the VM of the eggs of the studied birds. Clusterin is a strong ubiquitous extracellular protein that inhibits protein aggregation and precipitation caused by physical or oxidative stress [47]. Studies conducted by other authors showed the presence of clusterin in the shell matrix and white of hen eggs, but it was not described previously as a component of the VM proteome. The function of clusterin and alpha-macroglobulin has been suggested to support the process of egg formation [41]. Olfactomedin plays an important role in dorsal-central modeling during the early embryonic development of hen [48].

OCX-32 was found only in the VM of hen eggs. This protein is secreted in high concentration by the shell gland during the final stage of calcination and is localized mainly on the surface of the shell, forming a cuticle together with mucin [49]. Moreover, this protein belongs to the group of immune proteins with bactericidal characteristics, which increases permeability (bactericidal permeability-increasing protein—BPI) by binding to bacterial lipopolysaccharides. OCX-32 protein was not identified in the VM of the other four bird species, which may indicate that it is a protein that is specific for G. gallus species.

Compared to other proteomic studies of different morphological parts of the egg of birds [41], this study identified several new proteins, including vitellogenin 1 (VTG1) and vitellogenin 2 (VTG2). Vitellogenins take part in lipid movement [48]. In addition, these proteins are involved in the biosynthesis of lipovitellins and phosvitin. Phosvitin is an important element of the granular fraction of egg yolk and exhibits antioxidant properties. It also has the ability to chelate metal ions (favorable for the formation of free radicals). Such properties allow an effective inhibition of the oxidation of, for example, phospholipids. Moreover, according to Cordeiro and Hincke [49], these proteins are a source of nutrients for the developing bird embryo.

Another protein that was identified (hen, ring-necket pheasant, gray partridge, cockatiel parrot) in this study is the ZP. It should be noted that this protein was not found in the VM of domestic pigeon eggs. According to the literature, this protein can be located in the whole volume of an egg [50]. The ZP was originally identified in pigs and was named so because it binds to the transparent oocyte casing. It is made up of glycoproteins, and so far, three main glycoproteins of the transparent casing have been identified—ZP1, ZP2, and ZP3. The glycoproteins in the transparent casing are synthesized by oocytes during the growth phase. N-Glycans present on the surface of the proteins, especially the high-mannose structures and branched chains of the complex type, play a special role in the binding of the sperm to the transparent casing. These glycoproteins may show a high affinity for the glycoproteins present on the sperm membrane, which consequently suggests that they may be a potential receptor for sperm [51-53]. Ultrastructural studies have shown that the lack of ZP1 prevents the acrosomes from reaching the proper concentration, which results in their fragmentation and interruption of sperm penetration [54]. Moreover, the casing protects the developing embryo until it resides. The second protein (ZP2) was first identified in Limosa lapponica baueri, a medium-sized migratory bird belonging to the Sandpipers family. It is noteworthy that this protein was not observed in the VM of the eggs of cockatiel parrot and gray partridge.

Specific VM components that differed between particular bird species included about 10 protein fractions (Fig 4). Most of the proteins were isolated, but their sequences (Tables 2 and 3 and S2 Data and S3 Data) have not yet been adequately characterized in terms of their function. The majority of proteins are similar in the VM of different bird species (they belong to the same group of proteins), but their relative proportions are completely different. The VM proteome of the eggs of hen, ring-necket pheasant, and gray partridge contained proteins that were previously identified (Tables 2 and 3 and S2 Data and S3 Data). These included lysozyme C, ovalbumin, macroglobulins, ZPs, and cytoskeleton-building proteins.

The abundant nature of individual proteins obtained from avian VM demonstrated the association between the structure and the physical traits. In the case of domestic pigeon eggs, which had the highest number of proteins, we found that their abundance could be decisive about the different physical parameters. The proteins that were commonly found in ring-necked pheasant, gray partridge, and cockatiel parrot eggs did not have such a pronounced impact on the structure of the VM. Even in the case of cockatiel parrot eggs, the VM differed considerably in terms of morphology from the VMs isolated from the eggs of other birds. This can be confirmed by the presence of six proteins, which were absent in other birds. The similar structure of VM of ring-necked pheasant and gray partridge, which indicated the presence of five common proteins should be emphasized. However, it should be noted that in-depth proteomic analysis and physical linkages between individual avian membranes should be performed to obtain the highest possible amount of information on the differences between individual bird species. Such knowledge will enable us to identify numerous differences. Furthermore, it will allow noticing individual traits present between bird species, which can be of high significance from the proteome standpoint. Obtaining such information may be the basis for the definition of their functions and properties.

Among the proteins previously reported in the VM proteome, 41 similar fractions were determined. Unfortunately, no single hypothesis can explain the diversity of protein fractions in the VM of the eggs of the studied birds. This suggests that the ecology and life history of birds have most likely changed in the course of evolution. We can only speculate whether there VM constitutes any specific protein, but the complete proteomic data of VM is not yet available. It should be mentioned that the VM database (National Center for Biotechnology Information, ExPASy: SIB Bioinformatics Resource Portal) contains sequences of proteins that are very similar to those playing an important role in the structure of other morphological parts of bird eggs.

Conclusion

In this study, we demonstrated that the structure of VM differs between different species of birds. There were differences found in the number of VM layers, their course and connections, and in the fibers forming the membrane. Despite the fact that our data is based on a limited number of species, it cannot be definitely confirmed whether these differences are directly related to the nesting specificity of birds (precocial and superaltricial), several observations support the hypothesis. In particular, the structure of the VM of ring-necked pheasant and gray partridge eggs, which are precocial birds, differed from the structure of the VM of cockatiel parrot and domestic pigeon eggs, which are classified as superaltricial birds. The considerable difference in the structure of the VM between cockatiel parrot and domestic pigeon eggs suggests phylogenetic influences. Therefore, future studies on the structure of yolk VM should be conducted considering both the nesting specificity and the phylogenetic classification of bird species.

The proteomic analysis of the VM of precocial birds (cockatiel parrot and domestic pigeon) in relation to superaltricial birds (hen, ring-necked pheasant and gray partridge) showed differences in the presence of proteins characterized by low (<20 kDa) and high molecular weights (>210 kDa). Unidentified proteins were found in all VMs, the function of which has not been completely elucidated. Scientific knowledge on this subject is still inadequate and needs to be broadened by further research and experiments aiming at the meticulous identification of new protein fractions. The analyses conducted in this study showed the presence of protein fractions having an intensity of about 44 and 220 kDa, only in the VM of the precocial species.

This study is the first to report the differences in the protein composition and structure of the VM of precocial and superaltricial birds. The data presented here may broaden the existing knowledge by enabling a better understanding of the protein composition of the VM of birds. In the future, this knowledge of the differences in the structure and protein composition of VM may serve as a tool to identify species on a par with genetic analysis and support systematic differentiation.

Raw data for Tables 1 and 2.

(XLS)

Click here for additional data file.

All the proteins identified in whole VM by SDS-PAGE.

(XLSX)

Click here for additional data file.

Proteins identified from selected bands (according to Fig 5).

(XLSX)

Click here for additional data file.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

27 Sep 2019

PONE-D-19-16659

Structure and protein identification of some precocial and superaltricial birds eggs yolk vitelline membrane

PLOS ONE

Dear Dr Damaziak,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript has been assessed by 2 reviewers who have requested a number of revisions to improve the manuscript. In particular, Reviewer #2 felt there were significant concerns with the study and requests more data integration and discussion to explain the correlations between structure, physical characteristic and protein composition/abundance. Please revise the manuscript to address all the reviewer's comments in a point-by-point response in order to ensure it is meeting the journal's publication criteria.

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Reviewer #2: No

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Reviewer #1: In this manuscript, the authors analyzed the vitelline membranes of precocial and superaltricial birds in aspects of the ultrastructures or protein compositions, and compared the results to discuss whether the characteristics of VM are related to the hatching specification of birds. Zona pellucida, a mammalian homolog of bird VM, is essential for in-vivo fertilization and early embryo development, although the molecular mechanisms that underlie physiological functions of zona pellucida are not clearly resolved. Therefore, further investigations following this study could provide new insights into the zona pellucida-related infertility or disorders of embryo development not only in taxonomic studies of birds. From these point of view, I think this manuscript is worth being accepted after some revision.

I recommend that the authors revise and/or modify manuscript in some points mentioned below.

Major points:

1) lines 19-38

Syntactic complexity or errors throughout in "ABSTRACT" section should be improved.

2) lines 50-52 and 56-57

There might be some inconsistencies in "Introduction" section. I guess the IL is mainly composed of zona pellucida glycoproteins but not collagen.

3) lines 320 and 322

CM should be mentioned and explained in the "Introduction" section and be indicated in the Figure 3.

4) lines 331-335

In chicken and Japanese quail both being included in the precocial birds, either IL or OL appears to be the single layer. According to the authors hypothesis, they should have multiple layers. How to explain the previous observations in chicken and quail VM? Is it possible to explain that the VM of precocial birds are folded after OL formation?

5) lines 493-506

It might be better that "Conclusion" section contains the conclusion bringing together the results from all the experiments in this study not only from the proteomic ones.

Minor points:

1) line 59

"apovitellin" should be replaced with "lipovitellin".

2) lines 259-277

"protein fraction" might be replaced with "protein bands" or "protein signals".

3) line 262

"first line" should be replaced with "first lane".

4) lines 262-263

"egg whites VM" might be replaced with "egg yolks VM".

5) lines 328, 329, 334 and 335

"oocyte infundibulum" or "infundibulum" should be replaced with "ovarian follicle" or "follicle". "infundibulum" is a part of oviduct.

6) line 328

Is "they" mean ZP1? If so, "they" should be replaced for eample with "one of them".

7) line 484

"zona pellucida binding proteins" might be replaced with "zona pellucida sperm binding proteins".

8) lines 494 to 495

Bird species names of the precocial and altrical birds might be exchanged.

Reviewer #2: My comments and suggestions are summarized in the attached file entitled PONE-D-19-16659- 07242019

**********

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Reviewer #1: Yes: Hiroki Okumura

Reviewer #2: No

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Submitted filename: PONE-D-19-16659-072419.pdf

Click here for additional data file.

22 Oct 2019

Response to the Review

Manuscript: PONE-D-19-16659

Dear Editor

We would like to express our gratitude for the assessment of our manuscript entitled „Structure and protein identification of some precocial and superaltricial birds eggs yolk vitelline membrane”. Conducting of the research and preparing of the publication were made by us with the greatest possible precision and care. We would like to point out that for the authors: Krzysztof Damaziak and Marek Kieliszek the affiliations have been changed. This is due to the name changes of our University Faculties from October 1, 2019. Below are corrections for the specific remarks. All the amendments were made to the text and marked with a yellow background. Due to changes in the content of the text, line numbers may not match those before the review:

Reviewer: 1

R: lines 19-38: Syntactic complexity or errors throughout in "ABSTRACT" section should be improved.

A: English in the entire manuscript has been re-verified and corrected by a native speaker. We have attached a suitable certificate in the supplemental materials.

R: Lines 50-52 and 56-57: There might be some inconsistencies in "Introduction" section. I guess the IL is mainly composed of zona pellucida glycoproteins but not collagen.

A: It has been corrected

R: Lines 320 and 322: CM should be mentioned and explained in the "Introduction" section and be indicated in the Figure 3.

A: In the chapter Introduction we have added the following sentence ‘Between them lies a granular “continuous membrane” (CM – lamina continua) of unreported composition [1].’ We have marked CM in Figure 3 and introduced an explanation in the figure caption.

R: Lines 331-335: In chicken and Japanese quail both being included in the precocial birds, either IL or OL appears to be the single layer. According to the authors hypothesis, they should have multiple layers. How to explain the previous observations in chicken and quail VM? Is it possible to explain that the VM of precocial birds are folded after OL formation?

A: Unfortunately, at the moment we are unable to precisely explain the observations we obtained. The image of three-layer VM of pheasant and partridge was a great surprise to us. We repeated the analyses several times and the TEM image was always similar. It is surprising because in the case of chicken and partridge VM, the structure is indeed single, as pointed out by the reviewer. What is more, we are currently conducting VM analyses of other avian species, including Turdus merula, Turdus philomelos, Rhea americana, Cairina moschata and despite the considerable morphological diversity, VM only cosnsists of single layers. We suspect that this can be related to the evolutionary adaptation of nesting strategies (e.g. high number of eggs per clutch). As we have attempted to explain this in the discussion, the nesting behavior of pheasant and partridge differ from those observed in, among others, wild ancestors of the domestic hen. We suspect, that the answer may be brought by histological tests of the oviduct and VM with the use of various staining types. We will seek to clarify these observations in future studies.

R: Lines 493-506: It might be better that "Conclusion" section contains the conclusion bringing together the results from all the experiments in this study not only from the proteomic ones.

A: We have completed the „Conclusion” as suggested by the reviewer

R: Line 59: "apovitellin" should be replaced with "lipovitellin".

A: We replaced "apovitellin" to "lipovitellin"

R: Lines 259-277: "protein fraction" might be replaced with "protein bands" or "protein signals".

A: It has been corrected

R: Line 262: "first line" should be replaced with "first lane".

A: We replaced "line" to "lane”

R: Lines 262-263: "egg whites VM" might be replaced with "egg yolks VM".

A: We replaced "whites" to "yolks”

R: Lines 328, 329, 334 and 335: "oocyte infundibulum" or "infundibulum" should be replaced with "ovarian follicle" or "follicle". "infundibulum" is a part of oviduct.

A: It has been corrected

R: Line 328: Is "they" mean ZP1? If so, "they" should be replaced for eample with "one of them".

A: In the indicated fragment we have introduced the following sentence: “According to Bausek et al. [29] at least one of the major IL components - chkZP1 is synthesized in the liver and is transported via the bloodstream to the ovarian follicle.”

R: Line 484: "zona pellucida binding proteins" might be replaced with "zona pellucida sperm binding proteins".

A: It has been replaced

R: Lines 494 to 495: Bird species names of the precocial and altrical birds might be exchanged.

A: It has been corrected

Reviewer: 2

R: The authors aimed to correlate the protein and structure specificities of the vitelline membrane to these brooding/hatching characteristics. Although the concept is very interesting, the article deeply lacks convincing/striking conclusions and correlations between experimental observations are either missing or too hazardous.

A: We agree with the reviewer that the conclusions and discussion may be dangerous. However, this is the first report describing possible differences resulting from the structure and protein content in the VM of individual bird species. We've improved results discussions to provide new information. At the same time, we described the Venn diagram that will make the description of the results more attractive.

R: Moreover, the article needs to be edited by an native English speaker (grammatical errors + sentences to be rephrased) and to my opinion, it doesn’t fulfill quality requirements for a research article: figure legends are in the main text (at the end of each paragraph)

A: The article was corrected by Native English Speaker (www.translmed.com). Figures and legends have been included in the text in accordance with the requirements of the magazine PlosONE.

R: The SEM and MEB pictures are of poor quality and related figures need to be clarified and detailed in related legends. Some data related to chicken egg are missing in Table 1 and in SEM/TEM data considering that you performed proteomic analysis of VM from chicken eggs. The number of proteins identified by mass spectrometry is very low and it is very difficult to perform conclusions as the tables are quite confusing. A Venn diagram comparing results obtained from each species (number of proteins per species, common and specific proteins) would have been clearer.

A: TEM and SEM images were prepared in line with the PLOS ONE journal guidelines. We perceive them as a very high quality - clear and sharp. Their quality is deteriorated after sending them to the journal and PDF generation. Unfortunately, this is beyond our control. In line with the reviewer’s suggestion, we have developed two Venn diagrams: Figure 6 in which we have presented the amount of proteins defined for individual species and the common proteins. Figure 7 presenting specific proteins defined based on the selected bands. This study did not analyze the VM structure of chicken egg yolk with the use of SEM and TEM. This stems from the fact that such studies have been conducted and published on numerous occasions. Therefore, we assumed that this kind of information is well known and one can use the available knowledge with full confidence. Restriction of the analysis by excluding the hen VM structure analysis, which is expensive, labor-intensive and time-consuming, enabled us to better focus on VM analyses of the remaining 4 species. This was highly important for us, as none of the described analyses has ever been performed for either ring-necked pheasant, grey partridge, cockatiel parrot, or domestic pigeon. However, we decided to perform protein analyses for hen VM as such data have been published only on several occasions, and we were aiming at a comparison. In the study of protein identification, we have treated hen VM as reference and bridge between our results and the literature knowledge.

R: Figures/pictures are of poor quality and are not clear to readers.

A: All Figures/pictures have been prepared in line with the PLoS ONE Author Guidelines. Their deteriorated quality stems from processing and PDF file development in the journal's system. This is beyond author's control. The minimum photo resolution is 300x300 DPI.

R: Abstract: Please clearly indicate the name of the bird species studied and compared. The number of proteins identified for each species + common and specific proteins have to be mentioned. Replace “proteomic structure” by “proteomic composition or pattern”

A: We have added full avian species names in the abstract. We have provided the amount of proteins identified per species. We have not mentioned all proteins common between individual species because this prohibited by the editorial limitation of the word count for this chapter. This has been described in detail in the results chapter based on the attached Venn diagrams. We have replaced “proteomic structure” with “proteomic composition or pattern”. However, we suggest a complete deletion of this sentence. In our opinion it is not necessary in the abstract and we may not exceed the number of 300 words.

R: Introduction: Line 48. The vitelline membrane is the matrix for yolk sac expansion over the yolk.

A: It has been corrected

R: Introduction: Line 51. IL components are expressed by the liver of laying hens but also granulosa cells

A: The text has been completed

R: Introduction: Line 62. OCX36 is not specific to the VM but to the eggshell. Apolipoprotein is a yolk-protein. Proteins that seems to be more specifically found in the vitelline membrane are VMO-I and VMO-II (also known as AvBD11)

A: A: Of course, apolipoprotein is a yolk protein but it has also been identified in the hen's egg membrane (Mann, 2008). It should be noted that depending on the species of bird, the content of this protein (apolipoprotein) may be different in the vitelline membrane.

We agree with the reviewer. Of course (VMO-I and VMO-I) these are the most popular proteins found in vitelline membrane.

Research presented by Gautron (2011) showed that the OCX36 protein is eggshell-specific protein that is secreted by the regions of the oviduct responsible for eggshell formation. It has also been identified in vitelline mebrane.

Mann, K. (2008). Proteomic analysis of the chicken egg vitelline membrane. Proteomics, 8(11), 2322-2332.

Gautron, J., Rehault-Godbert, S., Pascal, G., Nys, Y. & Hincke, M. T. (2011). Ovocalyxin-36 and other LBP/BPI/PLUNC-like proteins as molecular actors of the mechanisms of the avian egg natural defenses. Biochemical Society Transactions, 39(4), 971-976, doi:10.1042/BST0390971

R: Introduction: It would be more convincing to explain the difference in VM structures/composition by phylogenetic analyses rather than on their affiliation to precocial and superaltar birds. Such a hypothesis would be better in the discussion and further prospects.

A: Our aim was to demonstrate differences depending on the nesting specificity of birds. Analysis of structure and composition of VM through phylogenetic analysis would be indeed highly interesting and we will seek to perform it in the future. However, it seems that it is too early for such an analysis. The available literature lacks information on VM of other birds than chicken, partridge and duck. Our team has recently expanded the available knowledge on the analysis of VM ratite birds (ostrich, emu and rhea). Now we have selected 4 further species for which the VM analyses are performed for the first time. We are currently analyzing the structure and protein composition of VM of Turdidae species. We hope that soon we will be able to conduct a phylogenetic meta-analysis. In the Material and methods chapter we have supplemented the information on the classification of birds to given families, but we would like to keep the main objective of the study without major changes.

R: Introduction: Please indicate that Gallus gallus, Perdix Perdix and Pahsioanus colchicus are galliforms, Nymphicus hollandicus is a psytaciform and Columba livia a columbiform.

A: We have supplemented the information. We have introduced names of systematic orders but we believe that this will be more appropriate in the M&M chapter. Of course, we agree with the reviewer this is the next stage (with more different species of birds) of the research we carry out. In this work we have added a special chart: Venn diagram - a diagram to illustrate the relationship between sets.

R: Material and methods: Egg collection. Indicate the strain of the laying hens used form proteomics.

A: Data has been corrected. Information about the hen strain (ISA BROWN) from which eggs were used analyzes was supplemented.

R: Material and methods: Scanning electron microscopy. Please insert a specific paragraph for VM preparation since these preparations were also used for proteomics.

A: We have inserted a separate chapter on VM preparation.

VM samples for SEM analysis were prepared following the methodology described by Kirund and McKee [20]. After breaking the shell, the egg content was poured onto a separator to separate the egg yolk from the white. After weight determination (± 0.1 g), yolk was placed on a glass pan so that the germ disc was visible on the surface. VM was cut with a scalpel around the egg yolk about halfway up. The VM was then rinsed in deionized water (~4°C) until all residues of the yolk visible to the naked eye were removed The weight of the whole VM (±0.1 mg) was determined and then the area of the embryonic disc (not analyzed area) was separated.

Protein identification is presented in a separate chapter: Protein identification

R: Material and methods: TEM. Indicate what Epon 182 is. Indicate the number of biological replicates

A: Eight biological replicates were performer.

R: Material and methods: Protein extraction and gel electrophoresis. Line 145. I do not understand the reason for using “15 μL of enzymatic proteins”. What is “enzymatic” ?

A: This is a mistake in translation. Of course, there should be only "proteins".

R: Results: Eggs morphology: Table1. use VM thickness instead of width. How did you get this value ? From SEM/TEM data ? Please indicate.

A: The VM thickness was measured on TEM images via Nis Elements version 5.10, Nikon optical microscope (type 104c, Japan).

R: Results: VM Structures. Do not distinguish between precocial and superaltrical birds here. These are results not discussion, please use the respective name of birds (chicken, pheasant, partridge, parrot and domestic pigeon). The use of precocial and superaltrical words may be confusing for non experts.

A: This has been corrected

R: Results: Line 219. Remove this legend at the end of the article + add details about magnification.

A: Figure legends are introduced in the text in place for the proposed presentation. They have been introduced in this manner because such are the requirements of the PLoS ONE Submission Guidelines.

„Figure captions

Figure captions must be inserted in the text of the manuscript, immediately following the paragraph in which the figure is first cited (read order). Do not include captions as part of the figure files themselves or submit them in a separate document.”

All magnifications SEM and TEM are present in the photographs with automatic caption. We understand that they were not visible due to the quality change of these files. Properly formatted micrograms are supplied to the editorial office, thus we believe that if the manuscript is published, they will be clearly visible.

R: Results: On the figures 1, please indicate A, B, C for each panel and their meaning with respective species to facilitate the reading. . What is the third panel? indicate it in the legend (it appears only in the text).

A: Photos must have been formatted incorrectly when building the PDF. In each photo, information is added: precocial or superaltrical. All photos have a resolution of 300x300 DPI.

R: Results: VM Proteome. Fig. 5. Remove 30 μL from the legend. This is not informative. The quantity 80 μg at the end of the legend is informative

A: It has been removed

R: Results: 287. Please start with the number of proteins per species with a Venn diagram showing common and specific proteins between species. Thus you need to perform blast and alignment analyses to identify homologous proteins that may have different protein names depending on the species.

A: This has been corrected. We have changed the discussion of the results obtained. We also made a Venn diagram.

The conducted characteristics of the proteins in the vitelline membrane with the use of Venn diagram (Fig. 6) for hen demonstrated 14 proteins, which have not been determined in the VM of all birds. In the case of pheasant VM 7 proteins were identified, which were absent from other birds. It should be emphasized that the protein structure of pheasant VM was most closely related in terms of the presence of proteins present in the VM of other birds as well. The most pronounced similarity of pheasant VM was determined for grey partridge (5 proteins) and chicken (2 proteins). The lowest number of proteins (6) among the analyzed avian VM was found in cockatiel parrot. It should be emphasized, that the presence of the same protein (A0A2IOTKM1) was found for cockatiel parrot, ring-necked pheasant and pigeon. Domestic pigeon VM analysis demonstrated the highest number of protein bands (23).

In addition, individual protein bands obtained through electrophoretic separation (Fig. 5) were selected in the study and were subjected to an in-depth analysis in terms of the presence of specific proteins (Fig. 7). The conducted analysis demonstrated the highest number of 4 proteins in the case of the protein band (>250 kDa) of VM isolated from cockatiel parrot egg and 3 proteins from the protein band with the weight of approx. 35 kDa.

R: Results: You infer that alpha2-M like 1 is specific to the parrot but at least in chicken you also have an alpha-2 M protein (the last protein of the chicken section in the table) but also in the pheasant (A0A091G4Q7) etc.

A: Thank you to the reviewer for the information. The data has been corrected. We have deleted invalid data. This protein was also confirmed in an additional analysis. Supplementary S3 Data file.

R: Discussion: The discussion has to be rewritten to better show the correlation between structure, physical characteristic and protein composition/abundance.

A: A: In the discussion, chapters about proteomics and physical features were written in separate chapters. After all, we've added new information. We believe that the combination of all this information could affect the occurrence of chaos and inconsistencies while reading the article. We have determined that such a division will be the most appropriate.

The abundance character of individual proteins obtained from avian VMs demonstrated association with the structure and individual physical traits. In the case of pigeon, for which the highest number of proteins was obtained, we determined that their abundance could be decisive for its different physical parameters. The identified single common proteins that have also been found in parrot, pheasant and partridge did not have such a pronounced impact on the similar structure towards the tested VM membranes. In the case of the structure of parrot VM, it also differed considerably in morphological terms from other VMs isolated from bird eggs. This can be confirmed by the presence of 6 proteins, which were absent from other birds. The similar structure of partridge and pheasant VM should be emphasized, which may indicate the presence of 5 common proteins. However, it should be emphasized that an in-depth analysis of proteomic and physical linkages between individual avian membranes should be continued to obtain the highest possible amount of information on the differences occurring between individual bird species. Such knowledge will enable us to understand and reveal numerous differences. Furthermore, it will allow noticing individual traits present between bird species, which can be of high significance from the proteome standpoint. Obtaining such information may be the basis for the definition of their functions and properties.

R: Discussion: You may have the same overall protein composition with some subtle difference in sequence but what make the VM structure different may results from proteins interaction that may differ between species and relative abundance.

A: Of course, we agree with the reviewer's suggestion. Protein interactions occurring in the VM membrane can change its structural structure. Determination of individual protein sequences and their conformations in the PyMol program and their impact on the VM structure - this is the topic for the next scientific article that we will prepare in the future. We want to perform an in-depth analysis of individual VM membranes. Many proteins have not yet been fully characterized and their function is still unknown. This is a new topic that will allow you to deepen knowledge about the construction of the VM and its role.

R: Discussion: The presence of “yolk” proteins such as vitellogenins, apolipoproteins” and white proteins (ovalbumin, ovomucin) may rather reflect some yolk/white contamination during VM preparations. Depending on the species, the viscosity of the white may be different and stickier. The conclusions are quite hazardous in this paragraph.

A: We agree with the reviewer that the conclusions may be dangerous. However, we think this is the first report on the analysis of proteins isolated from the VM of various birds. Vitellin membrane has been very carefully prepared. Several membrane extractions were performed.

R: Line 337. VMO-II is written three times.

A: It has been corrected

Submitted filename: Response to th review.docx

Click here for additional data file.

22 Nov 2019

PONE-D-19-16659R1

Structure and protein identification of some precocial and superaltricial birds eggs yolk vitelline membrane

PLOS ONE

Dear Dr. Damaziak,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

One of the reviewers suggested rejection so this Academic Editor did not send the revised version back to him/her.  Instead, this Academic Editor served as the second review.  Please see my comments below.

PONE-D-19-16659R1

The authors should be commended that they made major efforts in improving the English of the manuscript from professional editing and comments of the reviewers.  The manuscript, however, is still very rough in both description and English expression.  These deficiencies reduce the readers’ ability to understand the data/findings and undermine the perception of the importance of the work.  They may also likely induce the conception of poor work quality by the authors because readers only see the manuscript not the actual work.  It is, therefore, to the author’s best interest to present the best manuscript possible.  With all these said, the study encompasses large amount of electron microscopy and proteomics work.  The information presented will increase our understanding of the diversity of nature.  The data are therefore worthy of being seen by the scientific community and the general public.

The expertise of this Academic Editor is not in the field of Avian reproduction.  I therefore provided comments to improve the understanding of the manuscript/data from the view of an outsider.

Please change the title to: Structure and protein characterization of the egg yolk vitelline membranes of precocial (common names of the birds???) and superaltricial birds (??)

L23: “the species”: what species?

Also please change the statement to “we analyzed how the structure and protein composition of vitelline membrane (VM) differ among ?? species”.

L27: please change “enable to” to “be important for counteracting”. What do you mean by “complex” some specificity should be given here.

L29: “triple and three-layer”: are these different things or just redundancy?

L30: what is the difference between VM and VM sheets? If not, please use one term consistently throughout the text.

L32: are the results for all birds or for one type? Please specify. Please change “weights” to “molecular weights”. What are protein fractions? How are they fractioned?

L37-39: too much repeats.  Please delete them.

L56: Please change the first “is” to “of”.

L59: please change “so far” to “previously”.

L63: what do you mean by “which are known currently”? Each one of them has been identified?

L64: please remove “first” unless you want to say that they have been found elsewhere later and therefore no longer specific to VM.

L67: Please remove “available from studies”

L70: Please add “the” before “hen”.  When referring an animal as a species, please either use “hens” or “the hen”.  There are similar mistakes in the rest of the text.  Please change them all.

L72: what is the course of the fibers? Do you mean pattern?

L73: “Additional structures” such as ???

L76: “offspring ones”? do you mean “offspring”?

L77: “the authors”: are you still referring those that published the studies you mentioned earlier? From the context, “the authors” in L71 referred to them.

L80: Please change “The birds” to “Birds”. Please also add an explaining phrase after “precocial” (just like you did for the other three categories) to be parallel and coordinated.

L83: please change “first days” to “first few days”.

L85: please change “the chicks” to “chicks”.

L88: using % for egg yolk and water does not make sense.  Because water is part of the yolk.  At least change the “a large proportion” to “a high percentage” so people are not misled into thinking you are talking about the two proportions of the eggs.

L93: “a few “ and “several” are both unspecified and don’t differ much.  I suggest you just use one of them.

L94: please change “storage” to “delayed incubation”. By “storage” you gave the impression that the eggs were taken and stored deliberately.

L98: Please add “using ?? and ?? as models” before “We”.

L112: Please remove “to be” and add “because fertile males were present” (if this is the case).

L181: molecular weights?  Please change all such occurrences in the following sections.

L183: please remove “used”.

At the ends of the sections for SEM, TEM and Protein identification, please add a statement of the number of samples used.  For example, “Ten eggs from each of the 4 species were analyzed yielding a total samples of 40”.

L190: please change to “Egg and VM characteristics”

L191: Please change “morphological traits” to “weight characteristics”.  There were no morphology characterization.

Table 1: please change “Results of the comparative analysis of the morphological” to “Weights”.

Fig 3: From the labeling it appears that CM is a very thin layer of membrane with little characteristics to be seen from the figure.  What did the authors use to identify this layer?  Please describe its features and characteristics.

L246-247: “All the three layers were also characterized by a layered structure”.  This statement does not add anything unless the authors did not state it clearly.  They have already described in previous statements that the VM were triple-layered.

In the text, “Fig” was used but in the figure legend, “Fig.” was used, please be consistent and I suggest Fig. be used because it is abbreviated.

Fig 4 was not provided in the complied file.

L254: please change “corresponded to” to “formed”

L278: Please change “in” to “for”, please also remove “of proteins”

L298-300: Fig 5 legend. Please explain what the red arrows are.

L305-317: please move this section to after Table 3

L318: what bands were selected?  Are those the red arrowed ones?  Please specify.

L319: please remove “in the study”

L322 and 324: 4 proteins >250 kDa and 3 proteins of 35kDa were found.  Later only one of each were described.  Please explain the others.

L325: please add “ZP3” here

L310-311: In Fig 5, proteins of the complement system were not noted.  Which ones are referred to here?

L333: similarly, 3 proteins were observed but only one was described.  What happened to the other two?

Tables 2 and 3: please change pH, PLGS score, and % of coverage to contain one decimal point.  Please change the molecular weight to kDa to be consistent with the text.  Also in both tables for most protein entries, portions of the IDs were presented twice consecutively. For example: A0A2P4SB66 A0A2P4SB66.  What is the reason for this annotation?

Table 3: please indicate which section corresponds to which red arrow.  If all VM proteins were identified and presented in Table 2 (as indicated in the title of Table 2), why list them again in Table 3?  Are these presented for a second time?  If so, there is no need to have this Table.

Please note that this Academic Editor did not have sufficient time to review the discussion section.  Please use the above editing as a guideline and make revision on the discussion.

We would appreciate receiving your revised manuscript by Jan 17, 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Xiuchun Tian

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Compared to the previous version of manuscript, I think the revised version is much more sophisticated as a whole. Although there are many topics that are still remain unclear for example in the formation mechanism(s) of the structurally diverse VL among bird species, the authors will raise problems properly with this manuscript.

I found only one minor point as below.

1. "after ovulation in the ovarian follicle" in Line 373 in the corrected manuscript should be corrected to "after ovulation in the infundibulum".

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

15 Dec 2019

Response to the Reviewers

Manuscript: PONE-D-19-16659R1

Dear Editor,

We would like to express our gratitude for assessing our manuscript entitled “Structure and protein identification of some precocial and superaltricial birds eggs yolk vitelline membrane.” We conducted the research and prepared the manuscript with the greatest possible precision and care. We have stated the corrections for the specific remarks below. All the amendments have been made to the text and highlighted in yellow. Due to changes in the content of the text, the line numbers may not match those in the unreviewed manuscript.

R: Please change the title to: Structure and protein characterization of the egg yolk vitelline membranes of precocial (common names of the birds???) and superaltricial birds (??)

A: We have changed the title as per the suggestion.

R: L23: “the species”: what species?

Also please change the statement to “we analyzed how the structure and protein composition of vitelline membrane (VM) differ among ?? species”.

A: We have corrected the statement.

R: L27: please change “enable to” to “be important for counteracting”. What do you mean by “complex” some specificity should be given here.

A: We have modified the statement.

R: L29: “triple and three-layer”: are these different things or just redundancy?

A: We have removed the words “and three-layer.”

R: L30: what is the difference between VM and VM sheets? If not, please use one term consistently throughout the text.

A: We have removed this part of the sentence from the abstract, as it shall not provide any explanation. Sheets are the second structure found following fibers in the OL VM. This has also been discussed in an earlier study of Chung et al. (2010). We have mentioned this in the Results and Discussion (VM structure) sections.

R: L32: are the results for all birds or for one type? Please specify. Please change “weights” to “molecular weights”. What are protein fractions? How are they fractioned?

A: “We found the number of protein fractions to vary from 19 to 23, with molecular weights in the range of 15–250 kDa, depending on the species”—this information pertains to different species of birds.

Protein fractions are individual protein vacuums that are obtained after the SDS-PAGE electrophoresis and have different molecular weights (kDa).

SDS electrophoresis fractionates the proteins according to their mass. This type of electrophoresis is currently the most commonly used and can be applied either individually as an analytical method or as a part of a series of further, more complex studies (e.g. two-dimensional). Fractionation occurs depending on the length of the polypeptide chain, and you can determine the mass of a given protein by comparing with the appropriate standards. This method allows determining the protein mass with an accuracy of 5–10%.

R: L37-39: too much repeats. Please delete them.

A: We have changed the sentence to avoid repetition.

R: L56: Please change the first “is” to “of”.

A: We have corrected the word.

R: L59: please change “so far” to “previously”.

A: We have corrected as per the suggestion.

R: L63: what do you mean by “which are known currently”? Each one of them has been identified?

A: Yes. They are presented in the study of Mann (2008) as “the most comprehensive dataset available at present and complements proteomic analyses of chicken vitelline membrane compartments published previously.”

Mann K. Proteomic analysis of the chicken egg vitelline membrane. Proteomics. 2008; 8: 2322–2332. https://doi.org/10.1002/pmic.200800032 PMID: 18452232.

R: L64: please remove “first” unless you want to say that they have been found elsewhere later and therefore no longer specific to VM.

A: We have removed the word.

R: L67: Please remove “available from studies”

A: We have deleted this text.

R: L70: Please add “the” before “hen”. When referring an animal as a species, please either use “hens” or “the hen”. There are similar mistakes in the rest of the text. Please change them all.

A: All language errors have been corrected by a Native Speaker English (Translmed Publishing Group).

R: L72: what is the course of the fibers? Do you mean pattern?

A: We have substituted the word “course” by “pattern.”

R: L73: “Additional structures” such as ???

A: These structures do not have a name. They appear in the form of tabs, but this is not an official term. In the cited literature, they are depicted in the SEM micrographs.

R: L76: “offspring ones”? do you mean “offspring”?

A: We have deleted the word “ones.”

R: L77: “the authors”: are you still referring those that published the studies you mentioned earlier? From the context, “the authors” in L71 referred to them.

A: We have corrected the sentence.

R: L80: Please change “The birds” to “Birds”. Please also add an explaining phrase after “precocial” (just like you did for the other three categories) to be parallel and coordinated.

A: We have provided an explanation in the sentence.

R: L83: please change “first days” to “first few days”.

A: We have corrected as per the suggestion.

R: L85: please change “the chicks” to “chicks”.

A: We have corrected as per the suggestion.

R: L88: using % for egg yolk and water does not make sense. Because water is part of the yolk. At least change the “a large proportion” to “a high percentage” so people are not misled into thinking you are talking about the two proportions of the eggs.

A: We have removed the information on water content in eggs.

R: L93: “a few “ and “several” are both unspecified and don’t differ much. I suggest you just use one of them.

A: We have corrected the sentence.

R: L94: please change “storage” to “delayed incubation”. By “storage” you gave the impression that the eggs were taken and stored deliberately.

A: We have changed the word.

R: L98: Please add “using ?? and ?? as models” before “We”.

A: We have changed the sentence as follows:

“We also identified the proteins present in VM using the NanoAcquity Ultra Performance LC (Waters) system.”

R: L112: Please remove “to be” and add “because fertile males were present” (if this is the case).

A: We have changed the text.

R: L181: molecular weights? Please change all such occurrences in the following sections.

A: We have corrected as per the suggestion.

R: L183: please remove “used”.

A: We have removed the word.

R: At the ends of the sections for SEM, TEM and Protein identification, please add a statement of the number of samples used. For example, “Ten eggs from each of the 4 species were analyzed yielding a total samples of 40”.

A: We have corrected the data as follows:

“Six eggs from each of the 4 species were analyzed yielding a total sample of 24.”

R: L190: please change to “Egg and VM characteristics”

A: We have changed the subtitle.

R: L191: Please change “morphological traits” to “weight characteristics”. There were no morphology characterization.

A: We have changed the statement.

R: Table 1: please change “Results of the comparative analysis of the morphological” to “Weights”.

A: We have changed as per the suggestion.

R: Fig 3: From the labeling it appears that CM is a very thin layer of membrane with little characteristics to be seen from the figure. What did the authors use to identify this layer? Please describe its features and characteristics.

A: We agree that CM is poorly visible, and therefore, we did not determine it in the SEM images in the first version of our manuscript. However, one reviewer suggested that CM can be determined, as has been done in earlier studies. CM forms a very thin membrane between the IL and OL. Using an example, we would like to show the reviewer how CM has been determined in other studies, which were used as a model for ours. The below scan presents the CM determined in the publication of: Kido S, Doi Y. Separation and properties of the inner and outher layers of the vitelline membrane of hen’s eggs. Poult Sci. 1988; 67: 476–486. https://doi.org/10.3382/ps.0670476.

R: L246-247: “All the three layers were also characterized by a layered structure”. This statement does not add anything unless the authors did not state it clearly. They have already described in previous statements that the VM were triple-layered.

A: We have deleted the sentence.

R: In the text, “Fig” was used but in the figure legend, “Fig.” was used, please be consistent and I suggest Fig. be used because it is abbreviated.

A: We have changed “Fig” to “Fig.” consistently in the manuscript.

R: Fig 4 was not provided in the complied file.

A: We have provided Fig. 4.

R: L254: please change “corresponded to” to “formed”

A: We have changed the word.

R: L278: Please change “in” to “for”, please also remove “of proteins”

A: We have changed as per the suggestion.

R: L298-300: Fig 5 legend. Please explain what the red arrows are.

A: We have explained this in the caption of Fig. 5.

R: L305-317: please move this section to after Table 3

A: We have moved the entire text below Table 3.

R: L318: what bands were selected? Are those the red arrowed ones? Please specify.

A: Yes. We have provided this information in the text.

R: L319: please remove “in the study”

A: We have removed the text.

R: L322 and 324: 4 proteins >250 kDa and 3 proteins of 35kDa were found. Later only one of each were described. Please explain the others.

A: Proteomic analysis confirmed that the 250-kDa protein found in the VM of all the birds analyzed was alpha-2-macroglobulin-like 1 protein, which is an endopeptidase inhibitor or mucin 5B. In the case of partridge and pheasant, this protein was observed at the lowest intensity.

R: L325: please add “ZP3” here

A: We have completed the sentence.

R: L310-311: In Fig 5, proteins of the complement system were not noted. Which ones are referred to here?

A: In Fig. 5, we present the entire protein profile of the VM of individual cacti (supplementary materials). For further detailed proteomic analysis, we selected the individual numbered protein fractions.

R: L333: similarly, 3 proteins were observed but only one was described. What happened to the other two?

A: In the case of gray partridge and cockatiel parrot, only three and seven proteins were observed, respectively (Fig. 7). The 15-kDa protein band obtained after the separation of the VM of domestic pigeon demonstrated the presence of H0Z0C5 protein, the function of which has not yet been identified.

The data are presented in the supplementary materials and the Venn diagram.

R: Tables 2 and 3: please change pH, PLGS score, and % of coverage to contain one decimal point. Please change the molecular weight to kDa to be consistent with the text. Also in both tables for most protein entries, portions of the IDs were presented twice consecutively. For example: A0A2P4SB66 A0A2P4SB66. What is the reason for this annotation?

A: We have corrected everything and removed the IDs that were repeated by mistake.

R: Table 3: please indicate which section corresponds to which red arrow. If all VM proteins were identified and presented in Table 2 (as indicated in the title of Table 2), why list them again in Table 3? Are these presented for a second time? If so, there is no need to have this Table.

A: The red arrows represent the protein fractions that were selected for the detailed proteomic analysis of the VM birds. The detailed analysis of these fractions is presented in the supplementary material S3. We believe that all the results should remain in the article.

R: "after ovulation in the ovarian follicle" in Line 373 in the corrected manuscript should be corrected to "after ovulation in the infundibulum".

A: We have changed the text.

Submitted filename: Response to th reviewers.pdf

Click here for additional data file.

31 Dec 2019

PONE-D-19-16659R2

Characterization of structure and protein of vitelline membranes of precocial (ring-necked pheasant, gray partridge) and superaltricial (cockatiel parrot, pigeon) birds

PLOS ONE

Dear Dr. Damaziak,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please revise the manuscript by eliminating repetitions in the text as suggested by the reviewer.

We would appreciate receiving your revised manuscript by Jan 20, 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Xiuchun Tian

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I strongly recommend the authors to polish the draft more and more. For example, there are quite similar contents, "... is primarily composed of glycoproteins of the zona pellucida", "The components of IL (mainly glycoproteins of the zona pellucida)" and "The IL primarily consists of glycoproteins, five of which have been identified..." in lines 52, 55 and 60, respectively. For other example, the order of bird species in lines 39 to 40 do not depend on that in lines 36 to 37. The authors should arrange context to reduce such unnecessary repeats and disorders to make the arguments clear.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Jan 2020

Response to the Reviewers

Manuscript: PONE-D-19-16659R2

Dear Editor,

We would like to express our gratitude for assessing our manuscript entitled “Characterization of structure and protein of vitelline membranes of precocial (ring-necked pheasant, gray partridge) and superaltricial (cockatiel parrot, domestic pigeon) birds.” We conducted the research and prepared the manuscript with the greatest possible precision and care. We have stated the corrections for the specific remarks below. All the amendments have been made to the text and highlighted in yellow. Due to changes in the content of the text, the line numbers may not match those in the unreviewed manuscript.

R: I strongly recommend the authors to polish the draft more and more. For example, there are quite similar contents, "... is primarily composed of glycoproteins of the zona pellucida", "The components of IL (mainly glycoproteins of the zona pellucida)" and "The IL primarily consists of glycoproteins, five of which have been identified..." in lines 52, 55 and 60, respectively. For other example, the order of bird species in lines 39 to 40 do not depend on that in lines 36 to 37. The authors should arrange context to reduce such unnecessary repeats and disorders to make the arguments clear.

A: We have carefully checked the entire manuscript. All similar contents has been removed. The names of the birds have also been standardized and are now presented in the same order wherever possible.

Submitted filename: Response to th reviewers.DOCX

Click here for additional data file.

14 Jan 2020

Characterization of structure and protein of vitelline membranes of precocial (ring-necked pheasant, gray partridge) and superaltricial (cockatiel parrot, domestic pigeon) birds

PONE-D-19-16659R3

Dear Dr. Damaziak,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Xiuchun Tian

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

17 Jan 2020

PONE-D-19-16659R3

Characterization of structure and protein of vitelline membranes of precocial (ring-necked pheasant, gray partridge) and superaltricial (cockatiel parrot, domestic pigeon) birds

Dear Dr. Damaziak:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Xiuchun Tian

Academic Editor

PLOS ONE