# The role of multiple vitellogenins in early development of fishes

Ozlem Yilmaz, Craig V Sullivan, Julien Bobe, Birgitta Norberg

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Ozlem Yilmaz, Craig V Sullivan, Julien Bobe, Birgitta Norberg. The role of multiple vitellogenins in early development of fishes. General and Comparative Endocrinology, 2024, 351, pp.114479. 10.1016/j.ygcen.2024.114479. hal-04669839

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Submitted on 9 Aug 2024

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Research paper

# The role of multiple vitellogenins in early development of fishes

Ozlem Yilmaz<sup>a, *</sup>, Craig V. Sullivan<sup>b</sup>, Julien Bobe<sup>c</sup>, Birgitta Norberg<sup>a</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Vitellogenins

Yolk proteins

Free amino acids

Cathepsins

Early development

## ABSTRACT

Functions of vitellogenins have been in the limelight of fish reproductive physiology research for decades. The Vtg system of acanthomorph teleosts consists of two complete forms of Vtgs (VtgAa and VtgAb) and an incomplete form, VtgC. Insufficient uptake and processing of Vtgs and their yolk proteins lead to inadequate oocyte hydration ensuing failure in acquisition of egg buoyancy and early developmental deficiencies. This review presents a summary of our studies on utilization of multiple Vtgs in species with different egg buoyancy characteristics, as examples. Studies of moronids revealed limited degradation of all three forms of lipovitellin heavy chain derived from their three respective forms of Vtg, by which they contribute to the free amino acid pool driving oocyte hydration during oocyte maturation. In later studies, CRISPR/Cas9 was employed to invalidate zebrafish type I, type II and type III Vtgs, which are orthologs of acanthamorph VtgAa, VtgAb and VtgC, respectively. Results revealed type I Vtg to have essential developmental and nutritional functions in both late embryos and larvae. Genomic disturbance of type II Vtg led to high mortalities during the first 24 h of embryonic development. Despite being a minor form of Vtg in zebrafish and most other species, type III Vtg was also found to contribute essentially to the developmental potential of zebrafish zygotes and early embryos. Apart from severe effects on progeny survival, these studies also disclosed previously unreported regulatory effects of Vtgs on fecundity and fertility, and on embryo hatching. We recently utilized parallel reactions monitoring based liquid chromatography tandem mass spectrometry to assess the processing and utilization of lipovitellins derived from different forms of Vtg in Atlantic halibut and European plaice. Results showed the Lv heavy chain of VtgAa (LvHAa) to be consumed during oocyte maturation and the Lv light chain of VtgAb (LvLAB) to be utilized specifically during late larval stages, while all remaining YPs (LvLAa, LvHAb, LvHC, and LvLC) were utilized during or after hatching up until first feeding in halibut. In plaice, all YPs except LvHAa, which similarly to halibut supports oocyte maturation, are utilized from late embryo to late larval development up until first feeding. The collective findings from these studies affirm substantial disparity in modes of utilization of different types of Vtgs among fish species with various egg buoyancy characteristics, and they reveal previously unknown regulatory functions of Vtgs in maintenance of reproductive assets such as maternal fecundity and fertility, and in embryonic hatching. Despite the progress that has been made over the past two decades by examining multiple Vtgs and their functions, a higher complexity of these systems with much greater diversity between species in modes of Vtg utilization is now evident. Further research is needed to reveal novel ways each species has evolved to utilize these complex multiple Vtg systems and to discover unifying principles for this evolution in fishes of diverse lineages, habitats and life history characteristics.

## 1. Introduction

Progeny of oviparous species are dependent on maternal resources for their development. Egg yolk derived from multiple types of vitellogenins (Vtgs) constitutes a major portion of these resources, playing pivotal roles at different critical times during oocyte, early embryonic

and larval development. The process and utilization of different types of Vtgs appears to differ in a species-specific manner depending on the reproductive strategies of each species. This review presents findings on the utilization of multiple Vtgs from four different teleost fishes with different egg buoyancy characteristics as examples: European sea bass (*Dicentrarchus labrax*), a marine pelagic egg spawner; zebrafish (*Danio rerio*), a freshwater benthic egg spawner; Atlantic halibut (*Hippoglossus*

<footnote><sup>a</sup> Institute of Marine Research, Austevoll Research Station, 5392 Storebø, Norway</footnote>

<footnote><sup>b</sup> Carolina AquaGyn, P.O. Box 12914, Raleigh, NC 27605, USA</footnote>

<footnote><sup>c</sup> INRAE, LPGP, 35000 Rennes, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: ozlem.yilmaz@hi.no (O. Yilmaz).</footnote>
<table><thead><tr><th colspan="2">Nomenclature</th></tr></thead><tbody><tr><td colspan="2"><em>Abbreviations</em></td></tr><tr><td>Vtg</td><td>Vitellogenin</td></tr><tr><td>CRISPR/Cas9</td><td>clustered regularly interspaced short palindromic repeats guided CRISPR-associated protein 9</td></tr><tr><td>YP</td><td>yolk protein</td></tr><tr><td>LVH</td><td>lipovitellin heavy chain</td></tr><tr><td>LVL</td><td>lipovitellin light chain</td></tr><tr><td>Pv</td><td>phosvitin</td></tr><tr><td>β'c</td><td>beta component</td></tr><tr><td>Ct</td><td>C terminal domain</td></tr><tr><td>VtgR</td><td>vitellogenin receptor</td></tr><tr><td>VRBP</td><td>Vtg receptor binding peptide</td></tr><tr><td>VLDLR</td><td>very-low-density lipoprotein receptor</td></tr></tbody></table><table><tbody><tr><td>LDLR</td><td>low-density lipoprotein receptor</td></tr><tr><td>Lr8</td><td>lipoprotein receptor with eight ligand binding repeats</td></tr><tr><td>Lrp13</td><td>low-density lipoprotein receptor related-protein 13</td></tr><tr><td>OM</td><td>oocyte maturation</td></tr><tr><td>Cat</td><td>cathepsin</td></tr><tr><td>MVB</td><td>multivesicular bodies</td></tr><tr><td>FAA</td><td>free amino acid</td></tr><tr><td>NH4</td><td>ammonium</td></tr><tr><td>Hpf</td><td>hours post fertilization</td></tr><tr><td>Dpf</td><td>days post fertilization</td></tr><tr><td>KO</td><td>knock-out</td></tr><tr><td>PRM-LC-MS/MS</td><td>parallel reactions monitoring based liquid chromatography tandem mass spectrometry</td></tr><tr><td>PV</td><td>postvitellogenic oocytes</td></tr><tr><td>UF</td><td>unfertilized eggs</td></tr></tbody></table>

hippoglossus), a flatfish which spawns neutrally buoyant eggs in seawater; and another flatfish, European plaice (*Pleuronectes platessa*), a marine pelagic egg spawner. This review covers published information on sea bass and zebrafish, and introduces original unpublished data on

halibut and plaice.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Protein dendrogram nomenclature and domain structure of vitellogenins (Vtgs) in different fishes. The dendrogram (left panel) shows clustering by similarity of the Vtg polypeptide sequences deduced from cDNAs. YP domain structures are given in the right panel. Further details on Vtg nomenclature are given by Sullivan and Yilmaz (2018) (Figure modified from Yilmaz et al., 2018).</figcaption></figure>
## 2. Structure and multiplicity of vitellogenins in teleost fishes

In general, the Vtg system of teleost fishes consists of two paralogous complete forms of VtgA (VtgAa and VtgAb) bearing all yolk protein (YP) domains (heavy chain lipovitellin [LvH], light chain lipovitellin [LvL], phosvitin [Pv], β'-component [β'c], and C-terminal peptide [Ct]) (Finn 2007ab), and an incomplete Vtg (VtgC) lacking Pv and much of the C-terminus (β'c/Ct) (Finn and Kristoffersen, 2007). Some exceptions to this generalization are; the Atlantic salmon (Salmo salar) possessing a complete VtgAsa1, a truncated VtgAsb, and a phosvitin-less VtgC (Andersen et al., 2017), and zebrafish with a total of five type I vtg genes (vtg1, 4, 5, 6, and 7) encoding a truncated form of Vtg (orthologous to VtgAa), two type II vtg genes (vtg2 and vtg8) encoding a complete form of Vtg (orthologous to VtgAb), and one type III vtg gene (vtg3) encoding the incomplete form of Vtg (orthologous to VtgC), the Vtg I, Vtg II, and Vtg III, respectively (Yilmaz et al., 2018) (Fig. 1).

Complete forms of Vtg are made up of a linear series of five YP domains, as follows: NH2-LvH-Pv-LvL-β'c-Ct-COOH (Patiño and Sullivan, 2002). LvH is the major component, with an average mass of ~ 115 kDa, as compared to ~ 25 kDa for LvL (Lubzens et al., 2017). Once cleaved from the parent Vtg, lipovitellin (LvH + LvL) complex with amphipathic structures that form a basket lined with hydrophobic amino acid residues is formed to accommodate structural phospholipids

and carry them into growing oocytes (Romano et al., 2004). LvH bears a Vtg receptor (VtgR)-binding peptide (VRBP), which has the special shape and surface charge distribution required to bind to Vtgrs on the oocyte surface. Lv is the main source of the polar lipid and amino acid nutrients that serve as catabolic energy substrates, or in anabolic synthesis of membranes or proteins, during early development. The small Pv (~5–20 kDa) is made up mostly of phosphorylated serine residues that are thought to stabilize Lv structure, enhance lipid loading, promote Vtg solubility in the blood, and act as a carrier of mainly divalent cations (e.g., Ca2+, Mg2+, and Zn2+, also Fe3+ ) into the oocytes (Finn 2007b). These ions that are delivered to the embryos through phosphorylated Vtgs (with a Pv domain) (Babin et al., 2007) may be crucial as they are largely unavailable for uptake from the environment due to dilution by rainfall and runoff. However, some incomplete forms of Vtg lack a Pv domain altogether. The diminutive β'c and Ct domains possess a highly conserved motif of repeated cysteine residues forming an array of disulfide bond linkages that are thought to be important for the complicated folding of Vtg polypeptides, stabilizing Vtg structure, promoting Vtg dimerization, and enabling Vtg receptor (VtgR) binding but are of unknown nutritional function (Finn 2007b). When released from Vtg, β'c forms an ~ 16 kDa YP, but, with some exceptions, Ct (~13 kDa) may be degraded shortly after cleavage. Various Lv-Pv conjugates (LvH-Pv, Pv-LvL, LvH-Pv-LvL) have been detected in the yolk of several

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** General model for multiple Vtg synthesis, transport, uptake and processing. Top Panel. General model for multiple Vtg processing, including first and second proteolysis, in marine fishes spawning pelagic eggs. Panel A. Regulation of vitellogenesis in teleost fish via the brain-pituitary-gonad (BPG) axis. Gnrh, gonadotropin-releasing hormone; Fsh, follicle stimulating hormone; Lh, luteinizing hormone. Panel B. Primary proteolysis of Vtgs into their major YPs during oocyte growth. Complete forms of A-type vitellogenin (VtgAa and VtgAb) bind to the vitellogenin receptor (Vtgr) and are taken up into oocytes via clathrin-mediated endocytosis where they are cleaved bycathepsin D into product yolk proteins that are stored in the yolk vesicles. Panel C. Secondary proteolysis of Vtgs into free amino acids during oocyte maturation (Figure modified from Reading et al., 2009; Sullivan and Yilmaz, 2018).</figcaption></figure>
species (Finn 2007b).

### 3. Synthesis, uptake, processing, and utilization

Vitellogenins are synthesized by the liver in response to estrogen (17β-estradiol) and are secreted into the blood, from where they are taken up by growing oocytes via receptor-mediated endocytosis (Babin et al., 2007) Fig. 2B. The different forms of Vtg appear to disparately bind to different forms of VtgRs (Hiramatsu et al., 2013; Hiramatsu et al., 2015). Two forms of VtgR have been characterized to date. The classical form Lr8, an ortholog of mammalian very-low-density lipoprotein receptor (VLDLR), has been reported to function as a possible VtgR in various teleosts including rainbow trout (Oncorhynchus mykiss) (Stifani et al., 1990; Lancaster and Tyler 1994; Davail et al., 1998), coho salmon (Oncorhynchus nerka) (Stifani et al., 1990), white perch (Morone americana) (Tao et al., 1996; Hiramatsu et al., 2004; Reading et al., 2011), cutthroat trout (Oncorhynchus clarki) (Mizuta et al., 2013), Atlantic bluefin tuna (Thunnus thynnus) (Pousis et al., 2012), and greater amberjack (Seriola dumerili) (Pousis et al., 2019). In addition to the Lr8, other studies have suggested that multiple VtgR candidates are present in teleost ovaries (Hiramatsu et al., 2015). A novel receptor called LDLR related protein 13 (Lrp13) was characterized in distantly related teleosts (genus: Morone, Oncorhynchus and Seriola) and suggested to be the potential VtgR (Reading et al., 2014; Mushirobira et al., 2015; Hiramatsu et al., 2015; Reading et al., 2014; Reading et al., 2017). While both receptors seem to have affinity to a salmon A-type Vtg (VtgAsa and VtgAsb) in salmonids (Mushirobira et al., 2015; Hiramatsu et al., 2015), a recent study by Namgung et al. (2021) reported Lr8 to bind only VtgAb in medaka (Oryzias latipes). While proteins that bind to VtgC have been detected in several species, no specific VtgC receptor has been definitively identified and several lines of evidence suggest that VtgC may lack an oocyte receptor and enter oocytes via common endocytosis or in association with other proteins, possibly including Vtgs (Sullivan and Yilmaz, 2018).

Once Vtgs are taken up by the oocyte, their product YPs undergo different degrees of proteolysis by various lysosomal endopeptidases, cathepsins, during oocyte growth and oocyte maturation (OM) (Carnevali et al., 2006). During oocyte growth, Vtgs dissociate from the VtgRs due to slight vesicle acidification and the VtgRs are recycled to the oocyte surface. The vesicles, now called endosomes, fuse with lysosomes containing inactive cathepsin D (CatD) proenzymes to form multivesicular bodies (Fig. 2B). Vacuolar ATPases (i.e., proton pumps) located in the surface membrane further acidify the lumen of the multivesicular bodies (MVBs) and the decreased pH (~6.0) activates CatD, which cleaves Vtgs at specific recognition sites into its product YPs (LvH, LvL, Pv, β',c, and Ct) (Reading et al., 2009, Finn and Fyhn, 2010).

During OM, when fully-grown, postvitellogenic follicles undergo fusion and dissolution of the yolk granules, coalescence of the lipid droplets, ooplasm clarification, and oocyte hydration along with resumption of meiosis, YPs are subjected to a 'secondary proteolysis' (Finn 2007a). This process involves further acidification of MVBs, activating cathepsins B or L (CatB/L), which are co-localized with YPs in the yolk vesicle during oocyte growth. These cathepsins accomplish extensive proteolytic degradation of the YPs, generating a large pool of peptides and free amino acids (FAA). The FAA along with other osmolytes, including Pv-derived phosphate, ammonium (NH4 + ) from FAA metabolism, and potassium and chloride transported into the oocyte via the actions of Na+, K + -ATPase and pendrin, respectively, increase the osmotic pressure of the ooplasm, causing water to flow into the oocyte through aquaporin channels transiently expressed in the oocyte membrane (Cerdà et al., 2007). The net results are hydration of the oocyte, adjusting its density to provide for proper egg buoyancy, and provisioning of the oocyte with a pool of diffusible FAA nutrients, the preferred energy substrate of early embryos. Any remaining

polypeptides and Lv fragments are retained as nutrients for late-stage embryos and larvae, which have developed the machinery to process such macromolecules (e.g. the yolk syncytium) (Fig. 2C). Aside from gas exchange, the egg is largely a closed system during early stages of embryogenesis, and the water taken up during final maturation is required for embryo metabolism, dilution of metabolic wastes (e.g. NH4 + ), and provision of adequate space for further development (Finn and Fyhn, 2010).

The degree of maturational yolk proteolysis and the specific YP products of different types of Vtg that are subjected to degradation differ among species with eggs having disparate physiological properties leading to different environmental adaptations (i.e. demersal versus pelagic eggs). In many marine teleosts spawning pelagic eggs, virtually all YPs derived from VtgAa are proteolytically cleaved into FAAs during OM, whereas the major products of VtgAb (LvHAb) and VtgC (LvC), remain largely intact. In barfin flounder, (Verasper moseri) and red sea bream (Pagrus major) all YPs derived from VtgAa are nearly completely degraded into FAA while those derived from the corresponding VtgAb and VtgC are less susceptible to proteolysis (Hiramatsu et al., 2005, Matsubara et al., 2003, Sawaguchi et al., 2006). The relatively intact major VtgAb and VtgC polypeptides (LvHs) escaping proteolysis are ascribed to be specialized for delivering large lipoprotein nutrients to late stage larvae prior to first feeding without affecting the osmotically active FAA pool (Matsubara et al., 2003, Reading and Sullivan, 2011).

#### 3.1. European sea bass and other temperate basses

European sea bass (Dicentrarchus labrax; family Moronidae) is an acanthomorph teleost that spawns pelagic eggs in seawater (Carrillo et al., 1995; Saillant et al., 2003). As described by Yilmaz et al. (2016), like other temperate basses, such as striped bass (Morone saxatilis), which spawn nearly neutrally buoyant eggs in freshwater (Williams et al., 2014), and white perch (Morone americana), which spawn demersal adhesive eggs in fresh water (Reading et al., 2009), European sea bass also possess the three classical types of Vtgs: VtgAa, VtgAb, and VtgC. Comparisons of specific Vtgs contents in postvitellogenic oocytes and ovulated eggs via Western blotting shown that all three forms of LvH (LvHAa, LvHAb, LvHC) were detected intact in both PV oocytes and ovulated eggs and that degradation products of all three LvHs were observed in ovulated eggs (Fig. 3A-C) (Yilmaz et al., 2016). This indicates simultaneous but limited contribution of all three types of LvHs to OM in sea bass. Eggs of sea bass may not need extensive protolysis of Lvs to acquire buoyancy as they contain large oil droplets. Overall results from studies of other temperate basses (family Moronidae) have similarly revealed that maturational yolk proteolysis involves limited degradation of all three forms of LvH (LvHAa, LvHAb, LvHC) (Reading et al., 2009, Hiramatsu et al., 2002, Williams et al., 2014).

#### 3.2. Zebrafish

A member of Cypriniformes, zebrafish (Danio rerio) is an established biomedical model for research on vertebrate reproduction and developmental biology because they are small, easily bred in the laboratory with short generation time, and lay clutches of numerous large eggs every few days, with external fertilization of the transparent eggs in which embryonic development is easily observed (Ribas and Piferrer, 2013). These features facilitated our study of the complex Vtg system in zebrafish using the CRISPR/Cas9 technology (Yilmaz et al., 2019, Yilmaz et al., 2023). The zebrafish Vtg repertoire includes five type I Vtgs (Vtg 1, 4, 5, 6, and 7) that are incomplete, lacking β'c and Ct domains, two type II Vtgs (Vtg2 and Vtg8) that are complete, and one type III Vtg (Vtg3), which is a typical VtgC (Yilmaz et al., 2018). In an array of genome editing studies, we investigated the essentiality of different forms of Vtg, particularly focusing on the developmental stages when their functions become critical, via invalidation of all 5 genes encoding type I Vtg simultaneously, and genes encoding type II and type III Vtgs
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Western blotting detection of yolk proteins derived from specific types of Vtg in postvitellogenic oocytes and ovulated eggs of European sea bass. Western blotting was performed using primary antisera raised against purified graymullet A) Mugil cephalus, LvAa (anti-LvAa), B) LvAb (anti-LvAb), or C) LvC (anti-LvC) (Amano et al., 2008). Samples included prestained molecular mass marker proteins (M), and ovary extracts from postvitellogenic (PV) or ovulated (OV) females. Large numbers to the left of each blot image indicate the mass of marker proteins (kDa). Small numbers with arrow heads pointing to individual bands on the gel or blots indicate the estimated mass (kDa) of the protein in that band. Selected bands are tentatively identified with the abbreviation for the corresponding sea bass lipovitellin heavy chain (LvHAa, LvHAb, or LvHC) or lipovitellin light chain (LvLAa or LvLC, LvLAB not detected) likely to be present in that band(s) based upon the estimated mass of the respective LvH or Lvl primary peptide (Figure modified from Yilmaz et al., 2016).</figcaption></figure>

individually (Yilmaz et al., 2019, Yilmaz et al., 2023).

In the *vtg1*-KO study, *vtg6* and *vtg7* escaped Cas9 editing, however, simultaneous *vtg1*,4,5-KO was achieved and a pure line (F4) with the desired mutation was produced with significant effects of *vtg1*, 4, and 5 loss being observed. In the *vtg2*-KO study, a heterozygous mutant line (F3) with significant effects of *vtg2* genomic disturbance was produced (*vtg2*-mutant). In the *vtg3*-KO study, a pure line (F4) with striking effects of *vtg3* genomic loss was produced. In all cases, introduced mutations substantially impaired survival rates. During 22 days of embryo and larval development, mortalities were noted by 24 hpf for all lines, however, the changes in survival rates became statistically significant by 5 dpf, 8 hpf, and 2 hpf in *vtg1*-KO, *vtg2*-mutant and *vtg3*-KO line offspring, respectively. Final survival rates at 22 dpf were as low as 6 %, 18 % and 0 % for *vtg1*-KO, *vtg2*-mutant and *vtg3*-KO line offspring, respectively in comparison to 69 % survival in wild type progeny (Fig. 4). These findings indicate that type I Vtg is required after hatching during larval development, while type II Vtg is required during early embryonic development, and type III Vtg is required at all

developmental stages. Despite being a minor form of Vtg, type III Vtg was found to contribute importantly to the developmental potential of zebrafish zygotes and early embryos (Yilmaz et al., 2019, Yilmaz et al., 2023).

Additional findings from these studies indicated that zebrafish Vtgs also have reproductive regulatory functions. Fecundity, measured as the number of eggs per batch, was more than 2-fold higher in F3 *vtg1*-KO zebrafish females (N = 593) in comparison to wild type females (N = 280), while no significant fecundity effects of *vtg2* genomic disturbance or of *vtg3*-KO were observed (Fig. 5A). Fertilization rates of eggs from F2 *vtg2*-mutant females (37 %) and F3 *vtg3*-KO females (36 %) were more than halved in comparison to eggs from wild type females (82 %) (Fig. 5B). Embryo hatching rates were significantly higher in F3 *vtg2*-mutant embryos (62 %) in comparison to wild type embryos (24 %) (Fig. 5C), while no significant effects of *vtg1*-KO or *vtg3*-KO on embryo hatching rate were observed. In addition, morphological observations of yolk leaking disorder from the vitellin membrane in F3 *vtg2*-mutant egg batches with high mortalities (less than 10 % survival at 24 hpf) and

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Comparisons of survival percentages for homozygous F4 *vtg1*-KO (blue), F3 *vtg2*-mutant (red) and F4 *vtg3*-KO (green) zebrafish embryos and larvae versus wild type offspring (black). Line plots represent mean survival percentages and numbers on the x-axis underscored by dashed- or solid-lined arrows represent sampling times in hours or days postfertilization during the observation period. Asterisks indicate mean values that are statistically significantly different (p < 0.01) from corresponding mean wild type (Wt) values (Figure modified from Yilmaz et al., 2019; Yilmaz et al., 2023).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Effect of genomic disturbance of different types of zebrafish Vtgs on fecundity, fertilization rate, and hatching rates in zebrafish. Panel A. Vertical bars represent the number of eggs released per batch for homozygous F3 vtg1-KO, heterozygous F2 vtg2-mutant, homozygous F3 vtg3-KO and untreated wild type females. Panel B. Vertical bars represent the percentage of fertilization rate of eggs released by homozygous F3 vtg1-KO, heterozygous F2 vtg2-mutant, homozygous F3 vtg3-KO and untreated wild type females. Panel C. Vertical bars represent the hatching percentage of homozygous F4 vtg1-KO, heterozygous F3 vtg2-mutant, homozygous F4 vtg3-KO and untreated wild type zebrafish embryos. Asterisks above vertical bars indicate means significantly different from wild type values. (Figure modified from Yilmaz et al., 2019; Yilmaz et al., 2023).</figcaption></figure>

overrepresentation of histones, zona pellucida proteins, lectins, and protein degradation-related proteins in the F3 vtg2-mutant embryos provide evidence that Vtg2 is essential for proper vitellin membrane formation and function in zebrafish. (Yilmaz et al., 2019, Yilmaz et al., 2023).

### 3.3. Atlantic halibut and European plaice

Despite earlier research on proteolytic liberation of free amino acids and their contribution to oocyte hydration in Atlantic halibut (Finn et al., 2002), and on yolk resorption during early development in European plaice (Skjærven et al., 2003) detailed information on contributions of specific Vtgs to early development of these species was unavailable. Larval rearing bottlenecks currently restrain growth of the halibut farming industry, prompting our investigations of utilization of multiple Vtgs during early life stages in this species. Comparisons to Vtg

utilization in European plaice were also made. Due to its smaller size and taxonomic relation, this species may serve as a biological model for halibut and it is also currently being evaluated as an alternative species for aquaculture production. Newly completed genome sequencing projects (Atlantic halibut; fHipHip1.pri, 2019 and European plaice; fPlePla1.1, 2022) allowed us to identify the three classical types of Vtg (VtgAa, VtgAb and VtgC) in both species. This review contains previously unpublished original findings from our most recent study employing sequences of these Vtgs to design Vtg- and Lv-specific stable isotope labelled synthetic peptides as references to carry out relative quantification of Vtg-derived Lv yolk proteins (LvHs and LvLs) at different stages of early development using parallel reactions monitoring based liquid chromatography tandem mass spectrometry (PRM-LC-MS/MS). The study focused on the most critical and corresponding oocyte, egg and progeny development stages (PV: postvitellogenic oocytes (in halibut only), UF: unfertilized eggs (both species), 10 dpf: prior

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** PRM-based LC-MS/MS quantification of lipovitellin heavy chain (LvH) at different developmental stages in Atlantic halibut and European plaice. Three to four isotope labelled heavy peptides, specific to each type of Vtg, were utilized as reference peptides for quantification. Panels A. B and C demonstrate results from all peptides representing LvHs of VtgAa, Ab and C, respectively. Bar charts (in black at the on top of each panel for halibut and in gray at the bottom of each panel for plaice) present ratios of light peptides to heavy peptides (given on y axes) at different developmental stages (given on x axes). PV: postvitellogenic oocytes from ovarian biopsies (halibut only), UF: unfertilized eggs, 10dpf: 10 days post fertilization, 20dpf: 20 days post fertilization, 50dpf: 50 days post fertilization. Critical events such as the oocyte maturation, hatching for halibut (at 6 °C) and for plaice (at 8 °C), and first feeding are indicated by vertical arrows below each chart. Line curves indicate patterns of change and asterisks indicate significant changes in abundance profiles during development.</figcaption></figure>
to hatching at 6 °C in halibut and late embryonic development in plaice, 20 dpf; yolk sac stage in halibut and prior to hatching at 8 °C in plaice, 50 and 25 dpf; prior to first feeding in halibut and plaice, respectively (Yilmaz et al., *unpublished*). Findings of this study revealed critical information on the uptake, processing, and utilization of yolk proteins derived from specific forms of Vtg during oocyte, embryo, and larval development of these two closely related flatfish species.

Comparisons between halibut and plaice of the relative abundance of different types of LvH at early developmental stages are illustrated in Fig. 6 (Panel A: LvHAa, Panel B: LvHAb, Panel C: LvHC). In halibut, LvHAa, highly abundant at the PV oocyte stage, showed a significant drastic decrease, to nearly zero, prior to fertilization, indicating substantial utilization of this YP during oocyte maturation. Even though analyses of postvitellogenic oocytes in plaice were not included in this study the absence of LvHAa in any later stages indicates that, similar to halibut, this YP is consumed during oocyte maturation (Fig. 6A). In both species, LvHAb showed a significant increase up until 10 dpf and decreased thereafter until first feeding, with the decrease appearing to be more abrupt in plaice (Fig. 6B). This stage (10 dpf) approximately corresponds to hatching at 8 °C in halibut but is 10 days prior hatching at 6 °C in plaice (Fig. 6A). The LvHAb remained detectable at late larval stages, prior first feeding, in both halibut and plaice. In both species, LvHC levels showed a pattern of change similar to LvHAb with the only difference being that halibut LvHC remained at detectable levels up until first feeding while plaice LvHC was totally consumed before first feeding. These observations indicate that extensive utilization of LvHAb and LvHC is initiated much earlier in plaice, by embryos well before hatching, and it proceeds more rapidly, being complete or nearly so prior to first feeding, suggesting that these two LvHs may serve disparate purposes in the two flatfishes (Fig. 6B-C).

Comparisons between halibut and plaice of the relative abundance of different types of LvL at different stages are illustrated in Fig. 7 (Panel A: LvLAa, Panel B: LvLAb, Panel C: LvLC). Except for halibut LvLAb, all LvLs showed a significant increase until 10 dpf and a gradual decrease afterwards up until first feeding in both species. Halibut LvLAb did not

change in abundance until it decreased rapidly just prior to first feeding (Fig. 7 B). This portrait indicates that, except for halibut LvLAb, utilization of LvLs is initiated by late stage embryos or hatched larvae, while utilization of halibut LvLAb is initiated at late larval stages and proceeds until first feeding, by which time nearly all LvLs have been consumed. These findings, together with those on LvHAb, are in line with prior reports on products of VtgAb and VtgC being comparatively more resistant to secondary yolk proteolysis and serving late stage larval development in barfin flounder and red sea bream (Hiramatsu et al., 2005, Matsubara et al., 2003, Sawaguchi et al., 2006).

## 4. Summary and conclusions

A summary of findings from the studies reported in this review is illustrated in Fig. 8. In sea bass, all forms of LvH were degraded with some intact protein still present in ovulated eggs. This indicates that, although hydration of the oocytes during maturation is limited in sea bass compared to other marine fish spawning pelagic eggs, all three types of sea bass LvH undergo noticeable proteolysis during this time and likely contribute to the store of FAAs involved in oocyte hydration and early embryo nutrition (Yilmaz et al., 2015). Absence of LvLAb in ovulated eggs might indicate its use during oocyte maturation while the presence of other Lvs at this stage may signal a need for these YPs at later stages of embryonic and larval development. The pattern of multiple Vtg utilization among the Moronid species seems not to vary despite their different egg types and reproductive environmental salinities. The limited extent and unusual pattern of maturational proteolysis and oocyte hydration in moronids may be linked to their additional use of high levels of wax esters to regulate egg buoyancy (Yilmaz et al., 2016). In zebrafish, it was obvious that type I Vtgs were mainly required and utilized during larval development while type II Vtg was mainly utilized during early embryonic development, and type III Vtg is required for all developmental stages. The type II and III Vtgs seem to also have critical reproductive regulatory functions. In halibut, LvHAa is apparently utilized during oocyte maturation and perhaps embryonic development.

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** PRM based LC-MS/MS quantification of lipovitellin light chain (LvL) at different developmental stages in Atlantic halibut and European plaice. Three to four isotope labelled heavy peptides, specific to each type of Vtg, were utilized as reference peptides for quantification. Panels A, B and C demonstrate results from all peptides representing LvLs of VtgAa, Ab and C, respectively. Bar charts (in black at the top of each panel for halibut and in gray at the bottom of each panel for plaice) present ratios of light peptides to heavy peptides (given on y axes) at different developmental stages (given on x axes). PV: postvitellogenic oocytes from ovarian biopsies (halibut only), UF: unfertilized eggs, 10dpf: 10 days post fertilization, 20dpf: 20 days post fertilization, 50dpf: 50 days post fertilization. Critical events such as the oocyte maturation, hatching for halibut (at 6 °C) and for plaice (at 8 °C), and first feeding are indicated by vertical arrows below each chart. Line curves indicate patterns of change and asterisks indicate significant changes in abundance profiles during development.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Summary of findings on utilization of different types of vitellogenins in European sea bass, zebrafish, Atlantic halibut, and European plaice. Species are represented by virtual icons at the left of the figure, each with corresponding panel on the right. Critical developmental events such as oocyte maturation, fertilization, early/late embryonic development, hatching, early/late larval development, and first feeding are given for guidance in the top panel.</figcaption></figure>

LvHAa, LvHC, LvLAa and LvLC are required for larval development, and LvLAb is required for late larval development only. In plaice, LvHAa most probably has the same function as in halibut, and all other yolk proteins are required simultaneously from late embryonic to late larval stages.

In conclusion, the various major forms of Vtg have complex, species-specific and disparate functions. Independent from varying reproductive strategies, life histories and egg buoyancy characteristics of the different species, all forms of Vtgs are essential for development and survival of the respective progeny. Vtgs support oocyte hydration and nurture early development but they also have reproductive regulatory functions. The timing of processing and utilization of the different types of Vtgs differs even between closely related species, especially where there are different buoyancy characteristics of the eggs. Pelagic egg spawners utilize VtgAa for oocyte maturation while zebrafish, a benthic egg spawner, uses VtgAa to support larval development. Even though much progress has been made discerning molecular events involving multiple Vtgs over the past two decades, a higher complexity of these systems is now evident, with much greater diversity between species in modes of Vtg utilization at different reproductive and developmental stages. Further research is essential to discover the ways in which individual species have evolved to utilize this complex system.

## CRediT authorship contribution statement

**Ozlem Yilmaz:** Conceptualization, Funding acquisition, Visualization, Investigation, Methodology, Writing – original draft. **Craig V. Sullivan:** Conceptualization, Funding acquisition, Writing – review & editing. **Julien Bobe:** Conceptualization, Funding acquisition, Writing – review & editing. **Birgitta Norberg:** Conceptualization, Funding acquisition, Writing – review & editing.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

No data was used for the research described in the article.

## Acknowledgements

The European sea bass research was supported by awards to Ozlem Yilmaz from the International Fulbright Visiting Student Researcher Program for doctoral research in the United States, and to Craig V. Sullivan from the North Carolina Agricultural Foundation. The zebrafish research was supported by FP7 People 2013 Marie Curie Actions Intra-European Fellowships (FP7 PEOPLE 2013 MC IEF) Grant/Award Number: 626272; Region Bretagne in France (SAD-2013) Grant/Award Number: 13009218; and Agence Nationale de Recherches Grant/Award Number: ANR-13-BSV7-001. Atlantic halibut and European plaice research was funded by the Norwegian Nærings og Fiskeri Departement (Project # 15194) in the frame of support for sustainable production of marine fishes in aquaculture

## References

* Amano, H., Fujita, T., Hiramatsu, N., Kagawa, H., Matsubara, T., Sullivan, C.V., Hara, A., 2008. Multiple vitellogenin-derived yolk proteins in gray mullet (Mugil cephalus): disparate proteolytic patterns associated with ovarian follicle maturation. Mol. Reprod. Dev. 75, 1307–1317. https://doi.org/10.1002/mrd.20864.

* Andersen, Ø., Xu, C., Timmerhaus, G., Kirste, K.H., Næve, I., Mommens, M., Tveiten, H., 2017. Resolving the complexity of vitellogenins and their receptors in the tetraploid Atlantic salmon (Salmo salar): Ancient origin of the phosvitin-less VtgC in chondrichthyean fishes. Mol. Rep. Dev. Mol. Reprod. Dev. 84, 1191–1202. https://doi.org/10.1002/mrd.22881.

* Babin, P.J., Carnevali, O., Lubzens, E., Schneider, W.J., 2007. Molecular aspects of oocyte vitellogenesis in fish. In: Babin, P.J., Cerdà, J., Lubzens, E. (Eds.), The Fish Oocyte: from Basic Studies to Biotechnological Applications. Springer, Netherlands, pp. 39–76.

* Carnevali, O., Cionna, C., Tosti, L., Lubzens, E., Maradonna, F., 2006. Role of cathepsins in ovarian follicle growth and maturation. Gen. Comp. Endocrinol. 146, 195–203. https://doi.org/10.1016/j.ygcen.2005.12.007.

* Carrillo, M., Zanuy, S., Prat, F., Cerdà, J., Ramos, J., Mañanós, E., Bromage, N., 1995. Sea bass (Dicentrarchus labrax). In: Bromage, N.R., Roberts, R.J. (Eds.), Broodstock Management and Egg and Larval Quality. Blackwell Science, Oxford, pp. 138–168.

* Cerdà, J., Fabra, M., Raldúa, D., 2007. Physiological and molecular basis of fish oocyte hydration. In: Babin, P.J., Cerdà, J., Lubzens, E. (Eds.), The Fish Oocyte: from Basic Studies to Biotechnological Applications. Springer, Dordrecht, The Netherlands, pp. 349–396. https://doi.org/10.1007/978-1-4020-6235-3_12.
Davail, B., Pakdel, F., Bujo, H., Perazzolo, L.M., Waclawek, M., Schneider, W.J., Le Menn, F., 1998. Evolution of oogenesis: the receptor for vitellogenin from the rainbow trout. J. Lipid Res. 39 (10), 1929-1937. https://doi.org/10.1016/S0022-2275(20)32491-3.

Finn, R.N., 2007a. The maturationally disassembled and differential proteolysis of paralogous vitellogenins in a marine pelagophil teleost: a conserved mechanism of oocyte hydration. Biol. Reprod. 76, 936-948. 10.1095/biolreprod.106.055772.

Finn, R.N., 2007b. Vertebrate yolk complexes and the functional implications of phosvitis and other subdomains in vitellogenins. Biol. Reprod. 76, 926-935. 10.1095/biolreprod.106.059766.

Finn, R.N., Fyhn, H.J., 2010. Requirement for amino acids in ontogeny of fish. Aquac. Res. 41 (5), 684-716. https://doi.org/10.1111/j.1365-2109.2009.02220.x.

Finn, R.N., Kristoffersen, B.A., 2007. Vertebrate vitellogenin gene duplication in relation to the "3R hypothesis": correlation to the pelagic egg and the oceanic radiation of teleosts. PLoS One 2, e169. 10.1371/journal.pone.0000169.

Finn, R.N., Østby, G.C., Norberg, B., Fyhn, H.J., 2002. In vivo oocyte hydration in Atlantic halibut (Hippoglossus hippoglossus); proteolytic liberation of free amino acids, and ion transport, are driving forces for osmotic water influx. J. Exp. Biol. 205 (Pt2), 211-224. 10.1242/jeb.205.2.211.

Hiramatsu, N., Hara, A., Hiramatsu, K., Fukada, H., Weber, G.M., Denslow, N.D., Sullivan, C.V., 2002. Vitellogenin-derived yolk proteins of white perch, Morone americana: purification, characterization, and vitellogenin-receptor binding. Biol. Reprod. 67 (2), 655-667. https://doi.org/10.1095/biolreprod67.2655.

Hiramatsu, N., Chapman, R.W., Lindzey, J.K., Haynes, M.R., Sullivan, C.V., 2004. Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana). Biol. Reprod. 70 (6), 1720-1730. 10.1095/biolreprod.1 (Morone americana).

Hiramatsu, N., Cheek, A.O., Sullivan, C.V., Matsubara, T., Hara, A. 2005. Vitellogenesis and endocrine disruption. In: Mommsen, T.P., Moon, T. (Eds). Biochemistry and Molecular Biology of Fishes. Environmental Toxicology. Chapter 16 (s). Elsevier Science Press. Amsterdam, The Netherlands. Vol. 6. 562 pages. pp. 431-471. https://doi.org/10.1016/S1873-0140(05)80019-0.

Hiramatsu, N., Luo, W., Reading, B.J., Sullivan, C.V., Mizuta, H., Ryu, Y.-W., Nishimiya, O., Todo, T., Hara, A., 2013. Ovarian lipoprotein receptors in teleosts. Fish Physiol. Biochem. 39, 29-32. 10.1007/s10695-012-9612-6.

Hiramatsu, N., Todo, T., Sullivan, C.V., Schilling, J., Reading, B.J., Matsubara, R.-W., Mizuta, H., Luo, W., Nishimiya, O., Wu, M., Mushirobira, M., Yilmaz, O., Hara, A., 2015. Ovarian yolk formation in fishes: molecular mechanisms underlying formation of lipid droplets and vitellogenin-derived yolk proteins. Gen. Comp. Endocrinol. 221, 9-15. https://doi.org/10.1016/j.ygcen.2015.01.025.

Lancaster, P.M., Tyler, C.R., 1994. Developmental expression and modulation of the vitellogenin receptor in ovarian follicles of the rainbow trout. Oncorhynchus Mykiss. J. Exp. Zool. 269 (5), 458-466. https://doi.org/10.1002/jez.1402690509.

Lubzens, E., Bobe, J., Young, G., Sullivan, C.V., 2017. Maternal investment in fish oocytes and eggs: The molecular cargo and its contributions to fertility and early development. Aquaculture. 472, 107-143. https://doi.org/10.1016/j. aquaculture.2016.10.029.

Matsubara, T., Nagae, M., Ohkubo, N., Andoh, T., Sawaguchi, S., Hiramatsu, N., Sullivan, C.V., Hara, A., 2003. Multiple vitellogenins and their unique roles in marine teleosts. Fish Physiol. Biochem. 28, 295-299. https://doi.org/10.1023/B: FISH.0000030559.71954.37.

Mizuta, H., Luo, W., Ito, Y., Mushirobira, Y., Todo, T., Hara, A., Hiramatsu, N., 2013. Ovarian expression and localization of a vitellogenin receptor with eight ligand binding repeats in the cutthroat trout (Oncorhynchus clarki). Comp. Biochem. Physiol. B 166 (1), 81-90. https://doi.org/10.1016/j.cbpb.2013.07.005.

Mushirobira, Y., Mizuta, H., Luo, W., Todo, T., Hara, A., Reading, B.J., Hiramatsu, N., 2015. Molecular cloning and partial characterization of a low-density lipoprotein receptor-related protein 13 (Lrp13) involved in vitellogenin uptake in the cutthroat trout (Oncorhynchus clarki). Mol. Reprod. Dev. 82 (12), 986-1000. https://doi.org/10.1002/mrd.22579.

Namgung, J., Mizuta, H., Yamaguchi, Y., Nagata, J., Todo, T., Yilmaz, O., Hiramatsu, N., 2021. Knock out of a major vitellogenin receptor gene with eight ligand binding repeats in medaka (Oryzias latipes) using the CRISPR/Cas9 system. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2021 Jul;257:110967. https://doi.org/10.1016/j. cbpa.2021.110967.

Patino, R., Sullivan, C.V. 2002. Ovarian follicle growth, maturation and ovulation in teleost fish. Fish Physiol. Biochem. 26(1):57-70. https://doi.org/10.1239/A: 1023311613987.

Pousis, C., Santamaria, N., Zupa, R., De Giorgi, C., Mylonas, C.C., Bridges, C.R., de la Gándara, F., Vassallo-Agius, R., Bello, G., Corriero, A., 2012. Expression of vitellogenin receptor gene in the ovary of wild and captive Atlantic bluefin tuna (Thunnus thynnus). Animal Reproduction Science. 132 (1-2), 101-110. https://doi. org/10.1016/j.anireprosci.2012.03.014.

Pousis, C., Rodríguez, C., De Ruvo, P., De Virgilio, C., Pérez, J.A., Mylonas, C., C.C., Zupa, R., Passantino, L., Santamaria, N., Valentini, L., Corriero, A., 2019. Vitellogenin receptor and fatty acid profiles of individual lipid classes of oocytes from wild and

captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle. Theriogenology. 140, 73-83. https://doi.org/10.1016/j. theriogenology.2019.08.014.

Reading, B.J., Hiramatsu, N., Sawaguchi, S., Matsubara, T., Hara, A., Lively, M.O., Sullivan, C.V., 2009. Conserved and variant molecular and functional features of multiple vitellogenins in white perch (Morone americana) and other teleosts. Mar. Biotechnol. 11 (2), 169-187. 10.1007/s10126-008-9133-6.

Reading, B.J., Hiramatsu, N., Sullivan, C.V., 2011. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch. Biol. Reprod. 84 (2), 392-399. https://doi.org/10.1095/biolreprod.110.087981.

Reading, B.J., Sullivan, C.V., 2011. Vitellogenesis in Fishes, Chapter 257. In: Farrell, A.P., Stevens, E.D., Cech Jr., J.J., Richards, J.G. (Eds.), Encyclopedia of Fish Physiology: from Genome to Environment. Academic Press, p. 2272 p.

Reading, B.J., Hiramatsu, N., Schilling, J., Molloy, K.T., Glassbrook, N., Mizuta, H., Hara, A., 2014. Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes. J. Lipid Res. 55 (11), 2287-2295. https://doi.org/10.1194/jlr.M050286.

Reading, B.J., Sullivan, C.V., Schilling, J. 2017. Vitellogenesis in Fishes. In: Ferrel, A.P. (Ed.), Encyclopedia of Fish Physiology: From Genome to Environment. The Reproductive Organs and Processes. Reference Module in Life sciences, 2017 ed. Elsevier, Maryland Heights, MO. Available at: https://doi.org/10.1016/B978-0-12- 809633-8.03076-4.

Ribas, L., Piferrer, F., 2013. The zebrafish (Danio rerio) as a model organism, with emphasis on applications for finfish aquaculture research. Reviews in Aquaculture. 5, 1-32. https://doi.org/10.1111/raq.12041.

Romano, M., Rosanova, P., Anteo, C., Limatola, E., 2004. Vertebrate yolk proteins: a review. Mol. Reprod. Dev. 69 (1), 109-116. https://doi.org/10.1002/mrd.20146.

Saillant, E., Fostier, A., Haffray, P., Menu, B., Chatain, B., 2003. Saline Saline preferendum for the European sea bass, Dicentrarchus labrax, larvae and juveniles: effect of salinity on early development and sex determination. J. Exp. Mar. Biol. Ecol. 287 (1), 103-117. https://doi.org/10.1016/S0022-0981(02)00502-6.

Sawaguchi, S., Kagawa, H., Ohkubo, N., Hiramatsu, N., Sullivan, C.V., Matsubara, T., 2006. Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus major), a marine teleost spawning pelagic eggs. Mol. Reprod. Dev. 73, 719-736. https://doi.org/10.1002/mrd.20446.

Skjærven, K.H., Finn, R.N., Kryvi, H., Fyhn, H.J. 2003. Yolk resorption in developing plaice (Pleuronectes platessa). In: The Big Fish Bang. Proceedings of the 26th Annual Larval Fish Conference. 2003 (Eds. Howard I. Browman and Anne Berit Skiftesvik). Institute of Marine Research, Bergen, Norway. pp. 193-209. 2003 ISBN 82-7461- 059-8.

Stifani, S., Le Menn, F., Rodriguez, J.N., Schneider, W.J., 1990. Regulation of oogenesis: the piscine receptor for vitellogenin. Biochimica. Et Biophysica. Acta Lipids Lipid Metabol. 1045 (3):271-279 https://doi.org/10.1005/0005-2760(90)90130-P.

Sullivan, C.V., Yilmaz, O. 2018. Vitellogenesis and yolk proteins, fish. In: Encyclopedia of Reproduction 2nd edn (ed. Skinner, M.K.) 266-277 (Academic Press, 2018). https:// doi.org/10.1016/B978-0-12-809633-8.20567-0.

Tao, Y., Berlinsky, D.L., Sullivan, C.V., 1996. Characterization of a vitellogenin receptor in white perch (Morone americana). Biol. Reprod. 55(3):646-656. https://doi: 10.1095/biolreprod55.3.646.

Williams, V.N., Reading, B.J., Hiramatsu, N., Amano, H., Glassbrook, N., Hara, A., Sullivan, C.V., 2014. Multiple vitellogenins and product yolk proteins in striped bass, Morone saxatilis: molecular characterization and processing during oocyte growth and maturation. Fish Physiol. Biochem. 40 (2), 395-415. https://doi.org/10.1007/ s10695-013-9852-0.

Yilmaz, O., Prat, F., Ibáñez, A., Amano, H., Koksoy, S., Sullivan, C.V., 2015. Estrogen- induced yolk precursors in European sea bass, Dicentrarchus labrax: status and perspectives on multiplicity and functioning of vitellogenins. Gen. Comp. Endocrinol. 221, 16-22. https://doi.org/10.1016/j.ygcen.2015.01.018.

Yilmaz, O., Prat, F., Ibáñez, J.A., Koksoy, S., Amano, H., Sullivan, C.V., 2016. Multiple vitellogenins and product yolk proteins in European sea bass (Dicentrarchus labrax): Molecular characterization, quantification in plasma, liver and ovary, and maturational proteolysis. Comp. Biochem. Physiol. Part b. 194:71-86. https://doi. org/10.1016/j.cbpb.2015.11.010.

Yilmaz, O., Patinote, A., Nguyen, T., Bobe, J., 2018. Multiple vitellogenins in zebrafish (Danio rerio): quantitative inventory of genes, transcripts and proteins, and relation to egg quality. Fish Physiol Biochem. 44 (6), 1509-1525. https://doi.org/10.1007/ s10695-018-0524-y.

Yilmaz, O., Patinote, A., Nguyen, T., Com, E., Pineau, C., Bobe, J., 2019. Genome editing reveals reproductive and developmental dependencies on specific types of vitellogenin in zebrafish (Danio rerio). Mol. Reprod. Dev. 86 (9):, 1168-1188. https://doi.org/10.1002/mrd.23231.

Yilmaz, O., Com, E., Pineau, C., Bobe, J., 2023. Genomic disturbance of vitellogenin 2 (vtg2) leads to vitellin membrane deficiencies and significant mortalities at early stages of embryonic development in zebrafish (Danio rerio). Sci. Rep. 13, 18795. https://doi.org/10.1038/s41598-023-46148-2.