Characterization of zebrafish vitellogenin gene family for potential development of receptor mediated gene transfer method 1

Precursors of the major yolk protein components are called vitellogenins Vtgs, which are synthesized in the liver of oviparous vertebrates or in the fat body of most insects under the co

Chapter 1 General Introduction

1.1 Oogenesis and vitellogenesis

1.1.1 Oogenesis

The development of an egg is known as oogenesis, which can be divided into different phases including 1) proliferation of primordial germ cells, 2) mitotic division of oogonia within ovary, 3) progress through the early stage of meiosis and arrest in the prophase of meiosis I (primary oocyte), 4) further growth and development of the primary oocyte, 5) completion of division of meiosis I and arrest at metaphase of meiosis II (secondary oocyte), 6) completion of meiosis II (egg) before or after ovulation depending on species (Wolpert et al., 1998)

Eggs are filled with maternally provided building blocks (mostly RNAs and proteins) for the developing embryos and these materials are incorporated into the oocytes during the meiotic arrest stage before initiation of the division of meiosis I In particular, eggs of oviparous (egg-laying) animals, unlike those of mammals, accumulate enormous amounts

of proteins, lipids, glycogen (collectively called yolk) during this meiotic arrest period in order to support future embryonic development (Kalthoff, 1996) Precursors of the major yolk protein components are called vitellogenins (Vtgs), which are synthesized in the liver

of oviparous vertebrates or in the fat body of most insects under the control of hormones, released into the blood circulation or hemolymph, taken up by oocytes through receptor-mediated endocytosis and further processed inside oocytes (Wahli, 1988) The whole process from Vtg synthesis to the sequestration of processed Vtg products in the oocytes is termed vitellogenesis (Kalthoff, 1996)

In all teleosts studied to date, oocytes undergo the same basic pattern of growth (Tyler and Sumpter, 1996) Based on morphological criteria and physiological/biochemical events,

the oocyte development in the zebrafish (Danio rerio) can be divided into five stages

(Selman et al., 1993) In stage I (primary growth stage), an oocyte resides in the nest with other oocytes (stage IA), and then is surrounded by a single layer of follicle cells followed

by continuous growth before arrest in diplotene of the meiotic division I (stage IB) In stage II (cortical alveolus stage) and III (vitellogenesis), oocytes are distinguished by the appearance of cortical alveoli and yolk proteins, respectively In stage IV (oocyte maturation), meiosis is reinitiated and the nucleus (germinal vesicle) migrates towards the oocyte periphery After ovulation, a mature egg is formed (stage V) During the primary growth stage, an acellular vitelline envelop (referred to as zona pellucida, zona radiata or chorion vitelline envelop) develops around the oocyte and continues to differentiate and increase in complexity throughout the remainder of oocytes growth (Tyler and Sumpter, 1996) Since the early developmental stages of many species of fish last long periods of starvation before first exogenous feeding, the maternal production of Vtgs and the deposition of adequate supplies of yolk are essential to the survival of fish embryos and larvae

1.1.2 Vitellogenesis

The concept of hormonal control of vitellogenesis was first proposed by Bailey (1957) after studying the correlation of estradiol (E2) with blood Vtg content in goldfish The concept has been tested and modified after extensive studies of vitellogenesis in insects and amphibians The hormonal control of vitellogenesis in frogs and insects is similar

B

Fig 1-1 Hormonal control of vitellogenesis and basic structure of vitellogenin proteins

A: Hormonal control of vitellogenesis in frogs and insects (from Kalthoff, 1996) B:

Three basic Vtg organization schemes from three different phyla (from LaFleur, 1999) See text for detailed descriptions

(schematically shown in Fig 1-1A; from Kalthoff, 1996) Briefly, in amphibians, environmental cues stimulate the hypothalamus to secrete gonadotropin-releasing hormones which cause the pituitary gland to produce gonadotropins These hormones stimulate the ovarian follicle cells to produce estrogen, which in turn cause the liver to synthesize Vtgs In insects, the corpora allata (functionally analogous to the pituitary gland in amphibians) release juvenile hormone which stimulates the ovarian follicle cells

in dipterans to produce a steroid hormone, ecdysone The dipteran juvenile hormone or ecdysone stimulates the fat body (functionally analogous to the liver in amphibians) as well as follicle cells in to produce yolk protein precursors (Kalthoff, 1996) Vtgs or yolk protein precursors are secreted into the blood stream or hemolymph and then sequestered

in the oocytes by receptor-mediated uptake In Xenopus, Vtgs are further broken down

into lipovitellins (LVs) (I and II) and phosvitin (PV) or two phosvettes (due to an additional cleavage in the PV domain) after uptake into oocytes (Wiley and Wallace, 1981) Cathepsin D was found to be responsible for the cleavage at the C-terminal end of phosvitin (Opresko and Karpf, 1987)

Vitellogenesis is a major event responsible for the dramatic growth of oocytes in many teleosts and may account for 11-95% of the final egg size (Tyler and Sumpter, 1996) The duration of vitellogenic phase and the minimum size of oocytes before entering vitellogenic development vary in different fish species In most fish studied, hepatically-derived Vtgs are the principle precursors of yolk proteins and their synthesis in the liver is

in response to the circulating E2 derived from the ovary (Ng and Idler, 1983; Tyler, 1991)

Like other oviparous vertebrates, circulating Vtgs are selectively taken up by oocytes through receptor-mediated endocytosis (Chan et al., 1991; Tyler and Lancaster, 1993)

Studies indicated that the ability of an oocyte to sequester Vtgs depends on the development of patency or opening of intercellular channels, which allows Vtgs to pass through the follicular tissues to the oocyte surface (Wallace, 1985; Tyler et al., 1991) The presence of hormone and growth factor regulated Vtg receptors on the oocyte surface is also a key factor affecting vitellogenic growth of fish oocytes Hiramatsu et al (2002)

demonstrated that the LV domain of white perch (Morone americana) Vtg mediates its

binding to the oocyte receptor and the remaining domains may interact with the LV domain to facilitate the receptor binding Li et al (2003) further narrowed down the

receptor-binding region in tilapia (Oreochromis aureus) Vtg1 to an N-terminal 85-amino

acid fragment in LVI domain

After uptake into oocytes, Vtgs in teleosts, like those in amphibians, also undergo proteolytic cleavage to form three main products, LVI (heavy chain), PV and LVII (light chain) (Sharrock et al., 1992; Matsubara et al., 1999; Fig 1-1B) Additional proteolytic cleavages were observed either in LVI for lamprey Vtg (Sharrock et al., 1992) or in LVII for Vtgs of several other fish species, resulting in a C-terminal β-component of ~260 amino acids (Matsubara et al., 1995; Hiramatsu and Hara, 1996) One unique feature in fishes that produce floating eggs is that there is a second proteolytic cleavage event during oocyte maturation and hydration which causes an increase of free amino acid content and generates osmotic effectors needed for water influx (Carnevali et al., 1999) It has been

shown in seabream (Sparus aurata) that cathepsin D and B are involved in the first

proteolytic cleavage and cathepsin L is responsible for the second proteolytic cleavage, resulting in complete degradation of one LV component (Carnevali et al., 1999)

A variety of changes have been observed in hepatocytes of vitellogenic females or estrogen administrated male fish and these changes are consistent with substantial increases in the capacity of protein synthesis and export in the liver Briefly, vitellogenic hepatocytes are characterized by expanded nuclear envelope cisternae, swollen mitochondria, enhanced rough endoplasmic reticulum, Golgi apparatus and secretory vesicles, increased contents of proteins and total RNAs and increased amount of enzymes such as transaminases and those of the Krebs cycle and glycolysis (Mommsen and Walsh,

1988 and references within) The de novo synthesis of vtg mRNAs and increased amount

of rRNAs may account for the increase in total RNA, and enhanced translational activities are expected from the proliferation of translational machineries Thus, E2 is able to orchestrate cell metabolism and biosynthetic activities at various levels in the liver of teleost fish (Mommsen and Walsh, 1988)

1.2 Vitellogenin

1.2.1 Vitellogenin proteins

The term “vitellogenin” was first used to refer to a serum form of a yolk protein precursor

isolated from the Cecropia moth (Pan et al., 1969) Now, the term vitellogenin has been

reserved for yolk protein precursors that belong to an ancient gene family existing in a wide range of metazoans from nematodes to insects and vertebrates (LaFleur, 1999)

Vitellogenins produced by oviparous vertebrates are large lipophosphoglycoproteins, which are extensively modified with covalently linked carbohydrates, phosphates and sulfates and with noncovalently bound lipids, hormones, vitamins and metals (Chen et al.,

1997 and references within) Native Vtgs in the blood circulation of most oviparous

vertebrates studied so far are in the form of dimers with molecular weights between

326-550 kDa, which are composed of Vtg subunits of 140-220 kDa (Mommsen and Walsh, 1988; Byrne et al., 1989) The three major yolk proteins (LVI, LVII and PV) found in the eggs of oviparous vertebrates can be easily recognized as domains along the primary

structures of vtg cDNA translations At the N-terminal of Vtg lies a relatively large

domain representing yolk protein LVI At the C-terminal there is a relatively small domain representing yolk protein LVII and the middle polyserine domain represents yolk protein

PV (LaFleur, 1999; Fig 1-1B) It was suggested that the PV domain has undergone both contraction and expansion from low to high vertebrates (Bidwell and Carlson, 1995) Production, secretion and cleavage of Vtg precursors in different phyla of oviparous invertebrates are more heterogeneous than in oviparous vertebrates The native Vtgs in invertebrates is either in the form of monomer (170-195 kDa) or dimer (530-550 kDa), with or without posttranslational modifications and with (before or after uptake into oocytes) or without cleavage when forming the yolk (Wahli, 1988; Byrne et al., 1989; Fig 1-1B)

The most apparent difference between vertebrate and invertebrate Vtgs is that the invertebrate Vtgs lack the polyserine-rich phosvitin domain (Spieth et al., 1985; Nardelli

et al., 1987; Fig 1-1B) Interestingly, intact or vestigial polyserine domains were

identified in Vtgs of several insect species, including boll weevil (Anthonomus grandis), mosquito (Aedes aegypti) and silkmoth (Bombyx mori) (Trewitt et al., 1992; Chen et al.,

1994a; Yano et al., 1994) Further analysis indicated that the polyserine domains of arthropod and vertebrate Vtgs are unlikely to have originated from a common ancestor, since not only are the locations of these polyserine domains different, but also the usage of

serine codon differs between Vtgs in insects (mainly by TCX serine codons) and vertebrates (mainly by AGY serine codons) (Chen et al., 1997; LaFleur, 1999) The polyserine domains are heavily phosphorylated and may be important in maintaining tertiary structure of Vtgs or in carrying calcium phosphate in support of vertebrate embryonic bone formation (Wahli, 1988)

Although the divergence at the amino acid level is high, Chen et al (1997) have reported that there are five relatively conserved regions widespread along the Vtgs from nematodes, insects and vertebrates These five homologous subdomains (I-V) are located outside both the polyserine domains of insect Vtgs and the phosvitin domain of vertebrate Vtgs, and are aligned relatively easily among different phyla (Fig 1-1B) Further confirmation of the homology of Vtgs came from the observation that glycine, proline and cysteine, which are important in secondary structure, were the predominant residues among the strictly conserved residues (Chen et al., 1997) Thus, sequence analysis supports that Vtgs of nematodes, insects and vertebrates share common ancient ancestry (Chen et al., 1997)

Combining secondary structure features such as α-helixes and β-sheets, Babin et al (1999) further identified twenty-two N-terminal conserved sequence motifs (N1 to N22) covering the homologous subdomains I-III and seven C-terminal conserved motifs (C1 to C7) covering subdomains IV and V in invertebrate and vertebrate Vtgs Interestingly, the conserved motifs N1 to N22 were also identified in the N-terminal part of several nonexchangeable apolipoproteins, including insect apolipophorin II/I precursor (apoLp-II/I), human apolipoprotein B (apoB) and the large subunit of mammalian microsomal

triglyceride transfer protein (MTP), suggesting a derivation from a common ancestral functional unit, termed large lipid transfer (LLT) module (Babin et al., 1999) Furthermore, the seven conserved sequence motifs (C1 to C7) were also identified in the C-terminals of insect apoLp-II/I and human apoB (reminiscent sequence motifs), and named as the von Willebrand factor D (VWD) module previously characterized in von Willebrand factor (VWF) and several other proteins (Babin et al., 1999) In addition, there are four and one conserved ancestral exon boundaries in the LLT and VWD modules, respectively Thus, the same authors also concluded that genes coding for Vtg, apoLp-II/I, apoB and MTP large subunit were derived from a common ancestor and were members of the same multigene superfamily, named as large lipid transfer protein (LLTP) superfamily (Babin et al., 1999) Phylogenetic analysis also indicated that insect apoLp-II/I and mammalian apoB are paralogous to Vtgs (Babin et al., 1999) As a matter of fact, apoB and apoLp-II/I are implicated in the deposition of yolk reserves in birds and insects (Evans and Burley, 1987; Soulages and Wells, 1994)

1.2.2 Vitellogenin genes

Vitellogenin (vtg) genes constitute a multigene family in most oviparous animals For example, six vtg genes have been identified in the nematode (Caenorhabditis elegans), two in the migratory locust (Locusta migratoria), four in Xenopus laevis and three in chicken (Gallua gallus) (Spieth et al., 1991; Locke et al., 1987; Schubiger and Wahli, 1986; Wang et al., 1983) In teleost, vtg mRNA sequences derived from more than one vtg

have been reported in GenBank for many species, indicating that teleost genomes also

contain multiple members of vtg genes

The multiple copies of vtgs are located either on one chromosome, such as in L migratoria and rainbow trout (Oncorhynchus mykiss) or on two chromosomes, such as in

C elegans and X laevis (Bradfield and Wyatt, 1983; Trichet et al., 2000; Spieth et al., 1991; Wahli, 1988) Interestingly, most vtgs in nematode and the two vtgs in migratory

locust are located on the sex chromosome X (Spieth et al., 1991; Bradfield and Wyatt,

1983) Based on the genome organization of vtgs and sequence similarity, among different vtg members, it seems that vtgs in different species may have undergone gene duplication during evolution For example, the nematode vit-3 and vit-4 may arise from tandem

duplication of a precursor gene, since both genes are tandemly linked in a head-to-tail fashion and share high percentage of sequence identity (>99%) (Heine and Blumenthal,

1986; Wahli, 1988) In X laevis, it was speculated that after an early duplication of a primordial vtg, a more recent whole genome duplication took place, resulting in four vtgs (A1, A2, B1 and B2) in modern X laevis (Jaggi et al., 1982) Buisine et al (2002) postulated that the rainbow trout vtgs might be subjected to several rounds of amplification, including an initial tandem amplification of precursor vtgs, resulting in ~10 copies of vtg genes and pseudogenes, followed by amplification of the whole vtg cluster, leading to a three-fold increase in the total vtg copy number

Although the length of the coding sequence is similar, the intron number and the average

intron size of vtg increase from the nematode to vertebrates, resulting in considerable variation in the length of vtg genes For example, the nematode vtgs have 4-5 exons, while

35 exons are present in vtgs of Xenopus and chicken, and 34 exons in teleost vtgs due to the merging of the exons corresponding to exons 22 and 23 of the Xenopus and chicken It

is not clear whether the amphibians and birds split this domain by intron insertion after

radiation from fish or the teleost eliminated one of the introns during evolution (Mouchel

et al., 1997)

1.2.3 Regulation of vtg expression

1.2.3.1 Tissue specific expression of vtgs

The hormonal regulation of vitellogenesis and the site for vitellogenin (Vtg) synthesis differ in invertebrates from oviparous vertebrates In nematodes, Vtgs are synthesized in the intestine of hermaphrodites (Kimble and Sharrock, 1983); whereas in echinoderms such as sea urchin, Vtgs are synthesized in the intestine as well as in the gonads of both sexes (Shyu et al., 1986) In insects, the synthesis of yolk proteins is mainly controlled by juvenile hormone and also by ecdysteroids in Diptera (Wyatt, G.R., 1988; Byrne et al., 1989) Unlike the vertebrate system, female-specific synthesis of yolk proteins in insects

is also controlled by products of sexual differentiation genes (Belote et al., 1985) In terms

of the expression sites, Vtgs or yolk proteins are synthesized in the fat body of female insects or in both female fat body and ovarian follicle cells in the higher Diptera (Wahli, 1988) In oviparous vertebrates, Vtgs are synthesized mainly in the liver of female animals and under the control of estrogen (Byrne et al., 1989 and references within) In addition, different components of the ovarian follicle (somatic tissues surrounding an oocyte, including granulosa, theca and surface epithelium) were also implicated in the contribution of vitellogenesis in amphibians (Wallace, 1985) and squamate reptiles (Andreuccetti, 1992) Evidence from a recent report indicated that the ovarian follicle cells

in a cartilaginous fish the spotted ray (Torpedo marmorata) synthesize Vtgs (Prisco et al.,

2004)

1.2.3.2 Estrogen-dependent expression of vtgs in oviparous vertebrates

Vertebrate Vtgs are synthesized in hepatic parenchymal cells under the influence of estrogen Estrogen enters the liver and binds to the estrogen receptor (ER), and the hormone-bound receptor binds tightly at the estrogen-responsive element (ERE) located

upstream of, or within, estrogen-responsive genes such as ER and vtg, resulting in

activation or enhanced transcription of these genes (Lazier and MacKay, 1993)

Early observation of dynamic changes on the levels of both Vtgs and vtg mRNAs in the male Xenopus liver after estrogen treatment indicated that the expression of vtgs is

estrogen-dependent (Wallace and Jared, 1968; Baker and Shapiro, 1977) Furthermore, it

was first observed in male Xenopus that a second administration of estrogen caused a

quicker response, resulting in a higher level of Vtgs than that after the first administration

of estrogen (a so called “memory effect”) (Tata, 1988) The “memory effect” of estrogentreatment was also observed in rainbow trout and tilapia (Le Guellec et al., 1988; Lim et al., 1991) Lazier and MacKay (1993) proposed that after the primary estrogen exposure, long-term alterations in either chromatin conformation or in transcription factors may be responsible for the “memory effect” Interestingly, temperature also modulates the responsiveness of the teleost fish liver to estrogen treatment, with enhanced translational

or post-translational capacities at higher temperature (Lazier and MacKay, 1993)

A parallel increase in estrogen receptor (ER) was observed in male Xenopus liver after

estrogen treatment and the increased ER was equivalent to that in the female liver and persisted for several weeks (Hayward et al., 1980) Studies using primary cultured rainbow trout hepatocytes clearly showed that after estrogen treatment, a rapid increase