egg quality in fish- what makes a good egg

Egg quality in sh: what makes a good egg?

SUZANNE BROOKS , CHARLES R. TYLER and JOHN P. SUMPTER

Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex UB8 3PH, United

Kingdom

Contents

Abstract page 387

Introduction 388

The coordinated assembly of a sh egg 389

What is in an egg? 392

Hormones and egg quality 395

Egg size is bigger better? 398

Age of sh and egg quality 398

Environmental inuences on egg quality 399

Diet

Photoperiod and physiochemical properties of the water

Pollutants

Husbandry of captive sh

Genetic inuences on egg quality 404

Parental genes

Genetic markers for egg quality

Control of gene expression and egg quality

Acknowledgement 406

References 406

Abstract

Factors affecting egg quality are determined by the intrinsic properties of the egg itselfand the environment in which the egg is fertilized and subsequently incubated. Eggquality in sh is very variable. Some of the factors affecting egg quality in sh areknown, but many (probably most) are unknown. Components that do affect egg qualityinclude the endocrine status of the female during the growth of the oocyte in the ovary,the diet of the broodsh, the complement of nutrients deposited into the oocyte, and thephysiochemical conditions of the water in which the eggs are subsequently incubated. Incaptive broodsh, the husbandry practices to which sh are subjected are probably amajor contributory factor affecting egg quality. Our knowledge of the genetic inuenceson egg quality is very limited indeed. We know that parental genes strongly inuenceboth fecundity and egg quality, but almost nothing is known about gene expression

Reviews in Fish Biology and Fisheries 7, 387416 (1997)

09603166 # 1997 Chapman & Hall

Author to whom correspondence should be addressed (e-mail: [email protected]).

and=or mRNA translation in sh oocytes=embryos. This is surprising because theproducts synthesized in ovo and the mechanisms controlling their expression are likely toplay a central role in determining egg quality. The genetic mechanisms underpinningoocyte and embryo growth and development are a priority for research.

Introduction

Fish populations, both farmed and wild, are dependent upon the production of good-quality eggs. Poor egg quality is one of the major constraints in the expansion ofaquaculture of both marine and many freshwater sh species. In the sh farming industry,good-quality eggs have been dened as those exhibiting low mortalities at fertilization,eyeing, hatching and rst feeding (Bromage et al., 1992). Egg survival and hatchingrates, however, while being the ultimate measures of egg quality, tell us nothing aboutwhat factors determine egg quality. Morphological features of larvae have been used asindicators of gamete quality in some sh species (Kjorsvik, 1994). Other authors havesuggested that the appearance of the zona pellucida, the shape of the egg, itstransparency and distribution of oil globules can be related to quality (Kjorsvik et al.,1990; Bromage et al., 1994). There is little agreement, however, regarding reliablemethods for `quality' assessment in eggs of marine sh. Hatcheries culturing marinespecies often distinguish `good'-quality eggs from `poor'-quality eggs by virtue of theeggs' ability to oat or sink in sea water, respectively (McEvoy, 1984; Carrillo et al.,1989; Kjorsvik et al., 1990). However, the positive correlation between buoyancy and`good' quality does not hold true for a number of marine species for example, in theAtlantic halibut, Hippoglossus hippoglossus (Pleuronectidae). In this species, the onlyreliable indicator of egg quality established so far is based on assessment of cellsymmetry at early stages of cleavage (Bromage et al., 1994).

Developmental biologists would consider that the quality of an egg is determined bythe intrinsic properties of the egg itself, by its genes, and by the maternal mRNAtranscripts and nutrients contained within the yolk, all of which are provided by themother. After fertilization, of course, the quality of the egg (or rather the embryo) willalso be determined by the contribution of the paternal genes. Both in aquaculture and inthe wild, the environment in which the eggs are incubated also affects the success ofthe egg in producing a viable offspring. The conditions to which fertilized eggs areexposed, therefore, also often become encompassed in the term `egg quality'.

Our knowledge of the processes affecting egg quality in both wild and captive sh isextremely limited. In the few studies that have been carried out on wild sh, it hasbeen found that egg quality may show considerable variability from season to season(Kjorsvik et al., 1990). Egg quality in many farmed sh, particularly marine species, isthe major difculty in their culture (Kjorsvik et al., 1990). For example, in Europeansea bass (Dicentrarchus labrax, Percichthyidae) and gilthead seabream (Sparus auratus,Sparidae), hatching rates are often only 1015% of total eggs spawned (Carrillo et al.,1989). In Atlantic halibut, a very important commercial species with a considerablepotential for aquaculture, hatching rates are often less than 1% (Norberg et al., 1991;M. Bruce, pers. comm.). Some of the problems in egg quality in marine species areprobably a function of the difculties in providing optimum culture conditions for eggincubation. Even for salmonids, however, where the incubation methods for fertilizedeggs are well established, there may be losses of 50% up to hatching (Bromage et al.,

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1992). In multiple spawning sh, there are also considerable variations in the quality ofeggs produced in different batches over a single spawning season, even when thebatches of eggs are maintained under apparently identical culture conditions (yellowtailounder, Pleuronectes ferrugineus, Pleuronectidae Manning and Crim, 1995; Atlanticcod, Gadus morhua, Gadidae Kjesbu et al., 1996).

A great deal of anecdotal evidence exists concerning what are thought to be themajor determinants of egg quality in sh. There is widespread belief that the nutrientmaterials sequestered by the oocyte, and their processing during growth and maturationof the oocytes, are key factors affecting egg quality (Craik and Harvey, 1984; Kjorsviket al., 1990; Bromage et al., 1992). Diet has received the greatest attention with respectto its effect on egg quality, and recent studies indicate that the major inuences onquality are exerted by just a few of the many different dietary constituents (Washburnet al., 1990; Watanabe et al., 1991a,b; Harel et al., 1994). The physiology of thebroodsh and their hormonal status, which in turn affects the incorporation ofcompounds, including hormones, into eggs, are also likely to have some inuence onthe quality of the eggs (for example, stressing sh may have deleterious effects;Campbell et al., 1994). Other important determinants of egg quality, which in captivesheries may be a function of husbandry, include overripening (the process of ageing ofunfertilized eggs which have been retained in the body cavity after ovulation: Springateet al., 1984; Kjorsvik et al., 1990) and bacterial colonization of the fertilized eggs(Barker et al., 1989; Hansen and Olafsen, 1989). Genetic inuences on egg quality andthe possible roles of maternal RNAs have been the focus of studies on oocyte quality inhumans and agricultural animals for some years (Koenig and Stormshak, 1993; Navot etal., 1994). In sh, however, only very recently have attempts been made to assess anddene the genetic factors which may underlie and determine egg quality (Lam, 1994;Nagahama, 1994). This paper reviews the inuences on egg quality of bothenvironmental and genetic factors and attempts to highlight future research needs.

The coordinated assembly of a sh egg

In teleost sh, an egg is the nal product of oocyte growth and development, a processthat can take a year or more (Tyler and Sumpter, 1996). Once ovulated, sh eggs take upvery little, if any, nutrients: only water, and chemicals in the water, are known to passinto an ovulated egg (Holliday and Pattie Jones, 1967). Hence, all the contents of an egg,genetic and nutritive, that will determine its quality, must be incorporated into an eggwhen it is an oocyte within the ovary (Fig. 1). This situation is very different to that ineutherian mammals, where the nutrients contained in an egg simply provide the materialsnecessary to initiate embryonic development; once the embryo has implanted into theuterine wall, all the nutrients needed for development by the embryo are provided by themother as and when they are required. The eggs of sh, and other oviparous (egg-laying)animals, therefore, are considerably larger than in mammals; sh eggs measuring 1 mmin diameter are more than 23 000 times larger, by volume, than a human egg, and theeggs of a coelocanth (Latimeria chalumnae, Latimeriidae) are more than a million-foldlarger than a human egg. The developing oocyte autonomously makes most of thecomponents of the machinery for DNA and protein synthesis, as well as mRNAs neededimmediately after fertilization (Tata, 1986). However, specialized egg constituents such asyolk proteins and egg coat substances are synthesized in the liver (Ng and Idler, 1983;

Egg quality in sh 389

Fig. 1. A scheme showing the coordinated assembly of a sh egg.

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Hyllner et al., 1991, 1994; Oppen-Berntsen et al., 1991; Fig. 1). A knowledge of themechanisms underlying the processes of oocyte growth and development, and how theseprocesses are coordinated, is essential for understanding fully the factors affecting eggquality and fertilization. Oocyte growth and development, and subsequently embryonicdevelopment of the fertilized egg, however, are very complex processes and, despite itsimportance, knowledge on the coordinated assembly of the developing egg is far fromcomplete.

In all teleosts, oocytes appear to undergo the same basic pattern of growth, regardlessof their reproductive strategy. The major developmental events occurring during oocytedevelopment can be broadly classied into six phases, according to the state of oocytegrowth; they are: oogenesis, primary oocyte growth, cortical alveolus stage,vitellogenesis, maturation and ovulation (Nagahama, 1983; Selman et al., 1986, 1993;Bromage and Cumaranatunga, 1988; Tyler, 1991; Tyler and Sumpter, 1996). The geneticchanges and ultrastructural events accompanying oocyte development in teleosts aredescribed in Nakamura and Nagahama (1993) and Selman et al. (1993). Briey, duringthe early stages of oocyte development, DNA replication occurs (leptotene),homologous chromosomes pair (zygotene) and these pairs shorten and thicken(pachytene). The chromosomes then unpair into lampbrush congurations (diplotene),just before the oocyte enters a long period of cytoplasmic growth. The cytoplasmicgrowth of the oocyte is characterized by an enormous accumulation of yolk reserves(vitellogenesis). Meiosis resumes via a hormonal signal, and this leads to oocytematuration. During this period, the nucleus, arrested in meiotic prophase, breaks downand the chromosomes enter rst meiotic metaphase. The oocyte is then released fromthe ovary into the body cavity and it becomes an egg ready for fertilization (Nagahama,1995).

The production of a good-quality egg relies upon the correct progression througheach of these phases, and this coordinated assembly of an oocyte is controlled by aninterplay of endocrine and intra-ovarian factors (both paracrine and autocrine; Tyler andSumpter, 1996, Figure 1). The primary signal(s) triggering oocyte growth andmaturation are environmental and these are converted from electrical to chemicalsignals in the hypothalamus (Tata, 1986). In sh, gonadotrophin-releasing hormone(GnRH) is released by the hypothalamus in response to an external stimulus. TheGnRH activates the anterior pituitary to release gonadotrophins (GtH I and GtH II;probably representing sh FSH and LH, respectively; Prat et al., 1996). GtH I acts ongranulosa and thecal cells, stimulating the synthesis of oestradiol-17 (Suzuki et al.,1988), which in turn stimulates the production of yolk protein precursors and egg shellproteins by the liver (Ng and Idler, 1983; Oppen-Berntsen et al., 1994, see below). GtHI also effects uptake of vitellogenin (VTG) into trout oocytes (Tyler et al., 1991b).Furthermore, GtH I may play a role in recruitment of primary oocytes into the maturingpool of vitellogenic oocytes (Prat et al., 1996; our own unpublished observations). GtHII functions later in oocyte development than GtH I, acting on the follicle cells, tostimulate the synthesis of progesterones, which control the termination of oocyte growthand ovulation of the egg (Suzuki et al., 1988). The biosynthetic activity of thedeveloping oocyte, in contrast to the regulation of the extra-ovarian tissues involved inthe genesis of an egg, is believed to be autonomous and not regulated by hormones orany external signals (Tata, 1986). Very recently in mammals, the rst oocyte-specicgrowth factor has been identied (growth differentiation factor 9; GDF-9) and this


growth factor plays a vital role in somatic cell function (Dong et al., 1996). It is likelythat specic growth factors exist for sh oocytes, but as yet none have been identied.

What is in an egg?

An egg needs all the necessary information to direct the development of a functional,free-swimming embryo and all the `building blocks' to form the embryo; so, it needs allthe amino acids, lipids and carbohydrates that make up an embryo, together with calcium(for bones), vitamins and metals for enzyme and other metabolic actions, and many otherthings (Fig. 2). These specialized materials are derived from a number of maternalsources and must be incorporated during the growth of the oocyte in the ovary. If an eggdoes not contain a particular compound, or contains an inappropriate amount of acompound, it will not be able to sustain development of a viable embryo. Hence tounderstand egg quality, one needs to be able to understand what gets into oocytes, how itgets there, and the role of the various constituents.

In vertebrates' eggs, a large variety of macromolecules stored in the mature egg aresynthesized within the developing oocyte itself (Tata, 1986; Fig. 2). Many of thesemacromolecules constitute the machinery for post-fertilization cell division and proteinsynthesis, i.e. ribosomes, DNA and RNA polymerases, histone proteins, transcriptionand translation factors, etc. In Xenopus the embryo does not transcribe its ribosomalgenes until the late gastrula stage is reached (when the embryo has reached almost10 000 cells; Tata, 1986), illustrating the need for large stores of maternal ribosomes inoviparous eggs. A similar situation is likely to occur in sh. Another group ofmacromolecules synthesized in situ during oogenesis are messenger RNAs, includingthose coding for cytoskeletal and membrane proteins (Tata, 1986). In amphibians(review: Wallace and Selman, 1990), it appears that most of the RNA in full-grownoocytes is already present by the end of the primary growth phase (Anderson andSmith, 1978). In sh, although there is little information available on RNA syntheticactivity, a period of intense RNA synthesis occurs during the initial stages of primaryoocyte growth (Wallace and Selman, 1990). There is some evidence in oviparousanimals to suggest that mRNA transcripts may pass from the maternal circulation intothe developing oocyte and thus inuence oocyte growth and development, andsubsequently, the development of the embryo (Amanai et al., 1994; Hales et al., 1994).

Endogenous production of proteins has been shown to occur in Xenopus oocytes, butmost of these proteins appear to undergo turnover. Most, if not all, of these proteins areinvolved in general cell function (Wallace and Hollinger, 1979). Again, little is knownabout endogenous production of proteins in sh oocytes=eggs.

Vitellogenesis is the principal event responsible for the enormous growth of oocytesin many teleosts, and this is when most nutritive products are taken up and stored forfuture use by the developing embryo. In salmonid sh, for example, vitellogenesis mayaccount for over 90% of the nal volume of an oocyte (Tyler, 1991; Tyler et al.,1991a). In most sh studied, the `building blocks' for the subsequent embryo, includingthe amino acids, energy (from phosphate bonds), lipid and calcium, are derived fromthe plasma during vitellogenesis, most of them originating from the uptake of a largecomplex molecule, called vitellogenin (VTG; Tyler, 1991; Specker and Sullivan, 1994).VTG is synthesized by the liver in response to circulating oestradiol-17 from the ovary(Ng and Idler, 1983) and selectively sequestered by receptor-mediated endocytosis


Fig. 2. Uptake of materials into a developing oocyte and their subsequent mobilization for use in forming the developing embryo.

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(Chan et al., 1991; Le Menn and Nunez-Rodriguez, 1991; Tyler and Lancaster, 1993).Multigene families for VTG have been shown to exist in a number of oviparousvertebrates (Wang et al., 1983; Wahli and Ryf, 1985). To date, only one VTG mRNAhas been isolated from any sh (Chen, 1983; LaFleur et al., 1995). There are reports,however, in the zebra sh (Brachydanio rerio, Cyprinidae; Selman et al., 1993) and inthe blue tiliapia (Tilapia aurea, Oreochromis aureus, Cichlidae; Ding et al., 1989) thatmore than one type of VTG molecule may exist. These `different' forms of VTG mayarise from different genes, or differential splicing of the mRNA transcript. Alternatively,they may arise as a result of different post-translation modications of the same VTGmolecule. The functional signicance to the subsequent egg of these different forms ofVTG is not known. An array of other plasma lipoproteins distinct from VTG arepresent in the circulation during vitellogenesis (Babin, 1987), but most studies indicatethat they are not sequestered in signicant amounts by the growing oocyte (Tyler andSumpter, 1996).

The major yolk proteins in sh oocytes are lipoproteins, called lipovitellins, andphosphoproteins, which include phosvitins and phosvettes (reviews: Wallace, 1985;Specker and Sullivan, 1994). The number, types and forms of yolk proteins isolatedmay differ between different species of teleosts, but all seem to be derived from VTG(Specker and Sullivan, 1994). Materials present in an oocyte which are required for thedeveloping embryo must be positioned correctly within the oocyte and protected fromturnover and degradation until they are required. Yolk proteins are stored either in acrystalline platelet form (zebra sh, Selman et al., 1993), or, as is more common, inuid-lled yolk spheres or globules (Wallace and Selman, 1981). Lipids, in addition tothose derived from VTG, may be deposited from the circulation into the developingoocyte and in turn provide a nutrient source for the subsequent embryo. Acomprehensive review on the composition, accumulation and utilization of yolk lipidsin teleosts has recently been published (Wiegand, 1996a) and so the topic is notdiscussed here.

Although yolk proteins and lipids constitute the bulk of material in sh eggs, thereare other molecules that must be derived from sources outside of the oocyte, such asvitamins and metals that, although present in far lower quantities, are equally necessaryfor producing a viable egg and, subsequently, a viable offspring (Fig. 2). For example,some vitamins and metals are required for enzyme activities, including hatching (Brownand Lynam, 1981). Binding proteins and induction factors for embryonic developmentalso cross the oocyte membrane to support the development of the subsequent embryo(Uchiyama et al., 1994). Embryogenesis is inuenced by a variety of hormones andgrowth factors, and by RNAs deposited into the yolk which encode for hormonesgrowth factors and receptors. Vitamins, metals, binding proteins and hormones mayenter the oocyte adventitiously in the uid phase during receptor-mediated uptake ofVTG, others attach to VTG, or alternatively, they may enter quite specically, bound todistinct, specic receptors. In Xenopus, VTG has been shown to function as a carrierprotein for maternal factors involved in early development; activin, a candidate formesodermal induction factor (Smith et al., 1990) and follistatin, an activin-bindingprotein (Nakamura et al., 1990), preferentially bind to VTG in the plasma and both ofthese proteins localize in yolk platelets (Uchiyama et al., 1994). In the chicken, bothvitamin A and thyroid hormones (triiodothyronine and thyroxine) are transported intooocytes, attached to a transport protein called transthyretin, which has its own specic


receptor (Vieira et al., 1995). In sh, thyroid hormones attach to VTG in the blood andin turn are transported into the oocyte in this way (Babin, 1992; Cyr and Eales, 1992).Overall, however, very little is known in sh about the transport systems and theregulatory systems effecting uptake of hormones, vitamins and metals into the growingoocyte, despite the fact that they are likely to play key roles in determining egg quality.

Enzymes are present in the oocyte which catalyse a series of metabolic processesvital for the production of a viable offspring (Fig. 2). They include enzymes like thecathepsins that degrade VTG into yolk proteins for storage in the oocyte and, post-fertilization, which mediate the degradation of the stored yolk proteins into free aminoacids for use by the developing embryo (Sire et al., 1994). Very little is known aboutthe content of enzymes in sh oocyte=eggs, even though these enzymes will play acentral role in modifying free amino acids, fatty acids, etc. prior to their incorporationinto newly synthesized proteins and lipids in the developing embryo.

During ovarian growth, the oocyte becomes surrounded by an acellular envelope calledthe vitelline envelope (Brummett et al., 1982). The proteins forming the vitelline envelope,which are produced by the liver in response to oestrogen (Oppen-Berntsen et al., 1992a,b,1994), play important functions during fertilization and embryo development; whenhardened after fertilization (Yamagami, 1992), the vitelline envelope protects thedeveloping embryo against mechanical damage, desiccation and rapid chemical changesin the environment, and it also exerts bactericidal and fungicidal activity (Kudo, 1992).

Hormones and egg quality

Hormones play a role in larval development and thus may affect egg quality. Hormonescould be supplied to the egg before fertilization, in which case they must enter bymaternal transfer, or they could be synthesized in situ at any time after fertilization(Fig. 3). Studies looking at the ontogeny of the endocrine system (Leatherland andBarrett, 1993; Naito et al., 1993; Tanaka et al., 1995) have so far indicated that shlarvae are physiologically immature, with little or no capacity to produce certainenzymes, growth factors and hormones, until at or around the end of yolk resorption.Thus, sh larvae appear to be dependent on exogenous sources (mother and=or live food)for the supply of these regulatory factors, rather than their in situ synthesis in theegg=larvae (Lam, 1994). Indeed, hormones have been shown to pass into sh oocytesfrom the maternal circulation (Greenblatt et al., 1989). This store of maternal hormonesmay full the regulatory needs of sh larvae for growth, development, osmoregulation,stress responses and other physiological functions prior to the functional development oftheir own endocrine glands (Lam, 1994). Our knowledge on the hormonal content ofeggs prior to fertilization, however, is limited to a very few hormones, including thethyroid hormones, thyroxine (Tagawa and Hirano, 1991) and triiodothyronine (Tagawaand Hirano, 1987), cortisol (De Jesus et al., 1991: Hwang et al., 1992; Contreras-Sanchez et al., 1995; Brooks et al., 1995) and several sex steroids (Rothbard et al., 1987;Feist et al., 1990). A very limited number of studies (to date) have attempted tomanipulate the hormonal content of eggs and assess the subsequent effects (Brown et al.,1988, 1989; Tagawa and Hirano, 1991; Ayson and Lam, 1993; Brooks et al., 1995) andthese are discussed below.

Thyroid hormones of maternal origin are deposited in egg yolk, and they may havesignicant effects on sh embryo development (Lam, 1994). Triiodothyronine is


Fig. 3. Environmental and genetic factors that affect egg quality. Factors in light blue (pollutants, fungi, bacteria) are always detrimental to egg

quality.

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maintained in eggs in chum salmon (Oncorhynchus keta, Salmonidae) until hatching,and thereafter the amount in the egg gradually decreases, until completion of yolkabsorption (Tagawa and Hirano, 1987), whereas the amount of thyroxine remains moreor less constant throughout early development (Tagawa and Hirano, 1991). The onset ofendogenous thyroid hormone production usually occurs at or around the end of yolk sacresorption. There is conicting evidence linking the thyroid hormones and egg quality.Boosting thyroid hormone levels in sh eggs can enhance subsequent larval growth(striped sea bass, Morone saxatilis, Centrarchidae Brown et al., 1988, 1989).However a study by Tagawa and Hirano (1991), where eggs were made thyroidhormone-decient by treating female medaka (Oryzias latipes, Oryziidae), with thiourea which caused a reduction in egg hormone levels to a tenth of egg levels from femalesmaintained under normal conditions showed that there were no differences inhatchability, time of hatching or survival under starvation compared to eggs fromcontrol sh. Furthermore, there were no apparent differences between the two groups ofeggs after hatching, in the body weight, body length, condition factor and survival rateof the subsequent offspring (Tagawa and Hirano, 1991).

Cortisol is readily accumulated in growing sh oocytes and signicant amounts occurin fertilized eggs, embryos and larvae (De Jesus et al., 1991; Hwang et al., 1992;Contreras-Sanchez et al., 1995). The cortisol content of eggs, however, appears todecline rapidly between fertilization and hatching. Studies on the rainbow trout(Oncorhynchus mykiss, Salmonidae) indicate that at ovulation, as much as 90% of thecortisol contained within the egg may be eliminated during `water hardening' (Brookset al., 1995). Exposure of adult and juvenile sh to high concentrations of cortisol hasdeleterious effects (Carragher et al., 1989; Contreras-Sanchez et al., 1995); however, itis not known what the signicance of cortisol in eggs is, nor whether it affects eggquality. Studies on sh larvae indicate that cortisol may enhance the stimulatory actionof thyroid hormones (de Jesus et al., 1990), promote larval survival (in sea bass,Sampathkumar et al., 1993) and (in Mozambique tilapia, Tilapia mossambica,Cichlidae), stimulate larval growth (Lam, 1994).

Several sex steroids have been reported to be present in fertilized eggs of tilapia(Rothbard et al., 1987) as well as in eggs of salmonids (Feist et al., 1990). Thefunctional signicance of these hormones is unclear. If maternal transfer of testosteroneto the oocytes is enhanced, it leads to increased polyamine synthesis, and therefore,increased protein synthesis during embryonic development (Srivastava and Brown,1993). The egg content of sex steroids shows a rapid decline from fertilization tohatching (Feist et al., 1990), in a similar manner to that for cortisol, described above(Brooks et al., 1995). In birds, yolks of eggs may contain signicant amounts ofoestradiol-17, concentrations comparable to those in the maternal circulation (Adkins-Regan et al., 1995). Such maternal transport of a sex steroid to the egg yolk constitutesa potentially signicant source of maternal inuence over embryonic development andadult phenotype in oviparous species.

In mammals, tonically elevated levels of serum luteinizing hormone (LH) have beensuggested to affect oocyte quality and the development potential of the early embryo(Regan et al., 1990). Studies on humans have also demonstrated a decrease in oocytequality following ovarian stimulation with clomiphene citrate and human menopausalgonadotrophin (Fluker et al., 1993). Attempts to control the ovarian cycle withhormones in sh have met with mixed success. Induction of ovulation and spawning in


sea bream (S. auratus) with human chorionic gonadotrophin (hCG) resulted in eggs ofpoor quality (Gordin and Zohar, 1978). However, administration of GnRHa viacontrolled-release delivery systems, which stimulated long-term elevations of plasmaGtH II, have been successfully used to induce ovulation of captive white bass (Moronechrysops, Percichthyidae; Mylonas et al., 1996) and striped bass (Morone saxatilis;Hodson and Sullivan, 1993; Woods and Sullivan, 1993) without apparent detrimentalaffects on survival to hatching. Indeed, there have been numerous studies on articialinduction of spawning and its effects on egg quality in commercially important species.However, because of the very different hormone dosing regimes, the timing of thehormone injections=implantations and the subsequent variability in the egg quality(survival to hatching), interpreting the data is very difcult.

Egg size is bigger better?

In aquaculture, historically there has been the perception that, in terms of quality, biggereggs are better. Egg size may vary both within a species and between populations of thesame species (within the limits set by their genes: Beacham, 1982; Beacham and Murray,1985, 1987; West and Mason, 1987). Variations in egg weight between populations of cohosalmon (Oncorhynchus kisutch, Salmonidae), for instance, of comparable weights and age,may be up to 70%. Ecological explanations for differences in egg size of sh in differentpopulations include temporal and spatial changes in food particle size and in foodavailability to larvae, and predation. Age at maturity may also affect egg size in sh (Sargentet al., 1987), with a larger body size often resulting in the production of larger eggs(Bagenal, 1969; L'Abee Lund and Hindar, 1990; DeMartini, 1991). Egg size in sh is alsoaffected=modulated by the nutritional status of the female during ovarian recrudescence(Bromage et al., 1990; Tyler et al., 1994) and in asynchronous spawners, the size of eggsovulated in the later batches is often smaller, a phenomenon associated with the diminishingresources of the female (Hsiao et al., 1994). In Atlantic cod, their spawning strategyindicates that egg size takes priority over fecundity (Kjesbu et al., 1996). It has beensuggested that, in the wild, larger hatchlings that result from larger eggs, have a survivaladvantage during the rst few days of their lives, as they have larger yolk reserves (Blaxterand Hempel, 1963), may have higher growth rates (Moodie et al., 1989), are able to avoidpredators more effectively (Miller et al., 1988; Hinckley, 1990; Wootton, 1994), and eat awider variety of food items (Hunter, 1981; Webb and Weihs, 1986). Other authors, however,indicate that this is not necessarily so, arguing that larger offspring would be morenoticeable as prey (Kjesbu et al., 1996). Furthermore, as larger eggs often take longer tohatch than smaller eggs, they are at risk from predation or adverse abiotic conditions forlonger periods of time; the smaller eggs hatch earlier and the mobile larvae may then avoidadverse conditions (Miller et al., 1988). In cultured rainbow trout, egg size does not appearto be an important indicator of egg quality (Bromage et al., 1992; Brooks et al., unpublisheddata). Bromage et al. (1992) showed that larger eggs did produce larger fry, but this sizeadvantage was soon masked by other environmental determinants of growth.

Age of sh and egg quality

Studies on mammals have shown that reproductive age can affect egg quality (Koenigand Stormshak, 1993; Navot et al., 1994). For example, in pigs, the incidence of embryo


mortality in pubertal gilts (experiencing the rst oestrous cycle) is greater than that ofgilts mated after one or more oestrous cycles (Young and King, 1981; Archibong et al.,1992). Cytogenetic evaluation of ova from pubertal and third-oestrus gilts revealed thatgilts at rst oestrus ovulated a greater proportion of immature ova than gilts at thirdoestrus. In addition, the estimated frequency of meiotic nondisjunction was greater forgilts at rst oestrus than at third oestrus (Koenig and Stormshak, 1993). In older humans,the reverse is seen; there is a 50% decrease in human female fecundity from age 25 to35, which appears to be linked to a deterioration in oocyte quality (Navot et al., 1994).Studies of this nature in sh have not been reported, but our recent work in the rainbowtrout has indicated that over their rst two spawning seasons, females produce better-quality eggs in the second season (Brooks et al., unpublished data). Similarly, Bromageand Cumaranatunga (1988) found that the survival to eyeing of eggs from femalerainbow trout ovulating for the second time (as 3-year-olds) was signicantly highercompared with eggs from females spawning for the rst time (as 2-year-olds; 75% versus58% survival, respectively). A general improvement in egg quality, quantied as survivalto hatching, in successive spawning seasons has also been observed in European sea bass(D. labrax; Navas et al., 1995).

Environmental inuences on egg quality

Comparisons of wild and captive sheries show, consistently, that egg quality is higher inwild sh compared with that in captive stocks. As an example, studies on wild andcaptive Atlantic salmon (Salmo salar, Salmonidae) have shown that eggs from wildsalmon have up to 25% higher fertilization and hatching success, which is associatedwith greater size and survival of embryos and alevins, compared with their captivecounterparts (Srivastava and Brown, 1991). The superior quality of wild eggs overfarmed eggs is believed to be largely a function of environmental inuences.Environmental factors that may affect egg quality in sh include the diet of thebroodsh and the physiochemical conditions of the water in which the eggs are incubated(temperature, salinity and pH of the water, etc.). In aquaculture, the photoperiod to whichthe broodsh have been exposed and the quality of the husbandry factors such as thelevel of stress to which the broodstock are exposed, the fertilization procedures adopted,overripening of eggs in the body cavity and bacterial colonization of fertilized eggs can all affect egg quality (Fig. 3). In both wild and captive sheries, exposure ofmaturing females, or exposure of the eggs or developing embryos to envrionmentalpollutants may affect egg and fry survival (Smith and Cole, 1973; Mauck et al., 1978;Westin et al., 1985; van Leeuwen et al., 1986; Von Westernhagen et al., 1989; Matsui etal., 1992; Miller, 1993). More subtle features of the environment may also affectspawning. For example, in the ayu (Plecoglossus altivelis, Plecoglossidae), sensitivity tohormone signals is affected by the physical features of the spawning environment(Soyano et al., 1993). The known inuences of these environmental factors on eggquality are discussed in more detail below.

DIET

Difference in egg quality as a consequence of diet, especially the diet's lipid content, isone of the most researched aspects concerning egg quality and viability. Dietarycomponents as diverse as polar and non-polar lipids (Watanabe et al., 1991a,b), fatty


acids (Harel et al., 1994; Carrillo et al., 1995), protein (Washburn et al., 1990; Harel etal., 1995) and ascorbic acid (Dabrowski and Blom, 1994; Blom and Dabrowski, 1995)have all been shown to affect egg and embryo survival.

Egg quality was found to be improved in the European sea bass by altering the lipidcomposition of broodstock diet (Carrillo et al., 1995). Eggs considered to be of betterquality had a higher content of total n3 fatty acids, which included enhanced levels ofboth decosahexaenoic acid and eicosapentaenoic acid. In gilthead seabream, broodstockfed on a diet decient in n3 highly unsaturated fatty acids for a period of 10 daysshortly before spawning, produced eggs with a reduced viability. Further, it was shownthat the levels of n3 highly unsaturated fatty acids supplied in the diet were directlycorrelated with levels in both the polar and neutral fractions of egg lipid (Harel et al.,1994). In contrast to these studies, an analysis of the lipid and fatty acid composition ofAtlantic halibut eggs showed that batches of eggs with widely differing viabilities hadvery similar lipid compositions (Bruce et al., 1993).

Dietary proteins and carbohydrate also appear to inuence egg quality (Washburn etal., 1990; Harel et al., 1994, 1995), although these components have received less studythan the lipids. In sh, although carbohydrates are relatively poorly utilized, and themain sources of energy are protein and lipid (Walton and Cowey, 1982), in rainbowtrout, broodstock fed on a diet low in carbohydrate had a reduced relative fecundity(they produced fewer eggs per kg body weight), and the eggs had a reduced survival tothe eyeing stage and a reduced hatchability (Washburn et al., 1990). Proteins act as asource of amino acids and as a reservoir of materials used during the many biosyntheticactivities that are essential for the early stages of embryogenesis (Metcoff, 1986).Successful embryonic development in sh has been shown to be dependent on thebalance of amino acids present in the egg (Fyhn and Serigstad, 1987; Fyhn, 1989).

Most studies on diet and egg quality in sh have focused on the bulk dietarycomponents; that is, the proteins, fats and carbohydrates. There have been few studieson the `minor' dietary constituents. Some of the trace elements and vitamins have beenlinked with egg quality (Takeuchi et al., 1981; Hardy, 1983; Sandnes et al., 1984), butthere have been very few comprehensive studies on their importance. The most detailedstudies on trace dietary components have been conducted on ascorbic acid deposition inrainbow trout eggs (Dabrowski and Blom, 1994; Blom and Dabrowski, 1995). Theseauthors have shown that vitamin C is an essential nutrient, and that a deciency of thisvitamin in the diet results in eggs that show considerably higher mortalities than eggsfrom females fed on diets enriched with vitamin C. Watanabe et al. (1991a,b)demonstrated that red seabream (Pagrus major, Sparidae), which were fed oncommercial sh diets supplemented with phosphatidyl choline, astaxanthins andvitamin E shortly before spawning, produced eggs that had signicantly improvedviability and hatching rates. From these studies, the authors concluded that the principalfactors aiding red seabream reproduction were free radical scavengers, such as thesupplement they added to their diet.

There is little doubt that broodstock sh fed on `natural diets' often produce eggs ofbetter quality than those on formulated commercial diets, and sometimes thesedifferences are considerable. For example, studies in the gilthead seabream have shownthe sh fed on squid (which contains a very similar essential amino acid composition tothe main proteins in sea bream eggs) produced three times more viable eggs than shfed on a commercial, wheat-gluten-based diet (Harel et al., 1994). Unfortunately it is


not clear if egg quality was improved because of the amino acid composition of thediet or because of the other dietary constituents. Despite the considerable efforts thathave been directed towards unravelling the importance of dietary components indetermining egg quality, the evidence that diet can directly affect egg quality is verylimited; the different methods used to evaluate egg quality, and the different timings ofthe provision of the diets in the life of the sh, make it difcult to isolate any realimprovements that are a function of diet alone.

In formulating commercial diets, it would be benecial to look more closely at thenatural diet of the species in question, and what nutrients are contained within the eggsof that species. In the very few studies that have been carried out using this approach,there appear to be differences, sometimes substantial ones, in the nutrient compositionof eggs from different sh species. As an example, if we consider lipid in eggs, somesh eggs appear to be totally devoid of lipid droplets (Wallace and Selman, 1981),whereas in others, such as the gourami (Trichogaster cosby, Belontiidae), over one thirdof the weight of an oocyte may be wax ester (Kaitaranta and Ackman, 1981).Substantial differences in the patterns of utilization of the various lipid class byembryos and larvae have also been observed in different sh species (Ronnestad et al.,1994; Wiegand, 1996b). In summary, it appears that different sh species may havedifferent dietary requirements and that diets for broodstocks should be tailor-made toensure good egg quality. To assess the roles of the various dietary constituents on eggquality, however, requires experiments with large numbers of sh, extensive tank andhatchery facilities, and the studies are both labour and time intensive; there are veryfew research establishments that have these facilities.

PHOTOPERIOD AND PHYSIOCHEMICAL PROPERTIES OF THE WATER

It has been suggested that environmental factors such as photoperiod, temperature,salinity and pH of the water inuence egg quality (Carrillo et al., 1989; Bromage et al.,1992; Gillet, 1994; Brown et al., 1995). Photoperiod manipulation is commonly used inaquaculture as a method for advancing or delaying spawning, to obtain a year-roundsupply of eggs of salmonids and other sh species. Delaying spawning of pink salmon(Oncorhynchus gorbuscha, Salmonidae) by light manipulation led to increased eggmortality by the eyed stage from 5% (in the controls) to between 60% and 80%(Dabrowski and Blom, 1994). In contrast, Pohl-Branscheid and Holtz (1990) found onlyminor differences in egg quality estimated as percentage survival at the eyed stage infemale rainbow trout exposed to an articially compressed light regime that induced fourspawnings in 2 years; under a natural photoperiod, rainbow trout spawn once a year.Gillet (1994) found that delaying ovulation in the Arctic charr (Salvelinus alpinus,Salmonidae), by changing the photoperiod regime, improved egg quality. However, thiswas probably because the delay in ovulation meant that the eggs were ovulated when thewater was 2 8C colder than normal, which reduced over-ripening of the eggs. The effectof manipulating the rate of development of the ovary on the subsequent fertility and eggquality in non-salmonid species is also controversial (Girin and Devauchelle, 1978;Devauchelle, 1987; Carrillo et al., 1989; Gillet, 1994). Photoperiodic advancement ofovarian development in sea bass has resulted in slightly poorer egg survival in somestudies (Girin and Devauchelle, 1978; Devauchelle, 1987), but not in others (Carrillo etal., 1989). In summary, to what extent egg quality is affected by manipulating


photoperiod is not clear, but it may depend at what time of the year the advance=delay inspawning occurs.

Water temperatures during spawning and the incubation of the eggs are particularlyimportant in affecting egg quality. Temperature may affect metabolism, activity andstructure of the developing embryo (Kinne and Kinne, 1961). Brown et al. (1995)examined the effect of sea temperature on the spawning performance of Atlantic halibutover two spawning seasons, both in terms of egg morphology and viability of thesubsequent embryos. The viability of eggs from females kept at a constant temperatureof 6 8C was consistently higher than those from females maintained under ambient,uctating temperatures (Brown et al., 1995). In the wild, Atlantic halibut inhabit deepwaters that are characterized by fairly constant physical environmental conditions,including low, relatively stable temperatures (Haug, 1990). Similarly, in the Arctic charr,the viability of eggs from captive-reared females held under ambient temperatures waslower than that of wild-caught females. However, when the water in which the femalecharr were held was cooled to 5 8C where the sh spawn in the wild in Lake Geneva,the temperature does not exceed 6 8C the viability of eggs produced by captive-rearedfemales did not differ from that of eggs from wild females (Gillet, 1994). Thetemperature at which the eggs are incubated can affect not only their quality, but alsotheir growth rate and differentiation. Studies on salmonids have shown that bothvertebrae number and gill raker number have both a genetic and an environmentalcomponent, the environmental component being the temperature at which the eggs areincubated (Kubo, 1950; Kwain, 1975; Beacham and Murray, 1986). Thus, transfer ofdeveloping embryos to different temperature regimes can inuence the number ofmeristic elements formed (Ali and Lindsey, 1974; Fahy, 1976, 1983). In salmonids, thetemperature at which eggs are incubated has a signicant impact on survival and timeof hatching; excessively high or low incubation temperatures can induce substantialembryo mortality during early development stages. Different stages of embryodevelopment exhibit different thermal stabilities (Kinne and Kinne, 1961). In mostspecies of sh studied, it appears that there is a period of low thermal stability duringearly development (fertilization to gastrulation), followed by a phase of somewhatincreased stability and later, towards the end of embryonic development (heavy-pigmented-eye stage), there is a period of low stability again (Kinne and Kinne, 1961;Bailey and Evans, 1971). The basis of this susceptibility to temperature changes isunknown. It has been established, however, that profound changes in the pattern of geneexpression can be induced in cells and tissues by small temperature changes (Moreau etal., 1991). These changes in gene expression are likely to affect development.

Salinity may also affect egg quality, and is capable of modifying the physiologicaleffects of temperature on embryo development in both marine and brackish-water sh(Kinne and Kinne, 1961). For example, salinity affects the rate of embryo developmentin plaice (Pleuronectes platessa, Pleuronectidae; Berghahn and Karakiri, 1990).Hypertonicity can have major effects on embryo development and these changes mayresult from altered gene expression (Burg and Garcia-Perez, 1992).

POLLUTANTS

Fish oocytes and eggs are particularly sensitive to environmental pollutants. As anexample, sh have a 100-fold greater sensitivity compared with mammals to induction ofspecic mutations during oogenesis (Walker and Streisinger, 1983). There is considerable


experimental evidence that the quality of sh eggs can be adversely affected by pollution.Malformations and impaired development and viability may result from exposure to avariety of environmental contaminants, including insecticides (van Leeuwen et al., 1986),organochlorinated biphenyls (Smith and Cole, 1973) and polychlorinated biphenyls(Mauck et al., 1978; Matsui et al., 1992). Exposure to these pollutants, and theiraccumulation in the oocyte=egg, may occur when the oocytes are developing in the ovary(Miller, 1993), or subsequently, when the eggs are released into the aquatic environment.Gonads with high concentrations of chlorinated hydrocarbons yield eggs with lowerhatching success than less contaminated eggs (Westin et al., 1985; Von Westernhagen etal., 1989). Some persistant environmental chemicals, for example chlorinatedhydrocarbons, polychlorinated biphenols, and some known endocrine disrupters (e.g.DDT) are lipophilic and may be transported with lipid reserves into the developingoocyte (Knickmeyer and Steinhart, 1989; Miller and Amrhein, 1995; Ungerer andThomas, 1995). Rates of mortality and deformities become particularly pronounced whenlarvae from organochlorine-exposed females start to use their lipid reserves (Burdick etal., 1964). Bioconcentration factors for PCBs from the water column into oocytes may beup to 80 000 (Bruggeman et al., 1981). Studies on alligators, turtles and birds haveshown that exposure to oestrogenic xenobiotics (endocrine disruptors) during a specicperiod of embryonic development can affect not only embryo survival but sexualdevelopment too (Tyler et al., unpublished data). This is very likely to be the case fromembryonic development in sh also.

Reproduction in many commercial sheries takes place in waters that arecontaminated with pollution, for example, the coastal regions of the North Sea(Cameron and Berg, 1992). It has been proposed that this pollution of surface waters isa factor inuencing malformations and reduced viability in mackerel (Scomberscombrus, Scombridae) eggs off the Atlantic Coast of the US (Longwell and Hughes,1981; Longwell et al., 1992) and both cod and sprat (Sprattus sprattus, Clupeidae) eggsin the Baltic (Grauman and Sukhorukova, 1982).

HUSBANDRY OF CAPTIVE FISH

There is little doubt that poor husbandry results in poor reproductive success of culturedsh. It is surprising, therefore, that more attention has not been paid to the effects ofhusbandry practices on egg quality. Key husbandry factors that are likely to have majoreffects on egg quality are: to what extent the broodsh have been stressed, thefertilization practices adopted, egg over-ripening, and bacterial colonization of eggs(Bromage et al., 1992).

In captivity, connement and crowding of sh may affect egg quality. Both chronicconnement experienced during the nal stages of reproductive development, andperiods of acute stress, have been shown to disrupt the endocrinology underpinningnormal growth and development of the ovary in trout, and may result in signicantlylower progeny survival rates (Campbell et al., 1994). Stress can lead to irregularspawning intervals, low fertilization rates and increased occurrence of abnormalembryos in Atlantic cod (Kjesbu, 1989; Wilson et al., 1995). How stressing broodshleads to deleterious effects on egg quality has not been established.

Some sh, when reared in captivity, do not usually oviposit (release) their eggs; theeggs will then `age' or `over-ripen' within the body cavity. The time interval betweenovulation and fertilization will also affect the `ripeness' of the eggs. In rainbow trout,


there is approximately a one-week window for successful fertilization, in Atlantichalibut the window is 4 to 6 hours, but in tilapia, over-ripening of eggs occurs veryquickly and the window for successful fertilization is only an hour or so post-ovulation(Bromage et al., 1994). It is important, therefore, that broodsh are checked regularlyto ensure that they are stripped and the eggs are fertilized shortly after ovulation. Forboth cultured sh species that release (oviposit) their eggs, and for wild sh, the timeto fertilization will depend upon the presence of a mature male. Over-ripening of eggsis perhaps the most common reason for poor egg quality in captive broodsh.

After fertilization, dying or dead eggs become colonized with bacteria=fungus, and ifthese eggs are not removed quickly from the incubation trays, viable eggs may also becolonized. By incubating eggs in water of high quality, and by `picking' colonized eggsat regular intervals, egg survival (and, therefore, overall egg quality) can be enhancedconsiderably (Bromage et al., 1994).

Genetic inuences on egg quality

PARENTAL GENES

Fertility studies carried out both in humans and in domestic animals show that parentalgenetics can strongly inuence fecundity and egg quality (Ezra et al., 1992; Almeida andBolton, 1993). Work on humans has shown that a major cause of poor egg quality andinfertility is that ova are either immature or have chromosomal abnormalities (Almeidaand Bolton, 1993). Another proportion of women produce oocytes which are notimmature and do not appear morphologically abnormal, yet are in some way defective,perhaps because of defects in the zona pellucida or in the ooplasma (Ezra et al., 1992). Anumber of studies in sh also indicate that maternal genetics the DNA provided by thefemale may have signicant effects on egg quality (Reinitz et al., 1979; Brauhn andKincaid, 1982; Withler, 1987; Brooks et al., unpublished data). Preliminary data fromBrooks et al. (unpublished data) indicate that in a single population of rainbow trout,females that produce better-quality eggs in their rst spawning season year, do so in thesubsequent season, suggesting that there are genetic inuences on egg quality. However,genetic factors in sh that account for these differences have yet to be determined.

Studies on genetic inuences on egg quality need to take into account the `malefactor'; the complement of genes from the male will also affect embryo survival andtherefore, perceived `egg quality'. Designing experiments to separate the inuence onegg quality of the paternal genetic component from the maternal genetic component,however, is difcult. An experiment to examine the maternal genetic component wouldrequire that all the females in the study were fertilized with a single pool of male miltat the same point in time. In many sh species, however, in any one population,females do not all ovulate at exactly the same time; as an example, in the rainbowtrout, two females within a population may ovulate 6 or even 8 weeks apart. Similarly,separating the effects on egg quality of parental genetics and effects that are directlyattributable to the environment is also difcult. Some effects on egg quality that havebeen ascribed to parental genetics may, in fact, be a function of environmentalinuences. For example, different sh farms may report considerable differences in eggquality, and interpret this to mean that the basis for the variability was genetic females on some farms produce better eggs than females on other farms. This may notnecessarily be so, however, and the reported variability in egg quality could, equally, be


due to different husbandry practices on the different farms (i.e. an environmentalinuence). One possible experimental approach to assess the effects of parental geneticsalone on egg quality, removing variation as a function of the inuences of theimmediate environment, would be to place batches of fertilized eggs from the samefemales on different sh farms. The crucial elements in the design of such anexperiment are that each farm must be supplied with eggs from the same females andthat the eggs supplied from individual females to the different farms be derived fromthe same batch of eggs. The resulting variation in `egg quality' in eggs from anindividual female and the variation between the different females on the different farmswould make it possible to determine the genetic component of egg quality in that strainof sh. As with studies on the inuence of diet on egg quality, however, studies of thisnature require large numbers of sh, extensive hatchery facilities and are labourintensive.

GENETIC MARKERS FOR EGG QUALITY

Recently, it has been suggested that differences in the content of yolk proteins in sheggs may be used as a genetic `marker' for egg quality (sea bass; Carnevali et al.,unpublished data). This may not hold for all sh species, however, as preliminary studieson rainbow trout demonstrated that extracts of yolk from different eggs contained thesame major lipoprotein components, regardless of the subsequent survival of that batchof eggs. Furthermore, although there were differences in the phosphoprotein compositionof yolk extracts between batches of eggs, they did not appear to relate to egg quality(Brooks et al., unpublished data). Nevertheless, in Atlantic salmon alevins of differentparental origin, there do appear to be different pathways for processing yolk proteins andthese differences may be related to the proportion of the progeny surviving (Olin and Vonder Decken, 1990). These ndings have prompted work to look for markers of eggquality associated with the processing of yolk protein. Cathepsin D is the key enzyme inthe formation of yolk proteins, proteolytically cleaving vitellogenin into its componentpolypeptides and digesting the yolk proteins during the early stages of embryogenesis torelease free amino acids for the developing offspring (Retzek et al., 1992). Studies arenow underway in a number of teleosts, to isolate and sequence the cDNA for cathepsinD, with a view to its use as a potential genetic marker for egg quality.

CONTROL OF GENE EXPRESSION AND EGG QUALITY

RNA in oocytes may be derived from transcription of the oocyte's own genes orsynthesized by the mother during oogenesis and accumulated in the oocytes (reviews:Davidson, 1986; Amanai et al., 1994; Hales et al., 1994). The `maternal' RNA supportsoocyte protein synthesis during oogenesis and controls early embryonic development,until transcription of the embryonic RNA begins after fertilization (Kaani, 1973;Davidson, 1986). There is very little information on gene transcription and=or translationof mRNA in sh oocytes=embryos, despite the fact that the products synthesized and themechanisms controlling their expression are likely to play a central role in determiningegg quality. Indeed, the highest mortalities in sh embryos are often seen within severalweeks of hatching, at a time when many embryonic genes are being activated (Kimmel,1989).

In many animals, their early development within the egg is a result of changes in thepattern of protein synthesis via a change in the translational activity or stability of an


mRNA (Sheets et al., 1994). Translational control has been shown to be critical for avariety of developmental processes, including axis formation in Drosophila (Drieverand Nusslein-Volhard, 1988; Tautz and Pfeie, 1989) and sex determination inCaenorhabditis elegans (Ellis and Kimble, 1994). The actual mechanisms underlyingregulation of mRNAs encoding proteins involved in cell cycle regulation anddevelopment are virtually unknown in any animal. Experiments by various researchers(Gallie, 1991; Simon et al., 1992; Wickens, 1992; Sheets et al., 1994) have, however,demonstrated that increases in poly(A) tail length can activate translation, whereasremoval of poly(A) can prevent it. There is also evidence that mRNA translation isregulated by proteins associated with the maternal mRNA (Spirin, 1994). Thesemasking proteins bind to specic short sequence elements within the 39 untranslatedregion of the gene, acting as translational inhibitors until the proteins are modied ordestroyed at the appropriate stage during development, leading to normal translation ofthe previously dormant mRNA (Murray et al., 1992; Standart, 1992; Bouvet andWolffe, 1994).

Studies on gene transcription in sh oocytes have been initiated by Nagahama andco-workers, through the study of maturation promotion factor (MPF: Yamashita et al.,1992; Nagahama, 1994). MPF switches the developing oocyte from the rst meioticarrest into second meiotic division, and hence nal maturation. The activation of MPFin sh oocytes has been shown to be induced by maturation-inducing hormone throughthe de novo synthesis of protein initiators, which include cyclin B (Goetz, 1983; Maller,1985; in sh, MPF is a complex of cyclin B with cdc 2 kinase, Hirai et al., 1992).

In summary, little is known about transcription of the genes in sh oocytes or themechanism of regulation and translation of mRNAs. Studies on the role of maternalRNA during oogenesis and embryogenesis, and studies on how and when the messagesare masked and unmasked for their developmental expression in sh oocytes, eggs andembryos, have yet to be a focus for research. It is likely that hundreds, possiblythousands, of genes are transcribed and RNAs translated during embryo development.Some will be vital to development, whereas others will be less crucial. Inappropriateexpression of these genes, either in strength or timing, will likely lead to problemsduring embryogenesis, and hence to a `poor-quality egg'. Most, possibly all, of thefactors shown to affect egg quality will do so by inuencing gene expression and RNAtranslation in the egg. Studies of this nature on sh oocytes=eggs, therefore, are likelyto be of an increasing concern in unravelling what makes a `good-quality' egg.

Acknowledgement

Dr S. Brooks was supported by a grant from the European Union (AIR 2 93 1005).

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