Introduction

1. Introduction

In the forthcoming years one of the major issues facing Mankind will be food

security. It is hoped that the world population would be stabilized at around 11 billion

by the year 2050. A major challenge for the agriculture sector would be to double food

production by the year 2025 and triple it by the year 2050, under increasingly

challenging environmental conditions like lesser amount of land and water (Vasil,

1998). This will be further aggravated by the increasing deterioration of soil, water and

air quality, owing to global climatic changes, desertification, pollution and

industrialization, affecting land-based agricultural production. In many areas, in terms

of the output per hectare it has already reached maximal levels with the introduction of

fertilizers and selective breeding.

In the case of seafood production, the productivity needs to be increased

sevenfold by the year 2020 (Hew and Fletcher, 1997, 2001). Increased demands for

aquaculture production means increasing pressure for development of more efficient

production systems. Major improvements have already been achieved through better

management practices, in terms of nutrition, disease diagnostics and therapeutics, water

quality maintenance and genetic improvement of production traits. A common theme

running through all these diverse aspects is Genetics, which has actively been used to

meet many production challenges, such as enhanced feed conversion, disease

resistance, tolerance of handling and spawning manipulation, which are a few of the

requirements to which wild animals must adapt for productive "domestication".

Fish genetics programmes became more prevalent from the year 1900 onwards

with greater knowledge of breeding and quantitative inheritance. Genetic enhancement

programmes began in the 1960s and has continued to gain momentum from molecular

level that has been emerging since then. Efforts are now well established in areas of

traditional selective breeding, biotechnology and molecular genetics of fin fishes, and

the trend is rapidly growing for domestication of aquatic organisms for enhanced

overall production to meet the ever-increasing demand.

1.1. Indian major carps - An overview

India possesses nearly 11 percent of approximately 20,000 known species of

fish (Reddy, 1997), and is thus one of the rich nations with regard to genetic diversity

of fishes, distributed over a network of perennial rivers, lakes and reservoirs in the

country. Due to lack of emphasis on application of modern technology the average fish

production per hectare per year remained as low as 0.6 tonnes until the 1960s. In the

recent years the productivity has improved substantially with the initiation of better

husbandry practices. The· aquaculture systems in India constitute mainly Indian major

carps viz. Catla ( Catla catla, Hamilton), Rohu (Labeo rohita, Hamilton), Mrigal

( Cirrhinus mrigala, Hamilton) and occasionally Kalbasu (Labeo calbasu, Hamilton).

These carps contribute approximately 75 percent of the aquaculture production in India

(Reddy et al., 1997). As these carps are economically important, research on cultivating

these species was initiated in India during the early 1950s to study and understand their

biology to develop suitable technologies that enhance the aquaculture output in various

farming conditions.

Genetic work on Indian major carps was initiated in the late 1950s soon after

the success of induced breeding of these carps through hypophysation (Chaudhuri,

1959) with simple interspecific and intergeneric hybridization within the Indian carps

and later between these carps and the Chinese carps to increase the viability and

fertility of their hybrids. This was followed by cytogenetic work to study the

karyotypes of major carps and their hybrids on a comparative basis. During early 1980s

the modem method of genome manipulation (chromosomal engineering) was initiated

to induce and produce gynogens and polyploids so as to obtain homozygous inbred

lines and triploid sterile fish respectively.

Moreover, in India, majority of the water bodies disconnected from the river

systems are seasonal. Introduction of better husbandry practices and use of genetic

tools for the development of strains of Indian major carps that grow faster in such water

bodies will be a boon to aquaculture industry. Several attempts are being made in these

directions.

1.2. Husbandry practice

Polyculture is another strategy to enhance the output in an aquaculture system in

which all niches of water body is optimally used by the introduction of different species

of fishes in compatible combinations. The other strategy to increase aquaculture

production at a lower cost is composite culture in which several animals of agricultural

interest, like cow, pig, poultry are used such that the by-product of these organisms are

utilized by the organisms grown in aquaculture, in a manner that the feed cost is

reduced. Knowledge of nutrition, water quality management, detection of disease and

their prevention leads to increased production of Indian major carps in both polyculture

and composite culture. Both these approaches provide maximum utilization of a

variety of total niches and yield additional profit to the fish farmers.

1.3. Growth enhancement in Indian major carps

Various genetic approaches like hybridization, selective breeding, ploidy

manipulation have been adopted to enhance the aquaculture output of Indian major

carps with varying degrees of success.

1.3.1. Interspecific hybridization

Hybridization is referred to a cross between two species resulting in progeny

called hybrids that may be fertile or sterile. The hybrids are known to show better traits

than both the parent species, which is referred to as hybrid vigor. This simple

technique has been used extensively in fishes with varying degrees of success (Khan et

al., 1990; Reddy, 1997).

Natural occurrence of interspecific and intergeneric hybrids of Indian major

carps is reported mostly from reservoirs with intermediate morphometric traits to that

of their parent species. Occurrence of Rohu-Catla hybrids has been reported from

many perennial irrigation tanks of India. The natural hybrids of Rohu and Catla have a

general appearance of Catla with a conspicuously smaller head than Catla but with a

deeper body than Rohu (Natarajan et al., 1976; Majumdar and Ray-Chaudhuri, 1988).

The Rohu X Catla hybrid grows almost as fast as pure Catla, but has the small head of

the Rohu and is, therefore, useful in Indian aquaculture. Catla X L. fimbriatus hybrids

were reported to have small heads of L. fimbriatus and deep body and nearly equal

growth rate to that of Catla (Basavaraju, et al. 1995).

The intergeneric hybrids between common carp (Cyprinus carpio) and the

Indian major carps Catla (Catla catla), Rohu (Labeo rohita) and Mrigal (Cirrhinus

mrigala) are found to be triploid and sterile (Reddy et al., 1990a). Hybrids which

exhibit faster growth rate under monoculture and possess higher flesh content (Khan et

al., 1990) are produced in all the three combinations.

Hybridization between species can also result in offspring that are sterile or

have diminished reproductive capacity. As with monosex production, the production of

sterile hybrids can reduce unwanted reproduction or improve growth rate by energy

diversion from gametogenesis. Karyotype analysis can be used as a general predictor of

potential hybrid fertility. For example, hybrids of Indian major carps are generally

fertile and possess the same chromosome numbers (2N = 50). When crossed with

common carp (2N =100 or 102), the hybrids are triploid and sterile (Khan et al., 1990).

1.3.2. Selective breeding

Selective breeding has proved to be the most effective process for genetic

improvement of plants and animals. In aquaculture, ever since the success of induced

breeding of the Indian major carps through hypophysation (the technique is now

popular throughout the world), many hatcheries are using this technique regularly and

very effectively to meet the increasing demand of the hatchlings for aquaculture

purposes. Though the technique of hypophysation revolutionized the aquaculture

scenario, a survey of carp hatcheries in the state of Karnataka in southern India reveal

(Eknath and Doyle, 1990) the inbred nature of many hatchery fish stocks as evident

from their rapid deterioration of performance, but this observation could not be

supported by DNA finger printing analysis (Majumdar et al., 1997). Maheshwari and

Biradav (1998) reported decline in the fertility of hatchery bred Labeo rohita due to

inbreeding. A study conducted by the ICAR (Indian Council of Agricultural Research)

between 1982 and 1992 to assess fertility of Labeo rohita, revealed that inter breeding

of related individuals up to the sixth generation led to 71% decline in fertility levels

when compared to the ourbred population. It is also observed that the range, standard

deviation and coefficient of variation are highest for fecundity in the first generation.

This indicates that there is a scope for improvement through selective breeding (Reddy,

1997).

Selective breeding of Labeo rohita

The first ever project on selective breeding of Indian major carps was initiated

in the early 1990s, in India at the Central Institute of Freshwater Aquaculture,

Kausalyaganga (Bhubaneswar) in collaboration with the Institute of Aquaculture

Research (AKV AFORSK), Norway. Founder populations of Labeo rohita were

collected from five different rivers in north India, namely, Ganga, Gomati, Yamuna,

Sutlej and Brahmaputra, which are the native habitats of Rohu. Full-sibling groups

(families) were produced from all the stocks of Rohu and the subsequent communal

rearing in ponds under both mono and polyculture systems was done (Reddy et al.,

1998). Interestingly, the results revealed that growth and survival of the wild Rohu

stocks are equal to or better than the farmed stock. The selective breed of Rohu,

'Jayanthi' thus developed showed 30% more growth than the wild type. (Mahapatra, et

al., 2000).

1.3.3. Polyploidy

Fish provide a remarkable system for ploidy manipulation that can yield very

useful insights into the reproductive characteristics that are useful for commercial

aquaculture. In many fish species, triploid individuals are unable to reproduce due to

their inability to produce functional gametes. Triploid fishes are also known to exhibit

other traits, such as the differences seen between the female fish with decreased ability

to produce sex hormones and hence unable to reproduce although the triploid males

may develop secondary sexual characteristics and exhibit spawning behavior.

Triploidy can be induced either by temperature (low or high), pressure or chemical

(colchicine, cytochalasin etc) treatment soon after fertilization to block the extrusion of

second polar body. Tetraploid individuals can be produced by disrupting the first

mitotic cleavage using similar physical treatments and these animals produce diploid

gametes when crossed with normal diploid fishes would resulted in triploid individuals

(Reddy, 1997).

Induction of polyploidy in Indian major carps

Attempts have been made to induce triploidy and tetraploidy in Indian major

carps with varying degrees of success (Reddy et al., 1987; Reddy et al., 1990b). Reddy

et al. (1987), made the first attempt at inducing polyploidy in one of the Indian major

carps, Labeo rohita. By using colchicine they could only induce tetraploid and diploid

mosaics. However, Reddy et al. (1990) by using thermal shocks successfully induced

triploidy in, L. rohita, and tetraploidy in L. rohita and C. catla. Triploidy is induced in

Labeo rohita by giving heat shocks to zygotes (Reddy et al., 1990). Similarly

successful induction of tetraploidy was also reported in the case of Cirrhinus mrigala

and L. rohita (Zhang, 1990). Zhang (1990) induced tetraploidy in L. rohita and in C.

mrigala for the first time. However, the percentage of polyploids ranged from only 10-

40%. However, Islam et al. (1994) reported a 60% rate of induction of triploidy in L.

rohita.

1.4. Transgenics in Aquaculture

Compared to almost a thousand years of aquaculture and the genetic

improvement programmes, Aquaculture Genomics and gene mapping can be

considered to be truly in their infancy. However, the late 1990s have seen an explosion

in genomics and gene mapping of aquatic organisms. Many fish genes and regulatory

sequences have been identified and isolated, and the fish genome is now vastly better

understood (Kocher et al., 1998). The first reports of the application of modem

biotechnology to animals appeared in the 1980s. Genetic engineering enabled

researchers to insert novel genes into mice, rats, pigs, and fishes to achieve faster

growth rates, improved resistance to disease, and other desirable characters. In 1983,

mouse bearing novel genes that accelerated its growth rate was engineered (Palmiter et

al., 1983), which opened up a new area of applied research. Shortly thereafter,

scientists in China reported the first successful insertion of growth hormone genes into

fish (Zhu et al., 1985; 1986). It is now feasible to introduce foreign genes of interest

into the genome of fish by gene transfer technology. When the foreign gene is

integrated, and expressed, the transgenic fish acquires a new genotype, which can lead

to a changed phenotype depending on the nature of the gene introduced. Aquatic

animals, particularly fish, attract significant research attention for two primary reasons.

First, fish lay eggs in large quantities and these eggs are more easily manipulated,

making it easier for scientists to insert novel genes into the eggs of fish than into the

eggs of terrestrial livestock. Second, aquaculture is one of the fastest growing food­

producing sectors globally, suggesting a growing demand for increased fish production.

The Food and Agriculture Organization (FAO) of the United Nations in a report states

that close to 100 million metric tons of fish are consumed worldwide each year. Today,

fish is poised to become the first genetically modified animal grown for human food.

Researchers have genetically modified several varieties of carp, trout, salmon, channel

catfish, loach, tilapia, and pike for enhanced growth rates (Table 1) some of which are

awaiting commercialization.

Transgenesis, which is more effective than traditional breeding techniques, will

be equally beneficial in aquaculture to develop new fish strains (Hew and Fletcher,

1997). In principle, the technology can be used for (i) improvement in growth rate of

the fish; (ii) controlling sexual maturation, sterility and sex differentiation; (iii)

improving survival by increasing disease resistance against pathogens; (iv) improving

adaptation to extreme environments such as increased freeze-cold resistance; (v)

altering the biochemical characteristics of the flesh in order to enhance the nutritional

qualities. (vi) altering the biochemical or metabolic pathways to improve food

utilization. (vii) producing therapeutic proteins with pharmaceutical applications (Hew

and Fletcher, 2001; FAO, 2003). Though the technology involved in transgenesis is

sophisticated, it has advantage over classical selective breeding methods, which

depends on the life cycle of the individual species takes a longer time to complete.

Several factors determine the success of transgenic technology including the first step

of selecting the gene.

Fish species Promoter Source of GH gene Growth Reference enhance ment (fold increase)

Gold fish - Human GH gene - Zhu et al., 1985

Common carp mMT Human GH gene 1.1 Zhu et al., (1989) RSV Rainbow trout GH 1.2-1.4 Zhang et al., (1990)

eDNA Chen et al., (1993) Zebra fish - Rat growth hormone - Pimdian et al.,

(1991) Crucian carp mMT Human GH gene 1.7 Zhu, (l992) Pike RSV Bovine GH eDNA 0-1.12 Grosset al., (1992) Channel catfish RSV CohoGHcDNA 1.2 Dunham et al., (1992) Atiantic salmon op-AFP Saimon GH eDNA 3-10 Duet al., (1992b)

GH rninigene 3-10 Hew et al.,(1995) Loach mMT Human GH eDNA 2 Zhu et al., (1986)

op-AFP Chinook GH eDNA 2.5 Tsai et al., (1995) Tilapia op-AFP Chinook GH eDNA 2 Maclean et al., (1995)

Martinez et al., (1996) CMV Tilapia GH eDNA 1.81

Pacific salmon Sockeye MT Salmon GH gene 6-11 Devlin et al., (1994) Devlin et al., (1994)

Sockeye Salmon GH gene Devlin et al., (1995) histone op-AFP Salmon GH eDNA 3-10

Arctic charr CMV Salmon GH eDNA 14 Pitkanen et al., (1999) Salmon histone Pitkanen et al., (1999) Salmon Salmon GH eDNA 14 Pitkanen et al., (1999) metallothionein

Salmon GH eDNA 14 Heteropneustes RSV Rainbow trout growth - Sheela et al., (1999) fossil is hormone

Mud loach rnl-~actin rnl-GH gene 30 Nam et al., (2001) Labeo rohita Grass carp ~- Lab eo rohita GH - Venugopal et al.,

actin eDNA (2002a)

Table. 1. Different transgene constructs and growth enhancement in fishes. mMT-mouse metallothionein, RSV-Rous sarcoma virus, opAFP-ocean pout antifreeze protein, CMV­cytomegalo vius promoter, ml-~ actin-mud leach~ actin

1.4.1. Selection of genes

The genes that can provide superior traits, upon introduction into a desired

species has obviously gained significance in transgenic technology. These include

genes that code for growth hormone (enhanced growth rate), anti-freeze protein

(improved cold tolerance) and pathogen (bacterial/viral) resistance genes (increased

disease resistance) like lysozyme gene which acts against a broad range of pathogenic

bacteria.

Growth, being the single most important character governing success m

commercial aquaculture, is a strong target for transgenesis. The growth hormone (GH)

gene is thus an extremely feasible option, since exogenously administered GH is shown

to increase growth rate in fishes (McLean et al., 1990). GH is known to activate the

production of other cytokines like IGF-1 and IGF-ll, which are responsible for growth

enhancement.

1.5. Growth Hormone

Pituitary growth hormone (GH) or somatotropin, a single-chain polypeptide of

-22kD, is essential for normal growth, development and metabolism in all vertebrates

(Nicoll et al., 1999; Kaplan, 1999). With prolactin (PRL) and placental lactogen (PL),

it constitutes a family of structurally related proteins believed to have a common

ancestral origin (Moore et al., 1982; and Miller and Eberhardt, 1983) with overlapping

and related biological functions. GH has been conserved remarkably with respect to

amino acid content (Nicoll, 1987) among vertebrates. Apart from the primary role of

growth promotion (McLean and Donaldson, 1993), GH also has a role in

osmoregulatory action during seawater adaptation (Bolton et al., 1987), reproduction

(Van et al., 1990) and immune function (Calduch-Giner et al., 1997) in fishes.

The cellular action of GH (Fig. 1) is through its widely distributed growth

hormone receptor (GHR), especially in the liver where the GH-GHR interaction induce

the expression of another secretory peptide molecule i.e, insulin like growth factor I

(IGF-1) which in tum promotes growth in a variety of tissues (Jones and Clemmons,

1995). However, in fishes noticeable GH binding occurs in many other tissues like

testes, muscle, gill, adipose tissue, spleen, central nervous system and hemopoietic cells

(Perez-Sanchez, et al., 1991; Yao et al., 1991; Calduch-Giner et al., 1995). Though the

interaction of GH with a variety of cell types is demonstrated, the tissue specific

difference in GH response and the molecular mechanism of IGF-1 induction in the liver

is not known. The ligand GH interacts with its target receptor in monomeric form,

followed by receptor dimerization with the structural involvement of GH and a

subsequent signal transduction event which varies according to the cell type, is a

characteristic feature of all GHRs (Wang et al., 1995a). Similarly, the production of

GH from the pituitary is under the stringent control of hypothalamic and other

metabolic factors (Peter and Marchant, 1995).

1.5.1. Neuroendocrine regulation of growth hormone secretion

In vertebrates, the somatotroph cells of anterior pituitary gland constitute the

major site of GH expression. The neuroendocrine regulation of GH in fishes is

predominantly under the control of hypothalamus-pituitary-liver axis as reported for the

mammals. Various chemokines modulate the axis, which is schematically represented

in Fig. 1. The neuroendocrine regulation of GH secretion in gold fish and other carp is

multifactorial (Harvey, 1993), with a balance of stimulatory and inhibitory inputs to

somatotrophs.

Somatostatin-14 (somatotropin release inhibiting factor-SRIF-14), demonstrated

in the brain of several teleosts (Peter, 1986), is a potent inhibitor of GH secretion in

vivo (Cook and Peter, 1984) and in vitro (Marchant et al., 1987). Studies in goldfish

show that both norepinephrine and serotonin (5-HT) have inhibitory actions on GH

release in vitro (Peter, et al., 1990; Somoza and Peter, 1991; Wong, 1993).

Intraperitoneal injection of norepinephrine suppresses serum GH levels in goldfish

(Chang, et al., 1985). The actions of 5HT on suppression of GH release are dose­

dependent (Somoza and Peter, 1991), and both norepinephrine and 5HT suppress GH

release from goldfish pituitary cells in primary culture, indicating that the actions are

direct on somatotrophs (Wong, 1993). The inhibitory actions of norepinephrine are

similar to SRIF-14 as it can completely suppress the stimulatory actions of GnRH and

dopamine on GH in vitro (Wong, 1993).

GH secretion from the pituitary somatotrophs is stimulated by a variety of factors which

includes growth hormone release factor (GRF), Cholecystokinin (CCK), dopamine (DA),

gonadotropin releasing hormone (GnRH), Neuropeptide Y (NPY) and thyrotropin releasing

hormone (TRH) (for review, see Peter and Marchant, 1995).

9

Inhibition 1

Hypothalamus

GH

Liver

IGFI----....-

Connective tissue,

Muscle, Bone etc

Stimulation

Fig. 1. Schematic representation of neuroendocrine regulation of GH secretion in fishes. Abbreviations: IGF-1, insulin-like growth factor I; GH, growth hormone; DA, dopamine; NPY, neuropeptide Y; GnRH, gonadotropin releasing hormone; SRIF, somatostatin; GRF, GH releasing factor; TRH, thyrotropin releasing hormone; 5-HT, serotonin; NE, norepinephrine; CCK, cholecystokinin.

10

Growth hormone release factor (GRF) characterized from common carp

hypothalamic extracts is a 45 amino acid peptide (Vaughan et al., 1992) reported to be

a potent stimulator of GH release from gold fish pituitary following intra peritoneal

injection (Vaughan et al., 1992). Gonadotropin-releasing hormone (GnRH), seen in

mammals has also been demonstrated to have a stimulatory role in GH secretion in the

brain of gold fish (Yu et al., 1988) and common carp CAmano et al., 1992). Chang et al.

(199Gb) demonstrated that apomorphine, an agonist for dopamine (DA) receptor

stimulates GH release from the dispersed gold fish pituitary cells in culture, providing

direct evidence for dopamine stimulation of GH release, a unique discovery for the

vertebrate pituitary gland. Neuropeptide Y (NPY), a 36 amino acid peptide was first

demonstrated to be present in gold fish by immunological methods (Kah et al., 1989)

and has now been well characterized in other vertebrate systems. NPY is highly potent

in stimulating GH release from gold fish pituitary fragments (Peng et al., 1990).

Intraperitoneal injection of thyrotropin-releasing hormone (TRH) stimulates GH release

in gold fish (Cook and Peter, 1984), and common carp (Lin et al., 1993b) causing an

increase in serum GH levels. Cholecystokinin-8 (CCK-8) was also shown to be highly

effective in stimulating GH release from gold fish pituitary fragments in vitro (Himick,

et al., 1993).

1.5.2. Molecular components of the growth hormone-growth axis

The growth promoting action of exogenously administered GH in Cyprinids,

including the common carp (Fine et al., 1993) and other teleosts (McLean et al., 1990;

Sakata et al., 1993) is well established. However, there is not always a precise

relationship between blood GH levels and the rate of somatic growth (Merchant and

Peter, 1986; Merchant et al., 1986) and it is increasingly clear that a variety of

components within the GH-growth axis interact to determine growth rate in teleosts. At

tissue level, changes in the number and affinity of receptors for growth hormone (GHR)

have a profound influence on the sensitivity of target tissues to circulating GH.

Furthermore some of the actions of GH are mediated by insulin-like growth factor-!

(IGF-1), reported in all species from mammals to fishes. Other factors such as the

thyroid hormones, metabolic state, nutritional status, stress and environmental

variables, undoubtedly influence the endocrine-growth axis in teleosts (Eales, 1990;

Leatherland and Farbridge, 1992; Fine, et al., 1993; McLean and Donaldson, 1993).

11

1.6. Growth enhancement in fishes by growth hormone gene transfer

The ability to manipulate growth rates through the introduction of an additional

copy of growth hormone gene was demonstrated first in mice (Palmiter et al., 1982).

Such a method of transgenesis involving mammalian growth hormone gene constructs

did not enhance growth in the various fish species tried by different research groups

(Zhu et al., 1986; Enikolopov et al., 1989; Zhu, 1992; Gross et al., 1992; Wu et al.,

1994). Salmonids showed no effect (Guyomard et al., 1989a, 1989b; Penman et al.,

1991) despite the fact that they are very responsive to growth stimulation to

exogenously administered GH protein (McLean and Donaldson, 1993). Subsequent

gene constructs developed using fish GH sequences showed growth enhancement, to

the extent of doubling of body weight when compared with controls in carp, catfish,

zebrafish and tilapia (Zhang et al., 1990; Dunham et al., 1992; Duet al., 1992 Chen et

al., 1993; Zhao et al., 1993; Martinez et al., 1996;) providing convincing evidence that

growth enhancement in fish can be achieved by transgenesis. Dramatic growth

enhancement has been shown using this technique, especially in salmonids. 'All-fish'

gene construct consisting of ocean pout antifreeze protein (AFP) promoter fused to

Chinook salmon GH eDNA was injected into salmonid embryos. This particular

promoter was used as it was well characterized showing tissue specific expression

mostly in the liver, and lacking substantial seasonal variation in its activity. Moreover,

the transcription factors required for its activation appear to be present in a large variety

of fish species e.g. medaka, (Gong et al., 1991); salmon, (Shears et al., 1991; Devlin et

al., 1995); goldfish (Wang et al., 1995b; Tsai et al., 1995). Besides, in other teleosts

the presence of such a transgene, after manipulation can be easily detected through

simple PCR technique.

Transgenic adult salmon can grow to an average of 3-5 times the size of non­

transgenic controls, with some individuals reaching as much as 10-30 times the size of

the controls, especially during the first few months of their growth, (Du et al., 1992;

Devlin et al., 1994). Similarly, transgenic mud loach (Misgumus mizolepis) can attain a

body weight -30 times more than the sibling controls (Nam et al., 2001). Some of these

studies have also shown an increase in plasma GH levels, while the native pituitary GH

seems to have been down-regulated as a result of increased negative feedback, resulting

in smaller pituitaries and lower mRNA levels (Mori and Devlin, 1999). These fish

generally appeared healthy, and some produced second and third generation transgenic

offsprings (Saunders et al., 1998; Nam et al., 2001), showing that the enhanced growth

1')

phenotype is also inherited. The economic advantage of this kind of manipulation is

obvious and in comparison with selective breeding methods, the time frame is short for

achieving similar success rates.

When a gene is inserted into fish with the objective of improving a specific trait,

it may affect another trait. Transfer of growth hormone gene has been shown to affect

body composition, body shape, feed conversion efficiency, disease resistance,

reproduction, tolerance to low oxygen concentrations, carcass yield, swimming ability

and predator avoidance (reviewed in Dunham and Devlin, 1999; Dunham et al., 2000).

Such "pleiotropic" effects can be positive or negative, thus making it important to

evaluate all the important traits in transgenic fish other than the trait governed by the

trans gene.

1.6.1. Selection of promoters

Only a few well-characterized promoters are known from fishes. These include

metallothionein from trout and salmon (Zafarullah et al., 1988; Chan and Devlin,

1993), ~-actin from carp (Liu et al., 1990), histone from salmon (Chan and Devlin,

1993) and anti-freeze protein (AFP) genes from ocean pout (Hew et al., 1988; Du et

al., 1992), thus limiting the selection of promoters suitable for aquacultural purposes.

Compared to other promoters, the AFP promoters have several distinct features. First of

all, they are absent in most commercially important fish including salmon, carp, catfish

and tilapia, thus making its detection in the transgenic individuals by PCR simple and

at the same time, providing a useful marker for stock identification and proprietary

ownership. Furthermore, transient transfection studies in various cell lines (Chan et al.,

1997) and Japanese medaka embryos (Gong et al., 1991) as well as transgenic studies

in goldfish (Wang et al., 1995b), loach (Tsai et al., 1995) and salmon (Shears et al.,

1991; Du et al., 1992; Devlin et al., 1995) have further confirmed that the AFP

promoters are active in all fish species examined, suggesting that despite their limited

distribution, common transcription factors present in the transgenic hosts can actively

transcribe the AFP promoters. Unlike histone or ~-actin which are house-keeping

proteins or proteins with limited tissue specificity, the synthesis of the AFPs is either

liver-specific or liver-predominant and is thus more readily controlled. These promoters

also contain both positive and negative regulatory sequences. Lastly, there are at least

three types of AFP promoters with different degrees of tissue specificity and with

differing response to various signals. Thus, the AFP promoters offer a large repertoire

of candidates for transgenic studies. The disadvantage of these promoters is that it

cannot be used in tropical fishes as inducible promoter where the temperature of the

water does not go below 20°C.

1.6.2. Methods of gene transfer

Many methods of gene transfer developed for mammalian systems have been

applied to fish. The main method commonly used for the production of transgenic fish

is microinjection. However, electroporation, sperm-vector, gene-gun and liposome

mediated methods have also been shown to be effective in transferring DNA into

genome of the fish.

Microinjection

Microinjection is a well-established and accepted method of gene transfer in

fish (El-Badry, 1963; Proctor, 1992). The process of microinjection is straightforward

but technically tedious. Essentially, the process involves (i) collection of fertilized

embryos from natural spawning or after in vitro fertilization, (ii) holding the egg in

position using a specially designed apparatus called micro manipulator with an attached

microscope, (iii) injection of a very small volume of DNA solution into the embryo

using a glass micro needle and (iv) allowing the transgenic embryo to grow to adult

stage. The microinjection method of gene transfer has been successful as a tool for

transgenesis in a variety of fish species including common carp (Chen and Powers,

1990; Chen at al., 1991, 1992), gold fish and northern pike (Guise et al., 1992), medaka

(Chong and Vielkind, 1989; Ozato et al., 1992a, 1992b; Hong et al., 1993), sea bream

(Cavari et al., 1993), zebra fish (Moav et al., 1992; Khoo et al., 1993; Patil et al.,

1994), Atlantic salmon (Rokkones et al., 1985, 1989), rainbow trout (Penman et al.,

1990; Maclean et al., 1992), loach (Zhu et al., 1986; Zhu, 1992), mud loach (Nam et

al., 2001), arctic char (Shears et al., 1992), channel catfish (Dunham et al., 1992; Chen

et al., 1992) and tilapia (Rahman and Maclean, 1992; Maclean, 1993).

Unlike mammals, the egg nucleus is not visible in fertilized fish eggs. Thus the

best alternative is to introduce DNA as close to the pronuclei as possible.

Microinjecting through the micropyle (Szollosi and Billard, 1974; Kuchnow and Scott,

1977; Riehl, 1980) will achieve this objective since the pronuclei tend to lie below the

micropyle. Micropylar injection was successfully used in the production of transgenic

fish in salmonids (Shears et al., 1992) and tilapia (Brem et al., 1988; Rahman and

Maclean, 1992). Microinjection, though effective in fish transgenesis, is constrained by

a lot of limitations and is not ideal for large-scale gene transfer experiments. The

14

success of rnicroinjection depends to a large extent on the skill of the person carrying

out the procedure.

Electroporation

Electroporation is a mass gene transfer technique in which a large number of

eggs are simultaneously treated for transgenesis (Zhao et al., 1993). The technique

involves the use of sub lethal electrical pulses to permeabilize the egg cell membrane

thus allowing the entry of macromolecules into the egg cytoplasm. Essentially the

method is to place the fertilized eggs in a solution containing the DNA of interest in

between two electrodes, and to pulse them with electricity set at certain field strength.

The first successful gene transfer by electroporation was demonstrated in

medaka fertilized eggs (Inoue et al., 1990). Electroporation produced a greater number

of transgenic individuals than microinjection in zebrafish, common carp, channel

catfish (Powers et al., 1992a) and Indian catfish, Heteropneustes fossilis (Sheela et al.,

1999). Dechorionation of the eggs has improved DNA uptake after electroporation in

African catfish, zebrafish and rosy barb eggs (Muller et al., 1993). Electroporation of

spermatozoa was also efficient in the production of transgenic common carp, African

catfish, tilapia (Muller et al., 1992), chinook salmon (Sin et al., 1993, 1994) and Indian

major carps (Venugopal et al., 1998). The advantages of electroporation over

rnicroinjyction are that it is simple and allows treatment of many embryos

simultaneously. Moreover, electroporation being rapid allows the manipulation to be

completed before the first cleavage of the zygote.

Lipofection

Szelei et al. (1994) demonstrated liposome mediated gene transfer into African

catfish embryos. The eggs after fertilization are dechorionated by protease and the

embryo of two to four-cell stages are treated with liposome suspension pre-complexed

with DNA. Very efficient DNA uptake has been indicated by this method. Advantages

of this method include the relatively simple treatment procedure, extended shelf life of

liposomes and the ease of using large constructs of DNA and its use for treating large

number of eggs at a time. Disadvantages are the lengthy liposome preparation time,

lack of integration and problems associated with dechorionation of eggs leading to low

integration frequency.

Sperm-vector mediated gene transfer

Khoo (1992, 2000) demonstrated the successful sperm-mediated gene transfer

without electroporation in fish. This technique has previously been shown to be

effective in mouse (Lavitrano et al., 1989; Maione et al., 1998). Zebrafish spermatozoa,

after a simple incubation with plasmid DNA containing CAT reporter gene, was shown

to be effective as a vehicle to carry foreign DNA into the embryo (Khoo, 1992; 2000).

Simple incubation of DNA with sperm as an effective means of gene transfer is very

promising especially in aquaculture transgenesis where in vitro fertilization is routinely

practiced and highly successful. However, negative results are reported in the past by

using similar technique in common carp, African catfish and tilapia (Muller et al.,

1992). A plausible explanation for these discrepancies in the degree of transgenesis

could be due to variations in the DNA concentration used which could influence the

efficiency of gene transfer (Sin et al., 1994).

"Gene gun" mediated gene transfer

Another interesting mass gene transfer method is the micro projectile or "gene

gun" technique (Zelenin et al., 1991). Successful gene transfer in medaka (Yamauchi,

et al., 2000), rainbow trout (Lee et al., 2000) and zebra fish (Torgersen et al., 2000)

was obtained with high-velocity gold projectiles coated with DNA leading to transgenic

individuals. This method though simple and fast requires expensive equipment.

Embryonic stem cell mediated gene transfer

This is a powerful strategy for transgenesis. Cells are removed from the

developing blastocysts and are grown in culture in an undifferentiated state. Foreign

DNA of interest is introduced by electroporation or lipofection into the stem cells, and

these cells are then reintroduced into the blastocyst which are then allowed to develop.

If some of the cells having transgene in them get localized to form germ cells, then

subsequent breeding will produce transgenic individuals. Research in this area has

yielded some positive results (Collodi et al., 1992; Lin et al., 1992; Hagmann et al.,

1998; Hong et al., 2000; 2003; 2004) although more work needs to be done before it

can be an accepted method for gene transfer.

Retroviruses as tool for trans genesis

Retroviruses efficiently integrate their genetic materials into the genome of the

infected cells by precisely defined mechanism (Powers et al., 1992b). The advantage

of this system is that it allows integration of only a single copy of the provirus at a

given chromosomal site, and it does not induce rearrangement of the host genome

(Chong and Vielkind, 1989). Few reports (Burns et al., 1993; Lin et al., 1994; Chen et

al., 2002) deal with the effective usage of retroviral vectors developed for human gene

therapy for gene transfer in zebrafish cells and embryos. Very recently occurrence of

retroviruses have been reported in zebrafish (Shen and Steiner, 2004). Although the use

of retroviruses is very promising for the improvement of traits in commercial

aquaculture but since the species barrier for the retrovirus used in not discrete. The

usage of retroviral vectors may pose several consumer problems.

Nuclear localization sequence (NLS) mediated gene transfer

The efficiency of transgene integration is often lower due to (1) the introduced

DNA remaining as extra chromosomal, (2) degradation of the introduced DNA.

Besides, use of a large quantity of DNA can often be toxic to the egg resulting in poor

survival of the manipulated fishes. Collas et al. (1996) reported the first successful use

of NLS from SV40 T antigen in zebrafish transgenesis. Plasmid DNA encoding

luciferase gene when injected into the cytoplasm after complexing it with NLS was

shown to be transported into the nucleus, which was shown to express the protein. As

low as 103 copies of DNA when injected also showed integration. Thus NLS mediated

gene transfer can be used as an efficient tool for transgenesis especially in fishes where

egg-nucleus is not visible and is hence can be a major hurdle for microinjection.

1. 7. Problems associated with trans genesis

There are several advantages in using fishes as a model organism to study

transgene integration and expression (Pandian and Marian, 1994) when compared to

other vertebrates. First, external fertilization and development, of fish embryos allows

easy inspection and access to the developing embryo. Second, fish eggs are easy to

obtain in large quantities without any injury to the adult fish, and in some fishes like

zebra fish eggs can be obtained daily. Third, the embryos are hardy and can tolerate

repeated human handling. There are several bottlenecks in the production of transgenic

individuals in fishes, some of these are listed below.

1.7.1.Choice of the promoter

The expression of a transgene from a construct solely depends on the promoter

used. The criteria, which determine the promoter of choice, are its source, strength,

expressivity, size and ease of regulation and its size. Promoters are generally tissue

17

viral promoters are that they are small in size and express in a variety of animal types

including fishes. The only negative aspect for viral promoters is the consumer

nonacceptance due to an additional risk perception for human health especially since

even regular genetically modified organisms (GMO) are still not universally acceptable

for consumption as food. Other important promoters of choice include, metallothionein

promoter, which requires heavy metal induction in order to render the promoter active,

which again poses consumer problems. Another alternative is that of antifreeze protein

(AFP) promoter isolated from ocean pout (Hew et al., 1988; Chan and Devlin, 1993)

which is active in cold water fishes but reported to be active in tropical fishes like

medaka (Gong et al., 1991), goldfish (Wang et al., 1995b) and loach (Tsai et al., 1995)

under experimental conditions. Promoters like zona pellucida protein (Lyons et al.,

1993; Sheela et al., 1998; 1999), histone 3 (Connor et al., 1984; Chan and Devlin,

1993; Pitkanen et al., 1999), and ~ actin from carp (Liu et al., 1990) and mud loach

(Nam et al., 2001) have also been successfully used in fish transgenesis.

1.7.2.Choice of the gene

The structural gene can be classified into eDNA and chromosomal gene. In the

development of transgene constructs, the eDNA is an attractive option owing to its

overall small size, which enables simpler gene manipulation. The cloning of the eDNA

is simpler as it can be achieved by reverse transcription PCR (RT -PCR) approach

especially for genes like GH which has a very strong tissue specific expression in the

pituitary. On the other hand, chromosomal gene isolation is tedious and the size of the

gene mostly larger than its eDNA counterpart, which will make the manipulation

cumbersome. In spite of the difficulty in isolation, chromosomal gene has added

advantages like: amenability to isolation from a genomic library using heterologous

DNA probes, tissue specificity of the gene and the quantity of mRNA in a particular

tissue not becoming a limitation. Moreover, the suitability of chromosomal genes in

transgenesis is influenced by the presence of the full length gene along with the introns

which in turn influences its expression (reviewed by Hir et al., 2003), efficient

transcription and hence translation of the mRNA produced from the transgene (Garda

del Barco et al., 1994; Liu et al., 1995). Some reports of dramatic growth enhanced

18

phenotype is reported when the chromosomal gene is used in the transgene construct in

the case of mud loach (Nam et al., 2001) which substantiates the importance of

chromosomal gene as a prefered candidate for transgenesis.

1.7.3. Fate of the transgene in vivo

Studies have demonstrated that the transgenes upon introduction undergo

amplification by replication and degradation processes simultaneously (Pandian and

Marian, 1994). The presence of injected transgenes in supercoiled, open circular,

closed circular and multimeric forms has been detected throughout embryogenesis in

loach, goldfish, medaka and tilapia (eg- Winkler et al., 1991). Another most common

fate of the transgene is mosaicism in which the non-random distribution of the

transgene in various tissues is found. Spatial and temporal distribution of the transgene

has also been observed independently by two research groups (Stuart et al., 1988;

Winkler et al., 1991).

1.7.4. Expression of the transgene

The expression level of the desired protein derived from the transgene depends

on a variety of factors, including promoters used in the vector construct, number of

copies of the transgene in the target tissue and the source of the gene (homologous or

heterologous) (Pandian and Marian, 1994). In general viral promoters and other

promoters like the metallothionein promoter are highly active and even if the copy

number per cell of the transgene is low it can effectively bring about expression of the

desired phenotypic trait from the trans gene (Pan dian and Marian, 1994 ). If the

structural gene used in the trans gene construct is heterologous it can lead either to non­

responsiveness or to a drastic phenotypic alteration, such as lack of growth

enhancement seen when human GH transgenes are made in rabbits, pigs and sheep

(Hammer et al., 1985) or reduction in the number of eggs produced as seen in zebrafish

(Pandian et al., 1991) or even infertility as seen in the transgenic mouse bearing high

levels of human GH trans gene (Palrnitter et al., 1982).

1.7.5. 'All-Fish' constructs

In the 1990's however, a greater number of piscine genes as well as promoters

were sequenced which facilitated more specific transgenesis pertaining to a species.

The cloning of antifreeze protein promoter (AFP) from ocean pout (Hew et al., 1988;

Du et al., 1992), the ~-actin promoter from carp (Liu et al., 1990), histone 3,

metallothionein and protamine promoters from sockeye salmon (Chan ~md Devlin,

19

1993) allowed the production of an 'all-fish' construct. Besides the suitability of the

promoter of the construct, the success of transgenesis also depends on factors like the

structure of the gene and method of DNA delivery. Transgenesis experiments, where all

the components of the transgene are isolated from fish sources (Devlin et al., 1994).

'All-fish' trans gene constructs can also pose some difficulties like in the case of

expression of metallothioneins. These proteins can bind heavy metals in cells, in

particular cadmium, copper, zinc and mercury (Maclean and Penman, 1990)' and cause

adverse effects. Consumer problems arise especially when heavy metals are used to

induce such promoters. Problems can also arise in the case of other promoters like anti

freeze protein (AFP) promoter which when used in tropical fishes show less than

optimum biological activity when compared to its normal counter part. Hence a

feasible option is to develop expression vectors containing ubiquitously expressing

promoters like histone 3 or 0-actin, which combines small size and efficient

expressivity, often equivalent to viral promoters like the CMV promoter (Pitkanen ez

al., 1999).

1.7.6. Autotransgene and the use of chromosomal gene in transgene design

Considering the advantages and disadvantages of known transgenesi~

procedures, an ideal system in transgenesis would be to have an 'autotransgene'

construct. By definition an autotransgene construct should have the promoter

structural gene and the regulatory region in the transgene construct derived from the

same species (Beardmore, 1997). By the use of such a construct several problem~

associated with transgenesis may be eliminated. These include expressivity of the

transgene, consumer problems and undesired alteration of the host genome by th<

introduction of foreign genes/promoters. One published report deals with the

development of such an autotransgenic mud loach, Misgumus mizolepis having the Of

gene and the 0actin promoter both isolated from the same species and used ir

transgenic experiments (Nam et al., 2001). Some of the transgenic individuals havin~

autotransgene have been reported to show up to 35 fold increase in growth. Besides

Nam et al. (2004) also successfully developed a triploid sterile autotransgenic mu<

loach, which has a tremendous impact on the biological containment of these transgeni<

fishes.

Another important criterion in developing autotransgene construct is the use o

a chromosomal gene harboring the introns. This is because of many reports that dea

with the decisive role of the introns in transgene expression in contrast to the cDN.f

form of the same gene. There are reports which demonstrate that the transgenes

bearing introns in their structure transcribe 10-100 times more efficiently than their

eDNA counterparts (Brinster, et al., 1988) or eDNA constructs with the addition of a

single intron in the transgene as shown in mice (Palmiter et al., 1991). The

chromosomal gene thus has a lot of merits when compared to a construct with only _the

eDNA of the gene. Besides, the intronic sequence can base pair with its nuclear

counterpart due to the similarity in DNA sequence and length and can facilitate

homologous recombination. These factors point to the need for developing constructs

using the chromosomal gene and a constitutively expressing promoter from the sa~

species for effective 'autotransgenesis'. ~rft'-,:;.,' ,_( .··

1.8. Transgenesis: Indian scenario \:;> .:..)' In India, research on the development of transgenic fishes was initiate~~~£

Madurai Kamaraj University (MKU) by Prof. T.J. Pandian and colleagues. Such an

effort has resulted in transgenic zebrafish having rat growth hormone gene and a

mouse metallothionein promoter (Pandian et al., 1991). In this study, transgene

construct was introduced by microinjection into the cytoplasm of zebrafish eggs. After

injection, a survival percentage of 46% and genomic integration of 69% was reported.

The presence of a transgene construct was recorded inheritance up to F2 progeny,

though the gene and promoter used in this construct was not derived from fishes.

Because of the inherent problems associated with of microinjection in fish egg (tough

chorion, nonvisibility of pronuclei, shorter time before fertilization and first division of

the egg nucleus), Pandian and colleagues have attempted alternate gene transfer

technology methods like electroporation. Electroporation of eggs is often more

difficult, as one has to remove the chorion. The dechorionated eggs causes problems in

handling the eggs during electroporation. The number of eggs that can be

electroporated in the cuvette of the apparatus is also an added constraint. Pandian and

colleagues at MKU successfully overcame this hurdle by electroporating spermatozoa

used for transgenesis of Indian major carp species. They developed transgenic Indian

major carps for the first time using a transgene construct having a rainbow trout growth

hormone gene under the control of RSV promoter (pRSVrtGH) (Venugopal et al.,

1998). The presence of the transgene was proved by slot blot analysis of the DNA

isolated from the putative transgenic individuals. In another set of experiments, Sheela

et al. (1998) used GH gene isolated from rainbow trout as well as the yellow fin porgy

and placed them under the control of zona pellucida (Zp) gene promoter isolated from

21

winter flounder to obtain transgenic zebrafish. The use of a promoter (Zp) of pi seine

origin was expected to improve the transgene expression. This study was aimed also at

(i) determining the efficiency of electroporation as an alternative gene transfer method,

, (ii) evaluation of the Zp promoter in driving the transgene expression in vivo and

finally, (iii) the transmission of the transgene from transgenic founder population to

subsequent generations. The Zp promoter being active in the liver, and expressing

constitutively throughout the year without induction makes it an attractive choice for

use in fish transgenesis (Sheela et al., 1998). In their study, all the individuals from a

random analysis of surviving zebrafish transfected using one of the two constructs

(Zpj)ypGH and Zpj)rtGH) was found to be positive for the presence of transgene

possibly due to the stable integration facilitated by Zp homologous promoter (Sheela et

al., 1998). A F1 transmission frequency of 53% was shown for this transgenically

developed zebrafish, which was higher than the value reported for zebra fish (20%) by

Stuart et al. (1988). Pandian and colleagues have shown that the Zp promoter of winter

flounder used in their study was active in zebra fish by using a reporter gene lacZ,

which was cloned in-frame with the GH gene. This was the first report where

qualitative and quantitative expression of the putative transgenic individuals has been

described (Sheela et al., 1998). The GH gene expression under the control of Zp

promoter brought about an acceleration of growth rate in zebrafish an increase of the

order of 40% compared to the controls.

In a later study, Sheela et al. (1999) demonstrated the efficacy of the transgene

constructs Zpj)ypGH and Zpj)rtGH in the growth enhancement of Indian catfish

Heteropneustes fossilis. In this study, freshly fertilized eggs of the catfish were used

for electroporetic transfer of the trans genes. Founder population of the transgenic_

catfishes show a growth enhancement of 30-60% compared to the control group.

About 50% of the founder population was reported to have transmitted the transgene

into the F1 progeny (Sheela et al., 1999). Southern analysis of the DNA isolated from

the F1 transgenic catfishes at different time periods demonstrated the genomic

integration of the transgene (Sheela et al., 1999). In further studies on transgenic fish,

Anathy et al. (2001) expressed GH eDNA isolated from the Indian catfish,

Heteropneustes fossilis, under the control of viral regulatory elements like IRES in

zebrafish. This study also reports the cloning and expression of the fish GH protein in

E. coli.

Venugopal et al. (2002a, 2002b) isolated eDNA of growth hormone gene from

the pituitary glands of Indian major carps (Labeo rohita, Catla catla and Cirrhinus

mrigala) by RT-PCR, RACE and eDNA library screeing. They expressed GH eDNA

in E. coli and purified the GH protein to homogeneity. Bicistronic constructs having

GHcDNA and EGFP used for in vitro transcription of mRNA. The mRNA was then

injected into zebrafish embryos which showed expression of GFP "confirming the

cloned GH eDNA is functional in fish and the IRES element could be effectively used

in fish for bicistronic expression of foreign genes" (Venugopal et al., 2002a).

1.9. Requirements for a transgene in Indian context \

Literature cited above clearly indicate that to develop transgenic fish, emphasis

was given initially to standardize methodology especially the gene delivery system

irrespective of the transgene used for this purpose. Once successful, the efforts are now

been shifted to the construction of transgene, for which GH eDNA been successfully

isolated from several Indian major carps.

To obtain constitutively expressed GH gene in transgenesis,. the basic

requirement is the presence of a constitutive promoter and the transgene, which can act

as GH shunt as shown in the Fig. 2. The GH shunt works independently irrespective of

the pituitary-GH axis is active or not, as it contain promoter which is not under the

control of the GH and this transgene can express in all tissue provided the individuals

are not mosaic for the transgene. Besides, as mentioned earlier that in transgenesis,

where commercially important characters are to be manipulated, it is indeed preferred

to have 'autotransgene' construct for Indian situation. To obtain 'autotransgene' GH

gene construct, the basic requirements are the following: isolation of constitutive

promoter, isolation of GH gene (cDNNGH total) with 3' regulatory sequences. These

components can then be manipulated in such a manner that, the structural gene of the

construct express in vivo in transgenic individuals resulting in higher growth rate.

1.10. Objectives of the work

(i) Isolation and characterization of GH gene from genomic and pituitary sources of

Labeo rohita.

(ii) Comparative analysis of the GH gene in six species of the genus Labeo.

(iii) Expression and purification of the recombinant GH protein of L. rohita in E. coli.

(iv) Raising of antisera against recombinant GH protein.

(v) Cloning of constitutive promoters from L. rohita genome.

(vi) Development of autotransgene constructs with different combinations of the

promoters and growth hormone gene of L. rohita for transgenesis.

(vii)/n vitro analysis of the promoter activity and the expression of GH m cells

transfected with autotransgene.

(viii)Autotransgene delivery through electroporation of spermatozoa, leading to

transgenic L. rohita.

(ix) Studies on the integration of the autotransgene and its expression in transgenic L.

rohita.

24

Hypothalamus

GH SHUNT

Somatotrophs (Pituitary)

GH

Heart, intestine Kidney etc ..... ..,..

IGFI----...-

Inhibition 1 Connective tissue,

muscle and bone

Stimulation

Fig 2. Schematic representation of neuroendocrine regulation of GH secretion and GH shunt in an autotransgenic fish. Abbreviations: IGF-1, Insulin-like growth factor I; GH, Growth hormone; SRIF, Somatostatin;, GRF, GH releasing factor.