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Basis of sex determination and gonadal sex differentiation for sex control in aquaculture.

Objective

The project is an original contribution to sex control in fish. The objectives are threefold:

1.Identification of a fish Sex Determining factor (Sdf) by a reverse genetic approach.
2.Identification of genes involved in fish sex differentiation.
3.Transfert of knowledge in two important aquaculture species: sea bass and turbot.

This project will investigate the sex determining, cascade at the molecular level by, two complementary approaches:

First a Sex Determining factor (SDf will be searched in the platy fish as regions adjacent to the SDf have been already cloned and well characterised in that species. This will be performed through a walk on the Y chromosome of the platyfish.

Second, differentially expressed genes during gonadal sex differentiation will be searched using trout and tilapia monosex populations.

Finally, some of the most relevant -cries characterised by both approaches will be studied with respect to gonadal sex differentiation in a commercially important aquaculture species in which sex control is of particular importance and not yet achieved i.e. the sea bass. At the non-molecular level, sex determination will be analysed in turbot: balanced sex ratios are generally observed in turbot, suggesting a simple monofactorial sex determination. However, skewed male proportions could appear in some special cultured conditions, suggesting a possible involvement of other factors. The project plan to investigate a possible involvement of temperature in turbot sex differentiation by non-molecular approaches. In turbot, already available male and female breeders resulting from masculinizing and feminising. treatments will be used to analyse the genetic sex determination and a potential role of temperature on sex differentiation. Temperature/Genotype interactions will be studied in the tilapia model in which genetic sex determination and temperature influence on sex differentiation have been already demonstrated.

Sex control is almost always a concern in Aquaculture. Sex determination in vertebrates is considered as a switch mechanism, which determines the fate of the undifferentiated gonads through the activation of a cascade of sex determining genes (Mc Elvereavey et al., 1993). In mammals, sex is strictly dependent upon sex chromosomes : inheritance of a Y chromosome results in male differentiation. The sex-determining gene, SRY in man, has been cloned and proved to masculize XX transgenic female embryos in the mouse (Koopman et al., 1991). Some of the genes implicated in the early stages of gonadal differentiation have been shown to be sex linked. This is for instance the case for Sry and Zfy in mouse (Zwingman et al., 1993). Some other genes implicated in this cascade, have been shown to be conserved and expressed during sex differentiation in different classes of vertebrates i.e . Sox9 gene expression in differentiating testis of mouse and chicken (Kent et al., 1996). In fish the search for sex specific probes has been a challenge for many years now and despite many efforts this has been achieved in only a very few species: coho salmon (Forbes et al., 1994), chinook salmon (Devlin et al., 1991), Lake trout (Reed et al., 1995) and Leporinus elongatus (Nakayama et al., 1992). All these sequences do not show any homology with known genes involved in sex determining cascade. All approaches looking at already known sex specific sequences in others vertebrates have failed : repeat sequences, human HPRT gene, Bkm satellite, ZFY, SRY, polysat 3TM probe (Tiersch et al., 1992, Ferreiro et al., 1989, AIR2 -CT93-1543 project).

Our project will thus use a completely different strategy by investigating the sex determining cascade by two complementary approaches: First we will search for a Sex Determining factor (SDf) in the platyfish which is probably one of the best model for this investigation in non-mamalian vertebrates as regions adjacent to the SDf have been already cloned and well characterised in that species (Fornzler et al., 1996 ; Wittbrodt et al., 1989 ; 1992). This will be performed through a walk on the Y chromosome of the platyfish. Second, we will search for differentially expressed genes during gonadal sex differentiation. And finally, some of the most relevant genes characterised by both approaches will be studied with respect to gonadal sex differentiation in a commercially important aquaculture species in which sex control is of particular importance and not yet achieved i.e. the sea bass.
In the sea bass, genetic sex determination has not been understood by classic genetic analyses (AIR2 -CT93-1543 project), and in cultured conditions high male proportions are consistently found (Blazquez et al., 1995). On the contrary, balanced sex-ratios are generally observed in turbot, suggesting a simple monofactorial sex determination. However, skewed male proportions could appear in some special cultured conditions, suggesting a possible involvement of other factors. Temperature has been already shown to influence sex differentiation in tilapia (Baroiller et al., 1995). We thus plan to investigate this possible involvement of temperature in turbot sex differentiation by non-molecular approaches. In turbot, male and female breeders resulting from masculinizing and feminizing treatments are already available. They will be used to analyse the genetic sex determination and a potential role of temperature on sex differentiation in turbot. Temperature/Genotype interactions will be studied in the tilapia model in which genetic sex determination and temperature influence on sex differentiation have been already demonstrated.

These objectives will be facilitated by :
The use of suitable animal models:

- The platyfish (Xiphophorus maculatus) in which the Sex Determining (SD) gene is in a close vicinity with a specific pigmentation locus already cloned (Fornzler et al., 1996 ; Wittbrodt et al., 1989 ; 1992).
- A tilapia species (Oreochromis niloticus) in which genetic sex determination (XX/XY) and temperature influence on sex differentiation (TSD) have been already demonstrated (Mair et al., 1991).
- The rainbow trout (Oncorhynchus mykiss) in which there is a strict monofactorial sex determination, XX/XY (Johnstone et al., 1979).
In trout and tilapia, gonadal sex differentiation has been described and genetic male and female monosex populations are already available.

The use of two important commercial species :

Turbot and sea bass are two important commercial species for European aquaculture in which sex control is of particular importance (females grow better than males) and not yet achieved. They will be the target species of the project.

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Devlin et al., 1991. Can. J. Fish. Aquat. Sci., 48: 1606-1612.
Ferreiro et al., 1989. Aquaculture, 81: 245-251.
Fornzler et al., 1996. Genome Res. 6, 102-113,.
Forbes, et al., 1994. Proc. Natl. Acad. Sci. USA 91, 1628-1631.
Johnstone et al., 1979. Aquaculture, 18 : 13-19.
Kent et al., 1996. Development, 122: 2813-2822.
Koopman et al., 1991. Nature, 351 : 117-121.
Mair et al., 1991. Theor. Appl. Genet.
Mc Elvereavey et al., 1993. Proc. Natl. Acad. Sci., USA 90 : 3368-72.
Nakayama et al., 1994. Chromosoma 103, 31-39.
Reed et al., 1995.Chromosome Research, 3: 221-226.
Tiersch et al., 1992. Biology of Reproduction, 47: 185-192.
Wittbrodt et al., 1989. Nature 341, 415-421.
Wittbrodt, et al., 1992. EMBO J. 11, 4239-4246.
Zwingman et al., 1993. Proc. Natl. Acad. Sci. USA, 90: 814-817.

Coordinator

Centre de Cooperation International en Recherches Agronomiques pour le Developpement
Address
Campus International De Baillarguet
34032 Montpellier
France

Participants (5)

Institut National de la Recherche Agronomique
France
Address
Campus De Beaulieu, Av. Du General Leclerc
35042 Rennes
NATIONAL UNIVERSITY OF IRELAND, GALWAY
Ireland
Address
University Road
Galway
Noirmoutier Aquaculture Techniques Avancees
France
Address

85330 Noirmoutier-en-l'ile
Universitat Wurzburg Julius-Maximilians
Germany
Address
Am Hubland
97074 Wurzburg
University of Pisa
Italy
Address
Viale Delle Piagge 2
56124 Pisa