Tilapia (Oreochromis niloticus) vitellogenins: development of homologous and heterologous ELISAs and analysis of vitellogenin pathway through the ovarian follicle

Abstract

Vitellogenin (VTG) of Oreochromis niloticus was again purified, due to the conflicting results found in the literature. Three purification processes have been used: electrophoresis and electro-elution, double chromatography (gel filtration and ion-exchange chromatography) and single ion-exchange chromatography. Using SDS-PAGE we confirmed in all cases the presence of two polypeptidic forms of plasma VTG of 130 kDa (VTG1) and 170 kDa (VTG2). We raised polyclonal antibodies against each VTG form and we demonstrated the complete cross-reactivity of each antibody with both forms of VTG by Enzyme Immuno-Assay (EIA) and Western blots. The homologous ELISAs developed exhibited a detection limit of 6 ng x ml(-1), equivalent to 60 ng x ml(-1) of plasma VTG and allowed us to quantify the total plasma VTG of O. niloticus with high specificity and sensitivity. Using photonic and electron immunomicroscopy, we followed the pathway of VTG into the ovarian follicle (OF) demonstrating that VTG enters the oocyte at stage 3 of OF development, at the same time as cortical alveoli and lipid globules appear. Heterologous ELISAs performed on other cichlid species allowed us to quantify plasma VTG in Oreochromis aureus and Sarotherodon melanotheron and to detect it in Hemichromis fasciatus, Hemichromis bimaculatus and Tilapia zillii, constituting a reliable tool for monitoring the presence of xeno-estrogens in the environment of these fish species.

Towards a universal scale to assess sexual maturation and related life history traits in oviparous teleost fishes

“…Considering that in many species the cortical alveoli stage is considered to be a vitellogenic stage (Bazzoli and Godinho 1994;Gomes et al 2007) endogenously produced (Selman and Wallace 1989;Tyler and Sumpter 1996), the term ''endogenous vitellogenesis'' may be confusing, as cortical alveoli are not vitellogenic substances. Our observations in Tilapia (Oreochromis niloticus) have established the concomitance of both endogenous (oil droplets) and exogenous vitellogenesis (yolk granules and globules) (Ndiaye et al 2006), which seems to be a general pattern of marine and freshwater fish oocytes (Selman and Wallace 1989;Le Menn et al 2000). As previously mentioned, the physiological state of a previtellogenic and vitellogenic female are very different, and cortical alveoli and lipid droplets are clearly visible during this stage when observed by photonic microscopy (Selman and Wallace 1989).…”

“…The scale presented here is based on the synthesis of data from previous and ongoing studies on several fish species pertaining to some of the most important African (Nunez Rodriguez et al 1995;Duponchelle and Panfili 1998;Otemé et al 1996;Duponchelle et al 1999;Duponchelle and Legendre 2001;Duponchelle and Ribbink 2000), European Mañanos et al 1997;Le Menn et al 2000;Ndiaye et al 2006) and Neotropical taxa Muñoz et al 2006;Nuñez et al 2006a, b;Duponchelle et al 2007), including the Characiformes, Siluriformes, Osteoglossiformes and Perciformes. Nevertheless, the illustrations presented in this study will concern only Amazonian species for which histological or macroscopical illustrations have never been published.…”

Gender determination in the Paiche or Pirarucu (Arapaima gigas) using plasma vitellogenin, 17β-estradiol, and 11-ketotestosterone levels

“…The Paiche possesses two vitellogenin molecules (184 and 112 kDa apparent molecular masses), as observed in tilapia species: O. niloticus (Buerano et al 1995;Ndiaye et al 2006), O. mossambicus (Kishida and Specker 1993) and O. aureus (Ding et al 1989). The number of Vtg molecules varies in teleosts as a consequence of different conservation patterns of Vtg genes during evolutionary processes (Ndiaye et al 2006). From the two Vtg molecules, two different antibodies were raised and since both showed heterologous Vtg recognition, only anti-Vtg 1 was used in this study since it gave the best response/background ratio.…”

“…For this study, we chose an electrophoresis and electro-elution technique which is an efficient and more rapid technique that we previously used for the purification of African catfishes Vtg (Nuñez Rodriguez et al 1995). The Paiche possesses two vitellogenin molecules (184 and 112 kDa apparent molecular masses), as observed in tilapia species: O. niloticus (Buerano et al 1995;Ndiaye et al 2006), O. mossambicus (Kishida and Specker 1993) and O. aureus (Ding et al 1989). The number of Vtg molecules varies in teleosts as a consequence of different conservation patterns of Vtg genes during evolutionary processes (Ndiaye et al 2006).…”

“…Vtg, a pubertal female specific glycolipophosphoprotein, is correlated to oocyte development in sexually mature fish during the reproductive cycle (Nuñez Rodriguez et al 1989;Kishida et al 1992;Kishida and Specker 1993;Mañanos et al 1994a;Mourot and Le Bail 1995;Nuñez Rodriguez et al 1995;Sherry et al 1999;Nilsen et al 2004;Ndiaye et al 2006). A non-invasive sexing technique for A. gigas using both the Vtg detection in maturing females by enzyme imuno assay (EIA) and the sexual steroids technique based on 17b-estradiol (E 2 ) and 11-ketotestosterone (11KT) plasma levels in mature and immature individuals of both sexes was then developed.…”