Browsing by Author "Mylonas, Constantinos C."
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- Alternative transcripts of DMRT1 in the European sea bass: expression during gonadal differentiationPublication . Deloffre, Laurence A. M.; Martins, Rute S. T.; Mylonas, Constantinos C.; Canario, Adelino V. M.DMRT1 is conserved from invertebrates to human and has been implicated in sex differentiation and testis function in many organisms.We report the cloning of two DMRT1 transcripts, DMRT1a and DMRT1b, encoded by a single gene in the European sea bass, Dicentrarchus labrax, a teleost fish with polygenic sex determination influenced by temperature. DMRT1a and DMRT1b are specific to the testis and are present as transcripts of 2.7 and 4.6 kb, as determined by Northern blot, although the cloned cDNAs are 1.2 and 1.9 kb. DMRT1a, encodes a protein of 306-amino acids, of similar size to what has been found in other teleosts, while the DMRT1b encodes a unique protein which differ from DMRT1a by having a 26 amino acids insertion which separates the S and Y domains. This insertion is the result of an extra exon (exon 4), present in the genomes of teleosts but not in other vertebrate genomes. Furthermore, unlike in the European sea bass, the putative product of DMRT1b in other teleost species is a truncated peptide. In European sea bass, the two transcripts are expressed at a similar level and have a similar expression profile in developing gonads. They are first detected by reverse transcription-polymerase chain reaction at 150 days post-hatch coinciding with the start of sex differentiation. After 200 days, expression increases in testis and decreases in ovary. Although it is not clear whether the two transcripts have differential roles it is suggested that both are required for testicular function.
- Analysis of the Sox gene family in the european sea bass (Dicentrarchus labrax)Publication . Galay-Burgos, M.; Llewellyn, Lynda; Mylonas, Constantinos C.; Canario, Adelino V. M.; Zanuy, Silvia; Sweeney, Glen E.Sox (SRY-related genes containing a HMG box) genes encode a family of transcription factors that are involved in a variety of developmental processes including sex determination. Twenty Sox genes are present in the genomes of humans and mice, but far less is known about the Sox gene family in other vertebrate types. We have obtained clones representing the HMG boxes of twelve Sox genes from European sea bass (Dicentrarchus labrax), a fish species whose farming is complicated by a heavily skewed sex ratio, with between 70% and 99% of offspring typically being male. The cloned Sox genes are members of the SoxB, SoxC, SoxE and SoxF groups. Sequence analysis shows that some of the clones represent genes duplicated in sea bass with respect to the mammalian Sox gene family.
- A cDNA for European sea bass (Dicentrachus labrax) 11 beta-hydroxylase: Gene expression during the thermosensitive period and gonadogenesisPublication . Socorro, S.; Martins, Rute S. T.; Deloffre, Laurence A. M.; Mylonas, Constantinos C.; Canario, Adelino V. M.Steroid P450 11 -hydroxylase, encoded by the CYP11B gene, is a key mitochondrial enzyme for the production of 11-oxygenated androgens, which have been shown to be potent masculinising steroids in several Wsh species. In this study we have isolated a CYP11B cDNA of 1903 base pairs from the testis of European sea bass (Dicentrarchus labrax) encoding a predicted protein of 552 amino acids. The amino acid identities to other vertebrate 11 -hydroxylase proteins ranged from 66% to 72% to other Wsh; 45% to amphibian and 35–39% to mammalian. Southern blot indicated that a single CYP11B gene is present. Northern blot analysis detected two transcripts in testis and head kidney, one of the same size of the isolated clone and the other of 3.9 kb. Reverse transcriptase-polymerase chain reaction showed abundant mRNA expression only in testis and head kidney, being residual in a range of other tissues. Expression of CYP11B and CYP19A (which encodes for ovarian aromatase) was detected from at least 4 days post-hatching and did not appear to be aVected by rearing temperature (15 and 20 °C) during the Wrst 60 days, a period in which high temperatures promote masculinisation in European sea bass. Throughout, gonadogenesis (60–300 dph), a highly dimorphic pattern of CYP11B expression was consistent with a role of this gene in testicular development.
- Developmental expression of DAX1 in the European sea bass, Dicentrarchus labrax: lack of evidence for sexual dimorphism during sex differentiationPublication . Martins, Rute S. T.; Deloffre, Laurence A. M.; Mylonas, Constantinos C.; Power, Deborah; Canario, Adelino V. M.DAX1 (NR0B1), a member of the nuclear receptors super family, has been shown to be involved in the genetic sex determination and in gonadal differentiation in several vertebrate species. In the aquaculture fish European sea bass, Dicentrarchus labrax, and in the generality of fish species, the mechanisms of sex determination and differentiation have not been elucidated. The present study aimed at characterizing the European DAX1 gene and its developmental expression at the mRNA level.
- A genetic linkage map of the hermaphrodite teleost fish Sparus aurata L.Publication . Franch, Rafaella; Louro, Bruno; Tsalavouta, Matina; Chatziplis, Dimitris; Tsigenopoulos, C.; Sarropoulou, Elena; Antonello, Jenny; Magoulas, Andonis; Mylonas, Constantinos C.; Babbucci, Massimiliano; Patarnello, T.; Power, Deborah; Kotoulas, Georgios; Bargelloni, LucaThe gilthead sea bream (Sparus aurata L.) is a marine fish of great importance for fisheries and aquaculture. It has also a peculiar sex-determination system, being a protandrous hermaphrodite. Here we report the construction of a first-generation genetic linkage map for S. aurata, based on 204 microsatellite markers. Twenty-six linkage groups (LG) were found. The total map length was 1241.9 cM. The ratio between sex-specific map lengths was 1:1.2 (male:female). Comparison with a preliminary radiation hybrid (RH) map reveals a good concordance, as all markers located in a single LG are located in a single RH group, except for Ad-25 and CId-31. Comparison with the Tetraodon nigroviridis genome revealed a considerable number of evolutionary conserved regions (ECRs) between the two species. The mean size of ECRs was 182 bp (sequence identity 60–90%). Forty-one ECRs have a known chromosomal location in the pufferfish genome. Despite the limited number of anchoring points, significant syntenic relationships were found. The linkage map presented here provides a robust comparative framework for QTL analysis in S. aurata and is a step toward the identification of genetic loci involved both in the determination of economically important traits and in the individual timing of sex reversal.
- Induction of ovulation and spawning in the Mediterranean red porgy, Pagrus pagrus, by controlled delivery and acute injection of GnRHaPublication . Kokokiris, L.; Canario, Adelino V. M.; Mylonas, Constantinos C.; Pavlidis, M.; Kentouri, M.; Divanach, P.Gonadotropin-releasing hormone analogue (GnRHa) in the form of saline injections or sustained-release microspheres was used to induce oocyte maturation, ovulation, and spawning in captive red porgy (Pagrus pagrus). Individually tagged vitellogenic females (n = 9 or 10) were treated at the beginning of the spawning season (March) with 20 μg/kg body weight (bw) GnRHa-loaded microspheres, a single injection of 20 μg/kg bw dissolved in saline, or physiological saline (control). Females were placed in tanks (one tank per treatment) connected to overflow egg collectors and monitored for 11 days. In addition to the eggs collected from the tank overflow, eggs were stripped from the fish on a daily basis. Only one spawn was obtained from the control fish, probably from a single female, given the small relative fecundity (700 eggs/kg bw). On the contrary, treatment with a GnRHa injection produced two spawns (9 and 11 days after treatment) and 50% of the fish ovulated. Treatment with GnRHa microspheres induced seven spawns (3 and 6-11 days after treatment) and 100% of the females ovulated. Females did not spawn all the eggs ovulated on a particular day, evident from the significant number of eggs obtained by manual stripping. Egg quality did not significantly differ among treatments, whereas number of spawned eggs and total relative fecundity were significantly higher in fish treated with GnRHa microspheres (ANOVA, p<0.05). The results demonstrate the potential of GnRHaloaded microspheres to induce spawning in red porgy, as a method of overcoming spawning failures in commercial hatcheries.