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Copy of Intersex in Scrobicularia plana

new transcriptomes idetified in intersexed males

Corina Ciocan

on 23 November 2012

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Transcript of Copy of Intersex in Scrobicularia plana

In general, when we talk about sex determination, we involve 3 players.

1. Environmental sex determination – in species in which a direct association between specific environmental conditions (temperature, nutrients) and off spring sex has been observed.
2. Genetic sex determination the embryo sex is regulated by genes transferred by its parents during conception. This indicated a pre-existent commitment to a specific sex, which cannot normally be reversed later on.
3. Sex steroids control – varies from reptiles, where regulation of aromatase expression is the central phenomenon leading to sex determination; to mammals where sex steroids are only involved in subsequent sex differentiation. Molecular cloning and characterization of several sex related genes in the bivalve Scrobicularia plana (Veneroida) Sex determination and differentiation Merci pour votre patience et hospitalite!!! Different from vertebrates, sex determination and differentiation in marine bivalves depend less on genetic factors and more on hormones and environmental factors.

But what exactly do we know about sex determination?
Sex determination is an intriguing field of study, with various open questions and challenges. Several mechanisms of sex determination in molluscs are reported including
-a mammalian-like X/Y system in selected gastropods and surf clam Mulinia lateralis (Vitturi ei al., 1998; Guo et al., 1994)
- an X/O system in Littorina snails (Vitturi et al., 1988)
- a Drosophila-like mechanism of X/autosome balance in the clam Mya arenaria (Allen et al., 1986)
- a dominant allele for maleness and another for femaleness in C. gigas, also FoxL2 and dpy30 have been recently decribed (Guo et al., 1998; Dhelly et al., 2012)
- and a system where maternal nuclear genotypes influence gender in Mytiloidae and Veneroidae (Kenchington et al., 2002) Dr. Corina Ciocan
University of Sussex UK Additionally, the doubly uniparental mtDNA inheritance system (DUI) has been reported, whereby females pass on their mtDNA to all offspring, but males also inherit a second mt genome from their fathers.
In animal species, mtDNA is exclusively maternally inherited.
Mitochondria are multifunctional,
DNA bearing organelles found in eukaryotic cells.
Animal mtDNA normally encodes around 37 genes.

As an extreme deviation from this general rule, the double uniparental inheritance (DUI) delivers homoplasmic females (for the F genome) and heteroplasmic males (for both F and M genomes). As a results selection acts directly on the male mitochondrial genome!!! There are around 9,000 species of bivalves; for more than half of them the sexual cycle is unknown or unreported. From the other half, 96% are considered gonochoristic (separate sexes), 4% are hermaphrodites.
Veneroida family is the largest of the marine bivalves Intersex, the apparent abnormal appearance of female characteristics in male gonads, has been identified in a wide range of aquatic species worldwide.
In Scrobicularia plana it is characterised histologically by the occurrence of oocytes among normal testicular tissue (ovotestes).

Feminisation of males has thus become the suggested process, yet the precise underpinning molecular level etiology of the intersex condition, in any aquatic organism including fish and molluscs, still remains unclear.
Several causes of intersex have been suggested and include various factors,
such as genetic abnormality, parasites and water pollution. The presence of intersex has been shown to be a widespread phenomenon in aquatic organisms including fish, crustaceans and molluscs.
Focussing on molluscs, intersex has been reported in several species worldwide, including Ruditapes sp., Plaxiphora aurata, Mytilus galloprovincialis and Scrobicularia plana . Project Interreg IV DIESE 4040 WP2: Identification of mechanisms
University of Sussex team
(Dr. Jeanette Rotchell, Dr. Corina Ciocan, Dr. Elena Cubero-Leon,
Dr. Mika Peck)

Objective A: Analysis of normal and toxicant-induced genomes
Objective B: Gene isolation, informed by metabolomic result, and qPCR assay

Our study focuses on :
1) Investigation of any potential disruption of the reproductive cycle in
Mytilus edulis following exposure to estrogenic compounds;
2) Highlight cell pathway involved in the feminization of male clams
(Scrobicularia plana) sampled from natural populations;
3) Targeting key genes involved in endocrine disruption processes to use
as environmental monitoring analytical tools UK populations of the common estuarine bivalve Scrobicularia plana are considered to be inherently gonochoristic and their extensive distribution, sedentary lifestyle and deposit feeding habit make S. plana a potentially valuable monitoring species. Intersex in Scrobicularia gonad – photo kindly provided
by Dr. Langston, MBA. Varying degrees in the severity of ovotestis in male S. Plana have been reported.
The condition has been linked to endocrine disruptors present in the environment and has been induced in controlled experiments (Langston et al, 2006). The SSH procedure was used to isolate and enrich for genes differentially-expressed between normal male and intersex clams. Equal amounts of RNA (Nucleospin RNA II Macherey-Nagel, U.K.) were pooled from each group (10 clams/group). Forward- and reverse-subtracted libraries were produced using PCR-Select cDNA Subtraction reagents (Clontech, France). Transcripts generated by SSH were inserted in pCRr2.1 vectors transformed into competent TOP10 E.coli (Invitrogen). Target mRNAs, identified using SSH, were selected for quantitative RT-PCR. A normalization factor, calculated using geNorm software and based on the expression levels of the most stable reference mRNAs (actin and 18S) was used for accurate normalization of real-time PCR data. All statistical analyses were carried out using SPSS Inc. Chicago, U.S.A. (version 17.0). All data were tested for normality and homogeneity of variances. For non normally distributed data non-parametric Mann-Whitney U comparison tests were performed to compare the means. Statistical significance was accepted at p<0.05. Animals. S. plana (34-43 mm, n=10 for each of normal male and intersex clam) were sampled from locations on the South West coast of England between June-July 2009. Clams were depurated in 50% seawater for 2 days (to eliminate sediment and grit), before the gonads were excised. A small sample of gonad from each clam was examined under a light microscope to determine sex and reproductive development stage. Chi-squared test was performed on sex data to reveal any significant departure from a normal male: female ratio for clams samples at each location. A piece of gonad (approximately 20 mg) from each clam was fixed in RNALater (Qiagen Ltd., Crawley, U.K.) for later molecular analyses. Materials and methods Beta Tubulin Αlpha Tubulin PCNA + p53 - DNA replication
PCNA + p53 + DNA repair
PCNA - apoptosis Raport DIESE 2012 Determination of pertinant indicators for environmental monitoring - a strategy for Europe
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