Gonadal Atresia, Estrogen-Responsive, and Apoptosis-Specific mRNA Expression in Marine Mussels from the East China Coast: A Preliminary Study.

This preliminary survey analysed mussel atresia incidences, estrogen-responsive and apoptotic-specific molecular end points, and aqueous and gonadal levels of selected estrogens from the East China coast. Estrogen levels were low (e.g. < LOD-28.36 ng/L, < LOD-3.88 ng/g wet weight of tissue for BPA) relative to worldwide freshwater environments, but high oocyte follicle atresia incidences (up to 26.6%) occurred at selected sites. Expression of estrogen-responsive ER2 was significantly increased in males relative to females at sites with high atresia incidences in females. A second estrogen-responsive gene, V9, was significantly increased at two sites in April in females relative to males; the opposite was true for the remaining two sites. Apoptosis-specific genes (Bcl-2, fas) showed elevated expression in males relative to females at the site with the highest atresia incidence. These results provide coastal estrogen levels and the utility of several estrogen-specific molecular-level markers for marine mussels.

Intersex related gene expression profiles in clams Scrobicularia plana: Molecular markers and environmental application.

Intersex, the appearance of female characteristics in male gonads, has been identified in several aquatic species. It is a widespread phenomenon in populations of the bivalve, Scrobicularia plana, from the southwest coast of the U.K. Genes previously identified as differentially expressed (ferritin, testicular haploid expressed gene, THEG, proliferating cell nuclear antigen, PCNA; receptor activated protein kinase C, RACK; cytochrome B, CYB; and cytochrome c oxidase 1, COX1) in intersex clams relative to normal male clams, were selected for characterisation and an environmental survey of the Channel region. Transcripts were significantly differentially expressed at sites with varying intersex incidence and contaminant burdens. Significant correlations between specific gene expressions, key contaminants and sampling locations have been identified, though no single gene was associated with intersex incidence. The results highlight the difficulty in understanding the intersex phenomenon in molluscs where there is still a lack of knowledge on the control of normal reproduction.

Intersex in Scrobicularia plana: transcriptomic analysis reveals novel genes involved in endocrine disruption.

Intersex, the appearance of female characteristics in male gonads, has been identified in a wide range of aquatic species worldwide, yet the underpinning molecular etiology remains uncharacterized. The presence of intersex has been shown to be a widespread phenomenon in bivalve, S. plana, populations from the southwest coast of the U.K., as well as inducible in an experimental exposure regime using endocrine disrupting compounds (EDCs). Herein, we use the suppressive subtractive hybridization approach to isolate differentially expressed transcripts in S. plana males exhibiting intersex. Transcripts involved in cell signaling, cell cycle control, energy production/metabolism, microtubule assembly, and sperm physiology are all highlighted as differentially expressed in intersex male clams. These provide both an insight into the molecular mechanisms of action involved in the development of intersex, as well as facilitating potential molecular-level "early warning" biomarkers of the condition.

Identification of reproduction-specific genes associated with maturation and estrogen exposure in a marine bivalve Mytilus edulis.

BACKGROUND: While it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline. METHODOLOGY/PRINCIPAL FINDINGS: Using suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences. CONCLUSIONS: The differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure.

Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure.

PURPOSE: The aim of this study is to develop a normalization method for real-time PCR data by analyzing the most stably expressed control genes in mussel (Mytilus edulis) reproductive tissue. METHODS: To facilitate this, six candidate genes, including several commonly used in the literature, were investigated in mussels at different stages of gametogenesis and following experimental exposure to a model estrogen (17b-estradiol). GeNorm and NormFinder softwares were employed to assess the stability of the reference genes. RESULTS: Our results demonstrate that the most stable reference genes are not the same in mussels at different stages of gametogenesis and in experimentally E2-exposed mussels. Interestingly, HEL (helicase) and ACT (actin) mRNA expression levels were most affected by the stage of gametogenesis and yet, in molluscan studies, ACT is possibly the most frequently used reference gene. CONCLUSIONS: We demonstrate that the experimental results are highly dependent on the reference gene chosen and that statistically significant contrasting differences between sample groups are present or absent depending on the reference gene employed.

Effects of estrogen exposure in mussels, Mytilus edulis, at different stages of gametogenesis.

Mytilus edulis were exposed to 17beta-estradiol (E2) and the synthetic estrogens ethinyl estradiol (EE2) and estradiol benzoate (EB) for 10 days. Two exposures were performed to determine their effect on vitellogenin (VTG) and estrogen receptor 2 (ER2) mRNA expression at different stages of the reproductive cycle. Significant natural variation was not observed in VTG mRNA expression, though ER2 mRNA expression displayed significantly lower values during January, February and July compared with other times of the year. A significant increase in VTG and ER2 mRNA expression was observed in mussels exposed to estrogens at the early stage of gametogenesis. In contrast, mature mussels displayed no statistically significant change in the VTG or ER2 mRNA expression. The data presented suggests that the reproductive physiology of molluscs, in terms of VTG and ER2 mRNA expression, may be susceptible to damage by environmental estrogens at certain points in their gametogenesis process.

Estrogens disrupt serotonin receptor and cyclooxygenase mRNA expression in the gonads of mussels (Mytilus edulis).

Estrogenic contaminants in the aquatic environment are associated with feminisation of male fish, however their effects on some invertebrate species, such as bivalve molluscs, have yet to be characterised. Gametogenesis represents a critical step in the reproductive process and is subjected to hormonal control by serotonin (5-HT), prostaglandins (synthesised by cyclooxygenases-COX) and steroids such as 17beta-estradiol (E2). Here, we examine the responses of 5-HT receptor and COX mRNA expression in mussels, Mytilus edulis, exposed to estrogenic compounds during different stages of their reproductive cycle. In mature mussels, 5-HT receptor mRNA expression decreased following E2 exposure. The opposite trend was observed in mussels at early gametogenesis stages. COX mRNA expression levels at both stages were generally decreased by E2 exposure. Mussels at early gametogenesis stages were also exposed to ethynylestradiol (EE2) and estradiol benzoate (EB) and a significant increase in 5-HT receptor mRNA expression was observed with both xeno-estrogens. COX expression levels were increased with EB exposure but no significant effects were found with EE2 exposure. These results show that the natural estrogen, E2, as well as the synthetic estrogen, EE2, induce alterations, dependent on reproductive stage, in the mRNA expression levels of 5-HT receptor and/or COX in the marine bivalve M. edulis.

The role of ras gene in the development of haemic neoplasia in Mytilus trossulus.

Disseminated neoplasia has been reported in mussels (Mytilus spp) from numerous locations worldwide. This condition is progressive and fatal and the aetiology is unknown. In vertebrates, oncogenes such as ras, and tumour suppressor genes such as p53, play important roles in carcinogenesis. We have cloned a Mytilus trossulus homologue of the vertebrate ras gene, which shows conserved sequence in regions of functional importance. Neoplastic hemolymph samples derived from M. trossulus have been investigated for the presence of ras gene mutations and changes in expression.

Conservation of cancer genes in the marine invertebrate Mytilus edulis.

Mussels are susceptible to a wide range of environmental toxicants, including carcinogens, and thus are often employed as bioindicator species. To elucidate the molecular aetiology of such neoplastic damage, we have cloned Mytilus edulis homologues of the vertebrate ras proto-oncogene, and p53 tumor suppressor gene. The M. edulis ras cDNA encodes a predicted protein of 184 amino acids. The DNA sequence analysis with vertebrate ras sequences demonstrates that the M. edulis ras cDNA is highly conserved in regions of functional importance, including mutational hot spots. The partial p53 sequence also demonstrates that M. edulis p53 is highly conserved in two regions of functional importance and that these regions also include four of the five mutational hot spots for this gene. In contrast, the M. edulis p53 sequence shows little similarity to the other published invertebrate p53-like sequences. The cancer gene sequences characterized herein will allow development of specific biomarkers of genotoxic damage.

Cadmium induction of metallothionein isoforms in juvenile and adult mussel (Mytilus edulis).

Isoforms of metallothionein in the digestive gland of control and experimentally Cd-exposed mussels (Mytilus edulis) (200 microg L(-1) Cd2+ and 400 microg L(-1) Cd2+; 20 days) were studied using the reverse transcriptase-polymerase chain reaction. In addition, glutathione S-transferase (GSTpi) primers were designed to evaluate the reduction in the antioxidant defense systems (glutathione) accompanying the aging process in the same organisms. Following experimental exposure, an accumulation of Cd was observed in the digestive gland of exposed mussels, both adults and juveniles, up to 500 times higher than in the control. An induction of the dimeric form MT20 II was detected in 400 microg L(-1) exposed mussels, as well as a visible inhibition of the monomeric form MT10 IV. After 20 days of exposure juveniles expressed increased GSTpi compared with adults. Results reveal individual variation of both metallothioneins and GSTpi expression among control and Cd2+-exposed mussels of different ages. The ecotoxicological significance of MT utilization in biomonitoring of seawater for trace metals has been considered in light of these results.