Analysis of gene expression in female American lobsters (Homarus americanus) to determine reproductive status by oligonucleotide microarray analysis

Abstract

The American lobster (Homarus americanus) is harvested along the eastern seaboard of North America and forms the economic backbone of many small fishing communities in Atlantic Canada and the northeastern United States. The industry sets minimum size (carapace length) for harvesting American lobsters partially based on determining when females are sexually mature. Given the internal development of ovarian follicles (eggs) protected by a rigid exoskeleton, external assessment of sexual maturity is near impossible. Ovaries can be classified into 7 distinct developmental stages ranging from immature to mature based on a combination of factors including ovary colour, oocyte size, ovary factor, and time of year. Stage 4 ovaries are subdivided into stage 4a (developing) and 4b (mature) ovaries. Ovary stage 4b is important as it represents reproductive commitment. This means that once ovaries progress to stage 4b, they can no longer delay maturation and must continue through until extrusion of eggs onto the pleopods. To stage ovaries and accurately estimate the size at the onset of maturity, female American lobsters must be sacrificed to examine the ovary both grossly and histologically. The discovery of a non-lethal biomarker would result in more accurate ovary staging while allowing the return of the female to the spawning population. Several studies have examined specific reproductive endocrine processes in the American lobster, including the roles of vitellogenin, crustacean hyperglycemic hormone, and gonad-inhibiting hormone. However, no molecular approaches have been attempted to evaluate reproductive status in female American lobsters. This study was the first to examine genome-wide expression changes of reproductive female American lobsters using a novel 14,592 feature, spotted oligonucleotide microarray. The genetic information on the microarray was ~40% functionally annotated based on comparisons with publically available molecular databases. A reference design was used to allow stage specific comparisons between individual ovary stages. Four tissues (ovary, hepatopancreas, eyestalk, and haemocyte pellet) were pooled prior to cDNA synthesis to reduce reagents used and to capture gene expression data from these reproductively important tissues. Therefore, all results were total gene expression in the pooled samples. This study determined 1,774 genes to be statistically significant based on a one-way ANOVA, with 569 functionally annotated genes. The latter were involved in a variety of important biological processes including reproduction, development and growth. A total of 12 genes of interest were chosen for validation using reverse transcription quantitative polymerase chain reaction (RT-qPCR): four vitellogenin genes, two ovary development related proteins, egg-derived tyrosine phosphatase, estradiol-17-β-dehydrogenase 12-B-like, 97 kDa heat shock protein, quaking protein A, growth-hormone inducible transmembrane protein, and inhibitor of growth protein 1-like. Four genes represented distinct copies of vitellogenin, which is converted into vitellin, an important ovary/egg related protein involved in egg yolk. One of these genes was the complete Homarus americanus vitellogenin (HaVg1). A second gene, HaVg2, was 85% similar at a nucleotide level and 74% similar at a protein level with HaVg1. The third EST, given the name HaVg3, was 55% similar at nucleotide and protein levels with HaVg1. The final vitellogenin EST, named HaVg4, was 65% similar at a nucleotide level and 44% similar at a protein level with HaVg1. Three of these vitellogenins (HaVg2, HaVg3, and HaVg4) were unique to this study. The expression profiles of all 4 vitellogenins showed progression from downregulation at stage 2 to upregulation by stage 5, where stages 4a, 4b, and 5 are upregulated. This pattern was consistent between microarrays and RT-qPCR and agreed with previous literature in crustaceans. With the increased anthropogenic and environmental pressures on American lobster stocks, including harvesting and global climate change, this study has only begun to examine potential genetic variations in the American lobster. Future research should examine gene expression on an individual tissue level as genes are regulated on a tissue level. Only 32% of our statistically significant genes were functionally annotated. Further functional annotation may highlight genes associated with reproduction and commitment