In crustaceans, circulating hormones influence many physiological processes. Two neuroendocrine organs, the sinus gland (SG) and the pericardial organ (PO), are the sources of many of these compounds. As a first step in determining the roles played by hemolymph-borne agents in the crab Cancer productus, we characterized the hormone complement of its SG and PO. We show via transmission electron microscopy that the nerve terminals making up each site possess dense-core and/or electron-lucent vesicles, suggesting diverse complements of bioactive molecules for both structures. By using immunohistochemistry, we show that small molecule transmitters, amines and peptides, are among the hormones present in these tissues, with many differentially distributed between the two sites (e.g., serotonin in the PO but not the SG). With several mass spectrometric (MS) methods, we identified many of the peptides responsible for the immunolabeling and surveyed the SG and PO for peptides for which no antibodies exist. By using MS, we characterized 39 known peptides [e.g., beta-pigment-dispersing hormone (beta-PDH), crustacean cardioactive peptide, and red pigment-concentrating hormone] and de novo sequenced 23 novel ones (e.g., a new beta-PDH isoform and the first B-type allatostatins identified from a non-insect species). Collectively, our results show that diverse and unique complements of hormones, including many previously unknown peptides, are present in the SG and PO of C. productus. Moreover, our study sets the stage for future biochemical and physiological studies of these molecules and ultimately the elucidation of the role(s) they play in hormonal control in C. productus.

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MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy.

Herein we report the first application of Fourier transform mass spectrometry for the analysis of neuropeptides directly from neuronal tissues. Sample preparation protocols and instrumentation conditions are developed to allow in situ neuropeptide analysis of the neuroendocrine organs freshly isolated from a marine organism Cancer borealis. The utility of a previously developed in-cell accumulation (ICA) technique is extended for peptide analysis in complex tissue samples. With the ICA procedure, ion signals from multiple laser shots are accumulated in the analyzer cell prior to detection. This procedure allows the accumulation of ion signals without accumulating noise, thus improving the signal-to-noise ratio and enhancing the sensitivity for the detection of trace-level endogenous neuropeptides. De novo sequencing of peptides directly from tissue samples becomes more feasible through this improvement. Additionally, an integrated pulse sequence is constructed to cover a wide mass range from m/z 215 to 9000 by centering quadrupole collection of ions at different masses for successive laser shots. Finally, improved mass measurement accuracy (2 ppm) for tissue peptide analysis is achieved using ICA by incorporating calibrants on a separate spot from the sample of interest without premixing calibration standards with the analytes.

Imaging mass spectrometry (IMS) of neuropeptides in crustacean neuronal tissues was performed on a MALDI-TOF/TOF instrument. Sample preparation protocols were developed for the sensitive detection of these highly complex endogenous signaling molecules. The neuromodulatory complements of the pericardial organ (PO) and brain of the Jonah crab, Cancer borealis, were mapped. Distributions of peptide isoforms belonging to 10 neuropeptide families were investigated using the IMS technique. Often, neuropeptides of high sequence homology were similarly located. However, two RFamide-family peptides and a truncated orcokinin peptide were mapped to locations distinct from other members of their respective families. Over 30 previously sequenced neuropeptides were identified based on mass measurement. For increased confidence of identification, select peptides were fragmented by post-source decay (PSD) and collisional-induced dissociation (CID). Collectively, this organ-level IMS study elucidates the spatial relationships between multiple neuropeptide isoforms of the same family as well as the relative distributions of neuropeptide families.