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Bivalve biomineralization is a highly complex and organized process, involving several molecular components identified in adults and larval stages. However, information is still scarce on the ontogeny of the organic matrix before calcification occurs. In this work, first shell formation was investigated in the mussel Mytilus galloprovincialis. The time course of organic matrix and CaCO3 deposition were followed at close times post fertilization (24, 26, 29, 32, 48 h) by calcofluor and calcein staining, respectively. Both components showed an exponential trend in growth, with a delay between organic matrix and CaCO3 deposition. mRNA levels of genes involved in matrix deposition (chitin synthase; tyrosinase- TYR) and calcification (carbonic anhydrase; extrapallial protein) were quantified by qPCR at 24 and 48 hours post fertilization (hpf) with respect to eggs. All transcripts were upregulated across early development, with TYR showing highest mRNA levels from 24 hpf. TYR transcripts were closely associated with matrix deposition as shown by in situ hybridization. The involvement of tyrosinase activity was supported by data obtained with the enzyme inhibitor N-phenylthiourea. Our results underline the pivotal role of shell matrix in driving first CaCO3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis. [ABSTRACT FROM AUTHOR]

Coastal marine ecosystems experience dynamic fluctuations in seawater carbonate chemistry. The importance of this variation in the context of ocean acidification requires knowing what aspect of variability biological processes respond to. We conducted four experiments (ranging from 3 to 22 days) with different variability regimes (pHT 7.4-8.1) assessing the impact of diel fluctuations in carbonate chemistry on the early development of the mussel Mytilus galloprovincialis. Larval shell growth was consistently correlated to mean exposures, regardless of variability regimes, indicating that calcification responds instantaneously to seawater chemistry. Larval development was impacted by timing of exposure, revealing sensitivity of two developmental processes: development of the shell field, and transition from the first to the second larval shell. Fluorescent staining revealed developmental delay of the shell field at low pH, and abnormal development thereof was correlated with hinge defects in D-veligers. This study shows, for the first time, that ocean acidification affects larval soft-tissue development, independent from calcification. Multiple developmental processes additively underpin the teratogenic effect of ocean acidification on bivalve larvae. These results explain why trochophores are the most sensitive life-history stage in marine bivalves and suggest that short-term variability in carbonate chemistry can impact early larval development. [ABSTRACT FROM AUTHOR]

Study Question: Do the extreme conditions of vitrification affect mitochondrial health and reactive oxygen species (ROS) levels of human oocytes?Summary Answer: Vitrification of discarded human oocytes shifts the intracellular redox potential towards oxidation but does not alter the mitochondrial potential or intracellular ROS levels.What Is Known Already: Recent studies have reflected increased ROS levels in warmed young oocytes and have highlighted the temporal dynamic loss of mitochondrial potential that could, therefore, lead to a decrease in ATP production, impairing embryo development. Mitochondrial function can also be evaluated in vivo by the FAD/NAD(P)H autofluorescence ratio, which reflects the respiratory chain activity and is considered as a marker of the intracellular redox state.Study Design, Size, Duration: A total of 629 discarded Metaphase II (MII) oocytes collected from June 2013 to April 2014 were included in this control (fresh oocytes, n= 270) versus treatment (vitrified oocytes, n= 359) study.Participants/materials, Setting, Methods: Discarded MII oocytes were donated to research by young (<27 years old) and reproductively aged (>36 years old) women who underwent ovarian stimulation for IVF at a university-affiliated private fertility clinic. Redox state was assessed by measuring the FAD/NAD(P)H autofluorescence ratio, while ROS and mitochondrial activity were reported by in vivo labelling with carboxy-H2DCFDA and JC-1, respectively.Main Results and the Role Of Chance: Young and aged oocytes showed high and similar survival rates (81.8 versus 83.1%, not significant). Confocal microscopy revealed that the FAD/NAD(P)H ratio was significantly higher in vitrified oocytes than in fresh oocytes, suggesting a significant shift towards the oxidized state in oocytes after vitrification, regardless of the maternal age. Mitochondrial distribution was not affected by vitrification. Furthermore, it was not possible to resolve any difference in mitochondrial potential using JC-1 potentiometric dye or in reactive oxygen species (ROS) production (assessed with H2-DCFDA staining) between fresh and vitrified oocytes. Therefore, measurement of intracellular redox potential by autofluorescence imaging may be a more sensitive method to assess oxidative stress or mitochondrial demise in human oocytes because it showed a higher resolving power than JC-1 staining and displayed less variability than H2-DCFDA staining.Limitations, Reasons For Caution: Owing to sample availability, MII discarded oocytes (in vitro matured oocytes and unfertilized oocytes 20 h after ICSI) were included in the study. These discarded oocytes do not necessarily reflect the physiological condition of the MII human oocyte.Wider Implications Of the Findings: Although vitrified oocytes yield comparable clinical outcomes compared with fresh oocytes, lower cleavage and blastocyst rates can be observed during in vitro culture. Data here obtained suggest that the redox state of human oocytes could be affected by vitrification. Therefore, the importance of adding protective antioxidant molecules to the vitrification solution and to the post-warming culture medium to improve embryo cleavage deserves some research.Study Funding/competing Interests: This research project was supported by the Valencian Government (Val+i+D program, M.N.-C.), INCLIVA Foundation for health research (G.S.-A.) and by the University of L'Aquila and Regione Abruzzo ('Reti per l'Alta Formazione' - P.O.F.S.E. Abruzzo 2007-2013 G.D.E.). No conflicts of interest were declared. [ABSTRACT FROM AUTHOR]