Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction.

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Insights on the UV-Screening Potential of Marine-Inspired Thiol Compounds.

One of the major threats to skin aging and the risk of developing skin cancer is excessive exposure to the sun's ultraviolet radiation (UVR). The use of sunscreens containing different synthetic, organic, and inorganic UVR filters is one of the most widespread defensive measures. However, increasing evidence suggests that some of these compounds are potentially eco-toxic, causing subtle damage to the environment and to marine ecosystems. Resorting to natural products produced in a wide range of marine species to counteract UVR-mediated damage could be an alternative strategy. The present work investigates marine-inspired thiol compounds, derivatives of ovothiol A, isolated from marine invertebrates and known to exhibit unique antioxidant properties. However, their potential use as photoprotective molecules for biocompatible sunscreens and anti-photo aging formulations has not yet been investigated. Here, we report on the UVR absorption properties, photostability, and in vitro UVA shielding activities of two synthetic ovothiol derivatives, 5-thiohistidine and iso-ovothiol A, by spectrophotometric and fluorimetric analysis. We found that the UVA properties of these compounds increase upon exposure to UVA and that their absorption activity is able to screen UVA rays, thus reducing the oxidative damage induced to proteins and lipids. The results of this work demonstrate that these novel marine-inspired compounds could represent an alternative eco-friendly approach for UVR skin protection.

A Survey on the Distribution of Ovothiol and ovoA Gene Expression in Different Tissues and Cells: A Comparative Analysis in Sea Urchins and Mussels.

Ovothiols are histidine-derived thiols produced by a variety of marine invertebrates, protists and bacteria. These compounds, which are among the strongest natural antioxidants, are involved in controlling the cellular redox balance due to their redox exchange with glutathione. Although ovothiols were initially reported as protective agents against environmental stressors, new evidence suggests that they can also act as pheromones and participate in fundamental biological processes such as embryogenesis. To get further insight into the biological roles of ovothiols, we compared ovothiol biosynthesis in the sea urchin Paracentrotus lividus and in the mussel Mytilus galloprovincialis, the two species that represent the richest sources of these compounds among marine invertebrates. Ovothiol content was measured in different tissues and in the immune cells from both species and the expression levels of ovoA, the gene responsible for ovothiol biosynthesis, was inferred from publicly available transcriptomes. A comparative analysis of ovothiol biosynthesis in the two species allowed the identification of the tissues and cells synthesizing the metabolite and highlighted analogies and differences between sea urchins and mussels. By improving our knowledge on the biological roles of ovothiols and pointing out the existence of sustainable natural sources for their isolation, this study provides the basis for future biotechnological investigations on these valuable compounds.

The first genetic engineered system for ovothiol biosynthesis in diatoms reveals a mitochondrial localization for the sulfoxide synthase OvoA.

Diatoms represent one of the most abundant groups of microalgae in the ocean and are responsible for approximately 20% of photosynthetically fixed CO2 on Earth. Due to their complex evolutionary history and ability to adapt to different environments, diatoms are endowed with striking molecular biodiversity and unique metabolic activities. Their high growth rate and the possibility to optimize their biomass make them very promising 'biofactories' for biotechnological applications. Among bioactive compounds, diatoms can produce ovothiols, histidine-derivatives, endowed with unique antioxidant and anti-inflammatory properties, and occurring in many marine invertebrates, bacteria and pathogenic protozoa. However, the functional role of ovothiols biosynthesis in organisms remains almost unexplored. In this work, we have characterized the thiol fraction of Phaeodactylum tricornutum, providing the first evidence of the presence of ovothiol B in pennate diatoms. We have used P. tricornutum to overexpress the 5-histidylcysteine sulfoxide synthase ovoA, the gene encoding the key enzyme involved in ovothiol biosynthesis and we have discovered that OvoA localizes in the mitochondria, a finding that uncovers new concepts in cellular redox biochemistry. We have also obtained engineered biolistic clones that can produce higher amount of ovothiol B compared to wild-type cells, suggesting a new strategy for the eco-sustainable production of these molecules.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.