Transferrin maintains the motility rate, ATP content, and DNA integrity of common carp spermatozoa during short-term storage.

Short-term sperm storage is a straightforward and cost-effective method of managing logistics in large scale fish hatchery operations but may result in decline in sperm quality. For effective artificial reproduction of fish, use of an appropriate additive to optimize sperm storage conditions is essential. In this study, it was investigated the effect of purified seminal plasma transferrin (Tf) at 10 µg/ml on relevant parameters in common carp Cyprinus carpio sperm during short-term storage. We compared sperm motility and curvilinear velocity, adenosine triphosphate (ATP) content and DNA fragmentation of fresh spermatozoa to that stored for 24, 48, 72, and 144 h with or without Tf. The percentage of motile cells and the curvilinear velocity of spermatozoa in stored samples for 72 h with transferrin supplementation were greater compared to samples with no added protein. The ATP content in samples without added transferrin was reduced (P < 0.05) after 72 h of storage, in contrast to the levels observed in transferrin-supplemented sperm. A time-dependent increase in DNA fragmentation was observed. Significantly lower DNA damage, expressed as percent tail DNA (10.99 ±â€¯1.28) and olive tail moment (0.54 ±â€¯0.12), was recorded in Tf-supplemented samples stored for 48 h compared to that with no Tf. Hence, it is concluded that the beneficial effects of transferrin on common carp sperm could serve as an additional tool for developing and enhancing short-term sperm preservation procedures commonly used in aquaculture.

Utilization of Fast Photochemical Oxidation of Proteins and Both Bottom-up and Top-down Mass Spectrometry for Structural Characterization of a Transcription Factor-dsDNA Complex.

A combination of covalent labeling techniques and mass spectrometry (MS) is currently a progressive approach for deriving insights related to the mapping of protein surfaces or protein-ligand interactions. In this study, we mapped an interaction interface between the DNA binding domain (DBD) of FOXO4 protein and the DNA binding element (DAF16) using fast photochemical oxidation of proteins (FPOP). Residues involved in protein-DNA interaction were identified using the bottom-up approach. To confirm the findings and avoid a misinterpretation of the obtained data, caused by possible multiple radical oxidations leading to the protein surface alteration and oxidation of deeply buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly oxidized ions enabled their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed generation of complementary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The findings obtained by bottom-up and top-down approaches were highly consistent. Finally, FPOP results were compared with those of the HDX study of the FOXO4-DBD·DAF16 complex. No contradictions were found between the methods. Moreover, their combination provides complementary information related to the structure and dynamics of the protein-DNA complex. Data are available via ProteomeXchange with identifier PXD027624.