Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

The voltage-gated T-type Ca2+ channel is key to the sperm motility of Atlantic salmon (Salmo salar).

Ca2+ is a key element in the sperm activation process of Salmo salar. However, the molecular mechanisms by which this ion enters the sperm cell have been poorly studied. In this study, we examined, for the first time, the role of the voltage-gated T-type Ca2+ channel in the activation of sperm motility of Salmo salar. Using an in vitro inhibition assay, a significant decrease in total and progressive motility (P < 0.0001) was observed in Salmo salar sperm when they were treated with NNC-55-0396, a highly selective blocker. The in silico analysis showed that this blocker is docked with a strong affinity for the pore of the voltage-gated T-type calcium channel suggesting the blocking of Ca2+ ions. The results show that the T-type voltage-gated Ca2+ channel is key to sperm motility in Salmo salar.

Role of voltage-gated L-type calcium channel in the spermatozoa motility of Atlantic salmon (Salmo salar).

Ca2+ cations play a key role in the initiation of spermatozoa motility in Atlantic salmon (Salmo salar). In this study we assess the importance of the voltage-gated L-type calcium channels in the spermatozoa motility of Atlantic salmon by combined in vitro and in silico approaches. The results of this study showed that as in other fish species, voltage-gated L-type calcium channels are significant in the spermatozoa motility of Salmo salar. The in vitro assays showed that total and progressive motilities decrease significantly (****p < .001) when Salmo salar spermatozoa are treated with verapamil, which has its binding site in the pore of the voltage-gated L-type calcium channel according to the in silico analysis.

The CatSper channel is present and plays a key role in sperm motility of the Atlantic salmon (Salmo salar).

Among all the Ca2+ channels, CatSper channels have been one of the most studied in sperm of different species due to their demonstrated role in the fertilization process. In fish sperm, the calcium channel plays a key role in sperm activation. However, the functionality of the CatSper channels has not been studied in any of the fish species. For the first time, we studied the relationship of the CatSper channel with sperm motility in a fish, using Atlantic salmon (Salmo salar) as the model. The results of our study showed that the CatSper channel in Salmo salar has chemical-physical characteristics similar to those reported for mammalian CatSper channels. In this work, it was shown that Salmo salar CatSper 3 protein has a molecular weight of approximately 55-kDa similar to Homo sapiens CatSper 3. In silico analyses suggest that this channel forms a heterotetramer sensitive to the specific inhibitor HC-056456, with a binding site in the center of the pore of the CatSper channel, hindering or preventing the influx of Ca2+ ions. The in vitro assay of the sperm motility inhibition of Salmo salar with the inhibitor HC-056456 showed that sperm treated with this inhibitor significantly reduced the total and progressive motility (p < .0001), demonstrating the importance of this ionic channel for this cell. The complementation of the in silico and in vitro analyses of the present work demonstrates that the CatSper channel plays a key role in the regulation of sperm motility in Atlantic salmon.

Effects of cryopreservation on cAMP-dependent protein kinase and AMP-activated protein kinase in Atlantic salmon (Salmo salar) spermatozoa: Relation with post-thaw motility.

Sperm motility in fish with external fertilization is critical for reproductive efficiency in aquaculture, especially in salmonids. Gamete preservation techniques, such as cryopreservation, however, reduce sperm motility and fertilizing capacity. Very few studies have addressed cryodamage from energetic and cell signalling approaches. In this study, cAMP-dependent protein kinase (PKA) and AMP-activated kinase (AMPK) activities were quantified in fresh and cryopreserved spermatozoa of Atlantic salmon (Salmo salar); and the relation with motility was analysed. Results indicate there was a decrease in membrane integrity and motility in post-thawed spermatozoa compared to fresh samples, however, there was about 30% of cells with intact plasma membrane but incapable of motility. The PKA and AMPK activities were less after cryopreservation, indicating that loss of motility may be related to alteration of these key enzymes. Furthermore, PKA and AMPK activities were positively correlated with each other and with motility; and inhibition decreased motility, indicating there is a functional relationship between PKA and AMPK. The PKA inhibition also decreased AMPK activity, but results from protein-protein docking analyses indicated AMPK activation directly by PKA is unlikely, thus an indirect mechanism may exist. There have been no previous reports of these kinase actions in fish spermatozoa, making these findings worthy of assessment when there are future studies being planned, and may serve as base knowledge for optimization of cryopreservation procedures and development of biotechnologies to improve reproduction efficiency in the aquaculture industry.

Changes in Atlantic salmon (Salmo salar) sperm morphology and membrane lipid composition related to cold storage and cryopreservation.

The cold storage and cryopreservation of semen decrease sperm quality. Morphological and biochemical analyses of spermatozoa provide valuable information for the optimization of storage protocols to obtain a sufficient number of spermatozoa for in vitro fertilization. The aim of this study was to evaluate the morphology and lipid composition of Atlantic salmon (Salmo salar) spermatozoa after storage at 4 °C and cryopreservation. Semen samples were obtained by stripping. One aliquot was stored at 4 °C for 7 days, and another aliquot was cryopreserved. The morphology and ultrastructure were analysed using electron microscopy. The lipid composition was analysed by gas chromatography and a commercial kit. After cold storage, the mitochondrion was the most affected component; however, plasma membrane rupture and detachment of the flagellum were also observed. Morphological abnormalities were greater in cryopreserved spermatozoa. The head and mid-piece were dehydrated, sperm membranes were vesiculated, and alterations of mitochondria were observed. After cold storage and cryopreservation, there were less polyunsaturated and omega-3 fatty acids. Furthermore, there was an increase in saturated fatty acids and decrease in cholesterol concentration after cryopreservation (P < 0.05). Based on the results, cryopreservation drastically damaged sperm membranes; the cryogenic damage was associated with membrane lipid composition alterations. The sperm membranes were affected less by cold storage but there was also a decrease of some lipids; therefore, there is a need for improvement in cold storage processes to decrease structural damage of spermatozoa so that semen cryopreservation can be effectively used in the salmon industry.

From Synthesis to Characterization of Site-Selective PEGylated Proteins.

Covalent attachment of therapeutic proteins to polyethylene glycol (PEG) is widely used for the improvement of its pharmacokinetic and pharmacological properties, as well as the reduction in reactogenicity and related side effects. This technique named PEGylation has been successfully employed in several approved drugs to treat various diseases, even cancer. Some methods have been developed to obtain PEGylated proteins, both in multiple protein sites or in a selected amino acid residue. This review focuses mainly on traditional and novel examples of chemical and enzymatic methods for site-selective PEGylation, emphasizing in N-terminal PEGylation, that make it possible to obtain products with a high degree of homogeneity and preserve bioactivity. In addition, the main assay methods that can be applied for the characterization of PEGylated molecules in complex biological samples are also summarized in this paper.

Patagonian blenny (Eleginops maclovinus) spermatozoa quality after storage at 4 ºC in Cortland medium.

Patagonian blenny (E. maclovinus) is a marine species recently placed in captivity and which are potentially farmable. Understanding and improving its sperm capacity to withstand short-term storage conditions is a key element of initiating an artificial propagation program for this species. The aim of this study is to evaluate the ultrastructure and quality of E. maclovinus sperm during refrigerated storage. To address this objective, scanning electron microscopy (SEM), cytofluorimetric analysis (membrane integrity; reactive oxygen species generation; mitochondrial membrane potential) and cell respiration/mitochondrial-function analysis (ATP content; oxygen consumption) could be useful for optimizing or improving management for artificial reproduction of this species. Severe damage of plasma membranes was observed by SEM at day 7 and 14 of in vitro storage. Analyses of sperm quality were conducted during the 14-day cold storage period when sperm were in diluted (with Cortland solution) and undiluted conditions. When there were diluted conditions, there was greater preservation of motile capacity (from day-7; P < 0.05), membrane integrity (from day-7; P < 0.05), mitochondrial membrane potential (from day-10; P < 0.05) and ATP stores (from day-3; P < 0.05). Oxygen consumption indicators were 18.6% ±14.7% greater in the undiluted samples from day-3, and 32.1%±2.1% of the total spermatozoa had ample amounts of superoxide anion in both undiluted and diluted semen on day-0. The use of Cortland solution extended the viability of sperm when there were longer storage times. Factors that have a greater effect on the quality of semen during storage are reactive oxygen species generation and ATP depletion. In conclusion, Patagonian blenny spermatozoa can be stored at 4 °C between 7 and 10 days using Cortland solution.

Mitochondria in teleost spermatozoa.

There is an extraordinary diversity of reproductive modes in teleost and this variability is related to the phylogenetic relationships and adaption to very different biotopes. As in all vertebrates, sperm is produced as the end product of the process of spermatogenesis, and regarding teleost the spermatozoa lack an acrosome in almost all species and motility is activated as a response to osmolarity and ion content of the aquatic medium where the sperm is released. In this context, mitochondria possess a fundamental role for fish spermatozoa motility and integrity, hence, fertilizing potential; they are the energy supplier that allows flagellar movement and their dysfunction could play a main role in structural and functional damage to the spermatozoa. The ATP production through oxidative phosphorylation provides not only energy for cell activities, which includes Na+/K+ ATPase pump, endocytosis, protein synthesis and many other cell processes; but also produces reactive oxygen species, that under mitochondrial dysfunction causes oxidative stress. The assessment of mitochondrial function (e.g. through measurement of mitochondrial membrane potential) as well as ATP content (mostly supplied by mitochondrial respiration) can be useful as quality markers of fish spermatozoa. Also quantification of ROS and antioxidant status, strongly influenced by mitochondria, are used as complementary measurements. There is much information about sperm mitochondria and their function but studies of these aspects on fish reproduction are still required for applications in aquaculture. The real role of fish sperm mitochondria under short and long term storage and in vitro manipulation is not fully understood yet. Thus future research should focus on these matters.