Mariola A. Dietrich

Exposure of rainbow trout milt to mercury and cadmium alters sperm motility parameters and reproductive success

In the current work, seminal plasma was used for the first time as an incubation medium for monitoring short-time exposure effects of sublethal concentrations of mercury and cadmium ions on rainbow trout sperm. Sperm motility parameters (CASA) and hatching rates were used as gamete quality markers. Additionally live/dead sperm viability test and comet assay of DNA fragmentation were performed. We demonstrated that computer-assisted sperm motility analysis (CASA) may serve as a predictor of reproductive success, when milt contaminated with heavy metals is used. Results presented in this study demonstrate that mercury ions altered sperm motility characteristics at 1-10 mg Hg2+/l and 10 mg Cd2+/l and hatching rates at 10 mg Hg2+/l and 10 mg Cd2+/l after 4h of exposure. Although mercury ions affected sperm motility parameters immediately after dilution with milt as well as at 4h of exposure, no differences in sperm motility parameters were found between intact and mercury-treated milt after 24h of exposure. Our results suggest that rainbow trout seminal plasma has a protective role against the toxic effects of mercury ions of rainbow trout sperm motility.

Characterization of carp seminal plasma proteome in relation to blood plasma.

UNLABELLED The present study for the first time characterizes a diverse cohort of carp seminal and blood plasma proteins using the combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry. Using this approach, we identified 137 proteins in carp seminal plasma and 88 proteins in carp blood plasma, most of which were newly identified in fish. Transferrin, serine proteinase inhibitors, apolipoproteins, complement C3 and Wap65 were present in high abundance in carp seminal plasma. In carp blood plasma, besides these proteins, immunoglobulins and macroglobulins were identified as major proteins. Comparative analysis of carp seminal and blood plasma proteome performed using 2D-DIGE revealed that in contrast to mammals the majority (1014 from 1240 spots) of carp seminal plasma proteins are blood proteins. Moreover, proteins more abundant in seminal plasma (99 from 1240 spots) were identified, including parvalbumin, isoforms of apolipoproteins, heat shock proteins, components of antioxidative system, matrix metalloproteinases, cathepsin D, enzymes of glycolysis and sperm structural proteins. These proteins are involved in the regulation of sperm motility, spermatogenesis, maintenance of sperm membrane lipid stability and antioxidant protection. This study enhances the basic knowledge concerning fish seminal plasma protein composition and their potential role in fish reproduction. BIOLOGICAL SIGNIFICANCE Proteins similar or identical to blood plasma components are important for male reproductive physiology. Comparative study of blood and seminal plasma is especially justified in fish. Using 2D-DIGE we indicated that, in contrast to mammals, in carp seminal plasma most proteins are common for blood and seminal plasma, which possibly is related to a lack of accessory glands in reproductive tract of most fish. The proteins present in higher abundance in seminal plasma can be related to physiology of fish male reproduction including regulation of sperm motility, spermatogenesis, maintenance of sperm surface composition and antioxidant protection. Application of proteomics analysis to identify carp seminal and blood plasma proteins significantly extends current knowledge regarding the composition of fish seminal and blood plasma proteins and their relationship to higher vertebrates. Moreover, proteomic profiling of carp seminal plasma appears to be helpful for further understanding of the role of fish seminal plasma proteins in male reproductive tract as well as for identification of novel biomarkers for sperm quality.

Isolation and characterization of transferrin from common carp (Cyprinus carpio L) seminal plasma.

Transferrin (Tf) in fish is recognized as a component of non-specific humoral defense mechanisms against bacteria. It is a major protein of common carp seminal plasma but its structure and localization in carp testis is unknown. In this study we developed a simple and efficient three-step purification procedure consisting of affinity chromatography (Con A-Sepharose), hydrophobic interaction chromatography (Phenyl Sepharose) and gel filtration (Superdex 200). The molecular mass of Tf has been determined to be 73.6 kDa and isoelectric point 5.1. The peculiar characteristics of carp transferrin were the lack of carbohydrate component and binding of iron ions by only one functional iron-binding site. Western blot analysis revealed a strong similarity of carp seminal plasma Tf to carp blood Tf and Tf from seminal plasma of other cyprinids but a lower similarity to salmonid and percid fishes. Tf was localized to the blood vessels of the carp testis which strongly suggest that most Tf of carp seminal plasma originates from blood. In conclusion, seminal plasma Tf has a unique structure and is similar or identical to blood Tf.

Carp transferrin can protect spermatozoa against toxic effects of cadmium ions.

Cadmium is a widespread heavy metal that enters the aquatic environment and affects many processes involved in fish reproduction such as sperm motility. Fish seminal plasma proteins can protect spermatozoa against toxic effects of heavy metals. The objective of this study was to demonstrate the ability of a major carp seminal plasma protein-transferrin (TF) to bind cadmium ions and to neutralize the toxic effect of cadmium on carp sperm motility. To obtain a high quantity of carp seminal plasma TF necessary for the experiment, immunoaffinity chromatography as a one-step isolation procedure was established. The titration of TF with cadmium ions spectrophotometrically at 247nm revealed that TF binds cadmium ions at only one spectrophotometrically-sensitive binding site, which suggests that TF is capable of neutralizing the cadmium toxic effect. Indeed, the addition of carp TF to carp semen incubated with 50ppm cadmium for 48h led to about a four-times higher percentage of sperm motility (30.3±1.1%) in comparison to samples incubated with only 50ppm cadmium (8.2±5.2%). Similarly, higher values of other parameters of sperm movement measured by a computer-assisted sperm motility analysis system (VSL, VCL and ALH) were observed at the presence of transferrin. In conclusion, our study provides the first evidence that transferrin from carp seminal plasma can protect sperm motility from cadmium toxicity.

Isolation and identification of fetuin-B-like protein from rainbow trout seminal plasma and its localization in the reproductive system

Seminal plasma of rainbow trout (Oncorhynchus mykiss, Salmonidae) contains an inhibitory system consisting of three fractions (I-III) characterized by different electrophoretic migration rates. Using a two-step isolation procedure we purified (20- and 43-fold to homogeneity) and characterized the two subforms of inhibitor I (Ia and Ib). On the basis of the homology alignment of the amino acid sequences, inhibitor I was classified to the family of cysteine proteinase inhibitors - fetuins. The molecular masses were determined to be 61,146.5Da and 63,096.0Da, and the isoelectric points were estimated to be 6.04 and 6.22 for inhibitor Ia and Ib. Both inhibitors were glycoproteins with a carbohydrate content about 13% for inhibitor Ia and 19% for inhibitor Ib. The equilibrium association constant of inhibitor Ib with cod trypsin was determined to be 7.1×10(8)M(-1). Except for the cod trypsin inhibition, the inhibitor Ib effectively inhibited papain belonging to the cysteine proteainases. Comparative studies of the distribution of inhibitor I and the previously described inhibitor II were performed. The presence of inhibitor I in the seminal plasma was a common feature of several Salmoniformes, which was contrary to inhibitor II detected in seminal plasma of other fish families. Inhibitors I and II showed different expression patterns in the testes and spermatic duct of the rainbow trout.

The effect of reactive oxygen species on motility parameters, DNA integrity, tyrosine phosphorylation and phosphatase activity of common carp (Cyprinus carpio L.) spermatozoa.

The effect of reactive oxygen species production on the motility parameters, DNA integrity, acid phosphatase activity, and protein tyrosine phosphorylation in spermatozoa of the common carp (Cyprinus carpio) was investigated. Spermatozoa were exposed to different concentrations of xanthine and xanthine oxidase (X‐XO) either in the presence or absence of antioxidants for 15 and 60 min. A dose‐ and time‐dependent reduction in spermatozoa motility and velocity was observed. Comet assays showed a dramatic increase in DNA fragmentation after 15 min. Changes in tyrosine phosphorylation of spermatozoa proteins were observed by Western blotting with anti‐phosphotyrosine antibodies, and proteins of interest were identified by mass spectrometry. After a 60 min exposure to X‐XO, O‐linked N‐acetylglucosamine transferase, isoform 4 was phosphorylated and septin‐8‐A was dephosphorylated. Acid phosphatase activity also decreased in a dose‐dependent manner after a 60 min exposure to oxidative stress. The results demonstrate that oxidative stress impaired functional variables (sperm motility, velocity, DNA integrity) of carp spermatozoa, and altered intracellular signalling pathways through changes in tyrosine phosphorylation and acid phosphatase activity. Mol. Reprod. Dev. 82: 48–57, 2015.

Identification of parvalbumin-like protein as a major protein of common carp (Cyprinus carpio L) spermatozoa which appears during final stage of spermatogenesis

Parvalbumin is well known as the major fish allergen that is typically present in high amounts in muscles, where it functions in calcium buffering and is involved in the relaxation process in fast-twitch muscles. We show in our current study that parvalbumin-like protein is present in high amounts in carp spermatozoa. It is the first report to demonstrate the presence of parvalbumin-like protein in fish spermatozoa. Using antibodies produced against purified carp parvalbumin-like protein, we localized parvalbumin-like protein to spermatids and spermatozoa. Our results indicate that parvalbumin-like protein appeared during the final stage of spermatogenesis. We also detected high amounts of parvalbumin-like protein in carp seminal plasma but not in blood plasma which suggests that its function may be specific for the male reproductive tract. The activation mechanism of carp sperm movement is not fully understood, but in carp, Ca2+ influx is the prerequisite for the initiation of sperm motility. The appearance of parvalbumin-like protein in high amounts in mature spermatozoa coincides with their acquiring the ability to move. The presence of parvalbumin-like protein in spermatozoa and seminal plasma strongly suggests that parvalbumin-like protein is an important part of the Ca2+-mediated mechanism of sperm activation in carp.

Characterization, expression and antibacterial properties of apolipoproteins A from carp (Cyprinus carpio L.) seminal plasma.

Apolipoproteins A are multifunctional proteins that, in addition to contributing to lipid metabolism and transport, are associated with the innate immune system in fish. Using a three step isolation procedure consisting of affinity chromatography on Blue-Sepharose, delipidation and reverse phase HPLC we isolated apolipoproteins from carp seminal plasma and identified them as ApoA-I and Apo-14 kDa. Moreover, we provided the full-length cDNA sequence of ApoA-I encoding 257 amino acids including a 18 amino acid signal peptide and a 4 amino acid propeptide. Apolipoproteins corresponded to the most abundant proteins in carp seminal plasma. Both ApoA-I and Apo-14 kDa were represented by several proteoforms that differ both in molecular mass and isoelectric point. The proteoforms of ApoA-I characteristic for seminal plasma were distinguished from those of blood. Carp seminal plasma ApoA-I and Apo-14 kDa showed a high immunologic similarity to their counterparts in carp blood and seminal plasma of other Cyprinid species. The mRNA expression analysis and immunohistochemical study suggest synthesis and secretion of ApoA-I and Apo-14 kDa in the fish reproductive tract and suggest a role in spermatogenesis and the stabilization of sperm membrane. Moreover, ApoA-I displayed bactericidal activity against Escherichia coli and bacteriostatic activity against Aeromonas hydrophila which suggests that ApoA-I is associated with innate immune system of the fish reproductive tract.

Identification of apolipoprotein C-I in rainbow trout seminal plasma

Three distinct bands with high electrophoretic migration rates were isolated and purified from rainbow trout seminal plasma. The molecular masses of these bands were determined to be 5158.8, 4065.9 and 4929.0 Da. The N-terminal amino acids sequences were elucidated and were found to have high homology with Atlantic salmon apolipoprotein C-I. It can be concluded that apolipoprotein C-I is a major component of rainbow trout seminal plasma. Further studies are necessary to confirm the protective effects of apolipoprotein C-I on spermatozoa in terms of the stabilisation of the sperm membrane.

Proteomic analysis of extracellular medium of cryopreserved carp (Cyprinus carpio L.) semen

During freezing and thawing, spermatozoa are exposed to physical and chemical stressors that result in adverse changes in sperm structures and physiological functions. The present study provides, for the first time, a comprehensive description of protein changes in the extracellular medium of cryopreserved semen. Using 2D-DIGE and a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, 183 proteins released from sperm to an extracellular medium were identified. The majority of released proteins were involved in metabolism and energy production. Moreover, proteins associated with a response to stress, apoptosis, small GTPase mediated signal transduction, transcription, translation, protein folding and turnover, reproduction and DNA repair were identified. The dominant group of released proteins was related to cytoplasm. Moreover, specific proteins associated with the membrane, mitochondria and nucleus were identified. The identification of a high number of proteins released from sperm provides new insight into the mechanism of cryodamage to the particular sperm structure and to specific metabolic pathways, which were affected by cryopreservation. The availability of a catalog of carp sperm proteins altered by cryopreservation provides a crucial tool for the development of novel potential biomarkers of cryoinjuries and for the improvement of a long-term sperm preservation procedure.