Publications by authors named "Mariola Dietrich"

38 Publications

Differences in growth of Trypanoplasma borreli in carp serum is dependent on transferrin genotype.

Fish Shellfish Immunol 2021 Apr 20;114:58-64. Epub 2021 Apr 20.

Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture in Gołysz, Zaborze, 43-520, Chybie, Poland. Electronic address:

Kinetoplastid parasites require transferrin (Tf), being the main source of iron, for growth and multiplication. This group of parasites developed a unique receptor-mediated system for acquiring host Tf which bears no structural homology with the host transferrin receptor. Trypanoplasma borreli, a blood parasite of common carp, probably uses a similar mechanism to sequester iron from host transferrin. In this study, we demonstrate a critical role of Tf for parasite growth. For in vitro studies we isolated and purified Tf from carp homozygous for the D or G allele of Tf. We obtained Tf-depleted serum using specific antibodies to carp Tf and studied gene expression in vivo during T. borreli infection with Real Time-quantitative PCR. We demonstrate that T. borreli cannot survive in medium supplemented with Tf-depleted serum while reconstitution with Tf restores normal growth. The critical role of Tf for parasite survival was shown in incomplete medium (medium without serum): addition of purified Tf significantly increased parasite survival. We also demonstrate that Tf polymorphism has a significant impact on T. borreli multiplication. Cultured parasites die more quickly in an environment containing D-typed Tf, as compared to medium with G-typed Tf. Gene expression during T. borreli infection in carp did not show an acute phase response. We could, however, observe an increased transcription of Tf in the head kidney, which may be associated with an immunological function of the Tf protein.
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http://dx.doi.org/10.1016/j.fsi.2021.04.011DOI Listing
April 2021

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma.

Biol Reprod 2021 Mar 2. Epub 2021 Mar 2.

Department of Gamete and Embryo Biology.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRPV X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.
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http://dx.doi.org/10.1093/biolre/ioab032DOI Listing
March 2021

Characterization of carp seminal plasma Wap65-2 and its participation in the testicular immune response and temperature acclimation.

Vet Res 2020 Nov 25;51(1):142. Epub 2020 Nov 25.

Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748, Olsztyn, Poland.

Two functionally distinct isoforms of warm-temperature acclimation related 65-kDa protein (Wap65-1 and Wap65-2) with a role in the immune response are present in fish. To our knowledge, contrary to Wap65-1, Wap65-2 has neither been isolated nor functionally characterized in carp especially in reproductive system. The aim of this study was to characterize Wap65-2 and ascertain its functions in immune response and temperature acclimation within reproductive system. Wap65-2 corresponded to one of the most abundant proteins in carp seminal plasma, with a high immunologic similarity to their counterparts in seminal plasma of other fish species and a wide tissue distribution, with predominant expression in the liver. The immunohistochemical localization of Wap65-2 to spermatogonia, Leydig cells, and the epithelium of blood vessels within the testis suggests its role in iron metabolism during spermatogenesis and maintenance of blood-testis barrier integrity. Wap65-2 secretion by the epithelial cells of the spermatic duct and its presence around spermatozoa suggests its involvement in the protection of spermatozoa against damage caused by heme released from erythrocytes following hemorrhage and inflammation. Our results revealed an isoform-specific response of Wap65 to temperature acclimation and Aeromonas salmonicida infection which alters blood-testis barrier integrity. Wap65-2 seems to be related to the immune response against bacteria, while Wap65-1 seems to be involved in temperature acclimation. This study expands the understanding of the mechanism of carp testicular immunity against bacterial challenge and temperature changes, in which Wap65-2 seems to be involved and highlights their potential usefulness as biomarkers of inflammation and temperature acclimation.
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http://dx.doi.org/10.1186/s13567-020-00858-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688007PMC
November 2020

Identification of protein changes in the blood plasma of lung cancer patients subjected to chemotherapy using a 2D-DIGE approach.

PLoS One 2019 17;14(10):e0223840. Epub 2019 Oct 17.

Department of Clinical Molecular Biology, Medical University of Bialystok, Bialystok, Poland.

A comparative analysis of blood samples (depleted of albumin and IgG) obtained from lung cancer patients before chemotherapy versus after a second cycle of chemotherapy was performed using two-dimensional difference gel electrophoresis (2D-DIGE). The control group consisted of eight patients with non-cancerous lung diseases, and the experimental group consisted of four adenocarcinoma (ADC) and four squamous cell carcinoma (SCC) patients. Analyses of gels revealed significant changes in proteins and/or their proteoforms between control patients and lung cancer patients, both before and after a second cycle of chemotherapy. Most of these proteins were related to inflammation, including acute phase proteins (APPs) such as forms of haptoglobin and transferrin, complement component C3, and clusterin. The variable expression of APPs can potentially be used for profiling lung cancer. The greatest changes observed after chemotherapy were in transferrin and serotransferrin, which likely reflect disturbances in iron turnover after chemotherapy-induced anaemia. Significant changes in plasma proteins between ADC and SCC patients were also revealed, suggesting use of plasma vitronectin as a potential marker of SCC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0223840PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797170PMC
March 2020

Proteomic and metabolomic insights into the functions of the male reproductive system in fishes.

Theriogenology 2019 Jul 15;132:182-200. Epub 2019 Apr 15.

Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748, Olsztyn, Poland.

Proteomics and metabolomics are emerging and powerful tools to unravel the complex molecular mechanisms regulating reproduction in male fish. So far, numerous proteins and metabolites have been identified that provide us with valuable information to conduct a comprehensive analysis on seminal plasma and spermatozoa components and their functions. These analyses have allowed a better understanding of the blood-testis barrier functions, the molecular mechanisms underlying spermatogenesis, spermatozoa maturation, motility signaling, and competition as well as the mechanism of cryodamage to sperm structure and functions. To extend, proteins that undergo posttranslational modification, such as phosphorylation and oxidation in response to spermatozoa motility activation and cryopreservation, respectively, have been identified. Proteomic studies resulted in identification of potential proteins that can be used as biomarkers for sperm quality and freezability to enable the control of artificial reproduction, and to improve methods for long-term preservation (cryopreservation) of sperm. The different proteins expressed in the spermatozoa of neomales and normal males can also provide new insights into development of methods for separating X and Y fish sperm, and changes in the protein profiles in haploid and diploid spermatozoa will provide new perspectives to better understand the mechanism of male polyploidy. Overall, the knowledge gained by proteomic and metabolomic studies is important from basic to applied sciences for the development and/or optimisation of techniques in controlled fish reproduction.
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http://dx.doi.org/10.1016/j.theriogenology.2019.04.018DOI Listing
July 2019

Hormonal stimulation of carp is accompanied by changes in seminal plasma proteins associated with the immune and stress responses.

J Proteomics 2019 06 25;202:103369. Epub 2019 Apr 25.

Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland.

Hormonal stimulation in common carp is a routine practice to enhance sperm production and control gamete maturation. This study aimed to compare the proteome of carp seminal plasma between control and Ovopel-induced males using two-dimensional differential in-gel electrophoresis. Ovopel induction increased sperm volume, total sperm count, seminal plasma osmolality, and pH and decreased seminal plasma protein concentration. In total, 36 spots were identified (23 up- and 13 downregulated), corresponding to 23 proteins differentially abundant in seminal plasma after Ovopel induction (p < .05; fold change 1.2). The majority of proteins were associated with the immune and stress responses including the transport protein (hephaestin), antiproteases (fetuin, α2-macroglobulin, TIMP2), complement components (C3, complement factor B/C2A), regulator of the coagulation cascade (plasminogen), modulators of the innate immune response, such as intelectin, ApoA and ApoE, and the cathepsin/cystatin system, and stress response (enolase1). In addition, hormonal stimulation seems to be related to the proteins involved in lipid metabolism, signal transduction, and tissue remodeling. Our results suggest that hormonal stimulation is not just concomitant with the hydration of testis but also induces the synthesis and secretion of seminal plasma proteins involved in sperm maturation and protection against stress induced by administration of the exogenous hormone. SIGNIFICANCE: It is well known that hormonal stimulation of male fish induces the final maturation of spermatozoa. However, molecular and biochemical basis underlying hormone-induced changes in semen is unknown at present. This study for the first time reveals, using proteomic approach, that hormonal stimulation in addition to hydration of testis is accompanied by significant changes in seminal plasma proteins related mainly to immune and stress response, lipid metabolism, signal transduction and tissue remodeling. These changes are associated with gene expression and synthesis and secretion of seminal plasma proteins by reproductive tissues. Overall, our results provide a framework for understanding the molecular mechanism responsible for hormonal stimulation in the reproductive tract of fish males.
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http://dx.doi.org/10.1016/j.jprot.2019.04.019DOI Listing
June 2019

Seminal plasma transferrin effects on cryopreserved common carp Cyprinus carpio sperm and comparison with bovine serum albumin and antifreeze proteins.

Anim Reprod Sci 2019 May 23;204:125-130. Epub 2019 Mar 23.

CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China. Electronic address:

In the current study, there was evaluation of the cryopreservation effectiveness of common carp Cyprinus carpio sperm when cryopreservation medium was supplemented with proteins. Semen was diluted with Kurokura's extender composing 180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl, 2.38 mM NaHCO, and 10% dimethylsulfoxide (DMSO). Cryopreservation medium was supplemented with purified seminal plasma transferrin (Tf), bovine serum albumin (BSA) or antifreeze protein (AFP) Types I and III. Concentration of proteins evaluated was 0.1 μg/ml, 1 μg/ml, and 10 μg/ml. Motility and curvilinear velocity of spermatozoa was evaluated by the Computer Assisted Semen Analyzer (CASA). The percent of motile cells and spermatozoa curvilinear velocity of frozen-thawed sperm with supplementation of Tf and AFP III at all concentrations were greater compared to samples with no added proteins. The protective effect of BSA and AFP I was less and dose-dependent. Thus, it is concluded that incorporation of Tf in the extender before freezing improves crypreservation of common carp spermatozoa whereas supplementation with AFP III in greater concentrations was more effective.
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http://dx.doi.org/10.1016/j.anireprosci.2019.03.013DOI Listing
May 2019

Seasonal changes in the proteome of cryopreserved bull semen supernatant.

Theriogenology 2019 Mar 8;126:295-302. Epub 2018 Dec 8.

Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Tuwima 10, 10-748, Olsztyn, Poland.

Seasonal variability in cattle fertility may be attributed to male reproduction, including the quality of semen produced by males and its usefulness after cryopreservation. The exact molecular mechanisms responsible for such seasonal variation are not entirely known. The aim of our study was to evaluate the changes in the proteome of cryopreserved bull (Bos taurus) semen supernatant throughout the year. The quality of cryopreserved semen collected from the same Limousin bulls during spring, summer, autumn and winter (n = 5 in each group) was assessed by measuring sperm motility parameters using the CASA system and sperm viability and the level of sperm reactive oxygen species using flow cytometry. Two-dimensional difference in-gel electrophoresis analysis coupled with the MALDI-TOF mass spectrometry was used to detect seasonal changes in the proteome of cryopreserved bull semen supernatant. We found seasonal variability in the percentage of sperm motility (P < 0.05), which was the highest in autumn and winter and the lowest in spring and summer. There was an effect of season on sperm viability (P < 0.05), with the highest percentage of dead sperm recorded in summer. There was no effect of season on sperm levels of oxidation. Proteomic analysis identified 23 protein spots, representing 10 proteins that changed during the year (P < 0.05). Albumin, clusterin, spermadhesin 1 and platelet-activating factor acetylhydrolase precursor were most abundant during winter, which presumably reflected correct lipid composition and morphology of spermatozoa, as well as involvement in protection against premature capacitation. Moreover, osteopontin and nucleobindin 1 could also be connected to increased semen quality in this season. C-C motif chemokine 2 precursor, epididymal-specific lipocalin-5, peptidyl-prolyl cis-trans isomerase and seminal ribonuclease were most abundant during summer and probably reflected disturbances during spermatogenesis and sperm maturation. In summary, our study has shown that cryopreserved bull semen quality parameters were higher in winter than in summer. The identification of proteins connected to the variability of semen quality during the year have provided new insight into understanding the mechanisms underlying the seasonality of cattle breeding.
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http://dx.doi.org/10.1016/j.theriogenology.2018.12.015DOI Listing
March 2019

Differences in sperm protein abundance and carbonylation level in bull ejaculates of low and high quality.

PLoS One 2018 14;13(11):e0206150. Epub 2018 Nov 14.

Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Tuwima 10, Olsztyn, Poland.

In breeding and insemination centres, significant variation in bull ejaculate quality is often observed between individuals and also within the same individual. Low-quality semen does not qualify for cryopreservation and is rejected, generating economic loss. The mechanisms underlying the formation of low-quality ejaculates are poorly understood; therefore, the aim of the present study was to investigate the proteomic differences and oxidative modifications (measured as changes in protein carbonylation level) of bull ejaculates of low and high quality. Flow cytometry and computer-assisted sperm analysis were used to assess differences in viability, reactive oxygen species (ROS) level, and sperm motility. To analyse changes in protein abundance, two-dimensional difference gel electrophoresis (2D-DIGE) was performed. Western blotting in conjunction with two-dimensional electrophoresis (2D-oxyblot) was used to quantitate carbonylated sperm proteins. Proteins were identified using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight spectrometry. High quality ejaculates were characterised by higher sperm motility, viability, concentration, and a lower number of ROS-positive cells (ROS+). We found significant differences in the protein profile between high- and low-quality ejaculates, and identified 14 protein spots corresponding to 10 proteins with differences in abundance. The identified sperm proteins were mainly associated with energetic metabolism, capacitation, fertilisation, motility, and cellular detoxification. High-quality ejaculates were characterised by a high abundance of extracellular sperm surface proteins, likely due to more efficient secretion from accessory sex glands and/or epididymis, and a low abundance of intracellular proteins. Our results show that sperm proteins in low-quality ejaculates are characterised by a high carbonylation level. Moreover, we identified, for the first time, 14 protein spots corresponding to 12 proteins with differences in carbonylation level between low- and high-quality ejaculates. The carbonylated proteins were localised mainly in mitochondria or their immediate surroundings. Oxidative damage to proteins in low-quality semen may be associated with phosphorylation/dephosphorylation disturbances, mitochondrial dysfunction, and motility apparatus disorders. Our results contribute to research regarding the mechanism by which low- and high-quality ejaculates are formed and to the identification of sperm proteins that are particularly sensitive to oxidative damage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206150PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6241115PMC
April 2019

Cryopreservation effects on a viable sperm sterlet (Acipenser ruthenus) subpopulation obtained by a Percoll density gradient method.

PLoS One 2018 16;13(8):e0202514. Epub 2018 Aug 16.

Research Institute of Fish Culture and Hydrobiology, Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceske Budejovice, Vodnany, Czech Republic.

In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202514PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095596PMC
February 2019

Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing.

Sci Rep 2018 04 3;8(1):5425. Epub 2018 Apr 3.

Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland.

Recent studies have shown that the transcription factor Foxn1, which is expressed in keratinocytes, is involved in the skin wound healing process, yet how Foxn1 functions remains largely unknown. Our latest data indicate that Foxn1 drives skin healing via engagement in re-epithelization and the epithelial-mesenchymal transition (EMT) process. In the present study, 2D-DIGE proteomic profiling analysis of in vitro cultured keratinocytes transfected with adenoviral vector carrying Foxn1-GFP or GFP alone (control) revealed forty proteins with differential abundance between the compared groups. Among the proteins with Foxn1-dependent expression, several enable adaptation to hypoxia. Subsequent experiments revealed that hypoxic conditions (1% O) stimulate endogenous and exogenous (transfected Ad-Foxn1) Foxn1 expression in cultured keratinocytes. A proteomics analysis also identified proteins that can act as a factors controlling the balance between cell proliferation, differentiation and apoptosis in response to Foxn1. We also showed that in C57BL/6 keratinocytes, the stimulation of Foxn1 by hypoxia is accompanied by increases in Mmp-9 expression. These data corroborate the detected co-localization of Foxn1 and Mmp-9 expression in vivo in post-wounding skin samples of Foxn1::Egfp transgenic mice. Together, our data indicate that Foxn1 orchestrates cellular changes in keratinocytes in both physiological (self-renewal) and pathological (skin wound healing) contexts.
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http://dx.doi.org/10.1038/s41598-018-23794-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882803PMC
April 2018

Proteomic characterization of fresh spermatozoa and supernatant after cryopreservation in relation to freezability of carp (Cyprinus carpio L) semen.

PLoS One 2018 22;13(3):e0192972. Epub 2018 Mar 22.

Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima, Olsztyn, Poland.

Our recent studies suggested that the freezability of carp semen is related to seminal plasma protein profiles. Here, we aimed to compare the spermatozoa proteomes of good (GF) and poor (PF) freezability semen of carp. To achieve this, we used two-dimensional difference in gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. The semen was classified as GF or PF based on sperm motility after freeze/thawing. We identified proteins enriched in spermatozoa of GF (22 proteins) and PF (18 proteins) semen. We also identified 12 proteins enriched in the supernatant after cryopreservation of PF semen. Good freezability is related to high concentrations of proteins involved in the maintenance of flagella structure, membrane fluidity, efficient control of Ca2+ and sperm motility, energy production, and antioxidative protection, which likely reflects the full maturation status of spermatozoa of GF semen. On the other hand poor freezability seems to be related to the presence of proteins identified as released in high quantities from cryopreserved sperm of PF. Thus, the identified proteins might be useful bioindicators of freezing resilience and could be used to screen carp males before cryopreservation, thus improve long-term sperm preservation in carp. Data are available via ProteomeXchange with identifier PXD008187.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192972PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863941PMC
June 2018

Acclimation to cold and warm temperatures is associated with differential expression of male carp blood proteins involved in acute phase and stress responses, and lipid metabolism.

Fish Shellfish Immunol 2018 May 12;76:305-315. Epub 2018 Mar 12.

Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland.

The environmental temperature affects plasma biochemical indicators, antioxidant status and hematological and immunological parameters in fish. So far, only single blood proteins have been identified in response to temperature changes. The aim of this study was to compare the proteome of carp blood plasma from males acclimated to warm (30 °C) and cold (10 °C) temperatures by two-dimensional differential gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. A total of 47 spots were found to be differentially regulated by temperature (>1.2-fold change, p < 0.05): 25 protein spots were more abundant in warm-acclimated males and 22 were enriched in cold-acclimated males. The majority of differentially regulated proteins were associated with acute phase response signalling involved in: i) activation of the complement system (complement C3-H1), ii) neutralization of proteolytic enzymes (inter-alpha inhibitor H3, fetuin, serpinA1, antithrombin, alpha2-macroglobulin), iii) scavenging of free hemoglobin and radicals (haptoglobin, Wap65 kDa), iv) clot-formation (fibrinogen beta and alpha chain, T-kininogen) and v) the host's immune response modulation (ApoA1 and ApoA2). However, quite different sets of these proteins or proteoforms were involved in response to cold and warm temperatures. In addition, cold acclimation seems to be related to the proteins involved in lipid metabolism (apolipoproteins A and 14 kDa) and stress response (corticosteroid binding globulin). We discovered a strongly regulated protein Cap31 upon cold acclimation, which can serve as a potential blood biomarker of cold response in carp. These studies significantly extend our knowledge concerning mechanisms underlying thermal adaptation in poikilotherms.
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http://dx.doi.org/10.1016/j.fsi.2018.03.018DOI Listing
May 2018

Identification of oxidatively modified proteins due to cryopreservation of carp semen.

J Anim Sci 2018 Apr;96(4):1453-1465

Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Tuwima, Olsztyn, Poland.

During semen cryopreservation, spermatozoa are exposed to physical and chemical stressors that result in their functional and structural damage. Growing evidence suggests that most cryoinjuries result from oxidative stress accompanying sperm cryopreservation. Elevated amounts of reactive oxygen species (ROS) generated during cryopreservation can react with sperm macromolecules, including proteins. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of carp spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer-assisted sperm analysis were used to evaluate changes in viability, ROS level, and motility of spermatozoa. The spermatozoa proteins that were specifically carbonylated were identified and quantified by Western blotting, in conjunction with 2-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Cryopreservation decreased spermatozoa motility (P < 0.01) and viability (P < 0.0001) and significantly increased (P < 0.0001) the number of ROS-positive cells. We identified 25 protein spots, corresponding to 19 proteins, with increases (P < 0.05) in carbonylation level due to freezing/thawing. The identified proteins are involved in motility, metabolism, calcium-ion binding, signal transduction, protein folding, and intracellular transport. The results suggest that carbonylation of flagellar proteins can result in motility disorders and may contribute to the reduced percentage of motile spermatozoa and disturbances in movement trajectory after sperm cryopreservation. Moreover, cryopreservation may contribute to impaired cellular respiration, ATP regeneration, disturbances of Ca2+ turnover, unfolding of cytoplasmic or histone proteins, disturbances of cell signaling and intracellular transport, and reduced membrane stability. Our results contribute to the knowledge concerning cryoinjury and to further development of a modified cryopreservation procedure aimed at minimizing oxidative damage of carp sperm proteins.
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http://dx.doi.org/10.1093/jas/sky063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140970PMC
April 2018

DIGE Analysis of Fish Tissues.

Methods Mol Biol 2018 ;1664:203-219

Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748, Olsztyn, Poland.

Two-dimensional difference gel electrophoresis (2D-DIGE) appears to be especially useful in quantitative approaches, allowing the co-separation of proteins of control samples from proteins of treatment/disease samples on the same gel, eliminating gel-to-gel variability. The principle of 2D-DIGE is to label proteins prior to isoelectric focusing and use three spectrally resolvable fluorescent dyes, allowing the independent labeling of control and experimental samples. This procedure makes it possible to reduce the number of gels in an experiment, allowing the accurate and reproducible quantification of multiple samples. 2D-DIGE has been found to be an excellent methodical tool in several areas of fish research, including environmental pollution and toxicology, the mechanisms of development and disorders, reproduction, nutrition, evolution, and ecology.
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http://dx.doi.org/10.1007/978-1-4939-7268-5_16DOI Listing
May 2018

Protein phosphorylation in spermatozoa motility of and .

Reproduction 2017 11 29;154(5):653-673. Epub 2017 Aug 29.

Sorbonne Universités, UPMC Université Paris 06, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer, Observatoire Océanologique, Villefranche sur-mer, France.

Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp ( L.) are activated by hypoosmolality, whereas spermatozoa of sterlet () require Ca2+ and low concentration of K+ for motility activation. Intracellular signaling differs between these two species as well, particularly in terms of utilization of secondary messengers (cAMP and Ca2+), and kinase activities. The current study was performed in order to determine the importance of protein phosphorylation and protein kinases for activation of sperm motility in carp and sterlet. Treatment with kinase inhibitors indicates that protein kinases A and C (PKA and PKC) participate in spermatozoa motility of both species. Immunodetection of phospho-(Ser/Thr) PKA substrates shows that phosphorylated proteins are localized differently in spermatozoa of carp and sterlet. Strong phosphorylation of PKC substrate was observed in flagella of sterlet spermatozoa, whereas in carp sperm, PKC substrates were lightly phosphorylated in the midpiece and flagella. Motility activation induced either phosphorylation or dephosphorylation on serine, threonine and tyrosine residues of numerous proteins in carp and sterlet spermatozoa. Proteomic methods were used to identify proteins whose phosphorylation state changes upon the initiation of sperm motility. Numerous mitochondrial and glycolytic enzymes were identified in spermatozoa of both species, as well as axonemal proteins, heat shock proteins, septins and calcium-binding proteins. Our results contribute to an understanding of the roles of signaling molecules, protein kinases and protein phosphorylation in motility activation and regulation of two valuable fish species, and .
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http://dx.doi.org/10.1530/REP-16-0662DOI Listing
November 2017

Proteomic identification of seminal plasma proteins related to the freezability of carp semen.

J Proteomics 2017 06 24;162:52-61. Epub 2017 Apr 24.

Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland.

The variation in sperm freezability among individuals within a fish species is a major factor justifying the identification of useful predictive indicators of cryopreservation success. It is unknown at present whether the protein composition of fish seminal plasma affects sperm freezability. Therefore, the aims of this study were to compare the proteome of carp seminal plasma from semen rated as good (GF) and poor (PF) freezability by two-dimensional difference gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. The semen was classified as GF and PF based on sperm motility assessment after freeze/thawing. Five spots representing three proteins were more abundant in GF, while ten spots representing seven proteins were more abundant in PF seminal plasma. The majority of proteins present in higher abundance in PF seminal plasma were associated with the innate immune response. On the other hand, higher freezability was associated with proteins involved in the maintenance of sperm membrane integrity and antioxidative protection. These results indicate that carp semen freezability levels may be related to different seminal plasma protein profiles. Lower usefulness of spermatozoa in cryopreservation may be related to previous infection or stress leading to sublethal changes to sperm structure.

Significance: Sperm quality parameters such as motility, viability and sperm concentration have been used as predictive tools of sperm cryopreservation potential in fish species However, the usefulness of initial motility parameters as indicators of freezability varies among fish species and between individuals within a species. Recent studies in mammals revealed that male-to-male variability in cryoresistance can be attributed to differences in seminal plasma protein composition. To the best of our knowledge, no proteomic studies linking the protein composition of fish seminal plasma and freezing resilience have been performed in fish. Our results indicate for the first time that factors regulating how carp semen tolerate cryopreservation may be related to the different protein profiles of carp seminal plasma. The obtained results provide new insight into understanding the molecular mechanisms underlying cryoresistance of carp semen and provide a tool for the improvement of a long-term sperm preservation procedure.
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http://dx.doi.org/10.1016/j.jprot.2017.04.015DOI Listing
June 2017

Purification, characterization and expression of transferrin from rainbow trout seminal plasma.

Comp Biochem Physiol B Biochem Mol Biol 2017 Jun 11;208-209:38-46. Epub 2017 Apr 11.

Department of Gamete and Embryo Biology, Semen Biology Group, Institute of Animal Reproduction and Food Research, Tuwima 10, 10-748 Olsztyn, Poland.

Transferrin (TF) is recognized as a multifunctional protein and has been implicated in antioxidative, antimicrobial protection, growth, differentiation and cytoprotection effects. An efficient, original three-step isolation procedure for TF consisting in hydrophobic interaction chromatography, gel filtration and preparative electrophoresis was developed. Rainbow trout TF was found to be N-glycosylated (not O-glycosylated) and phosphorylated at all serine, threonine, and tyrosine residues. The protein consists of several proteoforms with an average molecular weight of 76.9kDa and isoelectric point ranging from 5.2 to 5.7. Rainbow trout TF has two functional iron-binding sites and appears to be quite distinct from carp TF regarding glycosylation and iron-binding properties. The highest gene expression of TF was detected in liver and testis, the lowest was detected in head kidney, spleen and efferent ducts. For the first time TF was identified in the semen of several salmonid species. TF was localized within testis, mainly in spermatozoa, Sertoli, Leydig cells, as well as in both columnar secretory and basal cells within the efferent duct. This work contributes to the existing knowledge information indicating significant variations in TF structure within teleost fish. The results obtained in this study provide valuable data on the TF from trout seminal plasma and the physiological role of this protein in the reproductive tract of salmonids. The results are important for our understanding of the role of TF in the antioxidant protection and resistance to pathogenic infections of reproductive cells. The protective role of TF against environmental pollution with heavy metals, especially during prolonged storage of spermatozoa in the spermatic duct, as well as regulation of spermatogenesis and providing Fe for developing germ cells is also postulated.
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http://dx.doi.org/10.1016/j.cbpb.2017.04.002DOI Listing
June 2017

Cryopreservation of bull semen is associated with carbonylation of sperm proteins.

Theriogenology 2017 Apr 10;92:95-102. Epub 2017 Jan 10.

Institute of Animal Reproduction and Food Research, Department of Gamete and Embryo Biology, Polish Academy of Sciences, Tuwima 10 Str., 10-748 Olsztyn, Poland.

Artificial insemination with cryopreserved semen enables affordable, large-scale dissemination of gametes with superior genetics. However, cryopreservation can cause functional and structural damage to spermatozoa that is associated with reactive oxygen species (ROS) production, impairment of sperm motility and decreased fertilizing potential, but little attention has been paid to protein changes. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of bull spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer-assisted sperm analysis were used to evaluate changes in viability, ROS level and motility of spermatozoa. Western blotting, in conjunction with two-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight spectrometry, was employed to identify and quantify the specifically carbonylated spermatozoa proteins. Cryopreservation decreased motility and viability but increased the number of ROS-positive cells. We identified 11 proteins (ropporin-1, outer dense fiber protein 2, glutathione S-transferase, triosephosphate isomerase, capping protein beta 3 isoform, actin-related protein M1, actin-related protein T2, NADH dehydrogenase, isocitrate dehydrogenase, cilia- and flagella-associated protein 161, phosphatidylethanolamine-binding protein 4) showing differences in protein carbonylation in response to cryopreservation. The identified proteins are associated with cytoskeleton and flagella organization, detoxification and energy metabolism. Moreover, almost all of the identified carbonylated proteins are involved in capacitation. Our results indicate for the first time that cryopreservation induces oxidation of selected sperm proteins via carbonylation. We suggest that carbonylation of sperm proteins could be a direct result of oxidative stress and potentially lead to disturbances of capacitation-involved proteins or could indicate cryopreservation-induced premature capacitation.
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http://dx.doi.org/10.1016/j.theriogenology.2017.01.011DOI Listing
April 2017

Serine protease inhibitor Kazal-type 2 is expressed in the male reproductive tract of carp with a possible role in antimicrobial protection.

Fish Shellfish Immunol 2017 Jan 17;60:150-163. Epub 2016 Nov 17.

Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, 10-748 Olsztyn, Poland.

The presence of the low-molecular-mass serine protease inhibitor Kazal-type (Spink) is a characteristic feature of vertebrate semen. Its main function is control of the serine protease in the acrosome, acrosin. Here we showed for the first time that Spink is present in the seminal plasma of carp, which have anacrosomal spermatozoa. Using a three-step isolation procedure that consisted in gel filtration and RP-HPLC and re-RP-HPLC, we isolated this inhibitor and identified it as serine protease inhibitor Kazal-type 2 (Spink2), a reproductive-derived member of the Spink family. The cDNA sequence of this inhibitor obtained from carp testis encoded 77 amino acids, including a 17 amino acids signal peptide; this sequence was distinct from fish Kazal-type inhibitors. The mRNA expression analysis showed that Spink2 is expressed predominantly in carp testis and spermatic duct. Immunohistochemical analysis demonstrated its localization in testis in Sertoli, Leydig and germ cells at all developmental stages (with the exception of spermatozoa) and in the epithelium of the spermatic duct. Aside from strong inhibition of trypsin, this inhibitor acts strongly against subtilisin and possesses bacteriostatic activities against Lactobacillus subtilis, Escherichia coli and Aeromonas hydrophila. The localization of Spink2 in carp reproductive tract suggests an important function in spermatogenesis and in maintenance of the microenvironment in which sperm maturation occurs and sperm are stored. Our results suggest that Spink2 from carp seminal plasma may play a role in antibacterial semen defense, protecting semen against unwanted proteolysis within the reproductive tract.
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http://dx.doi.org/10.1016/j.fsi.2016.11.041DOI Listing
January 2017

Motility of carp spermatozoa is associated with profound changes in the sperm proteome.

J Proteomics 2016 Apr 27;138:124-35. Epub 2016 Feb 27.

Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland.

Unlabelled: In freshwater cyprinids, spermatozoa are quiescent in seminal plasma and sperm motility is initiated by a decrease in osmolality (hypo-osmotic shock) after discharge into the aqueous environment. However, it is unknown at present if and to what extent changes in proteins are involved in carp sperm motility. Therefore, the aim of our study was to identify proteins related to carp sperm motility through a comparison of immobilized and activated carp spermatozoa using a 2D-DIGE approach. Our results, for the first time indicated that carp sperm motility is associated with changes in protein content. Seventy-two differentially expressed proteins were identified. These proteins are mainly involved in ubiquitin-proteasome pathways, glycolysis, the TCA cycle, remodeling and are putatively related to sperm energy metabolism and motility. Moreover proteins associated with oxidative stress responses, signal transduction by Ca(2+)-dependent MAPK cascades, and PKC and protein folding have been identified. The proteins involved in carp sperm motility were localized to the cytoplasm, mitochondria, cytoskeleton, nucleus and sperm membrane. The identification of a high number of proteins involved in carp sperm motility would contribute to current knowledge about the molecular mechanisms of sperm motility in freshwater fish.

Biological Significance: To the best of our knowledge, few changes in proteins involved in the initiation of fish sperm motility have been identified. This is a limited number of proteins compared with the 80 recently identified proteins involved in human sperm motility. However, no proteomic studies of sperm motility have yet been performed on freshwater fish. Our present study allowed for the first time a comprehensive characterization of the proteins associated with carp sperm motility and a better understanding of the molecular mechanisms underlying sperm motility activation and maintenance. The application of 2D-DIGE facilitated the identification proteins crucial for sperm structural organization and motility. The identification of a high number of proteins involved in carp sperm motility would contribute appreciably to the presently limited information available on the mechanisms of sperm motility in freshwater fish. Moreover the identified list of proteins will create a platform for future studies designed to assess the functional significance of specific proteins in sperm motility.
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http://dx.doi.org/10.1016/j.jprot.2016.02.029DOI Listing
April 2016

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

Comp Biochem Physiol Part D Genomics Proteomics 2015 Sep 6;15:49-57. Epub 2015 Jun 6.

Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland.

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.
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http://dx.doi.org/10.1016/j.cbd.2015.05.003DOI Listing
September 2015

Expression of apolipoprotein A-I and A-II in rainbow trout reproductive tract and their possible role in antibacterial defence.

Fish Shellfish Immunol 2015 Aug 2;45(2):750-6. Epub 2015 Jun 2.

Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn, Department of Gamete and Embryo Biology, Poland.

Antimicrobial proteins such as apolipoproteins A (ApoA-I and ApoA-II) play an important role in the primary defence barrier in vertebrates including fish. The aims of the present study were to isolate and characterise rainbow trout seminal plasma ApoA-I and ApoA-II, to examine the mRNA expression of each apolipoprotein in testis and spermatic ducts, and to test the antibacterial properties of the apolipoproteins. Using a three-step isolation procedure consisting of ion-exchange chromatography, gel filtration and preparative SDS-PAGE, apolipoproteins were purified and identified as ApoA-I and ApoA-II. Both apolipoproteins were represented by several proteoforms. The expression of ApoA-I and ApoA-II mRNA in the reproductive tract and their antibacterial properties against Escherichia coli suggest that seminal apolipoproteins play an important role in innate immunity in the rainbow trout reproductive tract. The functions of seminal ApoA can be related to protection of sperm and reproductive tissue from microbial attack and to the maintenance of sperm membrane integrity.
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http://dx.doi.org/10.1016/j.fsi.2015.05.048DOI Listing
August 2015

Proteomic analysis of porcine endometrial tissue during peri-implantation period reveals altered protein abundance.

J Proteomics 2015 Jul 12;125:76-88. Epub 2015 May 12.

Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland.

Unlabelled: In mammals, successful pregnancy depends upon the readiness of uterus for implantation, followed by correct communication between the endometrium and the developing conceptus. The objective of this study was to elucidate changes in protein abundance associated with progression of estrous cycle and pregnancy from Day 9 to Day 12. We analyzed porcine endometrial tissue lysates by 2D-DIGE. Abundance of several proteins was altered depending upon the pregnancy status of animals. MALDI-TOF/TOF was used to identify a number of these proteins. Endometrial proteins that increased from Day 9 to Day 12 of cycle included annexin A4, beta-actin, apolipoprotein, ceruloplasmin and afamin. Changes in protein abundances associated with conceptus secreted factors, including haptoglobin, prolyl-4-hydroxylase, aldose-reductase and transthyretin, were also observed. Functional analysis revealed that endometrial proteins with altered abundance on Day 12 irrespective of the reproductive status were related to growth and remodeling, acute phase response and free radical scavenging, whereas transport and small molecule biochemistry were the functions activated in the pregnant endometrium as compared to the cyclic endometrium. These data provide information on dynamic physiological processes associated with uterine endometrial function of the cyclic and pregnant endometrium during period of maternal recognition of pregnancy in pigs and may potentially demonstrate a protein profile associated with successful pregnancy.

Biological Significance: In pigs, the fertility rates are generally very high but the early embryonic loss that occurs during the second and third weeks of gestation critically affects the potential litter size. Temporal changes that take place in the uterine environment during the period of early pregnancy in pigs and a cross-talk between the uterus and the embryo play an important role in embryonic survival and successful pregnancy. A better understanding of the molecular changes associated with these processes will pave way for understanding of endometrial functions and help towards increasing embryo survival. In this study, we present a 2D-DIGE based analysis of changes in porcine endometrial proteome that are associated with progression of cycle and progression of pregnancy. The network analysis of the results clearly revealed the pathways that are involved in rendering the endometrium receptive to the presence of embryo and also the changes that are result of molecular communication between the endometrium and the conceptuses. This comprehensive identification of proteomic changes in the porcine endometrium could be a foundation for targeted studies of proteins and pathways potentially involved in abnormal endometrial receptivity, placentation and embryo loss.
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http://dx.doi.org/10.1016/j.jprot.2015.05.003DOI Listing
July 2015

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

Mol Reprod Dev 2015 Jan 30;82(1):48-57. Epub 2014 Dec 30.

Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, University of South Bohemia in Ceske Budejovice, Vodňany, Czech Republic.

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.
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http://dx.doi.org/10.1002/mrd.22442DOI Listing
January 2015

In-depth proteomic analysis of carp (Cyprinus carpio L) spermatozoa.

Comp Biochem Physiol Part D Genomics Proteomics 2014 Dec 28;12:10-5. Epub 2014 Sep 28.

Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Department of Gamete and Embryo Biology, Olsztyn, Poland.

Using a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography-electrospray ionization tandem mass spectrometry, we identified 348 proteins in carp spermatozoa, most of which were for the first time identified in fish. Dynein, tubulin, HSP90, HSP70, HSP60, adenosylhomocysteinase, NKEF-B, brain type creatine kinase, mitochondrial ATP synthase, and valosin containing enzyme represent high abundance proteins in carp spermatozoa. These proteins are functionally related to sperm motility and energy production as well as the protection of sperm against oxidative injury and stress. Moreover, carp spermatozoa are equipped with functionally diverse proteins involved in signal transduction, transcription, translation, protein turnover and transport. About 15% of proteins from carp spermatozoa identified here were also detected in seminal plasma which may be a result of leakage from spermatozoa into seminal plasma, adsorption of seminal plasma proteins on spermatozoa surface, and expression in both spermatozoa and cells secreting seminal plasma proteins. The availability of a catalog of carp sperm proteins provides substantial advances for an understanding of sperm function and for future development of molecular diagnostic tests of carp sperm quality, the evaluation of which is currently limited to certain parameters such as sperm count, morphology and motility or viability. The mass spectrometry data are available at ProteomeXchange with the dataset identifier PXD000877 (DOI: http://dx.doi.org/10.6019/PXD000877).
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http://dx.doi.org/10.1016/j.cbd.2014.09.003DOI Listing
December 2014

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

Fish Shellfish Immunol 2014 Dec 22;41(2):389-401. Epub 2014 Sep 22.

Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn, Department of Gamete and Embryo Biology, Poland.

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.
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http://dx.doi.org/10.1016/j.fsi.2014.09.020DOI Listing
December 2014

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

J Proteomics 2014 Feb 13;98:218-32. Epub 2014 Jan 13.

Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Department of Gamete and Embryo Biology, Olsztyn, Poland.

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.
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http://dx.doi.org/10.1016/j.jprot.2014.01.005DOI Listing
February 2014

Isolation, characterisation and cDNA sequencing of a new form of parvalbumin from carp semen.

Reprod Fertil Dev 2014 Oct;26(8):1117-28

Department of Gamete and Embryo Biology, Semen Biology Group, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, 10-747 Olsztyn, Poland.

Parvalbumins (Pv) are calcium-binding proteins present mainly in the muscle and nervous system where they act as a Ca(2+) buffer. Our previous work demonstrated the presence of Pv-I in carp semen and indicated the presence of a second Pv (Pv-II). The purpose of the present work was to identify, purify and determine the full-length cDNA sequence of Pv-II from carp testis. Pv-II from seminal plasma was purified by ion-exchange chromatography (IEC) and preparative electrophoresis, while the Pv-II from spermatozoa was purified by IEC, gel filtration and preparative electrophoresis. The purified Pv-II was submitted to an analysis of molecular mass, isoelectric point (pI), amino-acid sequence and oligomerisation ability. The amino-acid sequence was used to construct primers and obtain the full-length cDNA sequence of seminal-specific Pv-II from carp testis. Analysis of the cDNA sequence indicated that carp-testis Pv-II was distinct from carp-muscle parvalbumins. Pv-II was distinct from Pv-I regarding sequence, molecular mass and pI. Both parvalbumins had the ability to form oligomers or to bind to other proteins. Carp seminal plasma had a protective effect against parvalbumin oligomerisation. Pv-II underwent post-translational modification such as n-acetylation and cysteinylation. The present study is the first to report the full-length cDNA sequence of parvalbumin from carp testis.
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http://dx.doi.org/10.1071/RD13181DOI Listing
October 2014

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

Comp Biochem Physiol C Toxicol Pharmacol 2011 May 22;153(4):422-9. Epub 2011 Feb 22.

Department of Gamete and Embryo Biology, Semen Biology Group, Institute of Animal Reproduction and Food Research, 10-747 Olsztyn, Poland.

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.
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http://dx.doi.org/10.1016/j.cbpc.2011.02.003DOI Listing
May 2011