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Abstract

Phosphorylation and dephosphorylation of proteins are considered to be the most important processes in sperm maturation during epididymal transit. The main aim of this study was to isolate and identify phosphoproteins from the epididymal milieu obtained from reproductively mature stallions during and out of the breeding season. With the use of 1D-PAGE and nanoLC-MS/MS, we identified phosphoproteins that fulfil various functions: regulatory, transport, motility, ubiquitination, chaperone, antioxidant, apoptotic and enzymatic. Moreover, we characterized tyrosine, serine and threonine phosphorylation patterns, taking into consideration the seasonal and epididymal segment variables. The intensity of phosphorylation and profiles of phosphoproteins varied in subsequent regions of the epididymis. With the use of western and immunoblot tests, we demonstrated that fourteen proteins underwent phosphorylation both during and out of the breeding season. However, significant differences (p≤0.05) in the phosphorylation status were demonstrated in the case of 44 kDa (glutamine synthetase), 38 kDa (malate dehydrogenase), 34 kDa (clusterin/inorganic pyrophosphatase), 31 kDa (clusterin/ /ubiquitin thioesterase), 29 kDa (14-3-3 protein/purine nucleotide phosphorylase) for the season factor and 55 (Rab GDP dissociation inhibitor alpha) and 31 kDa ((clusterin/ubiquitin thioesterase) proteins for the segment factor. The occurrence of the other phosphoproteins was spontaneous among individuals and in both seasons.
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Authors and Affiliations

K. Dyrda
1
A. Orzołek
1
J. Ner-Kluza
2
P. Wysocki
1

  1. Department of Animal Biochemistry and Biotechnology, University of Warmia and Mazury, Oczapowskiego 5, 10-719 Olsztyn, Poland
  2. Department of Biochemistry and Neurobiology, Faculty of Materials Science and Ceramics, University of Science and Technology, A. Mickiewicza 30, 30-059 Krakow, Poland
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Abstract

During the rutting season, stag semen is accompanied by a sticky, dense secretion called yellow fraction (YF). There is little information about the role, biology, physiology, and most importantly, the composition of this fluid. The aim of this study was to isolate and identify zinc ions (ZnBPs) and heparin binding proteins (HBPs) from YF of the red deer ( Cervus elaphus L.). Using liquid chromatography, the presence of 6 fractions of ZnBPs (71, 65, 55, 16, 14 and 12 kDa) and 22 fractions of HBPs (163, 140, 96, 78, 71, 65, 55, 49, 33, 31, 26, 25, 24, 22, 18, 16, 13, 12, 11, 10, 9 and 8 kDa) in YF proteome was demonstrated. By means of two-dimensional electrophoreses and MALDI-TOF/TOF mass spectrometry some of them were then identified. Amongst ZnBPs the following were identified: glutaminyl-peptide cyclotransferase, inhibitor of carbonic anhydrase-like, potassium voltage-gated channel subfamily E member 2, WD repeat-containing protein 38 isoform X4. Amongst the HBPs metalloproteinase inhibitor 2 (TIMP2), seminal plasma glycoprotein PSP-I and adseverin (scinderin) were identified. Identifying all ZnBPs and HBPs present in YF may broaden up-to-date knowledge concerning the biology, physiology and preservation of red deer semen.
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Authors and Affiliations

A. Orzołek
1
K. Dyrda
1
K. Rafalska
1
P. Wysocki
1
W. Kordan
1
W. Giżejewski
2

  1. Department of Animal Biochemistry and Biotechnology, University of Warmia and Mazury, Oczapowskiego 5, 10-719 Olsztyn, Poland
  2. Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland

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