Isolation and identification of proteins from swine sperm chromatin and nuclear matrix
Guilherme Arantes Mendonça, Romualdo Morandi Filho, Elisson Terêncio Souza, Thais Schwarz Gaggini, Marina Cruvinel Assunção Silva-Mendonça, Robson Carlos Antunes, Marcelo Emílio Beletti
Abstract
The aim of this study was to perform a proteomic analysis to isolate and identify proteins from the swine sperm nuclear matrix to contribute to a database of swine sperm nuclear proteins. We used prechilled diluted semen from seven boars (19 to 24 weekold) from the commercial line Landrace x Large White x Pietran. The semen was processed to separate the sperm heads and extract the chromatin and nuclear matrix for protein quantification and analysis by mass spectrometry, by LTQ Orbitrap ELITE mass spectrometer (Thermo-Finnigan) coupled to a nanoflow chromatography system (LC-MS/MS). We identified 222 different proteins in the sample; a total of 159 (71.6%) were previously described as present in the somatic or sperm nuclei of other species, 41 (18.5%) did not have a previously reported nuclear presence and 22 (9.9%) had not been characterized. The most abundant family of proteins corresponded to ribosomal (13.1%), followed by cytoskeleton (12.2%), uncharacterized (9.9%), histones (5.4%), proteasome subunits (3.6%) and heat shock (1.8%). The other proteins clustered in other families accounted for 54% of the total proteins. The protein isolation of the nuclear matrix of the swine spermatozoa was satisfactory, thus demonstrating that the protocol used was efficient. Several proteins were identified and described. However, it was not possible to identify some protein structures. Therefore, this study helps to establish a starting point for future proteomic studies comparing fertile and sub-fertile animals.
Keywords
References
Almeida LO, Garcia CB, Matos-Silva FA, Curti C, Leopoldino AM. 2014. Accumulated SET protein upregulates and interacts with hnRNPK, increasing its binding to nucleic acids, the Bcl-xS repression, and cellular proliferation. Biochem Biophys Res Commun, 445:196-202.
Andrabi SMH. 2007. Mammalian sperm chromatin structure and assessment of DNA fragmentation. J Assist Reprod Genet, 24:561-569.
Applequist SE, Keyna U, Calvin MR, Beck-Engeser GB, Raman C, Jäck HM. 1995. Sequence of the rabbit glyceraldehyde-3-phosphate dehydrogenaseencoding Cdna. Gene, 163:325-326.
Beletti ME, Costa LF, Guardieiro MM. 2005. Morphometric features and chromatin condensation abnormalities evaluated by toluidine blue staining in bull spermatozoa. Braz J Morphol Sci, 22:85-90.
Beletti ME. 2013. Cromatina espermática: quebrando paradigmas. Rev Bras Reprod Anim, 37:92-96.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biochem, 72:248-254.
Chen M, Wang H, Li X, Li N, Xu G, Meng Q. 2014. PLIN1 deficiency affects testicular gene expression at the meiotic stage in the first wave of spermatogenesis. Gene, 543:212-219.
Codrington AM, Hales BF, Robaire B. 2007. Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa. Hum Reprod, 22:1431-1442.
DeMateo S, Castillo J, Estanyol JM, Ballescá JL, Oliva R. 2011. Proteomic characterization of the human sperm nucleus. Proteomics, 11:2714-2726.
D’Occhio MJ, Hengstberger KJ, Johnston SD. 2007. Biology of sperm chromatin structure and relationship to male fertility and embryonic survival. Anim Reprod Sci, 101:1-17.
Engel W, Keime S, Kremling H, Hameister H, Schluter G. 1992. The genes for protamine 1 and 2 (PRM1 and PRM2) and transition protein 2 (TNP2) are closely linked in the mammalian genome. Cytogenet Cell Genet, 61:158-169.
Galeraud-Denis I, Lambard S, Carreau S. Relationship between chromatin organization, mRNAs profile and human male gamete quality. Asian J Androl, 9:587-592.
Lambard S, Galeraud-Denis I, Martin G, Levy R. 2004. Analysis and significance of mRNA in human ejaculated sperm from normozoospermic donors: relationship to sperm motility and capacitation. Mol Hum Reprod, 10:535-541.
Lemos MS. 2013. Caracterização morfológica e bioquímica do ânulo nuclear de espermatozoide bovino. Uberlandia, Brazil: Federal University of Uberlândia. Thesis.
MacLeod G, Taylor P, Mastropaolo L, Varmuza S. 2014. Comparative phosphoproteomic analysis of the mouse testis reveals changes in phosphopeptide abundance in response to Ppp1cc deletion. EuPA Open Proteomics, 2:1-16.
Maier WM, Nussbaum G, Domenjoud L, Klemm U, Engel W. 1990. The lack of protamine 2 (P2) in boar and bull spermatozoa is due to mutations within the P2 gene. Nucleic Acids Res, 18:1249-1254.
Miller D, Brinkworth M, Iles D. 2010. Paternal DNA packaging in spermatozoa: more than the sum of its parts? DNA, histones, protamines and epigenetics. Reproduction, 139:287-301.
Morandi-Filho R. 2013. Análise da estrutura e identificação de proteínas da cromatina nuclear espermática de bovinos. Uberlandia, Brazil: Federal University of Uberlandia. Thesis.
Ocampo J, Mondragon R, Roa-Espitia AL, Chiquete-Felix N, Salgado ZO, Mújica A. 2005. Actin, myosin, cytokeratins and spectrin are components of the guinea pig sperm nuclear matrix. Tissue Cell, 37:293-308.
Oliva R. 2006. Protamines and male infertility. Hum Reprod Update, 12:417-435.
Oliva R, Ballescá JL. 2012. Proteomics of the spermatozoon. Balcan J Med Genet, 15:27-30.
Pirhonen A, Linnala-Kankkunen A, Maenpaa PH. 1994. Identification of phosphoseryl residues in protamines from mature mammalian spermatozoa. Biol Reprod, 50:981-986.
Rauch A. 2006. Computational Proteomics Analysis System (CPAS): an extensible, open-source analytic system for evaluating and publishing proteomic data and high throughput biological experiments. J Proteome Res, 5:112-121.
Takai H, Masuda K, Shirahige K, Akiyama T. 2014. 5-Hydroxymethylcytosine plays a critical role in glioblastomagenesis by recruiting the CHTOPMethylosome complex. Cell Rep, 9:48-60.
Uniprot.org [homepage on the internet]. 2015. Proteins data bank, Inc. Avaiable on: http://www.uniprot.org/. Accessed on: Jun 1st 2015.
Van Koningsbruggen S, Straasheijm KR, Sterrenburg E, Graaf N, Dauwerse HG, Frants RR, Van der Maarel SM. 2007. FRG1P-mediated aggregation of proteins involved in pre-mRNA processing. Chromosoma, 116:53-64.
Wright PC, Noirel J, Ow SY, Fazeli A. 2012. A review of current proteomics technologies with a survey on their widespread use in reproductive biology investigations. Theriogenology, 77:738-765.
Yamauchi Y, Shaman JA, Ward WS. 2011. Nongenetic contributions of the sperm nucleus to embryonic development. Asian J Androl, 13:31-35.
Zhong L, Belote JM. 2007. The testis-specific proteasome subunit Pros_6T of D. melanogaster is required for individualization and nuclearmaturation during spermatogenesis. Development, 134:3517- 3525.
Facebook
Google+
Twitter
LinkedIn
Mendeley
StumbleUpon
CiteULike
Reddit
Email