Fine resolution of human sperm nucleoproteins by two-dimensional electrophoresis

Abstract

Human sperm nucleoproteins consist of protamines and histones. Changes in composition of these proteins are thought to correlate with spermatogenesis and may be involved in some instances of male infertility. We sought to separate sperm nucleoproteins including variants of protamine using an improved two-dimensional electrophoretic method, with the aim of comprehensively analysing all sperm nucleoprotein constituents. After extracting nuclear basic proteins from the sperm of normal volunteers, we analysed these proteins on a gel sheet by a radical free, highly reducing method based on Kaltschmidt and Whittmann's two-dimensional electrophoresis. Basic proteins from sperm nuclei were separated clearly into 12 spots. By amino acid sequence analysis, these spots corresponded to protamine 1 (P1)- (five spots), protamine 2 (P2)-related proteins (six spots) and testis-specific histone H2B (one spot). The N-terminal amino acid sequences of the six P2-related proteins were compatible with those of HPI1, HPI2, HPS1, HPS2, HP2 and HP3, and quantitative comparison could be performed. In conclusion, human sperm nucleoproteins including all P2-related variants could be analysed quantitatively with high resolution on a single electrophoretic gel.

Proteomic identification of human sperm proteins

“…Protamine identification and analysis has been an extensively studied issue both in normal spermatogenesis and in spermatozoa from infertile patients [6,[14][15][16][17][18][19][20][21]43]. Recently an approach to specifically extract, separate and identify human protamines by MS has been described [8]. Other proteins not identified in the present work are A kinase anchoring proteins (AKAPs), possibly because their molecular weight is out of the range studied in the present work [11].…”

“…As we anticipated, in the present work we have not detected the protamines, and indeed the detection of protamines was not one of our goals. Human protamines contain approximately 50% arginine residues, representing the most basic proteins known in nature [1,5,6,8,43]. Because of their highly positive charge, they are focused at one end of the IEF strip, thus out of the range of pI that we decided to analyze in the present work.…”

“…The general lysis and extraction procedure used has the advantage that it provides a general idea of the types of proteins that constitute the sperm proteome. Therefore, this approach should be complementary to other proteomic approaches focusing on specific compartments or organelles such as the cytoplasm, membrane, cytoskeleton, tail, acrosome or sperm head [8][9][10][11][24][25][26][27][28].…”

“…Many efforts to identify sperm proteins have been directed to the nuclear proteins [1,[5][6][7][8], to identify the proteins required for sperm motility [9][10], and in the understanding of the membrane proteins required for capacitation, egg interaction and fertilization [11]. So far, most of the fundamental knowledge on the sperm protein composition has been gained using conventional protein purification and identification strategies [1-6, 12, 13].…”

The ’omics revolution and our understanding of sperm cell biology

“…Thus, 2D-PAGE applied to the nucleus of spermatozoa demonstrated 12 spots corresponding to protamines P1 and P2. 49 A quantitative analysis of these spots demonstrated a higher P1 to P2 level in two of the 184 men undergoing male infertility diagnosis. 50,51 A second way to overcome the inherent difficulties of 2D-PAGE is to use internally labeled fluorescent dyes, in the process now known as 'difference in gel electrophoresis' or DIGE.…”