Human sperm tail proteome suggests new endogenous metabolic pathways

Abstract

Proteomic studies are contributing greatly to our understanding of the sperm cell, and more detailed descriptions are expected to clarify additional cellular and molecular sperm attributes. The aim of this study was to characterize the subcellular proteome of the human sperm tail and, hopefully, identify less concentrated proteins (not found in whole cell proteome studies). Specifically, we were interested in characterizing the sperm metabolic proteome and gaining new insights into the sperm metabolism issue. Sperm were isolated from normozoospermic semen samples and depleted of any contaminating leukocytes. Tail fractions were obtained by means of sonication followed by sucrose-gradient ultracentrifugation, and their purity was confirmed via various techniques. Liquid chromatography and tandem mass spectrometry of isolated sperm tail peptides resulted in the identification of 1049 proteins, more than half of which had not been previously described in human sperm. The categorization of proteins according to their function revealed two main groups: proteins related to metabolism and energy production (26%), and proteins related to sperm tail structure and motility (11%). Interestingly, a great proportion of the metabolic proteome (24%) comprised enzymes involved in lipid metabolism, including enzymes for mitochondrial beta-oxidation. Unexpectedly, we also identified various peroxisomal proteins, some of which are known to be involved in the oxidation of very long chain fatty acids. Analysis of our data using Reactome suggests that both mitochondrial and peroxisomal pathways might indeed be active in sperm, and that the use of fatty acids as fuel might be more preponderant than previously thought. In addition, incubation of sperm with the fatty acid oxidation inhibitor etomoxir resulted in a significant decrease in sperm motility. Contradicting a common concept in the literature, we suggest that the male gamete might have the capacity to obtain energy from endogenous pools, and thus to adapt to putative exogenous fluctuations.

Fig. 1.

Human sperm tails can be physically isolated by means of sonication and sucrose gradient ultracentrifugation. At the top left of the figure, a low-magnification phase contrast microscopy image of sperm cells purified after percoll density selection and CD45-MACS purification is shown to demonstrate the absence of potentially contaminating cells. The nature of isolated sperm tail (and head) fractions was visualized via optical microscopy (A, tails; A', heads). The expression of alpha-tubulin was detected via immunofluorescence (B, B', Hoescht; C, C', anti-alpha-tubulin; B, C, tails; B', C', heads) and Western blotting (D, alpha-tubulin has a molecular weight of 55 kDa).

Fig. 2.

Electron microscope pictures of human sperm tail fractions isolated by means of sonication and sucrose gradient ultracentrifugation. All the typical tail structures were observed: axonemes (Ax), outer dense fibers (Odf), mitochondrial sheaths (Ms), and fibrous sheaths (Fs).

Fig. 3.

Similarity between the tail proteome described in the present study and those of other published human sperm proteomics descriptions. Martinez-Heredia et al., 2006 (3); Baker et al., 2007 (4); Siva et al., 2010 (50); de Mateo et al., 2011 (8); others (, , , , –, –55). Of the 1049 proteins identified here, 527 were not previously described in human sperm.

Fig. 4.

Classification of the sperm tail proteins according to the information available at the UniProtKB/Swiss-Prot web site. In the top panel (A), the tissue specificity of the proteins identified is shown. In the bottom panel (B), the known subcellular localization is indicated.

Fig. 5.

Classification of the sperm tail proteins according to their main function(s) using the information available at the UniProtKB/Swiss-Prot web site. It is interesting to note that 26% of the proteins belong to the “Metabolism and energy production” group. This group is shown in higher detail in the lower right part of the figure.

Fig. 6.

Human sperm express peroxisomal proteins in the midpiece. A, expression of peroxisomal membrane protein 11 (PEX11; upper panel, green) and peroxisomal 3-ketoacyl-CoA thiolase (ACAA1; lower panel, green) detected via immunocytochemistry. DNA was stained with Hoescht (blue). B, expression of ACAA1 different sperm samples (n = 8) detected via Western blotting. The predicted molecular weight of human ACAA1 is 44 kDa. Several molecular weight standard bands (75 kDa, 50 kDa, 37 kDa, and 25 kDa) are shown on the left.

Fig. 7.

Inhibition of mitochondrial fatty acid oxidation affects human sperm motility without significantly affecting viability. Sperm samples (n = 7) were incubated with different concentrations of etomoxir, and motility and viability were assessed according to World Health Organization guidelines (29) after short-term (3 h) and long-term (48 h) incubations. Data are expressed as mean ± S.E. of percentages relative to controls (i.e. absence of etomoxir). Asterisks denote significant differences relative to controls (p < 0.05).