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.
Highlights
From the ‡Human Genetics Research Group, IDIBAPS, Faculty of Medicine, University of Barcelona, Casanova 143, 08036 Barcelona, Spain, and Biochemistry and Molecular Genetics Service, Clinic Hospital, Villarroel 170, 08036 Barcelona, Spain; §Biology of Reproduction and Stem Cell Group, CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal; ¶Proteomics Unit, Scientific Technical Services, University of Barcelona, Casanova 143, 08036 Barcelona, Spain; ʈClinic Institute of Gynecology, Obstetrics and Neonatology, Clinic Hospital, Villarroel 170, 08036 Barcelona, Spain; **Department of Life Sciences, University of Coimbra, Largo Marques de Pombal, 3004-517 Coimbra, Portugal
The tail consists of a flagellum, responsible for sperm motility, and it contains a number of mitochondria in the midpiece (where the production of ATP through oxidative phosphorylation (OXPHOS)1 occurs)
Isolation of Sperm Tails—In order to choose the most appropriate approach to isolate human sperm tails, we first compared the efficiency of different methodologies described in the literature for mammalian sperm subcellular fractionation
Summary
Human sperm is a motile, differentiated haploid cell whose specialized function is to reach an oocyte and achieve fertilization. This amazing cell is composed of two main subcellular compartments, the head and the tail, with clear and distinct specific roles for each. Sperm Tail Proteomics would allow the identification of less concentrated proteins and suggest their probable cellular localization, providing further information about their biological roles [6, 7]. To this end, we have recently characterized the proteome of isolated human sperm nuclei and were able to identify 403 proteins, around half of which had not been detected in previous whole sperm proteome studies [8]. Others have described the proteome of human sperm membrane fractions [9, 10], and that work resulted in the identification of additional sperm proteins
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