Cytochrome c Upregulation during Capacitation and Spontaneous Acrosome Reaction Determines the Fate of Pig Sperm Cells: Linking Proteome Analysis

Abstract

Abstract.To identify the mechanisms underlying capacitation, we undertook a high-resolution differential proteomic analysis of pig sperm cells. Two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry analyses led to identification of 56 differentially expressed proteins. After induction of capacitation in vitro, the wellestablished markers of the capacitation (lactadherin P47, acrosomal protein SP-10 precursor, prohibitin, proteasomes, DJ-1 protein and arylsulfatase-A) and TCA cycle proteins (isocitrate dehydrogenase, malate dehydrogenase and pyruvate dehydrogenase) were identified. During induction, cytochrome c expression via the p53 pathway increased, however apoptotic executors, such as caspase-3, decreased significantly. Therefore, we tested the hypothesis that cytochrome c upregulation in spermatozoa is capable of activating tyrosine phosphorylation for capacitation, rather than apoptosis. Exposure of sperm cells to soluble Na2CrO4 [Cr (VI)], which induces cytochrome c upregulation, caused a dose-and time-dependent increase in tyrosine phosphorylation of sperm proteins in non-capacitating medium. In contrast, supplementation of cyclosporin A, which blocks cytochrome c upregulation, inhibited tyrosine phosphorylation of sperm proteins. Furthermore, spermatozoa in capacitation medium or non-capacitation media supplemented with soluble Cr (VI) showed similar levels of capacitation. These findings indicate that differential expression of many of these proteins has previously been unrecognized in sperm cells incubated in capacitation medium also suggest that a gradual increase of cytochrome c during incubation to induce capacitation determines sperm cell fate, i.e., apoptosis or further development for fertilization. Key words: Capacitation, Cytochrome c upregulation, 2-dimensional gel electrophoresis, Pig, Sperm maturation (J. Reprod. Dev. 54: [68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83] 2008) permatozoa are terminally differentiated and specialized cells [1]. The deficiencies of major transcriptional and translational activities of sperm are generally known [2] however, recent published evidence shows that sperm are capable of producing major nuclear gene expressions using 55S mitochondrial ribosomes during their residence in the female reproductive tract until fertilization [3]. Mature sperm cells are released from the testis with tightly supercoiled, transcriptionally inert DNA [4]. In addition, they do not exhibit progressive motility, but acquire this ability during passage through the epididymis. These processes are referred to as maturation; other maturational changes include completion of nuclear condensation and changes in the expression and distribution of molecules on the cell surface [1]. Therefore, freshly ejaculated sperm cells are incapable of fertilization and must spend some time in a suitable environment in order to capacitate.Capacitation in all mammals involves shedding of the sperm plasma membrane followed by hyperactivity of the tail, attachme...

Effects of Lunasia amara Blanco (Sanrego) on Male Fertility: A Preliminary Study on Sperm Proteomic Analysis

“…Following ejaculation, Eno1, ATP synthase, and tubulin regulate the sperm capacitation. Besides, a prior study demonstrated that sperm undergoing capacitation displayed increased expression of Eno1 (Choi et al, 2008). The level of sperm motility required in the female reproductive tract is determined by ODF2, PGK2, LDHC, DNAJB13, Sp17, and tubulin.…”

Sperm proteasome and fertilization

“…They include the ubiquitin activating enzyme UBE (Baker et al 2010), multiple proteasomal subunits (Arcelay et al 2008, Baker et al 2010, the valosine-containing protein, involved in ubiquitinated substrate presentation to the 26S proteasome (Geussova et al 2002), and ubiquitin itself (Arcelay et al 2008). Changes in the electrophoretic migration pattern of several 20S core subunits were observed by 2D electrophoresis of capacitated versus non-capacitated boar spermatozoa (Choi et al 2008).…”

HORMONE-DEPENDENT AND HORMONE-INDEPENDENT CONTROL OF METABOLIC AND DE­VEL­OP­MEN­TAL FUNCTIONS OF MALATE DEHYDROGENASE – Review

“…Also development and capacitation of mammalian sperm are accompanied by expression of several marker proteins including MDH and isocitrate dehydrogenase (representatives of TCA cycle), and several other proteins like lactadherin P47, acrosomal protein SP-10 precursor, prohibitin, DJ-1 protein, arylsulfatase-A, and cytochrome c (CHOI et al, 2008). At least expression of cytochrome c appears to be regulated via p53 pathway and may be involved in decision developmentallylinked programmed cell death or further spermiogenesis.…”