title: Membrane-associated proteins of ejaculated sperm from Morada Nova rams.
abstract: The objective was to describe the profile of membrane proteins from sperm of tropically adapted Morada Nova rams (N = 5). Samples from protein-enriched fractions of ejaculated sperm (containing 400 μg of protein) were separated by two-dimensional electrophoresis and respective maps analyzed using PDQuest software (version 7.3.0; Bio-Rad). Proteins were identified using tandem mass spectrometry. Also, membrane proteins were incubated with antibodies against binder of sperm protein (BSP) 1 and bodhesin 2 (Bdh-2), components of vesicular gland secretion. For membrane proteins of ejaculated sperm, an average of 133 ± 4.6 spots were detected per gel, of which, 107 spots were consistently present on all gels. Sixty-eight spots and 37 proteins were identified using mass spectrometry, corresponding to 71.6% of the intensity of all spots detected. Three major spots identified as ram seminal vesicle protein (RSVP) 14 represented approximately 30% of the intensity of all spots. Two of the most intense spots in the gel reacted against anti-BSP1, at 14 kDa. In addition, four low molecular weight spots reacted with anti-Bdh-2 antibodies. Proteins RSVP and Bdh-2 belong to the BSP and spermadhesin families, respectively, and were previously reported as major components of ram seminal proteins. Additional proteins identified in the sperm membrane two-dimensional maps included alpha-2-heparan sulfate-glycoprotein, plasma glutamate carboxypeptidase, arylsulfatase A, cathelicidin, heat shock protein 70 kDa, angiotensin-converting enzyme, leucine aminopeptidase, and clusterin. Some proteins were present as multiple isoforms, such as tubulin (12), alpha-2-heparan sulfate-glycoprotein (5), ATP synthase (5), Bdh-2 (4) and RSVP14 (3). Based on gene ontology analysis, the most common biological processes associated with the membrane proteins were cellular processes (34%), response to stimulus (14%), and metabolic processes (11%). Binding (37%) and catalytic activity (32%) corresponded to the most frequent molecular functions for those proteins. In conclusion, we identified a diverse cohort of components of membrane proteins in ram sperm. Major proteins previously reported in seminal plasma, such as RSVP14 and Bdh-2, were also extracted from sperm membranes. Knowledge of sperm proteins is crucial for elucidating mechanisms underlying their association with sperm function.
authors.affiliation: Department of Animal Science, Federal University of Ceará, Fortaleza, Ceará, Brazil.
authors.forename: M F
authors.initials: MF
authors.lastname: van Tilburg
authors.forename: M A M
authors.initials: MA
authors.lastname: Rodrigues
authors.forename: R A
authors.initials: RA
authors.lastname: Moreira
authors.forename: F B
authors.initials: FB
authors.lastname: Moreno
authors.forename: A C O
authors.initials: AC
authors.lastname: Monteiro-Moreira
authors.forename: M J D
authors.initials: MJ
authors.lastname: Cândido
authors.forename: A A
authors.initials: AA
authors.lastname: Moura
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D005786:Gene Expression Regulation / Q000502:physiology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D012756:Sheep / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology*; D059467:Transcriptome
chemical_list: D008565:Membrane Proteins