Bruno Ramires Macedo Costa1, Ronaldo Queiroz dos Santos1, Jorge André Matias Martins1, João Paulo Arcelino do Rego1, Ana Cristina de Oliveira Monteiro Moreira2, Arlindo de Alencar Araripe Moura1
The search for molecular markers of puberty and sexual maturity aims to elucidate the interactions between seminal plasma proteins and sperm cells from the moment of ejaculation to fertilization. It is therefore assumed that the proteomic composition of the seminal plasma in Guzerá bulls undergoes modifications throughout reproductive development, influencing the physiological processes of sexual maturation. Thus, the objective of this study was to investigate the profile of seminal plasma (SP) proteins during the transition from puberty to sexual maturity in Guzerá bulls. Five clinically healthy bulls, aged from 24 to 29 months, were used in this study. Animals were raised in the semi-arid region of Northeastern Brazi (Quixeramobim, Ceará) under native pasture. Semen was collected monthly via electroejaculation, and subsequently centrifuged to separate SP from sperm. Seminal plasma proteins (750 µg) were precipited (2-D Clean-Up® kit; GE Lifesciences, USA), solubilized in rehydration buffer and separated by 2D-SDS-PAGE. Gels were stained with Coomassie Brilliant Blue G-250 and analyzed using PDQuest software for spot detection and quantification. Spots of interest were excised, destained, digested with trypsin, and the resulting peptides, analyzed by NanoUPLC-MS/MS (Synapt G1 HDMS, Waters Corporation, USA). Protein identification was performed using the NCBInr database via Mascot software, with specific search parameters. Identified proteins were submitted to gene ontology analysis (PANTHER, UniProtKB) and protein–protein interaction mapping (STRING 9.0). There were 32 proteins identified from 22 spots, among which 15 were differentially expressed throughout the evaluated period (24 to 29 months). These included: carbonic anhydrase inhibitor, sulfhydryl oxidase, glucose-6-phosphate, (S)-3-amino-2-methylpropionate transaminase, plasma serine protease inhibitor, immunoglobulin gamma heavy chain, SLAM family member 9, glutathione peroxidase, ephrin-A1, metalloproteinase inhibitor 2, seminal plasma protein A3, lipocln_cytosolic_FA-bd_dom-containing protein, spermadhesin-1, PDC-109, and spermadhesin Z13, between the transition period of puberty and sexual maturity. Furthermore, proteins such as BSP-30 kDa, spermadhesin-1, PDC-109, Wnt protein, epididymal sperm-binding protein 1, 14-3-3 beta/alpha protein, and junction plakoglobin were expressed from 25 months until 29 months. From 29 months, proteins associated with sexual maturity included osteopontin, double-headed protease inhibitor, SLAM 9, BPI-fold-containing protein, sex hormone-binding globulin (SHBG), alpha-galactosidase, tissue factor pathway inhibitor 2, malate dehydrogenase, clusterin, apolipoprotein A-I, among others. These results suggest that the expressions of specific seminal plasma proteins vary with age and may be associated with reproductive maturation, indicating their potential role as molecular biomarkers of sexual maturity in Guzerá bulls.
Agradecimentos: The authors acknowledge and thank CAPES, CNPq, and FUNCAP for financial support. Canhotinho farm is also recognized for their great support and assistance of this study.