Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility

<p>Abstract</p> <p>Background</p> <p>Male infertility is a major problem for mammalian reproduction. However, molecular details including the underlying mechanisms of male fertility are still not known. A thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder.</p> <p>Results</p> <p>Using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) and identified 125 putative biomarkers of fertility. We next used quantitative Systems Biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. Compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. Our data also suggests a hypothesis that low fertility sperm DNA integrity may be compromised because cell cycle: G₂/M DNA damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa.</p> <p>Conclusion</p> <p>This is the first comprehensive description of the bovine spermatozoa proteome. Comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype.</p

Application of Proteomics to Identify Fertility Markers in Angus Bull Sperm

The goal of the study was to ascertain sperm proteins as fertility markers by identifying sperm proteins in Angus bull sperm using proteomics and validate the markers through comparative sperm biology between Angus and Holstein bulls for which there is reliable fertility data available. We aimed to determine proteins differentially expressed in sperm from Angus bulls with different fertility phenotypes. Two-dimensional differential gel electrophoresis with mass-spectrometry, functional gene clusters, canonical pathways and protein networks, using integrated discovery bioinformatics software and ingenuity pathway analysis were used to identify and analyze sperm proteome. We identified 80 proteins that were differentially expressed in sperm of our experimental population. Using computational biology approaches we demonstrated involvement of structural proteins such as outer dense fiber of sperm tails 2 and enzymes including kinases, and phosphatases having functions in essential pathways in glycolysis/gluconeogenesis and free scavenging. The results are significant because analyzed proteins in Angus sperm are determinants of fertility, gene-environment interactions, as well as potential biomarkers for animal breeding

Protein markers in Angus bull spermatozoa for fertility

In the field of mammalian reproduction, the success rate of fertilization largely relies on spermatozoa fertility potential. Total proteins change to a large degree during gametogenesis to activate gametes. There are some techniques that are most popular for proteomic studies of fertilization, including SDS-PAGE, 2D-DIGE, Western Blotting and Immunocytochemistry, as well as Mass Spectrometry. Proteins and their cofactors play important roles at different stages of gametogenesis, fertilization and embryo development. However, insufficiencies in the construction mode of fertility hinder the techniques in determination the sperm fertility. This study focused on identifying and tracking crucial proteins for fertility based on comparison between high- and lowertility sperms by means of 2D-DIGE and immunoblotting. We identified 18 proteins that varied significantly, including Outer dense fiber 2 (ODF2) of sperm tails and Manganous superoxide dismutase (MnSOD). Differences in these proteins suggest that posttranslational modification and deoxydation of sperm proteins might be associated with fertility

Evaluation of the proteomic profiles of ejaculated spermatozoa from Saanen bucks (Capra hircus).

Abstract: The Saanen goat breed has been widely explored in breeding programmes; however, there are few reports about the breed?s genetic and molecular composition. Thus, this study aimed to characterize the proteomic profile of spermatozoa from Saanen breeding goats. Five breeding animals with proven fertility were selected, the spermatozoa were collected, and the protein was extracted. Subsequently, the proteins were separated and analysed by two-dimensional electrophoresis and mass spectrometry; the proteins were then identified with the SwissProt database. A total of 31 proteins involved in reproduction were identified, including binding proteins on spermatozoa for fusion with the egg, acrosomal membrane proteins, metabolic enzymes, heat shock proteins, cytoskeletal proteins and spermatozoa motility proteins. The characterization of such proteins clarifies the molecular mechanisms of spermatogenesis and the modifications that ensure the success of fertilization

Expressão gênica de protaminas e proteínas nucleares de transição em testículos bovinos

Protamines (PRM) are the major DNA-binding proteins in the sperm nucleus and can pack the DNA into less than 5% of the volume of a somatic cell nucleus. It is already known that bulls only have the PRM1 protein on mature spermatozoa while most mammals also have the PRM2. Transition nuclear proteins (Tnps) and PRMs are fundamental to DNA integrity. It has already been reported the influence of PRM on chromatin structures, generating low fertility. However, molecular mechanisms underlying these effects are not known. The relative expression of PRM1, PRM2, PRM3, Tnp1 and Tnp2 was determined by real time RT-PCR, using bovine specific primers and β-actin as endogenous control. Quantification of mRNA relative expression showed a higher expression of PRM1 compared to the other genes. The PRM3 mRNA had the lowest relative expression. A significant (p &lt; 0.05) and positive correlation was found between PRM1 and PRM2 (r = 0.518), PRM2 and Tnp1 (r = 0.750), PRM2 and Tnp2 (r = 0.706), PRM3 and Tnp1 (r = 0.542), PRM3 and Tnp2 (r = 0.731) and between Tnp1 and Tnp2 (r = 0.820). Since most of the knowledge about protamine 2 in bovine is based on a work from 1990 and according to new studies we know that PRM1 and PRM2 are important to bull fertility, more research is needed to elucidate the real function of protamines on bovines.Protaminas (PRM) são as principais proteínas ligantes do DNA espermático e podem compactar o núcleo do espermatozoide em menos de 5% do volume de uma célula somática. Já se sabe que o touro produz apenas a PRM1 em espermatozoide maduro, enquanto a maioria dos mamíferos também produz a PRM2. As proteínas nucleares de transição (Tnps) e as PRMs são fundamentais para a integridade do DNA. Já foi descrita a influência das protaminas na estrutura da cromatina e a associação destas com a fertilidade. Entretanto, os mecanismos moleculares que geram mudanças na cromatina espermática são desconhecidos. A expressão relativa da PRM1, PRM2, PRM3, Tnp1 e Tnp2 foi determinada para dez testículos de touros oriundos de matadouros comerciais, utilizando a técnica de RT-PCR em tempo real, com primers específicos para bovinos e a β-actina como controle endógeno. Ao quantificar a expressão relativa do RNAm, detectou-se alta expressão relativa da PRM1, em comparação aos outros genes. A expressão relativa da PRM3 foi a menor de todos os genes. Foram encontradas correlações positivas e significantes (p &lt; 0,05) entre PRM1 e PRM2 (r = 0,518), PRM2 e Tnp1 (r = 0,750), PRM2 e Tnp2 (r = 0,706), PRM3 e Tnp1 (r = 0,542), PRM3 e Tnp2 (r = 0,731) e entre Tnp1 e Tnp2 (r = 0,820). Visto que a maioria dos conhecimentos sobre a PRM2 estão baseados em um trabalho de 1990 e, de acordo com recentes estudos se sabe que a PRM1 e a PRM2 são importantes para a fertilidade do touro, mais estudos são necessários para determinar a real função das protaminas em touros

ProAKAP4 Semen Concentrations as a Valuable Marker Protein of Post-Thawed Semen Quality and Bull Fertility : a Retrospective Study

Functional sperm quality markers to predict bull fertility have been actively investigated. Among them, proAKAP4, which is the precursor of AKAP4, the main structural protein in the fibrous sheath of spermatozoa; appears to be promising, especially since spermatozoa lacking AKAP4 expression were shown to be immotile, abnormal, and infertile. In this study, the objective was to evaluate proAKAP4 concentration values with the classic sperm motility descriptors and fertility outcomes (NRR at 90 days) in post-thawed conditions of 10 bulls' semen. ProAKAP4 expression was confirmed by Western blotting and proAKAP4 concentrations were determined by ELISA. Variations in proAKAP4 concentrations were observed independently of the motility sperm descriptors measured using computer-assisted semen analysis (CASA). A ProAKAP4 concentration of 38.67 ± 8.55 ng/10 million spermatozoa was obtained as a statistical mean of all samples. Threshold values of proAKAP4 were then determined between 19.96 to 96.95 ng/10 million spermatozoa. ProAKAP4 concentrations were positively correlated with progressive motility and the linearity coefficient. The sperm showing the lowest progressive motility were the samples exhibiting proAKAP4 concentrations below 20 ng/10 million spermatozoa. Furthermore, proAKAP4 concentrations were significantly higher in bulls with a higher NRR in the field. Our results demonstrate a correlation between the semen concentration of proAKAP4 and NRR-90d (p = 0.05) in post-thawed bull semen, highlighting the potential of proAKAP4 as a predictive marker of bull fertility

Diversity of ejaculated sperm proteins in Moxotó bucks (Capra hircus) evaluated by multiple extraction methods.

Abstract: This study aimed to develop protocols for the extraction of sperm proteins from Moxotó goats (Capra hircus) and to compare the resulting proteomic maps. The sperm proteins were isolated using an extraction buffer containing 7 M urea and 2 M thiourea, 20 mM DTT, and one of the following detergents: 1% or 4% CHAPS; 1% or 4% SDS; 1% or 4% Triton X-100; or a combination of CHAPS and SDS. The 1-DE and 2-DE profiles of the isolated proteins revealed that the various isolation methods were efficient. Qualitative and quantitative differences in the 1-DE and 2-DE profiles were observed. 2-DE maps indicated that the amount and diversity of proteins visualized depended on the detergent that was used. Furthermore, this work revealed that the combination of detergents increased the resolution of some spots and retained the characteristics of the individual detergents, depending on their concentrations

Sperm Proteomics: Road to Male Fertility and Contraception

Spermatozoa are highly specialized cells that can be easily obtained and purified. Mature spermatozoa are transcriptionally and translationally inactive and incapable of protein synthesis. In addition, spermatozoa contain relatively higher amounts of membrane proteins compared to other cells; therefore, they are very suitable for proteomic studies. Recently, the application of proteomic approaches such as the two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential in-gel electrophoresis has identified several sperm-specific proteins. These findings have provided a further understanding of protein functions involved in different sperm processes as well as of the differentiation of normal state from an abnormal one. In addition, studies on the sperm proteome have demonstrated the importance of spermatozoal posttranslational modifications and their ability to induce physiological changes responsible for fertilization. Large-scale proteomic studies to identify hundreds to thousands of sperm proteins will ultimately result in the development of novel biomarkers that may help to detect fertility, the state of complete contraception, and beyond. Eventually, these protein biomarkers will allow for a better diagnosis of sperm dysfunctions and aid in drug development. This paper reviews the recent scientific publications available from the PubMed database to address sperm proteomics and its potential application to characterize male fertility and contraception

A proteomic approach to identifying spermatozoa proteins in Indonesian native Madura bulls

Proteins assist sperm mature, transit the female reproductive tract, and recognise sperm oocytes. Indigenous Indonesian bulls, Madura bulls, have not been studied for reproductive proteomics. As local Indonesian beef livestock, Madura cattle assist in achieving food security; hence, their number must be improved. Thus, the identification of molecular proteomics-based bull fertility biomarkers is needed. This study aimed to characterise the sperm fertility function of the superior Madura bull (Bos indicus × Bos Javanicus) spermatozoa proteome. Frozen semen from eight Madura superior bulls (Bos indicus × Bos javanicus) aged 4–8 years was obtained from the artificial insemination centre (AIC) in Singosari and Lembang. Madura superior bulls are those that have passed the bull breeding soundness evaluation. Frozen sperm were thawed and centrifuged at 3000 × g for 30 min. Proteins in sperm were characterised through proteomic analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The resulting gene symbols for each protein were then subjected to bioinformatics tools, including UniProt, DAVID, and STRING databases. Regarding sperm fertility, the analysis revealed that 15 proteins were identified in the sperm of Madura bulls. Amongst the identified proteins, the superior Madura bull sperm contained several motilities, energy-related proteins, and chaperone proteins. A substantial portion of characterised proteins are linked to metabolic pathways and the tricarboxylic acid (TCA) cycle, contributing to sperm energy production. In conclusion, the first in-depth proteome identification of sperm related to sperm quality and bull fertility of a unique indigenous Madura breed of Indonesia was performed using the LC–MS/MS proteomic method. These findings may serve as a reference point for further studies related to the functions of bovine sperm and biomarkers of fertility and sperm quality

Studies on the composition variation and function of seminal plasma

The application of seminal plasma in vitro and its role in vivo during cervical transit has long been questioned due to the contradictory results published throughout the literature. This thesis examines the complex nature of ram seminal plasma, providing a link between seminal plasma composition and sperm function during cryopreservation and transit following deposition in the female tract. The results reported herein clearly show that exposure to seminal plasma is fundamentally important for the successful transit of ram spermatozoa through the cervix and its variable proteomic composition is responsible for its contradictory effect on sperm function, which is reported throughout literature. Additionally, its cryoprotective effect varies depending on the presence and concentration of individual proteins.. It has begun the arduous task of screening seminal plasma proteins for their effect on sperm function, but further research is necessary to unequivocally identify and confirm these markers within this biological fluid. Nonetheless, these findings lay the groundwork for the future supplementation of spermatozoa with seminal plasma proteins. These proteins could ameliorate the apparent alterations which occur to the sperm membrane during cryopreservation, which may prevent the cervical transit of frozen-thawed ram spermatozoa. Success in this endeavour would facilitate cervical artificial insemination of ewes with frozen-thawed semen and allow the Australian sheep industry to finally realise the rapid genetic progress capable from the widespread use of elite sires