Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility.
ABSTRACT: BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.
Project description:Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = -0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = -0.49, P<0.006 and r = -0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1-3 vs. Bull 4-5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.
Project description:MicroRNAs are small non-coding RNAs that regulate gene expression and thus play important roles in mammalian development. However, the comprehensive lists of microRNAs, as well as, molecular mechanisms by which microRNAs regulate gene expression during gamete and embryo development are poorly defined. The objectives of this study were to determine microRNAs in bull sperm and predict their functions.To accomplish our objectives we isolated miRNAs from sperm of high and low fertility bulls, conducted microRNA microarray experiments and validated expression of a panel of microRNAs using real time RT-PCR. Bioinformatic approaches were carried out to identify regulated targets.We demonstrated that an abundance of microRNAs were present in bovine spermatozoa, however, only seven were differentially expressed; hsa-aga-3155, -8197, -6727, -11796, -14189, -6125, -13659. The abundance of miRNAs in the spermatozoa and the differential expression in sperm from high vs. low fertility bulls suggests that the miRNAs possibly play important functions in the regulating mechanisms of bovine spermatozoa.Identification of specific microRNAs expressed in spermatozoa of bulls with different fertility phenotypes will help better understand mammalian gametogenesis and early development.
Project description:BACKGROUND: Fertility is one of the most critical factors controlling biological and financial performance of animal production systems and genetic improvement of lines. The objective of this study was to identify molecular defects in the sperm that are responsible for uncompensable fertility in Holstein bulls. We performed a comprehensive genome wide analysis of single nucleotide polymorphisms (SNP) for bull fertility followed by a second-stage replication in additional bulls for a restricted set of markers. RESULTS: In the Phase I association study, we genotyped the genomic sperm DNA of 10 low-fertility and 10 high-fertility bulls using Bovine SNP Gene Chips containing approximately 10,000 random SNP markers. In these animals, 8,207 markers were found to be polymorphic, 97 of which were significantly associated with fertility (p < 0.01). In the Phase II study, we tested the four most significant SNP from the Phase I study in 101 low-fertility and 100 high-fertility bulls, with two SNPs (rs29024867 and rs41257187) significantly replicated. Rs29024867 corresponds to a nucleotide change of C --> G 2,190 bp 3' of the collagen type I alpha 2 gene on chromosome 4, while the rs41257187 (C --> T) is in the coding region of integrin beta 5 gene on chromosome 1. The SNP rs41257187 induces a synonymous (Proline --> Proline), suggesting disequilibrium with the true causative locus (i), but we found that the incubation of bull spermatozoa with integrin beta 5 antibodies significantly decreased the ability to fertilize oocytes. Our findings suggest that the bovine sperm integrin beta 5 protein plays a role during fertilization and could serve as a positional or functional marker of bull fertility. CONCLUSION: We have identified molecular markers associated with bull fertility and established that at least one of the genes harboring such variation has a role in fertility. The findings are important in understanding mechanisms of uncompensatory infertility in bulls, and in other male mammals. The findings set the stage for more hypothesis-driven research aimed at discovering the role of variation in the genome that affect fertility and that can be used to identify molecular mechanisms of development.
Project description:Infertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing.Embryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate?<?1%. A total of 65 genes were upregulated in high fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions.Despite similar morphology and development to the blastocyst stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.
Project description:Conventional semen analyses are used to evaluate male factor fertility/infertility in humans and other animals. However, their clinical value remains controversial. Therefore, new tools that more accurately assess male fertility based on sperm function and fertilization mechanism are of interest worldwide. While protein markers in spermatozoa that might help differentiate fertile and infertile sperm have been identified, studies are in their infancy, and the markers require validation in field trials. In the present study, to discover more sensitive biomarkers in spermatozoa for predicting male fertility, we assessed protein expression in capacitated spermatozoa. The results demonstrated that cytochrome b-c1 complex subunit 2 (UQCRC2) was abundantly expressed in high-litter size spermatozoa (>3-fold). On the other hand, equatorin, beta-tubulin, cytochrome b-c1 complex subunit 1 (UQCRC1), speriolin, Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and seminal plasma sperm motility inhibitor were abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A and UQCRC1 expression negatively correlated with litter size, while UQCRC2 expression positively correlated with litter size. Finally, the putative biomarkers predicted litter size in field trials. Our study suggests that biomarkers present in spermatozoa after capacitation can help differentiate superior male fertility from below-average fertility with high sensitivity.
Project description:The objective of the study was to identify the fertility-associated metabolites in bovine spermatozoa using liquid chromatography-mass spectrometry (LC-MS). Six Holstein Friesian crossbred bulls (three high-fertile and three low-fertile bulls) were the experimental animals. Sperm proteins were isolated and protein-normalized samples were processed for metabolite extraction and subjected to LC-MS/MS analysis. Mass spectrometry data were processed using iMETQ software and metabolites were identified using Human Metabolome DataBase while, Metaboanalyst 4.0 tool was used for statistical and pathway analysis. A total of 3,704 metabolites belonging to various chemical classes were identified in bull spermatozoa. After sorting out exogenous metabolites, 56 metabolites were observed common to both the groups while 44 and 35 metabolites were found unique to high- and low-fertile spermatozoa, respectively. Among the common metabolites, concentrations of 19 metabolites were higher in high-fertile compared to low-fertile spermatozoa (fold change > 1.00). Spermatozoa metabolites with variable importance in projections score of more than 1.5 included hypotaurine, d-cysteine, selenocystine. In addition, metabolites such as spermine and l-cysteine were identified exclusively in high-fertile spermatozoa. Collectively, the present study established the metabolic profile of bovine spermatozoa and identified the metabolomic differences between spermatozoa from high- and low-fertile bulls. Among the sperm metabolites, hypotaurine, selenocysteine, l-malic acid, d-cysteine, and chondroitin 4-sulfate hold the potential to be recognized as fertility-associated metabolites.
Project description:Sperm carries information to the presumptive embryo upon fertilization in terms of epigenetic codes and transcripts along with the haploid genome. The epigenetic code includes DNA methylation and Histone modifications. During spermatogenesis the chromatin of sperm undergoes wide level of modifications and histone proteins are replaced by Protamine proteins. But some modified Histone forms still remain and they carry epigenetic codes essential for fertility and embryo development. Through this work we are trying to see the difference between H3K4me2 and H3K27me3 kind of histone modifications in spermatozoa of high and low fertility buffalo bulls. Overall design: Agilent two-color ChIP-on-Chip, 4X180K Microarray designed by Genotypic Technology Private Limited. (AMADID-040096). To see the differential H3K4me2 and H3K27me3 pattern between high and low fertile buffalo bull spermatozoa, the cryopreserved semen samples from the identified bulls were procured. The chromatin was isolated from all the samples, sheared with micrococcal nuclease treatment (NEB, Cat no. MO247S), immunoprecipitated and hybdidized on array to get information for all the probes printed on the array. The data was analyzed and the differentially enriched genes between the two group were identified for both kinds of histone modifications.
Project description:The xenoestrogen bisphenol-A (BPA) is a widespread environmental contaminant that has been studied for its impact on male fertility in several species of animals and humans. Growing evidence suggests that xenoestrogens can bind to receptors on spermatozoa and thus alter sperm function. The objective of the study was to investigate the effects of varying concentrations of BPA (0.0001, 0.01, 1, and 100 ?M for 6 h) on sperm function, fertilization, embryonic development, and on selected fertility-related proteins in spermatozoa. Our results showed that high concentrations of BPA inhibited sperm motility and motion kinematics by significantly decreasing ATP levels in spermatozoa. High BPA concentrations also increased the phosphorylation of tyrosine residues on sperm proteins involved in protein kinase A-dependent regulation and induced a precocious acrosome reaction, which resulted in poor fertilization and compromised embryonic development. In addition, BPA induced the down-regulation of ?-actin and up-regulated peroxiredoxin-5, glutathione peroxidase 4, glyceraldehyde-3-phosphate dehydrogenase, and succinate dehydrogenase. Our results suggest that high concentrations of BPA alter sperm function, fertilization, and embryonic development via regulation and/or phosphorylation of fertility-related proteins in spermatozoa. We conclude that BPA-induced changes in fertility-related protein levels in spermatozoa may be provided a potential cue of BPA-mediated disease conditions.
Project description:Male fertility is the ability of sperm to fertilize the egg and sustain embryo development. Several factors determine the fertilizing capacity of mammalian sperm, including those intrinsic to sperm and components of the seminal plasma. The present study analyzed the seminal fluid proteome of Bos taurus and potential associations between proteins and fertility scores. Mass spectrometry coupled with nano HPLC allowed the identification of 1,159 proteins in the dairy bull seminal plasma. There were 50 and 29 seminal proteins more abundant in high (HF) low fertility (LF) bulls, respectively. Based on multivariate analysis, C-type natriuretic peptide, TIMP-2, BSP5 and sulfhydryl oxidase indicated relationship with HF bulls. Clusterin, tissue factor pathway inhibitor 2, galectin-3-binding protein and 5'-nucleotidase were associated with LF bulls. Abundance of NAD(P)(+)-arginine ADP-ribosyltransferase, prosaposin and transmembrane protein 2 proteins had the highest positive correlations with fertility ranking. Quantities of vitamin D-binding protein, nucleotide exchange factor SIL1 and galectin-3-binding protein showed the highest negative correlations with fertility ranking. A fertility ranking score was calculated and the relationship with these proteins was significant (Spearman's rho?=?0.94). The present findings represent a major and novel contribution to the study of bovine seminal proteins. Indicators of fertility can be used to improve reproductive biotechnologies.
Project description:Bovine fertility remains a critical issue underpinning the sustainability of the agricultural sector. Phenotypic records collected on >7,000 bulls used in artificial insemination (AI) were used to identify 160 reliable and divergently fertile bulls for a dual strategy of targeted sequencing (TS) of fertility-related ?-defensin genes and whole exome sequencing (WES). A haplotype spanning multiple ?-defensin genes and containing 94 SNPs was significantly associated with fertility and functional analysis confirmed that sperm from bulls possessing the haplotype showed significantly enhanced binding to oviductal epithelium. WES of all exons in the genome in 24 bulls of high and low fertility identified 484 additional SNPs significantly associated with fertility. After validation, the most significantly associated SNP was located in the FOXJ3 gene, a transcription factor which regulates sperm function in mice. This study represents the first comprehensive characterisation of genetic variation in bovine ?-defensin genes and functional analysis supports a role for ?-defensins in regulating bull sperm function. This first application of WES in AI bulls with divergent fertility phenotypes has identified a novel role for the transcription factor FOXJ3 in the regulation of bull fertility. Validated genetic variants associated with bull fertility could prove useful for improving reproductive outcomes in cattle.