Male infertility and Perfluoroalkyl and poly-fluoroalkyl substances: Evidence for alterations in phosphorylation of proteins and fertility-related functional attributes in bull spermatozoa.

BACKGROUND: Perfluoroalkyl and poly-fluoroalkyl substances (PFAS) are pervasive environmental pollutants and emerging risk factors for reproductive health. Although epidemiological evidence supports the link between these substances and male infertility, their specific effects on male fertility remain poorly understood. OBJECTIVES: Investigate the effect of perfluorooctane sulfonic acid (PFOS), the most prevalent and prominent PFAS, on bull sperm protein phosphorylation, a post-translational modification process governing sperm functionality and fertility. METHODS: We exposed bull sperm to PFOS at 10 µM (average population level) and 100 µM (high-exposure level), and analyzed global proteome and phosphoproteome profile by TMT labeling and NanoLC-MS/MS. We also measured sperm fertility functions by flow cytometry. RESULTS: PFOS at 10 µM altered sperm proteins linked to spermatogenesis and chromatin condensation, while at 100 µM, PFOS affected proteins associated with motility and fertility. We detected 299 phosphopeptides from 116 proteins, with 45 exhibiting differential expression between control and PFOS groups. PFOS dysregulated phosphorylation of key proteins (ACRBP, PRKAR2A, RAB2B, SPAG8, TUBB4B, ZPBP, and C2CD6) involved in sperm capacitation, acrosome reaction, sperm-egg interaction, and fertilization. PFOS also affected phosphorylation of other proteins (AQP7, HSBP9, IL4I1, PRKAR1A, and CCT8L2) related to sperm stress resistance and cryotolerance. Notably, 4 proteins (PRM1, ACRBP, TSSK1B, and CFAP45) exhibited differential regulation at both the proteomic and phosphoproteomic levels. Flow cytometric analysis confirmed that PFOS increased protein phosphorylation in sperm as well as reduced sperm motility, viability, calcium, and membrane potential and increased mitochondrial ROS in a dose-dependent manner. CONCLUSIONS: This study shows that PFOS exposure adversely impacts phosphorylation of proteins critical for bull sperm function and fertilization. Moreover, the concentration of PFOS influences the severity of these effects. The comprehensive bull sperm phosphoproteomics data from this study can help us understand the molecular mechanisms of environmental exposure-related male infertility.

The Association Between Short-Acting ß2-Agonist Over-Prescription, and Patient-Reported Acquisition and Use on Asthma Control and Exacerbations: Data from Australia.

INTRODUCTION: In Australia, short-acting ß2-agonists (SABA) are available both over the counter (OTC) and on prescription. This ease of access may impact SABA use in the Australian population. Our aim was to assess patterns and outcome associations of prescribed, acquired OTC and reported use of SABA by Australians with asthma. METHODS: This was a cross-sectional study, using data derived from primary care electronic medical records (EMRs) and patient completed questionnaires within Optimum Patient Care Research Database Australia (OPCRDA). A total of 720 individuals aged ≥ 12 years with an asthma diagnosis in their EMRs and receiving asthma therapy were included. The annual number of SABA inhalers authorised on prescription, acquired OTC and reported, and the association with self-reported exacerbations and asthma control were investigated. RESULTS: 92.9% (n = 380/409) of individuals issued with SABA prescription were authorised ≥ 3 inhalers annually, although this differed from self-reported usage. Of individuals reporting SABA use (n = 546) in the last 12 months, 37.0% reported using ≥ 3 inhalers. These patients who reported SABA overuse experienced 2.52 (95% confidence interval [CI] 1.73-3.70) times more severe exacerbations and were 4.51 times (95% CI 3.13-6.55) more likely to have poor asthma control than those who reported using 1-2 SABA inhalers. Patients who did not receive SABA on prescription (43.2%; n = 311/720) also experienced 2.71 (95% CI 1.07-7.26) times more severe exacerbations than those prescribed 1-2 inhalers. Of these patients, 38.9% reported using OTC SABA and other prescription medications, 26.4% reported using SABA OTC as their only asthma medication, 13.2% were prescribed other therapies but not SABA OTC and 14.5% were not using any medication. CONCLUSION: Both self-reported SABA overuse and zero SABA prescriptions were associated with poor asthma outcomes. The disconnect between prescribing authorisation, OTC availability and actual use, make it difficult for clinicians to quantify SABA use.

Sperm proteomic landscape is altered in breeding bulls with greater sperm DNA fragmentation index.

Although, it is well understood that sperm DNA damage is associated with infertility, the molecular details of how damaged sperm DNA affects fertility are not fully elucidated. Since sperm proteins play an important role in fertilization and post-fertilization events, the present study aimed to identify the sperm proteomic alterations in bulls with high sperm DNA Fragmentation Index (DFI%). Semen from Holstein-Friesian crossbred breeding bulls (n = 50) was subjected to Sperm Chromatin Structure Assay. Based on DFI%, bulls were classified into either high- (HDFI; n = 6), or low-DFI (LDFI; n = 6) and their spermatozoa were subjected to high throughput proteomic analysis. Liquid chromatography and mass spectrometry analysis identified 4567 proteins in bull spermatozoa. A total of 2660 proteins were found common to both the groups, while 1193 and 714 proteins were unique to HDFI and LDFI group, respectively. A total of 265 proteins were up regulated and 262 proteins were down regulated in HDFI group. It was found that proteins involved in capacitation [heparin binding (molecular function), ERK1 and ERK2 cascade (biological process), PI3K-Akt signalling (pathway), Jak-STAT signalling (pathway)], spermatogenesis [TLR signalling (pathway), gamete generation (biological process)] and DNA repair mechanism (biological process) were significantly altered in the bulls with high DFI%.

Establishment of a repertoire of fertility associated sperm proteins and their differential abundance in buffalo bulls (Bubalus bubalis) with contrasting fertility.

Sperm harbours a wide range of proteins regulating their functions and fertility. In the present study, we made an effort to characterize and quantify the proteome of buffalo bull spermatozoa, and to identify fertility associated sperm proteins through comparative proteomics. Using high-throughput mass spectrometry platform, we identified 1305 proteins from buffalo spermatozoa and found that these proteins were mostly enriched in glycolytic process, mitochondrial respiratory chain, tricarboxylic acid cycle, protein folding, spermatogenesis, sperm motility and sperm binding to zona pellucida (p < 7.74E-08) besides metabolic (p = 4.42E-31) and reactive oxygen species (p = 1.81E-30) pathways. Differential proteomic analysis revealed that 844 proteins were commonly expressed in spermatozoa from both the groups while 77 and 52 proteins were exclusively expressed in high- and low-fertile bulls, respectively. In low-fertile bulls, 75 proteins were significantly (p < 0.05) upregulated and 176 proteins were significantly (p < 0.05) downregulated; these proteins were highly enriched in mitochondrial respiratory chain complex I assembly (p = 2.63E-07) and flagellated sperm motility (p = 7.02E-05) processes besides oxidative phosphorylation pathway (p = 6.61E-15). The down regulated proteins in low-fertile bulls were involved in sperm motility, metabolism, sperm-egg recognition and fertilization. These variations in the sperm proteome could be used as potential markers for the selection of buffalo bulls for fertility.

High-throughput proteomic characterization of seminal plasma from bulls with contrasting semen quality.

Seminal plasma proteins are the major extrinsic factors that can modulate the sperm quality and functions. The present study was carried out to compare the proteomic profiles of seminal plasma from breeding bulls producing good and poor quality semen in an effort to understand the possible proteins associated with semen quality. A total of 910 and 715 proteins were detected in the seminal plasma of poor and good quality semen producing bulls, respectively. A total of 705 proteins were common to both the groups, in which 380 proteins were upregulated and 89 proteins were downregulated in the seminal plasma of poor quality semen, while 236 proteins were co-expressed. The proteins negatively influencing sperm functions such as CCL2, UQCRC2, and SAA1 were among the top ten upregulated proteins in the seminal plasma of poor quality semen. Proteins having a positive role in sperm functions (NGF, EEF1A2, COL1A2, IZUMO4, PRSS1, COL1A1, WFDC2) were among the top ten downregulated proteins in the seminal plasma of poor quality semen. The upregulation of oxidation-reduction process-related proteins, histone proteins (HIST3H2A, H2AFJ, H2AFZ, H2AFX, HIST2H2AB, H2AFV, HIST1H2AC, HIST2H2AC, LOC104975684, LOC524236, LOC614970, LOC529277), and ubiquinol-cytochrome-c reductase proteins (UQCRB, UQCRFS1, UQCRQ, UQCRC1, UQCRC2) indicate deranged oxidation-reduction equilibrium, chromatin condensation and spermatogenesis in poor quality semen producing bulls. The expression of proteins essential for motile cilium (CCDC114, CFAP206, TEKT4), chromatin integrity (PRM2), gamete fusion (IZUMO4, EQTN), hyperactivation, tyrosine phosphorylation, and capacitation [PI3K-Akt signalling pathway-related proteins (COL1A1, COL2A1, COL1A2, SPP1, PDGFA, NGF)] were down regulated in poor quality semen producing bulls. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03474-6.

Single nucleotide polymorphisms cumulating to genetic variation for fertility in crossbred (Bos taurus × Bos indicus) bull spermatozoa.

Spermatozoa from high-fertile (HF) and low-fertile (LF) breeding bulls were subjected to high-throughput next-generation sequencing to identify important Single nucleotide polymorphisms (SNPs) and novel variants associated with fertility. A total of 77,038 genome-wide SNPs were identified, among which, 10,788 were novel variants. A total of 42,290 and 34,748 variants were recorded with 6115 and 4673 novel variants in in HF and LF bulls, respectively. Higher number of SNPs were identified in HF compared to LF bulls. GO analysis of filtered genes with significant variations in HF bulls indicated their involvement in oxidative phosphorylation and metabolic pathways. GO analysis of filtered genes with significant variation in LF bulls revealed their involvement in Ca2++ ion binding, structural constituent of ribosome, and biological processes like translation and ribosomal small subunit assembly. The study identified SNPs in candidate genes including TPT1, BOLA-DRA, CD74, RPS17, RPS28, RPS29, RPL14, RPL13, and RPS27A, which are linked to sperm functionality, survival, oxidative stress, and bull fertility. The identified SNPs could be used in selection of bulls for high fertility and the variation in these genes could be established as an explanation for the fertility differences in bulls upon validation in large number of bulls.

The cryopreservation process induces alterations in proteins associated with bull sperm quality: The equilibration process could be a probable critical control point.

The present study quantitatively characterized the proteomic changes in bull spermatozoa induced by the cryopreservation process. We performed high-throughput comparative global proteomic profiling of freshly ejaculated (before cryopreservation), equilibrated (refrigerated storage; during cryopreservation), and frozen (ultralow temperature; after cryopreservation) bull spermatozoa. Using the liquid chromatography-mass spectrometry (LC-MS/MS) technique, a total of 1,692, 1,415, and 1,286 proteins were identified in fresh, equilibrated, and cryopreserved spermatozoa, respectively. When the proteome of fresh spermatozoa was compared with equilibrated spermatozoa, we found that 166 proteins were differentially expressed. When equilibrated spermatozoa were compared with cryopreserved spermatozoa, we found that 147 proteins were differentially expressed between them. Similarly, we found that 156 proteins were differentially expressed between fresh and cryopreserved spermatozoa. Among these proteins, the abundance of 105 proteins was lowered during the equilibration process itself, while the abundance of 43 proteins was lowered during ultralow temperature preservation. Remarkably, the equilibration process lowered the abundance of sperm proteins involved in energy metabolism, structural integrity, and DNA repair and increased the abundance of proteins associated with proteolysis and protein degradation. The abundance of sperm proteins associated with metabolism, cGMP-PKG (cyclic guanosine 3',5'-monophosphate-dependent protein kinase G) signaling, and regulation of the actin cytoskeleton was also altered during the equilibration process. Collectively, the present study showed that the equilibration step in the bull sperm cryopreservation process was the critical point for sperm proteome, during which a majority of proteomic alterations in sperm occurred. These findings are valuable for developing efficient protocols to minimize protein damage and to improve the quality and fertility of cryopreserved bull spermatozoa.

Cryopreservation process alters the expression of genes involved in pathways associated with the fertility of bull spermatozoa.

In bovines, cryopreserved semen is used for artificial insemination; however, the fertility of cryopreserved semen is far lower than that of fresh semen. Although cryopreservation alters sperm phenotypic characteristics, its effect on sperm molecular health is not thoroughly understood. The present study applied next-generation sequencing to investigate the effect of cryopreservation on the sperm transcriptomic composition of bull spermatozoa. While freshly ejaculated bull spermatozoa showed 14,280 transcripts, cryopreserved spermatozoa showed only 12,375 transcripts. Comparative analysis revealed that 241 genes were upregulated, 662 genes were downregulated, and 215 genes showed neutral expression in cryopreserved spermatozoa compared to fresh spermatozoa. Gene ontology analysis indicated that the dysregulated transcripts were involved in nucleic acid binding, transcription-specific activity, and protein kinase binding involving protein autophosphorylation, ventricular septum morphogenesis, and organ development. Moreover, the dysregulated genes in cryopreserved spermatozoa were involved in pathways associated with glycogen metabolism, MAPK signalling, embryonic organ morphogenesis, ectodermal placode formation, and regulation of protein auto-phosphorylation. These findings suggest that the cryopreservation process induced alterations in the abundance of sperm transcripts related to potential fertility-associated functions and pathways, which might partly explain the reduced fertility observed with cryopreserved bull spermatozoa.

Integrated multi-omics analyses reveals molecules governing sperm metabolism potentially influence bull fertility.

Bull fertility is of paramount importance in bovine industry because semen from a single bull is used to breed several thousands of cows; however, so far, no reliable test is available for bull fertility prediction. In the present study, spermatozoa from high- and low-fertility bulls were subjected to high-throughput transcriptomic, proteomic and metabolomic analysis. Using an integrated multi-omics approach the molecular differences between high- and low-fertility bulls were identified. We identified a total of 18,068 transcripts, 5041 proteins and 3704 metabolites in bull spermatozoa, of which the expression of 4766 transcripts, 785 proteins and 33 metabolites were dysregulated between high- and low-fertility bulls. At transcript level, several genes involved in oxidative phosphorylation pathway were found to be downregulated, while at protein level genes involved in metabolic pathways were significantly downregulated in low-fertility bulls. We found that metabolites involved in Taurine and hypotaurine metabolism were significantly downregulated in low-fertility bulls. Integrated multi-omics analysis revealed the interaction of dysregulated transcripts, proteins and metabolites in major metabolic pathways, including Butanoate metabolism, Glycolysis and gluconeogenesis, Methionine and cysteine metabolism, Phosphatidyl inositol phosphate, pyrimidine metabolism and saturated fatty acid beta oxidation. These findings collectively indicate that molecules governing sperm metabolism potentially influence bull fertility.

Genom-wide analysis identifies single nucleotide polymorphism variations and altered pathways associated with poor semen quality in breeding bulls.

The reason for poor semen quality among the breeding bulls is not well understood. In the present study, we performed high-throughput RNAseq analysis of spermatozoa to identify the SNPs present in good and poor-quality semen-producing Holstein Friesian breeding bulls. A total of 21,360 and 44,650 SNPs were identified in good and poor-quality semen with a minimum read depth of 20, among which 4780 and 8710 novel variants were observed in good and poor-quality semen, respectively. Greater SNPs and indels variations were observed in poor compared to good-quality semen. In poor-quality semen, SNP variations were observed in ZNF280B, SLC26A2, DMXL1, OR52A1, MACROD2 and REV1 genes, which are associated with regulation of spermatogenesis, post-testicular maturation, Cl- channel activity, V-ATPase-mediated intracellular vesicle acidification, a mono-ADP-ribosyl hydrolase and ATR-Chk1 checkpoint activation. GO analysis of filtered genes with significant variations between good and poor-quality semen showed enrichment in important pathways related to semen quality such as MAPK signalling pathway, Akt signalling pathway, focal adhesion, cAMP signalling pathway, and Rap1 signalling pathway. Network analysis of filtered genes in poor-quality semen showed variations in pathways of purine metabolism, pyrimidine metabolism, prolactin signalling pathway and RNA cap-binding complex. It is inferred that SNP in genes involved in maintaining sperm functions could be the reason for poor-quality semen production in bulls, and the identified SNPs hold potential to be used as biomarkers for semen quality in bulls.

High throughput deep proteomic analysis of seminal plasma from stallions with contrasting semen quality.

Seminal plasma proteins and pathways associated with sperm motility have not been elucidated in stallions. Therefore, in the current study, using the high throughput LC/MS-MS approach, we profiled stallion seminal plasma proteins and identified the proteins and pathways associated with sperm motility. Seminal plasma from six stallions producing semen with contrasting sperm motility (n = 3 each high-and low-motile group) was utilized for proteomic analysis. We identified a total of 1687 proteins in stallion seminal plasma, of which 1627 and 1496 proteins were expressed in high- (HM) and low- motile (LM) sperm of stallions, respectively. A total number of 1436 proteins were co-expressed in both the groups; 191 (11%) and 60 (3.5%) proteins were exclusively detected in HM and LM groups, respectively. A total of 220 proteins were upregulated (>1-fold change) and 386 proteins were downregulated in SP from LM group stallions as compared to HM group stallions, while 830 proteins were neutrally expressed in both the groups. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed dysregulation of the important proteins related to mitochondrial function, acrosome, and sperm cytoskeleton in the seminal plasma of stallions producing ejaculates with low sperm motility. High abundance of peroxiredoxins and low abundance of seminal Chaperonin Containing TCP1 Complex (CCT) complex and Annexins indicate dysregulated oxidative metabolism, which might be the underlying etiology for poor sperm motility in LM group stallions. In conclusion, the current study identified the seminal plasma proteomic alterations associated with poor sperm motility in stallions; the results indicate that poor sperm motility in stallions could be associated with altered expression of seminal plasma proteins involved in oxidative metabolism.

Sperm Transcripts Associated With Odorant Binding and Olfactory Transduction Pathways Are Altered in Breeding Bulls Producing Poor-Quality Semen.

Spermatozoa carries a reservoir of mRNAs regulating sperm functions and fertilizing potential. Although it is well recognized that a considerable proportion of high genetic merit breeding bulls produce poor-quality semen, the transcriptomic alterations in spermatozoa from such bulls are not understood. In the present study, comparative high-throughput transcriptomic profiling of spermatozoa from good and poor-quality semen-producing bulls was carried out to identify the transcripts associated with semen quality. Using next-generation sequencing (NGS), we identified 11,632 transcripts in Holstein Friesian bull spermatozoa; after total hit normalization, a total of 544 transcripts were detected, of which 185 transcripts were common to both good and poor-quality semen, while 181 sperm transcripts were unique to good quality semen, and 178 transcripts were unique to poor-quality semen. Among the co-expressed transcripts, 31 were upregulated, while 108 were downregulated, and 46 were neutrally expressed in poor-quality semen. Bioinformatics analysis revealed that the dysregulated transcripts were predominantly involved in molecular function, such as olfactory receptor activity and odor binding, and in biological process, such as detection of chemical stimulus involved in sensory perception, sensory perception of smell, signal transduction, and signal synaptic transmission. Since a majority of the dysregulated transcripts were involved in the olfactory pathway (85% of enriched dysregulated genes were involved in this pathway), the expression of selected five transcripts associated with this pathway (OR2T11, OR10S1, ORIL3, OR5M11, and PRRX1) were validated using real-time qPCR, and it was found that their transcriptional abundance followed the same trend as observed in NGS; the sperm transcriptional abundance of OR2T11 and OR10S1 differed significantly (p < 0.05) between good and poor-quality semen. It is concluded that poor-quality semen showed altered expression of transcripts associated with olfactory receptors and pathways indicating the relationship between olfactory pathway and semen quality in bulls.

Modeling the trajectory of SARS-CoV-2 spike protein evolution in continuous latent space using a neural network and Gaussian process

Viral vaccines can lose their efficacy as the genomes of targeted viruses rapidly evolve, resulting in new variants that may evade vaccine-induced immunity. This process is apparent in the emergence of new SARS-CoV-2 variants which have the potential to undermine vaccination efforts and cause further outbreaks. Predictive vaccinology points to a future of pandemic preparedness in which vaccines can be developed preemptively based in part on predictive models of viral evolution. Thus, modeling the trajectory of SARS-CoV-2 spike protein evolution could have value for mRNA vaccine development. Traditionally, in silico sequence evolution has been modeled discretely, while there has been limited investigation into continuous models. Here we present the Viral Predictor for mRNA Evolution (VPRE), an open-source software tool which learns from mutational patterns in viral proteins and models their most statistically likely evolutionary trajectories. We trained a variational autoencoder with real-time and simulated SARS-CoV-2 genome data from Australia to encode discrete spike protein sequences into continuous numerical variables. To simulate evolution along a phylogenetic path, we trained a Gaussian process model with the numerical variables to project spike protein evolution up to five months in advance. Our predictions mapped primarily to a sequence that differed by a single amino acid from the most reported spike protein in Australia within the prediction timeframe, indicating the utility of deep learning and continuous latent spaces for modeling viral protein evolution. VPRE can be readily adapted to investigate and predict the evolution of viruses other than SARS-CoV-2 in temporal, geographic, and lineage-specific pathways.

Comparative Transcriptomic Analysis of Spermatozoa From High- and Low-Fertile Crossbred Bulls: Implications for Fertility Prediction.

Crossbred bulls produced by crossing Bos taurus and Bos indicus suffer with high incidence of infertility/subfertility problems; however, the etiology remains poorly understood. The uncertain predictability and the inability of semen evaluation techniques to maintain constant correlation with fertility demand for alternate methods for bull fertility prediction. Therefore, in this study, the global differential gene expression between high- and low-fertile crossbred bull sperm was assessed using a high-throughput RNA sequencing technique with the aim to identify transcripts associated with crossbred bull fertility. Crossbred bull sperm contained transcripts for 13,563 genes, in which 2,093 were unique to high-fertile and 5,454 were unique to low-fertile bulls. After normalization of data, a total of 776 transcripts were detected, in which 84 and 168 transcripts were unique to high-fertile and low-fertile bulls, respectively. A total of 176 transcripts were upregulated (fold change > 1) and 209 were downregulated (<1) in low-fertile bulls. Gene ontology analysis identified that the sperm transcripts involved in the oxidative phosphorylation pathway and biological process such as multicellular organism development, spermatogenesis, and in utero embryonic development were downregulated in low-fertile crossbred bull sperm. Sperm transcripts upregulated and unique to low-fertile bulls were majorly involved in translation (biological process) and ribosomal pathway. With the use of RT-qPCR, selected sperm transcripts (n = 12) were validated in crossbred bulls (n = 12) with different fertility ratings and found that the transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes was significantly (p < 0.05) lower in low-fertile bulls than high-fertile bulls and was positively (p < 0.05) correlated with conception rate. It is inferred that impaired oxidative phosphorylation could be the predominant reason for low fertility in crossbred bulls and that transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes could serve as potential biomarkers for fertility in crossbred bulls.

Sub-fertility in crossbred bulls: deciphering testicular level transcriptomic alterations between zebu (Bos indicus) and crossbred (Bos taurus x Bos indicus) bulls.

BACKGROUND: The incidence of poor semen quality and sub-fertility/infertility is higher in crossbred as compared to Zebu males. Several attempts have been made to understand the possible reasons for higher incidence of fertility problems in crossbred males, at sperm phenotype, proteome and genome level but with variable results. Since the quality of the ejaculated spermatozoa is determined by the testicular environment, assessing the testicular transcriptome between these breeds would help in identifying the possible mechanisms associated with infertility in crossbred bulls. However, such information is not available. We performed global transcriptomic profiling of testicular tissue from crossbred and Zebu bulls using Agilent Bos taurus GXP 8X60k AMADID: 29411 array. To the best of our knowledge, this is the first study comparing the testicular mRNAs between crossbred and Zebu bulls. RESULTS: Out of the 14,419 transcripts detected in bovine testis, 1466 were differentially expressed between crossbred and Zebu bulls, in which 1038 were upregulated and 428 were downregulated in crossbred bulls. PI4KB and DPY19L2 genes, reported to be involved in sperm capacitation and acrosome formation respectively, were among the top 10 downregulated transcripts in crossbred testis. Genes involved in ubiquitination and proteolysis were upregulated, while genes involved in cell proliferation, stem cell differentiation, stem cell population maintenance, steroidogenesis, WNT signalling, protein localization to plasma membrane, endocannabinoid signalling, heparin binding, cAMP metabolism and GABA receptor activity were downregulated in crossbred testis. Among the 10 genes validated using qPCR, expression of CCNYL, SOX2, MSMB, SPATA7, TNP1, TNP2 and CRISP2 followed the same trend as observed in microarray analysis with SPATA7 being significantly downregulated and transition proteins (TNP1, TNP2) being significantly upregulated in crossbred bulls. CONCLUSIONS: Abundant proteolysis by ubiquitination and downregulation of WNT signaling, cell proliferation, differentiation and steroidogenesis might be associated with higher incidence of poor semen quality and/or sub-fertility/infertility in crossbred bulls as compared to Zebu bulls. Downregulation of SPATA7 (Spermatogenesis Associated 7) and upregulation of transition proteins (TNP1 and TNP2) in crossbred bull testis might be associated with impaired spermatogenesis processes including improper chromatin compaction in crossbred bulls.