Muhammad Jasim Uddin

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.

Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues

BackgroundGene expression analysis using real-time RT-PCR (qRT-PCR) is increasingly important in biological research due to the high-throughput and accuracy of qRT-PCR. For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes or internal control genes is required. The stability of reference genes has a tremendous effect on the results of relative quantification of gene expression by qRT-PCR. The expression stability of reference genes could vary according to tissues, age of individuals and experimental conditions. In the pig however, very little information is available on the expression stability of reference genes. The aim of this research was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in varieties of porcine tissues at different ages.ResultsThe mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined in varieties of tissues collected from newborn, young and adult pigs. geNorm, NormFinder and BestKeeper software were used to rank the genes according to their stability. geNorm software revealed that RPL4, PPIA and YWHAZ showed high stability in newborn and adult pigs, while B2M, YWHAZ and SDHA showed high stability in young pigs. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that TBP was the most stable gene in newborn and young pigs, while PPIA was most stable in adult pigs. Moreover, geNorm software suggested that the geometric mean of three most stable gene would be the suitable combination for accurate normalization of gene expression study.ConclusionsAlthough, there was discrepancy in the ranking order of reference genes obtained by different analysing software methods, the geometric mean of the RPL4, PPIA and YWHAZ seems to be the most appropriate combination of housekeeping genes for accurate normalization of gene expression data in different porcine tissues at different ages.

Expression dynamics of Toll-like receptors mRNA and cytokines in porcine peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide

The Toll-like receptor (TLR)4 is critical for the recognition of Gram-negative bacterial lipopolysaccharide (LPS) but in porcine peripheral blood mononuclear cells (PBMCs) it may cooperate with other TLRs and lead to the production of inflammatory cytokines. Therefore, we analyzed TLR1-10 mRNA expression in porcine PBMCs stimulated with LPS over time (1-48 h) by using quantitative real-time PCR and cytokine proteins level by ELISA in culture supernatant. TLR1-10 mRNA was detectable in porcine PBMCs. When compared with the control (non-stimulated), TLR1 mRNA were increased (p<0.05) at 3 h after challenge with 1 μg/ml LPS, whereas TLR1 and TLR2 mRNA were increased (p<0.01) at 6 h after challenge with 10 μg/ml LPS. TLR4 increased (p<0.001) at 3h after challenge with LPS and remained constant. TLR5 and TLR6 mRNA increased (p<0.05) at 9 h and 1 h after of LPS stimulation, respectively. The mRNA of CD14 and MD2 were increased (p<0.001) at 1h after LPS stimulation. Additionally, at most of the time analyzed, the mRNA expression increased with the dose of LPS. The LPS concentration had influence (p<0.05) on all the TLRs expression except TLR10; whereas time had effect (p<0.05) on all TLRs expression except TLR2, 3, 6 and 10. When compared to the control, the cytokines IL1b, IL8 and TNFα proteins were increased (p<0.001) immediately at 1 h after LPS stimulation and remained constant till 48 h. IL12b was increased (p<0.001) 12 h after challenge with 10 μg/ml of LPS. Although IL8 level was the highest, the higher (p<0.05) expression of all these inflammatory cytokines indicate that upon interacting with TLRs, LPS exerted inflammatory response in PBMCs through the production of Th1 type cytokines. The production of cytokines was influenced (p<0.001) by both the dose of LPS and the stimulation time. Hence, the porcine PBMCs are likely able to express all members of TLRs.

Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA

BackgroundAs an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs.ResultsThe mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.ConclusionThere was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

Association study and expression analysis of porcine ESR1 as a candidate gene for boar fertility and sperm quality

Male fertility is impaired through the lack of ESR1 (Estrogen Receptor 1) but little is known about the ESR1 roles in boar spermatogenesis and fertility. Therefore, this research was aimed at investigating the association with sperm quality and boar fertility traits in a total of 300 boars both from purebred Pietrain and Pietrain × Hampshire crosses. A SNP in coding region of ESR1g.672C>T in exon 1 was associated with sperm motility (P<0.05) and plasma droplet rate (P<0.01) while the polymorphism in non-coding region of ESR1g.35756T>C in inton 1 was associated with non-return rate (P<0.05). Furthermore, to analyse the mRNA and protein expression of ESR1 in boar reproductive tissues, a total of six boars were divided into two groups [Group I (G-I) and Group II (G-II)], where G-I had relatively better sperm quality. ESR1 expression was higher in tissues collected from G-I boars than those of collected from G-II boars, and the difference in mRNA expression was significant (P<0.01) in head of epididymis. The ESR1 protein expression results from western blot coincided with the results of qRT-PCR. The ESR1 protein localization observed a strong staining in the cytoplasm of Sertoli cell in the testis, in the epithelial cells in head and tail of epididymis, in smooth muscle in tail of epididymis, and in the post acrosomal region and tail of the spermatozoa. These results will improve the understanding of the functions of the ESR1 in spermatogenesis within the reproductive tract and will shed light on ESR1 as a candidate in the selection of boar with good sperm quality and fertility.

ASSOCIATION STUDY AND EXPRESSION ANALYSIS OF CD9 AS CANDIDATE GENE FOR BOAR SPERM QUALITY AND FERTILITY TRAITS

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm-egg fusion, and was therefore selected as candidate gene for boar semen quality. The association of CD9 with boar sperm quality and fertility trait was analyzed using a total of 340 boars both from purebred Pietrain and Pietrain×Hampshire crosses. A single nucleotide polymorphism (g.358A>T) in intron 6 was significantly associated with sperm motility (MOT) (P<0.001), plasma droplet rate (PDR) (P<0.001) and abnormal spermatozoa rate (ASR) (P<0.01). Boars were divided into two groups with group 1 (G-I) boars having a higher SCON and SMOT, lower SVOL (sperm volume) and group 2 (G-II) having a lower SCON and SMOT, higher SVOL. The mRNA and protein expression levels were evaluated in reproductive, non-reproductive tissues and spermatozoa from G-I and G-II animals by using quantitative real-time PCR and western blotting. When both reproductive and non-reproductive tissues were examined, highest mRNA was expressed in prostate gland, then in the body of the epididymis, vas deferens and tail of the epididymis. In case of reproductive tissues, CD9 expression was higher in tissues and spermatozoa collected from G-I boars than those collected from G-II boars. The mRNA expression was significantly different (P<0.05) in body of epididymis from G-I and G-II boars. The CD9 protein expression results from western blot were coincided with the results of qRT-PCR. Moreover, CD9 protein localization in Leydig cells, Sertoli cells, epithelial cells and spermatozoa was remarkable which indicated the important role of CD9 in spermatogenesis process. By using mRNA and protein expression profiles, it could be shown that CD9 plays a crucial role during sperm development, especially within the epididymis where the maturation of the sperm, a key process for the sperm quality and motility takes place. These results will improve the understanding of the functions of the CD9 in spermatogenesis within the reproductive tracts and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.

RNA Deep Sequencing Reveals Novel Candidate Genes and Polymorphisms in Boar Testis and Liver Tissues with Divergent Androstenone Levels

Boar taint is an unpleasant smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone (5α-androst-16-en-3-one) and skatole (3-methylindole). It is crucial to understand the genetic mechanism of boar taint to select pigs for lower androstenone levels and thus reduce boar taint. The aim of the present study was to investigate transcriptome differences in boar testis and liver tissues with divergent androstenone levels using RNA deep sequencing (RNA-Seq). The total number of reads produced for each testis and liver sample ranged from 13,221,550 to 33,206,723 and 12,755,487 to 46,050,468, respectively. In testis samples 46 genes were differentially regulated whereas 25 genes showed differential expression in the liver. The fold change values ranged from −4.68 to 2.90 in testis samples and −2.86 to 3.89 in liver samples. Differentially regulated genes in high androstenone testis and liver samples were enriched in metabolic processes such as lipid metabolism, small molecule biochemistry and molecular transport. This study provides evidence for transcriptome profile and gene polymorphisms of boars with divergent androstenone level using RNA-Seq technology. Digital gene expression analysis identified candidate genes in flavin monooxygenease family, cytochrome P450 family and hydroxysteroid dehydrogenase family. Moreover, polymorphism and association analysis revealed mutation in IRG6, MX1, IFIT2, CYP7A1, FMO5 and KRT18 genes could be potential candidate markers for androstenone levels in boars. Further studies are required for proving the role of candidate genes to be used in genomic selection against boar taint in pig breeding programs.

Expression patterns of porcine Toll-like receptors family set of genes (TLR1-10) in gut-associated lymphoid tissues alter with age

The aim was to study the expression pattern of the porcine TLR family (TLR1-10) genes in gut-associated lymphoid tissues (GALT) of varying ages. A total of nine clinically healthy pigs of three ages group (1 day, 2 months and 5 months old) were selected for this experiment (three pigs in each group). Tissues from intestinal mucosa in stomach, duodenum, jejunum and ileum and mesenteric lymph node (MLN) were used. mRNA expression of TLRs (1-10) was detectable in all tissues and TLR3 showed the highest mRNA abundance among TLRs. TLR3 expression in stomach, and TLR1 and TLR6 expression in MLN were higher in adult than newborn pigs. The western blot results of TLR2, 3 and 9 in some cases, did not coincide with the mRNA expression results. The protein localization of TLR2, 3 and 9 showed that TLR expressing cells were abundant in the lamina propria, Peyers patches in intestine, and around and within the lymphoid follicles in the MLN. This expressions study sheds the first light on the expression patterns of all TLR genes in GALT at different ages of pigs.

Expression of Toll-like receptors and downstream genes in lipopolysaccharide-induced porcine alveolar macrophages.

The aim of the present study was to determine the age-related kinetic changes of Toll-like receptors (TLRs) and downstream genes expression, and secretion of cytokine in lipopolysaccharide (LPS) stimulated porcine alveolar macrophages (AM). For this purpose, AMs were isolated from 5-day-old newborn piglets and 120-day-old young pigs. mRNA expression and cytokine measurement was determined by quantitative real-time PCR and ELISA, respectively. First, AMs were incubated for 24 h in the absence or presence of increasing concentrations of LPS. Results showed the up-regulation of TLRs 2, 4, 5 and 9 mRNA from all concentrations of LPS used, as compared to non-stimulated cells, and TLR4 was the highest expression in both ages (P<0.05). Furthermore, quantitative analysis demonstrated increased expression of mRNAs encoding TLRs 2, 4, 5 and 9, LBP, CD14, MD2, MyD88, IRAK4 and TRAF6 in both ages in a time-dependant manner (P<0.05). Overall, LPS inducible mRNA for TLR4, LBP, CD14 and MyD88 had higher expression in newborn piglets compared with those of young pigs (P<0.05). The level of cytokine protein IL6 and TNFα in supernatant fluid significantly varied with time of incubation and age of animals. Their concentration increased immediately at 1 h after LPS stimulation and remained significantly higher up to 48 h in both ages. Production of pro-inflammatory cytokine protein IL6 and TNFα in supernatant was significantly higher in young pigs than those of piglets. This study suggests that differential age-related changes in the expression of TLRs and downstream genes, and pro-inflammatory cytokine could contribute to a different age-related innate immune response during pulmonary infection. Further investigation is warranted to determine the precise effects of LPS on porcine AMs by means of a functional study across a wider age range.

Mapping quantitative trait loci for innate immune response in the pig

The aim of the present study was to detect quantitative trait loci (QTL) for the serum levels of cytokines and Toll‐like receptors as traits related to innate immunity in pig. For this purpose, serum concentration of interleukin 2 (IL2), interleukin 10 (IL10), interferon‐gamma (IFNG), Toll‐like receptor 2 (TLR2) and Toll‐like receptor 9 (TLR9) were measured in blood samples obtained from F2 piglets (n = 334) of a Duroc × Piétrain resource population (DUPI) after Mycoplasma hypopneumoniae (Mh), tetanus toxoid (TT) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) vaccination at 6, 9 and 15 weeks of age. Animals were genotyped at 82 genetic markers covering all autosomes. QTL analysis was performed under the line cross F2 model using QTL Express and 33 single QTL were detected on almost all porcine autosomes. Among the single QTL, eight, twelve and thirteen QTL were identified for innate immune traits in response to Mh, TT and PRRSV vaccine, respectively. Besides single QTL, six QTL were identified by a two‐QTL model, of which two for TLR9_TT were in coupling phase and one for IL10_PRRSV was in repulsion phase. All QTL were significant at 5% chromosome‐wide level including one and seven at 5% genome‐ and 1% chromosome‐wide level significance. All innate immune traits are influenced by multiple chromosomal regions implying multiple gene action. Some of the identified QTL coincided with previously reported QTL for immune response and disease resistance, and the newly identified QTL are potentially involved in the immune function. The immune traits were also influenced by environmental factors like year of birth, age, parity and litter size. The results of this work shed new light on the genetic background of innate immune response and these findings will be helpful to identify candidate genes in these QTL regions related to immune competence and disease resistance in pigs.