Association and expression study of CD9, PLCz and COX-2 as candidate genes to improve boar sperm quality and fertility traits

The transcription factors tetraspanin CD9 (CD9), phospholipase C zeta (PLCz) and cyclooxygenase isoenzyme type 2 (COX-2) are believed to play a key role in spermatogenesis in rat, mouse and human. However, no study was devoted to associate the single nucleotide polymorphism (SNP) and expression pattern of three genes with sperm quality and fertility traits in boars. Therefore, this study aimed to investigate the polymorphism and expressional pattern of these genes with boar sperm quality and fertility. The association of SNP with sperm quality traits (sperm concentration [SCON], semen volume per ejaculate [VOL], sperm motility [MOT], plasma droplets rate [PDR] and abnormal spermatozoa rate [ASR]) and the fertility traits (non return rate [NRR] and number of piglet born alive [NBA]) were analyzed using 231 of purebred Pietrain (PI) boars, 109 of Pietrain × Hampshire crossbred (PIHA) boars and 340 of the combined analysis from PI and PIHA boars (COMBINED). In addition, to study the mRNA and protein expression pattern of those genes in boar reproductive and non reproductive tissue, three boars having higher sperm concentration, sperm motility and lower sperm volume (G-I groups) and three boars having lower sperm concentration, sperm motility, and higher sperm volume (G-II groups) were selected. The result showed that the SNP in intron 6 (A>T) of CD9 was significantly associated with MOT (p C) of PLCz was associated with SCON (p A) of COX-2 showed no association with sperm quality and fertility traits. In addition, the mRNA and protein expression of the CD9, PLCz and COX-2 were found to be different between reproductive tissues and non reproductive tissues of boars. Elevated CD9, PLCz and COX-2 proteins expression were found in Leydig cells, Sertoli cells, epithelial cells, germ cells and the acrosome and acrosomal membrane of mature spermatozoa. Importantly, higher CD9 and PLCz mRNA (p Assosiations- und Expressionsstudie der Kandidatengene CD9, PLCz und COX-2 zur Untersuchung ihres Effektes auf die Spermaqualität von Ebern und Fruchtbarkeitsmerkmake In Studien über Ratten, Mäuse und Menschen spielen die Transkriptionsfaktoren Tetraspanin CD9 (CD9), Phospholipase C zeta (PLCz) und Cyclooxygenase-Isoenzym Typ 2 (COX-2) eine wichtige Rolle in der Spermatogenese. Daher war das Ziel dieser Studie die Identifizierung von Polymorphismen und die Erstellung von Expressionsmuster dieser Gene für die Spermaqualität und Fruchtbarkeit, zu untersuchen. Die Assoziationanalysen der SNP mit Spermienqualitätsmerkmalen (Spermienkonzentration [SCON], Spermavolumen pro Ejakulat [VOL], Spermienmotilität [MOT], Plasmatröpfchenrate [PDR] sowie abnormale Spermienrate [ASR]) und den Fruchtbarkeitsmerkmalen (Non Return Rate [NRR] und Anzahl der Ferkel lebend geboren [NBA]) wurde mit 231 Tieren reinrassiger Pietrain (PI) Eber, 109 Tieren einer Pietrain × Hampshire Kreuzung (PIHA) und 340 Eber, die für die Analyse aus PI und PIHA (COMBINED) Ebern kombiniert wurden, durchgeführt. Darüber hinaus wurden die mRNA und Proteinexpressionsmuster. Dafür wurden Eber mit einer höheren Spermienkonzentration, Spermienmotilität und einem niedrigerem Spermienvolumen (G-I Gruppen) und Ebern mit einer geringeren Spermienkonzentration, Spermienmotilität und einem höheren Spermienvolumen (G-II Gruppen) ausgewählt. Das Ergebnis zeigte, dass der SNP im Intron 6 (A>T) von CD9 mit MOT (p C) von PLCz mit SCON (p A) von COX-2 zeigte keine Assoziation mit der Spermaqualität und den Fruchtbarkeitsmerkmalen. Eine erhöhte CD9, PLCz und COX-2 Proteineexpression konnte in Leydig-Zellen, Sertoli-Zellen, Epithelzellen, Keimzellen und im Akrosom und in der akrosomalen Membran reifer Spermien gefunden werden. Eine höhere CD9 und PLCz mRNA Expression (p < 0,05) wurden in Nebenhoden und Hoden in G-I im Vergleich zu G-II Ebern gefunden. Jedoch war die Expression von COX-2 im Hoden, Kopf und Nebenhodens in G-II (p < 0,05) höher als in G-I Ebern. Daher kann die vorliegende Studie zeigen, dass die Gene CD9, PLCz und COX-2 an der Spermatogenese von Ebern beteiligt sind

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

<p>Abstract</p> <p>Background</p> <p>Gene 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.</p> <p>Results</p> <p>The mRNA expression stability of nine commonly used reference genes (<it>B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP </it>and <it>YWHAZ</it>) 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 <it>RPL4, PPIA </it>and <it>YWHAZ </it>showed high stability in newborn and adult pigs, while <it>B2M, YWHAZ </it>and <it>SDHA </it>showed high stability in young pigs. In all cases, <it>GAPDH </it>showed the least stability in geNorm. NormFinder revealed that <it>TBP </it>was the most stable gene in newborn and young pigs, while <it>PPIA </it>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.</p> <p>Conclusions</p> <p>Although, there was discrepancy in the ranking order of reference genes obtained by different analysing software methods, the geometric mean of the <it>RPL4, PPIA </it>and <it>YWHAZ </it>seems to be the most appropriate combination of housekeeping genes for accurate normalization of gene expression data in different porcine tissues at different ages.</p

Expression analysis of porcine aromatase (CYP19) as a specific target gene in testis

Cytochrome P450 aromatase is the key enzyme in estrogen biosynthesis, encoded by the CYP19 gene. However, little is know about the CYP19 roles in boar spermatogenesis. Therefore, the aim of this work was to investigate the mRNA and protein expression of CYP19 in boar reproductive tissues from boars with different sperm quality. For mRNA and protein expression study, a total of six boars were divided into two groups with Group I (G-I) and Group II (G-II), where G-I is characterized for a relatively better sperm quality. For the expression study between reproductive and non-reproductive tissues by semi-quantitative PCR study, mRNA from all six boars was pooled together according to the tissues. On the other hand, mRNA and protein expression study in different reproductive tissues from two divergent groups of animals were performed by semi-quantitative PCR, qRT-PCR and western blot, respectively. Due to the limitations of fresh samples from G-I and G-II boars, different fresh testis from a healthy breeding boar was collected after slaughtering for protein localization by immunofluorescence. The remarkable CYP19 mRNA expression was detected only in testis. The mRNA expression of CYP19 was not detectable in other reproductive tissue (epididymis and accessory glands) and non-reproductive tissue (brain, liver and muscle) by semi-quantitative PCR

A Comparative Study of the Structural and Electrochemical Properties of Na3V2(PO4)3/C Cathode Materials for Na‐ion Batteries Prepared by Sol‐Gel vs. Solid‐State Methods

Abstract Na‐ion batteries have attracted much attention as one of the most promising alternatives to lithium‐ion batteries. Na3V2(PO4)3 has been widely investigated as a cathode material for Na‐ion batteries because of its high structural and thermal stability, leading to high cycling performance and safety. The cathode material also possesses a three‐dimensional open framework and consequently provides high ionic conductivity, as well as high‐rate capability. However, the cathode type experiences poor electronic conductivity, affecting its electrochemical properties. It is well established that preparation methods have remarkable effects on the crystal structure and morphology of electrode materials, altering their electrochemical performance. Thus, this work focuses on a comparative study of the morphology, structures, electrochemical performance, and kinetic properties of Na3V2(PO4)3/C cathodes prepared by sol‐gel and solid‐state methods, which are simple and scalable. The results reveal that sol‐gel and solid‐state reaction processes provide Na3V2(PO4)3/C cathode materials with different structural and morphological characteristics, significantly affecting their electrochemical performance and kinetic properties. This is a crucial key to finding a proper synthesis method to achieve Na3V2(PO4)3/C with high performance for commercial large‐scale production

Investigation on Association and Expression of ESR2 as a Candidate Gene for Boar Sperm Quality and Fertility

Contents ESR2 is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was aimed to investigate the association of ESR2 polymorphism with sperm quality and boar fertility traits and to analyse the ESR2 mRNA and protein expressions in boar reproductive tissues. DNA samples from 203 Pietrain (PI) and 100 Pietrain x Hampshire (PIHA) pigs with records of sperm quality [sperm concentration (SCON), motility (MOT), semen volume (VOL), plasma droplet rate (PDR) and abnormal spermatozoa rate (ASR)] and fertility [non-return rate (NRR) and number of piglet born alive (NBA)] traits were available. A SNP in coding region of ESR2 g.35547A>G in exon 5 was associated with MOT and PDR in the PI and with SCON, VOL, MOT and PDR in PIHA population. For mRNA and protein expression study, a total of six boars were divided into two groups with group I (G-I) and group II (G-II) where G-I characterized for relatively a better sperm quality according to the mean of two groups. mRNA expression was higher in brain and testis than that in all parts of epididymis. Both qRT-PCR and western blot analysis revealed that the ESR2 gene expression and protein expression were significantly higher in testis collected from G-II compared with that of G-I boars. Moreover, ESR2 protein localization in germ cell, Leydig and Sertoli cells, epithelial cells and spermatozoa was remarkable, which indicated the important role of ESR2 in spermatogenesis process. These results might shed new light on the roles of ESR2 in spermatogenesis as candidate for boar fertility, but still the lack of association across populations should be considered

Heterogeneous expression of Toll-like receptors genes in lymphoid tissues of different ages pigs

Toll-like receptors (TLRs), known as the pathogen recognition receptors, recognize the microbial components. The details expression kinetics of TLRs may help to understand the immune responsiveness of pigs, but the expression patterns of all TLRs (TLR1-10) have not yet been studied in pigs. Therefore, this study unravels the expression patterns of the TLR family set of genes (TLR1-10) in different lymphoid tissues collected from pigs at different ages. A total of nine clinically healthy Pietrain pigs at three age groups were selected for this experiment. Each age group consisted of three animals. Cervical lymph node (CLN), thymus, liver, spleen, lung, heart, skin tissue and peripheral blood mononuclear cells (PBMC) were collected for both mRNA and protein isolation. The mRNA expression of TLRs (1-10) was quantified in all tissues. TLR3 mRNA was the most abundant in all tissues. It was expressed highly in thymus, kidney, lungs and liver tissues. Western blot and immunofluorescence was performed for protein expression and localization of selected TLRs (TLR2, 3 and 9) in selected tissues (CLN, spleen and lungs). The Western blot results of TLR2, 3 and 9 in selected tissues appeared to be consistent with the mRNA expression results. Cells in lungs, spleen and CLN were positively immunostained for TLR2, 3 and 9. This study sheds light on the expression patterns of TLR (1-10) genes in important lymphoid tissues in pigs of different ages. The heterogeneous expression of TLRs with ages may contribute to the altered immune responsiveness of pigs with ages