Project description:Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low androstenone boars. The study allows identification of genes and pathways associated with elevated androstenone levels. Testicular tissue was collected from 60 boars, 30 with extreme high and 30 with extreme low levels of androstenone, from each of the two breeds Duroc and Norwegian Landrace. The samples were hybridised to porcine arrays containing 26.877 cDNA clones, detecting 563 and 160 genes that were differentially expressed (p < 0.01) in Duroc and Norwegian Landrace, respectively. Of these significantly up- and down-regulated clones, 72 were found to be common for the two breeds, suggesting both general and breed specific mechanisms in regulation of, or response to androstenone levels in boars. Ten of the most significant genes were chosen for verification of expression patterns by quantitative real competitive PCR and real-time PCR. As expected, our results point towards steroid hormone metabolism and biosynthesis as important biological processes for the androstenone levels, but other potential pathways were identified as well. Among these were oxidoreductase activity, ferric iron binding, iron ion binding and electron transport activities. Genes belonging to the cytochrome P450 and hydroxysteroid dehydrogenase families were highly up-regulated, in addition to several genes encoding different families of conjugation enzymes. Furthermore, a number of genes encoding transcription factors were found both up- and down-regulated. The high number of clones belonging to ferric iron and iron ion binding suggests an importance of these genes, and the association between these pathways and androstenone levels is not previously described. This study contributes to the understanding of the complex genetic system controlling and responding to androstenone levels in pig testis. The identification of new pathways and genes involved in the biosynthesis and metabolism of androstenone is an important first step towards finding molecular markers to reduce boar taint. Keywords: high vs low Testicle samples from animals with extreme androstenone values, 30 high and 30 low from each of the two breeds Norwegian Landrace and Duroc, were used for the experiment. Each microarray was hybridised with one high and one low androstenone sample from the same breed, giving a total of 30 arrays for each breed.

Project description:Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low androstenone boars. The study allows identification of genes and pathways associated with elevated androstenone levels. Testicular tissue was collected from 60 boars, 30 with extreme high and 30 with extreme low levels of androstenone, from each of the two breeds Duroc and Norwegian Landrace. The samples were hybridised to porcine arrays containing 26.877 cDNA clones, detecting 563 and 160 genes that were differentially expressed (p < 0.01) in Duroc and Norwegian Landrace, respectively. Of these significantly up- and down-regulated clones, 72 were found to be common for the two breeds, suggesting both general and breed specific mechanisms in regulation of, or response to androstenone levels in boars. Ten of the most significant genes were chosen for verification of expression patterns by quantitative real competitive PCR and real-time PCR. As expected, our results point towards steroid hormone metabolism and biosynthesis as important biological processes for the androstenone levels, but other potential pathways were identified as well. Among these were oxidoreductase activity, ferric iron binding, iron ion binding and electron transport activities. Genes belonging to the cytochrome P450 and hydroxysteroid dehydrogenase families were highly up-regulated, in addition to several genes encoding different families of conjugation enzymes. Furthermore, a number of genes encoding transcription factors were found both up- and down-regulated. The high number of clones belonging to ferric iron and iron ion binding suggests an importance of these genes, and the association between these pathways and androstenone levels is not previously described. This study contributes to the understanding of the complex genetic system controlling and responding to androstenone levels in pig testis. The identification of new pathways and genes involved in the biosynthesis and metabolism of androstenone is an important first step towards finding molecular markers to reduce boar taint. Keywords: high vs low

Project description:We investigated the nutritional effects on gene expression in testes of F0 boars from a three generation Large White pig feeding experiment. A group of experimental (E) F0 boars were fed a standard diet supplemented with high amounts of methylating micronutrients whereas a control (C) group of F0 boars received a standard diet. These differentially fed F0 boars sired F1 boars which then sired 60 F2 pigs which were investigated in a previous study. The aim of this project was to investigate if the nutrition affects gene expression in testis of differentially fed boars and thus impact on spermatogenesis. We found a small number of 70 genes that were differentially expressed (fc ≥ 1) on the P<0.01 significance level. The false discovery rate (FDR) was 0.82 indicating that only a small portion of these genes are real positives. Nevertheless, we performed a pathway analysis and found this moderate differential expression associated with pathways maps of development_A2B receptor: action via G-protein alpha s, cell adhesion_Tight junctions and cell adhesion_Endothelial cell contacts by junctional mechanisms. The gene ontology (GO) processes that matched the gene expression data in boars’ testes were positive regulation of nucleobase-containing compound metabolic process, cellular response to hormone stimulus and cellular process. The pathway maps and GO processes associated with gene expression differences do not indicate a simple relationship between nutritional influences and gene expression in testes. Nevertheless the Adenosine A2B receptor influences cell differentiation and proliferation and has thus far reaching consequences. Similar applies to those GO processes positive regulation of nucleobase-containing compound metabolic process, cellular response to hormone stimulus and cellular process that were associated with differentially expressed genes between the testes samples. The expression result is thus not conclusive of whether the diet affects processes related to transmittable epigenetic marks. The results, however, indicate that the extreme supplementation of methylating micronutrients from month one to month ten of age has a very moderate (if any) effect on gene expression in boar testes as measured by microarray analysis. Gene expression in testes from differentially fed F0 boars was measured. F0 boars received either a standard diet or a standard diet supplemented with methylating micronutrients. These boars were used to study transgenerational epigenetic inheritance in a three generation pig pedigree. Therefore it was of interest if the diet affects gene expression in testes and so could impact spermatogenesis.

Project description:Cryptorchidism and scrotal heating result in abnormal spermatogenesis but the mechanism(s) proscribing this temperature sensitivity are unknown. It was previously reported that the AKR/N or MRL/MpJ-+/+ mouse testis is more heat resistant than the testis from the C57BL/6 strain. We have attempted to probe into the mechanism(s) involved in heat sensitivity by examining global gene expression profiles of normal and heat-treated testes from C57BL/6, AKR/N and MRL/MpJ-+/+ mice by microarray analysis. In the normal C57BL/6 testis, 415 and 416 transcripts were differentially expressed (at least two-fold higher or lower) when compared to the normal AKR/N and MRL/MpJ-+/+ testis, respectively. The AKR/N and MRL/MpJ-+/+ strains revealed 268 differentially expressed transcripts between them. There were 231 transcripts differentially expressed between C57BL/6 and two purported heat-resistant strains, AKR/N and MRL/MpJ-+/+. Next, the testes of C57BL/6 and AKR/N mice were exposed to 43ºC for 15 min and harvested at different time points for TUNEL studies and microarrays. An increase of TUNEL-positive germ cell numbers was significant 8 hr after heat exposure in the C57BL/6 mouse. However, this increase was not observed in the AKR/N mouse until 10 hr after heat exposure. All tubules showed germ cell loss and disruption in C57BL/6 testis 24 hr after heat shock. In contrast, although a number of seminiferous tubules showed an abnormal morphology 24 hr post-heat shock in the AKR/N mouse, many tubules still retained a normal structure. Numerous transcripts exhibited differential regulation between the two strains within 24 hours after heat exposure. The differentially expressed transcripts in the testes 8 hr after heat exposure were targeted to identify the genes involved in the initial response rather than those due to germ cell loss. Twenty transcripts were significantly down-regulated and 19 genes were up-regulated by hyperthermia in C57BL/6 and did not show a parallel change in the AKR/N testis. Conversely, heat shock resulted in 30 up-regulated transcripts and 31 down-regulated transcripts in AKR/N that were not similarly regulated in C57BL/6. A number of genes shared similar differential expression patterns and differential regulation by hyperthermia in both strains of mice. Taken together, the present study indicates the diverse genetic backgrounds in the three strains lead to major differences in normal testis gene expression profiles while the differences in heat shock responses involves a significantly smaller number of genes. The data generated may provide insights regarding gene networks and pathways involved in heat stress and their relationship to spermatogenesis.

Project description:Cryptorchidism and scrotal heating result in abnormal spermatogenesis but the mechanism(s) proscribing this temperature sensitivity are unknown. It was previously reported that the AKR/N or MRL/MpJ-+/+ mouse testis is more heat resistant than the testis from the C57BL/6 strain. We have attempted to probe into the mechanism(s) involved in heat sensitivity by examining global gene expression profiles of normal and heat-treated testes from C57BL/6, AKR/N and MRL/MpJ-+/+ mice by microarray analysis. In the normal C57BL/6 testis, 415 and 416 transcripts were differentially expressed (at least two-fold higher or lower) when compared to the normal AKR/N and MRL/MpJ-+/+ testis, respectively. The AKR/N and MRL/MpJ-+/+ strains revealed 268 differentially expressed transcripts between them. There were 231 transcripts differentially expressed between C57BL/6 and two purported heat-resistant strains, AKR/N and MRL/MpJ-+/+. Next, the testes of C57BL/6 and AKR/N mice were exposed to 43ÂºC for 15 min and harvested at different time points for TUNEL studies and microarrays. An increase of TUNEL-positive germ cell numbers was significant 8 hr after heat exposure in the C57BL/6 mouse. However, this increase was not observed in the AKR/N mouse until 10 hr after heat exposure. All tubules showed germ cell loss and disruption in C57BL/6 testis 24 hr after heat shock. In contrast, although a number of seminiferous tubules showed an abnormal morphology 24 hr post-heat shock in the AKR/N mouse, many tubules still retained a normal structure. Numerous transcripts exhibited differential regulation between the two strains within 24 hours after heat exposure. The differentially expressed transcripts in the testes 8 hr after heat exposure were targeted to identify the genes involved in the initial response rather than those due to germ cell loss. Twenty transcripts were significantly down-regulated and 19 genes were up-regulated by hyperthermia in C57BL/6 and did not show a parallel change in the AKR/N testis. Conversely, heat shock resulted in 30 up-regulated transcripts and 31 down-regulated transcripts in AKR/N that were not similarly regulated in C57BL/6. A number of genes shared similar differential expression patterns and differential regulation by hyperthermia in both strains of mice. Taken together, the present study indicates the diverse genetic backgrounds in the three strains lead to major differences in normal testis gene expression profiles while the differences in heat shock responses involves a significantly smaller number of genes. The data generated may provide insights regarding gene networks and pathways involved in heat stress and their relationship to spermatogenesis. Experiment Overall Design: Total RNA of testes from two different strains of mice, C57BL/6 and AKR/N were analyzed using microarray 430.20 chip. Duplicates were used in controls and treated samples at 8hr, 10hr, 12hr, 24hr after heat shock at 43C for 15 min. All mice were 8 weeks old.

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Differential Gene Expression of Enriched Germ Cells Responding to a Short Heat Stimulus

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