Investigations on transgenerational epigenetic response down the male line in F2 pigs

We investigated the nutritional effects on carcass traits, gene expression and DNA methylation in 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 group (C) of F0 boars received a standard diet. These differentially fed F0 boars sired F1 boars which then sired 60 F2 pigs. Carcass traits were compared between 36 F2 descendants of E F0 boars and 24 F2 descendants of C F0 boars. The two F2 offspring groups differed with respect to backfat percentage (P = 0.03) and tended to differ with respect to adipose tissue (P = 0.09), fat thickness at the 10^th rib (P = 0.08) and at the croup (P = 0.09) as well as percentages of shoulder (P = 0.07). Offspring from the experimental F0 boars had a higher percentage of shoulder and were leaner compared to the control group. Gene expression profiles showed significant twofold differences in mRNA level between 8 C F2 offspring and 8 E F2 offspring for 79, 64 and 53 genes for muscle, liver and kidney RNA, respectively. We found that in liver and muscle respective pathways of lipid metabolism and metabolic pathway were over-represented for the differentially expressed genes between these groups. A DNA methylation analysis in promoters of differentially expressed genes indicated a significant difference in DNA methylation at the IYD gene. If these responses on carcass traits, gene expression and DNA methylation withstand verification and can indeed be attributed to transgenerational epigenetic inheritance, it would open up pioneering application in pork production and would have implications for human health.     

The effect of feeding level on semen quantity and quality of breeding boars

An experiment was carried out to investigate the influence of feeding level on semen quantity and quality in breeding boars in two successive periods of 12 and 8 weeks. Forty-two Yorkshire boars (13 months old) were fed for 12 weeks ad libitum (H=5.74 kg/day), a medium feeding level (M=3.62 kg/day) or a low feeding level (L=1.92 kg/day). After 8 weeks the number of ejaculated sperm cells for boars on treatment L was lower than for those on treatments H and M (P <0.05). In the last 2 weeks, the M and H boars ejaculated 46 and 69% more sperm cells, respectively, than the L boars. Differences between all three feeding levels were significant (P < 0.05). After this 12-week period the feeding level was altered for the H and the L boars. H boars were fed a low feeding level (HL) and L boars were fed the medium feeding level (LM). The medium-fed boars were kept at the same feeding level (MM). After 8 weeks in the second period, differences in the number of ejaculated sperm cells between the HL and MM boars and between the LM and MM boars were no longer significant (P >0.05). Throughout the experiment, no differences in the quality parameters, percentage of moving sperm cells in the ejaculate, vitality of the moving sperm cells and non-return percentage at 56 days were found between treatment groups.     

低压低氧对小鼠精子发生及其小RNA表达的影响

作为青藏高原最为关键的环境因子,低压低氧对高原非习服动物繁殖和生殖系统功能有不利影响。已有的研究表明,低氧环境会导致雄性生殖细胞凋亡、精子畸形率升高、精子质量下降,进而影响受精和早期胚胎发育,但目前缺乏低氧损伤精子功能的机理研究。小RNA (small RNA)是在转录后及翻译水平上调控基因表达的重要功能分子,不同类型的small RNA通过诱导基因沉默或调控翻译等方式参与调节精子发生。本研究通过低压氧舱模拟海拔5 000 m处理4周建立缺氧小鼠模型,发现低氧处理导致小鼠曲精细管中生精细胞排列紊乱,精子数量未发生显著变化但畸形率增加17.5倍(P <0.001)。通过small RNA测序发现,低氧组小鼠精子中的small RNA碱基偏好性与对照组一致,第一碱基对尿嘧啶(U)有很强的偏好性。低氧组小鼠精子中21 nt长度的small RNA比例显著减少4.4%(P <0.05)。低氧组小鼠精子中piRNA、tsRNA表达无差异,但miRNA表达上调21个,下调58个。对差异miRNAs靶基因与相同低氧处理小鼠睾丸组织差异基因比对,共比对到831个差异表达基因,其中上调miR...     

MicroRNA Alternations in the Testes Related to the Sterility of Triploid Fish

The knowledge of understanding the molecular traits of the sterile triploid fish is sparse. Herein, we analyzed the microRNA (miRNA) alternations in the testes of the sterile triploid fish produced by crossing the tetraploid fish with the diploid fish, compared with those of tetraploids and diploids used as the controls. A total of 136, 134, and 142 conserved miRNAs and 105, 112, and 119 novel miRNAs were identified in the diploid, triploid, and tetraploid fish, respectively. The genes targeted by the differentially expressed miRNAs were identified and were enriched in the GO term cell surface receptor signaling pathway, cellular process, G-protein coupled receptor signaling pathway, and metabolic process. KEGG pathway enrichment was also assessed to evaluate the target genes with differentially expressed miRNAs and these genes were enriched in four pathways (synthesis and degradation of ketone bodies, pentose and glucuronate interconversions, cyanoamino acid metabolic process, and ascorbate and aldarate metabolism). Nine differentially expressed miRNAs were verified by quantitative real-time PCR analysis (qPCR). The upregulated miRNAs in triploids, including miR-101a, miR-199-5p, miR-214, miR-222, and miR-193a, showed the same results with high-throughput sequencing. Among the selected downregulated miRNAs, miR-7b and miR-153b had significantly lower expression levels in triploids. Dnah3 and Tekt1 genes targeted by miR-199-5p showed lower expression in triploids by qPCR. These verified differentially expressed miRNAs may participate in testicular development and sperm activity by targeting functional genes, which were identified with differential expression in the triploid. This evidence provides insights into the epigenetic regulatory mechanisms of sterility in triploid cyprinids.     

A diet supplemented with l-carnitine improves the sperm quality of Piétrain but not of Duroc and Large White boars when photoperiod and temperature increase

It has been reported that a diet supplemented with l-carnitine can improve sperm quality in some mammalian species. Against this background, the current study seeks to determine the effects of feeding l-carnitine (625 mg·day⁻¹) on boar semen characteristics (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) in three different porcine breeds (Sus domesticus) (Piétrain, Duroc, and Large White) exposed to natural environmental changes in temperature and photoperiod over a 20-wk period (February to July 2007). One hundred twenty boars (40 per breed) were randomly separated into two groups (60 boars each): the first (20 boars per breed) was fed a control diet and the second (also 20 males per breed) the same diet supplemented with l-carnitine (625 mg·day⁻¹). Whereas the l-carnitine supplement did not affect ejaculate volume, concentration, motility, viability, or the osmotic resistance of spermatozoa, it did improve sperm morphology in Piétrain boars by reducing the percentage of immature spermatozoa when the temperature and the photoperiod increased. Conversely, no effect on sperm morphology from supplementing feed with l-carnitine was observed in both Duroc and Large White breeds. We can therefore conclude that the addition of l-carnitine to the diet of males may maintain the level of normal sperm morphology in Piétrain boars when a drop in sperm quality occurs (due to increases in photoperiod and temperature), without affecting the other sperm quality parameters.     

Reproductive organ weights and semen quality of pubertal boars fed dietary fumonisin B1

Fumonisins, a group of toxic metabolites produced by the genus Fusarium, are known to be consumed by farm animals and are the causative agent or a suspected contributing factor in farm animal diseases. Pigs are particularly susceptible to fumonisins. Reproductive inefficiency is recognized as the most costly limiting constraint to efficient animal production. To account for potential reproductive effects of fumonisin in boars, dietary fumonisin B1 (FB1) was fed to 24 male Large White weanling pigs, 8 to 9 weeks of age. The animals were randomly assigned to four diets containing 5.0, 10.0, 15.0 and 0.2 mg FB1/kg constituting diets 1, 2, 3 and control diet, respectively. After 6 months, semen samples were collected from the pubertal boars and analysed. After the semen collection, all the pubertal boars were killed by decapitation, their reproductive systems dissected and the weights of the testes and epididymides as well as the volumes of the testes recorded. Dietary FB1 did not influence both the relative weights of the testes and epididymides as well as the volumes of the testes. Except for the semen volume and spermatozoa morphological abnormalities, all the semen characteristics studied decreased in a dose-dependent manner and the decrease was statistically significant (P < 0.05). The sperm concentration, total sperm and motile sperm per ejaculate of the animals on diet 3 were 83.3%, 79.1% and 59.6% of the controls, respectively. The dietary FB1 levels influenced the mass activities of the semen, which ranged from very turbulent motion for animals on the control diet to absence of wave motion for those on diet 3. The study revealed that male weanling pigs for breeding should not be exposed to dietary FB1 higher than 5 mg/kg for optimum reproductive performance. The results of this present study suggest that the recommendation of 10 mg/kg by the United States Food and Drug Administration as the maximum level of total dietary fumonisins was above the no-observable-effect level of dietary fumonisin for swine.     

Deletion of porcine BOLL is associated with defective acrosomes and subfertility in Yorkshire boars

A recessive sperm defect of Yorkshire boars was detected more than a decade ago. Affected boars produce ejaculates that contain spermatozoa with defective acrosomes, resulting in low fertility. The acrosome defect was mapped to porcine chromosome 15 but the causal mutation has not been identified. We re-analyzed microarray-derived genotypes of affected boars and confirmed that the acrosome defect maps to a 12.24 Mb segment of porcine chromosome 15. To detect the mutation causing defective acrosomes, we sequenced the genomes of two affected and three unaffected boars to an average coverage of 11-fold. Read depth analysis revealed a 55 kb deletion that is associated with the acrosome defect. The deletion encompasses the BOLL gene encoding the boule homolog, an RNA binding protein which is an evolutionarily conserved member of the DAZ (Deleted in AZoospermia) gene family. Lack of BOLL expression causes spermatogenic arrest and sperm maturation failure in many species. Boars that carry the deletion in the homozygous state produce sperm but their acrosomes are defective, suggesting that lack of porcine BOLL compromises acrosome formation. Our findings warrant further research to investigate the role of BOLL during spermatogenesis and sperm maturation in pigs.     

A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression,amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice

Exosomes are nanoparticles that transfer cargos from donor cells to recipient cells where they elicit changes in gene expression and metabolism. Evidence suggests that exosomes and their cargos are also absorbed from dietary sources such as bovine milk, and bovine exosomes promote the growth of myofibers in murine C2C12 myotube cell cultures. The aim of the current study was to determine whether the dietary intake of bovine milk exosomes alters strength, gene expression and amino acid profiles in murine skeletal muscles. Male and female C57BL/6 mice, age three weeks, were fed an AIN93G-based, exosome and RNA-depleted (ERD) diet for six weeks; controls were fed an exosome and RNA-sufficient (ERS) diet. Variables of feeding behavior, metabolism, grip strength, liver and kidney function, amino acid profiles, and gene expression patterns were analyzed by using metabolic cages, grip strength analyzers, clinical chemistry analyzers, targeted LC/MS–MS, and RNA sequencing analysis. The diets had no effect on food and water intake, respiratory exchange rate, physical activity, grip strength, markers of liver and kidney dysfunction, and amino acid profiles in muscle. Only twelve and nine mRNAs were differentially expressed in skeletal muscle from female and male mice, respectively, fed ERD and ERS diets. The modest effect of the ERD diet on gene expression and levels of free amino acids in skeletal muscle is consistent with observations that bovine milk exosomes and their cargos accumulate in tissues other than skeletal muscle.     

Novel transcriptome data analysis implicates circulating microRNAs in epigenetic inheritance in mammals

Experimental evidence supports a role of mobile small non-coding RNAs in mediating soma to germline hereditary information transfer in epigenetic inheritance in plants and worms. Similar evidence in mammals has not been reported so far. In this bioinformatic analysis, differentially expressed microRNAs (miRNAs) or mRNAs reported previously in genome level expression profiling studies related to or relevant in epigenetic inheritance in mammals were examined for circulating miRNA association. The reported sets of differentially expressed miRNAs or miRNAs that are known to target the reported sets of differentially expressed genes, in that order, showed enrichment of circulating miRNAs across environmental factors, tissues, life cycle stages, generations, genders and species. Circulating miRNAs commonly representing the expression profiles enriched various epigenetic processes. These results provide bioinformatic evidence for a role of circulating miRNAs in epigenetic inheritance in mammals.     

Male germ cell-specific expression of a novel Patched-domain containing gene Ptchd3

The Hedgehog (Hh) signaling pathway plays an important role in various biological processes, including pattern formation, cell fate determination, proliferation, and differentiation. Hh function is mediated through its membrane receptor Patched. Herein, we have characterized a novel Patched-domain containing gene Ptchd3 in mouse. Messenger RNA of Ptchd3 was exclusively detected in the testis, and existed in two isoforms Ptchd3a and Ptchd3b. The expression of these two mRNA isoforms was shown to be developmentally regulated in testes, and specifically found in male germ cells. Further analysis revealed that the Ptchd3 protein was located on the midpiece of mouse, rat and human sperm. Collectively, these results indicate that Ptchd3 is a novel male germ cell-specific gene and may be involved in the Hh signaling to regulate sperm development and/or sperm function.