Analysis of the SNP loci around transcription start sites related to goat fecundity trait base on whole genome resequencing.

Genome flanking regions surrounding transcription start sites (TSSs) are critical for the regulation of gene expression, containing many translational regulatory elements. To investigate whether critical single nucleotide polymorphisms (SNPs) exist around TSSs in the dairy goat genome, we performed high throughput DNA sequencing to compare two dairy goat groups with discrepant litter sizes. After genome mapping, SNP calling, and annotation, we screened the SNPs within 2kb scales surrounding annotated TSSs in high fecundity (HF) and low fecundity (LF) groups, respectively. We attempted to identify distinct SNPs and motifs near the TSSs in both groups. The SNPs near the TSSs most were consistent; 318 new SNPs were uncovered in the HF group, of which 305 were heterozygote SNPs, 13 were homozygote SNPs, and majority of which were distributed on chromosome 2 and 29. After validation by Sanger sequencing we found that a SNP in CHI16: 27612330 C>A in the PSEN2 gene presented an A/A genotype in the HF group and an A/A or A/C genotype in the LF group. In conclusion, our study provides insightful information into the dairy goat genomic variations surrounding TSSs, which may contribute to enhanced litter size. Based on comparison studies of SNPs exist around transcription start sites between high fecundity group and low fecundity group. Our finding provides insights concerning the goat litter size phenotypic and will promote future goat breeding.

Whole-genome scanning for the litter size trait associated genes and SNPs under selection in dairy goat (Capra hircus).

Dairy goats are one of the most utilized domesticated animals in China. Here, we selected extreme populations based on differential fecundity in two Laoshan dairy goat populations. Utilizing deep sequencing we have generated 68.7 and 57.8 giga base of sequencing data, and identified 12,458,711 and 12,423,128 SNPs in the low fecundity and high fecundity groups, respectively. Following selective sweep analyses, a number of loci and candidate genes in the two populations were scanned independently. The reproduction related genes CCNB2, AR, ADCY1, DNMT3B, SMAD2, AMHR2, ERBB2, FGFR1, MAP3K12 and THEM4 were specifically selected in the high fecundity group whereas KDM6A, TENM1, SWI5 and CYM were specifically selected in the low fecundity group. A sub-set of genes including SYCP2, SOX5 and POU3F4 were localized both in the high and low fecundity selection windows, suggesting that these particular genes experienced strong selection with lower genetic diversity. From the genome data, the rare nonsense mutations may not contribute to fecundity, whereas nonsynonymous SNPs likely play a predominant role. The nonsynonymous exonic SNPs in SETDB2 and CDH26 which were co-localized in the selected region may take part in fecundity traits. These observations bring us a new insights into the genetic variation influencing fecundity traits within dairy goats.

Hydrogen Sulfide and/or Ammonia Reduces Spermatozoa Motility through AMPK/AKT Related Pathways.

A number of emerging studies suggest that air pollutants such as hydrogen sulfide (H2S) and ammonia (NH3) may cause a decline in spermatozoa motility. The impact and underlying mechanisms are currently unknown. Boar spermatozoa (in vitro) and peripubertal male mice (in vivo) were exposed to H2S and/or NH3 to evaluate the impact on spermatozoa motility. Na2S and/or NH4Cl reduced the motility of boar spermatozoa in vitro. Na2S and/or NH4Cl disrupted multiple signaling pathways including decreasing Na+/K+ ATPase activity and protein kinase B (AKT) levels, activating Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and phosphatase and tensin homolog deleted on chromosome ten (PTEN), and increasing reactive oxygen species (ROS) to diminish boar spermatozoa motility. The increase in ROS might have activated PTEN, which in turn diminished AKT activation. The ATP deficiency (indicated by reduction in Na+/K+ ATPase activity), transforming growth factor (TGFß) activated kinase-1 (TAK1) activation, and AKT deactivation stimulated AMPK, which caused a decline in boar spermatozoa motility. Simultaneously, the deactivation of AKT might play some role in the reduction of boar spermatozoa motility. Furthermore, Na2S and/or NH4Cl declined the motility of mouse spermatozoa without affecting mouse body weight gain in vivo. Findings of the present study suggest that H2S and/or NH3 are adversely associated with spermatozoa motility.

Regulation of MicroRNAs, and the Correlations of MicroRNAs and Their Targeted Genes by Zinc Oxide Nanoparticles in Ovarian Granulosa Cells.

Zinc oxide (ZnO) nanoparticles (NPs) have been applied in numerous industrial products and personal care products like sunscreens and cosmetics. The released ZnO NPs from consumer and household products into the environment might pose potential health issues for animals and humans. In this study the expression of microRNAs and the correlations of microRNAs and their targeted genes in ZnO NPs treated chicken ovarian granulosa cells were investigated. ZnSO4 was used as the sole Zn2+ provider to differentiate the effects of NPs from Zn2+. It was found that ZnO-NP-5 µg/ml specifically regulated the expression of microRNAs involved in embryonic development although ZnO-NP-5 µg/ml and ZnSO4-10 µg/ml treatments produced the same intracellular Zn concentrations and resulted in similar cell growth inhibition. And ZnO-NP-5 µg/ml also specifically regulated the correlations of microRNAs and their targeted genes. This is the first investigation that intact NPs in ZnO-NP-5 µg/ml treatment specifically regulated the expression of microRNAs, and the correlations of microRNAs and their targeted genes compared to that by Zn2+. This expands our knowledge for biological effects of ZnO NPs and at the same time it raises the health concerns that ZnO NPs might adversely affect our biological systems, even the reproductive systems through regulation of specific signaling pathways.

Regulation of neuroendocrine cells and neuron factors in the ovary by zinc oxide nanoparticles.

The pubertal period is an important window during the development of the female reproductive system. Development of the pubertal ovary, which supplies the oocytes intended for fertilization, requires growth factors, hormones, and neuronal factors. It has been reported that zinc oxide nanoparticles (ZnO NPs) cause cytotoxicity of neuron cells. However, there have been no reports of the effects of ZnO NPs on neuronal factors and neuroendocrine cells in the ovary (in vivo). For the first time, this in vivo study investigated the effects of ZnO NPs on gene and protein expression of neuronal factors and the population of neuroendocrine cells in ovaries. Intact NPs were detected in ovarian tissue and although ZnO NPs did not alter body weight, they reduced the ovary organ index. Compared to the control or ZnSO4 treatments, ZnO NPs treatments differentially regulated neuronal factor protein and gene expression, and the population of neuroendocrine cells. ZnO NPs changed the contents of essential elements in the ovary; however, they did not alter levels of the steroid hormones estrogen and progesterone. These data together suggest that intact ZnO NPs might pose a toxic effect on neuron development in the ovary and eventually negatively affect ovarian developmental at puberty.

Regulation of egg quality and lipids metabolism by Zinc Oxide Nanoparticles.

This investigation was designed to explore the effects of Zinc Oxide Nanoparticles (ZnO NP) on egg quality and the mechanism of decreasing of yolk lipids. Different concentration of ZnO NP and ZnSO4 were used to treat hens for 24 weeks. The body weight and egg laying frequency were recorded and analyzed. Albumen height, Haugh unit, and yolk color score were analyzed by an Egg Multi Tester. Breaking strength was determined by an Egg Force Reader. Egg shell thickness was measured using an Egg Shell Thickness Gouge. Shell color was detected by a spectrophotometer. Egg shape index was measured by Egg Form Coefficient Measuring Instrument. Albumen and yolk protein was determined by the Kjeldahl method. Amino acids were determined by an amino acids analyzer. Trace elements Zn, Fe, Cu, and P (mg/kg wet mass) were determined in digested solutions using Inductively Coupled Plasma-Optical Emission Spectrometry. TC and TG were measured using commercial analytical kits. Yolk triglyceride, total cholesterol, pancreatic lipase, and phospholipids were determined by appropriate kits. ß-carotene was determined by spectrophotometry. Lipid metabolism was also investigated with liver, plasma, and ovary samples. ZnO NP did not change the body weight of hens during the treatment period. ZnO NP slowed down egg laying frequency at the beginning of egg laying period but not at later time. ZnO NP did not affect egg protein or water contents, slightly decreased egg physical parameters (12 to 30%) and trace elements (20 to 35%) after 24 weeks treatment. However, yolk lipids content were significantly decreased by ZnO NP (20 to 35%). The mechanism of Zinc oxide nanoparticles decreasing yolk lipids was that they decreased the synthesis of lipids and increased lipid digestion. These data suggested ZnO NP affected egg quality and specifically regulated lipids metabolism in hens through altering the function of hen's ovary and liver.

Cloning and in vitro function analysis of codon-optimized FatI gene.

Currently, n-3 polyunsaturated fatty acids (n-3 PUFAs) have attracted great attention because of their biological significance to organisms. In addition, PUFAs show an obvious impact on prevention and treatment of various diseases. Because n-3 PUFAs cannot be endogenously synthesized by mammals, mammals have to rely on a dietary supplement for sufficient supply. The finding and application of the fatty acid dehydrogenase I (FatI) gene are expected to change the current situation because it can convert n-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. Meanwhile, the gradual maturation of transgenic technology makes it possible to produce transgenic animals that can synthesize n-3 PUFAs by themselves. In this study, the DNA coding sequence of FatI was synthesized by a chemical method after codon optimization according to the mammal's codon bias. The synthesized DNA sequence was introduced into Boer goat fetal fibroblasts by the constructed recombinant eukaryotic expression vector pcDNA3.1(+)-FatI. Boer goat fetal fibroblasts were transfected by electroporation, and the stable transfected cell lines were obtained by G418 selection. Genomic DNA PCR and Southern blot were applied to verify that the foreign gene FatI was integrated into the genome of the Boer goat fibroblasts. RT-PCR results showed the expression of FatI gene at the mRNA level. The fatty acid profile of cells carrying the FatI gene revealed an increase in total n-3 PUFAs (from 0.61 to 0.95), but a decrease in n-6 PUFAs (from 10.34 to 9.85), resulting in a remarkable increase in the n-3:n-6 ratio (from 0.059 to 0.096). The n-3:n-6 ratio had a 63.49 percent increase, which is a precursor of the response of n-3 desaturase activity of the FatI gene. The study may provide a practical tool for producing transgenic animals that can produce n-3 PUFAs by themselves, and we hope that the application will lay the foundation for animals producing n-3 PUFAs, which will benefit human nutrition and wellness.

Anti-senescence effect of FatI gene in goat somatic cells.

The fatty acid dehydrogenase I (FatI) is able to express in mammalian cells and convert n-6 polyunsaturated fatty acids (PUFAs) to n-3 PUFAs. n-3 PUFA is an important component of the cell membrane and plays an important role in the prevention and control of a variety of human diseases. However, n-3 PUFAs cannot be endogenously synthesized by mammals because they lack the dehydrogenase that converts n-6 to n-3 PUFA. For the time being, gradually matured transgenic technology makes it possible to produce transgenic animals that are able to synthesize n-3 PUFAs by themselves. However, the transgenic technology itself may bring negative impacts. In this study, the eukaryotic expression vector pcDNA3.1-FatI was introduced into the genome of Boer goat fetal fibroblasts cultured in vitro, and the influence of biological characteristics of the fetal fibroblast was studied via overexpression of FatI. The results showed that the proliferation and apoptosis of cultured fetal fibroblast were not affected significantly by the overexpression of FatI using BrdU and TUNEL staining methods, respectively. Moreover, the overexpression of FatI significantly inhibited the senescence of somatic cells compared with enhanced green fluorescent protein (EGFP) transgenic cells (P < 0.01). Quantitative PCR revealed that the mRNA expression of P16 and P53 in the FatI transgenic cell group was significantly lower than that in the EGFP transgenic cell group (P < 0.01). In conclusion, the senescence of goat somatic cells was inhibited by the overexpression of the FatI gene.

Maternal diabetes causes abnormal dynamic changes of endoplasmic reticulum during mouse oocyte maturation and early embryo development.

BACKGROUND: The adverse effects of maternal diabetes on oocyte maturation and embryo development have been reported. METHODS: In this study, we used time-lapse live cell imaging confocal microscopy to investigate the dynamic changes of ER and the effects of diabetes on the ER's structural dynamics during oocyte maturation, fertilization and early embryo development. RESULTS: We report that the ER first became remodeled into a dense ring around the developing MI spindle, and then surrounded the spindle during migration to the cortex. ER reorganization during mouse early embryo development was characterized by striking localization around the pronuclei in the equatorial section, in addition to larger areas of fluorescence deeper within the cytoplasm. In contrast, in diabetic mice, the ER displayed a significantly higher percentage of homogeneous distribution patterns throughout the entire ooplasm during oocyte maturation and early embryo development. In addition, a higher frequency of large ER aggregations was detected in GV oocytes and two cell embryos from diabetic mice. CONCLUSIONS: These results suggest that the diabetic condition adversely affects the ER distribution pattern during mouse oocyte maturation and early embryo development.

Growth of mouse oocytes to maturity from premeiotic germ cells in vitro.

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12-14 day-old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6-7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16-18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.

[Genetic co-adaptability among structural genes under the condition of genetic disequilibrium].

Genetic co-adaptability of colony gene bank is formed in the process of system evolvement, and it is a part of the attribute of gene bank, just as the gene frequency of every locus. With the genetic co-adaptability of loci combination under the condition of genetic disequilibrium, the genesis evolution and the system status of colony could be analyzed; through the study of genetic co-adaptability, the veracity of the economy character mark could be improved. We summarized about the concept, the basic and the actuality of genetic co-adaptability.

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

[Relationship between polymorphism of growth hormone gene and production traits in goats].

Growth hormone (GH) plays an important role in animal growth and development, and the growth hormone gene can be considered as a candidate gene for studying the weight in goats. The function of GH gene has been intensively studied in cattle and pigs, while in goats the study is still rare. In this paper 224 goats including LuBei White goat, Boer goat, hybrid generation 1 and backcross generation 1 of Boer goat and LuBei White goat were used. Two pairs of primers for 5' region of GH gene were designed according to the database of goat genomic sequence (Accession No. D00476) and polymorphisms were detected by PCR-SSCP. Homozygotes of the polymorphic fragment were cloned and sequenced. The result showed that there were five substitution mutations in the two fragments. Statistical analysis showed that in the fragment amplified by the first pair of primers, AA genotype had significant higher birth weight and weight of one year old than BB and AB genotypes in Boer goats (P<0.05). In hybrid generation 1 AA genotype also had higher birth weight and weight of one year old in significantly. While in LuBei White goats BB genotype had lower weight and the weaning weight was significant lower than the other two genotypes (P<0.05). In the fragment amplified by the second pair of primers there was no significant difference among different genotypes. From these results we can preliminarily draw the conclusion that GH gene may be a major gene or linked to the major gene to affect the weight traits and the polymorphic site could be used to select the goat weight in marker-assisted selection program.

[High-efficient gene targeting of goat mammary epithelium cell by the multi-selection mechanism].

Gene targeting is a more powerful method to produce mammary gland bioreactor and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe an efficient and reproducible gene targeting in goat mammary epithelium cell to place the GFP and neo at the beta-casein locus. The transgenic goat would be produced by nuclear transfer. To construct the gene targeting vector pGBC-GFP-neo, the milk goat beta-casein genomic DNA sequence for homologous arms was cloned first. The left arm was 2.1 kb fragment including goat beta-casein gene exon1 and part of exon 2, and the right arm was 5.1 kb fragment including beta-casein gene from exon 7 to 3'-flanking sequence. The bacterial neomycin (neo) gene as positive selection marker gene, with the promoter-trap GFP, was placed between two loxPs. The thymidine kinase (tk) as negative selection marker gene was just outside the right or left arms. Goat mammary epithelium cells were cultured to sub-confluence about 90% and transfected with linear pGBC-GFP-neo using Lipefectamin-2000. These transfected cells were cultured in collagen-coated 96-wellplate for 24 h without selection, then added the drug G418(600 microg/mL) and GANC(2 micromol/L). After nine days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate; 51 clones were selected; 17 clones were tested by GFP expression using promoter-trap strategy; only four clones grow well. After PCR confirmation the four targeting cell clones homologous recombination were used as the donor cell for nuclear transfer. 59.5% cloned embryos could develop up. Some could develop to morula or blastocyst in vitro.

[Polymorphism analysis of the goat growth hormone gene in the 5' regulatory sequence].

The polymorphism of goat growth hormone gene in 5'-region in 274 goats including Lubei white goat, introducted Boer goat, pure bred Boer goat , hybrid generation 1 of LuBei white goat and Boer goat , backcross of hybrid generation 1 and Boer goat were detected by PCR-SSCP with two different pairs of primers. The result showed that in 26-239bp most of the Boer goats and hybrid generation presented as AA genotype,while most LuBei white goats presented as BB genotype. In 225-429bp all breeds presented as more CC genotype. The amplified fragments of AA,BB and CC,DD of the two fragments were cloned and sequenced . The result showed that in 26-239bp AA genotype had one substitution mutation at 60 site (C-->T), and base C losing at 211 site and DD genotype had three substitution mutations at 264 site (T-->C), 292 site (T-->A) and 372 site (C-->T) in 225-429bp. The results above-mentioned first confirmed that there were polymorphisms in 5' region of GH gene.