Lingjiang Min

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.

Maternal imprinting during mouse oocyte growth in vivo and in vitro.

Epigenetic regulation of gene expression is critical for oogenesis in mammals. In this study, a simple and efficient method was used to obtain the oocytes from cultured fetal mouse ovaries of 12.5dpc. The methylation pattern of these oocytes was examined. The results showed that the establishment of imprinting of Igf2r and Peg3 in oocytes derived from cultured fetal mouse germ cells in vitro follows a slower time course than that of oocytes in vivo. However, oocytes in vitro and in vivo share similar methylation patterns. Igf2r was gradually de novo methylated, and the methylation covers 80% CpG sites in oocytes cultured for 28days. However, only 45% of the CpG sites is methylated in Peg3 at the same stage. Furthermore, it demonstrated that the degree of DNA methylation is positively correlated with the size of oocytes in vitro and in vivo, indicating a progressive methylation process during oocyte growth.

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. β-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 hens ovary and liver.

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 (TGFβ) 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 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.

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.

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.

Zinc Oxide Nanoparticles Influence Microflora in Ileal Digesta and Correlate Well with Blood Metabolites

Zinc oxide nanoparticles (ZnO NPs) are used widely in consumer and industrial products, however, their influence on gut microbiota and metabolism and their mutual interactions are not fully understood. In this study, the effects of ZnO NPs on ileal bacterial communities, plasma metabolites, and correlations between them were investigated. Hens were fed with different concentrations of ZnO NPs [based on Zn; 0 mg/kg (control), 25 mg/kg, 50 mg/kg, and 100 mg/kg] for 9 weeks. Subsequently, ileal digesta and blood plasma were collected for analysis of microflora and metabolites, respectively. The V3-V4 region of the 16S rRNA gene of ileal digesta microbiota was sequenced using the Illumina HiSeq 2500 platform. The predominant bacterial community in the ileum belongs to the phylum Firmicutes. The richness of the bacterial community was negatively correlated with increasing amounts of ZnO NPs (r = -0.636, P < 0.01); when ZnO NP levels were at 100 mg/kg, microbiota diversity was significantly decreased (P < 0.05). The community structure determined by LEfSe analysis indicated that Bacilli, Fusobacteria, and Proteobacteria were changed, and Lactobacillus was reduced by ZnO NPs. Moreover, metabolism as analyzed by nuclear magnetic resonance (NMR) indicated that glucose, some amino acids, and other metabolites were changed by ZnO NPs. Choline, lactate, and methionine were positively correlated with bacterial richness. In summary, ZnO NPs could influence the levels of microflora in ileal digesta, particularly Lactobacillus. Furthermore, the richness of the microbiota was related to changes in choline, lactate, and methionine metabolism.

Regulation of steroid hormones and energy status with cysteamine and its effect on spermatogenesis

Although it is well known that cysteamine is a potent chemical for treating many diseases including cystinosis and it has many adverse effects, the effect of cysteamine on spermatogenesis is as yet unknown. Therefore the objective of this investigation was to explore the effects of cysteamine on spermatogenesis and the underlying mechanisms. Sheep were treated with vehicle control, 10mg/kg or 20mg/kg cysteamine for six months. After that, the semen samples were collected to determine the spermatozoa motility by computer-assisted sperm assay method. Blood samples were collected to detect the levels of hormones and the activity of enzymes. Spermatozoa and testis samples were collected to study the mechanism of cysteamines actions. It was found that the effects of cysteamine on spermatogenesis were dose dependent. A low dose (10mg/kg) cysteamine treatment increased ovine spermatozoa motility; however, a higher dose (20mg/kg) decreased both spermatozoa concentration and motility. This decrease might be due to a reduction in steroid hormone production by the testis, a reduction in energy in the testis and spermatozoa, a disruption in the blood-testis barrier, or a breakdown in the vital signaling pathways involved in spermatogenesis. The inhibitory effects of cysteamine on sheep spermatogenesis may be used to model its effects on young male patients with cystinosis or other diseases that are treated with this drug. Further studies on spermatogenesis that focus on patients treated with cysteamine during the peripubertal stage are warranted.

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 mammals 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.