Yan-Kui Wang

Comparison of adipose tissue cellularity in chicken lines divergently selected for fatness

After 13 generations of divergent selection for abdominal fatness, 2 chicken lines (a fat line and a lean line) have been established. To clarify the cellular mechanism underlying the differences in fatness between the fat and lean lines, cellularity characteristics of the abdominal adipose tissues were analyzed during the first 7 wk of age by electron microscopy, proliferating cell nuclear antigen staining, and DNA content measurement. The abdominal fat percentage at 7 wk of age in the fat chicken line was 3.8 times that of the lean line, and was accompanied by a 1.3-fold increase in adipocyte diameter and a 2.4-fold increase in adipocyte number. The total cell number of the abdominal fat pad in the fat chicken line was 1.9 times that of the lean line at 7 wk of age. However, no significant difference was observed in the proliferation rate of stroma vascular fraction cells between the 2 chicken lines. These findings suggest that the divergently selected fat and lean chickens have different adipose tissue ontogeny.

Epigenetic DNA methylation in the promoters of peroxisome proliferator-activated receptor γ in chicken lines divergently selected for fatness.

Peroxisome proliferator-activated receptor γ is a master regulator of adipocyte differentiation and function. Expression of PPARγ in mammals is regulated by DNA methylation; however, it is currently unknown whether chicken PPARγ expression is regulated by DNA methylation. To enhance our understanding of molecular mechanisms underlying chicken adipose tissue development and adipogenesis, we investigated the promoter methylation status and gene expression of PPARγ gene in Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF). Deoxyribonucleic acid methylation was analyzed by bisulfite sequencing method, and mRNA expression was detected by real-time quantitative real time reverse-transcription polymerase chain reaction (RT-PCR). The analyzed region located from -1,175 to -301 bp upstream of the translation start codon ATG contains 6 CpG dinucleotides, which are located at positions -1,014, -796, -625, -548, -435, and -383 bp, respectively. The results revealed that the 3 CpGs at positions -548, -435, and -383 bp showed differential methylation between the lean and fat chicken lines, but the other 3 CpG sites at positions -1,014, -796, and -625 bp did not. PPARγ gene promoter methylation in both chicken lines decreased with age, and PPARγ promoter methylation levels were significantly higher in lean than fat broilers at 2 wk of age (79.9 to 64.5%; P < 0.0001), at 3 wk of age (66.7 to 58.3%; P < 0.0001), and at 7 wk of age (50.0 to 42.7%; P = 0.0004). Real-time quantitative RT-PCR analysis showed that, negatively correlated with DNA methylation (Pearsons r = -0.653, P = 0.0057), PPARγ expression was increased with age and significantly lower in lean than fat chicken lines at 2, 3, and 7 wk of age (P < 0.0001). In conclusion, our findings suggest that chicken PPARγ is regulated by DNA methylation during adipose tissue development.

Chicken adipocyte fatty acid-binding protein knockdown affects expression of peroxisome proliferator-activated receptor γ gene during oleate-induced adipocyte differentiation

Adipocyte fatty acid-binding protein (A-FABP) is expressed in both adipose cells and macrophages. As one of the downstream genes of peroxisome proliferator-activated receptor γ (PPAR-γ), A-FABP plays an important role in the lipid metabolism of adipocytes in mammal and poultry. However, studies in A-FABP null macrophages of mice showed that A-FABP was a critical regulator of PPAR-γ and could affect the expression of PPAR-γ. The current study was designed to investigate whether the same mechanism as that in macrophages exists in chicken adipocytes. After transfection with interference and overexpression plasmids of A-FABP in chicken adipocytes for 24 h, oleate was added to the medium. Then, lipid accumulation, nonesterified fatty acids (NEFA) in the medium, and expression of lipid metabolism-related genes were detected. The results showed that in the A-FABP knockdown adipocytes, lipid accumulation was decreased at 6 h and NEFA in the medium was higher at 1 and 6 h compared with that in the control group. Moreover, gene expression levels of lipoprotein lipase, perilipin, and PPAR-γ were higher than that of the control group (P < 0.05). In the A-FABP overexpression adipocytes, lipid accumulation and expression of lipid metabolism related genes were similar to that of the control group. However, NEFA in the medium was significantly lower in the A-FABP overexpression group 1 h after adding oleate (P < 0.05). The present study suggested that the A-FABP knockdown might lead to decreased lipid accumulation and upregulated expression of PPAR-γ in chicken adipocytes.

Dimethyl Sulfoxide Perturbs Cell Cycle Progression and Spindle Organization in Porcine Meiotic Oocytes

Meiotic maturation of mammalian oocytes is a precisely orchestrated and complex process. Dimethyl sulfoxide (DMSO), a widely used solvent, drug, and cryoprotectant, is capable of disturbing asymmetric cytokinesis of oocyte meiosis in mice. However, in pigs, DMSO’s effect on oocyte meiosis still remains unknown. We aimed to evaluate if DMSO treatment will affect porcine oocyte meiosis and the underlying molecular changes as well. Interestingly, we did not observe the formation of the large first polar body and symmetric division for porcine oocytes treated with DMSO, contrary to findings reported in mice. 3% DMSO treatment could inhibit cumulus expansion, increase nuclear abnormality, disturb spindle organization, decrease reactive oxygen species level, and elevate mitochondrial membrane potential of porcine oocytes. There was no effect on germinal vesicle breakdown rate regardless of DMSO concentration. 3% DMSO treatment did not affect expression of genes involved in spindle organization (Bub1 and Mad2) and apoptosis (NF-κB, Pten, Bcl2, Caspase3 and Caspase9), however, it significantly decreased expression levels of pluripotency genes (Oct4, Sox2 and Lin28) in mature oocytes. Therefore, we demonstrated that disturbed cumulus expansion, chromosome alignment, spindle organization and pluripotency gene expression could be responsible for DMSO-induced porcine oocyte meiotic arrest and the lower capacity of subsequent embryo development. Our results provide new insights on DMSO’s effect on porcine oocyte meiosis and raise safety concerns over DMSO’s usage on female reproduction in both farm animals and humans.

DMBA acts on cumulus cells to desynchronize nuclear and cytoplasmic maturation of pig oocytes

As an environmental pollutant and carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA) can destroy ovarian follicles at all developmental stages in rodents. However, the underlying molecular mechanism remains obscure. In the present study, we aim to address how DMBA affects the in vitro maturation and development of porcine oocytes. We discovered that for 20 μM DMBA-treated cumulus-oocyte complexes (COCs), the rate of oocyte germinal vesicle breakdown (GVBD) was significantly altered, and the extrusion rate of first polar body was increased. Moreover, oocytes from 20 μM DMBA-treated COCs had significant down-regulation of H3K9me3 and H3K27me3, up-regulation of H3K36me3, higher incidence of DNA double strand breaks (DSBs) and early apoptosis. In striking contrast, none of these changes happened to 20 μM DMBA-treated cumulus-denuded oocytes (CDOs). Furthermore, 20 μM DMBA treatment increased the reactive oxygen species (ROS) level, decreased mitochondrial membrane potential (Δ Ψm), and inhibited developmental competence for oocytes from both COC and CDO groups. Collectively, our data indicate DMBA could act on cumulus cells via the gap junction to disturb the synchronization of nuclear and ooplasmic maturation, and reduce the developmental competence of oocytes.

Expression and functional analysis of Krüppel-like factor 2 in chicken adipose tissue.

Studies in mammalian species showed that Krüppel-like factor 2 (KLF2) regulates adipogenesis. However, its role in birds is unclear. The objective of the current study was to explore the expression and function of KLF2 in chicken adipogenesis. Results showed that chicken KLF2 (Gallus gallus KLF2 [gKLF2]) was greatly expressed in abdominal adipose tissue, and its transcripts fluctuated during adipose tissue development. In addition, gKLF2 transcripts in abdominal adipose tissue of lean broilers were greater at 1 wk of age but lower at 3, 5, and 8 wk of age than those in fat broilers (P < 0.05). The gKLF2 was more greatly expressed in preadipocytes than in mature adipocytes (P < 0.05), and its expression level decreased during the preadipocyte differentiation in vitro (P < 0.05). The functional analysis showed that gKLF2 overexpression inhibited chicken preadipocyte differentiation (P < 0.05), accompanied by the reduced expression of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) and the elevated expression of GATA binding protein 2 (GATA2). Additionally, the luciferase reporter assays showed that gKLF2 overexpression suppressed the promoter activities of chicken C/EBPα and PPARγ (P < 0.05). In conclusion, our results indicated that gKLF2 inhibits chicken adipogenesis, at least in part, through inhibition of PPARγ and C/EBPα expression.

Heat shock protein 90α couples with the MAPK-signaling pathway to determine meiotic maturation of porcine oocytes1

Heat shock protein 90 (Hsp90) functions as a molecular chaperone in its interaction with clients to influence multiple cellular and physiological processes. However, our current understanding on Hsp90s relationship with mammalian oocyte maturation is still very limited. Here, we aimed to investigate Hsp90s effect on pig oocyte meiotic maturation. Endogenous Hsp90α was constantly expressed at both mRNA and protein levels in porcine maturing oocytes. Addition of 2 µM 17-allylamino-17-demethoxygeldanamycin (17-AAG), the Hsp90 inhibitor, to in vitro mature cumulus-oocyte complexes (COC) significantly decreased Hsp90α protein level (P < 0.05), delayed germinal vesicle breakdown (GVBD) (P < 0.05), and impeded the first polar body (PB1) extrusion (P < 0.01) of porcine oocytes. 2 µM 17-AAG treatment during in vitro maturation also decreased the subsequent development competence as indicated by the lower cleavage (P < 0.001) and higher fragmentation (P < 0.001) rates of parthenotes, whereas no effects on the percentage and average cell number of blastocysts were found. Immunodepletion of Hsp90α by antibody microinjection into porcine oocytes at germinal vesicle and metaphase II stages induced similar defects of meiotic maturation and parthenote development, to that resulted from 2 µM inhibitor 17-AAG. For oocytes treated by 2 µM 17-AAG, the cytoplasm and membrane actin levels were weakened (P < 0.01), and the spindle assembly was disturbed (P < 0.05), due to decreased p-ERK1/2 level (P < 0.05). However, the mitochondrial function and early apoptosis were not affected, as demonstrated by rhodamine 123 staining and Annexin V assays. Our findings indicate that Hsp90α can couple with mitogen-activated protein kinase to regulate cytoskeletal structure and orchestrate meiotic maturation of porcine oocytes.

Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation

The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent. However, the underlying molecular mechanism targeted by the BCB test is still unclear. To explore this question, we first confirmed that BCB-positive porcine oocytes had higher rates of meiotic maturation, better rates of cleavage and development into blastocysts, and lower death rates. Subsequent single-cell transcriptome sequencing on porcine germinal vesicle (GV)-stage oocytes identified 155 genes that were significantly differentially expressed between BCB-negative and BCB-positive oocytes. These included genes such as cdc5l, ldha, spata22, rgs2, paip1, wee1b, and hsp27, which are enriched in functionally important signaling pathways including cell cycle regulation, oocyte meiosis, spliceosome formation, and nucleotide excision repair. In BCB-positive GV oocytes that additionally had a lower frequency of DNA double-strand breaks, the CDC5L protein was significantly more abundant. cdc5l/CDC5L inhibition by short interference (si)RNA or antibody microinjection significantly impaired porcine oocyte meiotic maturation and subsequent parthenote development. Taken together, our single-oocyte sequencing data point to a potential new role for CDC5L in porcine oocyte meiosis and early embryo development, and supports further analysis of this protein in the context of the BCB test.