Zearalenone exposure impairs ovarian primordial follicle formation via down-regulation of Lhx8 expression in vitro.

Zearalenone (ZEA) is an estrogenic mycotoxin mainly produced as a secondary metabolite by numerous species of Fusarium. Previous work showed that ZEA had a negative impact on domestic animals with regard to reproduction. The adverse effects and the mechanisms of ZEA on mammalian ovarian folliculogenesis remain largely unknown, particularly its effect on primordial follicle formation. Thus, we investigated the biological effects of ZEA exposure on murine ovarian germ cell cyst breakdown and primordial follicle assembly. Our results demonstrated that newborn mouse ovaries exposed to 10 or 30µM ZEA in vitro had significantly less germ cell numbers compared to the control group. Moreover, the presence of ZEA in vitro increased the numbers of TUNEL and γH2AX positive cells within mouse ovaries and the ratio of mRNA levels of the apoptotic genes Bax/Bcl-2. Furthermore, ZEA exposure reduced the mRNA of oocyte specific genes such as LIM homeobox 8 (Lhx8), newborn ovary homeobox (Nobox), spermatogenesis and oogenesis helix-loop-helix (Sohlh2), and factor in the germline alpha (Figlα) in a dose dependent manner. Exposure to ZEA led to remarkable changes in the Lhx8 3'-UTR DNA methylation dynamics in oocytes and severely impaired folliculogenesis in ovaries after transplantation under the kidney capsules of immunodeficient mice. In conclusion, ZEA exposure impairs mouse primordial follicle formation in vitro.

[Genetic polymorphism of FSH b subunit gene and correlation with reproductive traits in Beijing Black Pig].

FSH beta subunit gene was regarded as a candidate gene for reproductive traits of Beijing Black Pig in this study. The polymorphism of two loci FSHbeta-1 and FSHbeta-2 was detected by electrophoretic method and PCR-RFLP with restriction endonuclease Hae III. Sequencing results showed that a 273 bp sequence, which was a retrotransponsons including a RNA Polymerase III inter promoter, was inserted between the 134th and 135th nucleotide of the PCR product in FSHbeta-1, and the mutation (C-->T) was revealed at the 173th nucleotide of the PCR product in FSHbeta-2. Both alleles (A and B) of both loci were found in the population that showed low polymorphism. Chi-square test indicated that the two polymorphism sites fitted Hardy-Weinberg equilibrium (P>0.05). The effects of polymorphism of FSH b subunit gene on total born number (TNB), number born alive (NBA), and birth weight (WB) were analyzed. For FSHbeta-1 locus, pigs of the first parity with genotype AA had 0.96 and 1.85 TNB more than those with genotypes AB and BB. The pigs of the first parity with genotypes AA and AB had 0.95 and 1.69 NBA more than those with genotype BB, respectively. For FSHbeta-2 locus, pigs of the first parity with genotype AA had1.57 and 2.15 TNB more than those with genotypes AB and BB. The pigs of the first parity with genotypes AA and AB had 1.00 and 0.94 NBA more than those with genotype BB, respectively. The pigs of multiparous with genotype AA had 0.25 kg of WB more than those with genotype BB. The results of combined genotype effects indicated that A allele of FSHbeta-1 locus in all the population and FSHbeta-2 locus in pigs of the first parity had the positive effective on TNB, NBA, and WB.

RNA-seq based gene expression analysis of ovarian granulosa cells exposed to zearalenone in vitro: significance to steroidogenesis.

Zearalenone (ZEA) is a natural contaminant of various food and feed products representing a significant problem worldwide. Since the occurrence of ZEA in grains and feeds is frequent, the present study was carried out to evaluate the possible effects of ZEA on steroid production and gene expression of porcine granulosa cells, using RNA-seq analysis. Porcine granulosa cells were administered 10 µM and 30 µM ZEA during 72 h of culture in vitro. Following ZEA treatment the gene expression profile of control and exposed granulosa cells was compared using RNA-seq analysis. The results showed that in the exposed granulosa cells ZEA significantly altered the transcript levels, particularly steroidogenesis associated genes. Compared with the control group, 10 µM and 30 µM ZEA treatment significantly increased the mRNA expression of EDN1, IER3, TGFß and BDNF genes and significantly reduced the mRNA expression of IGF-1 and SFRP2 genes. In particular, ZEA significantly decreased the expression of genes essential for estrogen synthesis including FSHR, CYP19A1 and HSD17ß in granulosa cells. Furthermore, Q-PCR and Western-blot analysis also confirmed reduced expression of these genes in ZEA exposed granulosa cells. These effects were associated with a significant reduction of 17ß-estradiol concentrations in the culture medium of granulosa cells. Collectively, these results demonstrated a concretely deleterious effect of ZEA exposure on the mRNA expression of steroidogenesis related genes and the production of steroid hormones in porcine ovarian granulosa cells in vitro.

[Study of single nucleotide polymorphism of A-FABP gene and its association with fatness traits in chicken].

In this experiment, Beijing-You chicken was used to detect single nucleotide polymorphisms (SNPs) in A-FBAP gene and to study the correlation between its genotype and the trait of fat accumulation. The first exon of the gene was amplified by one pair of primers, and SNPs were detected by the technique of single strand conformation polymorphism (SSCP) and finally confirmed by sequencing. Results of analysis of variance showed that a significant difference existed among abdominal fat percentage, subcutaneous fat thickness and intramuscular fat contents in breast musculature with different genotypes (P<0.01). It implied that A-FABP gene could be a major effector gene or could be linked to gene(s) which significantly affect fat metabolism in chicken.

[Relationship Between Molecular Marker of Western Main Pig H-FABP Gene and IMF Content.].

By using 265 pigs from eight breeds including Duroc,Landrace,Large White,Neijiang,Rongchang,Hanjiang Black,Hanzhong White,Bamei and wild ones, the genetic variations of 5'-upstream region from and the second intron in porcine H-FABP gene were checked by PCR-RFLP molecular marker with HinfI, Hae III and MspI,and effect of H-FABP gene on IMF content was then analyzed by least square analysis.The results showed as follows:(1) 8 pig breeds and wild pig had polymorphism at Hinf I-RFLP site. In above detected breeds,large white,Bamei pig, Hanjiang Black,Hanzhong White pig breeds and wild pig presented low polymorphism while other breeds have mediate polymorphism;(2)Among the tested breeds only 4 Chinese local pig breeds had no polymorphism at the Hae III-RFLP and Msp I-RFLP sites,but Duroc,Landrace,Largewhite, Hanzhong White pig breeds and wild pig had polymorphism. Wild pig at the Hae III-RFLP , Landrace,Largewhite and wild pig at the Hae III-RFLP and Msp I-RFLP sites were low polymorphism,others were mediate polymorphism;(3) H-FABP gene increased IMF content significantly(p0.05). Genetic effect of H-FABP gene on IMF content were HH>Hh>hh,DD.

[Effects of resveratrol on pig primary preadipocytes proliferation, differentiation and transcription expression of Sirt1 gene].

1 approximately 3 days old Piglet's primary preadipocytes in vitro were cultured and treated with 0micromol/L (control group), 10microlmol/L (lower dose group), 20micromol/L(middle dose group) and 50micromol/L, 100micromol/L (higher dose group) RES. Cell proliferation and viability were analyzed by MTT assay. The degree of differentiation and adipogenesis were measured by Oil Red O staining extraction assay and the expression of Sirt1 (sirtuin) mRNA were detected by RT-PCR. The results showed the optical density (OD) of MTT and Oil Red O staining were all decreased, especially treated by 50micromol/L, 100micromol/L RES at 72h and 96h (P < 0.01); the ratio of OD of the expression of Sirt1 mRNA to that of beta-actin mRNA were increased after treated by 100micromol/L RES (P < 0.01). RES can inhibit proliferation and differentiation of pig preadipocytes in certain degree. Higher dose of RES can markedly decrease adipogenesis and prevent preadipocytes differentiation into adipocytes, which may be in part associated with its effect on increasing the expression of Sirt1 mRNA.

[Three-dimensional spheroid model for cultivating WB-F344 cells in simulated microgravity].

Three-dimensional (3D) culture of cells could closely mimic the in vivo situation with regard to cell function and microenvironment compared with plane monolayer cultured cells. In this paper, we established 3D culture of rat WB-F344 cells with rotary cell culture system (RCCS) to simulate microgravity environment, and examined cells proliferation, morphology, microstructure, E-cadherin protein quantity and mRNA expression of adhesion molecules by count the number of cells, optical microscope, transmission electron microscope and reverse transcriptase-polymerase chain reaction (RT-PCR). The results demonstrated that cells were polyhedron with lots of micovilli and mitochondria, which grow well and packed together densely to form irregular aggregates. Adjacent cells were connected with desmosome and tight junction. With the regard, the aggregates behaved 3D growth characteristics. Moreover, compared with control, mRNA level of Fibronectin and E-cadherin protein were increased, the changes maybe is the part mechanism in this microgravity simulated cells culture models which strengthened cells junction. This rotating 3D model might facilitate the study of interactions of cell-cell, cell-matrix and the mechanisms.