title: Lipopolysaccharide and heat stress impair the estradiol biosynthesis in granulosa cells via increase of HSP70 and inhibition of smad3 phosphorylation and nuclear translocation.
abstract: LPS and heat stress have been shown to exert various toxic effects in animals, as they induce estradiol biosynthesis dysfunction in granulosa cells (GCs) and result in low reproductive performance. However, there is limited information regarding their detailed mechanisms. In the present study, primary cultured porcine GCs were treated with LPS (1000ng/mL for 48h), or heat stress (41°C for 3h), in vitro, with or without the HSP70 inhibitor VER155008 (10μM), to investigate their potential mechanisms. To mimic the spike in HSP70 from LPS and heat stress, treatments with only the HSP70 activator STA-4783 (10μM for 3h or 48h) were also performed. We found that LPS and heat stress treatments could significantly reduce the expressions of FSHR and CYP19A1; associated with a reduction in estradiol concentrations; and increased in HSP70 expression both at mRNA and protein levels. While, VER155008 attenuation of LPS and heat stress induced HSP70 upregulation can restore the expressions of FSHR and CYP19A1. Furthermore, STA-4783 treatment alone can mimic the effects of LPS and heat stress treatments. Following immunofluorescence staining and western blot analysis showed that Smad3 phosphorylation and nuclear translocation were also inhibited by LPS, heat stress and STA-4783 treatments. We also examined the interactions between HSP70 and Smad3 by yeast two-hybrid screening, the results revealed that HSP70 indirectly interacted with Smad3. Thus, our results suggested that LPS and heat stress could impair estradiol biosynthesis in GCs via increased HSP70 and indirect inhibition of Smad3 phosphorylation and nuclear translocation.
authors.affiliation: Key laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China.
authors.forename: Shuangshuang
authors.initials: S
authors.lastname: Guo
authors.affiliation: Key laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
authors.forename: Liuping
authors.initials: L
authors.lastname: Cai
authors.affiliation: College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian, China.
authors.forename: Weiming
authors.initials: W
authors.lastname: Ma
authors.affiliation: Key laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, China. Electronic address: zdshi@scau.edu.cn.
authors.forename: Zhendan
authors.initials: Z
authors.lastname: Shi
mesh_terms: D000818:Animals; D001141:Aromatase / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D004958:Estradiol / Q000096:biosynthesis*; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018869:Heat-Shock Response / Q000187:drug effects*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011962:Receptors, FSH / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism*; D034421:Sus scrofa; D020798:Two-Hybrid System Techniques
chemical_list: D018840:HSP70 Heat-Shock Proteins; D008070:Lipopolysaccharides; D011962:Receptors, FSH; D051900:Smad3 Protein; D004958:Estradiol; D001141:Aromatase
keywords: Estradiol; Granulosa cell; HSP70; Heat stress; Lipopolysaccharide; Smad3