title: Molecular mechanisms of enhancing porcine granulosa cell proliferation and function by treatment in vitro with anti-inhibin alpha subunit antibody.
abstract: BACKGROUND
  This study was conducted to clarify the effect of the inhibiting action of inhibin on porcine granulosa cell proliferation and function, and to investigate the underlying intracellular regulatory molecular mechanisms.
  
  
  METHODS
  Porcine granulosa cells were cultured in vitro, and were treated with an anti-inhibin alpha subunit antibody, with or without co-treatment of follicle-stimulating hormone (FSH) in the culture medium.
  
  
  RESULTS
  Treatment with anti-inhibin alpha subunit antibody led to a significant increase in estradiol (E2) secretion and cell proliferation. Anti-inhibin alpha subunit antibody worked synergistically with FSH at low concentrations (25 microg/mL) to stimulate E2 secretion, but attenuated FSH action at high concentrations (50 microg/mL). Immunoneutralization of inhibin bioactivity increased FOXL2, Smad3, and PKA phosphorylation, and mRNA expression of the transcription factors CEBP and c-FOS. The expression of genes encoding gonadotropin receptors, FSHR and LHR, and of those involved in steroidogenesis, as well as IGFs and IGFBPs, the cell cycle progression factors cyclinD1 and cyclinD2, and the anti-apoptosis and anti-atresia factors Bcl2, TIMP, and ADAMTS were upregulated following anti-inhibin alpha-subunit treatment. Treatment with anti-inhibin alpha subunit down regulated expression of the pro-apoptotic gene encoding caspase3. Although expression of the pro-angiogenesis genes FN1, FGF2, and VEGF was upregulated, expression of the angiogenesis-inhibiting factor THBS1 was downregulated following anti-inhibin alpha subunit treatment.
  
  
  CONCLUSIONS
  These results suggest that immunoneutralization of inhibin bioactivity, through augmentation of the activin and gonadotropin receptor signaling pathways and regulation of gene expression, permits the development of healthy and viable granulosa cells. These molecular mechanisms help to explain the enhanced ovarian follicular development observed following inhibin immunization in animal models.
authors.affiliation: Laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China. cailiupingclp@163.com.
authors.forename: Liuping
authors.initials: L
authors.lastname: Cai
authors.affiliation: Laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China. sunad2002@163.com.
authors.forename: Aidong
authors.initials: A
authors.lastname: Sun
authors.affiliation: Laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China. lhlydk@126.com.
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Life Science, Xijiao-Liverpool University, Suzhou, 215123, China. anastasia.tsigkou@xjtlu.edu.cn.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Tsinkgou
authors.affiliation: Laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China. 26460136@qq.com.
authors.forename: Jianning
authors.initials: J
authors.lastname: Yu
authors.affiliation: Laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China. 251501650@qq.com.
authors.forename: Shijia
authors.initials: S
authors.lastname: Ying
authors.affiliation: Laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China. chenzzju@163.com.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Laboratory of Animal Breeding and Reproduction, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China. zdshi@jaas.ac.cn.
authors.forename: Zhendan
authors.initials: Z
authors.lastname: Shi
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D051858:Forkhead Transcription Factors / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007265:Inhibins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000502:physiology*; D010766:Phosphorylation; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D013552:Swine / Q000502:physiology
chemical_list: D000906:Antibodies; D022762:CCAAT-Enhancer-Binding Proteins; D051858:Forkhead Transcription Factors; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D051900:Smad3 Protein; C094377:inhibin-alpha subunit; D004958:Estradiol; D007265:Inhibins; D005640:Follicle Stimulating Hormone; D017868:Cyclic AMP-Dependent Protein Kinases