title: Transgenic pig carrying green fluorescent proteasomes.
abstract: Among its many functions, the ubiquitin-proteasome system regulates substrate-specific proteolysis during the cell cycle, apoptosis, and fertilization and in pathologies such as Alzheimer's disease, cancer, and liver cirrhosis. Proteasomes are present in human and boar spermatozoa, but little is known about the interactions of proteasomal subunits with other sperm proteins or structures. We have created a transgenic boar with green fluorescent protein (GFP) tagged 20S proteasomal core subunit α-type 1 (PSMA1-GFP), hypothesizing that the PSMA1-GFP fusion protein will be incorporated into functional sperm proteasomes. Using direct epifluorescence imaging and indirect immunofluorescence detection, we have confirmed the presence of PSMA1-GFP in the sperm acrosome. Western blotting revealed a protein band corresponding to the predicted mass of PSMA1-GFP fusion protein (57 kDa) in transgenic spermatozoa. Transgenic boar fertility was confirmed by in vitro fertilization, resulting in transgenic blastocysts, and by mating, resulting in healthy transgenic offspring. Immunoprecipitation and proteomic analysis revealed that PSMA1-GFP copurifies with several acrosomal membrane-associated proteins (e.g., lactadherin/milk fat globule E8 and spermadhesin alanine-tryptophan-asparagine). The interaction of MFGE8 with PSMA1-GFP was confirmed through cross-immunoprecipitation. The identified proteasome-interacting proteins may regulate sperm proteasomal activity during fertilization or may be the substrates of proteasomal proteolysis during fertilization. Proteomic analysis also confirmed the interaction/coimmunoprecipitation of PSMA1-GFP with 13/14 proteasomal core subunits. These results demonstrate that the PSMA1-GFP was incorporated in the assembled sperm proteasomes. This mammal carrying green fluorescent proteasomes will be useful for studies of fertilization and wherever the ubiquitin-proteasome system plays a role in cellular function or pathology.
authors.affiliation: Division of Animal Sciences, National Swine Resource and Research Center, and Department of Obstetrics, Gynecology and Women's Health, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Edward L
authors.initials: EL
authors.lastname: Miles
authors.forename: Chad
authors.initials: C
authors.lastname: O'Gorman
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhao
authors.forename: Melissa
authors.initials: M
authors.lastname: Samuel
authors.forename: Eric
authors.initials: E
authors.lastname: Walters
authors.forename: Young-Joo
authors.initials: YJ
authors.lastname: Yi
authors.forename: Miriam
authors.initials: M
authors.lastname: Sutovsky
authors.forename: Randall S
authors.initials: RS
authors.lastname: Prather
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Wells
authors.forename: Peter
authors.initials: P
authors.lastname: Sutovsky
mesh_terms: D000177:Acrosome / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005298:Fertility / Q000502:physiology; D005307:Fertilization in Vitro / Q000662:veterinary; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D047468:Immunoprecipitation; D008297:Male; D008894:Milk Proteins / Q000378:metabolism; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D034421:Sus scrofa / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D008894:Milk Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex
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