title: Connexin 37 and 43 gene and protein expression and developmental competence of isolated ovine secondary follicles cultured in vitro after vitrification of ovarian tissue.
abstract: Cryoinjuries caused by vitrification of tissues and organs lead to the loss of membrane proteins that mediate intercellular communications, such as connexins 37 (Cx37) and 43 (Cx43). Thus, the present study aimed to evaluate ovine Cx37 and Cx43 gene and protein expressions and developmental competence by in vitro-cultured secondary follicles retrieved from vitrified ovarian tissue. Ovarian fragments for the same ovary pair were distributed into six treatments: (1) fresh ovarian tissue (FOT); (2) vitrified ovarian tissue (VOT); (3) isolated follicles from fresh ovarian tissue (FIF); (4) isolated follicles from vitrified ovarian tissue; (5) isolated follicles from fresh ovarian tissue followed by in vitro culture (CFIF); (6) isolated follicles from vitrified ovarian tissue followed by in vitro culture (CVIF). In all treatments, Cx37 and Cx43 gene and protein expression patterns were evaluated by reverse transcription polymerase chain reaction and immunocytochemistry. In addition, secondary follicles were analyzed according to follicular integrity and growth, apoptosis, and cell proliferation. In vitro-cultured secondary follicles (CFIF and CVIF) were evaluated based on morphology (extruded follicles), antrum formation, and viability. The percentage of intact follicles was higher, whereas antrum formation, oocyte extrusion rate, and follicle viability were lower in CVIF than in CFIF treatment (P < 0.05). Terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling assay demonstrated that apoptosis was absent in FIF, whereas follicles from all other treatments showed positive labeling. Cell proliferation index was higher in isolated follicles from vitrified ovarian tissue and CVIF treatments than in follicles from FIF. Expression of Cx43 messenger RNA was lower in CVIF treatment when compared with follicles from all other treatments (P < 0.05). Follicle Cx37 messenger RNA levels did not show alterations in any treatment (P > 0.05). Cx37 and Cx43 immunolabeling was localized mainly on granulosa cells and oocytes, respectively. In conclusion, isolation of ovine secondary follicles could be done successfully after vitrification of ovarian tissue, and the basement membrane integrity remained intact after in vitro culture. Although the gene and protein expression of Cx37 did not change after vitrification of ovarian tissue, Cx43 turned out to be altered in secondary follicles after vitrification and in vitro culture.
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Andréa Moreira
authors.initials: AM
authors.lastname: Sampaio da Silva
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Jamily Bezerra
authors.initials: JB
authors.lastname: Bruno
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Laritza Ferreira
authors.initials: LF
authors.lastname: de Lima
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Naíza Arcângela
authors.initials: NA
authors.lastname: Ribeiro de Sá
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Franciele Osmarini
authors.initials: FO
authors.lastname: Lunardi
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Anna Clara Accioly
authors.initials: AC
authors.lastname: Ferreira
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Hudson Henrique
authors.initials: HH
authors.lastname: Vieira Correia
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Francisco Léo Nascimento
authors.initials: FL
authors.lastname: de Aguiar
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Valdevane Rocha
authors.initials: VR
authors.lastname: Araújo
authors.affiliation: Group of Research in Biology of Reproduction, Department of Animal Science, Federal University of Ceará UFC, Fortaleza, Ceará, Brazil.
authors.forename: Carlos Henrique
authors.initials: CH
authors.lastname: Lobo
authors.affiliation: Group of Research in Biology of Reproduction, Department of Animal Science, Federal University of Ceará UFC, Fortaleza, Ceará, Brazil.
authors.forename: Arlindo
authors.initials: A
authors.lastname: de Alencar Araripe Moura
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: Cláudio Cabral
authors.initials: CC
authors.lastname: Campello
authors.affiliation: Follicle Biology Laboratory, Center for Reproductive Medicine, UZ Brussel, Brussels, Belgium.
authors.forename: Johan
authors.initials: J
authors.lastname: Smitz
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil.
authors.forename: José Ricardo
authors.initials: JR
authors.lastname: de Figueiredo
authors.affiliation: Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceara, Fortaleza, Ceará, Brazil. Electronic address: aprrodriguespapers@gmail.com.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Ribeiro Rodrigues
mesh_terms: D000818:Animals; D017209:Apoptosis; D018929:Cell Culture Techniques / Q000662:veterinary; D049109:Cell Proliferation; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D015925:Cryopreservation / Q000662:veterinary; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D006080:Ovarian Follicle / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012756:Sheep; D058989:Vitrification; D000097006:Gap Junction alpha-4 Protein
chemical_list: D018031:Connexin 43; D017630:Connexins; D012333:RNA, Messenger; D000097006:Gap Junction alpha-4 Protein
keywords: Connexin; Oocyte; Preantral follicle; Vitrification