Connexin 37 and 43 gene and protein expression and developmental competence of isolated ovine secondary follicles cultured in vitro after vitrification of ovarian tissue

• Published in: Theriogenology Vol. 85; no. 8; pp. 1457 - 1467
• Main Authors: Sampaio da Silva, Andréa Moreira, Bruno, Jamily Bezerra, de Lima, Laritza Ferreira, Ribeiro de Sá, Naíza Arcângela, Lunardi, Franciele Osmarini, Ferreira, Anna Clara Accioly, Vieira Correia, Hudson Henrique, de Aguiar, Francisco Léo Nascimento, Araújo, Valdevane Rocha, Lobo, Carlos Henrique, de Alencar Araripe Moura, Arlindo, Campello, Cláudio Cabral, Smitz, Johan, de Figueiredo, José Ricardo, Ribeiro Rodrigues, Ana Paula
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2016
• Subjects: Animals; Apoptosis; Cell Culture Techniques - veterinary; Cell Proliferation; Connexin; Connexin 43 - genetics; Connexin 43 - metabolism; Connexins - genetics; Connexins - metabolism; Cryopreservation - veterinary; Female; Fluorescent Antibody Technique; Gap Junction alpha-4 Protein; Gene Expression; Gene Expression Regulation, Developmental; Oocyte; Ovarian Follicle - cytology; Ovarian Follicle - growth & development; Ovarian Follicle - metabolism; Preantral follicle; RNA, Messenger - metabolism; Sheep; Vitrification; Connexin; Vitrification; Preantral follicle; Oocyte
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2016.01.001

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.