Open AccessRelative transcript level of BMP15, BAX and CASP3 with qRT-PCR in vitrified equine immature cumulus-oocytes complexes
Author(s) -
Mauro Flores Polenz,
Gabriel Ribas Pereira,
Vitor Braga Rissi,
Kalyne Bertolin,
Paulo Bayard Dias Gonçalves,
Geovani Celso Augusto,
Sandra Mara da Encarnação Fiala Rechsteiner
Publication year - 2022
Publication title -
semina. ciências agrárias
Language(s) - English
Resource type - Journals
eISSN - 1679-0359
pISSN - 1676-546X
DOI - 10.5433/1679-0359.2022v43n3p927
Subject(s) - andrology , vitrification , bone morphogenetic protein 15 , cryopreservation , biology , oocyte , embryo , gene , medicine , genetics , bone morphogenetic protein , bone morphogenetic protein 7
Research focused on female gamete vitrification has increased attention to develop a reliable cryopreservation method to preserve immature equine oocytes. Despite the intensive implementation of biotechnological procedures for horse breeding, vitrification of immature equine cumulus-oocyte complexes (COCs) remain to be clearly elucidated. We aimed to determine the relative transcript level of target genes Bone morphogenetic protein 15 (BMP15); Bcl-2-associated X protein (BAX); and Caspase 3 (CASP3) in equine COCs prior to and after vitrification. Ovarian follicles were aspirated from ovaries collected from an abattoir. A total of 240 COCs were collected and distributed into vitrified COCs (VIT, n=120) and non-vitrified (Non-VIT, n=120) groups. Then, COCs were preserved and relative transcript expressions of BMP15, BAX, CASP3 were measured and normalized against GAPDH performed by qRT-PCR. In addition, 38 COCs were evaluated to assess chromatin configuration of germinal vesicl e stage prior and after vitrification by exposure to 10 μg/ml of bisbenzimide. A difference was observed in the COCs’ mRNA level of abundance for the BAX gene between the VIT (2.05 ± 0.47) and (0.85 ± 0.08) Non-VIT groups. There was no difference in mRNA relative transcript level of CASP3 and BMP15 in Non-VIT (0.63 ± 0.20 and 1.55 ± 0.73, respectively) compared to VIT (0.64 ± 0.01 and 2.84 ± 2.20, respectively) equine COCs. All COCs where considered at immature stage of development even though COCs in Non-VIT group showed higher condensed chromatin configuration compared to VIT (100% vs 60.7%, respectively). We demonstrate that BMP15 and CASP3 are detected in VIT and Non-VIT immature COCs. In conclusion, BAX is expressed highly in vitrified immature equine COCs and indicates that activation of apoptosis signaling cascades in cells exposed to vitrification.