GDF9-β promotes folliculogenesis in sheep ovarian transplantation onto the chick embryo chorioallantoic membrane (CAM) in cryopreservation programs
- 30 Downloads
Ovarian tissue (OT) cryopreservation is a treatment option for fertility preservation among young cancer patients. However, the procedure may involve a reduction in the GDF9-β expression and a delay in follicular growth after thawing and transplantation. The aim of this study was to evaluate whether supplementation of GDF9-β can compensate the reduction of this factor during the cryopresevation process and promote folliculogenesis after transplantation of thawed sheep ovarian tissue.
Sheep OT was cryopreserved using two methods of vitrification and slow freezing. Fresh and thawed OTs were then transplanted onto chick embryo chorioallantoic membrane (CAM) and then divided into two groups based on the addition of GDF9-β to the grafted tissue. After 5 days of culture, both histological and immunohistological (Ki-67) assessments were performed to evaluate follicular structure, development, and proliferation. The fibrotic and necrotic areas were measured using MICROVISIBLE software.
Folliculogenesis took place in all culture groups, but was significantly improved only in the +GDF9-β cultured group. Also, better follicular structure was preserved in the aforementioned group (p < 0.05). When GDF9-β was supplemented to the culture medium, more neovascularization (p < 0.05) and better transplantation (p > 0.05) was observed. Furthermore, the areas of fibrosis and necrosis were lower in this group rather than the controls. Follicular proliferative activity was significantly higher only in the slow freezing +GDF9-β cultured group.
GDF9-β, as a stimulatory factor, not only promoted the folliculogenesis in the fresh ovarian transplant, but also compensated for its reduction during the cryopreservation process.
KeywordsGDF9-β Folliculogenesis Ovarian transplantation Cryopreservation
MV and MM: protocol/project development. MV and NY: data collection or management. NY: data analysis. BW: manuscript writing/editing. MAK: manuscript writing/editing
Compliance with ethical standards
Conflict of interest
The research did not get any financial support from any funding agency regarding the material. No conflicts of interest with the authors to be declared.
This article does not contain any studies with human participants or animals performed by any of the authors. The use of slaughterhouse derived ovaries and the CAM culture system does not raise ethical or legal concerns, nor does it violate the animal protection laws .
- 3.Lee D (2007) Ovarian tissue cryopreservation and transplantation: banking reproductive potential for the future. Oncofertility fertility preservation for cancer survivors. Springer, pp 110–129Google Scholar
- 9.Isachenko V, Mallmann P, Petrunkina AM, Rahimi G, Nawroth F, Hancke K, Felberbaum R, Genze F, Damjanoski I, Isachenko E (2012) Comparison of in vitro-and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue. PLoS One 7(3):e32549CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Isachenko V, Orth I, Isachenko E, Mallmann P, Peters D, Schmidt T, Morgenstern B, Foth D, Hanstein B, Rahimi G (2013) Viability of human ovarian tissue confirmed 5 years after freezing with spontaneous ice-formation by autografting and chorio-allantoic membrane culture. Cryobiology 66(3):233–238CrossRefPubMedGoogle Scholar
- 15.Aaltonen J, Laitinen MP, Vuojolainen K, Jaatinen R, Horelli-Kuitunen N, Seppä L, Louhio H, Tuuri T, Sjöberg J, Bützow R (1999) Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis. J Clin Endocrinol Metab 84(8):2744–2750PubMedGoogle Scholar
- 17.Celestino J, Lima-Verde I, Bruno J, Matos M, Chaves R, Saraiva M, Silva C, Faustino L, Rossetto R, Lopes C (2011) Steady-state level of bone morphogenetic protein-15 in goat ovaries and its influence on in vitro development and survival of preantral follicles. Mol Cell Endocrinol 338(1):1–9CrossRefPubMedGoogle Scholar
- 22.Mofarahe ZS, Salehnia M, Novin MG, Ghorbanmehr N, Fesharaki MG (2017) Expression of folliculogenesis-related genes in vitrified human ovarian tissue after two weeks in vitro culture. Cell J (Yakhteh) 19(1):18Google Scholar
- 24.Aguiar F, Lunardi F, Lima L, Rocha R, Bruno J, Magalhães-Padilha D, Cibin F, Rodrigues A, Gastal M, Gastal E (2016) Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture. Theriogenology 85(6):1063–1069CrossRefPubMedGoogle Scholar
- 30.Martins F, Celestino J, Saraiva M, Matos M, Bruno J, Rocha-Junior C, Lima-Verde I, Lucci C, Báo S, Figueiredo J (2008) Growth and differentiation factor-9 stimulates activation of goat primordial follicles in vitro and their progression to secondary follicles. Reprod Fertil Dev 20(8):916–924CrossRefPubMedGoogle Scholar