Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice

Stem Cell Research & Therapy

Ana Carolina Furlanetto Mançanares¹  Mario Binelli²  Angela Maria Gonella Diaza³  Juliana Barbosa Casals⁴  Naira Caroline Godoy Pieri⁵  Carlos Eduardo Ambrósio⁶  Fabiana Fernandes Bressan⁶  Daniele dos Santos Martins⁶  Aline Fernanda de Souza⁷  Hugo Fernandes⁷  Kelly Cristine Santos Roballo⁸ 

[1] 0000 0001 2298 9663, grid.5380.e, Department of Animal Science, Faculty of Veterinary Science, Universidad de Concepción, Chillan, Chile;0000 0004 1936 8091, grid.15276.37, Department of Animal Sciences, University of Florida, L.E. “Red” Larson Building, Bldg. 499, Room 122 C, 32611-0910, Gainesville, FL, USA;0000 0004 1936 8091, grid.15276.37, North Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Marianna, FL, USA;0000 0004 1937 0722, grid.11899.38, Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, Sao Paulo, SP, Brazil;0000 0004 1937 0722, grid.11899.38, Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, Sao Paulo, SP, Brazil;0000 0004 1937 0722, grid.11899.38, Department of Reproduction, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, Sao Paulo, SP, Brazil;0000 0004 1937 0722, grid.11899.38, Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, Sao Paulo, SP, Brazil;0000 0004 1937 0722, grid.11899.38, Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, Pirassununga, SP, Brazil;0000 0004 1937 0722, grid.11899.38, Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, Pirassununga, SP, Brazil;0000 0004 1937 0722, grid.11899.38, Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Av. Duque de Caxias Norte, 225, Pirassununga, SP, Brazil;0000 0001 2109 0381, grid.135963.b, School of Pharmacy at University of Wyoming, 1000 E. University Avenue, 82071, Laramie, USA;

关键词: Transplant;    Spermatogenesis;    Hormone;    Germ cells;   

DOI  :  10.1186/s13287-019-1250-9

来源: publisher

【 摘 要 】

BackgroundXenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis.MethodsSSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L−1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis.ResultsSupplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice.ConclusionsIn conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.

【 授权许可】

CC BY