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

Author(s):Show less -

Godoy Pieri, Naira Caroline ^{[1, 2]} ; Furlanetto Mancanares, Ana Carolina ^[3] ; de Souza, Aline Fernanda ^[4] ; Fernandes, Hugo ^[4] ; Gonella Diaza, Angela Maria ^[5] ; Bressan, Fabiana Fernandes ^{[4, 1]} ; Santos Roballo, Kelly Cristine ^{[4, 6]} ; Casals, Juliana Barbosa ^[1] ; Binelli, Mario ^[7] ; Ambrosio, Carlos Eduardo ^{[4, 1]} ; Martins, Daniele dos Santos ^{[4, 1]}

Affiliation

^[1] Univ Sao Paulo, Dept Surg, Fac Vet Med & Anim Sci, Av Prof Dr Orlando Marques de Paiva 87, Sao Paulo, SP - Brazil

^[2] Univ Sao Paulo, Dept Reprod, Fac Vet Med & Anim Sci, Av Prof Dr Orlando Marques de Paiva 87, Sao Paulo, SP - Brazil

^[3] Univ Concepcion, Fac Vet Sci, Dept Anim Sci, Chillan - Chile

^[4] Univ Sao Paulo, Fac Anim Sci & Food Engn, Dept Vet Med, Av Duque de Caxias Norte 225, Pirassununga, SP - Brazil

^[5] Univ Florida, North Florida Res & Educ Ctr, Inst Food & Agr Sci, Marianna, FL - USA

^[6] Univ Wyoming, Sch Pharm, 1000 E Univ Ave, Laramie, WY 82071 - USA

^[7] Univ Florida, Dept Anim Sci, LE Red Larson Bldg, Bldg 499, Room 122 C, Gainesville, FL 32611 - USA

• Document type: Journal article
• Source: STEM CELL RESEARCH & THERAPY; v. 10, MAY 20 2019.
• Web of Science Citations: 1

Abstract

Background: Xenotransplantation 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. Methods: SSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10IUL(-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. Results: Supplementation 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. Conclusions: In 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. (AU)

• FAPESP's process: 13/10542-5 - Epigenetic reprogramming of canine embryonic germ cells
• Grantee: Aline Fernanda de Souza
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 12/05609-0 - Xenogeneic-derived spermatogenesis after canine stem cell transplantation in murine testis
• Grantee: Naira Caroline Godoy Pieri
• Support Opportunities: Scholarships in Brazil - Doctorate