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

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 (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. 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 o...

Cryopreservation and Culture of Testicular Tissues: An Essential Tool for Biodiversity Preservation

“…To overcome the obstacle for reestablishing complete spermatogenesis in adult individuals, a recent study demonstrated the possibility of inducing infertility in mice and then injecting spermatogonia cells derived from dogs into mouse testes, where it was possible to verify the repopulation capacity of germ cells. 57 This study shows an alternative for the use of samples from domestic and wild adult animals that have not yet succeeded in the xenotransplantation of testicular fragments.…”

Actions and Roles of FSH in Germinative Cells

“…Rodents can complete spermatogenesis without FSH stimulation, but its deficiency significantly decreases sperm quantity. Xenotransplantation of canine spermatogonial stem cells (SSCs) into mice testicular tissues was found to promote spermatogenesis in infertile mice when regulated by FSH [ 162 ].…”

“…When cSSCs were transplanted in chemoablated mouse testes, cSSCs were found in seminiferous tubules. We discussed this paracrine effect of FSH as being possible via Sertoli cells, which express FSHR and secrete more GDNF in the presence of FSH, resulting in increased numbers of SSCs [ 162 ]. Other in vitro studies with bovine and mouse SSCs demonstrated the influence of FSH in the self-renewal and proliferation processes mediated by GDNF and FGF2 [ 237 ], confirmed by the expression of GFRα1 [ 238 , 239 ].…”

Weighted Gene Co-Expression Network Analysis Revealed The Cellular Senescence Signaling Pathway Associated With Non-Obstructive Azoospermia

“…NOA was quilt different form obstructive azoospermia which presents with obvious testis microstructure changes. NOA is a result of meiotic arrest, and it has been suggested that microenvironment changes may play critical roles [22] . Therefore, it is important to understand the biological characteristics of testis, and explore the gene expression status and identity of NOA-associated genes.…”