Oncofertility: Pharmacological Protection and Immature Testicular Tissue (ITT)-Based Strategies for Prepubertal and Adolescent Male Cancer Patients.

While the incidence of cancer in children and adolescents has significantly increased over the last decades, improvements made in the field of cancer therapy have led to an increased life expectancy for childhood cancer survivors. However, the gonadotoxic effect of the treatments may lead to infertility. Although semen cryopreservation represents the most efficient and safe fertility preservation method for males producing sperm, it is not feasible for prepubertal boys. The development of an effective strategy based on the pharmacological protection of the germ cells and testicular function during gonadotoxic exposure is a non-invasive preventive approach that prepubertal boys could benefit from. However, the progress in this field is slow. Currently, cryopreservation of immature testicular tissue (ITT) containing spermatogonial stem cells is offered to prepubertal boys as an experimental fertility preservation strategy by a number of medical centers. Several in vitro and in vivo fertility restoration approaches based on the use of ITT have been developed so far with autotransplantation of ITT appearing more promising. In this review, we discuss the pharmacological approaches for fertility protection in prepubertal and adolescent boys and the fertility restoration approaches developed on the utilization of ITT.

Generating Genetically Engineered Mice Using a Spermatogonial Stem Cell-Mediated Method.

Mouse spermatogonial stem cells (SSCs) can be grown in culture for long periods. Cultured SSCs, also called germline stem (GS) cells, maintain themselves by self-renewing proliferation while retaining the ability to differentiate into sperm. Thus, when transplanted into the seminiferous tubules of a host mouse testis, they settle in the basal compartment of the tubules and establish spermatogenenic colonies. The sperm produced in the host are competent to produce offspring. This can be exploited for the generation of genetically modified mice, through the transplantation of genetically modified GS cells. In this section, we describe a method of genome editing-mediated GS cell modification and transplantation.

Molecular markers of putative spermatogonial stem cells in the domestic cat.

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43 days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Identification of Germ-Line Stem Cells in Zebrafish.

Both male and female zebrafish have a population of germ-line stem cells that produce gametes throughout the life of the fish. These cells localize to specific regions in the gonads and can be identified because they uniquely express the nanos2 gene, which encodes a conserved regulator of translation. A method is presented here for identifying germ-line stem cells in the ovary and testis using a combined protocol for whole-mount fluorescent RNA in situ hybridization to detect nanos2 mRNA and immunofluorescence to detect the pan-germ cell marker Vasa.

Immunolocalization of proteins in the spermatogenesis process of canine.

Spermatogenesis is a process in which differentiated cells are produced and the adult stem cell population-known as spermatogonial stem cells (SSCs)-is continuously replenished. However, the molecular mechanisms underlying these processes are not fully understood in the canine species. We addressed this in this study by analysing the expression of specific markers in spermatogonia of seminiferous tubules of canine testes. SSCs at different stages of reproductive development (prepubertal and adult) were examined by immunohistochemistry and flow cytometry. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRA1), deleted in azoospermia-like (DAZL) and promyelocytic leukaemia zinc finger (PLZF) were expressed in SSCs, while stimulated by retinoic acid gene 8 (STRA8) was detected only in undifferentiated spermatogonia in prepubertal testis and differentiated spermatogonia and spermatocytes in adult canine. Octamer-binding transcription factor 4 (OCT4) showed an expression pattern, and the levels did not differ between the groups examined. However, C-kit expression varied as a function of reproductive developmental stage. Our results demonstrate that these proteins play critical roles in the self-renewal and differentiation of SSCs and can serve as markers to identify canine spermatogonia at specific stages of development.

Comparison of Diverse Differential Plating Methods to Enrich Bovine Spermatogonial Cells.

Spermatogonial stem cells (SSC) have important applications in domestic animal reproduction and advanced biotechnologies. Because differential plating is one of the most common methods used for SSC enrichment, the goal of this study was to compare three differential plating methods for the enrichment of bovine SSC. To achieve this goal, testicular parenchyma from pre-pubertal calves was minced and single cells were obtained after two enzymatic digestions. We compared three coating methods for differential plating: laminin (20 ng/ml), BSA (0.05 mg/ml) and PBS. Cells were incubated at 37°C, 5% CO2 in air for 15 min onto laminin-coated dishes or 2 h onto BSA- or PBS-coated dishes. Cell viability was assessed by trypan blue exclusion method. Recovered cells were analysed for the expression of SSC molecular markers by quantitative RT-PCR (GFRA1, CXCR4, ITGA6, THY1) and flow cytometry (GFRA1, CXCR4 and ITGA6). Cells at time 0, adherent cells on laminin and non-adherent cells from BSA and PBS groups had the same cell viability (p = 0.0655). GFRA1, CXCR4 and THY1 relative gene expression was higher (p = 0.0402, p = 0.0007, p = 0.0117, respectively) for non-adherent cells selected in PBS group. Flow cytometry analysis revealed that the presence of GFRA-positive (GFRA+) cells was higher in non-adherent cells from BSA and PBS groups (p < 0.001). However, laminin-adherent cells had higher number of ITGA6+ cells (p < 0.001) and lower presence of CXCR4+ cells (p = 0.0012). In conclusion, differential plating is an effective method for the enrichment of bovine undifferentiated spermatogonia and higher expression of SSC markers is obtained without laminin or BSA coating.