Timing of spermatogonial stem cell transplantation affects the spermatogenic recovery outcome in mice.

Spermatogonial stem cell transplantation (SSCT) is a strategy that has demonstrated to be feasible to restore spermatogenesis in animal models when it is performed shortly after the gonadotoxic onset to destroy their endogenous germ cells. However, in the case of boys subjected to fertility preservation, future transplantations will be performed with a delay of many years. In order to study how timing of SSCT affects donor-derived spermatogenic recovery in mice, we compared the percentage of spermatogenic tubule cross-sections within testes of 59 C57BL/6NCrl mice distributed in 6 groups: group 1, untreated mice controls (n = 9); group 2, mice that received a single dose of busulfan 40 mg/kg (n = 10); group 3, mice that received two additional doses of busulfan 10 mg/kg every 5 weeks (n = 10); group 4 (SSCT-A), mice subjected to a standard SSCT performed 5 weeks after a single injection of busulfan 40 mg/kg (n = 10); group 5 (SSCT-B), mice subjected to a delayed SSCT performed 15 weeks after a single injection of busulfan 40 mg/kg (n = 10); and group 6 (SSCT-C), mice subjected to a delayed SSCT with two additional doses of busulfan 10 mg/kg every 5 weeks (n = 10). Spermatogenic recovery in standard SSCT-A and SSCT-C groups ranged between 22.29 and 22.65%, compared with a lower recovery rate of 11.54% showed in the SSCT-B group. However, donor contribution resulted higher in standard SSCT-A, representing a 69.71% of cross-sections, compared with the rest of conditions ranging from 34.69 to 35.42%. Overall, we concluded that a delay in the SSCT from the gonadotoxic onset decreases the efficiency of donor-derived spermatogenic recovery in mice.

Comparative analysis of the germ cell markers c-KIT, SSEA-1 and VASA in testicular biopsies from secretory and obstructive azoospermias.

Testicular biopsy is needed to confirm diagnosis in azoospermic patients and to recover spermatozoa, if possible. This report aims to quantitatively analyse the germline markers stage-specific embryonic antigen (SSEA-1), c-KIT and VASA in testicular biopsies with distinct azoospermic aetiologies. Twenty-three testicular biopsies were analysed by flow cytometry and RT-qPCR for c-KIT, SSEA-1 and VASA. In all the Sertoli cell-only (SCO) samples, significantly lower VASA mRNA expression and fewer VASA+ cells were found compared with obstructive controls. Maturation arrest (MA) cases showed significant differences only with the non-mosaic SCO samples when compared for VASA mRNA expression and percentage of VASA+ cells, but not with the mosaics. However, the normalized VASA-KIT parameter obtained by subtracting the percentage of c-KIT+ cells from the percentage of VASA+ cells showed significant differences between the MA and all the SCO samples. RT-qPCR consistently found differences for the VASA expression between SCO mosaic and non-mosaic samples. However, by flow cytometry, only VASA-KIT showed significant differences between them. Conversely, the percentage of SSEA-1+ cells revealed no inter-group differences. In conclusion, testicular biopsies display different expression profiles for c-KIT and VASA depending on the azoospermic aetiology. These results can be used as a complementary tool to create new molecular categories for diagnoses in azoospermic patients, particularly useful to discriminate between mosaic and non-mosaic SCO patients.

Differentiation of germ cells and gametes from stem cells.

BACKGROUND: Advances in stem cell research have opened new perspectives for regenerative and reproductive medicine. Stem cells (SC) can differentiate under appropriate in vitro and in vivo conditions into different cell types. Several groups have reported their ability to differentiate SCs into germline cells, and some of them have been successful in obtaining male and female gamete-like cells by using different methodologies. METHODS: This review summarizes the current knowledge in this field and emphasizes significant embryological, genetic and epigenetic aspects of germ cells and gametes in vitro differentiation in humans and other species, highlighting major obstacles that need to be overcome for successful gametogenesis in culture: studies reporting development of germ cell-like cells from murine and human embryonic (ESC) and somatic SCs are critically reviewed. RESULTS: Published studies indicate that germ cells can be consistently differentiated from mouse and human ESC. However, further differentiation of germ cells through gametogenesis still has important genetic and epigenetic obstacles to be efficient. CONCLUSIONS: Differentiation of germ cells from SCs has the potential of becoming a future source of gametes for research use, although further investigation is needed to understand and develop the appropriate niches and culture conditions. Additionally, if genetic and epigenetic methodological limitations could be solved, therapeutic opportunities could be also considered.