Two allogeneic descendents derived from the high-dose busulfan-treated infertile mouse model after freeze-thawed spermatogonial stem cell transplantation

Abstract

Objective: To evaluate restoration of spermatogenesis and fertility of the recipients who underwent high-dose ablative treatment before freeze-thawed spermatogonial stem cell (SSC) allotransplantation.

Design: Prospective experimental study.

Setting: University-based teaching hospital.

Animal(s): Adult busulfan-treated BALB/c nude mice as recipients of C(57)BL/6 pup SSCs, oocytes of 4-to 6-week-old F(1) hybrid mice for IVF, adult closed group ICR mouse as pseudopregnant female.

Intervention(s): Isolation, purification, and fresh and freeze-thawed transplantation, IVF, embryo transplantation.

Main outcome measure(s): Cell viability, Western blot, real-time fluorescence quantitative-polymerase chain reaction (PCR), scanning electron microscope (SEM), outcome of IVF, and embryo transplantation.

Result(s): Western blot, real-time fluorescence quantitative-PCR, and SEM revealed different spermatogenesis where the fresh groups were uniquely higher than the freeze-thawed groups. A total of six developed two-cell embryos obtained by IVF were transplanted into the oviduct of a pseudopregnant ICR mouse, which resulted in a birth of two paternally different pups. Two female offspring grew into healthy adults with no apparent abnormalities.

Conclusion(s): The results provide the first solid evidence that spermatozoa generated from the busulfan-treated recipient and donor cryopreserved SSCs were both fertile. Ultimate evaluation of the SSC cryopreservation and transplantation technology rests on their functional capacity to generate desirable donor spermatogenesis and fertility in the recipient.