Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination

Abstract

Spermatogonial transplantation has demonstrated a unique opportunity for studying spermatogenesis and provided an assay for spermatogonial stem cells. However, it has remained unknown whether germ cells that matured in a xenogeneic environment are functionally normal. In this investigation, we demonstrate the successful production of xenogeneic offspring by using spermatogonial transplantation. Rat spermatogonial stem cells were collected from immature testis and transplanted into the seminiferous tubules of busulfan-treated nude mouse testis. Using rat spermatids or spermatozoa that developed in xenogeneic surrogate mice, rat offspring were born from fresh and cryopreserved donor cells after microinsemination with rat oocytes. These offspring were fertile and had a normal imprinting pattern. The xenogeneic offspring production by interspecies germ cell transplantation and in vitro microinsemination will become a powerful tool in animal transgenesis and species conservation.

Fig. 1.

Rat spermatogenesis in mouse testis. (A and B) Macroscopic appearance of a nude mouse recipient testis that received EGFP-expressing rat testis cell transplantation (left). The recipient mouse was killed 5 months after transplantation. (B) Note the extensive colonization of donor cells under UV light. Control testis without transplantation did not show fluorescence (right). (C) Histological appearance of a recipient testis that was immunostained with anti-SCP3 antibody (red). The section was counterstained with Hoechst 33258 dye (blue) for nuclei. (D–G) A round spermatid (D and E), elongated spermatid (F, arrow), and spermatozoan (G, arrow) released from a segment of seminiferous tubule that was used to inject eggs. (E) The round spermatid fluoresced under UV light. (H) Rat offspring from a mouse recipient that received cryopreserved rat testis cell transplantation. EGFP expression was observed in the offspring. (I) PCR analysis using DNA extracted from individual F₁ and F₂ offspring. All EGFP-positive offspring contained the EGFP transgene. (Scale bars: A and B, 1 mm; C, 50 μm; D–F, 15 μm; G, 150 μm.)

Fig. 2.

COBRA of genomic DNA from F₁ and F₂ pups derived from in vitro microinsemination. (A) Schematic diagram of the analyzed regions. (B) DNA methylation status of the analyzed locus. DNA was amplified by specific primers and the PCR products were digested with the indicated restriction enzymes. The percentage of methylation, which was estimated by the intensity of individual bands, is indicated below the gels. U, uncleaved; C, cleaved.