Figure 4: Micro-insemination experiments and testosterone production.

(A) Testis tissue cultured for 41 days included many spermatids (upper panels), which was dissected for harvesting haploid cells. ROSI and ICSI procedures produced healthy offspring (middle panels). Genomic PCR demonstrated that 7 out of 14 offspring carried the GFP gene, revealing their origin to be the transgenic sire, heterozygous for Acr-Gfp (lower left panel). They normally matured and were observed on 346 dpp (lower right panel). (B) Testis tissue cultured for 185 days was used for microinsemination (upper panels). Both ROSI and ICSI produced healthy offspring (middle panels). Genomic PCR demonstrated that all 11 offspring carried the GFP gene, revealing their origin to be the transgenic sire, homozygous for Acr-Gfp. Lanes P and N were loaded with a positive-control sample (genomic DNA of Acr-Gfp mouse) and a negative-control sample (genomic DNA of ICR mouse), respectively (lower left panel). The pups normally matured and were observed on 66 dpp (lower right panel). (C) Testosterone production by two cultured testis tissues, sample 1 & 2, was measured at 4 and 5 weeks by collecting medium flowing out of the device for 24 hours each. LH was added to the culture medium on the next day and medium samples were collected in the same way for testosterone measurement. (D) Testis tissue cultured for 120 days produced testosterone and responded to LH at 1 hour and onward. Scale bar: 1 cm (A,B middle panels), 500 μm (A upper left panel, B upper left & middle panels), 100 μm (A upper middle & right panels), 50 μm (B upper right panel).