Donor testis cells (including SSCs) are introduced into recipient seminiferous tubules via injection into the rete testis space. (A) The rete testis in rhesus can be visualized by ultrasound as a linear echo-dense structure and (B) ultrasound is used to guide an echo-dense injection needle into the rete testis space allowing cells to be injected by slow, positive pressure. (C) After this injection, presence of dye in the ductules of the caput epididymis (inset), which is contiguous via the efferent ducts with the rete testis, confirmed successful injection. (D) Bisection of the transplanted testis revealed that blue dye radiated from the rete testis into approximately 60–80% of seminiferous tubules. (E–F) Subsequent evaluation of intact seminiferous tubules confirmed the presence of blue dye in the lumen of seminiferous tubules. See also .

This cartoon shows relative timing of experimental procedures for recipient animals, including autologous transplants of peripheral blood stem cells (PBSCs) used to restore the hematopoietic system after busulfan chemotherapy. Indwelling central venous catheters were placed in the right internal jugular vein at the time of testicular tissue harvesting or approximately 5 weeks before PBSC harvest via apheresis. Autologous blood was collected for 5 weeks (red triangles) and pooled to prime the apheresis tubing set. Animals received daily subcutaneous injections with the cytokine G-CSF (and in some cases, SCF) (green triangles) for six days to mobilize hematopoietic stem cells from the bone marrow into the general circulation. PBSCs were collected on day 0 by apheresis using the indwelling central line for venous access. Twenty-four hours after completing apheresis, animals were treated with busulfan (labeled arrow). Approximately forty-two hours after completing apheresis (~18 hours after busulfan treatment), animals were transfused with autologous PBSCs collected by apheresis. Two days later, animals received one subcutaneous injection of neulasta (long-acting G-CSF) to stimulate rapid expansion of engrafted stem cells and hematopoietic recovery. Animals were monitored closely for hematopoietic deficits with weekly (or more frequent) complete blood count (CBC, orange triangles).
Approximately 10–12 weeks after busulfan treatment, animals received SSC transplants (when sperm counts were 0 for two consecutive weeks). Weekly ejaculated sperm counts (blue triangles) measured the effect of busulfan on spermatogenesis and the progression of spermatogenic recovery after transplant. See also and .

(A) Weekly sperm counts (total sperm per ejaculate) are shown for one autologous recipient (M037 treated with 10mg/kg busulfan). In this animal, busulfan was administered at week -14 (noted by blue arrow) relative to SSC transplant at week 0. (B) DNA from each ejaculate containing sperm was genotyped by PCR for a 1.1kb segment of the lentiviral backbone. Negative controls included pre-busulfan/pre-transplant (pre-TP) ejaculates, ejaculates from un-transplanted controls (negative) and H₂O. Positive controls included cultured testis cells treated with lentivirus (M306+, M307+) and dilutions of lentiviral plasmid DNA (100pg, 10pg, 1pg and 0.1pg). Histological (hematoxylin and eosin staining) comparison of testicular parenchyma before and after busulfan treatment (at necropsy) as well as the cauda epididymis after busulfan treatment reveals the degree of spermatogenesis in (C) M037, which exhibited successful transplant engraftment based on presence of sperm in the ejaculate (60% of tubule cross-sections contained spermatogenesis; necropsied 80 weeks after busulfan), (D) transplant recipient M214 which never exhibited sperm in the ejaculate after transplant (24% of tubule cross-sections contained spermatogenesis; necropsied 67 weeks after busulfan). (E) Histology from the testis and epididymis of an un-transplanted animal M104 (no spermatogenesis evident; necropsied 26 weeks after busulfan) illustrates the appearance of an azoospermic (empty) testis after busulfan treatment. Scale bars = 50μm. See also , and .

(A) Weekly sperm counts (total sperm per ejaculate) from two allogeneic recipients treated with 8mg/kg busulfan (M212; treated with busulfan on week -11, dark blue arrow) or 11mg/kg busulfan (M027; treated with busulfan on week -9, light blue arrow), (inset) sperm from M027 are shown as examples. DNA from each ejaculate containing sperm was genotyped by microsatellite DNA fingerprinting to determine presence of donor genotype. Both of these allogeneic recipients showed evidence of donor spermatogenesis. (B–D) Epididymal sperm obtained at necropsy from recipient M212 contained a mixture of M212 recipient and M214 donor signal at the two microsatellite loci examined. (E–G) Ejaculated sperm from M027 (collected 14 weeks after transplant) also demonstrated a mixture of M027 recipient and M092 donor signal at the two microsatellite loci examined. This result persisted for at least 12 months after transplant with analysis ongoing. Microsatellite loci are noted above each column of electropherograms and alleles for each animal or sample are indicated at the bottom-right of each electropherogram panel. Discriminating alleles for donor are noted by bold/colored text. (H) Allelic discrimination qPCR (TaqMan probes) was used for SNP genotyping to determine the degree of M092 donor spermatogenesis in M027 sperm samples between 3 and 17 months after transplant. Shown is the degree of M092 genotype (%) in each sperm DNA sample based on presence of SNPs in the rhesus CIITA locus. Percent donor genotype was determined by standard curve with known amounts of donor and recipient gDNA. Additional information about the specific samples used for SNP analysis are indicated in . See also .

Ejaculated sperm from allogeneic recipient M027 (collected 30 weeks after transplant) were used to fertilize rhesus oocytes by intracytoplasmic sperm injection (ICSI). (A) Pronuclear stage zygote produced using sperm from M027 (see ). (B–G) Subsequent in vitro culture resulted in embryos ranging from 2-cell to blastocysts. (H–O) Following whole-genome amplification, microsatellite DNA fingerprinting at two tetranucleotide repeat loci (DXS2506 and D15S823) confirmed SSC transplant donor (M092) paternity in 7 of 81 embryos generated from M027 sperm. Microsatellite profiles of four M092 donor-derived embryos are shown in panels L–O. Embryo 1 (L) was from dam 28510 and embryos 49, 51 and 63 (M–O) were from dam 25168. Microsatellite loci are noted above each column of electropherograms and alleles for each animal or sample are indicated in the upper right of each electropherogram panel. Discriminating alleles for donor are noted by bold/colored text. In cases where embryos were male (i.e., XY, panels L and O), paternal contribution at the X-linked DXS2506 locus was nill, and thus, simply noted by Y. In both cases M092 paternal origin could be confirmed by the D15S823 locus. See also and .