Maximal Donor Germline Transmission from DAZL-Deficient Rats.

<p>(A) Southern blot analysis of progeny from Wildtype (WT) and DAZL-Deficient (Dazl) recipient rats transplanted with 50,000 <i>GCS-EGFP</i> spermatogonia/right testis at passage 13 (i.e. 158 days in culture); left testes of each animal were not transplanted. At 75 days post-transplantation recipients (R) were paired with WT females (F) and allowed to produce pups by natural breeding (See R942-R949 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-t001" target="_blank">Table 1</a>). Shown are blots from representative litters probed for <i>EGFP</i> to distinguish progeny produced by donor cells. OMP = loading control. Genomic DNA Controls were from untreated <i>GCS-EGFP</i> and <i>DAZL</i>-Deficient transgenic rats. LTR = PCR primers specific for lentiviral transgene in <i>DAZL</i>-deficient rats. GAPDH = PCR loading control. (B) Bright field and green fluorescence images of testes from Wildtype (<i>Left</i>) and DAZL-deficient (<i>Right</i>) recipient rats at 212 days post-transplantation. Scale bar = 1 cm. (C) Graph of germline transmission rates for the donor, <i>GCS-EGFP</i> transgene from Wildtype and DAZL-deficient recipient rats transplanted with 50,000 <i>GCS-EGFP</i> spermatogonia/right testis at passage 13; left testes were not transplanted. <i>DAZL</i>-deficient recipients transmitted the <i>GCS-EGFP</i> transgene to 100%+/−0% of progeny (+/−SEM, n = 3 recipients; 9 litters), with 73 of 73 total F1 pups born from donor cells. Wildtype recipients transmitted the <i>GCS-EGFP</i> transgene to 14%+/−5.9% of progeny (+/−SEM, n = 3 recipients; 9 litters), with 16 of 116 total F1 pups born from donor cells. (D) Genealogy tree showing stable transmission of donor haplotypes from <i>DAZL</i>-Deficient recipients (F0) R989 and R990 to F1 and F2 progeny. Recipients were each transplanted with 150,000 rat spermatogonia/testis from line RSGL-GCS9 at passage 17 (See R988-R990 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-t001" target="_blank">Table 1</a>). Spermatogonial line RSGL-GCS9 was derived from a rat homozygous for the <i>GCS-EGFP</i> transgene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone.0006308-Wu1" target="_blank">[36]</a>. Thus, F1 progeny represent half-siblings; some of which were crossed to re-derive transgenic F2 progeny homozygous for the <i>tgGCS-EGFP</i> allele.</p

Long-Term Spermatogenic Potential of Donor Spermatogonia.

<p>(<i>Left</i>) Graph showing relative numbers of Round and Elongating Spermatids in seminiferous tubules of non-transplanted, non-busulfan-treated, Wildtype and <i>DAZL</i>-deficient rat lines at 4 months of age (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-g001" target="_blank">Fig. 1a</a>), in comparison to Spermatid numbers in busulfan-treated, <i>DAZL</i>-deficient recipient rats at 212 days (i.e. ∼8 months of age) after being transplanted with rat spermatogonial line, RSGL-GCS9, at passage 13 (i.e. culture day 158). Cell counts were normalized/1000 Sertoli cells. +/−SEM, n = 3 rats/group. (<i>Right</i>) Images of histological sections of seminiferous tubules from the <i>DAZL</i>-deficient recipient rats described in the “Left” panel after being transplanted with spermatogonia from RSGL-GCS9. <i>Bottom Right</i> shows a higher magnification image within the boxed region of the <i>Top Right</i> panel. Scale bars = 100 µm.</p

Analysis of Spermatogenic Failure in DAZL-Deficient Rats.

<p>(A) Relative numbers of spermatogenic cell types/1000 Sertoli cells in testes of 4 month old wildtype and transgenic <i>DAZL</i>-deficient rats (+/−SEM, n = 3 rats/group). Undifferentiated type A spermatogonia (Undif), differentiating type A spermatogonia (Type A), intermediate to type B spermatogonia (Int-Type B), preleptotene spermatocytes (PL), leptotene-zygotene spermatocytes (lept-zygo), pachytene-diplotene spermatocytes (Pachy-Di), secondary spermatocytes-round spermatids (SS-RS), elongating spermatids (ES). Significant differences (i.e. p<0.05) in testis cell types scored in wildtype and DAZL-deficient rats were determined for SS-RS (p = 0.022) and ES (p<0.0001) using unpaired two-tailed students t-test. (B) Histological cross-sections of seminiferous tubules from 4 month old wildtype (<i>Top left</i>) and transgenic <i>DAZL</i>-deficient rats (<i>Top right</i>). <i>Bottom left and right</i> show higher magnification images of boxed regions in the <i>Top</i> panels. Scale bars = 100 µm. (C) Images of spermatogonial types in testis sections from wildtype rats at stages VIII, XI, XIII, XIV, II and V of spermatogenesis. Types of undifferentiated (Undif) and differentiating spermatogonia (A1, A2, A3, A4, Int, B) were determined by analysis of staining patterns in nuclei at specific stages of spermatogenesis. Scale bar = 30 µm. (D) Images of spermatogonial types in testis sections from <i>DAZL</i>-deficient rats. Because stages of the epithelial cycle could not be classified in these rats, types of undifferentiated (Undif) and differentiating spermatogonia (A1-like, A2-like, A3-like, A4-like, Int-like, B-like) were estimated based on staining profiles of spermatogonia in wildtype rats. Scale bar = 30 µm.</p

Prospective Procedures for Curing Azoospermia with Stem Cells.

<p>In many cases of spermatogenic failure, healthy sources of spermatogonia could be obtained from either a biopsy of an azoospermic patient's own testis, or prospectively, from a biopsy of his own somatic cells following induction into a pluripotent state. Pure spermatogonial lines derived from either source would require that they are maintained in a spermatogenic lineage and prevented from acquiring a pluripotent state when in culture. If such protocols were established, naturally healthy spermatogonial lines could be selected for from a background of potentially unhealthy cell lines, such as from cancer patients, or azoospermic men diagnosed as germline mosaics (later example is shown). Once selected for, the healthy lines could then be induced to develop into sperm by transplanting them back into the patient's own testes. As potential alternatives to sterile-testis complementation that are currently being investigated, spermatogonial stem cells could be induced to develop through meiosis during culture <i>in vitro</i>, or within tissue grafts for production of haploid gametes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone.0006308-Honaramooz1" target="_blank">[50]</a>. The resulting haploid spermatids could then be used for assisted fertilization.</p

DAZL-Deficient Rats are Efficient Spermatogonial Recipients.

<p>(A) Relative abundance of EGFP in testes of Wildtype, <i>DAZL</i>-deficient and <i>GCS-EGFP</i> rats. <i>Left</i>: Data expressed as the equivalents of recombinant, histidine-tagged EGFP (rEGFP)/testis (+/−SEM, n = 3 testes/rat strain) as determined by fluorometry of testis extracts at 24 days of age. <i>Right</i>: Bright field (top) and green fluorescence (bottom) images of testes dissected from <i>GCS-EGFP</i>, wildtype (WT) and <i>DAZL</i>-deficient (Dazl-Def) rats at 24 days of age. Scale bar = 1 cm. (B) Spermatogenesis colony forming assays using <i>DAZL</i>-deficient rats as recipients. <i>Left</i>: Numbers of spermatogenic colonies formed/testis by donor <i>GCS-EGFP</i> rat spermatogonia in Wildtype (10.25+/−0.68 colonies/testis, +/−SEM, n = 8 testes) and <i>DAZL</i>-Deficient rats (30.69+/−0.62 colonies/testes, +/−SEM, n = 8 testes) at 30 days following transplantation; p<0.0001, unpaired two-tailed students t-test. Donor spermatogonia were transplanted at passages 15 and 17 (i.e. culture days 182 and 204) at 2000 <i>GCS-EGFP</i>⁺ cells/testis. <i>Right</i>: Images of individual colonies of spermatogenesis in Wildtype and <i>DAZL</i>-deficient recipient rats that were generated by the donor <i>GCS-EGFP</i> spermatogonia (green fluorescence is from donor cells). Images are representative of colonies scored and plotted in the <i>Left</i> panel. Scale bar = 100 µm.</p

Progeny from Wildtype and <i>DAZL</i>-Deficient Recipient Rats Transplanted with GCS-EGFP Rat Spermatogonia.

<p>Wildtype or DAZL-deficient (DAZL-def) recipient rats were transplanted with either 0.5 or 1.5×10⁵ EGFP⁺ cells/testis from rat spermatogonial line GCS9 at 12 days after busulfan treatment (i.e. 12 mg/kg i.p.) on postnatal day 24. At ∼75 days post-transplantation recipients were paired with 75–80 day old wildtype female rats. Spermatogonia line GCS9 was harvested from passages number 13 and 17, which corresponded to 158 and 204 days in culture, respectively, prior to their transplantation. Recipients R942-R949 were littermates born from a hemizygous, transgenic DAZL-deficient female and a wildtype Sprague Dawley male. No progeny were born from breeder pairs of un-transplanted, busulfan-treated DAZL-deficient males and wild-type females (n = 3 breeder pairs). Breeder pairs of untreated, wild-type male litter mates of DAZL-deficient rats and wild-type female rats from Harlan, Inc. produced 15.5±4.5 pups/litter (+/−SEM, n = 8 litters from 3 breeder pairs).</p>1<p>p = 0.0209 Average Group 1 versus Average Group 2; p = 0.0251 Average Group 2 versus Average Group 3.</p>2<p>p = 0.0031 Average Group 1 versus Average Group 2.</p>3<p>Percent <i>GCS-EGFP</i>⁺ F1 progeny.</p