Sterile Testis Complementation with Spermatogonial Lines Restores Fertility to DAZL-Deficient Rats and Maximizes Donor Germline Transmission

Despite remarkable advances in assisted reproductive capabilities ∼4% of all couples remain involuntarily infertile. In almost half of these cases, a lack of conception can in some measure be attributed to the male partner, wherein de novo Y-chromosomal deletions of sperm-specific Deleted-in-Azoospermia (DAZ) genes are particularly prevalent. In the current study, long-term cultures of rat spermatogonial stem cells were evaluated after cryo-storage for their potential to restore fertility to rats deficient in the DAZ-like (DAZL) gene. Detailed histological analysis of DAZL-deficient rat testes revealed an apparently intact spermatogonial stem cell compartment, but clear failure to produce mature haploid gametes resulting in infertility. After proliferating >1 million-fold in cell number during culture post-thaw, as few as 50,000 donor spermatogonia transplanted into only a single testis/recipient effectively restored fecundity to DAZL-deficient rats, yielding 100% germline transmission to progeny by natural mating. Based on these results, the potency and efficacy of this donor stem cell line for restoring fertility to azoospermic rodents is currently unprecedented. Prospectively, similar successes in humans could be directly linked to the feasibility of obtaining enough fully functional spermatogonial stem cells from minimal testis biopsies to be therapeutically effective. Thus, regeneration of sperm production in this sterile recipient provides an advanced pre-clinical model for optimizing the efficacy of stem cell therapies to cure a paradoxically increasing number of azoospermic men. This includes males that are rendered infertile by cancer therapies, specific types of endocrine or developmental defects, and germline-specific de novo mutations; all of whom may harbor healthy sources of their own spermatogonial stem cells for treatment

Genetic association and characterization of FSTL5 in isolated clubfoot

ACKNOWLEDGEMENTS: The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C). The authors thank the staff and participants of the ARIC study for their important contributions. Funding for GENEVA was provided by National Human Genome Research Institute grant U01HG004402 (E.Boerwinkle). We thank H. Hobbs and J. Cohen for contributing control samples for replication genotyping, Nadav Ahituv for sharing RNA-seq data for both bat and mouse embryonic limb buds, Tommy Hyatt for designing the custom genotyping assay, and members of the UT Southwestern Transgenic Core facility, including John Ritter, Mylinh Nguyen, and Robert Hammer. Publicly available mouse embryonic expression analysis results were provided online at https://oncoscape.v3.sttrcancer.org/atlas.gs.washington.edu.mouse.rna/landing (24). The authors acknowledge the contributions and support of the Center for Excellence in Clubfoot Research at Scottish Rite for Children, including Shawne Faulks and Kristhen Atala. Fstl5 mutant rats were produced by the NIH Mutant Rat Resource at UT Southwestern Medical Center (R24RR03232601, R24OD011108, R01HD036022, and (5R01HD053889). This study was supported by funding from the Scottish Rite for Children Research Fund (J.J.R.), Shriners Hospital for Children (J.T.H), and the National Institutes of Health award R01HD043342 (J.T.H.).Peer reviewedPostprin

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

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