Brian P. Hermann

Spermatogonial Stem Cell Transplantation into Rhesus Testes Regenerates Spermatogenesis Producing Functional Sperm

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a mans life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from four-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation.

Characterization, Cryopreservation, and Ablation of Spermatogonial Stem Cells in Adult Rhesus Macaques

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare Asingle spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant Adark and Apale spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high‐dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRα1), and cells expressing these markers were depleted following high‐dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high‐dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell‐depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.

Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

Spermatogonial stem cells in higher primates: are there differences from those in rodents?

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout the reproductive life of mammals. While A(single) spermatogonia comprise the rodent SSC pool, the identity of the stem cell pool in the primate spermatogenic lineage is not well established. The prevailing model is that primate spermatogenesis arises from A(dark) and A(pale) spermatogonia, which are considered to represent reserve and active stem cells respectively. However, there is limited information about how the A(dark) and A(pale) descriptions of nuclear morphology correlate with the clonal (A(single), A(paired), and A(aligned)), molecular (e.g. GFRalpha1 (GFRA1) and PLZF), and functional (SSC transplantation) descriptions of rodent SSCs. Thus, there is a need to investigate primate SSCs using criteria, tools, and approaches that have been used to investigate rodent SSCs over the past two decades. SSCs have potential clinical application for treating some cases of male infertility, providing impetus for characterizing and learning to manipulate these adult tissue stem cells in primates (nonhuman and human). This review recounts the development of a xenotransplant assay for functional identification of primate SSCs and progress dissecting the molecular and clonal characteristics of the primate spermatogenic lineage. These observations highlight the similarities and potential differences between rodents and primates regarding the SSC pool and the kinetics of spermatogonial self-renewal and clonal expansion. With new tools and reagents for studying primate spermatogonia, the field is poised to develop and test new hypotheses about the biology and regenerative capacity of primate SSCs.

Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques

BACKGROUND The spermatogonial stem cell (SSC) pool in the testes of non-human primates is poorly defined. METHODS To begin characterizing SSCs in rhesus macaque testes, we employed fluorescence-activated cell sorting (FACS), a xenotransplant bioassay and immunohistochemical methods and correlated our findings with classical descriptions of germ cell nuclear morphology (i.e. Adark and Apale spermatogonia). RESULTS FACS analysis identified a THY-1+ fraction of rhesus testis cells that was enriched for consensus SSC markers (i.e. PLZF, GFRα1) and exhibited enhanced colonizing activity upon transplantation to nude mouse testes. We observed a substantial conservation of spermatogonial markers from mice to monkeys [PLZF, GFRα1, Neurogenin 3 (NGN3), cKIT]. Assuming that molecular characteristics correlate with function, the pool of putative SSCs (THY-1+, PLZF+, GFRα1+, NGN3+/−, cKIT−) comprises most Adark and Apale and is considerably larger in primates than in rodents. It is noteworthy that the majority of Adark and Apale share a common molecular phenotype, considering their distinct functional classifications as reserve and renewing stem cells, respectively. NGN3 is absent from Adark, but is expressed by some Apale and may mark the transition from undifferentiated (cKIT−) to differentiating (cKIT+) spermatogonia. Finally, the pool of transit-amplifying progenitor spermatogonia (PLZF+, GFRα1+, NGN3+, cKIT+/−) is smaller in primates than in rodents. CONCLUSIONS These results provide an in-depth analysis of molecular characteristics of primate spermatogonia, including SSCs, and lay a foundation for future studies investigating the kinetics of spermatogonial renewal, clonal expansion and differentiation during primate spermatogenesis.

Eliminating malignant contamination from therapeutic human spermatogonial stem cells.

Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4-contaminated human testicular cell suspensions was performed to isolate EpCAM+/HLA-ABC-/CD49e- (putative spermatogonia) and EpCAM-/HLA-ABC+/CD49e+ (putative MOLT-4) cell fractions. The EpCAM+/HLA-ABC-/CD49e- fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to-nude mouse xenotransplantation. The EpCAM-/HLA-ABC+/CD49e+ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.

Separating spermatogonia from cancer cells in contaminated prepubertal primate testis cell suspensions

BACKGROUND Chemotherapy and radiation treatments for cancer and other diseases can cause male infertility. There are currently no options to preserve the fertility of prepubertal boys who are not yet making sperm. Cryopreservation of spermatogonial stem cells (SSCs, obtained via testicular biopsy) followed by autologous transplantation back into the testes at a later date may restore fertility in these patients. However, this approach carries an inherent risk of reintroducing cancer. METHODS To address this aspect of SSC transplantation safety, prepubertal non-human primate testis cell suspensions were inoculated with MOLT4 T-lymphoblastic leukemia cells and subsequently sorted for cell surface markers CD90 (THY-1) and CD45. RESULTS Cancer cells segregated to the CD90-/CD45+ fraction and produced tumors in nude mice. Nearly all sorted DEAD box polypeptide 4-positive (VASA+) spermatogonia segregated to the CD90+/CD45- fraction. In a preliminary experiment, a purity check of the sorted putative stem cell fraction (CD90+/CD45-) revealed 0.1% contamination with cancer cells, which was sufficient to produce tumors in nude mice. We hypothesized that the contamination resulted from mis-sorting due to cell clumping and employed singlet discrimination (SD) in four subsequent experiments. Purity checks revealed no cancer cell contamination in the CD90+/CD45- fraction from three of the four SD replicates and these fractions produced no tumors when transplanted into nude mouse testes. CONCLUSIONS We conclude that spermatogonia can be separated from contaminating malignant cells by fluorescence-activated cell sorting prior to SSC transplantation and that post-sorting purity checks are required to confirm elimination of malignant cells.

Transcriptional and Translational Heterogeneity among Neonatal Mouse Spermatogonia

ABSTRACT Spermatogonial stem cells (SSCs) are a subset of undifferentiated spermatogonia responsible for ongoing spermatogenesis in mammalian testes. Spermatogonial stem cells arise from morphologically homogeneous prospermatogonia, but growing evidence suggests that only a subset of prospermatogonia develops into the foundational SSC pool. This predicts that subtypes of undifferentiated spermatogonia with discrete mRNA and protein signatures should be distinguishable in neonatal testes. We used single-cell quantitative RT-PCR to examine mRNA levels of 172 genes in individual spermatogonia from 6-day postnatal (P6) mouse testes. Cells enriched from P6 testes using the StaPut or THY1+ magnetic cell sorting methods exhibited considerable heterogeneity in the abundance of specific germ cell and stem cell mRNAs, segregating into one somatic and three distinct spermatogonial clusters. However, P6 Id4-eGFP+ transgenic spermatogonia, which are known to be enriched for SSCs, were more homogeneous in their mRNA levels, exhibiting uniform levels for the majority of genes examined (122 of 172). Interestingly, these cells displayed nonuniform (50 of 172) expression of a smaller cohort of these genes, suggesting there is substantial heterogeneity even within the Id4-eGFP+ population. Further, although immunofluorescence staining largely demonstrated conformity between mRNA and protein levels, some proteins were observed in patterns that were disparate from those detected for the corresponding mRNAs in Id4-eGFP+ spermatogonia (e.g., Kit, Sohlh2, Stra8), suggesting additional heterogeneity is introduced at the posttranscriptional level. Taken together, these data demonstrate the existence of multiple spermatogonial subtypes in P6 mouse testes and raise the intriguing possibility that these subpopulations may correlate with the development of functionally distinct spermatogenic cell types.

Hormone suppression with GnRH antagonist promotes spermatogenic recovery from transplanted spermatogonial stem cells in irradiated cynomolgus monkeys

Hormone suppression given before or after cytotoxic treatment stimulates the recovery of spermatogenesis from endogenous and transplanted spermatogonial stem cells (SSC) and restores fertility in rodents. To test whether the combination of hormone suppression and transplantation could enhance the recovery of spermatogenesis in primates, we irradiated (7 Gy) the testes of 12 adult cynomolgus monkeys and treated six of them with gonadotropin‐releasing hormone antagonist (GnRH‐ant) for 8 weeks. At the end of this treatment, we transfected cryopreserved testicular cells with green fluorescent protein‐lentivirus and autologously transplanted them back into one of the testes. The only significant effect of GnRH‐ant treatment on endogenous spermatogenesis was an increase in the percentage of tubules containing differentiated germ cells (tubule differentiation index; TDI) in the sham‐transplanted testes of GnRH‐ant–treated monkeys compared with radiation‐only monkeys at 24 weeks after irradiation. Although transplantation alone after irradiation did not significantly increase the TDI, detection of lentiviral DNA in the spermatozoa of one radiation‐only monkey indicated that some transplanted cells colonized the testis. However, the combination of transplantation and GnRH‐ant clearly stimulated spermatogenic recovery as evidenced by several observations in the GnRH‐ant–treated monkeys receiving transplantation: (i) significant increases (~20%) in the volume and weight of the testes compared with the contralateral sham‐transplanted testes and/or to the transplanted testes of the radiation‐only monkeys; (ii) increases in TDI compared to the transplanted testes of radiation‐only monkeys at 24 weeks (9.6% vs. 2.9%; p = 0.05) and 44 weeks (16.5% vs. 6.1%, p = 0.055); (iii) detection of lentiviral sequences in the spermatozoa or testes of five of the GnRH‐ant–treated monkeys and (iv) significantly higher sperm counts than in the radiation‐only monkeys. Thus hormone suppression enhances spermatogenic recovery from transplanted SSC in primates and may be a useful tool in conjunction with spermatogonial transplantation to restore fertility in men after cancer treatment.

The regulatory repertoire of PLZF and SALL4 in undifferentiated spermatogonia

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout adulthood through balanced self-renewal and differentiation, yet the regulatory logic of these fate decisions is poorly understood. The transcription factors Sal-like 4 (SALL4) and promyelocytic leukemia zinc finger (PLZF; also known as ZBTB16) are known to be required for normal SSC function, but their targets are largely unknown. ChIP-seq in mouse THY1+ spermatogonia identified 4176 PLZF-bound and 2696 SALL4-bound genes, including 1149 and 515 that were unique to each factor, respectively, and 1295 that were bound by both factors. PLZF and SALL4 preferentially bound gene promoters and introns, respectively. Motif analyses identified putative PLZF and SALL4 binding sequences, but rarely both at shared sites, indicating significant non-autonomous binding in any given cell. Indeed, the majority of PLZF/SALL4 shared sites contained only PLZF motifs. SALL4 also bound gene introns at sites containing motifs for the differentiation factor DMRT1. Moreover, mRNA levels for both unique and shared target genes involved in both SSC self-renewal and differentiation were suppressed following SALL4 or PLZF knockdown. Together, these data reveal the full profile of PLZF and SALL4 regulatory targets in undifferentiated spermatogonia, including SSCs, which will help elucidate mechanisms controlling the earliest cell fate decisions in spermatogenesis. Summary: ChIP-seq in undifferentiated mouse spermatogonia identifies both unique and overlapping targets of PLZF and SALL4, and reveals preferential binding to gene promoters and introns, respectively.