Andrology

Background:Survivors of childhood cancer often suffer from infertility. While sperm cryopreservation is not feasible before puberty, the patient\u2019s own spermatogonial stem cells (SSCs), could serve as a germ cell reservoir, enabling these patients to father their own children in adulthood through the isolation, in vitro expansion, and subsequent transplantation of SSCs. However, this approach requires large numbers of stem cells and methods for successfully propagating SSCs in the laboratory are yet to be established for higher mammals and humans. The improvement of SSC culture requires deeper understanding of their metabolic requirements and the mechanisms that regulate metabolic homeostasis.Aim:This review gives a summary on our knowledge of SSC metabolism during maintenance and differentiation and highlights the potential influence of Sertoli cell and stem cell niche maturation on SSC metabolic requirements during development.Results and Conclusions:Fetal human SSC precursors, or gonocytes, migrate into the seminiferous cords and supposedly mature to adult stem cells within the first year of human development. However, the SSC niche doesn\u2019t fully differentiate until puberty, when Sertoli cells dramatically rearrange the architecture and microenvironment within the seminiferous epithelium. Consequently, prepubertal and adult SSCs experience two distinct niche environments potentially affecting SSC metabolism and maturation. Indeed, the metabolic requirements of mouse PGCs and pig gonocytes are distinct from their adult counterparts and novel single cell RNA sequencing analysis of human and porcine SSCs during development confirms this metabolic transition. Knowledge of the metabolic requirements and their changes and regulation during SSC maturation is necessary to implement laboratory-based techniques and enable clinical use of SSCs. Based on the advancement in our understanding of germline metabolism circuits and maturation events of niche cells within the testis we propose a new definition of spermatogonial stem cell maturation and its amendment in the light of metabolic change.R01 HD091068/HD/NICHD NIH HHSUnited States/R01 OD016575/OD/NIH HHSUnited States/R01 OD016575/CD/ODCDC CDC HHSUnited States

Improving in vitro culture of human male fetal germ cells

Stem cells & developmental biolog

NANOS2 is a sequence-specific mRNA-binding protein that promotes transcript degradation in spermatogonial stem cells

Summary: Spermatogonial stem cells (SSCs) sustain spermatogenesis and fertility throughout adult male life. The conserved RNA-binding protein NANOS2 is essential for the maintenance of SSCs, but its targets and mechanisms of function are not fully understood. Here, we generated a fully functional epitope-tagged Nanos2 mouse allele and applied the highly stringent cross-linking and analysis of cDNAs to define NANOS2 RNA occupancy in SSC lines. NANOS2 recognizes the AUKAAWU consensus motif, mostly found in the 3′ untranslated region of defined messenger RNAs (mRNAs). We find that NANOS2 is a regulator of key signaling and metabolic pathways whose dosage or activity are known to be critical for SSC maintenance. NANOS2 interacts with components of CCR4-NOT deadenylase complex in SSC lines, and consequently, NANOS2 binding reduces the half-lives of target transcripts. In summary, NANOS2 contributes to SSC maintenance through the regulation of target mRNA stability and key self-renewal pathways

The study and manipulation of piglet gonocytes

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model

The Role of CD40 in the Immune Response

A successful humoral immune response requires reciprocal signaling between T helper cells and B cells. The first signal is antigen specific and is mediated by the interaction between the T cell receptor and antigen in association with major histocompatibility complex on B cells. The subsequent signals are provided by the costimulatory molecules CD40 and CD28 and their respective ligands. CD40L which is expressed on activated T helper cells interacts with CD40 on B cells providing the essential signal for the induction of B cell activation and immunoglobulin production. The importance of the CD40-CD40L interaction has been shown in patients suffering from X-linked immunodeficiency with hyper-IgM (HIGM1). The disease is characterized by the inability of B cells to undergo immunoglobulin isotype switching. Affected males experience recurrent infections and most infections are of bacterial origin, but HIGM1 patients are also unusually susceptible to infections with opportunistic pathogens and often suffer from Pneumocystis carinii pneumonia and Cryptosporidium intestinal infection. Mutations in CD40L molecules are responsible for hyper IgM syndrome in humans. To study the role of the CD40-CD40L interaction in vivo and to derive an animal model for HIGM1, I generated CD40 deficient mice using homologous recombination in embryonic stem (ES) cells. A targeting vector was constructed using an 8 kb CD40 genomic fragment. The G418 resistance gene (NEO) was inserted into the 3rd exon to disrupt the CD40 gene and to allow selection. The targeting vector was transfected into ES cells and G418 resistant clones were isolated - and screened for homologous recombination by Southern blot analysis. Chimaeric mice were generated by injection of targeted ES cell clones into blastocysts. Germline transmission was obtained and heterozygous mutant mice bred to generate CD40 deficient mice. Flow cytometric analysis of lymphocytes in CD40 deficient mice revealed normal development of B and T lymphocytes. CD40 deficient mice were immunized with KLH and assessed for germinal centre formation. CD40 deficient mice did not generate germinal centres. Analysis of serum immunoglobulin levels showed that CD40 deficient mice displayed reduced levels of isotype switched immunoglobulins compared to wild-type mice. These results confirm the crucial role of the CD40-CD40L interaction in humoral immunity. CD40 is also expressed on other antigen presenting cells and the involvement of the CD40-CD40L interaction in cell-mediated immunity was determined in CD40 deficient mice infected with Mycobacterium Bovis (BCG). Although CD40 deficient mice survived mycobacterial infection, the increased numbers of bacilli in spleen and lungs and the reduced production of IFN-ɣ in response to mycobacterial infection indicates that CD40 deficient mice are more susceptible to infection with BCG than control mice

Isolation and characterisation of chick embryonic primordial germ cells

Embryonic stem cells (cESCs) can be isolated from chick embryos, with the ability to contribute to all somatic lineages in chimaeras, but not to the germ line. However, lines of chicken embryonic germ cells (cEGCs), which are able to contribute to the germ line, can be established from chicken primordial germ cells (cPGCs). However very little is known about these cells, or about the changes that accompany the establishment of gonadal cells as self-renewing cell lines. This thesis presents a detailed study of the properties of cPGCs and the parent tissue from which they are derived. Gene expression profiles for 30 genes related to pluripotency and/or differentiation were are compared between gonads at the indifferent stage (stage 26-28HH), in primary gonocytes, established PGCs and cESCs. The results reveal great heterogeneity in the expression of various markers in culture. Several genes associated with pluripotency change dramatically upon culture. The most salient of these changes is that while cSox3 (but not Sox2) is expressed in the gonads, whereas their expression becomes reversed upon culture (becoming more similar to mammalian stem cells). This suggests that these two SoxB1 class genes have swapped functions in chick. In the process of studying the expression pluripotency markers in later (stage HH35) gonads, we made an unexpected discovery: both male and female embryos show left-right asymmetric patterns of expression of some, but not all, of these markers. Expression of pluripotency (cPouV, cNanog, cSox2 and ERNI) in the left gonad is much higher than those in the right gonad of both sexes. The expression of pluripotency markers is irrespective of its colonisation by primordial germ cells, and it appears that this left-right decision is made independently of whether the gonad will regress or be retained. These findings offer a new model system for investigating the roles of pluripotency-related markers during normal development as well as in stem cell lines

Editing the genome of chicken primordial germ cells to introduce alleles and study gene function

With continuing advances in genome sequencing technology, the chicken genome assembly is now better annotated with improved accuracy to the level of single nucleotide polymorphisms. Additionally, the genomes of other birds such as the duck, turkey and zebra finch have now been sequenced. A great opportunity exists in avian biology to use genome editing technology to introduce small and defined sequence changes to create specific haplotypes in chicken to investigate gene regulatory function, and also perform rapid and seamless transfer of specific alleles between chicken breeds. The methods for performing such precise genome editing are well established for mammalian species but are not readily applicable in birds due to evolutionary differences in reproductive biology. A significant leap forward to address this challenge in avian biology was the development of long-term culture methods for chicken primordial germ cells (PGCs). PGCs present a cell line in which to perform targeted genetic manipulations that will be heritable. Chicken PGCs have been successfully targeted to generate genetically modified chickens. However, genome editing to introduce small and defined sequence changes has not been demonstrated in any avian species. To address this deficit, the application of CRISPR/Cas9 and short oligonucleotide donors in chicken PGCs for performing small and defined sequence changes was investigated in this thesis. Specifically, homology-directed DNA repair (HDR) using oligonucleotide donors along with wild-type CRISPR/Cas9 (SpCas9-WT) or high fidelity CRISPR/Cas9 (SpCas9-HF1) was investigated in cultured chicken PGCs. The results obtained showed that small sequences changes ranging from a single to a few nucleotides could be precisely edited in many loci in chicken PGCs. In comparison to SpCas9-WT, SpCas9-HF1 increased the frequency of biallelic and single allele editing to generate specific homozygous and heterozygous genotypes. This finding demonstrates the utility of high fidelity CRISPR/Cas9 variants for performing sequence editing with high efficiency in PGCs. Since PGCs can be converted into pluripotent stem cells that can potentially differentiate into many cell types from the three germ layers, genome editing of PGCs can, therefore, be used to generate PGC-derived avian cell types with defined genetic alterations to investigate the host-pathogen interactions of infectious avian diseases. To investigate this possibility, the chicken ANP32A gene was investigated as a target for genetic resistance to avian influenza virus in PGC-derived chicken cell lines. Targeted modification of ANP32A was performed to generate clonal lines of genome-edited PGCs. Avian influenza minigenome replication assays were subsequently performed in the ANP32A-mutant PGC-derived cell lines. The results verified that ANP32A function is crucial for the function of both avian virus polymerase and human-adapted virus polymerase in chicken cells. Importantly, an asparagine to isoleucine mutation at position 129 (N129I) in chicken ANP32A failed to support avian influenza polymerase function. This genetic change can be introduced into chickens and validated in virological studies. Importantly, the results of my investigation demonstrate the potential to use genome editing of PGCs as an approach to generate many types of unique cell models for the study of avian biology. Genome editing of PGCs may also be applied to unravel the genes that control the development of the avian germ cell lineage. In the mouse, gene targeting has been extensively applied to generate loss-of-function mouse models to use the reverse genetics approach to identify key genes that regulate the migration of specified PGCs to the genital ridges. Avian PGCs express similar cytokine receptors as their mammalian counterparts. However, the factors guiding the migration of avian PGCs are largely unknown. To address this, CRISPR/Cas9 was used in this thesis to generate clonal lines of chicken PGCs with loss-of-function deletions in the CXCR4 and c-Kit genes which have been implicated in controlling mouse PGC migration. The results showed that CXCR4-deficient PGCs are absent from the gonads whereas c-Kit-deficient PGCs colonise the developing gonads in reduced numbers and are significantly reduced or absent from older stages. This finding shows a conserved role for CXCR4 and c-Kit signalling in chicken PGC development. Importantly, other genes suspected to be involved in controlling the development of avian germ cells can be investigated using this approach to increase our understanding of avian reproductive biology. Finally, the methods developed in this thesis for editing of the chicken genome may be applied in other avian species once culture methods for the PGCs from these species are develope

TNF-α inhibits GDNF levels in Sertoli cells, through a NF-κB-dependent, HES1-dependent mechanism

Glial cell line-derived neurotrophic factor (GDNF) is a soluble molecule crucial for the regulation of the spermatogonial stem cells (SSC) of the testis. The effects of GDNF on target cells have been extensively described, but mechanisms underlying GDNF regulation are currently under investigation. In the nervous system, GDNF expression is regulated by pro-inflammatory cytokines including lipopolysaccharide (LPS), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α) but the effect of these cytokines on GDNF expression in the testis is unclear

Doctor of Philosophy

dissertationOur work focussed on how germ cell DNA is packaged and if it is poised by distinctive chromatin to influence embryo development. Finally, is misregulation of that poising a common theme observed in infertility and cancer? We profiled the epigenome in mature human sperm and found that packaging in mature sperm revealed the presence of two programs - a future program, involved in guiding embryo development and a past program, involved in spermatogenesis (Chapter 2). Next, the clearest place a chromatin problem can manifest is in infertility. We asked if the DNA methylation status of seven imprinted regions can serve as a diagnostic to inform two groups of infertile patients about the risk of their offspring developing a disorder. Although our results did not provide a causal link for the trans-generational inheritance of DNA methylation defects leading to imprinting disease, it showed a strong correlation between infertility in males and aberrations of DNA methylation at select imprinted loci (Chapter 3). Taken together, our data suggests that germ cell chromatin plays a significant role in early embryonic development and infertility. Finally, we investigated how defects in a metabolic enzyme, succinate dehydrogenase (SDH) can have an impact on chromatin packaging and transcriptome of paragangliomas. We also queried the epigenetic status of paragangliomas lacking mutations in SDH. We compared our two PGL subclasses to a progenitor cell type, neural crest cells (NCCs). Strikingly, we found that both subclasses of PGLs are phenotypically very similar. Furthermore, they share the majority of regions that gain and lose DNA methylation compared to neural crest cells. Whole exome sequencing of both PGL subclasses shows iv mutations in many epigenetic modifier genes and hence we speculate that in PGLs lacking SDH mutations, epigenetic enzymes may harbor mutations that could phenocopy the misregulation in SDH deficient tumors (Chapter 4). Together, we hope that by querying the epigenetic status in a normal system and comparing these findings to perturbed systems, we have gained more insight into the role of epigenetic misregulation in infertility and cancer