Knockout Pigs as Recipients for Spermatogonial Stem Cell Transplantation.

Spermatogonial stem cell (SSC) transplantation into the testis of a germ cell (GC)-depleted surrogate allows transmission of donor genotype via donor-derived sperm produced by the recipient. Transplantation of gene-edited SSCs provides an approach to propagate gene-edited large animal models. DAZL is a conserved RNA-binding protein important for GC development, and knockout (KO) causes defects in GC commitment and differentiation.

Comparing the adult and pre-pubertal testis: Metabolic transitions and the change in the spermatogonial stem cell metabolic microenvironment.

Background: Survivors of childhood cancer often suffer from infertility. While sperm cryopreservation is not feasible before puberty, the patient's own spermatogonial stem cells 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 spermatogonial stem cells. However, this approach requires large numbers of stem cells, and methods for successfully propagating spermatogonial stem cells in the laboratory are yet to be established for higher mammals and humans.

Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, a complex process supported by a specialized microenvironment, called the SSC niche. Postnatal development of SSCs is characterized by distinct metabolic transitions from prepubertal to adult stages. An understanding of the niche factors that regulate these maturational events is critical for the clinical application of SSCs in fertility preservation.

Metabolic transitions define spermatogonial stem cell maturation.

Study Question: Do spermatogonia, including spermatogonial stem cells (SSCs), undergo metabolic changes during prepubertal development?

Summary Answer: Here, we show that the metabolic phenotype of prepubertal human spermatogonia is distinct from that of adult spermatogonia and that SSC development is characterized by distinct metabolic transitions from oxidative phosphorylation (OXPHOS) to anaerobic metabolism.

What Is Known Already: Maintenance of both mouse and human adult SSCs relies on glycolysis, while embryonic SSC precursors, primordial germ cells (PGCs), exhibit an elevated dependence on OXPHOS. Neonatal porcine SSC precursors reportedly initiate a transition to an adult SSC metabolic phenotype at 2 months of development.

Organotypic Rat Testicular Organoids for the Study of Testicular Maturation and Toxicology.

An system to study testicular maturation in rats, an important model organism for reproductive toxicity, could serve as a platform for high-throughput drug and toxicity screening in a tissue specific context. maturation of somatic cells and spermatogonia in organ culture systems has been reported. However, this has been a challenge for organoids derived from dissociated testicular cells.

Targeted Gene Editing in Porcine Germ Cells.

As the genetic mutations driving human disease are identified, there is an increasing need for a biomedical model that can accurately represent the disease of interest and provide a platform for potential therapeutic testing. Pigs are a better model for human disease than rodents because of their genetic and physiological similarities to humans. However, current methods to generate porcine models are both technically challenging and expensive.

A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture.

Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood.

The Proliferation of Pre-Pubertal Porcine Spermatogonia in Stirred Suspension Bioreactors Is Partially Mediated by the Wnt/β-Catenin Pathway.

Male survivors of childhood cancer are at risk of suffering from infertility in adulthood because of gonadotoxic chemotherapies. For adult men, sperm collection and preservation are routine procedures prior to treatment; however, this is not an option for pre-pubertal children. From young boys, a small biopsy may be taken before chemotherapy, and spermatogonia may be propagated in vitro for future transplantation to restore fertility.

Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells.

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified.

PNKP is required for maintaining the integrity of progenitor cell populations in adult mice.

DNA repair proteins are critical to the maintenance of genomic integrity. Specific types of genotoxic factors, including reactive oxygen species generated during normal cellular metabolism or as a result of exposure to exogenous oxidative agents, frequently leads to "ragged" single-strand DNA breaks. The latter exhibits abnormal free DNA ends containing either a 5'-hydroxyl or 3'-phosphate requiring correction by the dual function enzyme, polynucleotide kinase phosphatase (PNKP), before DNA polymerase and ligation reactions can occur to seal the break.

Unique metabolic phenotype and its transition during maturation of juvenile male germ cells.

Human male reproductive development has a prolonged prepubertal period characterized by juvenile quiescence of germ cells with immature spermatogonial stem cell (SSC) precursors (gonocytes) present in the testis for an extended period of time. The metabolism of gonocytes is not defined. We demonstrate with mitochondrial ultrastructure studies via TEM and IHC and metabolic flux studies with UHPLC-MS that a distinct metabolic transition occurs during the maturation to SSCs.

Metabolic Requirements for Spermatogonial Stem Cell Establishment and Maintenance In Vivo and In Vitro.

The spermatogonial stem cell (SSC) is a unique adult stem cell that requires tight physiological regulation during development and adulthood. As the foundation of spermatogenesis, SSCs are a potential tool for the treatment of infertility. Understanding the factors that are necessary for lifelong maintenance of a SSC pool in vivo is essential for successful in vitro expansion and safe downstream clinical usage.

Targeted Gene Editing in Porcine Spermatogonia.

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype.

Regulation of Cell Types Within Testicular Organoids.

Organoids are 3-dimensional (3D) structures grown in vitro that emulate the cytoarchitecture and functions of true organs. Therefore, testicular organoids arise as an important model for research on male reproductive biology. These organoids can be generated from different sources of testicular cells, but most studies to date have used immature primary cells for this purpose.

Development and function of smooth muscle cells is modulated by in mouse testis.

In mammalian testis, contractile peritubular myoid cells (PMCs) regulate the transport of sperm and luminal fluid, while secreting growth factors and extracellular matrix proteins to support the spermatogonial stem cell niche. However, little is known about the role of testicular smooth muscle cells during postnatal testicular development. Here we report age-dependent expression of hypermethylated in cancer 1 (; also known as ) in testicular smooth muscle cells, including PMCs and vascular smooth muscle cells, in the mouse.

Transcriptional Profiling of the Adult Hair Follicle Mesenchyme Reveals R-spondin as a Novel Regulator of Dermal Progenitor Function.

The adult hair follicle (HF) undergoes successive regeneration driven by resident epithelial stem cells and neighboring mesenchyme. Recent work described the existence of HF dermal stem cells (hfDSCs), but the genetic regulation of hfDSCs and their daughter cell lineages in HF regeneration remains unknown. Here we prospectively isolate functionally distinct mesenchymal compartment in the HF (dermal cup [DC; includes hfDSCs] and dermal papilla) and define the transcriptional programs involved in hfDSC function and acquisition of divergent mesenchymal fates.

A reduction of primary cilia but not hedgehog signaling disrupts morphogenesis in testicular organoids.

Most mammalian cells possess a single, non-motile primary cilium that plays an important role in mediating cellular signaling pathways, such as Hedgehog (Hh) signaling. Primary cilia are present on testicular somatic cells and demonstrate a temporal expression during development; however, their role in testicular morphogenesis is not well characterized. To investigate the role of primary cilia and Hh signaling in Sertoli cells on morphogenesis, we inhibited assembly of primary cilia through CRISPR Cas9-mediated gene editing of ODF2, a structural component of primary cilia and siRNA-mediated gene silencing of IFT88, a functional component of the intraflagellar transport system.

Mammalian germ cells are determined after PGC colonization of the nascent gonad.

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated.

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture.

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology.

Three-dimensional testicular organoids as novel models of testicular biology and toxicology.

Organoids are three dimensional structures consisting of multiple cell types that recapitulate the cellular architecture and functionality of native organs. Over the last decade, the advent of organoid research has opened up many avenues for basic and translational studies. Following suit of other disciplines, research groups working in the field of male reproductive biology have started establishing and characterizing testicular organoids.

Testicular organoids to study cell-cell interactions in the mammalian testis.

Background: Over the last ten years, three-dimensional organoid culture has garnered renewed interest, as organoids generated from primary cells or stem cells with cell associations and functions similar to organs in vivo can be a powerful tool to study tissue-specific cell-cell interactions in vitro. Very recently, a few interesting approaches have been put forth for generating testicular organoids for studying the germ cell niche microenvironment.

Aim: To review different model systems that have been employed to study germ cell biology and testicular cell-cell interactions and discuss how the organoid approach can address some of the shortcomings of those systems.

Stirred Suspension Bioreactor Culture of Porcine Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) are an attractive cell source for regenerative medicine and the development of therapies, as they can proliferate indefinitely under defined conditions and differentiate into any cell type in the body. Large-scale expansion of cells is limited in adherent culture, making it difficult to obtain adequate cell numbers for research. It has been previously shown that stirred suspension bioreactors (SSBs) can be used to culture mouse and human stem cells.

Formation of organotypic testicular organoids in microwell culture†.

Three-dimensional (3D) organoids can serve as an in vitro platform to study cell-cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human.

Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring.

Testicular tissue cryopreservation is an experimental method to preserve the fertility of prepubertal patients before they initiate gonadotoxic therapies for cancer or other conditions. Here we provide the proof of principle that cryopreserved prepubertal testicular tissues can be autologously grafted under the back skin or scrotal skin of castrated pubertal rhesus macaques and matured to produce functional sperm. During the 8- to 12-month observation period, grafts grew and produced testosterone.