Spermatogonial Stem Cells: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Introduction: The Why´s and How´s for Studying Spermatogenesis and Spermatogonial Stem Cells
1 The Significance of Studying Spermatogenesis
2 What Are Spermatogonia and Why Should You Care?
3 Importance of Using Stringent and Definitive Definitions of SSCs
4 Organization of This Volume
References
Chapter 2: Optimization of Testicular Fixation-Embedding Techniques for Improved Evaluation of Mammalian Spermatogonial Morpho...
1 Introduction
2 Method #1: Aldehyde and Glycol Methacrylate (GMA)
2.1 GMA Materials
2.2 GMA Methods
2.2.1 GMA: Fixation and Storage
2.2.2 GMA: Embedding
2.2.3 GMA: Sectioning
2.2.4 GMA: Staining
3 Method #2: Aldehyde and Araldite (HRLM)
3.1 HRLM Materials
3.2 HRLM Methods
3.2.1 HRLM: Fixation and Storage
3.2.2 HRLM: Embedding
3.2.3 HRLM: Sectioning
3.2.4 HRLM: Staining
4 Notes
References
Chapter 3: Single-Molecule Fluorescence In Situ Hybridization for Spatial Detection of mRNAs in Sections of Mammalian Testes
1 Introduction
2 Materials
2.1 Testis Preparation and Freezing
2.2 RNAscope Reagents and Buffers
2.3 Immunofluorescence (If Necessary)
2.4 General Materials and Equipment
3 Methods
3.1 Testis Freezing and Embedding
3.2 Cryosectioning
3.3 Testis Pretreatment
3.4 Target Retrieval
3.5 Hybridization
3.6 Amplification
3.7 Develop C1 Probe (For Example)
3.8 Immunofluorescence (If Necessary)
3.9 DAPI Counterstain
4 Notes
References
Chapter 4: Bulk and Single-Cell RNA-Seq Analyses for Studies of Spermatogonia
1 Introduction
1.1 cDNA Library Preparation
2 Bionformatic Analysis of Bulk RNA-Seq Data
2.1 Quality Control
2.2 Read Alignment
2.3 Transcriptome Reconstruction and Expression Quantification
2.4 Normalization
2.5 Differential Gene Expression Analysis
2.6 Analysis of Bulk RNA-Seq Data Using DEseq2
2.6.1 Resources Required
2.6.2 Initialization, Quality Control, and Filtering
Box 1 Install BiocManager and load required packages
Box 2 Load read count matrix
Box 3 Construct DESeqDataSet object in DESeq2
Box 4 Perform data filtering
2.6.3 Differential Gene Expression Analysis
Box 5 Differential expression testing
Box 6 Summary of differential gene expression results
Box 7 Log fold change shrinkage
Box 8 Sort DE results by p-value
2.6.4 Sample Clustering and Visualization
Box 9 Plot MA-plot and counts
Box 10 Data transformation
Box 11 PCA Plot
Box 12 Plot Heatmap
3 Bioinformatic Analysis of Single-Cell RNA-Seq Data
3.1 Quality Control
3.2 Normalization
3.3 Differential Gene Expression Analysis
3.4 Single Cell Trajectory Inference
3.5 Analysis of Single-Cell RNA-Seq Data Using Seurat
3.5.1 Generation of a Gene Expression Count Matrix for Seurat Analysis
Box 13 Download FASTQ files
Box 14 Install Seurat
Box 15 Load single cell gene expression data in Seurat
Box 16 Create SeuratObject
Box 17 Merge datasets
3.5.2 Quality Control and Filtering Cells
Box 18 Calculate mitochondrial quality control metrics
Box 19 Visualize QC metrics
Box 20 Data filtering using QC metrics
3.5.3 SCTransform Normalization and Integration of Multiple Datasets
Box 21 Create list of Seurat objects
Box 22 Data transformation using SCTransform
Box 23 Feature selection for Data Integration
Box 24 Perform data integration
3.5.4 Cell Clustering and Visualization
Box 25 Perform dimensional reduction
Box 26 Perform cell clustering
3.5.5 Differential Gene Expression Analysis
Box 27 Perform differential expression testing
Box 28 Sort DE results by log fold-change
Box 29 Visualize differential gene expression using Dot plot, Violin plot and Feature plot
Box 30 Assign cell type identity to clusters
Box 31 Subset data by cell type identity
4 Notes
References
Chapter 5: Profiling the Epigenetic Landscape of the Spermatogonial Stem Cell-Part 1: Epigenomics Assays
1 Introduction
2 Materials
2.1 Whole-Genome Bisulfite Sequencing (WGBS)
2.1.1 Reagents
2.1.2 Equipment
2.2 Chromatin Immunoprecipitation-Sequencing (ChiP-seq)
2.2.1 Reagents
2.2.2 Equipment
2.2.3 Buffers
2.3 Assay for Transposase-Accessible Chromatin-Sequencing (ATAC-seq)
2.3.1 Reagents
2.3.2 Equipment
2.3.3 Buffers
2.4 High-Throughput Chromosome Conformation Capture-Sequencing Hi-C-seq
2.4.1 Reagents
2.4.2 Equipment
2.4.3 Buffers
3 Methods
3.1 Gene Expression: RNA-seq
3.2 DNA Methylation: Whole-Genome Bisulfite Sequencing (WGBS)
3.2.1 Bisulfite Conversion of Genomic DNA
3.2.2 Amplification with the PrepAmp Primer
3.2.3 Purification with DNA Clean-Up (DCC) (Zymo D4033)
3.2.4 Library Amplification
3.2.5 Amplification with the Index Primer
3.2.6 Sequencing, Validation, and Quantification of the Library
3.3 Histone Modifications-ChIP-seq (Protocol Modified from Brind´Amour et al.)
3.3.1 Sample Collection by Flow Cytometry
3.3.2 Chromatin Preparation
3.3.3 Chromatin Immunoprecipitation
3.3.4 DNA Extraction
3.3.5 DNA Purification Using AMpPure XP Beads
3.3.6 Library Construction
3.3.6.1 End Repair of Fragmented DNA
3.3.6.2 A-Tailing
3.3.6.3 Adapter Ligation
3.3.6.4 Library Amplification
3.4 Chromatin Accessibility: ATAC-seq
3.4.1 Cell Lysis
3.4.2 Transposition
3.4.3 PCR Amplification (Library Generation)
3.4.4 Library Purification
3.4.5 Assessing library Quality
3.5 Three-Dimensional Interactions: Hi-C-seq
3.5.1 Crosslinking of Cells
3.5.2 Cell Lysis and Chromatin Digestion
3.5.3 Marking of DNA Ends with Biotin-14-DCTP
3.5.4 Sonication
3.5.5 Biotin Pull-Down and Preparation for Illumina Sequencing
3.5.6 Library Amplification and Post-PCR Size Selection
3.6 Integration of Transcriptomic + Epigenomic Profiling Data Using ChromHMM
3.6.1 Installation
3.6.2 Binarization
3.6.3 File Preparation for Enrichment Analyses
3.6.4 Model Learning
3.6.5 Post-Model-Learning Analysis
References
Chapter 6: Profiling the Epigenetic Landscape of the Spermatogonial Stem Cell: Part 2-Computational Analysis of Epigenomics Da...
1 Introduction
2 Materials
2.1 Hardware
2.2 Software
3 Methods
3.1 Analysis of WGBS Data
3.1.1 Prepare Genome for Bismark
3.1.2 Align Reads to Reference Genome
3.1.3 Bismark BAM/SAM Output (Default)
3.2 Analysis of ChIP-seq Data
3.2.1 Alignment
3.2.2 Peak Calling
3.2.3 Peak Annotation
3.2.4 Motif Analysis
3.3 Analysis of ATAC-seq Data
3.4 Analysis of Hi-C Data
3.5 Integration of Transcriptomic + Epigenomic Profiling Data Using ChromHMM
3.5.1 Installation
3.5.2 Binarization
3.5.3 File Preparation for Enrichment Analyses
3.5.4 Model Learning
3.5.5 Post-Model-Learning Analysis
References
Chapter 7: Primary Cultures of Spermatogonia and Testis Cells
1 Introduction
2 Materials
2.1 Preparation of Feeder Layers
2.2 Preparation of Testicular Single-Cell Suspension
2.3 SSC Enrichment
2.4 Culture of SSCs
3 Methods
3.1 Preparation of Feeder Layers
3.2 Isolation of Mouse SSCs
3.2.1 Preparation of Single-Cell Suspensions from Mouse Testis
3.2.2 Enrichment of Mouse SSCs
3.3 Isolation of Rat SSCs
3.3.1 Preparation of Rat Testis Single-Cell Suspensions
3.3.2 Enrichment of Rat SSCs
3.4 Culture of SSCs
3.4.1 Culture of Mouse SSCs
3.4.2 Culture of Rat SSCs
4 Notes
References
Chapter 8: Reconstitution of Human Prospermatogonial Development from Human-Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Animals
2.2 Human iPSCs (hiPSCs)
2.3 Equipment
2.4 Tools
2.5 Reagents (See Note 3)
3 Methods
3.1 Preparation of Mouse Gonadal Somatic Cells (mGSC)
3.2 hPGCLC Induction
3.3 Cell Surface Marker Staining
3.4 Sorting
3.5 xrTestis
3.6 Harvest of xrTestis for FACS Analysis/Sorting
3.7 Fixation of xrTestis for IF Analyses
3.8 Immunofluorescence Analysis
4 Notes
References
Chapter 9: RNA Interference as a Method of Gene Knockdown in Cultured Spermatogonia
1 Introduction
1.1 Spermatogonial Stem Cell Biology
1.2 The Physiology of RNAi
2 Materials
2.1 siRNA or shRNA Selection
2.2 Reagents
3 Methods
3.1 Preparation of Spermatogonia for RNAi
3.2 Preparation of Transfection Reagents, Transfection, and Collection of Spermatogonia for Downstream Analyses
3.3 Assessing Knockdown Efficiency
3.4 Assessing Altered SSC Function in Response to RNAi
3.5 In Vivo Approaches for RNAi in Spermatogonia
3.6 Considerations When Adopting RNAi Technology: Off-Target Effects
3.7 RNAi Versus Knockout Techniques: Competing or Complementary Approaches?
4 Notes
References
Chapter 10: Isolation of Undifferentiated Spermatogonia from Adult and Developing Mouse Testes
1 Introduction
2 Materials
2.1 Mouse Testis Cell Isolation
2.2 Pre-Enrichment of Spermatogonia (Adult Testis Only)
2.3 Preparation for FACS Isolation of ID4-EGFP+ Undifferentiated Spermatogonia
2.4 FACS Analysis
3 Methods
3.1 Mouse Testis Cell Isolation
3.1.1 Preparation
3.1.2 Procedure for Testis Cell Isolation from Adult Mouse Testes
3.1.3 Procedure for Testis Cell Isolation from P6 Mouse Testes
3.2 Pre-Enrichment of Spermatogonia (from Adult Testes Only)
3.2.1 Preparation
3.2.2 Procedure
3.3 Preparation for FACS Isolation of ID4-EGFP+ Undifferentiated Spermatogonia
3.3.1 Preparation
3.3.2 Procedure for Adult Cell Staining and Preparation for FACS
3.3.3 Procedure for P6 Cell Preparation for FACS
3.4 FACS Analysis
3.4.1 Preparation
3.4.2 Procedure
4 Notes
References
Chapter 11: Spermatogonial Transplantation
1 Introduction
2 Materials
2.1 Host Preparation
2.2 Donor Cell Preparation
2.3 Transplantation
3 Methods
3.1 Preparation of Busulfan
3.2 Preparation of Host Mice
3.3 Preparation of Donor Cells
3.4 Microinjection of Donor Cells: Transplantation
3.5 Analysis of Host Testes
4 Notes
References
Chapter 12: Viral Transduction of Mammalian Spermatogonial Stem Cells
1 Introduction
2 Materials
2.1 Lentivirus Production
2.2 Lentiviral Transduction of SSCs In Vitro
2.3 AAV Virus Production
2.4 AAV Transduction of SSC In Vivo
3 Methods
3.1 Lentivirus Production
3.2 Lentiviral Transduction of SSCs In Vitro
3.3 AAV Virus Production
3.3.1 Transfection
3.3.2 Virus Harvesting
3.3.3 Iodixanol Gradient Centrifugation Purification
3.3.4 Virus Dialysis and Concentration
3.3.5 Determination of Viral Titers
3.4 In Vivo Microinjection of AAV
4 Notes
References
Chapter 13: Pharmacological Treatment of Neonatal and Juvenile Mice to Study Spermatogenesis
1 Introduction
2 Materials
2.1 Feeding of Mice, Aged Postnatal Day (P)1-P13
2.2 Subcutaneous Injections of Mice, Aged P7-P13
2.3 Intraperitoneal (IP) Injections of Juvenile Mice (>P14)
3 Methods
3.1 Preparing the Feeding Needle and the Treatment Station
3.2 Oral Feeding of Neonatal (P1-P13)
3.3 Subcutaneous Injections of Neonatal Mice (P7-P13)
3.4 Intraperitoneal (IP) Injections of Juvenile Mice (>P14)
3.5 Validation of Feeding and Injections Results
4 Notes
References
Chapter 14: Models and Methods for Evaluating Regeneration of Spermatogenesis After Cytotoxic Treatments
1 Introduction
2 Materials
2.1 Radiation Treatments
2.2 Chemotherapy Treatments
2.3 Spermatogenic Recovery Analysis
2.4 Gonadotropin and Testosterone Suppression
3 Methods
3.1 Mammalian Models for Radiation Therapy
3.1.1 Mouse Model for Radiation Therapy
3.1.2 Rat Model for Radiation Therapy
3.1.3 Non-human Primate Model for Radiation Therapy
3.2 Mammalian Models for Chemo Therapy
3.2.1 Mouse Model for Busulfan
3.2.2 Mouse Model for Cyclophosphamide
3.2.3 Non-human Primate Model for Chemotherapy (Busulfan)
3.3 Analysis of Spermatogenic Recovery After Cytotoxic Treatment
3.3.1 Determination of Sizes and Densities of a Spermatogonial Clones
3.3.2 Counts of Repopulated Tubules in the Testis
3.3.3 Histological Quantification of Spermatogonial Germ Cells Relative to Sertoli Cells After Cytotoxic Treatment
3.3.4 Sample Preparation for Testicular Sperm Head Counts
3.3.5 Mouse Epididymal Sperm Count
3.3.6 Rhesus ``Swim-Up´´ Sperm Preparation from the Epididymis
3.3.7 Freezing Monkey Sperm for Cryopreservation
3.3.8 Semen Collection and Analysis in Monkeys Using a Rectal Probe
3.4 Gonadotropin and Testosterone Suppression Treatments
3.4.1 Treatment with a GnRH Antagonist Acyline (Rodents)
3.4.2 Silastic Capsule Implants for Steroid Delivery
4 Notes
References
Chapter 15: Defining Gene Function in Spermatogonial Stem Cells Through Conditional Knockout Approaches
1 Introduction
2 Materials
2.1 Non-inducible Cre-Driver Strains
2.2 Inducible Cre-Driver Strains
2.3 SSC Niche Targeting Cre-Driver Strains
2.4 In Vivo Tamoxifen Treatment
2.5 In Vitro 4-Hydroxytamoxifen Induction
3 Selection of Mouse Strains for Conditional Knockout in SSCs
3.1 Conditional Alleles
3.2 Strains Expressing Non-inducible Recombinase During Embryonic and Fetal Germ Cell Development
3.2.1 Vasa-Cre
3.2.2 Prdm1-Cre
3.2.3 Tnap-Cre
3.3 Strains Expressing Non-inducible Recombinase at Postnatal SSC Developmental Stages
3.3.1 Stra8-iCre
3.3.2 Ngn3-Cre
3.4 Strains Expressing Inducible Recombinase Active in Spermatogonia
3.4.1 UBC-CreERT2
3.4.2 ROSA26-CreERT2
3.4.3 Oct4-MerCreMer
3.4.4 Vasa-CreERT2
3.5 Strains Expressing Recombinase in SSC Niche Cell Populations
3.5.1 Sertoli Cell-Expressed Cre Strains
3.5.2 Cyp17-iCre
3.5.3 Cx3cr1-Cre
3.5.4 Myh11-Cre
4 Methods for Using Conditional Knockout Lines to Study SSCs
4.1 Breeding Strategies
4.1.1 Paternal Transmission of Non-inducible Cre
4.1.2 Maternal Transmission of Non-inducible Cre
4.1.3 Inducible Conditional Knockout Models
4.1.4 Considerations for Sex Chromosome-Linked Conditional Alleles
4.2 Treatment Regimens for Inducible Conditional Knockouts
4.2.1 Tamoxifen Preparation
4.2.2 Tamoxifen Treatment
4.3 In Vitro SSC Conditional Knockout
4.3.1 4-OHT Treatment
4.4 Confirming and Improving Conditional Knockout Efficiency
4.4.1 Genotyping
4.4.2 Confirming Conditional Knockout Efficiency Using RNA- and Protein-Based Methods
4.4.3 Improving Conditional Knockout Efficiency
4.5 Other Uses for Conditional Alleles in the Study of SSCs
5 Conclusions
References
Chapter 16: Lineage Tracing of Spermatogonial Stem Cells Within the Male Germline
1 Introduction
2 Materials
2.1 Cre Recombinases
2.2 Tamoxifen Administration
2.3 Reporter Constructs
2.4 Reagents
3 Methods
3.1 Prepare Tamoxifen/4-OHT Solutions
3.2 Tamoxifen/4-OHT Injection
3.3 Whole-Mount Immunofluorescence Analysis
3.4 LacZ Staining for β-Galactosidase Reporters
3.5 Quantifying Whole-Mount Tubules
3.6 Calculating Labeling Efficiency
4 Notes
References
Chapter 17: Perspectives: Approaches for Studying Livestock Spermatogonia
1 Testicular Anatomy and Spermatogenesis in Mammals
2 Molecular Biomarkers of Livestock Spermatogonia
3 Primary Cultures of Livestock Spermatogonia
4 Transplantation of Spermatogonia in Livestock
5 Conclusions and Future
References
Chapter 18: Perspectives: Methods for Evaluating Primate Spermatogonial Stem Cells
1 Introduction
1.1 SSCs and the SSC Niche
1.2 Spermatogenic Lineage Development and Clonal Arrangement
1.2.1 Rodents
1.2.2 Higher Primates
1.2.2.1 Primate SSC Proliferation Dynamics
2 Molecular Characteristics of Primate SSCs
2.1 Cell Sorting Techniques for Isolating Primate SSCs
3 Functional Evaluation of Primate SSCs
3.1 SSC Transplantation
3.2 In Vitro Culture of Primate SSCs
3.3 hSSC Transplantation as a Therapy for Male Infertility
4 Conclusion
References
Index