Germline Development: Methods and Protocols (Methods in Molecular Biology, v825)

825_Germline development_Cover_9781617794353_161779435X......Page 1
FrontMatter......Page 2
Germline Development......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 10
Part I_Part I: Male Germline......Page 14
1. Introduction......Page 16
2. Materials......Page 18
3.2. Isolation of Gonads from Embryos at 12.5 dpc or Later......Page 19
3.3. Isolation of Gonads from Embryos Younger than 12.5 dpc......Page 21
3.5. Examples of Studies......Page 22
References......Page 27
1. Introduction......Page 30
2.1. Tissue Dissociation and Cell Isolation......Page 31
2.3. Cytospin and Immunocytochemistry......Page 32
3.1. Tissue Collection......Page 33
3.2. Tissue Dissociation......Page 34
3.3. Overnight Adhesion......Page 35
3.4. Cell Separation on a BSA Gradient......Page 36
3.6. Cell Fixation and Cytospin Centrifugation......Page 37
3.7. Immunocyto- chemical Analysis up to First Antibody Reaction......Page 38
3.8. Fluorescent Immunocyto- chemical Analysis......Page 39
3.9. Colorimetric Immunocytochemical Analysis......Page 40
4. Notes......Page 41
References......Page 42
1. Introduction......Page 44
1.2. In Vitro Use......Page 46
2.2. Buffers and Enzyme Solutions......Page 49
3. Methods......Page 50
3.1.3. Tubule Fragmentation and Cleanup (FACS Isolation of Pou5f1+ Cells Only)......Page 51
3.2.1. Tubule Fragmentation and Cleanup (FACS Isolation of Pou5f1+ Cells Only)......Page 52
3.2.4. FACS Controls......Page 53
4. Notes......Page 54
References......Page 56
1. Introduction......Page 58
2.2. Enzymatic Digestion of Human Testis Tissues......Page 60
2.4. Isolation of Human GPR125- and GFRA1-Positive Spermatogonia by MACS......Page 62
3.2. Enzymatic Digestion of Human Testis Tissue......Page 64
3.4. Isolation of Human GPR125- and GFRA1-Positive Spermatogonia by MACS......Page 67
4. Notes......Page 68
References......Page 69
1. Introduction......Page 72
2.3. Cell Identification and Counting......Page 73
3.1. Testis Isolation......Page 74
3.2. Tissue Digestion......Page 75
3.3. BSA Gradient......Page 76
3.4. Cell Identification and Counting......Page 77
4. Notes......Page 78
References......Page 79
1. Introduction......Page 80
2.1. Tissue Dissociation and Cell Isolation......Page 81
2.3. Percoll Gradient......Page 82
3.2. Tissue Dissociation......Page 83
3.3. Cell Separation on a BSA Gradient......Page 84
3.4. Pachytene Spermatocyte Enrichment on Percoll Gradient......Page 85
4. Notes......Page 86
References......Page 87
1. Introduction......Page 88
2.4. Synthesis, Amplification, Labeling, Cleanup, and Fragmentation of cDNA and cRNA......Page 91
2.8. Software......Page 92
3.1. Enriching mRNA Fraction from Total RNA Using RiboMinus Kit......Page 93
3.3. First-Cycle, Double-Stranded cDNA Synthesis......Page 94
3.4. In Vitro Transcription Amplification......Page 95
3.5. Second-Cycle, Double-Strand cDNA Synthesis......Page 96
3.7. Hybridization and Scanning......Page 98
3.8.3. Intensity Analysis......Page 99
3.8.4. Assessing Intensity Data......Page 100
3.8.5. Calculate Intervals......Page 101
4. Notes......Page 102
References......Page 105
1. Introduction......Page 106
2.2. Cell Culture and Transfection......Page 107
2.5. Western Blotting......Page 108
3.1. Preparation of Expression Vectors......Page 109
3.2. Cell Transfection for Co-immuno- precipitation......Page 110
3.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis......Page 111
3.4. Western Blotting......Page 112
3.5. Stripping and Reprobing Protein Blots......Page 114
3.6. NAT Assay......Page 115
4. Notes......Page 116
References......Page 117
1. Introduction......Page 118
2.3. Quantitative Real-Time PCR Validation......Page 119
2.5. Bioinformatics Tools and Databases......Page 120
3.2. Identification of Spermatid-Specific SAGE Tags from GermSAGE......Page 121
3.3. Validate Stage-Specific Expression by Quantitative Real-Time PCR......Page 122
3.4. Confirmation of Predicted ORF by Northern Blots......Page 123
4. Notes......Page 125
References......Page 127
1. Introduction......Page 128
2.2. MeDIP......Page 129
3.1. Extraction of Genomic DNA......Page 130
3.2. Sonication of Genomic DNA......Page 131
3.3. MeDIP......Page 132
3.4.1. Random Priming......Page 133
3.4.2. PCR Amplification......Page 134
3.6. Assessment of Amplified Product by qPCR......Page 135
3.7.1. Fragmentation of Amplified DNA......Page 136
3.7.3. Hybridization of Labeled DNA on Tiling Arrays......Page 137
4. Notes......Page 138
References......Page 139
1. Introduction......Page 140
2.1. Epidemiology......Page 141
2.2. Environmental Risk Factors......Page 142
3.1. Two Phases of Testicular Descent......Page 144
4.1. Cryptorchidism and Infertility......Page 146
4.2. Apoptosis in Cryptorchidism......Page 147
4.3. Somatic Cell Function in Cryptorchid Testis......Page 151
4.4. Gene Expression Studies in Cryptorchidism......Page 152
5. Cryptorchidism and Testicular Cancer......Page 153
6. Future Prospective......Page 155
References......Page 156
Part II: Female Germline......Page 162
1. Introduction......Page 164
2.1. Ovary Collection......Page 166
2.2.2. Complete Culture Medium (B)......Page 168
3.1. Preparation of the Base Culture Medium (A)......Page 169
3.3. Preparation of GPR30 siRNA......Page 170
3.4.2. Collection of Fetal Ovaries......Page 171
3.4.3. Culture of Fetal Ovaries......Page 172
3.4.4. Use of GPR30 siRNA in Culture......Page 173
3.4.5. Culture Termination and Processing of Ovaries......Page 175
3.5. Image Acquisition, Identification of Primordial Follicles in the Image, and Analysis......Page 177
3.6. Staging Developing Follicles in Adult Ovaries......Page 178
4. Notes......Page 180
References......Page 183
1. Introduction......Page 186
2.1. Dissociation of Ovarian Cells......Page 188
2.2. Microspread Nuclei Preparations......Page 189
3.2. Microspread Nuclei Preparations......Page 190
3.3. Dissociated Whole Cell Preparations......Page 191
4. Notes......Page 192
References......Page 194
1. Introduction......Page 196
2.1.1. Synthetic Oviductal Fluid (SOF)- 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid (HEPES) Bench Medium ( 11, 12)......Page 197
2.1.3. Purdue Porcine Medium (PPM) for Maturation ( 17)......Page 198
2.1.4. NCSU23 Maturation Medium ( 24)......Page 199
3.1.1. Medium Preparation......Page 200
3.1.3. Aspiration......Page 201
3.1.4. Oocyte Searching and Initiation of Maturation (Fig.  2) ( See Note 19)......Page 202
3.3.1. Denuding Oocytes......Page 204
3.3.2. Assessment of Meiotic Stage......Page 205
4. Notes......Page 206
References......Page 209
1. Introduction......Page 212
2.1. Dissection, Tissue Collection and Cell and Organ Culture......Page 215
2.3. Immunohis­tochemical Detection of Germ Cells......Page 216
3.1. Culture of Human First Trimester Fetal Ovaries......Page 217
3.2. Determination of Fetal Sex by PCR for SRY......Page 218
3.3. Immunohis­tochemical Detection of Germ Cells......Page 219
3.4. Stereological Determination of Germ Cell Number......Page 220
4. Notes......Page 221
References......Page 222
1. Introduction......Page 224
2.2. Tamoxifen Stock Solution......Page 226
2.4. Organ Culture......Page 227
3.1. Breeding of Animals and Tamoxifen Treatment......Page 228
3.3. Genotyping ( see Note 9)......Page 229
3.4. Immunohisto- chemistry ( see Note 12)......Page 230
4. Notes......Page 232
References......Page 233
1. Introduction......Page 236
2.2. Bisulfite Sequencing by the Sanger Dideoxy Method......Page 240
2.3. Bisulfite Shotgun Sequencing......Page 241
3.1. Bisulfite Sanger Sequencing......Page 242
3.2. High-Throughput Bisulfite Sequencing......Page 243
4. Notes......Page 245
References......Page 247
1. Introduction......Page 250
2.1. For Oocytes Isolation......Page 251
3.1.1. Oocyte Collection......Page 252
3.1.2. Removal of Cumulus Cells and Zonae......Page 253
3.1.3. RNA Extraction (Qiagen RNeasy Micro Kit)......Page 254
3.2.2. Second-Strand cDNA Synthesis......Page 255
3.2.5. Post-SPIA Amplification Modifications......Page 256
3.4. Making the Sequencing Library......Page 257
3.6. Agilent Bioanalyzer......Page 259
4. Notes......Page 260
References......Page 263
INDEX......Page 266