Germline Development: Methods and Protocols (eng)

Part I: Male Germline

1. Isolation of Fetal Gonads from Embryos of Timed-Pregnant Mice for Morphological and Molecular Studies

Yunmin Li, Teruko Taketo, and Yun-Fai Chris Lau

2. Neonatal Testicular Gonocytes Isolation and Processing for Immunocytochemical Analysis

Gurpreet Manku, Monty Mazer, and Martine Culty

3. Isolation of Undifferentiated and Early Differentiating Type A Spermatogonia from Pou5f1-GFP Reporter Mice

Thomas Garcia and Marie-Claude Hofmann

4. Isolation of Human Male Germline Stem Cells Using Enzymatic Digestion and Magnetic-Activated Cell Sorting

Zuping He, Maria Kokkinaki, Jiji Jiang, Wenxian Zeng, Ina Dobrinski, and Martin Dym

5. Isolation and Purification of Murine Male Germ Cells

Catherine Boucheron and Vanessa Baxendale

6. Preparation of Enriched Mouse Syncitia-Free Pachytene Spermatocyte Cell Suspensions

Yan Wang and Martine Culty

7. Revealing Transcriptome Landscape of Mouse Spermatogonial Cells by Tiling Microarray

Tin-Lap Lee, Owen M. Rennert, and Wai-Yee Chan

8. Biochemical Characterization of a Testis-Predominant Isoform of N-Alpha Acetyltransferase

Alan Lap-Yin Pang

9. Identification of Novel Long Non-Coding RNA Transcripts in Male Germ Cells

Tin-Lap Lee, Amy Xiao, and Owen M. Rennert

10. Methylation Profiling Using Methylated DNA Immunoprecipitation and Tiling Array Hybridization

Hoi-Hung Cheung, Tin-Lap Lee, Owen M. Rennert, and Wai-Yee Chan

11. Spermatogenesis in Cryptorchidism

Alexander I. Agoulnik, Zaohua Huang, and Lydia Ferguson

Part II: Female Germline

12. In Vitro Culture of Fetal Ovaries: A Model to Study Factors Regulating Early Follicular Development

Shyamal K. Roy, Cheng Wang, Anindit Mukherjee, and Prabuddha Chakraborty

13. Microspread Oocyte Preparations for the Analysis of Meiotic Prophase Progression with Improved Recovery by Cytospin Centrifugation

Teruko Taketo

14. In Vitro Maturation (IVM) of Porcine Oocytes

Ye Yuan and Rebecca L. Krisher

15. Experimental Approaches to the Study of Human Primordial Germ Cells

Andrew J. Childs and Richard A. Anderson

16. Investigating the Origins of Somatic Cell Populations in the Perinatal Mouse Ovaries Using Genetic Lineage Tracing and Immunohistochemistry

Chang Liu, Melissa Paczkowski, Manal Othman, and Humphrey Hung-Chang Yao

17. DNA Methylation Analysis of Germ Cells by Using Bisulfite-Based Sequencing Methods

Hisato Kobayashi and Tomohiro Kono

18. Gene Expression in Mouse Oocytes by RNA-Seq

Eric Antoniou and Robert Taft

The germline is unique in mammals as it is the only cell lineage that undergoes mitosis, meiosis, and differentiation, making these cells amenable to in-depth genomic and mechanistic studies. In Germline Development: Methods and Protocols, expert researchers provide a wide-ranging collection of techniques that can be applied to the study of both the male and female germline. As the techniques used for isolating and studying male and female germ cells are slightly dissimilar, the different technologies are grouped under two sections in this volume, namely, male germline and female germline. This collection brings the field of germline developmental studies together such that readers can find, in a single volume, techniques that allow the isolation of gonads with germ cells at different stages of development, the study of the genomics and epigenomics of germ cells, and an examination of the role of single genes in the developmental potential and function of germ cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and the avoidance of known pitfalls.

Comprehensive and cutting-edge, Germline Development: Methods and Protocols serves as a comprehensive text to individuals, particularly researchers in reproductive and developmental biology, who are interested in studying cells of the germ lineage.