The leading cause of infertility in men and women is quantitative and qualitative defects in human germ cell (oocyte and sperm) development. Yet, it has not been possible to examine the unique developmental genetics of human germ cell formation and differentiation due to inaccessibility of germ cells during fetal development. Although several studies have shown that germ cells can be differentiated from mouse and human embryonic stem cells, human germ cells differentiated in these studies generally did not develop beyond the earliest stages1-8. Here we used a germ cell reporter to quantitate and isolate primordial germ cells derived from both male and female hESCs. Then, by silencing and overexpressing genes that encode germ cell-specific cytoplasmic RNA-binding proteins (not transcription factors), we modulated human germ cell formation and developmental progression. We observed that human DAZL (Deleted in AZoospermia-Like) functions in primordial germ cell formation, whereas closely-related genes, DAZ and BOULE, promote later stages of meiosis and development of haploid gametes. These results are significant to the generation of gametes for future basic science and potential clinical applications.

Fission yeast mutants defective in DNA replication have widely varying morphological phenotypes. We designed a screen for temperature-sensitive mutants defective in the process of replication regardless of morphology by isolating strains unable to rereplicate their DNA in the absence of cyclin B (Cdc13). Of the 42 rereplication-defective mutants analyzed, we were able to clone complementing plasmids for 10. This screen identified new alleles of the APC subunit cut9 ϩ , the initiation/checkpoint factor rad4 ϩ /cut5 ϩ , and the first mutant allele of psf2 ϩ , a subunit of the novel GINS replication complex. Other genes identified are likely to play general roles in gene expression and protein localization.

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INTRODUCTIONThe genomic imprinting mechanism regulates differential expression of paternally and maternally derived genes. During development, primordial germ cells (PGCs) have a unique methylation pattern which undergoes demethylation, both globally and at imprinted loci. Later, the methylation status of imprinted genes is re-established during gametogenesis by de novo methylation. Errors in the methylation patterns of imprinted genes have been associated with syndromes such as Beckwith-Wiedemann, Angelman, and Prader-Willi in humans. Human embryonic stem cells (hESCs) have unique methylation patterns compared to somatic stem cells and cancer cells. The status of allelic expression or methylation of human imprinted genes can be studied in detail in human embryos/blastocysts and hESCs to understand early development and differentiation. This protocol describes the bisulfite genomic sequencing method for analysis of methylation patterns of imprinted loci in human embryonic stem cells. Multiple imprinted loci can be analyzed by performing the following protocol with gene-specific primers.

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