UCSF

Characterization of NANOS Expression and Function in Human Germ Cells

by

Vanessa Therese Angeles

ABSTRACT

Although errors during human germ cell development are the leading cause of birth defects and infertility, studies in this field are hindered by an inaccessibility of germ cells during fetal development. Thus, we implemented an in vitro system to differentiate human embryonic stem cells (hESCs) to the germ cell lineage. We evaluated the ability of in vitro-derived germ cells to erase genomic imprints as well as assessed the genetic dependency of germ cell development by investigating if the highly-conserved NANOS genes are required for human gametogenesis.

In hESCs, we observed mono-allelic expression of two imprinted genes, H19 and KCNQ1OT1, as well as a 1:1 ratio of methylated to unmethylated DNA at the H19 and PEG1 loci. Methylation assessment of in vitro-derived human germ cells revealed germ-cell specific hypomethylation at the H19 imprinted locus. This data suggests that hESC-derived germ cells have initiated erasure of genomic imprints, thereby recapitulating a landmark property of human germ cells in vitro.

We also assessed if human germ cell development is dependent on NANOS expression. We detected NANOS1, 2 and 3 mRNA and protein in human gonads, although NANOS1 was also detected in somatic tissues. We observed expression of NANOS3 in human germ cells throughout spermatogenesis and oogenesis, indicative of a germ cell function for NANOS3 in both sexes. Surprisingly, NANOS3 expression was highest in human germ cell nuclei, with the protein remaining bound to DNA during cellular division.

Functional analysis was also performed by knocking down NANOS3 expression in hESC-derived germ cells. We observed decreased expression of genes required for pluripotency, germ cell development and meiosis. NANOS1 and 2 expression was also reduced, implying a regulatory role for NANOS3 within the NANOS family. Moreover, we discovered decreased expression of the known germ cell factor DAZL, at both mRNA and protein levels, following knockdown of NANOS3 expression.

Together, this data demonstrates that human germ cells can be generated in vitro, and that NANOS3 modulates this fundamental process. These findings contribute to our understanding of the processes required for human germ cell development and likely have clinical applications, particularly in the field of human fertility studies.