Making Germ Cells from Human Embryonic Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 74

DAZ gene with human male infertility, it is of note that overexpression of DAZ in
the male line resulted in the greatest numbers of cells (more than 20%) reaching the
meiotic leptotene stage at day 7 and 14.
Finally, it was determined whether haploid gametes were produced in the
male ES cell line overexpressing all three genes. ACROSIN and TEKT1 , markers
of late spermatogenesis and mature sperm, were expressed at high levels by day
14. Additionally, FACS analysis to sort cells by DNA content using human
semen as a positive control showed that approximately 2% of ES-derived germ
cells overexpressing DAZ , DAZL , and BOULE were haploid (1N). Of these cells,
many stained positively for the sperm marker ACROSIN. The majority of cells
in the 1N population indeed had a haploid chromosome complement as deter-
mined by fluorescence in situ hybridization (FISH). It was noted that these 1N
cells were spherical or elliptical in morphology, lacked flagella, and were closest
in resemblance to round spermatids. Notably, with the overexpression of the
three genes, BMPs were not necessary to induce meiotic progression, suggesting
that DAZ , DAZL , and BOULE are intrinsic factors involved in this process and
may function downstream of the BMP signaling pathway. Taken together, these
results highlight the roles of the DAZ family genes in human germ cell develop-
ment and that each of the three genes may have distinct roles in this process and
sex-specific differences. DAZL may play a greater role in human primordial
germ cell formation, whereas DAZ and BOULE may modulate later stages of
gametogenesis including meiotic progression and development of haploid
gametes.
Other investigations in our laboratory have interrogated the genetic require-
ments of Dazl in mouse germ cell development both in vitro and in vivo (Haston
and Reijo Pera, unpublished). A double transgenic cross was used to produce mice
and mESC lines with genetic ablation of Dazl (Ruggiu et al. 1997 ), which also
contained a transgene expressing GFP from a germ cell-specific Oct4 promotor,
D PE-Oct4-GFP (Palmieri et al. 1994 ), described above in Sect. 3.4 . This germ cell-
specific DPE-Oct4-GFP was used to determine the effects of knocking out Dazl on
the development of germ cells produced both in vivo in mouse embryos and
in vitro from mouse ES cells. FACS analysis was used to isolate germ cells and
quantitatively characterize the loss of germ cells in Dazl null embryonic gonads
and mESC lines. The isolated Oct4-GFP-positive cells were utilized to determine
gene expression and imprinting status of these Dazl null germ cells. It was found
that Dazl null animals have significantly reduced primordial germ cell numbers
during embryonic development. Further, these isolated GFP-expressing PGCs
show sex-specific aberrant gene expression of pre-meiotic and meiotic germ cell
markers and failure to erase methylation at imprinted loci and reestablish sex-
specific methylation patterns, and they are unable to generate pluripotent EG cell
lines. Moreover, the mESCs generated from these mice lacking the Dazl gene have
a reduced number of GFP-positive germ cells produced in vitro and additional
defects in gene expression and methylation patterns (Haston and Reijo Pera,
unpublished). Therefore, the Dazl gene likely plays a key role in the development
of germ cells in both mice and man.