| dc.description.abstract | In vitro gamete derivation from stem cells has potential applications in animal
reproduction as an alternative method for the dissemination of elite animal
genetics, production of transgenic animals, and conservation of endangered species.
Mesenchymal stemcells (MSCs)may be suitable candidates for in vitro gamete derivation
considering their differentiative capacity and their potential for cell therapy. Due to its
relevance in gametogenesis, it has been reported that retinoic acid (RA) and bone
morphogenetic protein (BMP) 4 are able to upregulate the expression of specific markers
associated to the early stages of germ cell (GCs) differentiation in bovine fetal MSCs
(bfMSCs). In the present study, we used polycistronic vectors containing combinations
of GC genes DAZL, STRA8, and BOULE followed by exposure to BMP4 or RA to induce
GC differentiation of bovine fetal adipose tissue-derived MSC (AT-MSCs). Cells samples
at Day 14 were analyzed according to the expression of pluripotent genes NANOG and
OCT4 and GC genes DAZL, STRA8, BOULE, PIWI, c-KIT, and FRAGILIS using Q-PCR.
Fetal and adult testis and AT-MSCs samples were also analyzed for the expression
of DAZL, STRA8, and NANOG using immunofluorescence. Increased gene expression
levels in the adult testis and cell-specific distribution of DAZL, STRA8, and NANOG in
the fetal testis suggest that these markers are important components of the regulatory
network that control the in vivo differentiation of bovine GCs. Overexpression of DAZL
and STRA8 in bi-cistronic and DAZL, STRA8, and BOULE in tri-cistronic vectors resulted
in the upregulation of OCT4, NANOG, and PIWIL2 in bovine fetal AT-MSCs. While BMP4
repressed NANOG expression, this treatment increased DAZL and c-KIT and activated
FRAGILIS expression in bovine fetal AT-MSCs. Treatment with RA for 14 days increased
the expression of DAZL and FRAGILIS and maintained the mRNA levels of STRA8 in
bovine fetal AT-MSCs transfected with bi-cistronic and tri-cistronic vectors. Moreover,
RA treatment repressed the expression of OCT4 and NANOG in these cells. Thus,
overexpression of DAZL, STRA8, and BOULE induced the upregulation of the pluripotent markers and PIWIL2 in transfected bovine fetal AT-MSCs. The partial activation of GC
gene expression by BMP4 and RA suggests that both factors possess common targets
but induce different gene expression effects during GC differentiation in overexpressing
bovine fetal AT-MSCs. | |
