Abstracts - Society for Developmental Biology

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concentrations of glucose produced in different cell types changes in the synthesis and degradation of the ECM, because it
induces the formation of reactive oxygen species (ROS) and advanced glycation end products (AGEs) that alter the
expression of matrix metalloproteinases, therefore the effect of glucose on the expression of MMP-9 and TIMP-1 in
cultured blastocysts were evaluated and compared with the action of H2O2. Gestation day fourth blastocysts were cultured
in HAM-F10 and glucose 25 mM, orH2O2 10 μM were added in different schedules, glucose 6 mM was used as a control.
mRNAs of Mmp9 and Timp1 were measured using real time RT-PCR, MMP9 levels were analyzed by zymography in
SDS-PAGE-gels co-polymerized with gelatin. Both, glucose 25 mM or H2O210 μM induce higher levels of MMP9
protein and its mRNA, together with 85% lower concentration of Timp1 mRNA. In presence of high glucose or H2O2
aggregates of biggest giant trophoblast cells were observed. High glucose favored the development of trophoblast giant
cells, which invasiveness could be increased, since higher expression of MMP9, and lower expression of its inhibitor were
observed. The increased expression of MMP9 may be due to oxidative stress caused by glucose. Supported by PAPIT,
DGAPA, UNAM, grant IN230611.
Program/Abstract # 333
Effect of high glucose concentration on reactive oxygen species and the expression of urokinase plasminogen
activator and its inhibitor PAI-1 in cultured mouse blastocyst.
Sánchez Santos, Alejandra, FES Iztacala, UNAM, Tlalnepantla, Mexico; Martínez Hernández, María Guadalupe (FES
Iztacala, UNAM, Tlalnepantla, Mexico); Contreras Ramos, Alejandra (Laboratorio de Biología del Desarrollo y
Teratogénesis Experimental, Hospital Infantil de México, Federico Gómez., México, DF, Mexico); Ortega Camarillo,
Clara (Unidad de Investigación Médica en Bioquímica, Hospital de Especialidades, Centro Médico Nacional Siglo XXI,
IMSS, México, DF, Mexico); Baiza-Gutman, Luis Arturo (FES Iztacala, UNAM, Tlalnepantla, Estado de México, Mexico)
During embryo implantation, the blastocyst penetrates the uterine wall by an invasive process, involving proteases that
degrade the extracellular matrix (ECM), including urokinase plasminogen activator (PLAU), that catalyzes the formation
of plasmin, starting a proteolytic cascade that contributes to the breakdown of ECM, this enzyme is regulated by the
specific plasminogen activator inhibitor type 1(PAI-1). High concentrations of glucose induce chemical changes of
functional macromolecules (oxidation, formation of AGEs), inducing oxidative stress andmetabolic alterations that lead to
changes in gene expression. Reactive oxygenspecies (ROS) may regulate the activity of transcription factors such as
nuclear factor κβ (NF-κβ). Promoter region of PLAU contains an NF-κβ binding element, which is sensitive to redox
changes, therefore the effect of glucose on ROS levels and expression of PLAU and PAI-1 in cultured blastocysts were
evaluated and compared with the action of H2O2. Gestation day fourth blastocysts were cultured in HAM-F10 and glucose
25 mM, or H2O210 μM were added indifferent schedules, glucose 6 mM was used as a control, ROS, Plauand Pai1
mRNA and PLAU activity were evaluated using 2’-7’-dichloro dihydroflurescein, real time RT-PCR and an
amidolyticassay. Glucose 25 mM induces an increase of ROS, higher levels of Plau and Pai1 mRNA, higher activity of
PLAU and groups of biggest giant trophoblast cells. H2O2 induce similar changes, except that, Pai1 mRNA was
decreased. ROSS induced by high glucose favored formation of giant cells and PLAU expression, but the induction of
PAI1 by high glucose can be dependent on another mechanism, probably mediated by AGEs. Supported by PAPIT,
DGAPA, UNAM, grant IN230611.
Program/Abstract # 334
The role of Lkb1 in the control of cell polarity and epithelial morphogenesis in the pre-implantation mouse embryo
Krawchuk, Dayana; Yamanaka, Yojiro, McGill University Human Genetics, Montreal, Canada
The establishment of cell polarity and the orchestration of epithelial morphogenesis are critical processes in the formation
of the first lineages of the mouse embryo: epiblast (EPI), primitive endoderm (PE) and trophectoderm (TE). Lkb1, a
serine-threonine kinase and tumor suppressor, is involved in the establishmentof cell and epithelial polarity from worms to
humans. Whereas null zygotic Lkb1 mutants die at E9.0 with no obvious polarity defects, we hypothesize that the maternal
supply of Lkb1 in the oocytehas masked important roles it plays in the pre-implantation embryo. We generated maternalzygotic
(MZ) Lkb1 mutants by conditional excision with an oocyte-specific Cre line (Zp3-Cre), and analyzed cell
morphology, polarity and lineage in peri-implantation embryos. Time-lapse imaging showed numerous extruded cells from
the different cell layers of MZLkb1 embryos, which continued to divide, often formed trophoblast-like vesicles, and
occasionally migrated away from the embryo. Cell lineage markers confirmed that these cells could have TE, PE orEPI
identities. Furthermore, cell polarity markers showed that the extruded cells were not correctly polarized. Besides extruded
cells, we also observed some ICM cells occasionally nestled in the Cdx2-positive TE epithelium, and increased spreading
and flattening of the PE epithelium lining the blastocoel. Significantly, we found that embryos lacking maternal Lkb1, but
having paternal Lkb1, exhibited similar phenotypes but to a lesser frequency and severity. Together, these results suggest