PARENT SESSION
DEVELOPMENT OF THE EMBRYO
Harborside C
7:30 AM-10:00 AM

(144) INHIBITION OF GLYCOGEN SYNTHASE KINASE-3 ACTIVITY IS DETRIMENTAL TO MOUSE EARLY PRE-IMPLANTATION EMBYRO DEVELOPMENT.

Acevedo, Nicole^1,4, Swain, Jason^2,4, Dunn, Rodney³, Smith, Gary^1,2,3,4, ¹ Department of Physiology, Ann Arbor, MI⁴ Reproductive Sciences Program, Ann Arbor, MI² Department of OB/GYN, Ann Arbor, MI³ Department of Urology, Ann Arbor, MI

ABSTRACT- Lithium, the most prescribed antimanic agent, increases fetal developmental abnormalities. Lithium chloride (LiCl) regulates cellular events by inhibition of inositol monophosphatase (IMPase) or the serine/threonine protein kinase, glycogen synthase kinase-3 (GSK-3). We hypothesize that GSK-3 is expressed in mammalian embryos and continual exposure to LiCl compromises development by inhibition of GSK-3. Objectives were to identify and characterize GSK-3 in mouse pre-implantation embryos, to determine if continual LiCl exposure compromises embryo development, and to ascertain if effects of LiCl are caused by GSK-3 and/or IMPase inhibition. Western blot analysis showed GSK-3 and isoform expression in 1-cell embryos and blastocysts. One-cell embryos contain ~2X more compared to , whereas blastocysts have equivalent levels of each isoform. One-cell embryos were cultured for 72h in KSOM + 3% BSA (KSOM) alone or with 1,10, or 20 mM LiCl and examined daily microscopically. Logistic regression analysis indicated no significant treatment differences in 24h degeneration or cleavage rates. Embryo development was compromised significantly (P<0.001), in a dose-dependent manner, at 48h (>2-cell: KSOM-62%; 1 mM LiCl-33%; 10 mM LiCl-19%; 20 mM LiCl-0%) and 72h (>6-cell: KSOM-72%; 1 mM LiCl-35%; 10 mM LiCl-0%; 20 mM LiCl-0%). Embryos in 20 mM LiCl that failed to cleave past the 2-cell stage did not degenerate. To assess if repression of development by LiCl is due to GSK-3 or IMPase inhibition, 1-cell embryos were cultured for 72h in KSOM alone, or with 20mM LiCl, 20 mM KCl (molarity control), or 20 M L,690,330 (specific IMPase inhibitor). Results were analyzed using Fisher's Exact Test. At 24h embryos cultured in 20 mM LiCl had significantly reduced (P<0.001) cleavage rates (42%) compared to embryos exposed to KSOM (86%), KCl (73%), or L,690,330 (74%). Again, LiCl treatment blocked development at the 1- to 2-cell stage. By 48 and 72h of culture significantly (P<0.001) fewer embryos developed (>2- and >6-cell stages, respectively) in LiCl (0%; 0%) compared to KSOM (75%; 77%), KCl (58%; 52%), and L690,330 (61%; 67%). Results indicate that continual exposure to 20 mM LiCl blocks development of mouse embryos at the 1- or 2-cell stage without causing degeneration. This inhibitory effect of LiCl appears to act through GSK-3.

KEY WORDS: preimplantation embryo, glycogen synthase kinase-3, lithium chloride, inositol monophosphatase