Date of Award
Spring 2006
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Downs, Stephen M.
Second Advisor
Blumenthal, Edward
Third Advisor
Mynlieff, Michelle
Abstract
The present study examines the effects of heat pulsing on oocyte maturation and assesses the possible role of the stress activated enzyme, AMP-activated protein kinase (AMPK), during heat stress in the oocyte. Denuded oocytes (DO) from immature, eCGprimed mice were pulsed at increasing temperatures for 30 minutes in dbcAMPcontaining medium and put into the control bath for a total culture time of 17-18 h. Oocytes exposed to 42°C showed a significant increase in maturation. An extended pulse time course showed that a 60 minute pulse at 42°C resulted in optimal GVB. To ensure this heat pulse was not an artifact of dbcAMP-arrested DO, both DO and cumulus-cell enclosed oocytes (CEO) were arrested in three different meiotic inhibitors: dbcAMP, IBMX, and hypoxanthine. The heat treated DO and CEO in all three inhibitors showed a significant increase in maturation percentages compared to the controls, suggesting heat is acting to induce GVB in meiotically-arrested oocytes and that the cumulus cells are not protecting the oocyte from the heat-stimulated maturation. The heat pulse did not affect the progression to MIi as observed by polar body formation and chromosomal staining. The AMPK inhibitors, compound C and araA, both blocked the meiosis-stimulating effects of the heat pulse. Western blots showed that acetyl-CoA carboxylase (ACC), an important substrate of AMPK, was phosphorylated in heat-treated oocytes before GVB, supporting a role for AMPK in heat-induced maturation. The MEK inhibitor, PD98059, also prevented stress-induced maturation, but western analysis revealed that ERKl/2 did not become phosphorylated, and thus activated until after GVB, supporting our previous findings that PD98059 can suppress meiosis in fashion unrelated to MAPK activation. The stress activated member of the MAPK family, p38, is not found to be phosphorylated, and therefore activated, with heat treatment; and only high concentrations of the p38 inhibitor, SB203580, are able to block heat-stimulated maturation, suggesting p38 is not involved. However, the JNK MAPK protein does become activated with a heat pulse, and the JNK inhibitor, SP600125, blocks heatinduced maturation, suggesting JNK could be an important protein involved in the induction of maturation caused from a heat pulse. Spontaneous maturation (3h culture time) was blocked by pulsing with heat; however, the heat-treated groups were able to undergo spontaneous maturation if cultured for 17-18 h, suggesting the heat pulse slows down spontaneous resumption of meiosis but does not block it. Polar body formation in DO undergoing spontaneous maturation was also delayed with the heat pulse. Together, these data show that heat pulsing stimulates GVB in meiotically-arrested DO and CEO in vitro and suggest this effect is mediated through the activation of AMPK.