No abstract

Sperm-induced activation of mammalian eggs is associated with a transient increase in Ca2+ concentrations thought to be derived from inositol 1,4,5-trisphosphate-sensitive and -insensitive intracellular stores. Whereas the importance of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores has been evaluated, the identity and role of inositol 1,4,5-trisphosphate-insensitive stores are poorly understood. To explore the role of the ryanodine-sensitive Ca2+ store, we first used reverse transcription-polymerase chain reaction to identify transcripts of the ryanodine receptor in eggs and determined that transcripts for the type 2 and 3 receptor were present. Immunoprecipitation of radioiodinated egg extracts with an antibody that recognizes both type 2 and 3 receptors detected specifically a band of Mr = 520,000. Immunolocalization of the receptor(s) using laser-scanning confocal microscopy revealed that the receptor(s) was uniformly distributed in the cortex of the germinal vesicle-intact oocyte, but became asymmetrically localized to the cortex in a region apposed to the meiotic spindle in the metaphase II-arrested egg; this asymmetrical localization developed by metaphase I. The role of the ryanodine receptor in mouse egg activation was examined by determining the effects of microinjected ryanodine or cyclic ADP ribose on endpoints of egg activation in either uninseminated or inseminated eggs. Ryanodine induced the conversion of the zona pellucida glycoprotein ZP2 to its postfertilization form ZP2f in a biphasic concentration-dependent manner; nanomolar concentrations stimulated this conversion, whereas micromolar concentrations had no stimulatory effect. Cyclic ADP ribose also promoted the ZP2 conversion, but with a hyperbolic concentration dependence. Neither of these compounds induced cell cycle resumption. Inhibiting the inositol 1,4,5-trisphosphate-sensitive Ca2+ store did not inhibit the ryanodine-induced ZP2 conversion and, reciprocally, inhibiting the ryanodine-sensitive Ca2+ store did not inhibit the inositol 1,4,5-trisphosphate-induced ZP2 conversion. Last, treatment of eggs under conditions that would block the release of Ca2+ from the ryanodine-sensitive store had no effect on any event of egg activation following fertilization. Results of these experiments suggest that although ryanodine receptors are present and functional, release of Ca2+ from this store is not essential for sperm-induced egg activation.

Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms. We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation. Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated. Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation. Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation. Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm. Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation. Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm. Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation. In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage. These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

Protein expression in human umbilical vein endothelial cells (HUVECs) is a useful indicator of maternal condition and the intrauterine environment during pregnancy. Therefore, we investigated protein expression in HUVECs obtained from patients with gestational diabetes mellitus (GDM). HUVECs were prepared from the umbilical cords of GDM patients and controls who underwent planned cesarean section between 2013 and 2014 at Teikyo University Hospital (Tokyo, Japan). There were no differences in blood glucose levels between the GDM patients and controls at admission. However, pre-pregnancy body mass index (BMI) was higher in GDM patients, although the changes in gestational BMI were smaller during hospitalization. To evaluate the state of the endothelium, we examined the protein expression levels of vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, thrombomodulin (TM), endothelial nitric oxide synthase, plasminogen activator inhibitor-1 (PAI-1), cyclooxygenase-2 (COX-2), and VE-cadherin, which are altered by various factors in endothelial tissue. VCAM-1, PAI-1, and COX-2 expression was higher in HUVECs from patients with GDM than the controls. Because the pre-pregnancy BMI was higher in GDM patients, we examined the relationship between BMI and protein expression. However, the expression levels of these proteins were not correlated with pre-pregnancy BMI and were higher in HUVECs from BMImatched GDM patients than from BMI-matched controls. Intriguingly, TM expression was also higher in HUVECs from BMI-matched GDM patients. Thus, expression of VCAM-1, PAI-1, COX-2, and TM may reflect certain factors in the intrauterine environment that are altered in hospitalized GDM patients with controlled body weight.