WEE2 Is an Oocyte-Specific Meiosis Inhibitor in Rhesus Macaque Monkeys1

Abstract

WEE1 homolog 2 (WEE2, also known as WEE1B) is a newly identified member of the WEE kinase family that is conserved from yeast to humans. The aim of the present study was to determine the spatiotemporal expression pattern and the function of WEE2 during oocyte maturation in a nonhuman primate species, the rhesus macaque. Among 11 macaque tissues examined, WEE2 transcript is predominantly expressed in the ovary and only weakly detectable in the testis. Within the ovary, WEE2 mRNA is exclusively localized in the oocyte and appears to accumulate during folliculogenesis, reaching the highest level in preovulatory follicles. Microinjection of a full-length WEE2-GFP (green fluorescent protein) fusion mRNA indicates a specific nuclear localization of WEE2 protein in both growing and fully grown germinal vesicle (GV)-intact oocytes. Taking the long double-stranded RNA-mediated RNA interference approach, we found that down-regulation of WEE2 led to meiotic resumption in a subset of GV oocytes even in the presence of a phosphodiesterase 3 inhibitor. On the other hand, overexpression of WEE2 delays the reentry of oocytes into meiosis in both mice and monkeys. These findings suggest that WEE2 is a conserved oocyte-specific meiosis inhibitor that functions downstream of cAMP in nonhuman primates.

Analyses of the Regulatory Mechanism of Porcine WEE1B: The Phosphorylation Sites of Porcine WEE1B and Mouse WEE1B Are Different

“…3Ab), indicating the presence of NLS in pWEE1B as reported for the mouse, human and rhesus monkey WEE1Bs [1,3,5]. We found a putative NLS sequence, KRK, at 173-175 in pWEE1B (Fig.…”

“…The localization of pWEE1B was examined in the present study, and exclusive nuclear localization was revealed in the fully grown meiotically arrested oocytes. The nuclear localization of WEE1B has already been reported in mice, humans and rhesus monkeys [1,3,5], and this is also the case in pigs, indicating the presence of a nuclear localization signal (NLS) in pWEE1B. In the present study, we mutated a KRK sequence, corresponding to the NLS of mWEE1B [5], to KTT and overexpressed it in oocytes maintained at the GV stage.…”

“…Phosphorylation of inhibitory phosphorylation sites in CDC2, a catalytic subunit of MPF, plays a principle role in inhibiting the MPF activity of these oocytes, and WEE1B, also called WEE2, has been identified as an oocyte-specific kinase involved in this phosphorylation in mice, humans, rhesus monkeys and pigs [1][2][3][4]. The induction of germinal vesicle break down (GVBD) by WEE1B inhibition using RNA interference or antisense oligonucleotide injection has been shown in mouse, rhesus monkey and porcine immature oocytes, in which spontaneous meiotic resumption was pharmacologically prevented [2][3][4][5]. Successful meiotic resumption was also induced in porcine growing oocytes, which could not start meiosis autonomously, by the injection of WEE1B antisense RNA [6].…”

“…This fact indicates the phosphorylation of other amino acids by PKA for the regulation of pWEE1B activity, and suggests a difference in the WEE1B-regulation mechanism between mice and pigs, although the phosphorylation site in pWEE1B has never been identified. In addition, nuclear localization of WEE1B has been reported in mice, humans and rhesus monkeys [1,3,5], although the requirement of nuclear localization for the function of WEE1B has been demonstrated only in mWEE1B [5]. It is thus necessary to obtain more information on WEE1B regulation in species other than mice in order to improve our understanding of the molecular mechanisms of meiotic resumption.…”

Stage-Specific Proteome Signatures in Early Bovine Embryo Development

“…To increase analytical depth, the sample complexity was reduced by a prefractionation step using strong cation exchange chromatography (SCX), which separated the iTRAQ-labeled peptides on the basis of their charge. Peptides were eluted from the SCX column (Biobasic, Thermo Scientific, Rockford, IL, USA) stepwise using eight different potassium chloride (KCl) concentrations (0, 45,75,90,110,135,175, and 350 mM KCl) and subsequently desalted using C18 spin columns (Pierce, Thermo Scientific, Rockford, IL, USA). SCX fractions were evaporated to dryness under vacuum (vacuum concentrator, Bachofer) and stored at −80°C until LC−MS/MS analysis.…”