Dormant blastocysts during delayed implantation exhibit heightened autophagic activation. Activation of autophagy, the self-eating process within cells, was suggested as an adaptive response to unfavorable environment of prolonged survival in utero. During the course of this study, we observed by transmission electron microscopy that multivesicular bodies (MVBs) accumulate in the trophectoderm of dormant blastocysts upon activation of implantation by estrogen. MVBs are the late endosomes which are characterized by the presence of diverse internal vesicles within a large vesicle. Autophagosomes fuse with MVBs during autophagic activation, and efficient autophagic degradation requires functional MVBs. Biogenesis of MVBs depends on a dynamic network of ESCRT complexes 0, I, II, and III. Tsg101 (a component of the ESCRT-I complex) and CD63 are often used as a marker of MVBs. Lysobisphosphatidic acid (LBPA) is an abundant lipid in MVBs and required for the formation of MVBs. In this study, we performed immunofluorescence staining for detection of MVB makers in dormant and activated embryo. In dormant blastocysts, expression of Tsg101 and LBPA exhibited a uniform pattern throughout the trophectoderm. In contrast, expression of both markers prominently increased in the mural trophectoderm of activated blastocysts. To investigate the relationship with MVB formation and autophagy activation in activated blastocyst, 3-MA, a widely used inhibitor of autophagy, was daily injected intraperitoneally to ovx mice. Interestingly, 3-MA injection to block autophagy during delayed implantation led to a reduction of the signal of MVB markers, suggesting that prolonged activation of autophagy in dormant blastocysts is associated with MVB formation upon activation of implantation. Collectively, these results show that expression of MVB makers increase in the trophectoderm of blastocysts upon activation of implantation and that the formation of MVB is associated with heightened autophagy during delayed implantation.

Vitrification uses cryoprotectants and liquid nitrogen, which may cause osmotic stress and cryodamage to oocytes. Autophagy is widely considered as a survival or responsive mechanism to various environmental and cellular stresses. However, the status of autophagy in vitrified-warmed oocytes has not been studied. In this work, we investigated if vitrification-warming process induces autophagy in mouse oocytes. Four-week-old female ICR mice and GFP-LC3 transgenic mice were used. The mice were superovulated with 5IU PMSG and 5IU hCG and ovulated MII oocytes were collected from oviducts. Oocytes obtained from several mice were pooled and divided into three groups. Group1: fresh oocytes. Group2: oocytes treated with vitification solutions (1.3 M EG+1.1 M DMSO and 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 2.5 min) and warming solutions (0.5 M, 0.25, 0,125, and 0 M sucrose at intervals 2.5 min). Group3: vitrified-warmed oocytes (loaded onto an EM copper grid, and were stored in LN2 for 2 weeks). RT-PCR and confocal live imaging of GFP-LC3 were performed to examine the effects of vitrification-warming process on autophagy in oocytes. In RT-PCR analyses, expression of autophagy related (Atg) genes, such as Atg5, Atg7, Atg12, LC3a, LC3b, and Beclin1 was examined. Expression of Atg7 and Atg12 was slightly reduced in Group 3 (vitrified-warmed oocytes). The expression levels of other Atg genes did not change. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that some vitrified-warmed oocytes showed green puncta which indicate autophagic activation. All oocytes of Group 1 and Group 2 show no puncta formation. Our results suggest that induction of autophagy may serve as an indicator of conditions of vitrification-warming process. Moreover, it offers the possibility that development of methods to modulate autophagic response during cryopreservation could improve efficacy of oocyte cryopreservation.

The Egr family of zinc finger transcription factors consisting of 4 members (Egr1 to -4) regulates critical genetic programs involved in cellular growth, differentiation, and function. Especially, the critical role for Egr1 in regulating luteinizing hormone responsiveness was demonstrated by using gene-targeted mouse models. Other members of Egr family were shown to be involved in other cellular and developmental processes. To understand if Egr3 is implicated in ovarian functions, we focused on identifying cell type-specific and subcellular localization of Egr3 in cycling mouse ovaries and oocytes. RT-PCR analyses show that Egr3 mRNA is expressed in the mouse ovary and oocytes. By immunofluorescence staining, we observed that Egr3 is weakly expressed in subsets of granulosa cells. Interestingly, Egr3 seems to be co-localized with meiotic spindle in some oocytes in the ovarian section. Therefore, we examined Egr3 localization in MI oocytes cultured in vitro. We confirmed co-localization of Egr3 and microtubule in the mouse oocyte during meiosis I. Egr3 localization is noted around condensing chromosomes during prometaphase I (PMI). At metaphase I (MI) and MII, Egr3 is localized on meiotic spindle and also around each cytosolic microtubule organizing centers (MTOCs) in a punctate pattern. To examine if microtubule is required for correct positioning of Egr3 on this structure, we observed the pattern of Egr3 in oocytes matured under taxol or nocodazole. In taxol-treated oocyte, Egr3 and gamma-tubulin complex are enlarged. In nocodazole-treated oocyte, Egr3 localization on spindle and MTOCs are abolished. Thus, Egr3 localization seems to require the presence of intact microtubule. Collectively, our result shows for the first time that Egr3, a transcription factor, is localized on meiotic spindle of maturing mouse oocytes. The work suggests a novel role for Egr3 as a factor involved in MTOC dynamics during meiosis.