The aim of this study was to investigate the effects of culture in isolated oviducts relative to meiotic maturation, the time required to resume meiosis and the viability of the canine oocytes. For this purpose, cumulus-oocyte complexes and isthmus-ampullar tracts of the oviducts were collected from bitches undergoing ovariohysterectomies and destined to two experiments of culture. In experiment 1, the oocytes were cultured for 24 or 30 h: (1) in 100 micro l drops under oil; (2) on the mucosal epithelium of the open oviducts; (3) in the ligated oviducts. In experiment 2, oocytes were cultured in the ligated oviduct for 24, 30 and 48 h. A group of control oocytes was not cultured (0 h). The results showed that within 30 h of culture, a higher proportion of oocytes (p < 0.001) resumed meiosis in the ligated oviduct (63.8%) than in drop (20.4%) or in the open oviduct (27.1%). Moreover, 24 and 30 h of culture assured higher proportions of meiosis resumption than 48 h (69.2 and 59.1% vs 35.8%, p < 0.005). Oocyte resumption of meiosis was mainly determined by oocytes at meiotic stages preceding metaphase I, while stages between metaphase I and II in the ligated oviduct ranged between 12.5 and 31.9%. The extension of the culture time up to 48 h in the oviduct increased oocyte degeneration significantly (59.3%, p < 0.0001) compared with 24 and 30 h (18.7 and 27.3%, respectively) and the oviductal epithelium showed nuclear picnosis and degeneration following culture. The present study suggests that the close physical interaction between the canine oocytes and the oviductal tract positively affects oocyte maturation, and meiosis is resumed within 30 h of culture. Moreover, the oocyte survival is better preserved within 30 h in the ligated oviduct compared with the conventional culture in drop or to the culture in the open oviduct, but the ligated oviduct does not assure viability of the oocytes up to 48 h of culture.
Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood. Here, we uncovered an ATR- and CHK1-mediated transcriptional response to replication stress (RS) in mouse embryonic stem cells (ESCs) that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GRSF1-dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extra-embryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS.
Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood.Here, we uncovered an ATR-and CHK1-mediated transcriptional response to replication stress (RS) in ESCs that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GSRF1 dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extraembryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS. BackgroundESCs are characterized by self-renewal and the ability to propagate for several cycles in vitro and in vivo 1 . Even if ESCs exhibit several markers of RS 2 , they are able to maintain genome integrity more efficiently than differentiated cells 1 . The mechanisms underlying such distinctive feature are largely unknown. ESC colonies harbor a small transient subpopulation of cells (2C-like cells) with functional andtranscriptional features of totipotent 2C-stage embryos 3-5 . Transition to 2C-like cells has been shown to promote maintenance of genome integrity and survival of ESCs in long-term culture 6-8 . In addition, several studies have demonstrated that transition to the 2C-like state confers expanded developmental potential to ESCs, making them capable of contributing to both embryonic and extra-embryonic tissues (also referred to as bidirectional cell fate potential) 3-5 . However, the molecular players underlying the transition to the 2C-like state in ESC culture and its possible physiological relevance in vivo in maintaining genome integrity and expanding cell fate potential in a developing embryo are not fully understood.Here we provide several lines of evidence that ATR and CHK1-mediated response to RS triggers the activation of 2C-specific genes in ESCs and mouse embryos. This transition is hampered in ESCs derived from ATR-deficient Seckel and CHK1 haploinsufficient mouse models and following ATR or CHK1 inhibition. Significantly, we show that ETAA1-mediated ATR activation is sufficient to trigger the formation of 2C-like cells in the absence of RS.Mechanistically, ATR-induced transition to 2C-like state is mediated by post-transcriptional regulation of the Dux gene, which shapes the transcriptional signature of 2C-like cells and totipotent 2C-stage embryos in placental mammals 9-11 . ATR-dependent regulation of Du...
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