X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.

X-box-binding protein 1 (XBP1) is an important regulator of a subset of genes active during endoplasmic reticulum (ER) stress. In the present study, we analyzed XBP1 level and location to explore the effect of ER stress on oocyte maturation and developmental competency of porcine embryos in an in vitro culture system. First, we examined the localization of XBP1 at different meiotic stages of porcine oocytes and at early stages of parthenogenetic embryo development. Fluorescence staining showed that expression of functional XBP1 was weak in mature oocytes and at the 1-, 2-, and 8-cell stages of embryos but abundant at the germinal vesicle (GV), 4-cell, morula, and blastocyst stages. In addition, RT-PCR revealed that both spliced XBP1 (XBP1-s) and unspliced XBP1 (XBP1-u) were expressed at the GV, 4-cell, morula, and blastocyst stages. Tunicamycin, an ER stress inducer, induced active XBP1 protein in nuclei of 4-cell embryos. Next, porcine embryos cultured in the presence of tauroursodeoxycholate, an ER stress inhibitor, were studied. Total cell numbers and the extent of the inner cell mass increased (P < 0.05), whereas the rate of nuclear apoptosis decreased (P < 0.05). Moreover, expression of the antiapoptotic gene BCL2 increased, whereas expression of the proapoptotic genes BCL2L1 (Bcl-xl) and TP53 decreased. The results indicated that inhibition of ER stress enhanced porcine oocyte maturation and embryonic development by preventing ER stress-mediated apoptosis in vitro.

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BackgroundTwo stages of genome activation have been identified in the mouse embryo. Specifically, minor transcriptional activation is evident at the one-cell stage and a second major episode of activation occurs at the two-cell stage. Nuclear translocation of RNA polymerase II and phosphorylation of the C-terminal domain (CTD) of the largest enzyme subunit are major determinants of embryonic genome activation. P-TEFb, the Pol II CTD kinase, regulates transcriptional elongation via phosphorylation of the serine 2 residues of the CTD.ResultsHere, we show that the CDK9 and cyclin T1 subunits of P-TEFb are present in mouse oocytes and preimplantation embryos. Both proteins translocate to pronuclei at the late one-cell stage and are predominantly localized in nuclei at the two-cell stage. We additionally examine the effects of the CDK9-specific inhibitor, flavopiridol, on mouse preimplantation development. Our data show that treatment with the drug results in mislocalization of CDK9, cyclin T1, and phosphorylated Pol II, as well as developmental arrest at the two-cell stage.ConclusionsA change in CDK9 localization from the cytoplasm to the pronucleus occurs at the time of minor embryonic genome activation, and CDK9 accumulation at the two-cell stage is evident, concomitant with major transcriptional activation of the embryonic genome. Moreover, CDK9 inhibition triggers a developmental block at the two-cell stage. Our findings clearly indicate that CDK9 is essential for embryonic genome activation in the mouse.

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