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The enzyme telomerase is active in germ cells and is critically involved in maintenance of telomere length in successive generations. In preimplantation mammalian embryos, telomerase activity is present from the morula stage onward and is associated with an increase in telomere length in blastocysts. Herein, we show that telomere length regulation in murine and bovine blastocysts differed between trophectodermal and inner cell mass cells in a species-specific manner. Ectopic expression of human telomerase reverse transcriptase (TERT) in bovine embryos increased telomerase activity and in turn increased telomere length. Transient expression of human TERTcould be targeted to the 4-cell to morula stages and to the morula to blastocyst stages using unmodified and cytosine-methylated expression plasmids, respectively. Introduction of human TERTconstructs in bovine embryos resulted in functional telomerase expression and effective telomere elongation, allowing us to study the effects on embryonic development. Ultimately, these studies may lead to a large-animal model for telomere regulation and aging.

In porcine oocytes, acquisition of meiotic competence coincides with a decrease of general transcriptional activity at the end of the oocyte growth phase and, specifically, of ribosomal RNA (rRNA) synthesis in the nucleolus. The present study investigated the regulation of rRNA synthesis during porcine oocyte growth. Localization and expression of components involved in regulation of the rRNA synthesis (the RNA polymerase I-associated factor PAF53, upstream binding factor [UBF], and the pocket proteins p130 and pRb) were assessed by immunocytochemistry and semiquantitative reverse transcription-polymerase chain reaction and correlated with ultrastructural analysis and autoradiography following [3H]uridine incubation in growing and fully grown porcine oocytes. In addition, meiotic resumption, ultrastructure, and expression of p130, UBF, and PAF53 were analyzed in growing and fully grown porcine oocytes cultured with 100 μM butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, to gain insight concerning the regulation of rRNA transcription during meiotic arrest. Immunocytochemical analysis demonstrated that p130 became colocalized with UBF and PAF53 and that the intensity of the PAF53 labeling decreased toward the end of the oocyte growth phase. These data suggest that the decrease in rRNA synthesis is regulated through inhibition of UBF by p130 as well as by decreased availability of PAF53. Moreover, expression of mRNA encoding PAF53 was decreased at the end of the oocyte growth phase. At the morphological level, these events coincided with inactivation of the nucleolus, as visualized by the transformation of the fibrillogranular nucleolus to an electron-dense fibrillar sphere with remnants of the fibrillar centers at the surface. Meiotic inhibition with 100 μM BL-I had a detrimental effect on the ability of porcine oocytes to resume meiosis and on nucleolus morphology, resulting in a lack of RNA synthetic capability as the fibrillar components, where rRNA transcription and initial processing occur, condensed or even disintegrated.