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Global reduction of DNA methylation, a part of genome reprogramming processes, occurs in a gradual manner until before implantation and is recognized as a conserved process in mammals. Here, we reported that in bovine, satellite regions exhibited varied patterns of methylation changes when one-cell egg advanced to the blastocyst; a maintenance methylation was observed in satellite I sequences, a decrease in alpha satellites, and an increase in satellite II regions. Cloned embryos exhibited similar changes for DNA methylation in the satellite I and alpha. We also observed that the satellite I and alpha sequences were methylated more in inner cell mass region of the blastocyst whereas the satellite II showed selective demethylation in this region. Together, these findings point that individual satellite sequences carry their own methylation patterns under the pressure of global demethylation, suggesting that local methylation control system acts on the satellite regions in early bovine embryos.

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell–cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa–cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP]i was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP]i in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP]i, and cumulus and granulosa cells. Mol. Reprod. Dev. 65: 336–343, 2003. © 2003 Wiley-Liss, Inc.

Development of mammalian early embryos relies on stored maternal messenger RNAs (mRNAs) that have been synthesized during oogenesis until embryonic genome activation. Although embryonic genome acti vation in bovine embryos has been proposed to start at the late 4-cell stage, recent evidences suggest that embryonic genome activation starts earlier than the 4-cell stage, and molecular details of this event are not known. RNA polymerase II in eukaryotes is responsible for transcription of mRNA and most of the small nuclear RNAs. The unphosphorylated form of RNA polymerase II (IIA) has been shown to function in transcriptional initiation, and the hyperphosphorylated form (IIO) functions in translational elongation and mRNA splicing. In this study, we examined the changes in the amount of RNA polymerase IIA by immunoblotting in immature oocytes; mature oocytes; and 2-, 4- and 8-cell bovine embryos. We also examined the levels of IIO and the multiple intermediately phosphorylated form in the same oocytes and embryos. The IIA reached the highest level at the 2-cell stage and decreased gradually at the 4- and 8-cell stages, and IIO was at very low levels in mature oocytes and 2-cell stage embryos and was not detectable at later stages. The multiple intermediately phosphorylated form was present at the highest level in mature oocytes and was detectable at the other stages. We demonstrate that RNA polymerase IIA, which is responsible for initiation of transcription, is present in oocytes and preimplantation embryos and reaches the highest levels in the 2-cell stage embryos. Inhibition of RNA polymerase II-dependent transcription during any of the first four embryonic cell cycles has detrimental effects on progression of embryonic development beyond the 16-cell stage, indicating the importance of early transcripts for continuation of development. The results indicate that expression of all the genes whose transcription is inhibited by alpha-amanitin is essential for embryo development.

The transition from the maternal to embryonic control of early embryonic development (MET) in mammals is not fully understood. The objective of this study was to determine the amount of transcriptional activity in immature oocytes containing germinal vesicle (GV), mature metaphase II arrested oocytes (MII), 2-, 4- and 8-cell bovine embryos by labeling with 35S-UTP followed by isolation of total RNA and autoradiography. Expression of counts per minute (CPM) per cell showed that incorporation of 35S-UTP in GV oocytes was significantly higher than the background (P0.01) and decreased sharply by the time the oocytes reached MII arrest. Incorporation significantly increased during the 2-cell stage and remained at the same level during the 4- and 8-cell stages. Uptake remained constant throughout different development stages (P0.05) with the highest variability observed during the 2-cell stage. When CPM were expressed per oocyte or embryo incorporation remained high at the GV stage, decreased to the background levels at the time of MII and increased again at the 2-cell stage. It remained at the same level during the 4-cell stage but increased significantly for the second time during the 8-cell stage. Uptake remained at the same level until the 8-cell stage when a significant increase was observed. The negative controls showed a significantly lower amount of incorporation compared to the positive control (P0.05). Similar results were observed by autoradiography. Our observations suggest that MET starts as early as the 2-cell stage in bovine embryos.

This study was designed to investigate the effect of the kinetics of nuclear maturation in bovine oocytes on early embryo development and to examine whether the time of insemination of mature oocytes affects the oocytes' ability to support events of early embryo development. The time required for completion of nuclear maturation was influenced by gonadotropins used to supplement the maturation medium. Luteinizing hormone (LH) enhanced the speed of nuclear maturation when compared to follicle-stimulating hormone (FSH). Oocytes completing their nuclear maturation early (by 16 hours after the initiation of culture) were more likely to complete the first embryonic cell cycle (78% in LH vs. 43% in FSH) and develop to the blastocyst stage (47% in LH vs. 34% in FSH). As the age of the oocytes at the time of MII arrest increased (extrusion of the polar body by 20 or 24 hours), a decrease in their ability to cleave and develop to the blastocyst stage was observed. Differences in the oocyte's ability to decondense chromatin and form pronuclei were also observed. Early maturing oocytes started forming pronuclei earlier than their later maturing counterparts. The time of insemination of mature oocytes played an equally important role. Generally, when insemination of mature oocytes was delayed for 8 hours, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after metaphase II arrest.

Bovine sperm motility and respiration were stimulated by the triazine dye Cibacron Blue F3GA (CB), which may operate as a nucleotide mimic. CB stimulation of respiration was half-maximal at about 35 μM and respiration reached maximal levels about 1.5 minutes after CB addition. Respiratory stimulation was preceded by a transient increase in cytosolic cAMP. Sperm cAMP titers were elevated from 5 to 10 pmoles/108 cells within 30 seconds of CB addition, but rapidly dropped to a stable level of about 7.5 pmoles/108 cells. CB was a potent inhibitor of sperm membrane adenylyl cyclase and inhibited respiration in permeabilized cells. Taken together, the data indicated that CB stimulation was not manifested via the cytosol. In addition, a nonpermeant blue dextran preparation synthesized with CB also stimulated sperm respiration and motility. CB inhibited sperm membrane phosphodiesterase activity, suggesting that the transient pulse of cAMP resulted from CB interaction with this enzyme in the sperm membrane. © 1995 wiley-Liss, Inc.

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat β-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli β-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli β-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli β-galactosidase gene under a β-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes. © 1993 Wiley-Liss, Inc.