Developmental potential of bovine oocytes cultured in different maturation and culture conditions.

Diverse groups of chemicals in culture media are needed for successful bovine oocyte maturation and embryo development during which dramatic cytoplasmic and nuclear reprogramming events take place. In vitro embryo production (IVP) procedures frequently include supplements such as serum and/or co-culture with various types of somatic cells. However, the presence of undefined serum in culture media introduces a variation from batch to batch, increases viral or prion contamination risk, and leads to problems during fetal development. The aim of the present study was to investigate the possibility of using chemically defined-synthetic serum substitute (SSS) in place of fetal calf serum (FCS) during maturation and long-term culture to stimulate in vitro maturation (IVM), fertilization (IVF) and subsequent embryo development. In Experiment I, the effect of the protein source on in vitro maturation was tested by maturing oocytes in culture media supplemented with 10% FCS (Control Group), 10% SSS (Group I) and 10% SSS+10 ng/ml epidermal growth factor (EGF) (Group II). In Experiment II, effects of SSS on both oocyte maturation and embryo development during in vitro culture (IVC) were tested by maturing oocytes in media supplemented with 10% FCS (FCS Group) or 10% SSS+10 ng/ml EGF (SSS Group), followed by IVF and IVC in SOF media supplemented with 10% FCS and 10% SSS on day 4 for FCS and SSS Groups, respectively. Even though rates for cleavage and development to blastocyst stage were not different, blastocyst cell numbers were higher in Group II containing SSS and EGF. The SSS supplementation group had higher apoptotic nuclei as compared to the FCS Group in Experiment II. Transcripts for heat shock protein 70 (Hsp70), interferon tau (IF-tau), DNA methyltransferase 3a (Dnmt3a), desmosomal glycoprotein desmocollin III (DcIII) and insulin-like growth factor II receptor (Igf-2r) were altered in different culture conditions in Experiment I. However, only glucose transporter-1 (Glut-1) mRNA was different in the SSS and FCS Groups in the second experiment. In summary, SSS and EGF in maturation medium and replacement of FCS with SSS alone in culture medium on day 4 of IVC support oocyte maturation and embryo development in vitro. However, significance of culture condition induced changes on the genome-wide abundance of messenger ribonucleic acid and the significance of the apoptotic nuclei during fetal development still remain to be determined.

Developmental and molecular correlates of bovine preimplantation embryos.

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were cultured in vitro in three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR. In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P < 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P < 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P < 0.001). Expression of interferon tau (IF-tau) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P < 0.001). Gene expression did not differ between in vivo-derived blastocysts and their in vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

Varied patterns of DNA methylation change between different satellite regions in bovine preimplantation development.

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.

Meiotic state of bovine oocytes is regulated by interactions between cAMP, cumulus, and granulosa.

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.

Genome-wide epigenetic alterations in cloned bovine fetuses.

To gain a better understanding of global methylation differences associated with development of nuclear transfer (NT)-generated cattle, we analyzed the genome-wide methylation status of spontaneously aborted cloned fetuses, cloned fetuses, and adult clones that were derived from transgenic and nontransgenic cumulus, genital ridge, and body cell lines. Cloned fetuses were recovered from ongoing normal pregnancies and were morphologically normal. Fetuses generated by artificial insemination (AI) were used as controls. In vitro fertilization (IVF) fetuses were compared with AI controls to assess effects of in vitro culture on the 5-methylcytosine content of fetal genomes. All of the fetuses were female. Skin biopsies were obtained from cloned and AI-generated adult cows. All of the adult clones were phenotypically normal and lactating and had no history of health or reproductive disorders. Genome-wide cytosine methylation levels were monitored by reverse-phase HPLC, and results indicated reduced levels of methylated cytosine in NT-generated fetuses. In contrast, no differences were observed between adult, lactating clones and similarly aged lactating cows produced by AI. These data imply that survivability of cloned cattle may be closely related to the global DNA methylation status. This is the first report to indicate that global methylation losses may contribute to the developmental failure of cloned bovine fetuses.