Yanfeng Dai

Nuclear transfer in sheep embryos: The effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes

The developmental ability of nuclear transplant sheep embryos derived from in vitro matured oocytes was studied by controlling cell‐cycle coordination of donor embryonic nuclei and recipient cytoplasts. Oocytes were recovered from nonatretic antral follicles of adult sheep ovaries and cocultured with follicle shells in M199‐based medium supplemented with gonadotrophins in a nonstatic system. Effective activation of IVM oocytes was obtained by applying two pulses of 1.0 kv/cm 22 min apart in inositol‐based electroporation medium to oocytes matured in vitro for 27 hr. Synthesis of DNA (S‐phase) was assessed by BrdU incorporation and was found to initiate around 5 hpa (hours postactivation) and to persist until 18 hpa. Mitotic blastomeres were induced by treating embryos with 6.6 μM nocodazole for 14–17 hr. Three types of transfers were compared directly: “S → S,” early embryonic nuclei (mostly in S‐phase) were transferred to presumptive S‐phase cytoplasts; “M → MII,” nocodazole‐treated embryonic nuclei (most in M‐phase) were transferred to MII‐phase cytoplasts; and control (S → MII), conventional nuclear transfer of fusion and activation simultaneously. The results showed that fusion and recovery rates did not differ among the three groups. However, after 6 days of in vivo culture, the morula and blastocyst formation rate was significantly higher for the M → MII combination than for the control (28.3% vs. 8.1%, P < 0.05), while no significant differences in developmental rate were observed between S → S and M → MII, and between S → S and control, though developmental rate was also increased for S → S compared to control (20.9% vs. 8.1%, P > 0.05). Transfer of blastocysts derived from M → MII or S → S nuclear cytoplasmic reconstitution to synchronized recipient ewes resulted in the birth of lambs. These data suggest that in vitro matured oocytes can support full‐term development of nuclear transplant sheep embryos when the cell cycle of nucleus and cytoplasm is coordinated, and that M → MII nuclear transfer might be an efficient and simple way to improve the developmental competence of the reconstituted embryos. Mol. Reprod. Dev. 47:255–264, 1997.

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes

To understand the mechanisms which regulate meiosis‐specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP‐E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP‐E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP‐E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP‐E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP‐E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP‐E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP‐E survived through the transition even in cycloheximide‐treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP‐E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side‐by‐side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI. Mol. Reprod. Dev. 56:51–62, 2000.

Selective Requirement for Cdc25C Protein Synthesis During Meiotic Progression in Porcine Oocytes

Abstract Fundamental differences between meiosis and mitosis suggest that the shared central cell cycle machinery may be regulated differently during the two division cycles. This paper focuses on unique features of Cdc25C protein function during meiotic progression. We report on the existence of oocyte-specific CDC25C transcripts that differ from their somatic counterparts in the 3′ untranslated region. While CDC25C mRNA levels remain constant in fully-grown oocytes, corresponding protein levels increase progressively during maturation to a maximum at metaphase II. Elevation of Cdc25C protein levels in G2-oocytes by mRNA injection failed to increase MPF-kinase levels or to induce premature entry into M-phase. Likewise, antisense-induced arrest of translation (translational arrest) had no effect on chromosome condensation, nucleolar disassembly, or nuclear membrane contraction. By contrast, translational arrest inhibited subsequent events including membrane disassembly and spindle formation. Neither up- nor down-regulation of Cdc25C synthesis after metaphase I plate formation influenced progression to metaphase II. However, translational arrest during metaphase resulted in incomplete chromosome decondensation and abnormal pronuclear membrane assembly after activation. We conclude that Cdc25 protein, translated from unique transcripts, is preferentially located in the oocyte nucleus and is essential for progress through late diakinesis. Subsequently, new synthesis of Cdc25C protein is required for the orderly transition from meiotic to mitotic cell division.

Role of secreted proteins and gonadotrophins in promoting full maturation of porcine oocytes in vitro

Experiments were designed to identify the extent to which follicle cells and hormones contribute to the developmental competence of porcine oocytes matured in vitro. Oocyte‐cumulus complexes were collected from ovaries by dissection and cultured in 2 ml of TCM199‐based medium in 5% CO2 in humidified air at 38.5°C. This basic maturation system was supplemented, for either the first 24 hr only or for the 48‐hr culture period, with 1) everted follicle shell alone, 2) gonadotrophic hormones alone, or 3) both follicle shells and hormones. The effect of these treatments was evaluated on 1) meiotic maturation rates, 2) the capacity of matured eggs to undergo activation and early cleavage, and 3) changes to the profile of proteins secreted into the culture medium. The results showed that 1) supplementation with either follicle shell or hormones alone increased the rates of meiotic maturation over the nonsupplemented control group, and 2) combined follicle shell and hormonal supplementation yielded the highest rates for maturation, activation, and cleavage but only when hormonal supplementation was removed after the first 24 hr of culture. Proteins of 30, 37, 45, and 46 kD, but of unknown function, were secreted during the first 24 hr into the culture medium in groups supplemented with follicle shells. The addition of hormones did not affect this pattern of secreted proteins. It is possible that some secreted proteins may act to facilitate full maturation of pig oocytes. Mol. Reprod. Dev. 47:191–199, 1997.

Antral follicles confer developmental competence on oocytes.

This paper addresses the proposition, first advanced by Wilson (1925), that successful embryogenesis depends on an ordered series of events in oogenesis. It is at the completion of this varied set of intracellular changes that the oocyte finally acquires its full capacity to support fertilisation and development. Amongst the earliest nuclear events are those associated with chromosome pairing and meiotic recombination. During the growth phase cell volume increases 300-fold and the cytoplasm becomes the storage site for RNA and protein which will be mobilised during early development. Finally, a short phase of intracellular reprogramming, or maturation, completes the series of events during oogenesis that confer developmental competence upon the oocyte. Follicle cell support is an indispensable requirement for ordered oocyte development and provides the early germline cell with many of the essential nutrients and growth regulators required to ensure progression through the protracted growth phase (see contributions by Cecconi & Rosella and De Felici et al. this issue). Although different, the interactions between the full-grown oocyte and the antral follicle are no less crucial to the acquisition of competence than those involved in the earlier stages of oogenesis.

Transcription of c-mos protooncogene in the pig involves both tissue-specific promoters and alternative polyadenylation sites

The function of the c‐mos gene has been intensively studied, but its role in the mammal is still a subject for debate. For this reason, and because the gene is regulated posttranscriptionally, further study of the gene from other mammalian species is timely. The pig c‐mos gene has been cloned, and the genomic sequence is presented here. The gene has no introns and shows close similarity to human and monkey genes, with striking sequence similarities in both the 5′ and 3′ flanking regions.The significance of this similarity in the context of gene regulation is discussed. c‐mos expression was found to be restricted to gonadal tissues in the pig. The major start sites for transcription initiation in ovary and testis were identified by primer extension and found to be distinct, as in the mouse. Within the ovary, expression is confined to oocytes. Messenger RNA is synthesized in growing oocytes, and remains stable during oocyte maturation, but begins to be degraded in electrically stimulated eggs. Unexpectedly, RNase protection assays revealed that the 3′ ends of transcripts in the pig ovary are heterogeneous, and this, together with the identification of three distinct cDNA clones, shows that multiple polyadenylation sites are used. The significance of these transcripts in terms of translational control is discussed.

Tyrosine phosphorylation of p34cdc2 in metaphase II-arrested pig oocytes results in pronucleus formation without chromosome segregation

At the G2‐M boundary, maturation‐promoting factor (MPF) activation is usually induced in one or both of two ways; tyrosine dephosphorylation of p34cdc2 or synthesis of cyclin B according to cell type and species. At the end of M‐phase, however, MPF inactivation is normally triggered only by cyclin degradation. We investigated whether tyrosine phosphorylation of p34cdc2 can inactivate MPF and what kinds of events are induced in pig metaphase II (MII)‐arrested oocytes. First, cyclin B1 in MII‐arrested oocytes is degraded upon fertilization. Second, when MII oocytes were treated with vanadate, an inhibitor of tyrosine phosphatases, they were released from MII arrest, but MPF was inactivated by further tyrosine phosphorylation of p34cdc2 rather than cyclin B1 degradation. The vanadate‐induced exit from M‐phase is distinct from normal M‐phase exit, which is accompanied by cyclin B1 degradation; the former lacks both sister chromatid separation and second polar body emission. Vanadate itself has no inhibitory effect on chromosome segregation since calcium ionophore induced chromosome segregation in the presence of vanadate. Furthermore, when MII oocytes were treated with olomoucine, an inhibitor of cyclin‐dependent kinases, they exited from MII arrest in a manner similar to vanadate‐treated MII oocytes. Finally, we propose that MPF inactivation by tyrosine phosphorylation of p34cdc2 enables MII oocytes to form an interphase nucleus, but not to segregate sister chromatid due to the absence of the mechanisms required to trigger sister chromatid separation such as anaphase‐promoting complex (APC)‐mediated proteolysis, on the signaling pathway from intracellular Ca2+ increase to MPF inactivation. Mol. Reprod. Dev. 52:107–116, 1999.

Translational Regulation of MOS Messenger RNA in Pig Oocytes

Abstract The temporal and spatial translation control of stored mRNA in oocytes is regulated by elements in their 3′-untranslated region (3′-UTR). The MOS 3′-UTR in pig oocytes is both heterogeneous (180, 480, or 530 nucleotides), and it contains multiple U-rich elements and extensive A-rich sequences (CA13CA5CA5CA6). We have examined the role of these potential regulatory elements by fusing wild-type or mutant MOS 3′-UTRs to luciferase mRNA and then injecting these chimeric transcripts into oocytes. We draw six main conclusions. First, the length of the MOS 3′-UTR tightly controls the level of translation of luciferase during oocyte maturation. Second, two U-rich (U5A) elements and the hexanucleotide signal (AAUAAA) are required for translation. Third, mutations, duplications, or relocations of the A-rich sequence reduce or block translation. Fourth, the relative importance of the A-rich and U-rich elements in controlling the level of translation differs. Fifth, none of our MOS 3′-UTR manipulations relieved translational repression before germinal vesicle breakdown. Sixth, all the MOS mRNA variants underwent polyadenylation during maturation. Whereas mutations to the hexanucleotide signal block both polyadenylation and translation, mutations to either the A-rich sequence or the U-rich elements block translation without fully blocking polyadenylation. We conclude that MOS mRNA translation in pig oocytes is subject to a more extensive series of controls than that in lower vertebrates.

Degradation of pig cyclin B1 molecules precedes MAP kinase dephosphorylation during fertilisation of the oocytes.

Pig oocytes at metaphase II were activated by penetration of spermatozoa in cycloheximide-free and cycloheximide-containing fertilisation media. The precise nuclear stage, and the kinetics of degradation of cyclin B1 and dephosphorylation of MAP kinase were assessed after insemination. After maturation culture, 96% of oocytes reached metaphase II. At 6 h after insemination in cycloheximide-free medium, 68% of the oocytes were activated and had progressed to anaphase II or beyond. After 8 h, 89% of the oocytes were activated: a female pronucleus had formed and the heads of penetrating spermatozoa had enlarged and changed to male pronuclei. In the cycloheximide-containing medium, activation of oocytes started earlier than in cycloheximide-free medium. After 4 h, 43% of the oocytes were activated, and the percentage increased to 97% after 6 h. Pig cyclin B1 disappeared in the oocytes at 6 h after insemination in both cycloheximide-containing and cycloheximide-free media. Pig oocytes at metaphase II contained two types of MAP kinase--ERK 1 and ERK 2--in their active phosphorylated forms. At 8 h after insemination ERK 2 changed to the fast-migrating inactive form in the oocytes cultured in both cycloheximide-containing and cycloheximide-free media, although the shift-down was not complete. The change was delayed by 2 h after the degradation of cyclin B1 molecules. These results demonstrate that degradation of pig cyclin B1 molecules corresponds to the transition of the oocytes from metaphase II arrest to anaphase II/telophase II and was followed by MAP kinase dephosphorylation.