Simone Cristina Méo

Pronounced Segregation of Donor Mitochondria Introduced by Bovine Ooplasmic Transfer to the Female Germ-Line

Abstract Ooplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offsprings oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygotes ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% ± 9.6% versus 12.1% ± 4.5%; P < 0.0001). We also characterized the proliferation of mitochondria from the OT parents—the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r2 = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.

Ooplast-mediated developmental rescue of bovine oocytes exposed to ethidium bromide

Ooplasm transfer has been used successfully to treat infertility in women with ooplasmic insufficiency and has culminated in the birth of healthy babies. To investigate whether mitochondrial dysfunction is a factor in ooplasmic insufficiency, bovine oocytes were exposed to ethidium bromide, an inhibitor of mitochondrial DNA replication and transcription, during in-vitro maturation (IVM). Exposure of immature oocytes to ethidium bromide for 24h during IVM hampered meiotic resumption and the migration of cortical granules. However, a briefer treatment with ethidium bromide during the last 4h of IVM led to partial arrest of preimplantation development without affecting oocyte maturation. Ooplasm transfer was then performed to rescue the oocytes with impaired development. In spite of this developmental hindrance, transfer of normal ooplasm into ethidium bromide-treated oocytes resulted in a complete rescue of embryonic development and the birth of heteroplasmic calves. Although this study unable to determine whether developmental rescue occurred exclusively through introduction of unaffected mitochondria into ethidium bromide-damaged oocytes, e.g. ethidium bromide may also affect other ooplasm components, these results clearly demonstrate that ooplasm transfer can completely rescue developmentally compromised oocytes, supporting the potential use of ooplasm transfer in therapeutic applications.

The effect of interaction between macromolecule supplement and oxygen tension on bovine oocytes and embryos cultured in vitro.

SummaryAiming to improve in vitro production of bovine embryos and to obtain supplements to replace serum for in vitro maturation (IVM), this study evaluated the effects of macromolecular supplementation of IMV medium (bovine serum albumin - BSA, polyvinyl alcohol - PVA, polyvinyl pyrrolidone - PVP, Ficoll, KnockoutSR, or fetal calf serum - FCS) and oxygen tension [5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2)] on oocyte maturation and embryo development. Nuclear progression to germinal vesicle breakdown, metaphase I and metaphase II stages were evaluated and overall results revealed that undefined (FCS) and semi-defined (BSA) media gave better results at 20% O2 and defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, IVM at 20% O2 was considered optimal for nuclear maturation. To evaluate embryo development, oocytes matured in the previously described conditions were fertilized and cultured at the same oxygen tension used for IVM and assessed for cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). Apart from oxygen tension, all treatments, except Knockout (22.7%), gave similar results for blastocyst development (26.5 to 38.7%). Independently of macromolecule supplement, higher development rates were obtained in an oxygen tension of 20% O2 (67.4% cleavage, 29.2% morulae, 40.8% blastocyst and 34.0% hatched blastocyst) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively). This study indicates that BSA, PVA, PVP and Ficoll can replace serum during IVM and that the optimal atmospheric condition for in vitro production of bovine embryos is 5% CO2 and 20% O2.

Demecolcine Effects on Microtubule Kinetics and on Chemically Assisted Enucleation of Bovine Oocytes

This study aimed to evaluate the effect of demecolcine, a microtubule-depolymerizing agent, on microtubule kinetics; to determine the best concentration of demecolcine as a chemically assisted enucleation agent in metaphase I (MI) and metaphase II (MII) bovine oocytes, and to evaluate the embryonic development after nuclear transfer (NT) using chemically assisted enucleation of recipient oocytes. Oocytes in vitro matured for 12 h (MI) and 21 h (MII) were exposed to several concentrations of demecolcine and evaluated for enucleation or membrane protrusion formation. Demecolcine concentration of 0.05 microg/mL produced the highest rates of enucleation in group MI (15.2%) and protrusion formation in group MII (55.1%), and was employed in the following experiments. Demecolcine effect was seen as early as 0.5 h after treatment, with a significant increase in the frequency of oocytes with complete microtubule depletion in MI (58.9%) and MII (21.8%) compared to initial averages at 0 h (27.4% and 1.9%, respectively). Microtubule repolymerization was observed when MII-treated oocytes were cultured in demecolcine-free medium for 6 h (42.4% oocytes with two evident sets of microtubules). Chemically assisted enucleated oocytes were used as recipient cytoplasts in NT procedures to assess embryonic development. For NT, 219 of 515 oocytes (42.5%) formed protrusions and were enucleated, and reconstructed, resulting in 58 nuclear-transferred one-cell embryos. Cleavage (84.5%) and blastocyst development (27.6%) rates were assessed. In conclusion, demecolcine can be used at lower concentrations than routinely employed, and the chemically assisted enucleation technique was proven to be highly efficient allowing embryonic development in bovine.

Xenooplasmic transfer between buffalo and bovine enables development of homoplasmic offspring.

Nuclear-mitochondrial incompatibilities may be responsible for the development failure reported in embryos and fetuses produced by interspecies somatic cell nuclear transfer (iSCNT). Herein we performed xenooplasmic transfer (XOT) by introducing 10 to 15% of buffalo ooplasm into bovine zygotes to assess its effect on the persistence of buffalo mitochondrial DNA (mtDNA). Blastocyst rates were not compromised by XOT in comparison to both in vitro fertilized embryos and embryos produced by transfer of bovine ooplasm into bovine zygotes. Moreover, offspring were born after transfer of XOT embryos to recipient cows. Buffalo mtDNA introduced in zygotes was still present at the blastocyst stage (8.3 vs. 9.3%, p = 0.11), indicating unaltered heteroplasmy during early development. Nonetheless, no vestige of buffalo mtDNA was found in offspring, indicating a drift to homoplasmy during later stages of development. In conclusion, we show that the buffalo mtDNA introduced by XOT into a bovine zygote do not compromise embryo development. On the other hand, buffalo mtDNA was not inherited by offspring indicating a possible failure in the process of interspecies mtDNA replication.

Karyoplast exchange between strontium- and 6-DMAP-parthenogenetically activated zygotes of cattle.

Ooplasmic factors drive nuclear organization after fertilization and are also important for re-programming in nuclear transfer procedures, in which artificial activation is essential for reconstructed embryos to progress in development. The present research evaluated the effect of pronuclear transfer (PT) between zygotes parthenogenetically activated with ionomycin followed by strontium (S) or 6-DMAP (D) on early embryonic development. PT was performed in the same zygote to obtain embryos in control groups (S-PT and D-PT) and between zygotes activated with S and D to achieve embryos with differentially activated cytoplasm (C) and nucleus (N) (SCDN and DCSN). PT procedure did not affect cleavage and blastocyst rates, respectively, in PT control groups compared to non-manipulated control (S-PT: 73.6% and 7.3% compared with S-Control: 77.9% and 7.8%; and D-PT: 73.3% and 31.7% compared with D-Control: 83.1% and 41.5%). Cleavage, eight-cell, and blastocyst rates, respectively, were similar between SCDN (76.5%, 36.4%, and 6.8%) and DCSN (69.5%, 25.0%, and 4.9%) embryos. Developmental rates in SCDN were similar to S-PT, but inferior to D-PT. Developmental arrest up to eight-cell stage was greater in SCDN and DCSN than in S-PT and D-PT. In conclusion, karyoplast exchange between parthenogenetic zygotes activated with strontium and 6-DMAP can lead to nuclear-cytoplasmic incompatibilities and affect embryonic development to the eight-cell and blastocyst stages.

53 EFFECTS OF DEMECOLCINE ON MICROTUBULE COMPOSITION AND CHEMICALLY ASSISTED ENUCLEATION OF BOVINE OOCYTES

The developmental competence of enucleated oocytes is a key factor that determines the overall success of animal cloning. Enucleation is an invasive procedure in traditional nuclear transfer (NT). The objective of this work was to evaluate the effects of demecolcine, a microtubule-depolymerizing agent, on metaphase II (MII) bovine oocytes and to verify the capacity of embryonic development after NT using chemically assisted enucleation. In the first experiment, oocytes after 21 h of IVM were exposed for 2 h to several concentrations of demecolcine: 0 (control), 0.025, 0.05, 0.2, and 0.4 µg mL–1, and evaluated in relation to membrane protrusion formation. After the best concentration of demecolcine was determined, the nuclear and microtubular dynamics of the treated oocytes were evaluated by immunofluorescence microscopy of tubulin and chromatin (Liu et al. 1998 Biol. Reprod. 5, 537–545) in a second experiment. The results were analyzed by Duncan and Tukey tests in Experiments I and II, respectively, and a level of 5% significance was used. In Experiment III, embryonic development following NT was determined using adult donor cells, derived from a skin biopsy. After 19 h of IVM, oocytes were exposed to demecolcine (0.05 µg mL–1) for 2 h, and enucleation was performed in oocytes that presented membrane protrusions. Samples of cytoplasts were stained with Hoechst 33342 for 10 min (10 µg mL–1) for evaluation of enucleation effectiveness. The remaining oocytes were reconstituted by NT, fused (two pulses of 2.0 kV cm–1 for 20 µs each, in 0.28 m mannitol solution), chemically activated (5 mm ionomycin for 5 min and 2 mm 6-DMAP for 4 h), and cultured in SOF medium with 2.5% FCS and 0.5% BSA. The 0.05 µg mL–1 concentration resulted in greater protrusion rates (55.1%; 211/388) in comparison to 0, 0.025, 0.2, and 0.4 µg mL–1 concentrations (0, 42.6, 45.1, and 39.3%, respectively). In Experiment II, at the beginning of treatment, the majority of the oocytes were in MII (40.7%) or anaphase I/telophase I (AI/TI; 22.4%). Effects of demecolcine occurred within only 0.5 h of treatment, with a significant increase in oocytes with complete depletion of microtubules (21.8%; initial average: 1.9%) and a reduction in the proportion at MII (13.5%) and AI/TI (8.2%). New polymerization of microtubules was observed when treated oocytes were then cultured in drug-free medium for 6 h (42.4% oocytes with two evident sets of microtubules). In Experiment III, we verified the effectiveness of the chemically assisted enucleation technique (90.6%; 77/85). We evaluated 515 oocytes, of which 219 (42.5%) had protrusions and were enucleated. After losses in the NT procedure and fusion, 58 reconstructed 1-cells were cultured resulting in cleavage and blastocyst rates of 84.5% (49/58) and 27.6% (16/58), respectively, with great variation in blastocyst production between the three replicates (12.5% to 47%). In conclusion, demecolcine can be used at lower concentrations than those used routinely, and the chemically assisted enucleation method has proven highly efficient and does not appear to inhibit embryonic development in the bovine. This work was supported financially by FAPESP.

187 Imprinted gene expression in in vivo-and in vitro-produced bovine fetuses and placentas

Some gestational alterations associated with bovine somatic cell nuclear transfer (SCNT) are presumably consequences of abnormal imprinted gene expression. This work aimed to evaluate the expression patterns of imprinted genes IGF2 and IGF2R in bovine fetuses and chorioallantoic membranes derived from in vivo- and in vitro-produced embryos. Fetuses were produced by AI (in vivo group, n = 3), IVF (n = 3), parthenogenesis (n = 3), or SCNT (n = 2). Cows with positive pregnancy diagnosis after ultrasonographic examination were slaughtered between Days 33 and 36 of gestation. The reproductive tract was transported on ice to the laboratory, where fetuses and chorioallantoic fragments were collected and stored in liquid nitrogen. Total RNA extraction was performed using TRIzol, according to manufacturers instructions, and the reverse transcription reaction was carried out with 1 µg of total RNA, 6.75 µm oligo pd(T)12–18, and 50 U of reverse transcriptase (Improm-II, Promega, Madison, WI, USA). The relative quantification of IGF2 and IGF2R transcripts was done using real-time PCR with SYBR Green dye. The average efficiency of PCR amplifications was estimated for each gene using a linear regression on the logarithm of fluorescence per cycle (Ramakers et al. 2003 Neurosci. Lett. 339, 62–66), and the expression ratios were calculated according to the method described previously by Livak and Schmittgen (2001 Methods 25, 402–408). To verify statistical differences, a pair-wise fixed reallocation randomization test (Pfaffl et al. 2002 Nucl. Acids Res. 30, e36) was used. All expression ratios were normalized by glyceraldehyde 3-phosphate dehydrogenase expression and calibrated by the in vivo group (expression assumed as 1.00 for all genes and tissues). The analysis of relative differences on transcript levels of imprinted genes in fetuses revealed IGF2 down-regulation (P < 0.05) in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to the in vivo group and IVF fetuses (2.02). In chorioallantois, IGF2 was down-regulated (P < 0.001) in parthenotes (0.001) when compared to the in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was down-regulated (P < 0.001) in SCNT chorioallantois (0.25) when compared to the in vivo group. Low expression of IGF2 in parthenogenetic fetuses and chorioallantois confirms its imprinted status in bovine. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in bovine SCNT-derived fetuses and chorioallantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes for the low efficiency of SCNT procedures in this species. Such alterations suggest modifications in DNA methylation patterns at IGF2 and IGF2R imprinting centers.