Developmental competence of different quality bovine oocytes retrieved through ovum pick-up following in vitro maturation and fertilization

In the present study, oocytes retrieved from cross bred Karan Fries cows by ovum pick-up technique were graded into Group 1 and Group 2, based on the morphological appearance of the individual cumulus–oocyte complexes (COCs). To analyze whether the developmental potential of the COCs bears a relation to morphological appearance, relative expression of a panel of genes associated with; (a) cumulus–oocyte interaction (Cx43, Cx37, GDF9 and BMP15), (b) fertilization (ZP2 and ZP3), (c) embryonic development (HSF1, ZAR1 and bFGF) and (d) apoptosis and survival (BAX, BID and BCL-XL, MCL-1, respectively) was studied at two stages: germinal vesicle (GV) stage and after in vitro maturation. The competence was further corroborated by evaluating the embryonic progression of the presumed zygotes obtained from fertilization of the graded COCs. The gene expression profile and development rate in pooled A and B grade (Group 1) COCs and pooled C and D grade (Group 2) COCs were determined and compared according to the original grades. The results of the study demonstrated that the morphologically characterized Group 2 COCs showed significantly (P<0.05) lower expression for most of the genes related to cumulus–oocyte interplay, fertilization and embryonic development, both at GV stage as well as after maturation. Group 1 COCs also showed greater expression of anti-apoptotic genes (BCL-XL and MCL1) both at GV stage and after maturation, while pro-apoptotic genes (BAX and BID) showed significantly (P<0.05) elevated expression in poor quality COCs at both the stages. The cleavage rate in Group 1 COCs was significantly higher than that of Group 2 (74.46±7.06 v. 31.57±5.32%). The development of the presumed zygotes in Group 2 oocytes proceeded up to 8- to 16-cell stages only, while in Group 1 it progressed up to morulae (35.38±7.11%) and blastocyst stages (9.70±3.15%), indicating their better developmental potential.     

Effects of exogenous hexoses on bovine in vitro fertilized and cloned embryo development: Improved blastocyst formation after glucose replacement with fructose in a serum-free culture medium

To evaluate the embryotrophic role of three hexoses (glucose, fructose, and galactose), bovine embryos derived from somatic cell nuclear transfer (SCNT) or in vitro-fertilization (IVF) were cultured in a modified synthetic oviductal fluid (mSOF), which contained either glucose (1.5 or 5.6 mM), fructose (1.5 or 5.6 mM), or galactose (1.5 or 5.6 mM). Compared to 1.5 mM glucose, use of 1.5 mM fructose significantly enhanced blastocyst formation in both SCNT (23 vs. 33%) and IVF embryos (26 vs. 34%), while 5.6 mM fructose did not improve blastocyst formation. Using 1.5 mM galactose did not improve blastocyst formation in SCNT embryos (22 vs. 23%), whereas it significantly inhibited blastocyst formation in IVF embryos (26 vs. 0%). In both SCNT and IVF embryos, 5.6 mM glucose or galactose significantly inhibited embryo development. In a second experiment, in glucose-free mSOF, fructose at concentrations of 0.75, 1.5, 3.0, or 5.6 mM was able to support to morula (32-42 vs. 12%) and blastocyst formation (30-38 vs. 12%) compared to 0 mM fructose. In Experiment 3, addition of fructose (1.5, 3.0, or 5.6 mM) to mSOF containing 1.5 mM glucose did not further promote blastocyst formation in SCNT embryos compared with replacement with 1.5 mM fructose only. Replacement of glucose with 1.5 mM fructose significantly increased total blastomeres (143 vs. 123 cells) and trophectodermal (TE) cells (116 vs. 94 cells) and decreased inner cell mass (ICM) to TE cell ratio (0.24 vs. 0.31) in blastocysts, compared to 1.5 mM glucose. The combined addition of 1.5 mM fructose and glucose significantly increased ICM cell number (36.7 cells) and ICM/TE ratio (0.46). In conclusion, fructose might be a more efficient energy substrate than glucose for producing large number of transferable blastocysts derived from SCNT.     

Differential responses of bovine oocytes and preimplantation embryos to heat shock

The authors sought to determine whether developmental differences in the magnitude of embryonic mortality caused by heat stress in vivo are caused by changes in resistance of embryos to elevated temperature. In this regard, responses of oocytes, two-cell embryos, four- to eight-cell embryos, and compacted morulae to heat shock were compared. An additional goal was to define further the role of cumulus cells and glutathione in thermoprotection of oocytes. In experiment 1, heat shock (41°C for 12 hr) decreased the number of embryos developing to the blastocyst stage for two-cell (26% vs. 0%) and four- to eight-cell (25% vs. 10%) embryos but did not affect morulae (37% vs. 42%). In experiment 2, exposure of two-cell embryos to 41°C for 12 hr reduced the number of four- to eight-cell embryos present 24 hr after the end of heat shock (88% vs. 62%). In experiment 3, heat shock reduced the number of two-cell embryos developing to blastocyst (49% vs. 8%) but did not affect subsequent development of oocytes when heat shock occurred during the first 12 hr of maturation (46% vs. 41% development to blastocyst); membrane integrity was not altered. In experiment 4, oocytes were cultured with an inhibitor of glutathione synthesis, _DL-buthionine-[S,R]-sulfoximine (BSO), for 24 hr and exposed to 41°C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39°C), 18% (41°C), 17% (39°C+BSO), and 11% (41°C+BSO). For experiment 5, oocytes were either denuded or left with cumulus intact and were then radiolabeled with [³⁵S]methionine and [³⁵S]cysteine at 39°C or 41°C for 12 hr. Exposure of oocytes to 41°C for 12 hr reduced overall synthesis of ³⁵S-labeled TCA-precipitable intracellular proteins (18,160 vs. 14,594 dpm/oocyte), whereas presence of cumulus increased synthesis (9,509 vs. 23,246). Analysis by two-dimensional SDS PAGE and fluorography revealed that heat shock protein 68 (HSP68) and two other putative heat shock proteins, P71 and P70, were synthesized by all oocytes regardless of treatment. Heat shock did not alter the synthesis of HSP68 or P71 but decreased amounts of newly synthesized P70. Cumulus cells increased synthesis of P71 and P70. Results indicate there is a biphasic change in resistance to elevations in temperature as oocytes mature, become fertilized, and develop. Resistance declines from the oocyte to the two-cell stage and then increases. Evidence suggests a role for cumulus cells in increasing HSP70 molecules and protein synthesis. Data also indicate a role for glutathione in oocyte function. Mol Reprod Dev 46:138–145, 1997. © 1997 Wiley-Liss, Inc.     

Effect of individual heifer oocyte donors on cloned embryo development in vitro

The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7+/-0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos.     

GnRH antagonist enhance follicular growth in FSH-treated sheep but affect developmental competence of oocytes collected by ovum pick-up

This study evaluates the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from sheep treated with GnRH antagonists (GnRHa) and high doses of FSH. Eighteen Sarda ewes were treated with progestagen sponges (day 0). On day 7, 10 ewes received 3 mg of GnRHa s.c., while 8 served as control receiving saline. On day 10, all animals were treated with 96 IU of ovine FSH in four equal doses given i.m. every 12 h. We monitored follicular development by ultrasonography, twice daily from day 7 to 11, and found that GnRHa induced a significant increase in the number of total follicles in 72 h (11.7+/-0.9 to 21+/-2.4, r(2)=0.598, P<0.0001), while this number remained stable in control sheep. We found that FSH induced a significant rise in the number of follicles in both groups; but always higher (P<0.05) in GnRHa treated sheep, confirming that GnRHa enhances ovarian response to exogenous FSH stimulation. Twelve hours after the last FSH dose, oocytes were collected by OPU. Recovery percentage, morphological quality, ability to resume meiosis, fertilization and cleavage were similar in oocytes from treated and untreated sheep. However, the final blastocysts output was lower in GnRHa group (10.1% versus 27.4% in control group; P<0.05). In addition, re-expansion rates after vitrification, thawing and in vitro culture were lower in GnRHa treated ewes, although differences did not reach statistical significance (55.5% versus 74.1% in GnRHa treated and in control sheep, respectively).     

Hyperglycemia-induced apoptosis affects sex ratio of bovine and murine preimplantation embryos

The effect of glucose in the medium used during in vitro culture on both cell death by apoptosis and the sex ratio of bovine blastocysts derived from in vitro-matured and in vitro-fertilized oocytes was evaluated. Oocytes were matured, inseminated, and cultured in vitro in mSOF medium with 10% FCS with or without glucose supplementation. Exposure to high concentrations of glucose (10, 20, and 30 mM) during bovine embryo development in vitro from zygote to blastocyst resulted in a decrease in the number of cells per embryo and an increase in the frequency of apoptotic cells. A significantly higher proportion of females was found among those embryos that developed under hyperglycemic conditions in vitro. Moreover, both murine and bovine blastocysts incubated for 6 hr in 20 mM glucose had a significantly higher number of apoptotic cells in comparison to control. In this study, we also determined whether blastocyst production of the X-linked inhibitor of apoptosis protein (XIAP) differs between the sexes. Our results show that female bovine blastocysts produce significantly higher amounts of XIAP mRNA than males and this could be crucial in explaining the higher proportion of female blastocysts observed following in vitro culture under hyperglycemic conditions which induce apoptosis. Moreover, a higher proportion of female murine blastocysts cultured under hyperglycemic conditions were implanted in the uterus (65.3 of implantations from embryos cultured with 20 mM of glucose are females vs. 49% in control). This mechanism provides an explanation for the significant reduction of male children born to diabetic mothers.     

Effects of donor cells on in vitro development of cloned bovine embryos

The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The ooeytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer.The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastoeyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129)had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P<0.05), and the rate was 62.3%, 37.0%, 35.1%, and 15.6%, respectively. There was no significant difference among the rate of fusion, cleaved and blastocyst in donor cells with different foreign genes (P>0.05). It was concluded that the genetic background of the donor cells could affect the effi-ciency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.     

Ceramide inhibits development and cytokinesis and induces apoptosis in preimplantation bovine embryos

Ceramide is a second messenger induced by various cellular insults that plays a regulatory role in apoptosis. The objective of the present study was to determine whether ceramide signaling can occur in the preimplantation embryo by testing (1) effects of ceramide on development, cytokinesis, and apoptosis and (2) whether heat shock, which can induce apoptosis in embryos, causes activation of neutral or acidic sphingomyelinases responsible for generation of ceramide. Treatment of embryos > or =16 cells collected at Day 5 after insemination with 50 microM C(2)-ceramide increased caspase-9 activity and the proportion of blastomeres undergoing apoptosis but did not increase caspase-8 activity. Induction of apoptosis was more extensive when culture with ceramide was for 24 hr than for 9 hr. Ceramide also reduced the proportion of embryos that developed to the blastocyst stage when exposure was for 24 hr. At the two-cell stage, a period in development when apoptosis responses are blocked, culture of embryos with ceramide did not increase caspase-9 activity or the proportion of blastomeres that were apoptotic. However, culture with ceramide for 24 hr reduced cell proliferation and caused an increase in multinucleated cells because of inhibition of cytokinesis. Exposure of Day 5 embryos to a heat shock of 41 degrees C for 15 hr increased neutral sphingomyelinase activity but did not change acid sphingomyelinase activity. In conclusion, ceramide can regulate embryo development and apoptosis in a time and stage-of-development dependent manner and ceramide generation can be activated by cellular insult. Thus, the ceramide signaling pathway is present in the preimplantation embryo.     

Improving in vitro development of cloned bovine embryos with hybrid (Holstein-Chinese Yellow) recipient oocytes recovered by ovum pick up

In the present study, oocytes from F1 hybrid cattle, as well as their parental lines, were recovered by ovum pick up (OPU) and used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Four F1 hybrid (Holstein dam x Chinese Yellow sire), 10 Holstein and four Chinese Yellow cattle were subjected to OPU once weekly. There were no significant differences among breeds for number of recovered oocytes per session (overall average, 7.8+/-0.5; mean+/-S.E.M.), quality of the recovered oocytes, or oocyte maturation rate (72-73%). Matured oocytes were all used as recipient cytoplasm (without selection) and a single batch of cumulus cells collected from a Holstein cow were used as donor cells. Although reconstructed embryos initiated cleavage sooner when the recipient cytoplasm was from hybrid cattle versus the two parental breeds, the overall cleavage rate was indistinguishable among breeds. At Day 8, the blastocyst rate from the cleaved embryos (51% versus 37% and 27%), the total number of cells per blastocyst (135+/-4.1 versus 116+/-3.6 and 101+/-4.2), and the percentage of Grade-A (excellent quality) blastocysts (54% versus 42% and 29%) in the hybrid group were all higher than that of Holstein and Yellow groups. Furthermore, the proportion of blastocysts obtained at Day 7 (as a percentage of the total number of blastocysts) was greater in the hybrid group than in Holstein and Yellow groups (89% versus 71% and 63%). In conclusion, the use of F1 hybrid oocytes as recipient cytoplasm significantly improved in vitro development of cloned bovine embryos relative to oocytes derived from the parental lines.     

FSH different regimes affect the developmental capacity and cryotolerance of embryos derived from oocytes collected by ovum pick-up in donor sheep

The objective of the present study was to compare the developmental capacity of sheep oocytes obtained by OPU after two different ovarian stimulations, and cryotolerance to vitrification procedures of in vitro derived embryos after in vitro maturation, fertilisation and culture of these oocytes. Sheep were divided into three groups: (A) no treatment (control); (B) constant doses of FSH (FSH-c); (C) decreasing doses of FSH (FSH-d). Ovine groups FSH-c and FSH-d were synchronised by the insertion of intravaginal sponges left in situ for 7 days; FSH (total dose: 96IU) was administered in four doses given every 12h starting on Day 5. Twelve hours after the last FSH administration oocytes were collected by OPU technique. The control group showed a significantly lower number ( P<0.05 ) of follicles (166) than FSH-c (294) and FSH-d (317) groups, while the number of follicles >5mm was significantly higher ( P<0.01 ) in FSH-d group, showing that this protocol stimulates the growth of a different follicle population compared to FSH-c group. The control group showed a higher number of <2mm follicles ( P<0.01 ). We did not find any difference in oocyte quality between the three groups and therefore the percentage of discarded oocytes was similar. No significant differences were found between control, FSH-c and FSH-d groups in terms of maturation (90.9, 85.7 and 87.7%, respectively) and fertilisation rates (75.2, 80.9 and 83.7%, respectively) while a significantly higher ( P<0.01 ) blastocyst rate was observed in the FSH-c group than in the FSH-d and control groups (20.4% versus 11.8 and 13.7%, respectively). After vitrification, warming and 72 h in vitro culture, the hatching rate was significantly higher ( P<0.01 ) in the control (87.5%) and FSH-c (90.5%) groups than in the FSH-d group (66.7%). Control and FSH-c groups showed a significantly higher ( P<0.001 ) number of total cells than FSH-d group ( 217.6+/-26.5 and 203.0+/-33.2 versus 147.5+/-20.2 ), while no differences were observed in ICM cell rates in the control ( 35.6+/-3.8 ), FSH-c ( 37.1+/-4.6 ) and FSH-d ( 36.6+/-6.7 ) groups. These results indicate that donor sheep stimulated with FSH-c produced better quality oocytes and blastocysts showing better cryotolerance than ewes given the decreasing doses treatment.     

Heterogeneous nuclear transfer embryos reconstructed by bovine oocytes and camel (Camelus bactrianus) skin fibroblasts and their subsequent development

Summary This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation. This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.     

Melatonin supplementation during in vitro maturation of oocyte enhances subsequent development of bovine cloned embryos

Oocyte quality, which is directly related to reprogramming competence, is a major important limiting factor in animal cloning efficiency. Compared with oocytes matured in vivo, in vitro matured oocytes exhibit lower oocyte quality and reprogramming competence primarily because of their higher levels of reactive oxygen species. In this study, we investigate whether supplementing the oocyte maturation medium with melatonin, a free radical scavenger, could improve oocyte quality and reprogramming competence. We found that 10⁻⁹ M melatonin effectively alleviated oxidative stress, markedly decreased early apoptosis levels, recovered the integrity of mitochondria, ameliorated the spindle assembly and chromosome alignment in oocytes, and significantly promoted subsequent cloned embryo development in vitro. We also analyzed the effects of melatonin on epigenetic modifications in bovine oocytes. Melatonin increased the global H3K9 acetylation levels, reduced the H3K9 methylation levels, and minimally affected DNA methylation and hydroxymethylation. Genome-wide expression analysis of genes in melatonin-treated and nontreated oocytes was also conducted by high-throughput RNA sequencing. Our results indicated that melatonin ameliorates oocyte oxidative stress and improves subsequent in vitro development of bovine cloned embryos.     

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.