Developmental capacity, energy metabolism and ultrastructure of mature oocytes from prepubertal and adult sheep

1996 ◽  
Vol 8 (7) ◽  
pp. 1029 ◽  
Author(s):  
JK O'Brien ◽  
D Dwarte ◽  
JP Ryan ◽  
WM Maxwell ◽  
G Evans
Keyword(s):  
Volume Fraction ◽  
Blastocyst Stage ◽  
Glutamine Metabolism ◽  
Cortical Granules ◽  
Developmental Potential ◽  
Adult Sheep ◽  
Ultrastructural Studies ◽  
Developmental Capacity

Development to the blastocyst stage was assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The proportion of cleaved oocytes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal sheep than for those from adult sheep (7.4% and 24.6% respectively). There were no differences in the metabolism of glucose, glutamine or pyruvate between oocytes matured in vivo and in vitro, or of glucose or pyruvate between oocytes from prepubertal and adult sheep. Glutamine metabolism by mature oocytes from prepubertal sheep was significantly lower than that by oocytes from adult sheep. Ultrastructural studies revealed no differences in the morphology of cytoplasmic organelles of oocytes matured in vitro from prepubertal and adult sheep, but differences in the volume fraction and size of mitochondria and cortical granules were observed. These data suggest that mature oocytes from prepubertal sheep do not possess the developmental potential of their adult-derived counterparts, and this phenomenon may be associated with metabolic and ultrastructural anomalies.

Relationship between bovine oocyte morphology and in vitro developmental potential

Zygote ◽  
2006 ◽  
Vol 14 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Masashi Nagano ◽  
Seiji Katagiri ◽  
Yoshiyuki Takahashi
Keyword(s):  
Small Diameter ◽  
Blastocyst Stage ◽  
Cortical Granules ◽  
Developmental Potential ◽  
Developmental Capacity ◽  
Sperm Penetration ◽  
Developmental Rates ◽  
Large Clusters

We investigated the relationship between the morphology of oocytes collected from small antral follicles and their developmental capacity. Immature oocytes were classified into seven groups and cultured in vitro for maturation (IVM), fertilization (IVF) and development to blastocysts (IVC). After IVF, sperm penetration and normal fertilization rates were higher in the oocytes whose cytoplasm appeared brown. The rate of polyspermy was highest in the oocytes whose cytoplasm was black. After IVC, the rates of cleavage and of development to the blastocyst stage were also higher in the brown oocytes. Although the oocytes with dark clusters in a pale cytoplasm showed lower cleavage rates, cleaved zygotes had high developmental rates the same as the oocytes with a brown cytoplasm. Transmission electron microscopy showed that the oocytes with a pale or black cytoplasm had organelles arranged differently from other oocytes before IVM. Most of the oocytes with a brown and homogeneous cytoplasm or small diameter had the characteristics of immature cytoplasm (large clusters of cortical granules) even after IVM. On the other hand, the brown oocytes with a dark zone at the periphery or with dark clusters showed the same arrangement of organelles as in vivo matured oocytes. The oocytes with a pale or black cytoplasm appeared to be degenerating and/or ageing. In conclusion, a dark ooplasm indicates an accumulation of lipids and good developmental potential, while a light-coloured ooplasm indicates a low density of organelles and poor developmental potential. A black ooplasm indicates ageing and low developmental potential.

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection

2004 ◽  
Vol 16 (6) ◽  
pp. 617 ◽  
Author(s):  
Genevieve M. Magarey ◽  
Karen E. Mate
Keyword(s):  
Glucose Metabolism ◽  
Intracytoplasmic Sperm Injection ◽  
Tammar Wallaby ◽  
Blastocyst Stage ◽  
In Vitro Fertilisation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Developmental Capacity

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte–1 h–1), in vitro-matured (0.93 pmol oocyte–1 h–1) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte–1 h–1) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17–19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

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 culturedin vitroin 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-τ) 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 betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

Morphokinetics and in vitro developmental potential of monopronucleated ICSI zygotes until the blastocyst stage

Zygote ◽  
2020 ◽  
Vol 28 (3) ◽  
pp. 217-222
Author(s):  
Silvia Mateo ◽  
Francesca Vidal ◽  
Beatriz Carrasco ◽  
Ignacio Rodríguez ◽  
Buenaventura Coroleu ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
Control Group ◽  
Study Group ◽  
Comprehensive Understanding ◽  
Developmental Potential ◽  
Morphokinetic Parameters ◽  
Developmental Capacity ◽  
Blastocyst Rate ◽  
Kinetics Of

SummaryThe aim of this study was to provide a more comprehensive understanding of 1PN intracytoplasmic sperm injection (ICSI) zygotes. To achieve this objective, we assessed whether all 1PN-derived embryos showed a similar morphokinetic pattern, and if the morphokinetic behaviour of 1PN-derived embryos was comparable with that of 2PN-derived embryos. In total, 149 1PN ICSI zygotes (study group) and 195 2PN ICSI zygotes (control group) were included in the study. Embryo development potential was evaluated in terms of blastocyst rate. Morphokinetic parameters, including the pronucleus diameter and kinetics of in vitro development, were also analyzed. Embryos derived from 1PN ICSI zygotes showed impaired development compared with 2PN-derived embryos, with blastocyst rates of 28.9% and 67.2%, respectively. The diameter of the pronucleus of 1PN zygotes was larger than that of 2PN zygotes. When compared with 2PN-derived embryos, those derived from 1PN zygotes had a visible pronucleus for a shorter time, in addition to a longer syngamy time and slower kinetic behaviour from two to nine cells. When 1PN-derived blastocysts and 2PN-derived blastocysts were compared, the developmental kinetics were similar in both groups, except for a delayed and longer duration of the compaction phase in 1PN-derived embryos. In conclusion, monopronucleated ICSI zygotes present differences in developmental capacity and morphokinetic behaviour compared with 2PN ICSI zygotes, showing particular morphokinetic parameters related to pronucleus formation. Only the 1PN ICSI-derived embryos that reached the blastocyst stage have similar morphokinetic development to blastocysts from 2PN zygotes.

94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
A. Velasquez ◽  
D. Veraguas ◽  
F. O. Castro ◽  
J. F. Cox ◽  
L. l. Rodriguez-Alvarez
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Gene Markers ◽  
Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

Abstract 4: Porcine Parthenotes as a Model to Evaluate Developmental Potential of Human Inducible Pluripotent Stem Cells

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Naoko Koyano-Nakagawa ◽  
James Dutton ◽  
Mary G Garry ◽  
Daniel J Garry
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cellular Proliferation ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Parthenogenetic Embryos

The use of human induced pluripotent stem cells (hiPSCs) has tremendous potential for regenerative medicine by providing an unlimited source of personalized cells. A number of protocols have been established for efficient differentiation of hiPSCs to the desired lineage in vitro, such as cardiomyocytes and blood. However, the field lacks an in vivo system to evaluate the differentiation potential and quality of hiPSCs. Developmental potential of stem cells derived from experimental animals can be readily assessed by generating blastocyst chimeras and examination of the contribution to the embryos, or by the potential of teratoma formation. However, this is not possible in the case of humans. As a potential solution for this issue, we examined whether porcine parthenotes could be used as an experimental model to test the developmental potential of the hiPSCs. Parthenotes are generated by electrical activation of the oocytes collected at the abattoir and will develop up to gestational day 53 if transferred to a pseudo-pregnant sow. The embryonic culture conditions have also been established and the zygotes can develop normally to the expanded blastocyst stage (day 7 post fertilization/activation), in vitro. We took advantage of this in vitro system and examined the ability of hiPSCs to proliferate and integrate into the parthenogenetic embryos. Parthenogenetic embryos were injected with ten undifferentiated hiPSCs at day 4 (8 cell ~ morula stage) and cultured up to 72 hours. During this period, parthenotes underwent blastocoel cavity formation and hatching. Cell tracing experiments demonstrated that hiPSCs proliferated and integrated into the parthenotes. They retained pluripotency marker expression during this period. hiPSCs and their derivatives were found both in trophoectoderm and embryo proper. We further observed that the hiPSCs underwent cellular proliferation and promoted developmental progression of the parthenote in vitro. In summary, the porcine parthenote model system is an efficient high throughput system to examine the developmental capacity of human stem cell populations.

scholarly journals Effects of heat stress during in vitro maturation on cytoplasmic versus nuclear components of mouse oocytes

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 181-189 ◽  
Author(s):  
Jun-Zuo Wang ◽  
Hong-Shu Sui ◽  
De-Qiang Miao ◽  
Na Liu ◽  
Ping Zhou ◽  
...  
Keyword(s):  
Heat Stress ◽  
Similar Rate ◽  
Blastocyst Formation ◽  
Cortical Granules ◽  
Cell Stage ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Mouse Oocytes

The objectives of this study were to investigate the effect of heat stress duringin vitromaturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 °C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 °C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles maturedin vivoorin vitroat 37, 40 or 40.7 °C were transplanted intoin vivomatured cytoplasts, no blastocyst formation was observed whenin vivospindles were transferred into the 40 °C cytoplasts. While oocytes reconstructed between 37 °C ooplasts and 37 or 40 °C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 °C ooplasts and 37 °C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 °C compared with oocytes matured at 37 °C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 °C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 °C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 °C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.

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