Developmental competence of human in vitro aged oocytes as host cells for nuclear transfer.

BACKGROUND: Improving human nuclear transfer (NT) efficiencies is paramount for the development of patient-specific stem cell lines, although the opportunities remain limited owing to difficulties in obtaining fresh mature oocytes. METHODS: Therefore, the developmental competence of aged, failed-to-fertilize human oocytes as an alternate cytoplasmic source for NT was assessed and compared with use of fresh, ovulation-induced oocytes. To further characterize the developmental potential of aged oocytes, parthenogenetic activation, immunocytochemical analysis of essential microtubule proteins involved in meiotic and mitotic division, and RT-PCR in single oocytes (n = 6) was performed to determine expression of oocyte-specific genes [oocyte-specific histone 1 (H1FOO), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zygote arrest 1 (ZAR1)] and microtubule markers [nuclear mitotic arrest (NuMA), minus-end directed motor protein HSET and the microtubule kinesin motor protein EG5]. RESULTS: For NT, enucleation and fusion rates of aged oocytes were significantly lower compared with fresh oocytes (P < 0.05). Cleavage rates and subsequent development were poor. In addition, parthenote cleavage was low. Immunocytochemical analysis revealed that many oocytes displayed aberrant expression of NuMA and EG5, had disrupted meiotic spindles and tetrapolar spindles. One of the six oocytes misexpressed GDF9, BMP15 and ZAR1. Two oocytes expressed EG5 messenger RNA (mRNA), and HSET and NuMA were not detectable. RT-PCR of mRNA for oocyte specific genes and microtubule markers in single aged oocytes. CONCLUSIONS: Thus, aneuploidy and spindle defects may contribute to poor parthenogenetic development and developmental outcomes following NT.

Effects of high concentrations of hyaluronan in culture medium on development and survival rates of fresh and frozen-thawed bovine embryos produced in vitro.

Hyaluronic acid (HA) is the main glycosaminoglycan present in follicular, oviductal and uterine fluids. The main functions of HA include dynamic processes that are mediated through interaction with extracellular matrix components, regulation of gene expression, cell proliferation and cell differentiation. HA increases the viscosity of solutions and also has several physiological functions, including regulation of water distribution and water-binding capacity. The addition of 6 mg HA ml(-1) to synthetic oviduct fluid (SOF; SOF-HA) culture medium on day 5 (IVF = day 0) significantly (P < 0.001) increased the viscosity of the medium in comparison with SOF culture medium containing BSA (SOF-BSA). On day 8, rate of blastocyst development in SOF-HA culture medium was significantly (P < 0.05) higher than in SOF-BSA culture medium (38.2 versus 29.3%). The number of trophectoderm cells and the total number of cells of expanded blastocysts cultured in the presence of HA were significantly (P < 0.01) higher in comparison with expanded blastocysts cultured in the presence of BSA (88.9 +/- 7.3 versus 67.6 +/- 3.0 and 130.1 +/- 10.9 versus 104.8 +/- 2.5, respectively). After freezing and thawing, the percentage of day 8 embryos that re-expanded and hatched when cultured with SOF-HA was greater than that of embryos cultured with SOF-BSA (11.3 and 10.5% versus 75.5 and 36.8%, respectively). After thawing, the ATP contents of in vivo-derived, SOF-HA and SOF-BSA expanded blastocysts were similar. The embryos cultured with HA showed less ultrastructural deviation and de-differentiation after freezing and thawing than the embryos cultured with BSA. This study demonstrates that HA improves the developmental capacity of bovine embryos under in vitro conditions and is warranted as a supplement for in vitro production of bovine embryos, particularly if they are to be cryopreserved.

Energy status of nonmatured and in vitro-matured domestic cat oocytes and of different stages of in vitro-produced embryos: enzymatic removal of the zona pellucida increases adenosine triphosphate content and total cell number of blastocysts.

In this study, we evaluated the adenosine triphosphate (ATP) content of individual domestic cat oocytes before and after in vitro maturation and of different stages of in vitro-produced embryos. To investigate the effects of assisted-hatching technique on the ATP content and total cell number, the zona pellucida of in vitro-produced blastocysts and expanded blastocysts (recovered 144 h postinsemination [hpi]) was completely removed by pronase treatment. The average (mean +/- SEM) ATP content of nonmatured oocytes (3.47 +/- 0.18 pmol) was significantly (P < 0.01) higher than that of in vitro-matured oocytes (2.17 +/- 0.10 pmol). After in vitro fertilization and culture, the ATP content of two-cell stages (24 hpi) was 1.17 +/- 0.08 pmol, which increased to 1.47 +/- 0.19 and 1.88 +/- 0.32 pmol at the four- (40 hpi) and eight-cell (48 hpi) stages, respectively. The ATP content then decreased to 1.48 +/- 0.10 pmol in 16-cell embryos (64 hpi), reaching a minimum of 0.49 +/- 0.04 pmol at the morula stage (120 hpi). Blastocysts, expanded blastocysts (both 144 hpi), and hatching blastocysts (192 hpi) revealed ATP levels of 1.05 +/- 0.09, 1.79 +/- 0.01, and 4.17 +/- 0.21 pmol, respectively. After enzymatic removal of the zona pellucida (ERZP) at 144 hpi, ATP content and total cell numbers of blastocysts (4.15 +/- 0.37 pmol of ATP, 328.3 +/- 48.5 cells) and expanded blastocysts (5.81 +/- 0.54 pmol of ATP, 430.1 +/- 29.7 cells) analyzed at 192 hpi were significantly (P < 0.001) higher than in their nontreated counterparts (blastocysts: 1.00 +/- 0.09 pmol of ATP, 65.3 +/- 4.6 cells; expanded blastocysts: 1.79 +/- 0.11 pmol of ATP, 121.4 +/- 6.5 cells). Our study describes, to our knowledge for the first time, changes in the energy status of domestic cat oocytes before and after maturation and during in vitro development after fertilization. The ERZP markedly increased the ATP content and total cell number of blastocyst stages, suggesting that this technique may improve the quality and viability of in vitro-produced domestic cat embryos.

Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture.

In this study, we evaluated mitochondrial distribution and ATP content of individual bovine oocytes before and after in vitro maturation (IVM). Cumulus-oocyte complexes were classified according to morphological criteria: category 1, homogeneous oocyte cytoplasm, compact multilayered cumulus oophorus; category 2, cytoplasm with small inhomogeneous areas, more than five layers of compact cumulus; category 3, heterogeneous/vacuolated cytoplasm, three to five layers of cumulus including small areas of denuded zona pellucida; category 4, heterogeneous cytoplasm, completely or in great part denuded. In immature oocytes, staining with MitoTracker green revealed mitochondrial clumps in the periphery of the cytoplasm, with a strong homogenous signal in category 1 oocytes, a weaker staining in category 2 oocytes, allocation of mitochondria around vacuoles in category 3 oocytes, and poor staining of mitochondria in category 4 oocytes. After IVM, mitochondrial clumps were allocated more toward the center, became larger, and stained more intensive in category 1 and 2 oocytes. This was also true for category 3 oocytes; however, mitochondria maintained their perivacuolar distribution. No mitochondrial reorganization was seen for category 4 oocytes. Before IVM, the average ATP content of category 1 oocytes (1.8 pmol) tended to be higher than that of category 2 oocytes (1.6 pmol) and was significantly (P < 0.01) higher than in category 3 (1.4 pmol) and 4 oocytes (0.9 pmol). The IVM resulted in a significant (P < 0.01) increase in the average ATP content of all oocyte categories, with no difference between oocytes extruding versus nonextruding a polar body. After in vitro fertilization (IVF) and culture, significantly (P < 0.05) more category 1 and 2 than category 3 and 4 oocytes developed to the morula or blastocyst stage (determined 168 h after IVF). Total cell numbers of expanded blastocysts derived from category 1 and 2 oocytes were significantly (P < 0.05) higher than of those originating from category 3 and 4 oocytes. These data indicate that mitochondrial reorganization and ATP levels are different between morphologically good and poor oocytes and may be responsible for their different developmental capacity after IVF.

Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.

Coenzyme Q(10) (CoQ(10)) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ(10) is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ(10) is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ(10), inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 microM CoQ(10) (66.5 +/- 0.8% and 68.7 +/- 1.1%, respectively) and lowest in 10 microM CoQ(10) (55.3 +/- 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 +/- 0.6% and 25.2 +/- 0.3% in medium supplemented with 10 microM and 30 microM CoQ(10), respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 +/- 0.4% and 11.3 +/- 0.4%). In the presence of 30 microM CoQ(10), significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 +/- 1.3 vs. 8.4 +/- 2.2). Expanded blastocysts produced in the presence of 30 microM CoQ(10) had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer. Our results show that noncrystalline CoQ(10) in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system.