Increasing carbon dioxide from five percent to ten percent improves rabbit blastocyst development from cultured zygotes.

One-cell rabbit zygotes were cultured at 39 degrees C in basal synthetic medium II (BSM-II) with 5%, 10%, or 15% CO2 and humidified air to determine the effect of CO2 concentration on development in vitro. After 4 days in culture, 37% of the embryos grown in 10% or 15% CO2 had reached the hatching blastocyst stage, but only 10% of the embryos were hatching when cultured under 5% CO2 (P = 0.01). Over all blastocysts, cell numbers were 207, 246, and 205 for the 5%, 10%, and 15% CO2 treatments, respectively. In a second experiment to determine if there was a beneficial effect, particularly at the blastocyst stage, of a higher concentration of CO2, embryos were cultured 4 days in either 5% or 10% CO2 or for 2 days in 5% CO2 followed by 2 days in 10% CO2. The numbers of blastomeres per embryo and embryo diameter were greater (P < 0.05) in embryos cultured continuously in 10% CO2 or in 10% CO2 only during days 3 and 4 of culture than in embryos cultured continuously in 5% CO2. In a third experiment, one-cell rabbit zygotes were cultured with 5% or 10% CO2 in a defined, protein-free medium consisting of 1:1 RPMI 1640 and Dulbecco's modified Eagle's medium. The proportion of embryos hatching and cell counts were significantly greater (P < 0.01) when cultured in the presence of 10% CO2. These data indicate that a 10% CO2 atmosphere exerts a beneficial effect on the development of zygotes into expanding and hatching rabbit blastocysts in vitro.     

An improved culture medium for mouse blastocysts

Summary Eagle's basal medium, modified to contain essential amino acids at the concentrations optimal for mouse blastocyst hatching, attachment, and outgrowth, supported in vitro development of the mouse blastocyst better than other tissue culture media tested. This medium was improved for growth and differentiation of the inner cell mass by doubling the concentration of amino acids and glucose and by adding uridine (10⁻⁵ M) and β-mercaptoethanol (10⁻⁵ M). In this improved medium nearly all blastocysts grown from the two-cell stage hatched and formed trophoblast outgrowths, and 62% developed into two-layer egg cylinders. This work was supported by the U.S. Department of Energy.     

The stimulating effect of recombinant cytokine LIF on the mouse blastocysts during implantation

The effect of recombinant LIF cytokine (Leukemia inhibitory factor) on the isolated mouse embryos at the stages of middle and late blastocyst has been investigated. We have demonstrated here that this agent is necessary in vitro at the stage of normal trophoblast formation after the blastocysts hatch from zona pellucida. This cytokine (10 ng/ml) caused intensification of adhesion and proliferative activity of the trophoblast cells. This is important for intercellular interactions with endometrium and for invasion of embryos into the uterus. The recombinant LIF insignificantly influenced cells of the inner cell mass.     

Morphological development,cell number,and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: The effect of IGF-I

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5–9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 ± 21.4, which significantly decreased to 85.6 ± 17.4, 62.0 ± 14.5, and 17.0 ± 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5–5.5. Blastocysts developed, at the earliest, between days 5.0–6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 ± 11.3, including 59.2 ± 8.4 cells of TE and 42.1 ± 6.9 cells of ICM. © 1996 Wiley-Liss, Inc.     

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved (P < 0.05) by the addition of 10 μM Y-27632 (94.9 ± 2.4%, mean ± SEM) compared to a control (78.0 ± 6.0%). Conversely, after 48 h of culture, there were no significant differences in hatching rate (62.8 ± 11.1 vs. 59.6 ± 9.4%) and mean total cell number (135.2 ± 13.1 vs. 146.7 ± 13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7 ± 3.8 vs. 54.7 ± 8.9%, P < 0.05), with no difference in mean total cell number of blastocysts (230.0 ± 23.0 vs. 191.2 ± 22.2, P = 0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5 ± 2.1, 31.4 ± 2.3, 36.2 ± 3.2, and 28.6 ± 6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.     

Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age

The objective was to investigate the effects of developmental stage (fully-expanded or expanding blastocysts) and/or age (harvested on Days 7 or 8) on post-vitrification in vitro survival of bovine blastocysts derived from intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Post-warming survival (re-expansion of blastocoele within 24 h) of ICSI-derived fully-expanded blastocysts (80%) was similar to that of their IVF-derived counterparts (88%). However, the ability of ICSI-derived expanding blastocysts to survive vitrification procedures (61%) was lower than that of IVF-derived blastocysts (85%; P < 0.05), although the ICSI- and IVF-derived fresh blastocysts were of similar quality. The age of the blastocysts before vitrification did not affect cryotolerance for either ICSI-derived (73 and 59% for Days 7 and 8 embryos, respectively) or IVF-derived blastocysts (86% for both Days 7 and 8 embryos). At 24 h of post-warming culture, ICSI-derived blastocysts surviving vitrification contained a higher proportion of dead cells than their IVF-derived counterparts (5-13% vs. 2-4%; P < 0.05), but these proportions were not different from those of fresh control embryos. There was an adverse effect of vitrification on the ability of blastocysts to hatch within 72 h of culture only in IVF-derived Day 8 blastocysts (41 and 70% in vitrified and fresh control groups, respectively). In conclusion, the proportion of blastocysts that survived vitrification procedures was similar for ICSI- and IVF-derived bovine blastocysts if the former were cultured to the fully-expanded stage prior to vitrification, with no significant difference between embryos harvested on Day 7 versus Day 8.     

Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation.     

Predictive variables of in vitro fertilization and pre-implantation embryo development in the mouse

The present study aims to analyze the cause-effect relationships among several in-vitro fertilization and pre-implantation embryo development variables in the mouse. Superovulation of hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of pregnant mare's serum gonadotropin at the estrus stage of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Ovulated cumulus-enclosed oocytes were inseminated with sperm from hybrid males of 12-16 weeks of age. The multiple linear regression analyses performed indicated that (a) total number of ovulated oocytes is a good predictor of both fertilization frequency and total number of cells in day-5 blastocysts; (b) fertilization frequency predicts percentage of day-5 blastocysts; (c) total number of cells in day-5 blastocysts is predicted by percentage of day-5 blastocysts; and (d) total number of cells in day-5 blastocysts predicts percentage of apoptotic cells, number of inner cell mass (ICM) and trophectoderm (TE) cells, and ICM/TE ratio in day-5 blastocysts. Mitotic index in day-5 blastocysts was positively correlated with total number of ovulated oocytes, percentage of ovulated cumulus-enclosed oocytes, fertilization frequency, percentage of day-5 blastocysts and total number of cells in day-5 blastocysts. On the contrary, it was negatively correlated with percentage of apoptotic cells in day-5 blastocysts.