Succinate and malate improve development of hamster eight-cell embryos in vitro: Confirmation of viability by embryo transfer

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% ± 3.9 vs. 54.5% ± 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% ± 7.2), but not pyruvate (85.8% ± 6.2) or glutamine (84.1% ± 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%–58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% ± 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 ± 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20–24). The MCN was the highest (33.4 ± 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 ± 1.1) or malate (24.7 ± 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (±4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% ± 2.0) or blastocysts (28.9% ± 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium. Mol. Reprod. Dev. 47:440–447, 1997. © 1997 Wiley-Liss, Inc.     

Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: comparison of culture media

To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P < 0.03) higher in M16 medium (72.5 +/- 2.4%) than that in CZB (13.2 +/- 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 +/- 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 +/- 2.6%) and CZBG (83.9 +/- 3.9%) media than in CZB (31.9 +/- 2.8%) medium (P < 0.0001). However, percentages of morulae at 72 hr were comparable in all treatments. Developed blastocysts were better in M16 than in CZB or CZBG media. Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P < 0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos. Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P < 0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts. Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blastocysts from M16 medium produced live transgenic "green" pups (11%) following embryo transfer. Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFP-transgene-injected mouse embryos.     

One-minute exposure of 4-cell mouse embryos to glucose overcomes morula block in CZB medium

One-cell mouse embryos that block at the 2-cell stage can progress to the morula stage in CZB medium, but fail to cavitate and then swell and lyse. A 1-min exposure to 27 mM glucose at the 4-cell stage (～42 hr) will support a high frequency of development to the blastocyst stage (75%) in the same medium. A glucose exposure is beneficial anytime between 30 and 54 hr of culture (67–73% blastocysts). Of a group of additional sugars and glucose analogues tested for their ability to replace glucose, only galactose was equivalent in promoting embryo development to the blastocyst stage (64% blastocysts). © 1994 Wiley-Liss, Inc.     

Effects of amino acids and α-amanitin on bovine embryo development in a simple protein-Free medium

Five experiments, utilizing 3741 embryos produced in vitro, were designed to test the effects of Eagle's nonessential amino acids, and combinations of Eagle's essential amino acids and the RNA polymerase inhibitor α-amanitin on the development of preimplantation bovine embryos in a modified protein-free KSOM medium. Embryos were cultured in 5% O₂:5% CO₂:90% N₂ at 39°C for the first 40–44 hr in modified KSOM, and embryos with ≥4 cells were cultured in modified KSOM-PVA with different amino acids in experiments 1–4, and with the addition of α-amanitin in experiment 5. In experiment 1, addition of 0.5× of the essential amino acids, with different concentrations of nonessential amino acids significantly increased hatching of blastocysts and decreased blastocyst degeneration, but increasing the nonessential amino acids from 1× to 5×, did not stimulate embryo development. In experiments 2–4, increasing only the glycine concentration, or adding each of the 12 essential amino acids singly or several in combination to the medium containing nonessential amino acids, did not significantly improve embryo development. Taurine (0.4 mM) in the modified KSOM medium reduced blastocyst degeneration. In experiment 5, α-amanitin (20 μM) completely inhibited further embryo development when it was added at several stages from 4-cell embryos to morulae. The study with protein-free KSOM plus amino acids provided a completely defined simple medium for culturing bovine embryos, with evidence that continuous mRNA activity and presumed protein synthesis was obligatory to meet the complex and continuous requirements for proteins by the developing blastocyst. Mol. Reprod. Dev. 46:278–285, 1997. © 1997 Wiley-Liss, Inc.     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

Influence of ovine oviducal amino acid concentrations and an ovine oestrus-associated glycoprotein on development and viability of bovine embryos

This study examined the effects of incorporating an ovine oviducal oestrus-associated glycoprotein (oEGP) and amino acids, at the concentrations present in the ovine oviduct around the time of oestrus, on in vitro production and subsequent viability of bovine embryos. The first experiment compared the influence of ovine oviducal concentrations of amino acids with MEM and BME amino acids. There was no treatment effect on cleavage rate (74.9% vs. 75.5%), but there was a higher (P < 0.05) blastocyst yield (30.4 vs. 25.2) and a shorter time (P < 0.05) to blastocyst formation (7.16 ± 0.64 vs. 7.27 ± 0.56 days) following use of oviducal concentrations of amino acids. Experiment 2 examined the influence of oEGP in combination with each of the amino acid treatments. oEGP had no effect on cleavage or blastocyst yield within amino acid treatments. Day of blastocyst formation significantly influenced nuclei numbers (P < 0.001) with higher numbers being obtained on day 7 than on either day 6 or day 8. There was also a significant (P < 0.01) interaction between day of blastocyst formation and amino acid treatment on blastocyst nuclei numbers. The third experiment studied the effects of the amino acid treatments on embryo viability. There was no effect of amino acid treatment of embryos on pregnancy rates (34.5 vs. 44.4%) following transfer of days 6 and 7 blastocysts to synchronized recipients. oEGP did not influence any of the parameters of bovine embryo development that were measured, suggesting that effects of this protein observed on ovine embryos are species specific. It is concluded that ovine oviducal amino acid concentrations are beneficial to blastocyst development in vitro but do not have any further beneficial effect following transfer of blastocysts to recipients. Mol. Reprod. Dev. 47:164–169, 1997. © 1997 Wiley-Liss, Inc.     

Total protein content and protein synthesis within pre-elongation stage bovine embryos

Protein content was measured in zona-free bovine oocytes and pre-elongation stage embryos, following in vitro maturation, fertilisation, and then culture in Synthetic Oviduct Fluid medium supplemented with amino acids and 8 mg ml^-1 bovine serum albumin (BSA). Values (ng embryo^-1) of 122 ± 7.8, 137 ± 8.6, 111 ± 8.8, 115 ± 10.4, 139 ± 9.0 and 152 ± 10.1 were obtained for zona-free mature oocytes, 2-cell (day 2), 8-cell (day 3), compact morula (day 6), blastocyst (day 7), and expanded blastocyst (day 8) stage embryos, respectively. The protein content of day 7 zona-enclosed blastocysts was 337 ± 58.0 ng embryo^-1. These values suggest that prior to compaction and blastulation, the early cleavage stage bovine embryo has a higher rate of protein degradation than that of synthesis. Net growth is observed only after initiation of compaction. The protein content of day 7 blastocysts was measured in embryos following in vitro production and culture in the same media supplemented with either 0.5% w/v polyvinyl alcohol (PVA), 8 mg ml^-1 BSA, 8 mg ml^-1 BSA and further supplemented with 10% fetal calf serum (FCS) from the beginning of culture (FCS-D1), 8 mg ml^-1 BSA and 10% FCS from the fourth day of culture (day 5 of development) or from in vivo-derived day 7 blastocysts. Protein content was significantly (P< 0.05) lower in PVA-cultured embryos than other treatments. To determine if this difference in PVA-cultured embryos was due to a difference in the rate of protein synthesis, comparisons were made between day 7 embryos derived from BSA-culture and either PVA-culture, FCS-D1 culture or in vivo-derived embryos. Despite differences in diameter, no significant difference was observed in the incorporation of L-[2,3,4,5,6-³H]-phenylalanine into the TCA-precipitable fraction in any of the three comparisons made. However, incubation in the presence of FITC-labelled BSA or β-casein and examination under either fluorescence or confocal microscopy revealed that protein in the extra-embryonic environment was actively taken up by the trophectoderm of day 7 blastocysts, most likely by endocytosis. These results suggest that exogenous protein is an important nutritive source, probably maintaining intracellular amino acid pools. Results obtained from the production of embryos in protein-free medium should be viewed with the knowledge that such embryos differ metabolically from those embryos grown in the presence of protein, including in vivo-derived embryos. Mol. Reprod. Dev. 50:139–145, 1998. © 1998 Wiley-Liss, Inc.     

Relationship between age of blastocyst formation and interferon-τ secretion by in vitro–derived bovine embryos

This study was designed to examine the relationship between the speed at which bovine embryos reach the blastocyst stage, their cell number, and interferon-τ production. A total of 800 oocytes were fertilized by frozen-thawed semen. On day 2, 44 hr after exposure to sperm, 78, 320, and 296 embryos were at the two-, four-, and eight-cell stages, respectively, with an overall cleavage rate of 86.8%. Within these three groups 15 (19.2%), 106 (33.1%), and 158 (53.4%) embryos proceeded to the blastocyst stage. Of these 46.7%, 65.1%, and 63.3% hatched in the three groups, respectively. Blastocysts began to appear at day 7, but a few did not form until as late as day 13. Expanded blastocysts (n = 279) were cultured individually for 48 hr in 50-μl droplets of medium, fixed for cell counts, and the concentration of interferon-τ in the medium was determined. Blastocysts originating from two-cell embryos had significantly fewer cells (46.5 ± 23.3) than either four-cell- (97.2 ± 13.5) or eight-cell-derived embryos (113.8 ± 13.6; P < 0.05). Hatching was accompanied by an increase in cell number (129.8 ± 15.5 versus 41.9 ± 14.4; P < 0.01). Blastocysts derived from embryos that had reached the eight- or four-cell stage 44 hr after insemination produced significantly more interferon than embryos derived from two-cell embryos (941.7 ± 92.1, 930.1 ± 163.1, versus 232.8 ± 70.1 pM). In contrast, hatching, ovary batch, the speed of early cleavage, cell number, and quality grade had no effect on interferon-τ secretion. The embryo's age at blastocyst formation was not related to the number of its cells but did have a significant effect (P < 0.001) on interferon-τ production, with mean concentrations in the medium of 294.8 ± 57.9, 563.3 ± 82.0, 1126.3 ± 133.6, 1778.5 ± 297.2, 512.9 ± 82.0, 315.0 ± 157.5, and 157.5 pM among blastocysts appearing from days 7 to 13, respectively. These data suggest that blastocysts that form at days 7 and 8 produce less interferon-τ than those that form on days 9 or 10. Since early-forming blastocysts are generally considered more developmentally competent than those which form late, there may be a negative relationship between early interferon-τ production and competence. Mol. Reprod. Dev. 49:254–260, 1998. © 1998 Wiley-Liss, Inc.     

Effect of prooxidant agents added at the morula/blastocyst stage on bovine embryo development,cell death and glutathione content

Two prooxidant agents, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), a generator of free radicals in the culture medium, and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, were used to reinforce from the morula stage (day 5 post-insemination, p.i.) the oxidative stress encountered by bovine embryos in culture. Exposure to increasing concentrations of both prooxidants from the morula stage did not affect blastocyst formation but some blastocysts were found degenerated on day 8 in a dose-dependent manner (0, 0.001, 0.01, 0.1 mM AAPH gave respectively 0, 10%, 32%, 48% degeneration, while 0, 0.1, 0.2, 0.4 mM BSO led respectively to 0, 14%, 30%, 41% degeneration). Hatching rates and cell numbers of surviving blastocysts were not affected. Morulae and early blastocysts exposed from day 5 to day 6 p.i. appeared more resistant than expanded blastocysts (75-80% survival vs 20-65%; p < 0.05). Treatment with BSO significantly decreased the level of reduced glutathione in day 7 blastocysts (0.02 vs 0.42 pmol per embryo in the control) while AAPH had no effect (0.38 pmol per embryo). The proportion ofcells showing membrane lesions was increased in degenerated blastocysts from day 7.5 p.i. In AAPH-treated, but not in BSO-treated embryos, cell membrane permeabilisation seems to occur before blastocyst degeneration. DNA fragmentation evaluated by the TUNEL technique was increased in day 7 blastocysts by both prooxidants (2.8 +/- 0.4 in the control group vs 4.5 +/- 0.4 and 6.0 +/- 0.4 respectively in the AAPH- and BSO-treated groups). Addition of an inhibitor of caspase-3, DEVD-CHO, partially prevented DNA fragmentation, indicating that prooxidant treatment induced a caspase-dependent pathway of apoptosis.     

Comparison of hybrid and purebred in vitro-derived cattle embryos during in vitro culture

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P<0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P<0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.     

Nuclear transfer in cattle using in vivo-derived vs. in vitro-produced donor embryos: Effect of developmental stage

To determine the best developmental stage of donor embryos for yielding the highest number of clones per embryo, we compared the efficiencies of nuclear transfer when using blastomeres from morulae or morulae at cavitation, or when using inner-cell-mass cells of blastocysts as nuclear donors. This comparison was done both on in vivo-derived and in vitro-produced donor embryos. In experiment 1, with in vivo-derived donor embryos, nuclei from morulae at cavitation supported the development of nuclear transfer embryos to the blastocyst stage (36%) at a rate similar to that of nuclei from morulae (27%), blastomeres from morulae at cavitation being superior (P < 0.05) to inner-cell-mass cells from blastocysts (21%). The number of blastocysts per donor embryo was significantly (P < 0.05) higher when using nuclei from morulae at cavitation (15.7 ± 4.1) rather than nuclei from morulae (9.8 ± 5.5) or blastocysts (6.3 ± 3.3). With in vitro-produced donor embryos (experiment 2), nuclei from morulae yielded slightly more blastocysts (32%) than nuclei from morulae at cavitation (29%), both stages being superior to nuclei from blastocysts (15% development to the blastocyst stage). Morulae at cavitation yielded a higher number of cloned blastocysts per donor embryo (11.5 ± 5.9) than did morulae (9.3 ± 3.2) and blastocysts (3.3 ± 1.4). Transfer of cloned embryos originating from in vivo-derived morulae, morulae at cavitation, and blastocysts resulted in four pregnancies (10%), three pregnancies (7%), and one (17%) pregnancy on day 45. The corresponding numbers of calves born were 3 (4%), 3 (7%), and 0, respectively. After transfer of blastocysts derived from in vitro nuclear donor morulae (n = 16) and morulae at cavitation (n = 7), two (20%) and two (50%) recipients, respectively, were pregnant on day 45. However, transfer of seven cloned embryos from in vitro donor blastocysts to three recipients did not result in a pregnancy. Using in vitro-produced donor embryos, calves were only obtained from morula-stage donors (13%). Our results indicate that the developmental stage of donor embryos affects the efficiency of nuclear transfer, with morulae at cavitation yielding a high number of cloned blastocysts. © 1996 Wiley-Liss, Inc.     

Phosphate is required for inhibition by glucose of development of hamster 8-cell embryos in vitro

The influence of sodium dihydrogen phosphate (Pi) and glucose on the development of hamster 8-cell embryos mediated by pyruvate (P) or amino acids (A) or lactate (L) was investigated using modified Tyrode's medium, TLP-PVA. When pyruvate was tested as the only energy substrate in medium TP-PVA for embryo development, blastocyst formation ranged from 81.3 to 90.9% whether or not the medium contained 0.35 mM Pi or 5 mM glucose; but, when these two compounds were present together, blastocyst formation fell to 51.8%. Similarly, in TA-PVA medium containing four amino acids: Phe, Ile, Met, and Gln), embryo development to blastocyst ranged from 74.1% to 90.4% whether or not the medium contained 0.35 mM Pi or 5 mM glucose; but, when these compounds were present together, blastocyst formation fell to 16.0%. In TL-PVA medium, 10 mM sodium lactate supported embryo development (84.4% blastocysts); the addition of 0.35 mM Pi decreased blastocyst development to 65.6%. However, addition of glucose to Pi-free TL-PVA medium did not decrease blastocyst formation (81.3%); when the medium contained 0.35 mM Pi, glucose curtailed blastocyst development to 7.5%. When glucose and Pi interactions were studied at different concentrations, glucose up to 1 mM was not inhibitory in Pi-free TL-PVA medium (74.3% blastocysts), but 0.25 mM glucose in the presence of 0.35 mM Pi markedly inhibited embryo development (7.7% blastocysts). Phosphate at a relatively high concentration (1 mM) was inhibitory (37.9% blastocysts), even in the absence of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo

The intracellular magnesium and calcium ion concentrations of in vivo-developed 2-cell hamster embryos were measured using ratiometric fluorometry. Intracellular magnesium and calcium ion concentrations were found to be 0.369 ± 0.011 mM and 129.3 ± 7.5 nM respectively. Culture of 1-cell hamster embryos for 24 hr to the 2-cell stage in control medium containing 0.5 mM magnesium and 2.0 mM calcium resulted in approximately a threefold increase to 343.5 ± 8.0 nM in intracellular calcium ion concentration, while magnesium ion levels were not altered (0.355 ± 0.007 mM). Increasing medium magnesium concentrations to 2.0 mM significantly increased intracellular magnesium ion concentrations of cultured 2-cell embryos with a concomitant reduction in intracellular calcium ion concentrations. Furthermore, increasing the medium magnesium concentration to 2.0 mM significantly increased development of 1-cell embryos collected at either 3 or 9 hr post-egg activation to the morula/blastocyst and blastocyst stages. Resultant blastocysts had an increased total cell number and increased development of the inner cell mass. Most important, however, culture with 2.0 mM magnesium increased the fetal potential of cultured 1-cells twofold. Therefore, because highest rates of development were observed in a medium that resulted in reduced intracellular calcium ion concentrations, it appears that altered calcium homeostasis is associated with impaired developmental competence of 1-cell embryos in culture. Mol. Reprod. Dev. 50:443–450, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Expression and function of amphiregulin during murine preimplantation development

Amphiregulin (Ar) is an EGF receptor ligand that functions to modulate the growth of both normal and malignant epithelial cells. We asked whether mouse preimplantation embryos express Ar, and if so, what the function of Ar is during preimplantation development. We used RT-PCR to show expression of Ar mRNA in mouse blastocysts, and using a polyclonal anti-Ar antibody and indirect immunofluorescence, we detected the presence of Ar protein in morula- and blastocyst-stage embryos. Ar protein was present in both the cytoplasm and nucleus in both morulae- and blastocyst-stage embryos, which is similar to Ar distribution in other cell types. Embryos cultured in Ar developed into blastocysts more quickly and also exhibited increased cell numbers compared to control embryos. In addition, 4-cell stage embryos cultured in an antisense Ar phosphorothioate-modified oligodeoxynucleotide (S-oligo) for 48 hr exhibited slower rates of blastocyst formation and reduced embryo cell numbers compared to embryos exposed to a random control S-oligo. TGF-α significantly improved blastocyst formation, but not cell numbers, for embryos cultured in the antisense Ar S-oligo. From these observations, we propose that Ar may function as an autocrine growth factor for mouse preimplantation embryos by promoting blastocyst formation and embryo cell number. We also propose that blastocyst formation is stimulated by Ar and TGF-α, while Ar appears to exert a greater stimulatory effect on cell proliferation than does TGF-α in these embryos. Mol. Reprod. Dev. 47:271–283, 1997. © 1997 Wiley-Liss, Inc.     

Effect of amino acids and α-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES

Four experiments were conducted to test the effects of Eagle's non-essential amino acids (NEAA) and essential amino acids (EAA), glycine, and the RNA polymerase inhibitor α-amanitin, on the development of preimplantation rabbit embryos in modified protein-free KSOM medium. Embryos were distributed randomly into different treatments and cultured in 5% O₂:5% CO₂:90% N₂. In experiment 1, 100% of the embryos became blastocysts in the medium with Eagle's IX NEAA and 0.5X EAA, but 100% stopped development at the morula stage in KSOM without amino acids. These morulae failed to develop further when transferred to amino acid supplemented medium after 72 hr of culture. Glycine alone in modified KSOM (experiment 2) was ineffective in supporting development of 8–16-cell stage embryos past the morula stage. In experiment 3, the addition of IX NEAA and 0.5X EAA at 0, 12, 24, 36, and 48 hr of culture resulted, respectively, in 57, 65, 65, 44, and 14% blastocysts on Day 3 (P<0.05) and 86, 77, 77, 78, and 69% on Day 5 (P<0.05). Omission of Eagle's amino acids until 48 hr clearly delayed embryo development. In experiment 4, when α-amanitin (20 μM) was added to the medium containing Eagle's amino acids after 0, 12, 24, 36, and 48 hr of culture most embryos cleaved only once or twice after adding the α-amanitin. Without the inhibitor, 94% of the zygotes developed into blastocysts. These results indicate that modified KSOM or KSOM plus glycine could not support rabbit embryo development past the morula stage, but this block was overcome by adding Eagle's amino acids. An exogenous source of amino acids was not critical for embryo development during the first 24 hr of culture, but was required after that for development to equal controls. Addition of α-amanitin at multiple pre-blastocyst stages limited further embryo development to one or two cleavage divisions, with no blastocyst development. © 1996 Wiley-Liss, Inc.     

Expression of miR-Let-7a,miR-15a,miR-16-1, and their target genes in fresh and vitrified embryos and its surrounding culture media for noninvasive embryo assessment

microRNAs (miRNAs) play a critical role in implantation and development of mouse embryos. In this study, we aim to evaluate the possibility of miRNAs as potential biomarkers in the blastocyst culture to assess embryo quality. We also intend to investigate whether improved clinical outcomes of vitrified embryos agree with altered miRNA expressions. Mouse embryos from in vitro fertilization were vitrified at the two-cell stage. After thawing, the embryos were individually cultured and developed to the blastocyst stage. We used quantitative real-time polymerase chain reaction to evaluate miRNA expression levels in both vitrified and fresh groups, and culture medium (CM). The fibronectin binding assay was performed to examine for blastocyst attachment. The findings showed reduced expressions of miR-16-1 (0.2 ± 0.06) and miR-Let-7a (0.65 ± 0.1) after vitrification compared to fresh embryos. We observed significant upregulation of the target genes Vav3 (4.33 ± 0.25), integrin β-3 (Itg β3; 4.73 ± 0.2), and Bcl2 (2.29 ± 0.16) in the vitrified embryos compared to the fresh groups. Evaluation of blastocyst CM showed upregulation of miR-Let-7a (15.68 ± 0.89), miR-16-1 (16.18 ± 0.75), and miR-15a (13.36 ± 0.73) in the vitrified group in comparison to the fresh blastocysts (P < .05). The expression levels of miR-16-1 (3.28 ± 0.63), miR-15a (5.91 ± 0.38), and miR-Let-7a (9.07 ± 0.6) in CM of the vitrified blastocysts conducted on fibronectin were significantly higher than the fresh group (P < .05).This study showed that vitrification of embryos changes implantation and proliferation biomarkers. In addition, upregulated miRNAs in CM could be potentially used for noninvasive early assessment of embryo quality.     

Colcemid‐treatment of heifer oocytes enhances nuclear transfer embryonic development,establishment of pregnancy and development to term

Four experiments were designed to examine the effects of colcemid, a microtubule assembly inhibitor, on the development of bovine nuclear transfer (NT) embryos in vitro and in vivo. Recipient oocytes matured at different times were exposed to colcemid. Approximately 80–93% of the exposed oocytes, with or without the first polar body (PB1), developed obvious membrane projections. In Experiment 1, oocytes matured for either 14–15 or 16–17 hr, treated with colcemid and used as recipient cytoplasm for NT resulted in over 40% blastocyst development. In Experiment 2, oocytes matured for 16–17 hr were treated with either 0.2 or 0.4 µg/ml colcemid for 2–3 or 5–6 hr, respectively. The percentages of blastocyst development (39–42%) were not statistically different among the different colcemid treatment groups, but were both higher (P < 0.05) than the control group (30%). Colcemid concentrations and length of colcemid treatment of oocytes did not affect their ability to support NT embryo development to the blastocyst and hatched blastocyst stages. Results from Experiment 3 indicate that semi‐defined medium increases morula and blastocyst development of NT embryos derived fromcolcemid‐treated oocytes under 5% CO₂ in air atmosphere. In addition, cell numbers of blastocysts in colcemid‐treated groups were numerically higher than the control groups. After embryo transfer, higher (P < 0.05) pregnant rates were obtained from the colcemid‐treated group than the nontreated group. Five of 40 recipients (12.5%) which received embryos from colcemid‐treated oocytes delivered healthy calves, significantly higher than those recipients (3.3%) that received embryos derived from nontreated oocytes. Mol. Reprod. Dev. 76: 620–628, 2009. © 2009 Wiley‐Liss, Inc.     

Protein content and volume of early porcine blastocysts

Mean protein and volume of 222 blastocysts collected on 6 to 9 days of pregnancy were measured. Embryo protein differed (P < 0.05) for each day of development studied. Protein content of embryos doubled between days 6 and 7 and days 7 and 8 (1.2 ± 0.04, 2.0 ± 0.14, and 3.7 ± 0.2 μg, respectively). A dramatic increase from 3.7 ± 0.2 to 56.0 ± 3.4 μg was observed between days 8 and 9. Blastocyst volume increased (P < 0.05) from 0.56 ± 0.03 × 10^?2mm³ to 1.11 ± 0.04 × 10^?2mm³ between days 6 and 7, and then increased 10-fold on day 8 and five-fold on day 9. Blastocyst volume was not correlated with protein for days of development and females studied. Approximately 20% of all blastocysts within a single female contained less protein than the average protein content of all embryos from the same uterus. The results indicate that day 6 of development marks the onset of an exponential increase in embryo protein. Also, blastocyst volume is not correlated with blastocyst protein, suggesting that embryo viability is difficult to estimate by size alone. Further, approximately 20% of the blastocysts collected from a single female may exhibit reduced viability, based on reduced protein content, as early as day 6 of development.     

Effect of stage of development on survival of mouse embryos frozen-thawed rapidly

Embryos were recovered on Day 4 of pregnancy from superovulated random-bred OF₁ Swiss albino mice. They were classified into four categories based on their stage of development: expanding blastocyst, blastocyst, early blastocyst, and compacted morula. They were then cooled at 2 °C/min from ?7 to ?25 °C in a freezing medium containing 1.36 M glycerol and 0.25 M sucrose in phosphate-buffered saline (PBS). At ?25 °C, they were plunged into LN₂ and thawed a few hours later in water at 20 °C. After washing in PBS, recovered embryos were cultured for 20 to 24 hr and the number of embryos that had developed normally was recorded. The results showed a clear effect of the stage of development on survival. Survival of expanding blastocysts and blastocysts was very low (1.4 and 21.8%, respectively) compared to that of early blastocysts and compacted morulae (69.4 and 73.5%). The more differentiated stage of the blastocyst (two kinds of cells) and the presence of a blastocoelic cavity may explain the differences observed under our cooling conditions. As a further test of viability, 93 blastocysts that had developed in culture for 20 hr from 153 frozen-thawed early blastocysts and compacted morulae (60.8%) were transferred to 8 recipient mice. Seven became pregnant, yielding $\text{38}\text{82}$ normal live young (46.3%).