The amino acid requirements of the preimplantation mouse embryo in culture changes as development proceeds from the zygote to the blastocyst stage. Eagle's non-essential amino acids and glutamine significantly increased cleavage rates during the first four cell cycles, while Eagle's essential amino acids without glutamine did not confer any benefit to embryo development before the eight-cell stage. After the eight-cell stage, non-essential amino acids and glutamine no longer stimulated cleavage rates but significantly increased blastocoel development and blastocyst hatching. In contrast, after the eight-cell stage essential amino acids increased cleavage rates as well as stimulating development of the inner cell mass of the resultant blastocysts. Fetal development after transfer of blastocysts was also significantly increased by culture with essential amino acids from the eight-cell stage. Consequently highest rates of development in vitro and viability after transfer were achieved when embryos were cultured with non-essential amino acids and glutamine to the eight-cell stage followed by development to the blastocyst stage in the presence of all 20 amino acids. Analysis of the parameters measured revealed a significant relationship between number of blastocyst cells and inner cell mass development with viability after transfer. Blastocyst formation and hatching could not be used to assess subsequent developmental potential.
The effects of amino acids and ammonium on the postimplantation development of cultured preimplantation mouse zygotes were assessed. Development after transfer revealed that the mouse embryo undergoes a switch in nitrogen requirements during the preimplantation period. Although Eagle's nonessential amino acids and glutamine supported the highest implantation and fetal development rates per embryo transferred when zygotes were cultured for 48 h, by 93 h of culture the highest implantation rate was observed when all 20 amino acids were in the culture medium. Furthermore, fetal development per implantation at 69 and 93 h of culture was increased only in the presence of essential amino acids without glutamine. The beneficial effects of amino acids on postimplantation development when embryos were cultured for 4 days required that the medium be renewed after 48 h (at the 6-8-cell stage) to alleviate the build-up of ammonium. Ammonium was shown to induce fetal retardation and exencephaly in a time- and concentration-dependent manner. Renewal of amino-acid-free culture medium reduced fetal mass, providing indirect evidence for the production of an embryo-derived growth factor capable of stimulating postimplantation development. These data demonstrate that inclusion of amino acids in the culture medium for preimplantation embryos significantly increases postimplantation development the preimplantation mouse embryo changes its nitrogen requirement as development proceeds, nonessential amino acids increase the implantation rate while the essential amino acids enhance fetal development, and ammonium in the medium retards fetal development and induces the neural tube defect exencephaly.
Summary. Large White gilts, 9 to 18 months old, that had exhibited at least two natural oestrous cycles were divided into three groups (phases): unmated pre-ovulatory, unmated post-ovulatory and mated post-ovulatory (n = 16, 20 and 18). Oviductal luminal fluid samples were collected under anaesthesia by micropipette from the ampulla and ampullary\p=n-\isthmicjunction and analysed by an ultramicrofluorometric technique. Glucose concentrations (mmol l \ m=-\ 1, means combining regions; mean \ m=+-\ sem)were significantly higher in blood plasma than in oviductal fluid (4\m=.\56 \ m=+-\0\m=.\20versus 0\m=.\59\ m=+-\0\m=.\16;P < 0\m=.\0001; n = 27), whereas lactate was higher in the oviduct (5\m=.\71\ m=+-\ 0\m=.\53versus 2\m=.\480\m=.\24;P < 0\m=.\0001; n = 27). No significant differences were found between the ampulla and the ampullary\p=n-\isthmicjunction. However, the concentration of glucose was significantly higher (P < 0\m=.\05) in the ampulla of the pre-ovulatory group (0\m=.\97 \m=+-\0\m=.\20;n = 13) compared with the mated group (0\m=.\25 \m=+-\0\m=.\05;n = 14) and its concentration in the ampullary\p=n-\isthmicjunction in the pre-ovulatory group (1\m=.\65\ m=+-\ 0\m=.\63; n = 13) was significantly greater (P < 0\m=.\05) than in the post-ovulatory (0\m=.\43 \ m=+-\0\m=.\11 ; n = 11) or mated groups (0\m=.\17 \ m=+-\ 0\m=.\02; n = 14). Lactate in the ampulla of mated animals was higher than in the pre-ovulatory group (6\m=.\83 \m=+-\0\m=.\70versus 3\m=.\86\ m=+-\0\m=.\38;P < 0\m=.\05;n = 15 and 13), but neither was significantly different from the post-ovulatory group. Furthermore, no change was seen at the ampullary\p=n-\isthmic junction in lactate concentration with phase. Pyruvate concentrations showed no differences with phase, region or with plasma (oviduct 0\m=.\21\ m=+-\ 0\m=.\02;plasma 0\m=.\14\ m=+-\ 0\m=.\01; n = 27 and 26). Glucose concentration within the oviduct decreased at a time when gametes or embryos, with their liberated cumulus cells, were present. Consequently it is evident that the microenvironment of the gametes and embryos is changing and, in the light of these measurements, the composition of the media used for in vitro culture experiments may require modification to reflect the physiological levels more closely.
Vitrification appears to be a viable method for the cryopreservation of human metaphase II (MII) oocytes, but concerns regarding the concentration of cryoprotectants used during vitrification have been raised. In an attempt to circumvent this potential problem, the majority of protocols are carried out at room temperature. Exposing oocytes to temperatures below 37 degrees C, however, leads to rapid microtubule depolymerization. Polarized light microscopy was used to measure meiotic spindle retardance following exposure to cryoprotectants and vitrification in human and mouse oocytes. To quantify the extent of depolymerization, spindle retardance was determined before and after each treatment. Exposure to vitrification and warming solutions at room temperature (21-22 degrees C) caused the spindle of mouse MII oocytes to depolymerize. In contrast, no measurable changes in the meiotic spindle were detected by maintaining the temperature at 37 degrees C during the exposure regimen. By carrying out the entire vitrification and warming procedure at 37 degrees C, the spindle was also unaffected. Comparable results were obtained with vitrification of human MII oocytes at 37 degrees C. Analysis of sibling human oocytes demonstrated that slow freezing, in contrast to vitrification, was unable to preserve the meiotic spindle. Using a vitrification protocol employing 37 degrees C impacts negligibly on the meiotic spindle. Thus, fertilization can proceed without having to await spindle reformation.
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