Isolation and Maturation of Germinal Vesicle Stage Mouse Oocytes
Immature germinal vesicle stage oocytes are obtained when mice are around 22-26 days of age. It has been reported that using mice between 4 and 5 weeks old results in a low yield of quality oocytes 19 . Oocytes surrounded by granulosa cells are aspirated from ovaries 44-46 h after PMSG injection. Ovaries are removed from the female in warmed handling media. The cumulus-enclosed oocytes can then aspirated from the follicles, washed, and placed into maturation media to mature overnight see...
Injection
The oocyte is held in place by the suction applied through the holding pipette Figure 5.2 . During the injection procedure, a better grip of the egg is established when the inferior pole of the oocyte is touching the bottom of the dish. The injection pipette is lowered, and the outer right border of the oolemma on the equatorial plane at 3 o'clock is brought Figure 5.1. Immobilized human spermatozoon. The sperm flagellum is kinked by mechanical action 600x . Figure 5.1. Immobilized human...
Metabolic Pathway Activity
1. SIGNIFICANCE OF METABOLISM IN EMBRYO DEVELOPMENT The early development of the mammalian embryo is directed by its inherent genetic programming and affected by its environment. Both the genetic program and the environment act as stimuli to the early embryo to induce developmental responses, initially in its intracellular biochemical activity, and ultimately in cell division, differentiation, and function. Techniques for manipulating gene expression and development, such as gene transfer,...
Freefloating Organ Cultures Rodent and Domestic Species
In this system, either whole ovaries from mice 1 or cortical sections bovine, primate are placed in culture medium, free floating with or without a collagen membrane. 1. Dissect mouse ovaries on the day of birth day 0 and cut in half. 2. Alternatively, remove thin strips of ovarian cortex from the ovaries of domestic species using a scalpel blade and watchmaker's forceps. Trimmed strips to 0.5 x 0.5 x 0.2 mm. 3. Transferred ovaries in a drop of culture medium Protocol 1.1 using a Pasteur...
Conclusions
We have outlined the three methods of embryo biopsy in this chapter. Worldwide, the majority of clinics are performing cleavage-stage biopsy at the six- to eight-cell stage, using acid Tyrodes to drill the zona and aspiration to remove the blastomeres 3 . There has been an increase in the use of the laser for zona drilling 2, 3 , but the cost of the laser is still high, and its use has not been approved in every country. For those performing cleavage-stage biopsy, the decision whether to take...
Contents
1. Follicle Development in Vitro 3 Norah Spears, Evelyn E. Telfer, and Alison A. Murray 2. Preparation of Gametes, in Vitro Maturation, in Vitro Fertilization, and Embryo Recovery and Transfer 24 3. Culture of the Mammalian Preimplantation Embryo 41 4. Assessment of Preimplantation Embryo Development and Viability 62 Denny Sakkas, Michelle Lane, and David K. Gardner 5. Micromanipulation of Gametes 76 Gianpiero D. Palermo, Queenie V. Neri, Takumi Takeuchi, Lucinda L. Veeck, and Zev Rosenwaks 6....
Microinjection 241 Setting
Immediately before injection, 1 ml of the sperm suspension is diluted with 4 ml of a 10 PVP solution approximately 300 mOsmol PVP-K 90, molecular weight 360,000 ICN Biochemicals, Cleveland, OH in M-HEPES medium placed in the middle of the plastic Petri dish. The viscosity of this solution slows down the aspiration and prevents sperm cells from sticking to the injection pipette. Avoidance of too many spermatozoa in the PVP-containing droplet also reduces the level of contaminants that can be...
Methods of Zona Drilling 231 Acid Tyrodes Drilling
Acid Tyrodes has been used in embryology since 1986 52 and has been the best choice to date for cleavage-stage embryo biopsy 3 Figures 6.2a see also Figure 6.4 . Acid Tyrodes solution is commercially available Medicult, Denmark, or Vitrolife, Sweden or can be made in house. The pH of acid tyrodes should not be lower than 2.2, as this can be detrimental in lowering the pH of the biopsy drop when drilling. Cleavage-stage embryo biopsy using acid tyrodes usually incorporates the use of a double...
Methods of Blastomere Removal 241 Aspiration
Aspiration is the most commonly used method to remove blastomeres for clinical embryo biopsy after drilling with either acid tyrodes, laser, or PZD 3 Figure 6.3a and 6.4 . A finely fire polished biopsy pipette 30-40 mm internal diameter containing biopsy medium is inserted close to the aperture, and the blastomere in contact with the pipette is aspirated using gentle suction with either hydraulic, pneumatic, or mouth-controlled syringes. The most important factor determining a successful biopsy...
Advantages and Disadvantages of Polar Body Biopsy
The advantages of polar body biopsy are that, first, it is ethically more acceptable to certain countries e.g., Malta and Germany where research and discarding embryos is prohibited. Second, the first and second polar bodies may not be required for further embryonic development and hence the viability of the oocytes and resulting embryos may be equally maintained. One disadvantage of polar body biopsy is that it can only be applied to diagnose maternally inherited diseases. Recombination and...
Assessment of Nutrient Uptake Metabolite Production and Enzyme Activity
The ability to measure the uptake and production of substrates by single embryos is an important tool for understanding how embryos control metabolism and embryo production. Little is known about the control of metabolism in the mammalian embryo compared to somatic cells or embryos from marine species or Xenopus. One of the main reasons for this is that the small amounts of material in the early embryo make measurement technically difficult. In recent years microfluorometric techniques have...
Contributors
Departments of Obstetrics and Gynecology Division of Reproductive Medicine , and Cellular and Molecular Medicine University of Ottawa, Canada Molecular and Cellular Biology Program School of Life Sciences Arizona State University Tempe, Arizona, USA Department of OB GYN and Physiology The University of Western Ontario London, Ontario, Canada Parvathi K. Basrur Department of Biomedical Sciences Ontario Veterinary College University of Guelph Guelph, Ontario, Canada Department of Biomedical...