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Research studies using animal gametes led to the first documented human IVF in 1969. A key contribution was the development of a reliable culture medium for IVF of hamster oocytes, which was then used successfully with human gametes in the laboratory. This article describes how this was accomplished. Hamster IVF was obtained using a bicarbonate-buffered culture medium based on Tyrode's solution. A dose-response experiment showed that IVF in this species was highly dependent on the pH of the culture medium. At pH 7.2 or less, very few oocytes were penetrated and spermatozoa did not undergo acrosome reactions. Over the pH range 7.3-7.5, 40% of oocytes were fertilized in vitro, while at pH 7.6 or higher, more than 75% of oocytes were fertilized and most spermatozoa exhibited acrosome reactions. There was no apparent effect of pH on sperm motility. These experiments showed that in the hamster, fertilization in vitro is highly pH-dependent, most likely through pH effects on the sperm acrosome reaction and sperm:zona binding. The data led to formulation of a culture medium, Tyrode-B, that consistently supported high levels of IVF of hamster oocytes. When this medium was used for human gametes, IVF was observed and documented for the first time. Spermatozoa penetrating through the zona pellucida and sperm components within the ooplasm were detected, and some oocytes exhibited two pronuclei. These observations suggested that human IVF might be useful for alleviation of infertility. [ABSTRACT FROM AUTHOR]

Vitrification of hamster 2-cell embryos impairs the activity of both the Na(+)/H(+) antiporter and HCO(3)(-)/Cl(-) exchanger; the two transport proteins responsible for the regulation of intracellular pH (pHi). The activities of both the Na(+)/H(+) antiporter and HCO(3)(-)/Cl(-) exchanger were significantly reduced at 4 h following warming compared to freshly collected embryos. Normal levels of activity of both transporters were not restored until 6 h after warming. Thus, cryopreservation of cleavage stage hamster embryos has a detrimental effect on their ability to maintain intracellular ionic homeostasis. Impairment of these pHi regulatory proteins resulted in the pHi of embryos being significantly elevated from the control values of 1.2 to 7.35 for approximately 4 h after warming. In addition, an elevated pHi value significantly impaired oxidative metabolism. Therefore, the loss in developmental competence of embryos following cryopreservation may in part be explained by a reduced ability to regulate intracellular pH that results in perturbations in metabolism and disruption of energy production. [ABSTRACT FROM AUTHOR]

The effects of water-soluble vitamins, singly or in combinations, on development of hamster 1-cell embryos were examined in a protein-free, chemically defined culture medium, HECM-6. Pantothenate significantly stimulated blastocyst development compared to the vitamin-free control and to every other single vitamin, except thiamine. Ascorbic acid, biotin, choline, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine had no detectable stimulation or inhibition on cleavage stage development or morula/blastocyst formation. When combinations of vitamins were tested, embryo development was either unchanged or significantly greater than in the control, but never significantly greater than development with pantothenate alone. A dose response to pantothenate indicated that 3 micromol/l was the optimum concentration. After embryo transfer, the percentage of live fetuses recovered per 100 1-cell embryos cultured in HECM-6 plus pantothenate (now designated HECM-9) was 24%, significantly higher than the 11% recovered from 100 1-cell embryos cultured in HECM-6 alone. This is the first report to show a stimulatory effect of a single vitamin on in-vitro development of preimplantation embryos in any mammalian species. [ABSTRACT FROM AUTHOR]

A model to study mechanisms controlling nuclear and cytoplasmic maturation of primate oocytes is being developed in our laboratory. The high incidence of pregnancy failure in women following in-vitro fertilization (IVF) may be partly attributed to inadequate cytoplasmic maturation of oocytes. Advancement of knowledge of mechanisms controlling primate oocyte maturation would have important implications for treatment of human infertility, and would potentially increase numbers of viable non-human primate embryos for biomedical research. Use of a non-human primate model to study oocyte and embryo biology avoids legal, ethical and experimental limitations encountered in a clinical situation. Using this model, the meiotic and developmental capacity of oocytes from three sources have been compared: (i) in-vivo matured oocytes from monkeys stimulated with follicle-stimulating hormone (FSH) and human chorionic gonadotrophin, (ii) in-vitro matured oocytes from monkeys primed with FSH, and (iii) in-vitro matured oocytes from non-stimulated monkeys. This work demonstrates that oocyte developmental competence is likely acquired both during follicle development, before meiotic resumption, and during meiotic progression, concurrent with nuclear maturation. Potential causes of developmental failure of in-vitro matured oocytes, implications for human infertility, and future strategies to study the regulation of primate oocyte maturation are discussed. [ABSTRACT FROM AUTHOR]

The behaviour of bovine, equine and human blastocysts was studied in vitro by time-lapse videomicrography and computer imaging. This study revealed that cytoplasmic extensions of the trophectoderm ['trophectoderm projections' (TEP)] were expressed by embryos of all three species, prior to or during zona escape. Bovine and human blastocysts escaped their zonae with a combination of blastocoele expansion, collapse and re-expansion coupled with the penetration of the zona pellucida by TEP. In equine embryos, after several cycles of blastocoele expansion and collapse, trophectoderm ruptured the zona with the concomitant appearance of TEP. This study provides documentation that TEP are expressed by a diverse range of mammalian species, bringing the total number of species in which this phenomenon is found to six, since TEP are also known to be expressed by guinea-pig, hamster and rhesus monkey blastocysts, representing rodents, ungulates and primates. In all species studied, the dynamic nature (extension, retraction, and angular movement) of the TEP was similar, moving in an undulating manner with rapid cycles of extension and retraction. Because TEP appear to be a general feature of mammalian blastocysts, they are implicated in one or more key events in early development, namely zona escape, attachment and/or implantation. [ABSTRACT FROM AUTHOR]

The formulation of chemically defined culture media that support primate embryo development would facilitate studies on primate preimplantation embryogenesis. The specific aims of this study were (i) to evaluate the development of the macaque embryos in a simple, chemically defined, protein-free medium developed for a rodent embryo model, and (ii) to determine if a two-step progressive culture system could enhance blastocyst development and zona escape. In experiment 1, in-vitro-fertilized pronucleate-stage embryos (n = 81) from nine monkeys were randomly allocated to one of three treatments: (a) hamster embryo culture medium-6 (HECM-6; chemically defined, protein-free medium), (b) CMRL-BCS medium (modified CMRL-1066 medium containing 20% bovine calf serum; BCS), and (c) a two-step culture procedure (HECM-6 through to the 8- to 12-cell stage, and CMRL-BCS medium beyond that stage). Optimal development was attained equally (P > or = 0.05) with embryos cultured in CMRL-BCS medium or the two-step procedure (48 and 61%), but not to the blastocysts respectively). HECM-6 alone supported development to the morula stage (72%) equally as well as CMRL-BCS medium (80%) or the two-step procedure (69%), but not to the blastocyst stage (22 versus 48 and 61% respectively). Hatching of the blastocysts was essentially limited to the serum-containing media (CMRL-BCS medium, 31%; two-step procedure, 44%). In experiment 2, in-vitro-fertilized pronucleate-stage embryos (n = 87) from nine monkeys were randomly placed in each of four two-step treatments: (a) HECM-6 through to the 8- to 12-cell stage and CMRL-BCS medium beyond that stage, (b) HECM-6 through the 8- to 12-cell stage and HECM-6-BCS beyond that stage, (c) HECM-6 through to the morula stage and CMRL-BCS medium beyond that stage, and (d) HECM-6 through to the morula stage and HECM-6-BCS beyond that stage. Greater (P < or = 0.05) percentages of embryos developed into blastocysts, expanded blastocysts and hatched blastocysts when switched at the 8- to 12-cell versus the morula stage in the second step medium. When transferred into BCS-containing medium at either the 8- to 12-cell or morula stage, embryos underwent blastulation and expansion equally well in CMRL-BCS medium versus HECM-6-BCS. However, when embryos were switched to the second step medium at the 8- to 12-cell stage, hatched blastocysts were obtained more (P < or = 0.05) frequently in CMRL-BCS medium (50.9%) than in HECM-6-BCS (37%). This work is the first to produce in-vitro-fertilized primate blastocysts cultured from the pronucleate stage in chemically defined, protein-free medium, and demonstrates that while primate embryos can form morulae in such a medium, their requirements for blastocoel formation and zona escape appear to be more demanding, and may be acquired as early as the 8-cell stage. [ABSTRACT FROM AUTHOR]

The specific aims of this study were to determine if culture with freshly recovered granulosa cells obtained from follicle stimulating hormone (FSH)-primed versus non-stimulated monkeys during in-vitro maturation (IVM) improves the meiotic and development capacity of oocytes from non-stimulated macaque monkeys and confers on them competence to develop into blastocysts in vitro. Antral follicles > or = 1 mm in diameter were dissected from the excised ovaries of 11 non-stimulated rhesus monkeys. Cumulus-enclosed germinal vesicle-stage oocytes (n = 282) were randomly cultured in each of three IVM treatments; (i) control (no granulosa cells), (ii) non-stimulated granulosa cells (5 x 10(6) live cells/ml) from non-stimulated monkeys, or (iii) FSH-primed granulosa cells (5 x 10(6) live cells/ml) from monkeys primed with purified FSH for 8.5 days. All treatments contained gonadotrophins (5 micrograms/ml FSH; 10 micrograms/ml luteinizing hormone). Oocytes were cultured for 36-41 h in 25 microliters drops (one to 10 oocytes per drop) of modified CMRL-1066 medium containing 20% bovine calf serum, and then inseminated and cultured in the same medium (minus gonadotrophins) until developmental arrest or zona escape. Nuclear maturation, fertilization and cleavage to the 8-cell stage did not differ for the three treatment methods. However, development to the 9- to 15-cell stage was improved (P < or = 0.01) by culture with either non-stimulated granulosa or FSH-primed granulosa cells compared with control. The culture of oocytes with FSH-primed granulosa but not non-stimulated granulosa cells enhanced (P < or = 0.05) development to the morula stage compared with control (23, 11 and 5% respectively). Oocytes cultured with FSH-primed granulosa cells were more (P < or = 0.05) competent to develop into blastocysts (6.5%) than controls (0.5%). In conclusion, the culture of immature oocytes from non-stimulated monkeys with fresh granulosa cells recovered from FSH-primed monkeys enhanced their competence to develop into morulae and blastocysts in vitro, and resulted in the first blastocysts produced in vitro from in-vitro-matured/in-vitro-fertilized oocytes of non-stimulated macaques. [ABSTRACT FROM AUTHOR]

Specific aims of this study were to determine effects of granulosa cells and gonadotrophins on the meiotic and developmental competence in vitro of oocytes from non-stimulated rhesus monkeys. Oocytes (368) were obtained from small antral follicles (class 1: 700-999 microns; class 2: 1000-2500 microns) dissected from excised ovaries of 10 follicular and three luteal phase monkeys. Oocytes were cultured in one of four treatment groups: (i) granulosa cells (4 x 10(6) live cells/ml) + gonadotrophins (5 micrograms/ml follicle stimulating hormone and 10 micrograms/ml luteinizing hormone), (ii) granulosa cells alone, (iii) gonadotrophins alone, or (iv) controls (no granulosa cells or gonadotrophins). Mature oocytes were inseminated and cultured until arrest of embryo development. Meiotic and developmental capacity was greater (P < or = 0.05) for oocytes from class 2 compared with class 1 follicles and from luteal compared with follicular phase monkeys. Culture of oocytes with gonadotrophins alone during in-vitro maturation improved (P < or = 0.01) activation and cleavage through the 5-8-cell stage (46.5%). Culture with granulosa cells alone during in-vitro maturation augmented progression of embryos to the morula stage (6.3%). The combination of granulosa cells+gonadotrophins enhanced (P < or = 0.01) nuclear maturation, but had no effects on developmental capacity beyond those of either treatment alone. In conclusion, both granulosa cells and gonadotrophins during in-vitro maturation have stimulatory effects on the meiotic and developmental competence of oocytes from non-stimulated macaques in vitro. [ABSTRACT FROM AUTHOR]

Development of embryos from the 1-cell stage into blastocysts in vitro is generally slower than the time-course for development in vivo. It was the objective of this work to determine whether embryos that reach the 8-cell stage within a normal time-frame have a developmental advantage (both in vitro and post-embryo transfer) over slower embryos. Hamster 1-cell embryos were collected 10 h post-egg activation (PEA) and cultured for 48 h (58 h PEA = t50 for 8-cell embryo development in vivo) in hamster embryo culture medium-6. Embryos were sorted according to stage reached, culture was continued in fresh medium and stage of development was observed at 78, 82 and 86 h PEA. At 58 h PEA, embryos were < 4-cell (4%), 4-cell (19%), 5- to 7-cell (16%) or 8-cell (61%). The 58 h 8-cell embryos had a significantly greater ability to develop to the blastocyst stage than 58 h 4-cell embryos at 78, 82 and 86 h PEA (74 versus 13%, 69 versus 25% and 65 versus 37% respectively). The percentages of 14-day-old fetuses collected after embryo transfer indicated that morulae and blastocysts derived from 58 h 4-cell embryos were, on average, less viable (26% fetuses) than morulae and blastocysts from 58 h 8-cell embryos (51% fetuses). Thus morulae and blastocysts developing in vitro from faster or slower cleaving embryos can be qualitatively as well as quantitatively different. These data indicate that the timing of development in vitro, specifically the timing of completion of the third cell cycle, is a critically important parameter for predicting successful embryogenesis in the hamster. [ABSTRACT FROM AUTHOR]

Energy substrate requirements (pyruvate, lactate and amino acids) were determined for in-vitro development of hamster 1- and 2-cell embryos to blastocysts, using a chemically defined, protein free medium (hamster embryo culture medium, HECM). One-cell embryos were very sensitive to energy substrate type and concentration. Pyruvate alone could not support development of 1-cell embryos to greater than 4-cells, whereas lactate as sole energy substrate supported 14% development into morulae/blastocysts. Pyruvate, with lactate and 20 amino acids, inhibited 1-cell embryo development into blastocysts relative to lactate and 20 amino acids. The highest development of 1-cell embryos to blastocysts (up to 27%) occurred with reduced lactate concentration (less than 10 mM) and either 20 amino acids or 0.2 mM glutamine. Hamster 2-cell embryos were much less sensitive to energy substrates, requiring only lactate for development to blastocysts (53%). Lactate with 20 amino acids supported 70-75% of 2-cell embryos to blastocysts. Glutamine as sole energy and nitrogen source supported development to morulae and blastocysts of some 2-cell, but not 1-cell, embryos. Pyruvate did not enhance development of 2-cell embryos. We conclude that (i) altering the types and concentrations of available energy substrates drastically changes the developmental responses of 1-cell hamster embryos in vitro and (ii) energy substrate requirements for hamster embryo development in vitro are markedly different from those of mouse embryos, the standard model for studies on preimplantation development. This is the first report of successful in-vitro culture of hamster 1-cell embryos to the blastocyst stage. [ABSTRACT FROM AUTHOR]

Reviews the status of reported genetic abnormalities in human oocytes and embryos and discuss how non-human primates including the baboon are necessary models for reproductive senescence and human infertility. Importance of understanding mechanisms of cytoplasmic and nuclear anomalies underlying reproductive senescence; Role of chromosomal aneuploidies in fetal abnormalities; Mitochondria in oogenesis and aging; Murine models versus non-human primate models in evaluating reproductive senescence.