Barry D. Bavister

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.

Containerless vitrification of mammalian oocytes and embryos

Adapting a proven method for flash-cooling protein crystals to the cryopreservation of live cells.

Translocation of active mitochondria during hamster preimplantation embryo development studied by confocal laser scanning microscopy

The role of mitochondrial metabolism in the development of preimplantation embryos is unclear. To clarify the importance of mitochondria in early development, the fluorescent probes rhodamine 123 (Rh123: stains active mitochondrial membrane) and nonyl acridine orange (NAO: stains active and inactive mitochondrial membrane) were used with confocal laser scanning microscopy to study the distribution of mitochondria in hamster unfertilized follicular and oviductal eggs (11.5 hr and 16 hr post‐hCG, respectively) and preimplantation embryos (1‐cell to blastocyst). Rh123 staining indicated that active mitochondria were homogeneously distributed in unfertilized follicular and oviductal eggs. At 3 hr post egg activation (PEA) by sperm, active mitochondria were still found throughout the cytoplasm of the activated egg although they were slightly clustered around the pronuclei and were intensely active in the second polar body. During the next 9 hr, the majority of active mitochondria encircled the apposing pronuclei. By this time the second polar body no longer stained. In 2‐cell, 4‐cell and 8‐cell embryos, there was a striking constancy in the pattern of active mitochondria which were clustered around the nuclei and delineating the cytocortex subjacent to the plasma membrane. In the blastocyst, active mitochondria were most readily detected in the trophectoderm cells in a homogeneous distribution. Staining mitochondria with NAO showed the same distribution patterns as Rh123, indicating that perinuclear clustering of active mitochondria involves the physical movement of these organelles rather than simply changes in their activity. Distribution of actin microfilaments and microtubules showed similar patterns to mitochondria and may be involved in their movement. This migration of mitochondria, beginning during the early stages of fertilization in the hamster egg and persisting until blastocoel formation, must have some functional correlation with successful preimplantation development.

Development of in vitro matured/in vitro fertilized bovine embryos into morulae and blastocysts in defined culture media

Abstract The techniques of in vitro maturation (IVM) and in vitro fertilization (IVF) of bovine oocytes, with development of the resulting embryos to advanced preimplantation stages in vitro, have gained widespread popularity in recent years because of the potential for obtaining information about regulatory mechanisms, and for inexpensively mass-producing embryos for research or for transfer. However, the efficiency of the techniques (blastocyst yield) is unsatisfactory, due to inadequate information about the requirements of bovine embryos for development in culture, and of oocytes for achieving normal maturation. An experiment was designed to determine effects of using different media for bovine oocyte maturation on subsequent embryo development. Five of seven media tested on oocyte maturation resulted in higher levels of fertilization and/or first cleavage division. In addition, the data indicated that culture conditions for oocyte maturation also affect development to the morula/blastocyst stages. A second experiment evaluated culture conditions for embryo development, using two media (HECM-1 and TCM-199) with or without supplementation by serum and/or oviduct cell conditioning, in a 2×2×2 factorial design. Neither serum supplementation nor conditioning nor the two together increased morula/blastocyst formation (35–46% in all treatments). Serum had a biphasic effect, suppressing the first cleavage division but enhancing morula compaction. No block was observed at the 8- to 16-cell stage with TCM-199 + serum, contrary to many other studies. The data support the hypothesis that the reported stimulation of bovine IVM/IVF embryo development by somatic cell conditioning is due to removal of inhibitory influences from the culture environment. The ability to support development of IVM/IVF embryos to the morula and blastocyst stages in defined media (i.e., without serum or somatic cell conditioning) will help to elucidate the metabolic and nutritional requirements of bovine oocytes and embryos, and to formulated improved culture media. These advances will increase the utility of bovine IVM/IVF for both basic and applied resarch, and for commercial embryo production.

Differentiation-related changes in mitochondrial properties as indicators of stem cell competence.

Several methods may be used to assess stem cell competence, including the expression of cell surface markers and telomerase activity. We hypothesized that mitochondrial characteristics might be an additional and reliable way to verify stem cell competence. In a multipotent, adult monkey stromal stem cell line, previously shown to differentiate into adipocytes, chondrocytes, and osteocytes, we found that several mitochondrial properties change with increasing passage number in culture. Cells from the earliest passage (P11) versus those from a later passage (P17) are characterized by: (a) a much higher percentage of cells (85% vs. 18%) with a perinuclear arrangement of mitochondria; (b) a much lower percentage of cells (1% vs. 57%) with an aggregated mitochondrial arrangement, in which mitochondria appear to coalesce into large clumps; (c) a much lower percentage of cells with lipid droplets (1% vs. 36%), suggesting less differentiation into adipocytes; (d) a 5.6‐fold lower ATP content per cell (0.45 vs. 2.51 pmoles ATP/cell; and (e) a 10‐fold higher rate of oxygen consumption (37.8 vs. 3.8 nmoles O2/min/103 cells), indicating a higher metabolic activity. Collectively, these data indicate that the perinuclear arrangement of mitochondria, accompanied by a low ATP/cell content and a high rate of oxygen consumption, may be valid indicators of stem cell differentiation competence, while departures from this profile indicate that cells are differentiating or perhaps becoming senescent. These results represent the first characterization of mitochondrial properties reported for a primate stem cell line. J. Cell. Physiol.

Preimplantation embryo development

This volume contains the proceedings of the Serono Symposium on Preimplantation Embryo Development, held in Newton, Massachusetts in 1991. A recent resurgence of interest has occurred in this fundamental phase of embryogenesis and its relevance to basic research and applied fertility studies in both humans and animals. In particular, these proceedings focus on the regulation of cellular and molecular events underlying blastocyst formation, which is the culmination of preimplantation development. The papers herein present the considerable advances made in understanding regulatory mechanisms such as oocyte maturation and the genetic control of embryo development as well as the differentiation of the embryo and embryo-maternal interactions.

Responses of oocytes and embryos to the culture environment

Embryo development is strongly influenced by events occurring during oocyte maturation. Although many immature oocytes are capable of completing meiosis in vitro, only a small percentage of the original pool of immature oocytes is competent to continue development to the blastocyst stage and subsequently result in a pregnancy. This indicates that maturation of oocytes in vitro may not be occurring in an entirely normal manner. Cytoplasmic changes occurring during maturation, collectively termed cytoplasmic maturation, are essential for embryonic development. The cytoplasm of the oocyte may play a crucial role in assembling the correct metabolic machinery for production of sufficient energy for cellular functions during maturation, cleavage and blastocyst formation. A better understanding of the structural, functional and metabolic characteristics of the oocyte during maturation, and the consequence of changes in these parameters on developmental competence is needed. Understanding the role of cytoplasmic changes during oocyte maturation will help increase the efficiency of in vitro embryo production. Better embryo production strategies will facilitate basic research into the control of early development, improve implementation in endangered species, provide a source of high quality oocytes for nuclear transfer and transgenic technologies and benefit the commercial embryo transfer industry.

Enhanced glycolysis after maturation of bovine oocytes in vitro is associated with increased developmental competence

The effect of maturation in vitro on metabolism of individual bovine oocytes was examined. Three maturation media were used: standard, consisting of tissue culture medium 199 supplemented with serum and pyruvate, and a chemically defined medium supplemented with either amino acids or lactate. Development to blastocyst was significantly higher (P < 0.05) after maturation in standard medium (47%) than in defined medium with lactate (17%) but was not different than maturation in defined medium with amino acids (29%). Glucose metabolism through the Krebs cycle was not different after maturation in standard or defined medium with amino acids or lactate (0.48, 0.43, 0.38 pmol/oocyte/3 hr, respectively) but was affected by the removal of unlabeled pyruvate from the metabolic measurement medium (0.16, 0.21, 0.27 pmol/oocyte/3 hr, respectively). When physiological concentrations of glucose (0.52 mM) and pyruvate (0.5 mM) were used, oxidation of pyruvate was not different after maturation in standard or defined medium with amino acids or lactate (1.38, 1.13, 1.13 pmol/oocyte/3 hr, respectively); however, glycolysis was significantly increased (P < 0.05) in treatments that supported higher blastocyst development (standard medium, 1.77 pmol/oocyte/3 hr; defined medium with amino acids, 1.58 pmol/oocyte/3 hr; defined medium with lactate, 1.32 pmol/oocyte/3 hr). Metabolism of glucose through the Krebs cycle was low in all media. In contrast, oxidation of pyruvate readily occurred after maturation in vitro. Metabolism of glucose through the Embden‐Meyerhof pathway is important during oocyte maturation in vitro, and higher glycolytic rates in in vitro matured oocytes may reflect increased developmental competence. Mol. Reprod. Dev. 53:19–26, 1999.

Development of bovine embryos in a cell-free culture medium: Effects of type of serum, timing of its inclusion and heat inactivation

Bovine embryos, derived from in vitro matured (IVM)/in vitro fertilized (IVF) ova, were used to investigate the effects of timing of serum inclusion in the culture medium and different types of blood sera and heat inactivation of the serum on embryo development. In Experiment 1, oocytes at 18 h post insemination were allocated to 1 of the following 4 treatments: 1) TCM-199 + 0.1 mg/ml polyvinylalcohol (PVA), 2) TCM-199 supplemented with 10% bovine calf serum (BCS), 3) PVA medium followed by BCS medium at 47 h, or 4) PVA medium followed by BCS medium at 82 h. Supplementation with BCS at 18 h post insemination suppressed (P<0.05) development of morulae/blastocysts (17.6%) when compared with PVA (30.5%) or with serum supplementation at 47 or 82 h post insemination (32.4 and 27.6%, respectively). However, inclusion of BCS at 18, 47 or 82 h post insemination produced more blastocysts (16.8, 29.3 and 22.1%, respectively; P<0.05) than medium +PVA (8.8%). In Experiment 2, ova were cultured from 18 h to 42 h post insemination in PVA-medium, then >/=2-cell embryos were transferred into serum-supplemented medium for another 168 h. Fetal bovine serum (FBS) +/- heat-inactivation (56 degrees C for 30 min, = heated FBS) suppressed morula/blastocyst development compared with medium + PVA, medium + BCS or medium + heated BCS (P<0.05). Bovine calf serum was superior to FBS in supporting blastocyst development (35.1 and 15.2%, respectively), but there was no difference between BCS and heated BCS. However, heated FBS increased the proportion of blastocysts/>/=8-cell embryos compared with that of FBS (51.0 and 31.4%, respectively; P<0.05). These results indicate that the type of serum supplementation and the timing of its inclusion in the culture medium markedly affect bovine embryo development in vitro, and that heat inactivation of serum with high embryotrophic properties is not necessary.

Different lots of bovine serum albumin inhibit or stimulate in vitro development of hamster embryos

Dear Editor: In recent years, much emphasis has been placed on eliminating serum from culture media for somatic cells and preimplantation embryos, in part because of the high cost and inconsistency of this product, and in part to examine effects of specific growth factors and hormones without experiments being compromised by serum factors (3). In many studies, serum has been replaced with bovine serum albumin (BSA) (8,14). However, serum albumin, in general, binds biological substances such as fatty acids, steroids and many trace metals, provides an accessible protein reserve, serves as a colloid osmotic regulator and acts as a transport protein (1). Thus, BSA is not an inert constituent of culture media and may provide low molecular weight growth-promoting factors (9) as well as chelate heavy metals and other potentially toxic contaminants (4,7,10). Considerable progress has been made in supporting development of hamster preimplantation embryos in vitro using a simple proteinfree, chemically defined medium generically designated Hamster Embryo Culture Medium (HECM), from 0% 2-cell embryo development a few years ago (12) to approximately 50% blastocysts currently (11). However, this culture medium may lack important factors found in vivo that could improve development; accordingly, we examined the effect of incorporating bovine serum albumin into the medium. We tested the effect of four Fraction V BSA preparations on the development of hamster 2-cell embryos in vitro. Hamster 2-cell embryos, collected 32 h post egg-activation from PMSG-stimulated females mated to fertile males (13), were cultured in 100 #1 drops of HECM1 (13) containing 0.1 mg/ml polyvinyl alcohol (PVA) under a silicone oil overlay in an atmosphere of 10% CO2:10% 02:80% N 2 at 37.5 ° C for 72 h (11). The experiment consisted of five treatments, arranged as five drops of media in a 60 mm petri dish. The control treatment contained no BSA, while the four experimental treatments each contained a different batch of commercial Fraction V BSA at 3 mg/ml. BSA-1, BSA-2 and BSA-3 were obtained from Sigma Chemical Co., all purchased under catalog number A-9647, with lot numbers 41-F-0593, 106-F-0580 and 26-F-0202, respectively. BSA-4 was obtained from Armour Pharmaceuticals: catalog number 0140-00, lot number B-10012. The experiment was repeated four times. Embryo development, expressed as a percentage of the total number of embryos in each treatment, was determined at 48 h. The number of embryos that developed to the hatching blastocyst stage was recorded at 72 h. Transformed data were subjected to a 2-way ANOVA. F values were determined, and when they were significant, levels of treatments were compared using Newman-Keuls multiple range test. The four lots of BSA were examined using one-dimensional polyacrylamide gel electrophoresis. In the control treatment, containing no BSA, 49% of the 2-cell embryos cultured developed to the blastocyst stage; 14% were