A method of culturing preimplantation mouse embryos

A modification was proposed for the method of cultivation of preimplantation mouse embryos which does not require mineral oil and strict maintenance of CO2 content of gaseous phase.     

Progress on methods of gene detection in preimplantation embryos

The advent of the polymerase chain reaction (PCR) and the development of fluorescence in situ hybridization (FISH) have had a tremendous impact on preimplantation genetic diagnosis (PGD). While PCR is a powerful tool in detecting genetic diseases or molecular markers affecting quantitative trait loci, the main use of FISH is screening for chromosomal aberrations. This presentation reviews the recent progress in preimplantation genetic diagnosis with an emphasis on bovine embryos. In particular the importance of biopsy size and strategies to avoid PCR contamination are discussed. Alternative DNA amplification and detection methods as well as methods to meet the challenge of multiple locus detection for marker assisted selection are presented.     

Use of PCR-based methods for selection of integrated transgenes in preimplantation embryos

The production of transgenic animals from ungulate species is an inefficient and expensive procedure. The development of selection methods to identify the small number of transgenic preimplantation embryos produced following DNA microinjection of one-cell embryos would greatly reduce both the cost and effort of these procedures. This study has examined the fate of the ovine β-lactoglobulin-human α₁-antitrypsin (AATB) minigene construct or a subfragment of this following microinjection into one-cell mouse embryos. It has examined two PCR-based methods that were designed to identify a biochemical difference between microinjected DNA constructs to select preimplantation stage embryos in which chromosomal integration of exogenous DNA has occurred. The two methods involved the modification of the AATB DNA construct either by dam-methylation or the substitution of dTTP by dUTP. The dam-sensitive DNA endonuclease Dpnl, that was used to digest nonintegrated AATB sequences at sites located between PCR oligonucleotide sequences, was found to interfere with the activity of the subsequent PCR reaction. Analyses of the fate of dUTP-DNA indicated that either repair or replication of microinjected DNA interfered with the ability to distinguish between integrated and nonintegrated DNA constructs in the mid-preimplantation stage embryo. The distribution of microinjected AATB DNA between the blastomeres of individual four and eight-cell stage embryos was also examined by the PCR reaction. Microinjected DNA was not found to be evenly distributed between all the blastomeres of individual embryos. © 1994 Wiley-Liss, Inc.     

Pathogenicity of mouse hepatitis virus for preimplantation mouse embryos

Mouse embryos which were hatched from the zona pellucida in vitro in the presence of mouse hepatitis virus (MHV) or outgrown on coverslips and then exposed to MHV were shown by immunohistochemical staining to have virally infected trophoblast cells. Zona-intact embryos incubated with MHV for 48 h (2-cell embryos) or 1.5 h (blastocysts) were resistant to infection. Morulae and early blastocysts collected from donor mice experimentally infected with MHV were not infected, but the medium in which they were flushed from the uterine horns was contaminated with virus. No virus was detected after embryos were washed through three changes of uncontaminated medium. MHV was transmitted to foster mothers when embryos were transferred in medium contaminated with the virus. Fetal and decidual tissues were not infected. We suggest that embryo transfer is an effective and simple alternative to Caesarian rederivation of MHV-contaminated mice.     

Lectin-mediated agglutination of preimplantation mouse embryos

Plant lectins were used to monitor qualitative changes in carbohydrate-containing receptors during preimplantation mouse development. Beginning at the morula stage, an age-related decline was observed in agglutination of early mouse embryos by concanavalin A (ConA). In contrast, wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) agglutinated embryos strongly throughout preimplantation development.     

Studies on preimplantation development of buffalo embryos

A total of 71 lactating and nonlactating buffalo-cows of the Murrah breed and F(1)-F(3) crossbreds of Murrah x Bulgarian buffalo were used for a year as donors of embryos after a preliminary treatment for superovulation induction with pregnant mare serum gonadotrophin (PMSG) or follicle stimulating hormone (FSH) in combination with prostaglandin F-2 alpha analog (PGF-2 alpha) according to general application procedures in cows. From 36 to 72 h following prostaglandin injection, the buffalo-cows were checked with the help of a teaser bull for detection of estrus. The animals in estrus were inseminated twice either naturally or artificially with frozen semen. Nonsurgical flushing of the uterine horns was done in 45 of the buffalo-cows between 108 and 162 h after the onset of estrus. After slaughter the uterine horns and oviducts of the other 26 animals were flushed separately between 74 and 108 h after the beginning of estrus. Seven late morulae and eight hatched blastocysts were recovered between 114 and 116 h from the onset of estrus as a result of nonsurgical flushing. All of the 40 embryos recovered after 117 h were in the hatched blastocyst stage. As a result of flushing the oviducts and the uterine horns of slaughtered donors between 74 and 100 h, eggs were obtained only from the oviducts, while flushing conducted between 102 and 108 yielded eggs from both the oviducts and the uterine horns.     

Transition from academia to industry: a personal account

A career in industry has become a widely accepted alternative for those of us trained in medicine and/or science who have traditionally focused on careers in academia. Like any career decision, consideration of a position in industry should include asking yourself a series of fundamental questions. A few of the key questions should include: 1) What kind of work environment do you find most enjoyable? (e.g., patient care setting, basic research lab, team-oriented setting); 2) What are you focused on accomplishing in your career? (basic research discoveries, contributions to clinical medicine, compensation); 3) Are you team oriented in your interactions or are you more of an individual contributor? A successful career in any endeavor, including industry, starts with a careful and honest examination of what you are best suited for and inspired to do.     

Decompaction and recompaction of mouse preimplantation embryos

Summary Early (non-compacted) and late (compacted) 8-cell embryos were observed after few hours of culture in vitro. The former embryos underwent compaction and the latter embryos were found decompacted. Cell counting suggested that decompaction preceded fourth cleavage division of any blastomere and lasted until the blastomeres divided.About one third of mouse morulae, which had about twenty cells, were found non-compacted upon obtaining from females. After few hours of culture in vitro these embryos underwent recompaction and cavitation. Increasing the contributions of mitosis-arrested and cytokinesisarrested cells within the morulae by culture with nocodazole and cytochalasin B respectively, did not delay recompaction.The data show that periods of decompaction and recompaction alternate in preimplantation development.     

Academic genealogy and direct calorimetry: a personal account

Each of us as a scientist has an academic legacy that consists of our mentors and their mentors continuing back for many generations. Here, I describe two genealogies of my own: one through my PhD advisor, H. T. (Ted) Hammel, and the other through my postdoctoral mentor, Knut Schmidt-Nielsen. Each of these pathways includes distingished scientists who were all major figures in their day. The striking aspect, however, is that of the 14 individuals discussed, including myself, 10 individuals used the technique of direct calorimetry to study metabolic heat production in humans or other animals. Indeed, the patriarchs of my PhD genealogy, Antoine Lavoisier and Pierre Simon Laplace, were the inventors of this technique and the first to use it in animal studies. Brief summaries of the major accomplishments of each my scientific ancestors are given followed by a discussion of the variety of calorimeters and the scientific studies in which they were used. Finally, readers are encouraged to explore their own academic legacies as a way of honoring those who prepared the way for us.     

Coats of preimplantation mammalian embryos as a target of reproductive technologies

The structure and function of the mammalian oocyte and preimplantation embryo coverings are described in this review. The integrity of embryonic coverings is the main prerequisite for the success of such technology as preimplantation embryo freezing and, especially, for successful rederivation. On the other hand, results of in vitro fertilization and, sometimes, the results of embryo freezing are improved after perforation of the oocyte/embryonic coverings. Modern reproductive technologies focusing on oocyte/embryonic coverings, such as preimplantation embryo freezing/cryopreservation, in vitro fertilization, intracytoplasmic sperm injection, assisted hatching, immunocontraception, and rederivation, are reviewed. Application of these technologies to different mammalian species is discussed with a special emphasis on the oocytes/preimplantation embryos coverings.