Human embryo culture and assessment for the derivation of embryonic stem cells (ESC).

The culture and critical assessment of early human embryos during the first week of human development are reviewed for the derivation of ESC. Both normal and abnormal features are assessed by phase contrast microscopy of whole embryos and in serial sections of fixed material by light and electron microscopy (TEM). Normal embryos follow a time table of development and have equal blastomeres with minimal fragmentation and nuclear defects. Abnormal embryos show more fragmentation and nuclear aberrations such as micronucleation and multinucleation, reflected by aneuploidy, polyploidy, and mosaicism. The selection of normal embryos and the hardiest of embryos that survive to blastocysts is recommended for the derivation and culture of ESC.

Rediscovering Boveri's centrosome in Ascaris (1888): its impact on human fertility and development.

We rediscover and review the brilliant work of Theodore Boveri, over a 100 years ago, on the centrosome of the round worm Ascaris and show how it impacts on our understanding of human fertilization and embryogenesis. Boveri was able to make fundamental predictions on the mechanics of fertilization and the dominant role of the sperm centrosome (Boveri's rule), which is now applicable to most animals. Using advanced digital imaging by light and electron microscopy, we explore centrosomal dynamics during Ascaris fertilization and the first cell cycle during cleavage. Twenty figures are presented in this visual publication. Humans follow Boveri's rule, as do most mammals excluding some rodents, and there is a remarkable similarity of the events of fertilization and cleavage in Ascaris and humans, the latter of which has been documented since 1991. The role of the sperm centrosome (centriole) in egg activation, polarity, embryogenesis, infertility and cancer is discussed. An attempt is made to portray the images Boveri may have visualized, in his painstaking drawings presented in his thesis in 1888. We now know the origins of the centrosome in human somatic cells--predominantly from the sperm cell. The impact of Boveri's work on human development is highlighted in this age of assisted reproductive technology.

Nine-day-old human embryo cultured in vitro: a clue to the origins of embryonic stem cells.

The aims of this study were to investigate whether the human embryo could sustain development beyond the blastocyst stage in vitro and to identify the precise origins of embryonic stem cells (ES cells) from the embryoblast. A frozen-thawed 4-cell embryo was cultured to the post-blastocyst stage. This 9-day-old embryo presented a solid mass of inner cells (resembling a tumour) surrounded by surface trophoblast cells. Clumps of multinucleated syncytiotrophoblast cells were evident at one pole. Most cells resembled those of blastocysts. However, there were groups of comparatively undifferentiated cells within the inner cell mass somewhat resembling ES cells documented previously, that might give a clue as to their origins. The embryo attempted to form an amnion with a cavity, but did not present a bilaminar, discoidal structure as expected in week 2 of development, and hence was abnormal.

Fine structure of human oogonia in the foetal ovary.

Foetal ovarian tissue is now being cultured or frozen, to generate oocytes for assisted reproduction, an emerging technology. This study examines the ultrastructure of oogonia at 13-15 weeks of gestation, which could be used as a control for culture and freezing of foetal ovaries. Oogonia are largely located in the ovarian cortex, whilst primordial germ cells (PGC) and somatic follicle cells compose the surface epithelium. Oogonia and PGC have large vesicular nuclei with clear cytoplasm, compared to dense follicle cells, which have polymorphic nuclei. Follicle cells intermingle with oogonia and establish close contacts - beginning of folliculogenesis. Nuclei of oogonia contain one to three highly reticulated nucleoli, reflecting high levels of RNA synthesis at the onset of growth. Rough endoplasmic reticulum (RER) form stacks of cisternae associated with numerous ribosomes. Prominent organelles in the ooplasm are elongated mitochondria with dense matrices and tubular cristate presenting a multilocular appearance. Typical Golgi complexes, dense bodies and clear vacuoles are present and microfilaments are located beneath the plasma membrane. The most remarkable feature of oogonia is that they have typical juxtanuclear centrioles (diplosomes) with dense pericentriolar material, which nucleate microtubules, characteristic of functional centrosomes organizing the cytoskeleton. The mature oocyte has no centrioles, since the maternal centrosome is inactivated or reduced, while the paternal is dominant. Centrioles are most likely involved in mitosis of oogonia.