Stage 2 begins with the division of the zygote into two cells and ends with the appearance of the blastocystic cavity after the embryo is composed of more than 16 cells. The embryo has an estimated postfertilization age of two to three days and is approximately 0.1 to 0.2 mm in diameter. The zygote undergoes cleavage during this period whereby it divides mitotically into an increasing number of cells that become progressively smaller with each division. The individual cells are called blastomeres and sometimes are referred to as founder cells because they give rise to embryonic stem cells. True growth does not occur during cleavage since the volume of the entire protoplasmic mass actually diminishes. However, the DNA content increases dramatically duplicating with each cell division.

Cleavage divisions do not occur synchronously so that stage 2 embryos are comprised of anywhere from one to sixteen blastomeres which can differ in their size, staining ability and electron density. During the early cleavage period the fate of each blastomere is not yet determined. The advanced embryo of 16 blastomeres or more is frequently called a morula as it resembles a mulberry.

Because of the scarcity of in vivo stage 2 specimens in the Carnegie collection the database for this stage is supplemented with previously published images of in vitro specimens. Only images of specimens that the authors considered healthy and show best the progression of development were selected for inclusion in the database. Grateful appreciation is expressed to the authors and the publishers for their generosity in permitting the use of their images. The author(s) is acknowledged in each figure legend. The complete references are listed here. A measuring bar is present in many of the light micrographs (LM). The length of the bar was calculated based on a 175 µm diameter of the zona (capsula) pellucida before fixation. Original magnification is given in the legend of most scanning (SEM) and transmission (TEM) electron micrographs.

IN VIVO STAGE 2 SPECIMEN

This disk contains the sections a 2-cell embryo (Carnegie embryo #8698) previously described by Hertig A.T. et al. (1954) The two-cell embryo is spherical and surrounded by a transparent zona pellucida . Before fixation the embryo had a diameter of 178.5 microns, including the zona pellucida (Table of Dimensions). Two polar bodies are present. Each blastomere is nearly spherical, and the larger blastomere probably divides first and becomes trophoblasts.>

The specimen was collected and prepared for microscopic examination in 1949. It was fixed in 70% alcohol and Bouin’s fluid, embedded in celloidin paraffin, and serially sectioned at 6 µm. The sections were mounted on a glass slide and stained with H & E. About 13 serial sections were obtained from the embryo, however, only 7 of these are visible on the slide. Six of the seven sections are included in this database. The sections have been digitally restored and labeled, and can be viewed at two magnifications. Structures are identified on every section image. The morphology of this embryo is well documented in the literature. It was first described by Hertig et al. in 1954.

IN VITRO STAGE 2 SPECIMENS

Stage 2 in vitro begins when cell membranes form along the anterior-posterior polar axis of the zygote. The zygote is thereby divided into two cells. The stage ends with the appearance of the blastocystic cavity when stage 3 begins. The post insemination age of in vitro stage 2 specimens is one to two days. Stage 2 in vitro is characterized by cleavage and compaction.

CLEAVAGE

The first cleavage occurs sometime between 24 and 36 hours after sperm penetration of the oocyte. Two cells are present at one and one-half days, four cells at two days and eight cells at two and one-half days. The cell doubling time in healthy embryos is thought to average 18-20 hrs. Each cell is called a blastomere.

Cleavage is very clearly seen in the in vitro specimens up through the 12-cell embryo (Fig. 1-7). The second polar body in the 2-cell embryo determines the location of the polar, or anterior-posterior (A-P), axis and the first cleavage plane (Fig. 2). A second axis that is perpendicular and equidistant to the polar axis marks the second cleavage plane that forms the 4-cell embryo (Fig. 4). Prior to implantation the embryo expresses predominantly gap junctions that first form in opposing cell membranes at the 4-cell stage. The junctions become increasingly organized as development proceeds (Hardy et. al., 1996).

COMPACTION

On day 3 at about eight cells (third mitotic division) a phenomenon called compaction begins (Fig. 8) and lasts for about one day. Tight intercellular junctions form between blastomeres. Blastomeres become closely apposed and flatten as the areas of contact increase. A variety of cell junctions are formed in sequential order. The contacts and junctions are dynamic and appear to change to accommodate the loss of coupling during mitosis which is necessary for continued development. Each type of junction plays an important role in cell communication, adhesion and differentiation. During compaction the cleaving embryo changes from a collection of individual cells into a smooth mass with indistinguishable cell boundaries. The surface of the embryo is covered with densely distributed microvilli. What triggers compaction is unknown but the process does not appear to require a strict number of cell divisions.

The compaction phenomenon is divided into six phases: 1) initiation (Fig. 8), 2) moderate cell-cell contact (Fig. 10), 3) increased cell-cell contact (Fig. 12), 4) closely apposed cell membranes (Fig. 13), 5) disappearing cell membranes (Fig. 14) and 6) cell membranes are not longer visible (Fig. 15).

The time-line for optimal in vitro development of stage 2 and the hypotheses of cell allocation and differentiation are given in the stage 3 database.

Gualtieri, R., Santella, L., and Dale, B. (1992)Tight junctions and cavitation in the human pre-embryo. Mol. Reprod. Dev. 32:81-87.

Hertig, A.T., Rock, J., Adams, E.C., and Mulligan, W.J. (1954) On the preimplantation stages of the human ovum: a description of four normal and four abnormal specimens ranging from the second to the fifth day of development. Carnegie Instn. Wash. Publ. 603, Contrib. Embryol. 35:199-220.

Hardy, K., Warner, A., Winston, R.M.L., and Becker, D.L.(1996) Expression of intercellular junctions during preimplantation development of the human embryo. Mol. Hum. Reprod. 2:621-32.

Makabe, S., Naguro, T., Nottola, S.A., and Motta, P.M. (2001) Ultrastructural dynamic features of in vitro fertilization in humans. Ital. J. Anat. Embryol. 106:11-20.

Ménézo, Y.J.R., Kauffman, R., Veiga, A., and Servy, E.J. (1999) A mini-atlas of the human blastocyst in vitro. Zygote 7:61-65.

Nikas, G., Ao, A., Winston, R.M.L., and Handyside, A.H. (1996) Compaction and surface polarity in the human embryo in vitro. Bio. Reprod. 55:32-37.

Pereda, J., and Croxatto, H.B. (1992) Human preimplantation development in vivo: ultrastructural observations. Ann. Acad. Med. Singapore, 21:480-8.

Sathananthan, A.H. (1984) Ultrastructureal morphology of fertilization and early cleavage in the human. In Trounson, A.O. and Wood, C. (eds.). In Vitro Fertilization and Embryo Transfer. Churchill Livingstone, London.

Sathananthan, A.H. (2005) Video clips of cleaving embryos in vitro. Personal communication.

Sathananthan, A.H., and Trounson, A.O. (1982) Ultrastructure of cortical granule release and zona interaction in monospermic and polyspermic human ova fertilized in vitro. Gamete Res., 6:225-234.

Sathananthan, A.H., and Trounson, A.O. (2000) Mitochondrial morphology during preimplantational human embryogenesis. Human Repro. 15:148-159.

Sathananthan, A.H., Trounson, A.O., and Wood, C. (1986) Atlas of fine structure of human sperm penetration, eggs and embryos culture in vitro. Praeger Publisher, New York.

Sathananthan, A.H., Wood, C., and Leeton, J. F. (1982) Ultrastructural evaluation of 8- to 16-cell human embryos cultured in vitro. Micron 13:193-203.

Trounson, A.O., and Sathananthan, A.H. (1984) The application of electron microscopy in the evaluation of two- to four-cell embryos cultured in vitro for embryo transfer. J. In Vitro Fert Embryo Transf. 1:153-165.

Veeck, L.L., and Zaninovic΄, N. (2003) An atlas of human blastocysts. Parthenon Publishing Group, New York.