BIOLOGY OF REPRODUCTION 55, 32-37 (1996)
Compaction and Surface Polarity in the Human Embryo In Vitro
George Nikas, Asangla Ao, Robert M.L. Winston, and Alan H. Handyside
Human Embryology Laboratory, Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School,
Hammersmith Hospital, London W12 ONN, United Kingdom
and the related cell mechanisms is sparse. Ultrastructural
studies have reported the presence of primitive intercellular
junctions at the 8-cell stage [27], and at the 16-cell stage
these junctions are more elaborate and extend peripherally
[28]. Scanning electron microscopy (SEM) demonstrated
that the human embryo does not develop surface polarity
before the 8-cell stage [29]. Totipotency of the blastomeres
at this stage has been confirmed by lineage tracing [30].
Immature tight junctions have been observed from the 6cell stage, and these junctions become functional in cavitating morulae [31]. Gap junctional communication was
first observed at the blastocyst stage [32].
To gain more insight into early morphogenetic phenomena in humans, we carried out SEM studies on oocytes and
embryos fertilized and grown in vitro to the morula stage
on Day 4 postinsemination. The aim of this study was to
investigate the timing and features of compaction of the
embryo and surface polarization of disaggregated blastomeres. SEM has been shown previously to be a useful tool
for this purpose in other mammalian species studied [21,
33].
ABSTRACT
The surface morphology of the human ovum fertilized and
cultured in vitro to the morula stage was studied by scanning
electron microscopy with the specific aim of investigating embryo compaction and polarity. Unfertilized oocytes examined
one day after attempted insemination (Day 0) were evenly and
densely covered by long microvilli. The length and density of
microvilli appeared to decrease in fertilized polypronuclear oocytes; a further decrease was observed in Day 2 and Day 3 embryos with 2-12 cells. No evidence of compaction or surface
polarity was observed in any of these stages. On Day 4, compaction was evident in the majority of embryos with 10 or more
cells, and the microvilli appeared dense again with a polarized
distribution over the free surface of the compacted blastomeres.
This study provides ultrastructural evidence that the human conceptus undergoes a relatively marked compaction at the morula
stage during Day 4 postinsemination development in vitro.
INTRODUCTION
Mammalian development after fertilization consists of
mitotic proliferation and differentiation into distinct cell lineages. Unique cell surface changes are reported to occur
during this period, expressing the activation of fundamental
morphogenetic mechanisms. In the extensively studied
mouse model, the first differentiative event occurs at the 8cell stage with compaction of the embryo and development
of cell polarity [1, 2]. The blastomeres flatten against neighbors, and the distribution of microvilli (MV) and various
plasma membrane components become restricted to the free
surface [3, 4]. Cell-to-cell adhesion [5, 6], gap and tight
junctions [7-10], and cytoplasmic polarization [11] first appear at this stage. The expression of these functions requires
zygotic transcription [12, 13], which in this species occurs
at the 2-cell stage [14, 15]. As a result of these processes,
the blastomeres arising from the next cleavage segregate to
inside apolar and outside polar cells. Several studies have
shown that the developmental fate of the cells is mostly
determined by their position: the inside cells form the inner
cell mass, while the outside cells form the trophectoderm
[16-19].
The onset of compaction varies among species. In the
mouse and rat, compaction appears at the 8-cell stage [20],
in bovine species at the 16- to 32-cell stage, and in the
rabbit at the 32- to 64-cell stage [21]; in pig embryos compaction is not established until shortly before blastocyst formation [22]. Compaction is most prominent in the mouse,
while it is less marked in the rhesus monkey and baboon,
occurring at the 16- to 32-cell stage in these species [2326].
Precise information about human embryo compaction
MATERIALS AND METHODS
Recovery and Culture of Human Oocytes and Embryos
The human eggs and embryos used for these studies
were donated with informed consent by couples undergoing
in vitro fertilization (IVF) at the infertility clinic at Hammersmith and the Royal Masonic Hospitals. The embryos
were surplus after transfer of up to three of the best quality
to the patient on the second or third day postinsemination.
This research was carried out under license from the Human Fertilization and Embryology Authority of the United
Kingdom and with the approval of the research ethics committee of the Royal Postgraduate Medical School, Hammersmith Hospital.
Patients were undergoing IVF because of various infertility disorders. Details of the ovarian stimulation protocol
have been published previously [34]. Briefly, after pituitary-gonadal suppression with a GnRH agonist, follicular
growth was induced by human menopausal gonadotropin.
After adequate stimulation, hCG was administered, followed by transvaginal follicle aspiration 34-36 h later. The
oocytes were preincubated for 4-6 h and then inseminated
(Day 0) with the husband's motile sperm obtained by a
swim-up procedure.
Sixteen-eighteen hours after insemination the oocytes
were washed to remove sperm and cumulus cells to check
fertilization. With use of a stereomicroscope, oocytes were
classified as nonfertilized, normally fertilized, or polyspermically fertilized according to the number of pronuclei
present (0, 1, or 2 or more, respectively). Subsequently the
embryos were cultured individually in 1 ml of Earle's Balanced Salt Solution (GIBCO BRL, Life Technologies Ltd,
Middlesex, UK) supplemented with 10% heat-inactivated
human maternal serum, 0.47 mM pyruvate (Sigma, Poole,
Accepted February 19, 1996.
Received November 27, 1995.
'Correspondence: George Nikas, Human Embryology Laboratory, Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical
School, Hammersmith Hospital, Du Cane Road, London, W12 ONN, UK.
FAX: 0181 259 8065.
32
COMPACTION OF THE HUMAN EMBRYO
33
TABLE 1. Numbers and groups of embryos and associated measurements of microvilli.
Developmental
stage
Unfertilized
day 1 oocytes
Polyspermic
day 1 oocytes
Day 2 2-6 cell
embryos
Day 3 6-12 cell
Mechanically
after fixation
DisagIntact
gregated
AT before
fixation
Trypsin
before
fixation
11
9
10
10
7
9
8
5
18
9
7
embryos
Total
MV length
(im)
MV density
(MV/Im 2)
-
30
1.0-2.0
10-20
-
26
0.5-1.5
5-15
13 (38)a
44
0.5-1.5
0.1-10
16
12 (55)a
44
0.5-1.5
0.1-10
19
12 (71)a
31
0.5-1.5
1-15
Day 4 10-18 cell
embryos
a
-
-
Numbers of blastomeres available for observation.
UK), 25 mM sodium bicarbonate (BDH, Lutterworth, UK),
and 37.5 U/ml of streptomycin (Sigma) in an atmosphere
of 5% CO 2:5% 02:90% N2 at 37°C [35]. On Day 2 (4244 h after insemination), the quality of the embryos was
scored according to morphological criteria for five grades
[36]. Only good-quality (grade one or two) normally fertilized embryos with even blastomeres and no or little fragmentation were used in this study. The embryos were kept
in culture until fixed for electron microscopy. The ages of
the embryos were calculated in days after insemination,
with the day of insemination designated as Day 0. Oocytes
and embryos were allocated to groups according to age and
cell number, as summarized in Table 1. All unfertilized oocytes had a visible polar body at the time of fixation.
Removal of the Zona Pellucida and Disaggregation of
Blastomeres
Major consideration was given to the method used for
removing the zona pellucida (ZP), as various enzymatic or
chemical treatments have been reported to affect the surface
properties of the plasma membrane and the MV [37, 38].
In our series we have used acid Tyrode's solution (AT) [39],
trypsin, and mechanical removal. For AT and trypsin treatment, the eggs were placed in 5 0-1l microdrops of AT (pH
2.2) or 0.1% trypsin (Sigma) in PBS, respectively, and repeatedly aspirated in a glass pipette under continual observation through a dissecting microscope. As soon as the eggs
appeared free from ZP remnants, they were removed from
the microdrop, washed several times in fresh medium, and
cultured for 1 h to allow them to recover before being fixed.
For mechanical removal of the ZP, the eggs were fixed first,
and the ZP of each specimen was cut manually with fine
metal needles under a dissecting microscope. Then the ZP
was pressed by the needle, and the oocyte/embryo came
out through the slit, either intact or disaggregated depending on the amount of pressure applied. Mechanical removal
of the ZP after fixation was employed most often in this
study, as this approach was believed to more likely represent the undisturbed state of the plasma membrane. For the
same reason, all embryos to be disaggregated were fixed
first and then mechanically manipulated.
Fixation and Electron Microscopy
For fixation, the eggs were transferred into 0.I M sodium
cacodylate (BDH) buffer containing 1% glutaraldehyde
(Sigma) and stored in the fixative at 4C for several days
or weeks until processed for SEM. All fixatives were fil-
tered in Millipore filters (0.2 lm pore size; Millipore Corp.,
Bedford, MA) prior to use. For SEM, the specimens were
dehydrated in acetone series and then dried according to
the critical point method using CO 2. After drying, the eggs
were mounted on the specimen holder, sputter-coated with
gold (14 nm thickness), and observed under an accelerated
voltage of 10.0 kV at a short working distance in a Cambridge Stereoscan 360 (Cambridge Instruments, Cambridge,
UK) scanning electron microscope. For MV measurements,
the screen magnification was increased to 20 000, and three
representative areas of 4 Lm2 were examined for each specimen, using a layer put on the screen. The specimens were
processed all at once in small groups. Each group comprised oocytes and cleaving embryos to allow comparative
evaluation of MV findings at different stages of development with the same treatment (see Table 1).
RESULTS
Oocytes
Unfertilized oocytes appeared evenly covered with MV
of 1.0- to 2.0-pLm length, with a density of 10-20/Vpm 2 . No
polarized regions were found apart from long projections
under the first polar body (3 specimens), which was smooth
and sometimes fragmented (Fig. 1). In fertilized polypronuclear oocytes, the plasma membrane showed shorter MV
(0.5-1.5 um), with decreased density (5-15/pLm 2 ) (Fig. 2).
The MV in both fertilized and unfertilized oocytes were
always present, and no difference arising from the method
of ZP removal was observed.
Cleavage Stage
None of 53 embryos examined on Day 2 and Day 3 was
found to be compacted. Day 2 and Day 3 intact embryos
at the 2- to 12-cell stage generally had sparse MV (0.5-1.5
pum length, density of 0.1-10/pxm 2) scattered all over the
surface of the blastomeres. Large variations in MV density
were seen between embryos of the same stage and grade,
as well as between blastomeres of the same embryo; some
blastomeres were completely smooth. The blastomeres of
Day 2 embryos were of similar size (Fig. 3), while in Day
3 embryos relatively large or small blastomeres were frequently seen (Fig. 4). Cytoplasmic fragments were localized to the clefts between the blastomeres and usually did
not have MV. Adjacent blastomeres were connected by focal areas of close membrane apposition or long cytoplasmic
bridges (Fig. 5). Blastomeres of disaggregated embryos
were round, and no polarized distribution of MV was no-
34
NIKAS ET AL.
FIG. 1. Detail of unfertilized oocyte fixed one day after insemination. Surface is covered by dense and long MV. Note presence of long protrusions
under first polar body, which is devoid of MV. ZP was removed by AT.
FIG. 2. Detail of fertilized oocyte fixed at pronuclear stage. Three pronuclei were present in this specimen. Length and density of MV appear reduced,
compared with those of unfertilized oocyte in Figure 1. One polar body is visible. ZP was removed by AT.
FIG. 3. Four-cell embryo fixed on Day 2. MV are sparse. Close contact between blastomeres is seen, but flattening of blastomeres is not. ZP was
removed by AT, but remnants are still present.
FIG. 4. Ten-cell embryo fixed on Day 3. Note unequal size of blastomeres and presence of pronounced intercellular clefts. ZP was removed mechanically.
COMPACTION OF THE HUMAN EMBRYO
35
FIG. 7. Compacted morula with 10 cells
fixed on Day 4. Note complete absence of
clefts between several blastomeres. Not all
of them are compacted to the same degree, and they show differences in size
and MV density. ZP was removed mechanically.
ticed in any of the 38 blastomeres examined at Day 2. This
appearance was found also in 47 of 55 (85%) Day 3 blastomeres (Fig. 6), but the remaining 8 (15%), derived from
8- to 12-cell embryos, showed early signs of polarization,
with the interblastomeric areas flattened and free from MV.
The MV of Day 2 and Day 3 embryos were easily damaged by excessive chemical or enzymatic treatment for ZP
removal or faulty preparation during processing; such specimens were not considered in this study. In our experience,
prolonged exposure to trypsin affected the integrity of the
MV, leading to smooth membranes, while the AT was apparently less harmful and more effective for ZP removal.
Morulae
In Day 4 intact morulae with 10-18 cells, compaction
and polarity of the blastomeres were apparent. Compaction
was evident by the presence of blastomeres flattened
against each other, with the clefts between them being
greatly reduced. However, noncompacted cells were frequently encountered, and they generally had fewer MV
compared to the compacted ones (Fig. 7). Fifteen of 19
embryos examined were classified as compacted (79%),
while the remaining 4 were noncompacted and resembled
the Day 3 embryos.
In preparations with partially disaggregated blastomeres,
polarization was seen as a flattened MV-free area with distinct borders, at the sites of contact between the cells, which
frequently contained small (1- to 3-pLm) cytoplasmic blebs
(Fig. 8). The free surfaces of the blastomeres were covered
by MV of an increased density (5-15/Rm2) compared with
that of cleavage-stage embryos. Polarization was noticed in
53 of 71 blastomeres examined (75%). The size of the blas-
FIG. 5. Detail of 8-cell embryo fixed on Day 3, showing cleft between
two adjacent blastomeres. Blastomeres are connected by long and large
MV. ZP was removed by AT.
FIG. 6. Blastomere of 7-cell embryo fixed on Day 3. MV are sparse and
no polarization is seen. ZP was removed mechanically.
tomeres varied from large (> 40 pm) to small (< 20 pm).
Tables 1 and 2 summarize these morphological findings.
DISCUSSION
Morphological analysis of our specimens clearly demonstrated that striking changes in cell shape occur during
preimplantation development of the human embryo. The
unfertilized oocyte is covered by long and dense MV, similar to those observed in other mammalian species [40, 41]
and as previously described in the human [37]. The long
protrusions under the first polar body seen in 3 oocytes
were reminiscent of those seen in the hamster and may
represent an activated membrane area overlying the meiotic
spindle [41]. In some rodents, the oocyte develops an MVfree area as the second meiotic spindle moves cortically
[40, 42].
Reduction of MV density and length after fertilization,
to our knowledge, has not been described in other mammalian species. When mouse, rat, and hamster embryos
were treated and prepared for SEM in a series of experiments by one of the present authors (George Nikas, unpublished), the embryos were always found to be covered with
abundant MV, as previously reported by others [43]. In the
present study, the sensitivity of the MV in cleavage stage
TABLE 2. Incidence of compaction and polarity in intact and disaggregated embryos.
Intact embryosa
Developmental stage
Day 2 2-6
cell embryos
Day 3 6-12
cell embryos
Day 4 10-18
cell embryos
aZP
Noncompact
Compact
Disaggregated
blastomeresb
Nonpolar
Polar
31
0 (0%)
38
0 (0%)
32
0 (0%)
47
8 (15%)
15 (79%)
18
53 (75%)
4
removal by all techniques.
b ZP removal by mechanical means only.
36
NIKAS ET AL.
FIG. 8. Disaggregated blastomeres from a
12-cell embryo fixed on Day 4. Distinct
border in MV distribution is seen. Note
abundance of small blebs at sites of contact between blastomeres. ZP was removed mechanically.
embryos to chemicals applied during preparation suggests
that the MV are not structurally stable at these stages. Furthermore, a large variation in MV density was noticed between and within embryos in this study, although all embryos were scored as being of good quality under the light
microscope. Therefore, it is difficult to know whether the
low MV density reflects degenerative changes or occurs
normally at these stages.
Reduced MV density during the first cleavages has already been reported in bovine [21] and human embryos
[29]. Since human embryonic gene activation first occurs
at the 4- to 8-cell stage [44], the embryo presumably relies
on maternal supplies of plasma membrane and cytoskeletal
elements needed for MV assembly until this time. However,
exhaustion of the maternal stocks cannot directly account
for the reduction in MV seen in Day 1 fertilized oocytes.
Compaction assessed by SEM occurred on Day 4 postinsemination, at the 10- to 18-cell stage, as suggested by
previous light and transmission electron microscopic
(TEM) studies on embryos grown in vitro [28, 45, 46]. In
a small number of rhesus monkey and baboon embryos
examined mainly by TEM, compaction in vivo did not occur until the 16- to 32-cell stage [23-26]. Although less
prominent than in the mouse, compaction of human embryos developing in vitro appeared to be more pronounced
than in these primate embryos in vivo. Nevertheless, the
presence of noncompacted blastomeres or blebs preventing
contact of adjacent cell membranes (Fig. 8) has also been
observed in primate embryos [23-26].
In this study, it was possible to examine the relationship
between embryonic age, cell number, and compaction. Interestingly, compaction was related to both total blastomere
number and embryonic age. Day 3 embryos with 10-12
blastomeres showed some polarization, but none of them
was classified as compact. In contrast, most Day 4 embryos
were compact, including some with only 10 blastomeres.
The uneven blastomere size in embryos with 8 or more
cells is probably due to the asynchrony of cell divisions
from this stage onwards. SEM provides an easy and accurate way to count blastomeres in cleaving embryos. However, the developmental potential of each blastomere cannot
be assessed simply in terms of surface morphology. For
instance, blastomeres with nuclear abnormalities are common at these stages in embryos grown in vivo [47] or in
vitro [48, 49]. These abnormalities could impair the viability of the blastomere and may affect the plasma membrane
and its ability to participate in compaction.
To conclude, surface ultrastructural changes during early
human embryogenesis in vitro follow a pattern similar to
those extensively studied in rodents. Compaction is morphologically distinct in the human embryo in vitro and occurs on the fourth day postinsemination when the majority
of normally developing embryos have ten or more blastomeres. Compared with other primate species studied in
vivo, the human embryo undergoes an earlier and more
pronounced compaction.
ACKNOWLEDGMENTS
The authors wish to thank Dr Timothy A. Ryder and Miss Margaret
A. Mobberley from the electron microscopy unit of the Queen Charlotte's
Hospital for help and advice concerning scanning electron microscopy and
Karin Dawson and all the staff of the infertility clinic of the Hammersmith
and the Royal Masonic Hospitals for their help in IVE
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