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This Article Abstract Full Text (PDF) All Versions of this Article: 69/4/1208    most recent biolreprod.103.018283v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Alarcón, V. B. Articles by Marikawa, Y. Search for Related Content PubMed PubMed Citation Articles by Alarcón, V. B. Articles by Marikawa, Y. Agricola Articles by Alarcón, V. B. Articles by Marikawa, Y.

Deviation of the Blastocyst Axis from the First Cleavage Plane Does Not Affect the Quality of Mouse Postimplantation Development¹

Department of Anatomy and Reproductive Biology, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Several researchers have suggested recently that the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst is orthogonal to the first cleavage plane of the two-cell embryo. To determine the universality of this relationship, we used embryos of two different genotypes, F₁ (C57BL/6 x DBA/2) and CD-1. The position of the first cleavage plane in the early blastocyst was determined by labeling a blastomere with the fluorescent lineage tracer DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) at the two-cell stage. Approximately one quarter of the blastocysts from both genotypes possessed an Em-Ab axis that respected the orthogonal relationship with the first cleavage plane. However, the remainder of the blastocysts deviated from the orthogonal relationship. This result indicates that the orthogonal orientation of the Em-Ab axis to the first cleavage plane is not a universal phenomenon. We also tested whether the angular relationship between the Em-Ab axis and first cleavage plane influences postimplantation embryo development. We sorted the blastocysts that had the Em-Ab axis orthogonal to the first cleavage plane from the ones that did not. These two types of blastocysts were transferred separately into surrogates, and fetal development was examined in late gestation. The results revealed that both types of blastocysts produced normal fetuses at a similar frequency. Thus, the relationship of the blastocyst axis to the first cleavage plane does not significantly influence later development.

assisted reproductive technology, conceptus, developmental biology, early development, embryo

	INTRODUCTION

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In many organisms, patterns of cell division are critical for cell fate specification and body axis formation during early embryogenesis [1, 2]. Mammals had long been thought to be exempt from this mode of development. However, recent reports have revealed that in the mouse zygote, the plane of the first cleavage tends to lie orthogonal to the embryonic-abembryonic (Em-Ab) axis of the blastocyst [3–6]. This observation predicted the model in which the progeny of one blastomere of the two-cell embryo contributes mainly to mural trophectoderm and inner cell mass (ICM) that becomes primitive endoderm, whereas the other blastomere develops into polar trophectoderm and ICM that becomes epiblast. These studies were carried out with mouse embryos obtained from F₁ (CBA x C57BL/6) [3], PO [3], and F₁ (C57BL/6 x CBA) [4, 5] females that were mated with males of the same genotype. Currently, it is unknown whether the orthogonal relationship between the Em-Ab axis and first cleavage plane is found in other mammalian species such as humans or in other genotypes of mice.

Although previous researchers reported the predominance of the orthogonal relationship of the Em-Ab axis to the first cleavage plane, a significant number of blastocysts in which the Em-Ab axis deviates from this orthogonal relationship have been observed [3, 5]. However, it is not known whether postimplantation development for blastocycsts with such an axis deviation is different from that for blastocysts that respect the orthogonal relationship.

To test whether the orthogonal relationship between the Em-Ab axis and the first cleavage plane is a universal phenomenon, we performed lineage tracing on embryos obtained from two different genotypes of mice. We then tested whether the deviation of the Em-Ab axis from the first cleavage plane influences development, specifically body axis formation in the postimplantation embryo.

	MATERIALS AND METHODS

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Animals and Collection of Embryos

B6D2F₁ (C57BL/6 x DBA/2; National Cancer Institute, Frederick, MD) and CD-1 (Charles River Laboratories, Wilmington, MA) mice were used in this study. Females were superovulated by i.p. injections of 10 IU eCG (Calbiochem, La Jolla, CA) followed by 10 IU hCG (Calbiochem) 48 h later and were then mated with males of the same genotype. About 40 h after the injection of hCG, two-cell embryos were flushed from the oviducts with FHM^PVA medium (modified from FHM [7] with 5 mM NaHCO₃, 0.01% [w/v] polyvinyl alcohol, and no BSA) and cultured in mKSOM^AA medium (modified from KSOM/AA [8] with 5.56 mM glucose and 5 mg/mL BSA) under mineral oil (Roberts Pharmaceutical, Eatontown, NJ) at 37°C in 4.5% CO₂ in air. Animals were maintained according to the guidelines of the Laboratory Animal Services at the University of Hawaii and those prepared by the Committee to Revise the Guide for the Care and Use of Laboratory Animals of the National Research Council [9]. The protocol of animal handling and treatment was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Hawaii.

Fluorescent Labeling of Blastomeres

Labeling of the blastomere of two-cell embryos was performed as previously described [5]. The membrane-soluble fluorescent dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; Molecular Probes, Eugene, OR) was dissolved in virgin olive oil at 2.5 mg/ml. Prior to labeling, the microinjection needle was backfilled with DiI. The needle tip was pushed through the zona pellucida and pressed against the blastomere, where a DiI microdroplet was deposited on the plasma membrane. Labeled embryos were cultured in mKSOM^AA up to the early blastocyst stage (about 48 h). Most of the labeled two-cell embryos developed into blastocysts; DiI appeared to be nontoxic.

Scoring DiI-Labeled Blastocysts

Labeled embryos were scored at the early blastocyst stage when the ratio of the size of the ICM to blastocoel cavity was from 1:1 to 1:2. The blastocysts were placed in FHM^PVA and observed under an Axiovert 200 fluorescence microscope with a rhodamine filter (Carl Zeiss, Thornwood, NY). Initially, the blastocyst was held with a holding pipet to position its equatorial plane (parallel to the blastocoel cavity floor) perpendicular to the plane of view. In this position, the Em-Ab axis of the blastocyst ran parallel to the plane of view (Fig. 1A). With the aid of a glass needle attached to a Model MWO-202 micromanipulator (Narishige, Tokyo, Japan), the blastocyst was then carefully rotated along the Em-Ab axis to view DiI-labeled cells. The plane of the first cleavage was identified as the boundary between fluorescent and nonfluorescent cells (Fig. 1, B–E). Blastocysts were sorted into two groups, depending on the angular departure of the first cleavage plane from the equatorial plane. When the angular departure was 30°, the blastocyst was scored as orthogonal. Conversely, when the angular departure was >30°, the blastocyst was scored as deviant. Sorted blastocysts were further cultured in mKSOM^AA up to the late blastocyst stage (16–20 h).

View larger version (70K): [in this window] [in a new window]   FIG. 1. Criteria for scoring the orientation of the Em-Ab axis of the mouse blastocyst relative to the first cleavage plane of the two-cell embryo. The schematic diagram (A) of the early blastocyst depicts the Em-Ab axis and the equatorial plane of the blastocyst, which lies parallel to the blastocoel cavity floor. The first cleavage plane is identified as the boundary between the progeny of DiI-labeled and nonlabeled blastomeres of the two-cell stage. Two representative B6D2F1 x B6D2F1 blastocysts are shown as bright field (B and D) and fluorescent (C and E) images. When the angle between the first cleavage plane (dotted white line) and the cavity floor was within 30°, the blastocyst was scored as orthogonal (B and C). By contrast, when the angle was >30°, the blastocyst was scored deviant (D and E). In E, the Em-Ab axis is nearly parallel to the first cleavage plane. Bar (B–E) = 50 µm

Embryo Transfer

Orthogonal and deviant late blastocysts were transferred separately into uteri of pseudopregnant CD-1 females that had been mated with vasectomized CD-1 males the previous three nights. Six to ten blastocysts were transferred per uterine horn, using a glass capillary as previously described [10]. Numbers of implantation sites and normal developing fetuses were recorded on Day 14.5 of gestation because these numbers provide information on the extent of early embryonic loss after implantation, and normal fetuses at Day 14.5 of gestation rarely fail to develop to full term [11]. Group comparisons of data were made using the chi-square test.

	RESULTS

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To locate the position of the first cleavage plane relative to the Em-Ab axis in the mouse blastocyst, we used the nonperturbing cell-lineage tracer DiI, which was originally employed for the same purpose by Piotrowska et al. [5]. By labeling a blastomere of the two-cell stage embryo, the first cleavage plane can be identified as the boundary between labeled and nonlabeled cells at the later stages of development. First, to ensure that the lineage tracer did not spread beyond the labeled blastomere and its progeny, we followed the DiI fluorescent signal over two cell divisions after labeling. Soon after the DiI microdroplet was deposited onto a blastomere at the two-cell stage, a fluorescent stain spread through the plasma membrane of the blastomere without crossing the first cleavage plane to stain the other blastomere (Fig. 2, A and B). Consequently, only two blastomeres at the four-cell stage and four blastomeres at the eight-cell stage had a fluorescent signal (Fig. 2, C–F), which indicates that the DiI label in the two-cell embryo was restricted to only one blastomere and its progeny.

View larger version (93K): [in this window] [in a new window]   FIG. 2. Restriction of the DiI staining to the labeled blastomere and its progeny. B6D2F1 x B6D2F1 mouse embryos at the two-cell (A and B), four-cell (C and D), and eight-cell (E and F) stages are shown as bright field (A, C, and E) and fluorescent (B, D, and F) images. The DiI label does not cross the first cleavage plane during early development; only two blastomeres at the four-cell stage (n = 10) and four blastomeres at the eight-cell stage (n = 5) exhibit fluorescence. Bar = 50 µm

Other researchers have reported that the first cleavage plane tends to have an orthogonal relationship with the Em-Ab axis of the early mouse blastocyst [3–5]. To test the universality of this orthogonal relationship, we examined two different genetic backgrounds of mice, B6D2F₁ and CD-1. The labeled embryos were observed at the early blastocyst stage and categorized into two groups, orthogonal and deviant, based on the angular relationship between the first cleavage plane and the Em-Ab axis (Fig. 1). For the B6D2F₁ mice, 25.0% of the blastocysts possessed an Em-Ab axis that was orthogonal to the first cleavage plane (Table 1). However, 75.0% of the blastocysts did not show the orthogonal relationship. Similarly, in the CD-1 mice, 27.5% of the blastocysts were categorized as orthogonal, and 72.5% were categorized as deviant (Table 1). Among the deviant group of blastocysts, the position of the Em-Ab axis relative to the first cleavage plane was variable, e.g., in some of these blastocysts the axis and first cleavage plane were nearly parallel (Fig. 1, D and E). These results demonstrate that the Em-Ab axis of the blastocyst has no tendency to be orthogonal to the first cleavage plane in these genotypes of mice.

We also examined whether the angular relationship between the first cleavage plane and the Em-Ab axis of the blastocyst has an effect on the quality of postimplantation development. Specifically, we tested the possibility that the blastocysts whose Em-Ab axis respected the orthogonal relationship with the first cleavage plane exhibit more successful development after implantation than do the deviant blastocysts. The blastocysts were sorted into orthogonal and deviant groups and were transferred separately into the uteri of surrogate mothers. When the surrogate mothers reached 14.5 days postcoitum, we dissected the uteri and counted the numbers of embryos and implantation sites. In both the B6D2F₁ and CD-1 mice, the frequencies of implantation and embryo development were not significantly different for the orthogonal and deviant blastocysts (Table 2). Most embryos in both groups were normal in terms of size and morphology. These results suggest that the angular relationship of the blastocyst axis to the first cleavage plane does not affect the quality of postimplantation development.

	DISCUSSION

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Other researchers have reported that the Em-Ab axis of the mouse blastocyst tends to be orthogonal with respect to the first cleavage plane [3–5]. In the present study, we examined whether this orthogonal relationship is a universal phenomenon by evaluating two different genotypes of mice. Our cell-lineage tracing experiment showed that only one quarter of the blastocysts respected the orthogonal relationship between the Em-Ab axis and the first cleavage plane, suggesting that the relationship is not universal.

To perform cell-lineage tracing, we used the membrane-soluble fluorescent dye DiI to label a blastomere at the two-cell stage. This method was previously employed by Piotrowska et al. [5] and appears to be nonperturbing to early development. By contrast, the intracytoplasmic injection of lineage-tracing substances may disturb the normal pattern of development by delaying the cell division cycle of the injected blastomeres [5]. Other nonperturbing methods have been used previously to assess the topographical relationship between the two-cell embryo and the blastocyst axis. Gardner [3] injected the zona pellucida with small oil droplets to mark the plane of first cleavage. Alternatively, denuded two-cell embryos were encapsulated in alginate gel to visualize the first cleavage plane as a constriction in the resulting blastocyst. Both methods produced results that suggested that the Em-Ab axis of most blastocysts was within 30° of orthogonal to the first cleavage plane [3]. Piotrowska and Zernicka-Goetz [4] marked the sperm entry point of fertilized eggs with a fluorescent bead. The sperm entry point tended to be located near the first cleavage plane and the boundary between the embryonic and abembryonic regions in the blastocyst, implicating an orthogonal relationship between the first cleavage plane and the Em-Ab axis. Although all of these methods [3–5] are technically different, they are apparently less invasive to embryos than the intracytoplasmic injection of a marker and may be suitable for the lineage-tracing study of nonperturbed development of early mouse embryos. In the present study, we employed the DiI labeling method because it is the most direct way to trace the progeny of the two-cell blastomeres and requires minimum manipulation of the embryos.

The previous studies of the angular relationship between the Em-Ab axis and the first cleavage plane were carried out using mouse embryos derived from F₁ (CBA x C57BL/6) [3], PO [3], and F₁ (C57BL/6 x CBA) [4, 5] females, which were mated with males of the same genotype. In the present study, we used embryos from F₁ (C57BL/6 x DBA/2) and CD-1 females mated with males of the same genotype. No researchers have reported any specific difference in the pattern of early development among these genotypes of mice. However, many physiological and developmental properties vary among different genotypes of mice [12]. The results of the present study suggest that the angular relationship between the first cleavage plane and the blastocyst Em-Ab axis is not a universal rule to be applied to all mouse or other mammalian embryos.

Alternatively, the apparent contradiction between our result and those of previous studies [3–5] may be due to the difference in the experimental procedures or interpretation of data. For example, in the alginate encapsulation experiment [3], the embryos were forced to develop in a dumbbell-shaped cast that had been molded around the denuded two-cell embryo. If the blastocoel cavity behaves like a bubble, the shape of the blastocoel should be more stable in a spherical form rather than an elongated form. Because of this physical property, a growing blastocoel may shift to one side of the dumbbell cast rather than being stretched over both sides of the cast. As a result, the blastocoel cavity tends to form in one end of the dumbbell while the ICM forms in the other end, which may have led to the interpretation that the first cleavage divided the embryo into the blastocoel (abembryonic) side and ICM (embryonic) side. This explanation can also be applied to the zona pellucida-labeling experiment [3]. Before the first cleavage, the shape of the zona pellucida is spherical. However, at the two-cell stage the sum of the diameter of the two blastomeres is larger than the internal diameter of the zona pellucida, so the zona pellucida is slightly stretched outward along the axis that is orthogonal to the first cleavage plane (Fig. 1, D and F; [3]). If the zona pellucida maintains this distorted shape until the blastocyst is formed, it may serve as a cast to bias the location of the blastocoel. Although the distortion of the zona pellucida is very subtle, the physical property of the blastocoel may be sensitive enough to shift the position of the cavity. The majority of the embryos that were encapsulated by alginate had the orthogonal relationship of the first cleavage plane to the Em-Ab axis, whereas a smaller proportion of the embryos in the zona pellucida-labeling experiment had this relationship [3].

In another study [5], the cell membrane of the blastomeres at the two-cell stage was labeled with lipophilic dye, such as DiI, to trace the progeny, as in the present study. However, the scoring of the angular relationship of the first cleavage plane to the Em-Ab axis was different from that in our study. The researchers counted the number of cells derived from a blastomere at the two-cell stage that crossed the boundary zone. The boundary zone was defined as a cell layer that is one cell deep and parallel to the roof of the blastocoel cavity. When the number of crossing cells was 3, the Em-Ab axis was considered orthogonal to the first cleavage plane [5]. The potential problem with this scoring system is that the size of the boundary zone may be too large. The number of cells that constitute the boundary zone is nearly one third of the total cell number of the blastocyst (Table 2, A–C; [5]). Thus, even when only a few cells cross this large boundary zone, the Em-Ab axis may be significantly deviated from an orthogonal relationship with the first cleavage plane. By contrast, we examined the topographical distribution of the blastomere progeny in the blastocyst to determine the position of the first cleavage plane (Fig. 1). We believe that our method yields a better view of assessing the angular relationship between the first cleavage plane and the blastocyst Em-Ab axis.

We also examined whether the angular relationship of the first cleavage plane to the Em-Ab axis of the blastocyst influences the quality of later development. This issue was not addressed in the previous studies [3–5], in which embryo development was observed only up to the blastocyst stage. Our results indicate that the efficiency of implantation and postimplantation development was not significantly different between the orthogonal and deviant blastocysts (Table 2), suggesting that the quality of postimplantation development is independent from the angular relationship between the first cleavage plane and the blastocyst axis. Results of other cell-lineage studies, in which the animal-vegetal axis of the blastocyst was traced to the egg cylinder stage, suggested that the animal-vegetal axis corresponds to the anterior-posterior axis of the postimplantation embryo [13, 14]. Studies are needed to determine whether deviation of the anterior-posterior axis from the animal-vegetal axis has a significant impact on the quality of embryo development.

Studies that have produced data suggesting that information in mammalian zygotes pattern later development (reviewed in [15, 16]) inevitably raise concerns regarding human assisted reproductive technology. During in vitro fertilization, the culture condition of the fertilized egg may influence patterns of cell division and thereby impact on the blastocyst polarity and the body axis. Although we report here that the deviation of the blastocyst axis from the first cleavage plane had no effect on mouse development, further study is required to determine whether this lack of effect also applies to human embryos.

	ACKNOWLEDGMENTS

 
We give special thanks to R. Yanagimachi and both past and present members of the Institute for Biogenesis Research for their enthusiastic encouragement. We are grateful to S. Varmuza and L. Oppedisano (Department of Zoology, University of Toronto, Toronto, ON, Canada) who first taught Y.M. basic mouse techniques.

	FOOTNOTES

 
¹ This study was supported by grants to V.B.A. from the University of Hawaii Research Council and the Victoria S. and Bradley L. Geist Foundation and grants to Y.M. from the Harold K.L. Castle Foundation and NIH (HD40208).

² Correspondence: Vernadeth B. Alarcón, Department of Anatomy and Reproductive Biology, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Rd., Honolulu, HI 96822. FAX: 808 956 7316; vernadet{at}hawaii.edu

Received: 12 April 2003.

First decision: 26 April 2003.

Accepted: 21 May 2003.

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This Article Abstract Full Text (PDF) All Versions of this Article: 69/4/1208    most recent biolreprod.103.018283v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Alarcón, V. B. Articles by Marikawa, Y. Search for Related Content PubMed PubMed Citation Articles by Alarcón, V. B. Articles by Marikawa, Y. Agricola Articles by Alarcón, V. B. Articles by Marikawa, Y.