Interactions of macaque blastocysts with epithelial cells in vitro

Allen C. Enders1,4, Stuart Meyers2, Catherine A. VandeVoort3 and Gordon C. Douglas1

1 Department of Cell Biology and Human Anatomy, 2 Department of Anatomy, Physiology and Cell Biology and 3 California National Primate Research Center, University of California, Davis, California, USA

4 To whom correspondence should be addressed at: Department of Cell Biology and Human Anatomy, University of California, One Shields Avenue, Davis, CA 95616 USA. E-mail: acenders{at}ucdavis.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Early in vitro studies of blastocyst formation in several primate species have demonstrated the feasibility of such studies. Initial studies of in vitro-fertilized oocytes cultured with buffalo rat liver cells suggested that other epithelial cells might be used to assess blastocyst adherence and penetration in vitro. METHODS: Macaque blastocysts were incubated with different epithelial cell lines or with Matrigel. The interaction was studied using light and transmission electron microscopy. RESULTS: In general, zona-free blastocysts attached 2 days after placing on the substrates. MDCK cells provided optimal conditions for blastocyst development. The best preparations showed some development of an amniotic cavity and distribution of cytotrophoblast and syncytial trophoblast. Distribution of syncytial trophoblast at the margin of the site and cytotrophoblast centrally was similar to that seen at the trophoblastic plate stage in this species. However, there was less syncytial trophoblast than is normally found at this stage, and total time from fertilization to the trophoblastic plate stage was delayed 2 days. CONCLUSIONS: While in vitro studies with blastocysts cannot completely mimic the intrauterine environment, they can illustrate some of the potential interactions and provide a situation in which parameters may be manipulated.

Key words: animal model/implantation/MDCK/trophoblast


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Early in vitro studies of blastocyst formation in the marmoset, baboon, rhesus monkey and human demonstrated the possibility of developing blastocysts in vitro (Pope et al., 1982Go). Such studies also showed that in vitro-cultured preimplantation human embryos could produce chorionic gonadotrophin (Fishel et al.,1984Go; Lopata et al.,1997Go) as could marmoset blastocysts (Lopata et al.,1995aGo). It was also found that many primate blastocysts could produce syncytial trophoblast when cultured beyond the time of normal implantation (Pope et al.,1982Go; Enders et al.,1989Go; Lopata et al.,1995bGo).

Lindenberg et al. (1986)Go and Bentin-Ley et al. (2000)Go pioneered the in vitro study of human blastocysts cultured on cells derived from the endometrium. In an important ultrastructural study (Bentin-Ley et al.,2000) it was demonstrated that trophoblast cells could intrude between uterine epithelial cells under co-culture conditions. These authors also demonstrated that trophoblast cells could share desmosomes with the epithelial cells. There was, however, a peculiar development of stromal cells within the blastocyst cavity in these conditions. Lopata and Borg (1998)Go demonstrated that marmoset blastocysts cultured on uterine epithelial cells produced areas of polar cellular and syncytial trophoblast adjacent to the uterine cells and that trophoblast protrusions from this area displaced uterine epithelial cells from a Matrigel substrate.Go

Meseguer et al. (2001)Go showed that human blastocysts apparently modify the glycocalyx of endometrial epithelial cells when co-cultured. The blastocysts adhered to these uterine epithelial cells but showed no further development. It also appeared that a single blastocyst could induce apoptosis in underlying human endometrial epithelial cells when adhering (Galan et al.,2000aGo,bGo). Carver et al. (2003)Go demonstrated that hatched human blastocysts co-cultured on human endometrial stromal cell monolayers showed trophoblast outgrowth and invasion into the stromal layer. Co-culture of in vitro-fertilized ova to the blastocyst stage in the presence of endometrial cells has also been used to enhance blastocyst development prior to transfer in the human (Mercader et al.,2003Go).

While in vitro studies with blastocysts cannot completely mimic the intrauterine environment, they can illustrate some of the potential interactions that may occur, as well as possibly providing a situation in which some of the parameters of interaction may be manipulated.

In an unpublished study, in vitro-fertilized oocytes were cultured with buffalo rat liver cells since these cells had been shown to improve blastocyst development during in vitro culture (Zhang et al.,1994Go; Weston et al.,1996Go). Although the blastocysts varied somewhat in morphology, they tended to hatch at ~10–11 days after IVF. One of the specimens was left an additional 48 h; when this specimen was examined it was found that the trophoblast had penetrated the otherwise intact epithelial layer of hepatic cells. This suggested that a variety of epithelial cells from different sources and species might be used to assess the ability of blastocysts to adhere to and penetrate epithelial cells in vitro. Subsequently we examined the interaction of blastocysts with MDCK cells, UtMVEC cells, ME-180 cells, CaSki cells, and Matrigel plated on micropore filters.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Blastocyst culture methods
All procedures involving animals were performed in accordance with the NIH Guide for the Care and Use Laboratory Animals and under the approval of the University of California, Davis, Animal Care and Use Committee. Rhesus monkey oocytes were obtained as previously described (VandeVoort and Tarantal, 1991Go, 2001Go). Beginning on cycle day 1 to 4, adult female rhesus monkeys (Macaca mulatta) were injected twice daily for 7 days with 37.5 IU recombinant human (r)FSH to support multiple follicular development. On day 8, 1000 IU of rHCG was given at 07:00. On day 9, oocytes were obtained by ultrasound-guided follicular aspiration between 07:00 and 10:00 and cultured in 70 µl drops under oil of HECM-9 culture medium (McKiernan and Bavister, 2000Go) at 37°C in 5% (vol/vol) CO2 in air until just before insemination. Semen collection and washing, sperm capacitation and IVF were performed as previously described (Bavister et al.,1983Go; Sarason et al.,1991Go). Oocytes were transferred to 70 µl drops of TL-PVA medium (modified Tyrode medium containing 10mM Sodium lactate and polyvinyl alcohol (0.10mg/ml) (Boatman, 1987Go) under oil and inseminated between 16:00 and 17:00. The final concentration of sperm in each medium drop was 300 000/ml. The oocyte/sperm culture drops were incubated at 37°C in 5% CO2 in air. The next morning oocytes were rinsed in HECM-9 medium and moved to fresh HECM-9 medium drops (70 µl under oil) at 37°C in 5% CO2, 5% (vol/vol) O2 and 90% (vol/vol) N2 for 48 h. Embryos were changed to HECM-9 with 5% (vol/vol) bovine calf serum and media changes continued every other day until blastocyst formation and hatching. Most embryos first formed blastocysts on day 6 and began zona escape on day 8. Hatched blastocysts were then placed on a variety of substrates.

Substrates and co-culture conditions
The following cell lines were obtained from the American Type Culture Collection, Manassas, VA, USA; Madin Darby Canine Kidney cells (MDCK, ATCC# CCL34), ME-180 (human cervical carcinoma, ATCC# HTB33), CaSki (human cervical carcinoma, ATCC# CRL1550), buffalo rat liver cells (BRL3A). Human uterine microvascular endothelial cells (UtMVEC) were purchased from Cambrex. These cells were cultured on collagen-coated filters (5 µm pore size) in Dulbecco’s modified Eagle’s medium with 5% (vol/vol) bovine calf serum (Hyclone) and penicillin/streptomycin. Hatched blastocysts were then added. An additional group of blastocysts was placed on Matrigel matrix (9) or just the uncoated filter (1). The cultures were examined periodically to assess development of the blastocyst and whether or not the blastocyst was adherent to the substrate. When blastocysts adhered to the substrate sufficiently to withstand gentle movement of the culture dishes, the culture fluid was withdrawn and Karnovsky’s fixative was added to the well. Subsequently the blastocysts together with the substrate were cut from the wells, postfixed in 1% (wt/vol) osmium tetroxide, dehydrated in ethanol, and embedded in Araldite epoxy resin. Thick sections were stained with Azure B and thin sections stained for electron microscopic examination. Oocytes from more than one female were used to provide blastocysts for the Matrigel, MDCK cell, and UtMVEC cell experiments.

A single blastocyst was obtained from an aspirated oocyte, injected with a spermatozoon, and cultured continuously in the presence of buffalo rat liver cells. The blastocyst was adherent to the cell layer at 14 days after fertilization, and was fixed and prepared as described above.

Statistical analysis
Blastocysts cultured on different cellular substrates and on Matrigel were examined for the presence or absence of selected morphological features. Results for MDCK cells or UtMVEC were separately compared to the Matrigel data using 2x2 contingency tables. Statistical significance (P < 0.05) was tested using Fisher’s exact test (Prizm; GraphPad, San Diego, CA, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Zona-free blastocysts placed on different substrates were examined and gently rocked to determine whether or not they were adherent to the substrate. Fixed adherent blastocysts were cut from the wells. If they remained attached during the processing, they were considered attached and were examined in relationship to the substrate. If they came free at this stage, they were considered adherent but not attached. In general, 2 days after placing blastocysts on the substrates the blastocysts tended to be attached, although two of the blastocysts on MDCK cells were attached after 38 h and the blastocysts on CaSki and ME-180 cells were not adherent for nearly 3 days rather than 2 days.

Blastocysts on MDCK cells
In general the conditions of the blastocysts on MDCK cells (Figures 1Go3) were superior to those on other substrates. Of nine blastocysts plated on these cells, eight were attached and one was adherent. All of the blastocysts had intact mural trophoblast. Two of the blastocysts had rather poor inner cell mass (ICM) development. The blastocysts cultured for 38 h on MDCK cells had good ICM but no evidence of the polarization of ICM cells that precedes amnion formation in this species (Figure 2A). Two of the blastocysts that were incubated for 48 h had the beginnings of amnion formation with polarized epiblast cells radiating around a small amniotic cavity (Figures 2B, C and 3B).



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Figure 1. Macaque blastocyst incubated on MDCK cells, photographed in the epoxy block. Note the circular holes in the Millipore filter. The inner cell mass and associated trophoblast rest on the filter, and the abembryonic trophoblast arches above it. Magnification x200.

 


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Figure 2. Blastocysts incubated on MDCK cells. Magnification x325. (A) Blastocyst incubated for 38 h after zona shedding. The MDCK cells can be seen extending on either side of the blastocyst. The inner cell mass (ICM) and associated trophoblast lie directly on the Millipore filter. (B) Blastocyst incubated on MDCK cells for 48 h, showing an ICM with the beginning of an amniotic cavity (arrow). There is some thickening in the abembryonic trophoblast. (C) Blastocyst incubated on MDCK cells for 48 h. This blastocyst has a good amniotic cavity beneath the epiblast and a partitioned primary yolk sac (YS). The left side was against the side of the chamber; at the extreme right the trophoblast is attached to an MDCK cell. (D) Blastocyst on MDCK cells for 48 h. This blastocyst does not have a good ICM but has a small yolk sac cavity. It is adherent to the MDCK cells rather than resting on the filter as did the other blastocyst at this age. Note the continuity of the MDCK cells.

 


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Figure 3. Electron micrographs of blastocysts incubated on MDCK cells. (A) The inset shows the margin of the blastocyst in Figure 2C, where the syncytial trophoblast abuts an MDCK cell (magnification x800). The electron micrograph shows syncytial trophoblast on the left, with irregular projections on the free surface. It abuts an MDCK cell on the right and shares a desmosome with this cell (arrow). Magnification x7800. (B) Electron micrograph of the inner cell mass and underlying trophoblast of the blastocyst shown in Figure 2B. The epiblast cells radiate around a forming amniotic cavity (*). They are overlain by endodermal cells, and trophoblast cells lie against the filter surface. x3950

 

All of the attached blastocysts had penetrated though the MDCK cell layer. All but one of these had extensive areas of trophoblast resting on the underlying Millipore filter. The MDCK layer was otherwise intact and unilaminar in all specimens, and in some regions MDCK cells had migrated though the filter pores, forming stretches of unilaminar epithelium on the bottom of the filter. Most of the blastocysts had regions of syncytial trophoblast, commonly situated at the margins, but mostly cytotrophoblast rested on the Millipore filter. At the periphery of the area of attachment, the trophoblast abutted MDCK cells, and shared desmosomes with these cells (see Figure 3A). One of the blastocysts that did not have an ICM penetrated the MDCK cells in only two places, and was overlying some of the MDCK cells (Figure 2D). This blastocyst also did not show any regions of syncytial trophoblast.

Blastocysts on UtMVEC cells
Of the six blastocysts placed on UtMVEC cells, two were adherent and four were attached. All but one of these blastocysts had a discernible ICM. However, none of them showed amnion formation, although all were incubated a minimum of 48 h and one was incubated for 70 h after hatching.

Of those blastocysts that were attached, there was always contact with UtMVEC cells (Figure 4A). However, the UtMVEC cells formed a discontinuous layer, and many of the UtMVEC cells migrated though the pores in the Millipore filter, some of them adhering to the far side. It could therefore not be determined whether the blastocysts penetrated the UtMVEC cells as well as adhering to them.



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Figure 4. (A) Blastocyst incubated for 48 h on UtMVEC cells. Although there is an inner cell mass (ICM) there is no amniotic cavity. A UtMVEC cell appears at the left in contact with the trophoblast. Other cells have penetrated though the pores in the Millipore filter. Magnification x325. (B) Blastocyst on CaSki cells for 72 h. Although this blastocyst was adherent, it became dislodged in processing. Note the syncytial trophoblast mass beneath the poorly formed ICM. Magnification x325.

 

ME-180 and CaSki
Two other epithelial cell lines, ME-180 and CaSki, were also used. Both of these cell lines formed incomplete unilaminar stretches of cells under our culture conditions. The two blastocysts on CaSki cells that were adherent after ~72 h post-hatching became displaced during processing. Of these two blastocysts the larger appeared healthy, with a good ICM and a mass of syncytial trophoblast adjacent to the ICM, but no indication of incipient amnion formation (Figure 4B).

Both of the blastocysts on ME-180 cells (Figure 5 and 6) were attached 72 h after hatching. One of these blastocysts had a compact ICM but rather small blastocyst cavity, and was underlain by endothelial cells. It had a mass of syncytial trophoblast at the margin of the attachment and was underlain by epithelial cells (Figure 5A). The other attached blastocyst, although adhering to underlying ME-180 cells, had no well-formed ICM and extensive debris within the blastocyst cavity (Figure 5B, C).



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Figure 5. Blastocysts Incubated on ME-180 cells for 72 h. Magnification x325. (A) This blastocyst has an abnormally small cavity, has some inner cell mass (ICM) cells, a syncytial trophoblast mass (arrow) and is in contact with some isolated ME-180 cells. Note on the right where an ME-180 cell has penetrated though the filter, joining other cells beneath the filter. (B) Although the trophoblast is complete, there is no ICM, only cell fragments in the blastocyst cavity. Note the linear patch of ME-180 cells to which the trophoblast is adhering.

 


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Figure 6. Electron micrograph of trophoblast cells (above) in contact with an ME-180 cell, which is resting on the filter. Note the chomosomes in the trophoblast cell on the right. Magnification x6000.

 

Blastocysts on Matrigel
Nine blastocysts were placed on Matrigel. Two of the nine blastocysts were not adherent but were partially collapsed although they had been incubated only a little over 1 day. Two blastocysts that were incubated for 2 days were adherent but came free during processing. One of these appeared reasonably well developed, the other looked more like a morula. Of the four attached blastocysts, none had appropriate development; they had little or no blastocyst cavity (Figure 7A). Only one of the four showed outgrowth along the Matrigel (Figure 7B) but all four were slightly indented into the Matrigel.



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Figure 7. Blastocysts incubated on Matrigel. Magnification x325. (A) This blastocyst, which lacks a cavity, indents the Matrigel, but does not show any processes. (B) This blastocyst shows no cavity but has a few trophoblast cells growing out along the Matrigel.

 

Blastocyst on a hepatocyte cell layer
The blastocyst placed on buffalo rat hepatic cells was large, and had penetrated through the hepatocyte layer which was otherwise continuous (Figure 8). In places the trophoblast of the blastocyst was in contact with connective tissue cells; in other places it was in contact with the support membrane. However, since the distribution of connective tissue cells under the hepatocyte layer was irregular, it could not be determined whether the trophoblast penetrated the connective tissue. At the periphery of the area of attachment the trophoblast cells shared desmosomal-type junctions with the hepatocytes. Although there was an appreciable endodermal layer underlying the trophoblast, there was no discernible ICM in this specimen.



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Figure 8. Electron micrograph of a blastocyst incubated on buffalo rat liver cells. The syncytial trophoblast has penetrated the hepatocyte layer and is in contact with striated collagen fibrils of the underlying connective tissue. A thin process of endodermal cells (arrow) overlies the basal lamina of a cytotrophoblast cell process. Magnification x7900.

 

Summary of results
Table I summarizes the incidence of major morphological features of the blastocysts when cultured on different substrates. Statistical analyses of these data using contingency tables showed that blastocysts cultured on MDCK cells or on UtMVEC had significantly greater incidence of blastocyst cavity and inner cell mass formation than blastocysts cultured on Matrigel. Blastocysts cultured on ME-180 cells or CaSki cells were not included in the analyses due to the small sample numbers.


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Table I. Characteristics of blastocysts cultured on different substrates

 


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Of the substrates used in this series of experiments, MDCK cells clearly provided the most dependable complete surface layer as well as the best conditions for development of the blastocyst. The data also suggest that UtMVEC provide a substrate that allows significantly improved blastocyst development compared to Matrigel. However, unlike MDCK cells, the UtMVEC did not consistently provide a uniform monolayer and they do not offer the same advantages in terms of in vitro manipulation. Although some caution is warranted due to the small sample size, future work will focus on extending the MDCK cell model. Future studies should also use a supporting membrane with a smaller pore size since all cell types were able to penetrate though the 5 µm pores. Changes in culture conditions that might promote better ICM development such as the use of granulocyte cell stimulating factor (Sjoblom et al., 2002Go) might produce a higher percentage of good ICM development.

The blastocysts that showed the most advanced development had an amniotic cavity as well as cytotrophoblast and syncytial trophoblast situated along the supporting membrane. The distribution with the syncytial trophoblast at the margin of the site and cytotrophoblast centrally is similar to that seen normally at the trophoblastic plate stage in this species (Enders, 1995Go). However, there was less syncytial trophoblast than is normally found at this stage, and the total time from IVF to the trophoblastic plate stage was delayed by ~2 days. Although syncytial trophoblast is the initial type of trophoblast invading the endometrium by penetrating between uterine epithelial cells in vivo, these studies did not demonstrate whether cytotrophoblast without syncytium could or could not penetrate the epithelial cell layer.

Of particular interest is the observation that trophoblast can attach to and even penetrate diverse heterologous epithelial sheets from different organs and species. These results suggest, as does the presence of ectopic implantation in the human, that the limited opportunities for implantation in vivo are not a result of the lack of invasiveness by the trophoblast but rather modification of the endometrial environment, including the glycocalyx of the luminal epithelial cells, to decrease the facility of trophoblast to attach appropriately. Extensive expression of MUC1 may be deleterious to attachment of trophoblast to epithelial cells in a number of species (Carson et al., 2000Go), and a recent study suggests that human blastocysts may induce paracrine cleavage of endometrial cell MUC1 at the implantation site (Meseguer et al., 2001Go). In this respect it is noteworthy that normal hepatocytes do not express MUC1 histochemically (Cao et al., 1999Go), and that MDCK cells can be transfected to produce abundant expression of MUC1 (Lavelle et al.,1997Go).

The use of a cell line such as MDCK cells which has been thoroughly studied and can be manipulated has a number of advantages as a means of identifying factors that may increase or decrease adhesion and epithelial invasion by intact blastocysts. Use of such cells does not completely substitute for the use of uterine epithelial cells, which have been used to model implantation in the human (Dominguez et al., 2001Go). However, it avoids the need to provide macaque endometrial luminal epithelial cells that respond in culture to hormones in a similar fashion to endometrium at implantation. Since even short-term supravital culture of endometrium results in changes including increased cell death, which in itself produces adhesion, this method also has severe limitations.

Further studies of co-culture of blastocysts on variously transfected MDCK cells and on macaque uterine cells or human luminal epithelial cell lines could provide further information on possible limits to trophoblast penetration of epithelial cell layers.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
These studies were supported by NIH grants R03HD43863, RR001699 and RR14093.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on March 22, 2005; resubmitted on May 6, 2005; accepted on May 31, 2005.





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