Preimplantation embryo morphology following early luteal phase anti-nidatory treatment with mifepristone (RU486) in the rhesus monkey

D. Ghosh1, P.G.L. Lalitkumar1, Viviana J. Wong2, A.G. Hendrickx2 and Jayasree Sengupta1,3

1 Department of Physiology, All India Institute of Medical Sciences, New Delhi 110029, India and 2 California Regional Primate Research Center, University of California, Davis, CA 95616, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ultrastructural characteristics of peri-implantation stage embryos recovered on day 6 after ovulation from rhesus monkeys with or without mifepristone (RU486) treatment during the early luteal phase were examined in the present study. Monkeys were randomly allocated to two groups; group 1 animals were injected s.c. with 2 ml vehicle (1:4, benzyl benzoate: olive oil, v/v, n = 21) and group 2 animals received a single dose of mifepristone (2 mg/kg body weight, w/v, n = 30) in the same volume of vehicle on day 2 after ovulation in mated cycles. On day 6 after ovulation, female monkeys of both groups were laparotomized and their reproductive tracts were flushed to retrieve preimplantation stage embryos. Embryos that showed frank degeneration or desynchrony on gross microscopical examination were not included in the present study. Preimplantation embryo growth on day 6 after ovulation was significantly (P < 0.05) affected in the morula–blastocyst transition stage in mifepristone-treated monkeys compared with that in the control group of monkeys. Ultrastructurally, administration of mifepristone on day 2 after ovulation depressed preimplantation stage embryo development, characterized by loss of cell polarity, lack of mitochondrial maturity, and lack of differentiation in trophoblast cells. Furthermore, preimplantation embryos from mifepristone-treated animals displayed a higher occurrence of inter-blastomere space, intra-cytoplasmic vacuoles, myelinoid bodies, accumulation of lipid droplets, lysosomes, lipofuscins, autophagosomes and multivesicular bodies. Collectively, it appears that the developmental potential of preimplantation embryos was significantly compromised in mifepristone-treated cycles.

Key words: blastocyst/inner cell mass/mifepristone/morula/trophoblast


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Progesterone secretion during the luteal phase influences oviductal and endometrial functions that are essential for embryo viability and implantation in a number of species including primates. Administration of mifepristone (RU486), which is a potent antiprogestin, during the early luteal phase blocks implantation in the rhesus monkey (Ghosh and Sengupta, 1993Go) and in the human (Gemzell-Danielson et al., 1993). The anti-nidatory effect of early luteal phase mifepristone administration was not associated with any change in the serum concentrations of oestradiol-17ß and progesterone during the luteal phase, and menstrual cyclicity was also not affected (Swahn et al., 1990Go; Ghosh and Sengupta, 1993Go). Early luteal phase administration of mifepristone inhibits progesterone-induced typical endometrial secretory transformation, resulting in endometrial desynchronization and loss of receptivity for blastocyst implantation (Li et al., 1988Go; Johannisson et al., 1989Go; Swahn et al., 1990Go; Berthois et al., 1991Go; Ghosh et al., 1996Go, 1998Go; Ghosh and Sengupta, 1998Go; Lalitkumar et al., 1998Go).

While endometrial hostility in early luteal phase mifepristone-treated conception cycles may mediate the observed inhibition of blastocyst implantation (endometrial contraception), it is also possible that the viability of preimplantation embryos is compromised as a result of anti-progesterone mediated endometrial desynchronization. In fact, hormonally regulated oviductal and uterine milieu can influence embryo growth and differentiation. To this effect, growth factors and cytokines of oviductal and endometrial origin have been suggested to play crucial roles in establishing synchronous development and differentiation of embryo and endometrium during the pre- and peri-implantation stages of gestation (Harvey et al., 1995Go; Tabibzadeh and Babaknia, 1995Go; Ghosh and Sengupta, 1998Go). The development and viability of 2-cell mouse embryos were adversely affected when embryos were grown in oviductal fluid collected from donors treated with oestradiol-17ß, while the addition of the steroid directly into the culture medium failed to produce any deleterious actions (Cline et al., 1977Go). Progesterone is thought to maintain embryo viability, indirectly through its action on the uterus (Mead, 1989Go). Heterologous anti-progesterone monoclonal antibody administered to mice and ferrets during the preimplantation stage blocks normal cleavage and embryonic development (Wang et al., 1984Go; Rider and Heap, 1986Go). It has been reported that the growth rate and the implantation ability of preimplantation stage embryos from cynomolgus monkeys were not affected by direct exposure to RU486 in vitro (Wolf et al., 1990Go). However, peri-implantation stage embryos recovered from monkeys subjected to mifepristone treatment during the early luteal phase failed to implant on transferring to naturally synchronous recipients (Ghosh et al., 1997Go). We have no information about the morphological characteristics of preimplantation stage embryos following early luteal phase mifepristone treatment in conception cycles. In the present study, our aim was to examine the ultrastructural characteristics of peri-implantation stage embryos recovered from rhesus monkeys with or without early luteal phase treatment with mifepristone.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Healthy, adult female and male rhesus monkeys (Macaca mulatta) of proven fertility were used in this study. The details of animal housing, handling and management have been described elsewhere (Ghosh and Sengupta, 1992Go, 1993Go; Ghosh et al., 1996Go). Monkeys were individually housed in cages in a semi-natural condition in the Primate Research Facility of the All India Institute of Medical Sciences (New Delhi, India). The study was performed with the approval of the Ethics Committee on the Use of Non-Human Primates in Biomedical Research, A.I.I.M.S.

Treatment procedure
Females (n = 56) showing at least two consecutive ovulatory menstrual cycles of normal lengths (26–32 days) were allowed to cohabit with males during cycle days 8–19. Vaginal smears were examined daily for the presence of spermatozoa. Peripheral blood samples were collected twice daily at 10.00 a.m. and 3.00 p.m. respectively. Days of ovulation were detected from the peripheral serum concentrations of immunoreactive oestrogen and progesterone, as described earlier (Ghosh and Sengupta, 1992Go). The details of radioimmunoassays for oestrogen and progesterone have been given earlier (Ghosh and Sengupta, 1988Go, 1989Go). The antisera and other chemicals for RIAs were obtained from the WHO Matched Assay Reagents Programme (Sufi et al., 1988Go). Monkeys were randomly allocated to two groups; group 1 monkeys were injected s.c. with 2 ml vehicle (1:4, benzyl benzoate: olive oil, v/v, n = 21) and group 2 monkeys received a single dose of mifepristone (RU486; 2 mg/kg body weight, w/v, n = 30) in the same volume of vehicle on day 2 after ovulation in mated cycles. Females (n = 5) which failed to show typical peri-ovulatory oestradiol rise were not used in the present study.

Processing of preimplantation stage embryos
On day 6 after ovulation, female monkeys of both groups were laparotomized under ketamine (12 mg/kg body weight) anaesthesia and their reproductive tracts were flushed with Dulbecco's minimum Eagle's medium (pH 7.4) containing pyruvate (11 mg%, w/v), glutamate (58 mg%, w/v), glucose (100 mg%, w/v) and neomycin (40 µg/ml) to retrieve preimplantation stage embryos (Ghosh et al., 1997Go). After flushing and collection of embryos from the reproductive tract, the collection dishes were examined under stereozoom microscope, and the embryos were staged according to a previously published method (Enders et al., 1990aGo). Embryos with abnormalities or frank degeneration or desynchrony on gross microscopical examination (Table IGo) were not included in the present study. Nine synchronous embryos from group 1 and seven synchronous embryos from group 2 were employed in a heterologous transfer study, the results of which have been reported previously (Ghosh et al., 1997Go). As shown in Table IIGo, five embryos from group 1 and five embryos from group 2 were used in the present study. The embryos that were processed for ultrastructural studies were immediately fixed in 3% glutaraldehyde in 0.1 mol/l phosphate buffer (pH 7.2) overnight at 4°C, washed in cold phosphate buffer and post-fixed in 1% osmium tetroxide in the same buffer for 30 min, followed by dehydration in graded alcohol, and embedded in Spurr's resin. Semi-thin (0.5 µm) and thin (70–80 nm) sections were cut and stained as detailed previously (Sengupta et al., 1990Go; Ghosh et al., 1992Go, 1996Go). The necessary supplies were obtained from Electron Microscopy Sciences (Fort Washington, PA, USA). Light and transmission electron microscopy were performed using a Leica microscope and a Zeiss transmission electron microscope, EM 10.


View this table:
[in this window]
[in a new window]
 
Table I. Preimplantation embryos recovered on day 6 of gestation in monkeys with and without mifepristone treatment
 

View this table:
[in this window]
[in a new window]
 
Table II. Details of recovered embryos used for the ultrastructure study
 
Statistical analysis
Comparisons between groups for different parameters were performed using the {chi}2 test (Samuels, 1991Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As shown in Table IGo, the recovery of embryos from mifepristone (RU486)-treated monkeys (group 2; 53.4%) was less than that from the control group (group 1; 76.2%); however, this difference was not statistically significant. Table IGo also shows the growth status of the preimplantation embryos recovered on day 6 after ovulation from monkeys in groups 1 and 2. In mifepristone-treated animals (group 2), 25% (4/16) embryos were desynchronized or degenerated, 44% (7/16) were at the morula stage, and 31% (5/16) were at the blastocyst stage on day 6 of gestation. In the control group (group 1), more than 68% (11/16) of preimplantation embryos recovered were at the blastocyst stage, 19% (3/16) were at the morula stage and 13% (2/16) were desynchronized or degenerated. Thus, on day 6 of gestation, preimplantation embryo growth was significantly (P < 0.05) affected in the morula–blastocyst transition stage in mifepristone-treated monkeys compared with that in control group monkeys. There was, however, no significant change in the numbers of degenerated or desynchronized embryos between the two treatment groups. Table IIGo shows the stages of embryos collected from group 1 and group 2 animals that were subjected to ultrastructural examination in the present study.

Morula stage
Figure 1Go shows morulae collected from group 1 and group 2 animals. Morulae from group 1 showed blastomeres with apical microvilli, and junctional complexes in apical, as well as basal domains (Figure 1AGo). In these blastomeres, round to ovoid-shaped mitochondria with dense matrices and without clear cristae were present in clusters. Nuclei were generally large and round, bearing dispersed heterochromatin. Focal areas of cytoplasm lacked any organelles. Morulae collected from monkeys exposed to early luteal phase mifepristone (group 2) showed large intercellular spaces, minimum degree of apposition of lateral cell membranes of blastomeres which, however, had retained their rounded cell shape with few apical microvilli, poorly developed junctional complexes and relatively small, pleiomorphic mitochondria present in clusters (Figure 1BGo). Occasional intracytoplasmic degenerative Lafora body-like inclusions were seen in the blastomeres of morulae recovered from group 2 animals (Figure 1BGo). Subzonal space and spaces between blastomeres generally contained large amounts of cellular debris.



View larger version (151K):
[in this window]
[in a new window]
 
Figure 1. Blastomeres from morulae recovered from group 1 (A) and group 2 (B) animals. (A) In the control group (group 1) morulae, blastomeres show closely apposed lateral cell border, primitive junctional complexes in apical and basal domains, microvilli on apical surfaces, and round nuclei (N) with dispersed heterochromatin. Note clusters of round to ovoid mitochondria (M) and areas of cytoplasm devoid of organelles. (B) Blastomeres of morulae collected from group 2 animals, showing rounded appearance of cells with marginal apposition between cells, and mitochondria (M) clustered in groups. Lafora body-like intracytoplasmic inclusion (asterisk) is seen. Z, zona pellucida. Bars = 2 µm.

 
Early blastocyst
Transitional stage embryos from both group 1 and group 2 animals revealed distinct spaces among blastomeres (Figure 2Go). Mitochondria were elongated and contained cristae, and at this stage of development, they were generally not clustered in the cytoplasm. The blastomeres of transitional stage embryos collected from group 1 animals revealed Golgi complexes in the perinuclear region, and in the central regions of blastomeres, and were often associated with mitochondria, and they had large nuclei bearing prominent nucleoli and dispersed heterochromatin (Figure 2A, BGo).



View larger version (163K):
[in this window]
[in a new window]
 
Figure 2. Several blastomeres of transitional stage embryos collected from group 1 (A, B) and group 2 (C, D) animals. In both groups, mitochondria (M) are no longer clustered, they are pleiomorphic and are distributed throughout the cytoplasm, showing some degree of cristae formation; however, U-, C- and O-shaped mitochondria and large, cristae-free electron dense mitochondria are numerous in group 2 blastomeres (D). In addition, signs of cellular damage and occurrence of intracellular vacuoles appear distinctive in blastomeres from group 2 animals (C). G, Golgi bodies. Arrow, annulate lamellae. Bars = 2 µm (A, C), and 1 µm (C, D).

 
Blastomeres of transitional stage embryos collected from group 2 animals showed relatively less development and very few junctional complexes between apposing blastomeres (Figure 2CGo), and an overt lack of mitochondrial maturity (Figure 2DGo). A considerable degree of pleiomorphism (ovoid to curved shapes) was observed in mitochondria. In addition, very large mitochondria bearing electron dense matrix and irregular cristae were seen (Figure 2DGo).

Zonal blastocyst
Figures 3 and 4GoGo show some of the ultrastructural characteristics of zonal blastocysts recovered from control (group 1) animals. At the blastocyst stage, embryos had an enlarged cavity which was often irregular in shape. Trophoblast cells in embryos collected from group 1 animals exhibited marked polarity with numerous microvilli on apical surfaces (Figure 3A, BGo). Polar trophoblast cells overlying inner cell mass (ICM) cells were generally squamous, had distinctive apical junctions, infoldings of lateral cell membranes and desmosomes (Figure 3A, BGo). Mitochondria were large and ovoid with lamellar cristae, and were often located in close association with granular endoplasmic reticulum. The conspicuous presence of intracytoplasmic bundles of filaments appeared to be a typical feature of trophoblast cells (Figure 3BGo). Discontinuous portions of basal lamina were present beneath trophoblast cells (Figure 3BGo). Endodermal cells were characterized by the presence of well-differentiated mitochondria bearing lamellar cristae, and rough endoplasmic reticulum (RER) with engorged cisternae (Figure 3A, BGo).



View larger version (153K):
[in this window]
[in a new window]
 
Figure 3. Ultrastructural characteristics of blastocysts recovered from group 1. Zona pellucida (Z) is rarefied at this stage (A, B). Polar trophoblast cells show the presence of complex and extensive apical junctional complexes ({blacktriangleright}) with margination and plication of lateral membrane, and characteristic bundles of filaments (F) (A, B). Mitochondria (M) at this stage are now more electron-lucent with several showing lamellar cristae (B). Discontinuous patches of basal lamina (->) are seen underlying polar trophoblast cells beneath which a cytoplasmic extension of an endodermal cell is visible (B). Some typical ultrastructural features found in inner cell mass blastomeres are shown in C and D. Nuclei bearing distinctive nuclear pores and intranuclear annulate lamellae, mitochondria close to granular endoplasmic reticulum and distinctive Golgi complexes (G) are evident. Note the desmosomal junctions (arrowhead) between blastomeres in (B). Bars = 2 µm (A), 1 µm (BD).

 


View larger version (180K):
[in this window]
[in a new window]
 
Figure 4. Trophoblast cells from blastocysts recovered from group 1. (A) Mural trophoblast having numerous caveolae, and microvilli bearing fine filaments on apical surface. Long bundles of filaments (F), large mitochondria with typical numerous cristae (M) and a few lipid droplets are seen. The basal domains of such cells facing the blastocyst cavity are generally flattened. (B) Trophoblasts showing extensive junctional complexes and rich arborization of cytoplasmic filaments (F), and lysosomes (L). Bars = 1 µm (A) and 0.5 µm (B).

 
Trophoblast cells and cells of the inner cell mass (ICM) showed numerous polyribosomes and short strands of RER (Figures 3 and 4GoGo). They also had mitochondria that were either elongated or large ovoid-shaped with typical lamellar cristae. Within the ICM, discontinuous patches of basal lamina were found. The nuclei of ICM blastomeres were large with dispersed heterochromatin, intranuclear annulate lamellae, and prominent nuclear pores (Figure 3C, DGo). Numerous coated caveolae and cytoplasmic vesicles were seen on the free surfaces of blastomeres at intercellular spaces (Figure 3C, DGo).

As shown in Figure 4AGo, mural (outermost) trophoblast cells showed mitochondria with less dense matrix and numerous lamellar cristae, long bundles of cytoplasmic filaments, numerous plasma membrane-associated coated caveolae and cytoplasmic vesicles, and numerous microvilli having fine filaments within their cores on apical cell surfaces. Basal domains of trophoblast cells facing the blastocyst cavity remained flattened.

Figures 5–7GoGoGo show several characteristics observed in blastocysts collected from group 2 animals treated with mifepristone on day 2 after ovulation. Blastomeres of these blastocysts frequently exhibited mitochondria, endoplasmic reticulum and junctional complexes, with cytological features that were more often seen in cleavage stage embryos (Figure 5A, BGo). Trophoblast cells of blastocysts retrieved from mifepristone-treated animals lacked the typical features generally found in such differentiated cells. For example, junctional complexes, though present, were not as well developed or as extensive as observed in trophoblast cells of blastocysts collected from group 1 animals (Figure 6Go). In addition, the cytoplasm of polar and mural trophoblasts had clustering of pleiomorphic mitochondria around lipid droplets, of which very few had electron-lucent matrices and lamellar cristae (Figure 6Go). Furthermore, a conspicuous absence of intracytoplasmic filaments was a marked feature in trophoblasts of group 2 blastocysts. Additionally, few morphological features, which were seen very infrequently in blastocysts of group 1 animals, were seen frequently in blastocysts from mifepristone-treated animals (group 2). These included binucleate cells, inter-blastomere spaces, intra-cytoplasmic vacuoles, autolysosomes, lipofuscin, erythrophagolysosomes, myelinoid and multivesicular bodies. Blastocysts obtained from group 2 animals showed large inter-blastomere spaces (Figures 5 and 7GoGo) suggestive of lack of compaction, as was seen in morulae obtained from the same group of animals. Again, several blastomeres showed large intra-cytoplasmic vacuoles containing cellular debris, as well as various cytoplasmic contents (Figure 5AGo). In addition, numerous multivesicular bodies were seen in blastomeres of group 2 blastocysts (Figures 5B, 7AGoGo). Myelinoid bodies were also frequently seen as stacks or whorls of myelinated membranes in concentric and reticuloid patterns in blastomeres of zonal blastocysts collected from group 2 animals (Figure 7AGo). Overall, there was a higher tendency towards accumulation of lipid droplets, lysosomes, autophagosomes, and multivesicular bodies in embryos collected from group 2 animals (Figures 5–7GoGoGo).



View larger version (157K):
[in this window]
[in a new window]
 
Figure 5. Blastomeres of blastocysts from group 2. (A) Large inter-blastomere spaces and lack of contiguity between blastomeres are evident. Areas of cytoplasm are devoid of organelles, close clustering of electron-dense mitochondria, erythrophagosome-like structure (large asterisk) and typical autolysosomes with lipid droplets (small asterisks) are seen. (B) Blastomeres showing pleiomorphism of mitochondria (M) with electron-dense matrix; few show beginning of cristae. Coated caveolae are found on free surfaces of blastomeres. Large, electron-dense multivesicular bodies (mb) are also seen. Bars = 5 µm (A) and 1 µm (B).

 


View larger version (114K):
[in this window]
[in a new window]
 
Figure 6. Trophoblast cells of blastocysts from group 2 monkeys. (A) Polar trophoblasts showing apical junctional complexes (arrowhead), numerous microvilli and lysosomes (L). These cells show primitive cytology with many large undifferentiated mitochondria (M), lack of filaments and endoplasmic reticulum. (B) Mural trophoblast cells of blastocyst showing apical junctional complexes (arrowhead). The basal domains of these cells, however, are rounded unlike the flattened domains found in mural trophoblast cells of blastocysts from group 1 animals. Several mitochondria (M) are differentiated, with the presence of lamellar cristae; however, in many mitochondria cristae are lacking. Z, zona pellucida. Bars = 1 µm (A) and 2 µm (B).

 


View larger version (151K):
[in this window]
[in a new window]
 
Figure 7. Blastomeres of blastocysts from group 2 animals showing (A) myelin bodies ([]) and multivesicular bodies (mb), (B) lipid dropletes, lipofuscin bodies (lb) and lysosomes (L). One blastomere shows typical karyorrhexis (B). Bars = 1 µm (A) and 2 µm (B).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a previous study, we have reported that preimplantation stage embryos recovered on day 6 of gestation from monkeys treated with early luteal phase mifepristone (RU486) failed to implant on transferring to naturally synchronized, untreated, surrogate monkeys (Ghosh et al., 1997Go). Now, we report that the loss of implantation ability in preimplantation-stage embryos retrieved from female monkeys treated with an anti-nidatory dose of mifepristone on day 2 after ovulation was associated with the lack of: (i) compaction at the morula stage as distinguished by a lower degree of apposition and polarity of blastomeres, (ii) normal sequential structural and organizational changes in mitochondria, and (iii) cytological differentiation features in trophoblast cells.

Features such as typical junctional complexes, occasional desmosome-like structures, and appearance of microvilli on free surfaces, which are considered signs of increasing cell differentiation and determination (Ducibella et al., 1977Go), were seen in morula and transition stage embryos retrieved from control group animals. The presence of apical tight junctional complexes in outer cells constitutes the beginning of an outer epithelial layer, and it can then function to separate the outer environment from the inner one; this process of early compartmentalization in the embryo is indeed a primary event in embryogenesis (Ducibella et al., 1977Go). Interestingly, junctional complexes in apical domains and along lateral cell borders of blastomeres in morula and transition stage embryos recovered from mifepristone-treated monkeys remained primitive. Cellular polarization of blastomeres, which is important for initiating differentiation of inner cell mass and trophectoderm in normal compacting morula, was clearly delayed in embryos recovered from mifepristone-exposed monkeys.

The differentiation of the trophoblast and the inner cell mass (ICM) and expansion of the blastocoele are important events in the development of the preimplantation mammalian embryo. Microscopical evidence suggests that cell–cell junctions have specific and indispensable roles in these processes (Schlafke and Enders, 1967Go; Enders and Schlafke, 1981Go). There is experimental evidence to suggest that the position and environment of blastomeres at the morula stage could account for their determination to become either trophoblast or ICM cells (Hillman et al., 1972Go; Johnson, 1981Go). Trophoblast cells of zonal blastocysts collected from control group monkeys revealed cellular characteristics typical of this stage of development and differentiation, which were not generally observed in trophoblast cells lying beneath the zona pellucida and overlying the blastocoele cavity, and adjoining the inner cell mass in zonal blastocysts collected from monkeys treated with early luteal phase mifepristone.

Change in shape and adhesion of blastomeres of the morula initiates the formation of the blastocyst cavity and marks the earliest sign of differentiation. However, the presence of a cavity and segregation of cells to one side as seen in blastocysts collected from mifepristone-treated cycles by itself may not be a reliable indicator of either the stage of development or its developmental potential (Dokras et al., 1994Go). It has also been shown in the mouse that the morphological event of cavitation could occur even when either DNA replication was inhibited (Dean and Rossant, 1984Go), or cytokinesis was inhibited (Pratt et al., 1981Go). It appears, therefore, critical to analyse whether cavitation observed in embryos following mifepristone exposure reflects vacuole formation and segregation of cells or true blastocoel formation.

In early cleavage stage embryos, mitochondria are structurally undifferentiated and generate ATP at relatively low levels when compared to those observed at late morula and blastocyst stages (Van Blerkom, 1989Go). Maturation of mitochondria from large convoluted electron-dense structures to short, elongated electron-lucent forms with distinctive lamellar cristae is a feature of maturation of this cellular organelle in rhesus monkey and baboon morulae (Enders et al., 1990bGo) and one of the earliest features of maturation from ooplasm to embryonic cytoplasm (Stern et al., 1971Go). In transitional stage embryos and in zonal blastocysts collected from control animals, typically differentiated mitochondria with lamellar cristae were found, while mitochondrial maturation was markedly diminished in transitional stage embryos and in zonal blastocysts from mifepristone-exposed monkeys. Since any potential adverse influence on normal mitochondrial function and differentiation during the preimplantation stages could be of direct developmental significance for the embryo (Van Blerkom et al., 1998Go), it is possible that the loss of embryonic viability in mifepristone-exposed animals could arise from inadequate mitochondrial maturation in these embryos.

Additionally, in transitional stage embryos and in zonal blastocysts from mifepristone-exposed monkeys several membrane-bound electron-dense granulated bodies or multivesicular bodies were often found in close association with large unbranched electron-dense mitochondria. Such associations have been reported for mouse embryos in early developmental stages and less frequently in morulae (Calarco and Brown, 1969Go). Aggregates of dense bodies probably comprising multivesicular bodies have also been observed in pre-compaction stage human embryos but not in later stages of development (Lopata et al., 1983Go). Mitochondria–vesicle complexes have also been described for human embryos fertilized and grown in culture, but were found in early but not in later stages of development (Sundstrom et al., 1981). Along with the appearance of these structures, autolysosomes, lipofuscins, residual bodies, myelinoid bodies, erythrophagosomes and degenerative Lafora body-like intracytoplasmic inclusions consisting of complex glycoprotein and acid mucopolysaccharide were more often found in embryos recovered from mifepristone-treated females. These cytoplasmic features are generally associated with cytotoxicity (Ghadially, 1988Go). Whether such cytotoxicity is linked with the observed higher number of degenerated and desynchronized embryos in the mifepristone treatment group remains an open question, because it was not statistically higher compared with the control group.

Collectively, it appears that the developmental potential of embryos was significantly compromised in mifepristone-treated cycles. Thus, the question arises as to how early luteal phase administration of mifepristone to monkeys on day 2 of potential conception cycles induces embryo growth arrest. Mifepristone may directly affect the early embryo (Juneja and Dodson, 1990Go; Yang and Wu, 1990Go); however, this does not appear to be the case in the present study, because a very low dose of the anti-progestin was used in the present study. Moreover, it has been shown (Wolf et al., 1990Go) that the growth rate and implantation ability of preimplantation-stage embryos of cynomolgus monkeys were not affected directly by RU486 in vitro.

The possibility that mifepristone inhibits the secretion of progesterone-dependent factors from oviductal and endometrial cells, and thereby inhibits embryonic development appears intriguing. This notion is supported by the fact that receptors for many growth factors and cytokines are present in pre-embryos, and that these growth factors and cytokines are secreted by oviductal and endometrial cells (Edwards, 1995Go; Tabibzadeh and Babaknia, 1995Go; Smotrich et al., 1996Go; Ghosh and Sengupta, 1998Go). Interestingly in a study designed to evaluate the potential metabolic requirements for macaque embryo development in vitro, it has been shown (Schramm and Bavister, 1996Go) that primate embryos can form morulae in a chemically defined, protein-free medium; however, blastocyst formation and its hatching require some components from the serum. Compacting mouse embryos co-cultured with uterine cells showed improved development in the presence of progesterone added to culture medium (Sakkas and Trounson, 1990Go). Progesterone-dependent stimulation of specific gene expression and repression in the uterus has been linked with embryo developmental processes (Ding et al., 1994Go; Psychoyos et al., 1995Go). It is noteworthy, in this connection, that the contragestional effect of early luteal phase mifepristone has been shown to be associated with a decrease in endometrial concentration and secretion of placental protein 14 (Lalitkumar et al., 1998Go), which belongs to a family of glycoproteins known as glycodelins and functions to transport small hydrophobic molecules across tissue boundaries, and thus may influence embryo growth before the development of the placental circulation (Huhtala et al., 1987Go).

Additionally, it is to be noted that mifepristone (RU486) also possesses anti-glucocorticoid activity (Gaillard et al., 1985Go). It has been reported over the years that various tissues of the body possess glucocorticoid receptors (Agarwal, 1982Go) and the permissive action of cortisol is required for normal function (Feldman, 1989Go). Thus, mifepristone may block the permissive action of cortisol, and thereby cause endometrial stress and shock, and initiate a local cascade of pro-inflammatory events in the endometrial bed (Antonakis et al., 1994Go; Critchley et al., 1996; Ghosh et al., 1996Go). In that case, the associated mediators such as interleukin-1 and tumour necrosis factors produced in the endometrium (Hunt et al., 1992Go; Laird et al., 1996Go) may inhibit embryonic growth (Hill et al., 1987Go; Pampfer et al., 1994Go, 1995Go).

Besides inhibitory cytokines, endometrial stress under the action of anti-progestin may also produce reactive oxygen species (Sugino et al., 1996aGo), which may cause cellular damage (Mates and Sanchez-Jimenez, 1999Go), and inhibit embryo growth (Paszkowski and Clark, 1996Go). This appears particularly important because progesterone inhibits superoxide radical formation (Sugino et al., 1996bGo); also, superoxide dismutase, which is a scavenging system for free radicals, is very high in endometrial cells at the mid-secretory stage and is found in human uterine fluid at the pre- and peri-implantation stages (Narimoto et al., 1990Go). Thus, this mechanism could play a role in protecting embryos from superoxide damage. Indeed, many of the observed changes in preimplantation embryos recovered from mifepristone-treated animals displayed morphological characteristics that are known to be associated with cellular oxidative stress (Reiter et al., 1998Go) and cellular ageing (Robbins, 1974Go), such as damage to cell membranes (Mates and Sanchez-Jimenez, 1999Go), lack of mitochondrial maturation (Polla et al., 1996Go; Tandler et al., 1996Go) and the presence of erythrophagosomes (Ghadially, 1988Go), lipofuscin and other types of residual bodies (Robbins et al., 1970Go), and myelinoid bodies (Hruban et al., 1972Go; Ghadially, 1979Go) in cells.

In summary, administration of mifepristone on day 2 after ovulation depressed preimplantation stage embryo development at the morula to blastocyst transition characterized by loss of cell polarity, lack of mitochondrial maturity, and lack of differentiation in trophoblast cells. It appears that altered endocrine and paracrine signals from desynchronized endometrium following anti-progestin treatment cause inadequate embryo growth and development. However, the precise nature of causative factors responsible for such developmental defects in preimplantation stage embryos resulting in their loss of viability remains to be investigated.


    Acknowledgments
 
The study was supported by funds from the Rockefeller Foundation, CONRAD, the NIH, US-held India Rupee Fund, the Indian Council of Medical Research and the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization (to D.G. and J.S.G.) and NIH grant PR 00169 (to A.G.H.).


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Agarwal, M.K. (1982) Antagonism of glucocorticoid action in vivo. In Agarwal, M.K. (ed.), Hormone Antagonists. Walter de Gruyter, Berlin, pp. 381–390.

Antonakis, N., Georgoulias, V., Margioris, A.N. et al. (1994) In vitro differential effects of the antiglucocorticoid RU 486 on the release of lymphokines from mitogen-activated normal human lymphocytes. J. Steroid Biochem. Mol. Biol., 51, 67–72.[ISI][Medline]

Berthois, Y., Brux, J.D., Salat-Baroux, J. et al. (1991) A multiparametric analysis of endometrial estrogen and progesterone receptors after the postovulatory administration of mifepristone. Fertil. Steril., 55, 547–554.[ISI][Medline]

Calarco, P.G. and Brown, E.H. (1969) An ultrastructural and cytological study of preimplantation development of the mouse. J. Exp. Zool., 171, 253–284.[ISI][Medline]

Cline, E.M., Randall, P.A. and Oliphant, G. (1977) Hormone mediated oviductal influence on mouse embryo development. Fertil. Steril., 28, 766–771.[ISI][Medline]

Crichley, H.O.D., Jones, R.L., Lea, R.G. et al. (1996) Role of inflammatory mediators in human endometrium during progesterone withdrawal and early pregnancy. J. Clin. Endocrinol. Metab., 84, 240–248.[Abstract/Free Full Text]

Dean, W.L. and Rossant, J. (1984) Effect of delaying DNA replication on blastocyst formation in the mouse. Differentiation, 26, 134–137.[ISI][Medline]

Ding, Y.Q., Zhu, L.J., Bagchi, M.K. and Bagchi, I.C. (1994) Progesterone stimulates calcitonin gene expression in the uterus during implantation. Endocrinology, 135, 2265–2274.[Abstract]

Dokras, A., Sargent, I.L. and Barlow, D.H. (1994) Human blastocyst grading: an indicator of developmental potential. Hum. Reprod., 8, 2119–2127.[Abstract]

Ducibella, T., Albertini, D.F., Anderson, E. and Biggers, J.D. (1977) The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development. Dev. Biol., 45, 231–250.

Edwards, R.G. (1995) Physiological and molecular aspects of human implantation. Hum. Reprod., 10, 1–13.[ISI][Medline]

Enders, A.C. and Schlafke, S. (1981) Differentiation of blastocyst of the rhesus monkey. Am. J. Anat., 162, 1–21.[ISI][Medline]

Enders, A.C., Lantz, K.C. and Schlafke, S. (1990a) The morula–blastocyst transition in two Old World primates: the baboon and rhesus monkey. J. Med. Primatol., 19, 725–747.[ISI][Medline]

Enders, A.C. Lantz, K.C. and Schlafke, S. (1990b) Differentiation of the inner cell mass of the baboon blastocyst. Anat. Rec., 226, 237–248.[ISI][Medline]

Feldman, D. (1989) Mechanism of action of cortisol. In DeGroot, L.J. (ed.), Endocrinology, volume 2. Saunders, Philadelphia, pp. 1557–1571.

Gaillard, R.C., Riondel, A., Muller, A.F. et al. (1985) RU 486: studies of its antiglucocorticoid activity in man. In Baulieu, E.E. and Segal, S.J. (eds), The Antiprogestin Steroid RU 486 and Human Fertility Control. Plenum Press, New York, pp. 331–337.

Gemzell-Danielsson, K., Swahn, M.L., Svalander, P. and Bygdeman, M. (1993) Early luteal phase treatment with mifepristone (RU 486) for fertility regulation. Hum. Reprod., 8, 870–873.[Abstract]

Ghadially, F.N. (1979) Haemorrhage and haemosiderin. J. Submicrosc. Cytol., 11, 121–129.

Ghadially, F.N. (1988) Ultrastructural Pathology of the Cell and Matrix, vol. II. Butterworths, London, UK.

Ghosh, D. and Sengupta, J. (1988) Patterns of estrogen and progesterone receptors in rhesus monkey endometrium during the secretory phase of normal menstrual cycle and preimplantation stages of gestation. J. Steroid Biochem., 31, 223–229.[ISI][Medline]

Ghosh, D. and Sengupta, J. (1989) Endometrial responses to a deciduogenic stimulus in ovariectomized rhesus monkeys treated with oestrogen and progesterone. J. Endocrinol., 120, 51–58.[Abstract]

Ghosh, D. and Sengupta, J. (1992) Patterns of ovulation, conception and pre-implantation embryo development during the breeding season in rhesus monkeys kept under semi-natural conditions. Acta Endocrinol., 127, 168–173.[ISI][Medline]

Ghosh, D. and Sengupta, J. (1993) Anti-nidatory effect of a single, early post-ovulatory administration of mifepristone (RU 486) in the rhesus monkey. Hum. Reprod., 8, 552–558.[Abstract]

Ghosh, D. and Sengupta, J. (1998) Recent developments in endocrinology and paracrinology of blastocyst implantation in the primate. Hum. Reprod. Update, 4, 153–168.[Abstract/Free Full Text]

Ghosh, D., De, P. and Sengupta, J. (1992) Effect of RU 486 on the endometrial response to deciduogenic stimulus in ovariectomized rhesus monkeys treated with oestrogen and progesterone. Hum. Reprod., 7, 1048–1060.[Abstract]

Ghosh, D., Sengupta, J. and Hendrickx, A.G. (1996) Effect of a single dose, early luteal phase administration of mifepristone (RU486) on implantation stage endometrium in the rhesus monkey. Hum. Reprod., 11, 2026–2035.[Abstract]

Ghosh, D., Kumar, P.G.L.L. and Sengupta, J. (1997) Early luteal phase administration of mifepristone inhibits preimplantation embryo development and viability in the rhesus monkey. Hum. Reprod., 12, 575–582.[ISI][Medline]

Ghosh, D., Liu, N., Zhu, Z.M. and Sengupta, J. (1998) Immunolocalization of Ley oligosaccharide in endometrium during menstrual cycle and effect of early luteal phase mifepristone administration on its expression in implantation stage endometrium of the rhesus monkey. Hum. Reprod., 13, 1374–1379.[Abstract]

Harvey, M.B., Leco, K.J., Aecellana-Panlilio, M.Y. et al. (1995) Roles of growth factors during preimplantation development. Hum. Reprod., 10, 712–718.[Abstract]

Hill, J.A., Haimovici, F. and Anderson, D.J. (1987) Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro. J. Immunol., 139, 2250–2254.[Abstract/Free Full Text]

Hillman, N. Sherman, M.I. and Graham, C.F. (1972) The effect of spatial arrangement on cell determination during mouse development. J. Embryol. Exp. Morphol., 28, 263–278.[ISI][Medline]

Hruban, Z., Slesers, A. and Hopkins, E. (1972) Drug-induced and naturally occurring myeloid bodies. Lab. Invest., 27, 62–70.[ISI][Medline]

Huhtala, M., Seppala, M., Narvanen, A. et al. (1987) Amino acid sequence homology between human placental protein 14 and ß-lactoglobulins from various species. Endocrinology, 120, 2620–2622.[Abstract]

Hunt, J.S., Chen, H.L., Hu, X. and Tabibzadeh, S. (1992) Tumour necrosis factor-{alpha} messenger ribonucleic acid and protein in human endometrium. Biol. Reprod., 47, 141–147.[Abstract]

Johannisson, E., Oberholzer, M., Swahn, M.L. and Bygdeman, M. (1989) Vascular changes in the human endometrium following the administration of the progesterone antagonist RU 486. Contraception, 39, 103–117.[ISI][Medline]

Johnson, M.H. (1981) The molecular and cellular basis of preimplantation mouse development. Biol. Rev., 56, 463–498.[ISI][Medline]

Juneja, S.C and Dodson, M.G. (1990) Effect of RU486 on different stages of mouse preimplantation embryos in vitro. Can. J. Physiol. Pharmacol., 68, 1457–1460.[ISI][Medline]

Laird, S.M., Tuckerman, E.M., Saravelos, H. and Li, T.C. (1996) The production of tumour necrosis factor-{alpha} (TNF-{alpha}) by human endometrial cells in culture. Hum. Reprod., 11, 1318–1323.[Abstract]

Lalitkumar, P.G.L., Sengupta, J., Karande, A.A. and Ghosh, D. (1998) Placental protein 14 in endometrium during menstrual cycle and effect of early luteal phase mifepristone administration on its expression in implantation stage endometrium in the rhesus monkey. Hum. Reprod., 13, 3478–3486.[Abstract]

Li, T.C., Rogers, A.W., Dockery, P. et al. (1988) The effects of progesterone receptor blockade in the luteal phase of normal fertile women. Fertil. Steril., 50, 732–742.[ISI][Medline]

Lopata, A., Kohlman, D. and Johnston, I. (1983) The fine structure of normal and abnormal human embryos developed in culture. In Beier, H.M. and Lindner, H.R. (eds), Fertilization of the Human Egg In Vitro. Springer-Verlag, Berlin, pp. 189–210.

Mates, J.M. and Sanchez-Jimenez, F. (1999) Antioxidant enzymes and their implications in pathophysiologic processes. Front. Biosci., 15, D339–345.

Mead, R.A. (1989) Role of the mammalian corpus luteum in the maintenance of early pregnancy. In Yoshinaga, K. (ed.), Blastocyst Implantation. Serono Symposia, Adams Publishing Group, Boston, pp. 189–194.

Narimoto, K. Noda, Y., Shiotani, M. et al. (1990) Immunohistochemical assessment of superoxide dismutase expression in the human endometrium throughout the menstrual cycle. Acta Histochem. Cytochem., 24, 85–91.[ISI]

Pampfer, S., Vanderheyden, I., Vesela, J. and Hertogh, R. De (1995) Neutralization of tumor necrosis factor-{alpha} (TNF-{alpha}) action on cell proliferation in rat blastocysts by antisense oligodeoxyribonucleotides directed against TNF-{alpha} p60 receptor. Biol. Reprod., 52, 1316–1326.[Abstract]

Pampfer, S., Wuu, Y.D., Vanderheyden, I. and Hertogh, R. De (1994) Expression of tumour necrosis factor-{alpha} (TNF-{alpha}) receptors and selective effect of TNF-{alpha} on the inner cell mass in mouse blastocysts. Endocrinology, 134, 206–212.[Abstract]

Paszkowski, T. and Clark, R.N. (1996) Antioxidative capacity of preimplantation embryo culture medium declines following the incubation of poor quality embryos. Hum. Reprod., 11, 2493–2495.[Abstract]

Polla, B.S., Kantengwa, S., Francois, D. et al. (1996) Mitochondria are selective targets for the protective effects of heat shock against oxidative injury. Proc. Natl Acad. Sci. USA, 93, 6458–6463.[Abstract/Free Full Text]

Pratt, H.M., Chakraborty, J. and Surani, M.A.H. (1981) Molecular and morphological differentiation of the mouse blastocyst after manipulations of compaction with cytochalasin D. Cell, 26, 279–292.[ISI][Medline]

Psychoyos, A., Nikkas, G. Sarantis, L. and Gravanis, A. (1995) Hormonal anti-implantation agents: antiprogestins. Hum. Reprod., 10, 140–150.[ISI][Medline]

Reiter, R.J., Guerrero, J.M., Garcia, J.J. and Acuna-Castroviejo, D. (1998) Reactive oxygen intermediates, molecular damage and aging. Ann. N. Y. Acad. Sci. USA, 854, 410–424.[Abstract/Free Full Text]

Rider, V. and Heap, R.B. (1986) Heterologous anti-progestrone monoclonal antibody arrests early embryonic development and implantation in the ferret (Mustela putorius). J. Reprod. Fertil., 76, 459–470.[Abstract]

Robbins, E., Levine, E.M. and Eagle, H. (1970) Morphologic changes accompanying senescence of cultured human diploid cells. J. Exp. Med., 131, 1211–1222.[ISI][Medline]

Robbins, S.L. (1974) Pathologic Basis of Disease. Saunders, Philadelphia.

Sakkas, D. and Trounson, A.O. (1990) Co-culture of mouse embryos with oviduct and uterine cells prepared from mice on different days of pseudo-pregnancy. J. Reprod. Fertil., 90, 109–118.[Abstract]

Samuels, M.L. (1991) Statistics for the Life Sciences. Dellen Publishing Co., San Francisco.

Schlafke, S. and Enders, A.C. (1967) Cytological changes during cleavage and blastocyst formation in the rat. J. Anat., 102, 13–32.[ISI]

Schramm, R.D. and Bavister, B.D. (1996) Development of in-vitro fertilized primate embryos into blastocysts in a chemically defined, protein-free culture medium. Hum. Reprod., 11, 1690–1697.[Abstract]

Sengupta, J., Given, R.L., Talwar, D. and Ghosh, D. (1990) Endometrial resonse to deciduogenic stimulus in ovariectomized rhesus monkeys treated with oestrogen and progesterone: an ultrastructural study. J. Endocrinol., 124, 53–57.[Abstract]

Smotrich, D.B., Stillman, R.J., Widra, E.A. et al. (1996) Immunocytochemical localization of growth factors and their receptors in human pre-embryos and Fallopian tubes. Hum. Reprod., 11, 184–190.[Abstract]

Stern, S., Biggers, J.D. and Anderson, E. (1971) Mitochondria and early development of the mouse. J. Exp. Zool., 176, 179–191.[ISI][Medline]

Sufi, S.B., Donaldson, A. and Jeffcoate, S.L. (1988) Method Manual: WHO Programme for the Provision of Matched Assay Reagents. World Health Organization, Geneva, pp. 57–83.

Sugino, N., Shimamura, K., Takiguchi, S. et al. (1996a) Changes in activity of superoxide dismutase in the human endometrium throughout the menstrual cycle and in early pregnancy. Hum. Reprod., 11, 1073–1078.[Abstract]

Sugino, N., Shimamura, K. and Tamura, H. et al. (1996b) Progesterone inhibits pseudopregnant superoxide radical formation by mononuclear phagocytes in pseudopregnant rats. Endocrinology, 137, 740–754.

Sundsrom, P., Nilsson, O. and Liedholm, P. (1981) Cleavage rate and morphology of early human embryos obtained after artificial fertilization and culture. Acta Obstet. Gynecol. Scand., 60, 109–120.[ISI][Medline]

Swahn, M.L., Bygdeman, M., Cekan, S. et al. (1990) The effect of RU 486 administered during the early luteal phase on bleeding pattern, hormonal parameters and endometrium. Hum. Reprod., 5, 402–408.[Abstract]

Tabibzadeh, S. and Babaknia, A. (1995) The signals and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion. Hum. Reprod., 10, 1579–1602.[Abstract]

Tandler, B., Horne, W.I., Brittenham, G.M. and Tsukamoto, H. (1996) Giant mitochondria induced in rat pancreatic exocrine cells by ethanol and iron. Anat. Rec., 245, 65–75.[ISI][Medline]

Van Blerkom, J. (1989) Development failure in human reproduction associated with preovulatory oogenesis and pre-implantation embryogenesis. In Van Blerkom, J. and Motta, P. (eds), Ultrastructure of Human Gametogenesis and Embryogenesis. Kluwer, Dordrecht, pp. 125–180.

Van Blerkom, J., Sinclair, J. and Davis, P. (1998) Mitochondrial transfer between oocytes: potential applications of mitochondrial donation and the issue of heteroplasty. Hum. Reprod., 13, 2857–2868.[Abstract/Free Full Text]

Wang, M.-Y., Rider, V. and Heap, R.B. (1984) Action of anti-progesterone monoclonal antibody in blocking pregnancy after postcoital administration in mice. J. Endocrinol., 101, 95–100.[Abstract]

Wolf, J.P., Chilik, C.F., Dubois, C. et al. (1990) Tolerance of perinidatory primate embryos to RU 486 exposure in vitro and in vivo. Contraception, 41, 85–92.[ISI][Medline]

Yang, Y.Q. and Wu, J.D. (1990) RU 486 interferes with egg transport and retards in vivo and in vitro development of mouse embryos. Contraception, 41, 551–556.[ISI][Medline]

Submitted on July 12, 1999; accepted on October 1, 1999.