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
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Abstract |
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Key words: blastocyst/inner cell mass/mifepristone/morula/trophoblast
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Introduction |
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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., 1995; Tabibzadeh and Babaknia, 1995
; Ghosh and Sengupta, 1998
). 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., 1977
). Progesterone is thought to maintain embryo viability, indirectly through its action on the uterus (Mead, 1989
). Heterologous anti-progesterone monoclonal antibody administered to mice and ferrets during the preimplantation stage blocks normal cleavage and embryonic development (Wang et al., 1984
; Rider and Heap, 1986
). 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., 1990
). 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., 1997
). 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.
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Materials and methods |
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Treatment procedure
Females (n = 56) showing at least two consecutive ovulatory menstrual cycles of normal lengths (2632 days) were allowed to cohabit with males during cycle days 819. 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, 1992). The details of radioimmunoassays for oestrogen and progesterone have been given earlier (Ghosh and Sengupta, 1988
, 1989
). The antisera and other chemicals for RIAs were obtained from the WHO Matched Assay Reagents Programme (Sufi et al., 1988
). 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., 1997). 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., 1990a
). Embryos with abnormalities or frank degeneration or desynchrony on gross microscopical examination (Table I
) 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., 1997
). As shown in Table II
, 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 (7080 nm) sections were cut and stained as detailed previously (Sengupta et al., 1990
; Ghosh et al., 1992
, 1996
). 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.
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Results |
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Morula stage
Figure 1 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 1A
). 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 1B
). Occasional intracytoplasmic degenerative Lafora body-like inclusions were seen in the blastomeres of morulae recovered from group 2 animals (Figure 1B
). Subzonal space and spaces between blastomeres generally contained large amounts of cellular debris.
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Zonal blastocyst
Figures 3 and 4 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, B
). 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, B
). 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 3B
). Discontinuous portions of basal lamina were present beneath trophoblast cells (Figure 3B
). Endodermal cells were characterized by the presence of well-differentiated mitochondria bearing lamellar cristae, and rough endoplasmic reticulum (RER) with engorged cisternae (Figure 3A, B
).
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As shown in Figure 4A, 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 57 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, B
). 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 6
). 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 6
). 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 7
) 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 5A
). In addition, numerous multivesicular bodies were seen in blastomeres of group 2 blastocysts (Figures 5B, 7A
). 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 7A
). Overall, there was a higher tendency towards accumulation of lipid droplets, lysosomes, autophagosomes, and multivesicular bodies in embryos collected from group 2 animals (Figures 57
).
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Discussion |
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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., 1977), 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., 1977
). 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 cellcell junctions have specific and indispensable roles in these processes (Schlafke and Enders, 1967; Enders and Schlafke, 1981
). 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., 1972
; Johnson, 1981
). 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., 1994). 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, 1984
), or cytokinesis was inhibited (Pratt et al., 1981
). 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, 1989). 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., 1990b
) and one of the earliest features of maturation from ooplasm to embryonic cytoplasm (Stern et al., 1971
). 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., 1998
), 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, 1969). 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., 1983
). Mitochondriavesicle 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, 1988
). 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, 1990; Yang and Wu, 1990
); 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., 1990
) 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, 1995; Tabibzadeh and Babaknia, 1995
; Smotrich et al., 1996
; Ghosh and Sengupta, 1998
). Interestingly in a study designed to evaluate the potential metabolic requirements for macaque embryo development in vitro, it has been shown (Schramm and Bavister, 1996
) 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, 1990
). Progesterone-dependent stimulation of specific gene expression and repression in the uterus has been linked with embryo developmental processes (Ding et al., 1994
; Psychoyos et al., 1995
). 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., 1998
), 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., 1987
).
Additionally, it is to be noted that mifepristone (RU486) also possesses anti-glucocorticoid activity (Gaillard et al., 1985). It has been reported over the years that various tissues of the body possess glucocorticoid receptors (Agarwal, 1982
) and the permissive action of cortisol is required for normal function (Feldman, 1989
). 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., 1994
; Critchley et al., 1996; Ghosh et al., 1996
). In that case, the associated mediators such as interleukin-1 and tumour necrosis factors produced in the endometrium (Hunt et al., 1992
; Laird et al., 1996
) may inhibit embryonic growth (Hill et al., 1987
; Pampfer et al., 1994
, 1995
).
Besides inhibitory cytokines, endometrial stress under the action of anti-progestin may also produce reactive oxygen species (Sugino et al., 1996a), which may cause cellular damage (Mates and Sanchez-Jimenez, 1999
), and inhibit embryo growth (Paszkowski and Clark, 1996
). This appears particularly important because progesterone inhibits superoxide radical formation (Sugino et al., 1996b
); 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., 1990
). 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., 1998
) and cellular ageing (Robbins, 1974
), such as damage to cell membranes (Mates and Sanchez-Jimenez, 1999
), lack of mitochondrial maturation (Polla et al., 1996
; Tandler et al., 1996
) and the presence of erythrophagosomes (Ghadially, 1988
), lipofuscin and other types of residual bodies (Robbins et al., 1970
), and myelinoid bodies (Hruban et al., 1972
; Ghadially, 1979
) 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.
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Acknowledgments |
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Notes |
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References |
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Submitted on July 12, 1999; accepted on October 1, 1999.