MOLECULAR REPRODUCTION A N D DEVELOPMENT 32:81-87 (1992)
Tight Junctions and Cavitation in the Human Pre-Embryo ROBERTO GUALTIERI, LUIGIA SANTELLA, AND BRIAN DALE Stazione Zoologica, Naples, Italy.
ABSTRACT In the human morula, tight junctions are found between all cell pairs, at all levels of cellular apposition, associated with underlying masses of microfilaments. In cavitating morula, lanthanum tracer gained access to the intercellular spaces, except at the intersections with nascent extracellular cavities, marking the first assembly of zonulae occludentes. Presumptive trophectoderm cells contained vacuoles and larger cavities often associated with secondary lysosome-likebodies. Since the vacuoles and intracellular and extracellular cavities contain electron-dense polygranules of about 23 nm diameter, they may have common origins. In trophectoderm cells of the early blastocyst, the large intracellular vacuoles and cavities were absent, and the zonulae occludentes were located apically. Mechanisms for nascent blastocoele formation are discussed. o 1992 Wiiey-ks, Inc. Key Words: Morula, Blastocoele, Zonulae occludentes
INTRODUCTION Compaction in the mammalian pre-embryo is a fundamental event that leads to the formation of the trophectoderm, the inner cell mass and the blastocoele cavity. Polarization of early blastomeres is thought to be at the basis of compaction, and much work has been dedicated to studying the mechanism of polarization particularly in the mouse (see review by Fleming and Johnson, 1988, for references). Cell polarity has been shown to be necessary for the formation of the nascent blastocoele (Wiley and Eglitis, 1981; Wiley, 19841, while compaction is essential for blastocoele expansion (Johnson et al., 1979). All three processes are spatially and temporally interdependent. Intercellular devices, in particular tight junctions and gap junctions (Ducibella and Anderson, 1975; Caveney, 1985; Fleming and Johnson, 1988), are considered fundamental elements in these cell-cell interactions. While there has been much work on the physiological mechanisms of blastocoele enlargement (Benos and Biggers, 1981; Wiley, 1984; Manejwala and Schultz, 1989), little is known about the initiation of cavitation in the mammalian embryo. It has been proposed that the early blastocoele is derived from the expansion of preexisting intercellular spaces (Calarco and Brown, 1969), while Wiley and Eglitis (1981) suggest that both mitochondria and lipid droplets are responsible for the origin of the nascent blastocoele fluid. We have carried 0 1992 WILEY-LISS, INC.
out a preliminary ultrastructural study on the mechanism of cavitation in the human pre-embryo by following the distribution of tight junctions and their assembly into zonulae occludentes.
MATERIALS AND METHODS Oocyte Collection and Embryo Culture Spare embryos were obtained from a n in vitro fertilization (IVF) programme in Naples, Italy, from consenting patients. The follicular stimulation protocol, described previously (Dale e t al., 1991), was Gn-rh analogue down-regulation (Buserelin, Hoechst) + human menopausal gonadotropin daily, from day 2 to day 9 of stimulation (Pergonal; Serono, Rome, Italy), followed by 5,000 IU human chorionic gonadotropin (hCG; Profasi; Serono) for ovulation induction. The oocytes were collected by transvaginal ultrasonography about 34 hr following hCG administration. Oocytes were cultured for 3-6 h r in minimum essential medium (Earle's; Gibco, Grand Island, NY) supplemented with 8% heat-inactivated human serum and maintained at 37°C in a gas mixture of 5% CO, in air. The same culture medium was used for preparing semen, which was used at a final concentration of 105/ml for insemination. Embryos were cultured in MEM + 16% HS, in 5% CO, in air, a t 37°C until use. Electron Microscopy A previously reported fixation method (Dale e t al., 1991) was modified to improve the preservation of membranes and microfilaments. Samples were processed at room temperature or at 4°C where indicated. Embryos were prefixed in 3% glutaraldehyde, 5 mM CaCl,, 0.1 M cacodylate buffer, pH 7.3, containing 0.05% OsO, (added 5 min before starting) for 10-15 rnin at 4°C in the dark. Fixation was then continued for 45 min in the same fixative without OsO,. After a 3 x 10 min, or overnight, wash at 4°C in the same buffer with CaCl,, embryos were postfixed in 1% OsO,, 0.8% K,Fe(CN)Gin buffer for 1h r at 4"C, washed 3 x 10 rnin in buffer at P C , treated with 0.15% tannic acid in buffer for 3 min, quickly washed in distilled water, and block stained in 1%uranyl acetate for 1hr. After dehydration, embryos were embedded in Epon 812. Received November 2,1991; accepted December 16,1991. Address reprint requests to Brian Dale, Stazione Zoologica, Villa Comunale, 80121 Naples, Italy.
82
R. GUALTIERI ET AL.
For studies with lanthanum tracer, embryos were fixed in 2% glutaraldehyde, 2% La(N0,)36H,0 in 0.1 M cacodylate buffer, pH 7.4, for 2 hr, washed overnight in the same medium without glutaraldehyde, postfixed in 1%OsO, in buffer plus lanthanum for 1hr, dehydrated for a total of 30 min at 4"C, and embedded in Epon 812. Semithin sections were stained with 1% toluidine blue in 1%sodium borate. Grey to white serial thin sections were cut with Diatome diamond knives on a Reichert Ultracut microtome, collected on thin bar grids, and stained with methanolic saturated uranyl acetate and Reynolds lead citrate. Thin sections were observed with a Philips EM 400 electron microscope at 80 kV. The embryos, assessed under a dissecting microscope, showed normal morphology and developmental progression. Cleavage-arrested and fragmented embryos were not used for experiments. From a total of 13 embryos fixed at days 4-5 after insemination and serially sectioned, three morula and two blastocysts were fixed conventionally, and five morula and two blastocysts were infiltrated with lanthanum.
RESULTS
resembling secondary lysosomes. The latter were often found associated or actually within vacuoles and intracellular cavities (Fig. 2a). Vacuoles and intracellular and extracellular cavities contained electron-dense polygranules of about 23 nm in diameter (Fig. 2a-e), morphologically indistinguishable from granules found intracellularly (Fig. 2e). At the nascent blastocyst stage, tight junctional regions were usually observed apically between trophectoderm (TE) cells (Figs. 2e, 31, and access to lanthanum was also blocked apically (Fig. 2f). The blastocoele was irregularly shaped, and TE cells were more attenuated than in the morula (Fig. 3, right inset). Normal-sized vacuoles and secondary lysosome-like bodies were observed in both the TE and the inner mass cells, while larger vacuoles and intracellular cavities were no longer present at this stage (Fig. 3). TE cells had numerous microvilli on their apical surfaces (Fig. 3). Tight junctional regions showed the same ultrastructural features as in the morula and were only rarely encountered in basal regions of the intercellular space between TE cells (Fig. 4). Points of tight membrane apposition were more frequently seen than in the earlier stages (Fig. 4b,c). Apically, they were sometimes found to delimit strongly electron-dense, amorphous material filling the intercellular space (Fig. 4b). This material may be a product of fixation; however, the nature and physiological meaning of this material were not investigated. At the early blastocyst stage, gap junctions were common (see also Dale et al., 1991) and sometimes annular (Fig. 4a).
At the morula stage, tight junctions were found at the apposed cell surfaces of polar, apolar, and polarapolar cell pairs. Since points of tight membrane apposition were found associated with underlying masses of microfilaments (Fig. l ) , the latter were used a s an indication for following tight junctional distribution at low magnifications. Small gap junctions were sometimes observed and were often intercalated with the tight junctional regions (Fig. 1). Gap junctions (gap = 2-3 DISCUSSION nm) were associated with a n electron-dense plaque, 4-7 In this study we have shown that in the human emnm thick, clearly different from the cytoplasmic aspect of the tight junctional region (see transitional zones in bryo assembly of tight junctions, ultrastructurally simFig. 1).Desmosome-like junctions, or fascia adherens- ilar to those observed in the mouse (Ducibella et al., like junctions, as they are often called, previously ob- 1975; Magnuson e t al., 1977; Fleming et al., 19841, is served a t six- to eight-cell stages (Dale et al., 1991), well advanced a t the morula stage. As was reported in a previous paper (Dale et al., 1991), assembly probably were not found at morula or blastocyst stages. In the five morulae fixed with lanthanum, the tracer starts at the six- to eight-cell stage in regions where gained access to the intercellular spaces, showing that fascia adherens-like junctions are present. The distrithe tight junctions were not zonular at this stage. Two bution of tight junctions in the morula a t all levels of of these morulae were found to have large cavities in cellular apposition is similar to that in the mouse successive sections. By serial sectioning, it was seen morula (Magnuson et al., 19771, a s is the observation that some of these cavities were intracellular and lo- that gap junctions are often found intercalated with cated in polar cells, while others, often appearing intra- tight junctional regions (Magnuson et al., 1977). In concellular in a n earlier section, were in fact extracellular. trast to the mouse embryo, however, in which the first For example, Figure 2a shows one of these cavitating gap junctions develop at compaction (late eight-cell morulae in a section in which a large extracellular cav- stage) (Ducibella and Anderson, 1975; Ducibella et al., ity is flanked by two other large cavities, which seem to 1975), we did not observe gap junctions prior to the be intracellular. The inset in Figure 2a shows a deeper morula stage. Since in a previous study (Dale et a]., section in which the cavity on the left disappeared, 1991) we did not detect spread of Lucifer yellow beconfirming its intracellular location, while that on the tween blastomeres until the blastocyst stage, the quesright became larger and was continuous with two inter- tion arises of whether the gap junctions found in the cellular spaces (one of which is shown in Fig. 2d). At morula are fully functional. The gap junctions reported this stage, lanthanum gained access to the intercellular by Tesarik (1989) to be present in the eight-cell human spaces, stopping only at the intersections with the ex- embryo are not dissimilar to the early tight junction tracellular cavities (two for each cavity) (Fig. 2a-d). included in a fascia adherens-likejunction in our prepThe cytoplasm of polar cells was filled by organelles arations (see Dale et al., 1991).
PRE-EMBRYO TIGHT JUNCTIONS AND CAVITATION
Fig. 1. a: Apposed cell surfaces of two polar cells at apical level in a human morula, showing an obliquely cut tight junctional region (TJR) (within two bars) adjacent to a gap junction (GI. x75,500. b Apposed cell surfaces of a polar and an apolar cell in a morula. A long tight junctional region (TJR),part of which is obliquely cut, with a n interca-
83
lated gap junction(G) is shown. Note the association of the tight junctional region with masses of microfilaments, which are substituted by a n electron-dense plaque a t the boundaries (bars) with the gap junction. Arrowheads point to sites of tight membrane contact. x 158,000.
84
R. GUALTIERI ET AL.
Fig. 2
PRE-EMBRYO TIGHT JUNCTIONS AND CAVITATION
85
Fig. 3. Micrographs showing a n early human blastocyst. Arrowheads point to apical tight junctional regions in TE cells. Note the polarized distribution of microvilli. V, vacuoles. SL, secondary lysosome-like bodies. ZP, zona pellucida. X3,lOO. Right inset: Thick section of the blastocyst cut at the edge between inner cell mass and blastocoele. X440. Left inset: Enlargement of an apical tight junction between TE cells. ~ 1 2 , 8 0 0 . Fig. 2. Cavitating human morula (a+) and midblastocyst (0 infiltrated with lanthanum. a:Three large cavities are evident: the one on the left (IC,) is intracellular,that in the middle (EC,) is extracellular, and that on the right (EC,) is also extracellular as seen in successive sections (thick section in inset). Smaller intracellular cavities (IC, and IC.J, one ofwhich (IC,) is filled by lysosomal material (SL),are closely apposed to EC,. ~ 2 , 8 0 0Inset: . x520. V, large vacuole. SL, secondary lysosome-like bodies. M, mitochondria. IC and EC, intra- and extracellular cavities. b,c: Micrographs showing the two intercellular spaces included in boxes in a. d Micrograph showing the region included in the box in inset in a. Arrowheads in b-d point to sites where lanthanum is arrested. b-d. x 16,000. e: High magnification of cytoplasmic and extracellular granules. x 36,000. fi Intercellular space with interdigitating microvilli between two TE cells in a mid blastocyst stage. Lanthanum is arrested apically (arrowhead). X 28,600.
The establishment of an intercellular high-resistance seal is a prerequisite for the development of the blastocoele (Magnuson et al., 1978; Wiley, 1984), although in the mouse the trophectoderm becomes a tight epithelium only a t blastocoele expansion (Wiley, 1984). Our experiments with lanthanum show that an intercellular permeability seal of unknown resistance is formed in the early cavitating human morula, at the intersection of the intercellular spaces with the early extracellular cavities. The complex geometry of extracellular cavities in the cavitating morula and the later assembly of apical zonulae occludentes in the TE of the
86
R. GUALTIERI ET AL.
Fig. 4. Micrographs showing appositional regions between TE cells in a n early human blastocyst. a: High magnification of the tight junctional region included in the box in the inset. Asterisks delimit the thickness of underlying masses of microfilaments. G, annular gap junction. x 103,000. Inset: Low magnification showing the distribution of tight junctional regions between TE cells. Asterisks mark the
basal position of some tight junctional regions. ~ 5 , 0 0 0b. Apical tight junction between TE cells. Arrowhead points to a site of tight membrane contact which delimits strongly electrondense amorphous material. ~90,000.c: Sites of tight membrane contact (arrowheads) in a tight junctional region more basally located than in b. x 146,000.
PRE-EMBRYO TIGHT JUNCTIONS AND CAVITATION blastocyst suggest that early zonulae occludentes lining the extracellular cavities are subject to assemblydisassembly in the human pre-embryo. For the human cavitating morula, we have shown that large vacuoles and intracellular and extracellular cavities contain granules indistinguishable in electron density, size, and arrangement from granules found in corresponding cavities in the mouse morula and shown to consist of glycogen (Aziz and Alexandre, 1991). Furthermore, secondary lysosomes are found in abundance in presumptive TE cells in association with the intrablastomeric vacuoles and cavities. In a n ultrastructural and cytochemical study on cavitation in the mouse, Aziz and Alexandre (1991) suggested that a lysosomemediated mechanism leads to the formation of intracellular vacuoles. These vacuoles coalesce, forming intracellular cavities, which, by a sort of exocytosis, form the initial extracellular cavities of the nascent blastocoele. Although our present studies are preliminary, the data are consistent with a n intrablastomeric fluid accumulation mechanism operating in the cavitating human morula. The absence of large vacuoles and intracellular cavities in the early human blastocyst lends support to the hypothesis that these transitory structures are involved in the formation of the blastocoele. The developmental heterogeneity in human pre-embryos (Hardy et al., 1989; Sathananthan et al., 19901, resulting in variability in cell numbers at the blastocyst stage, will lead to differences in the geometrical pattern of early extracellular cavities in the cavitating morula. Similarly, intrablastomeric cavities may be relatively large or small depending on cell number. In fact, experimental manipulation of the cavitating morula of the mouse with a variety of agents leads to the formation of large intrablastomeric vacuoles (Ducibella and Anderson, 1979; Johnson et al., 1979; Wiley and Eglitis, 1980). Nonetheless, since Tesarik (1989) has shown that the development of zonulae occludentes and the formation of a nascent blastocoele require the enhancement of embryonic transcription at the eightcell stage, these two parameters may be regarded as markers of “normal development” in the human.
ACKNOWLEDGMENTS This work was supported in part by a grant from Ares-Serono (to B.D.). REFERENCES Aziz M, Alexandre H (1991): The origin of the nascent blastocoele in preimplantation mouse embryos: Ultrastructural cytochemistry
87
and effect of cloroquine. Rouxs Arch Dev Biol200:77-85. Benos DJ, Biggers J D (1981): Blastocyst Fluid formation. In Mastroianni L J r , Biggers J D (eds):“Fertilization and Embryonic Development.” New York: Plenum. Calarco PG, Brown EH (1969): An ultrastructural and cytological study of preimplantation development of the mouse. J Exp Zoo1 171:253-284. Caveney S (1985):The role of gap junctions in development. Annu Rev Physiol47:319-335. Dale B, Gualtieri R, Talevi R, Tosti E, Santella L, Elder K (1991): Intercellular communication in the early human embryo. Mol Reprod Dev 29:22-28. Ducibella T, Albertini D, Anderson E, Biggers J D (1975):The preimplantation mammalian embryo: Charaderization of intercellular junctions and their appearance during development. Dev Biol 45231-250. Ducibella T, Anderson E (1975): Cell shape and membrane changes in the eight-cell mouse embryo: Prerequisites for morphogenesis of the blastocyst. Dev Biol47:45-58. Ducibella T, Anderson E (1979): The effects of calcium deficiency on the formation of the zonula occludens and blastocoel in the mouse embryo. Dev Biol73:46-58. Fleming TP, Johnson MH (1988): From egg to epithelium. Annu Rev Cell Biol4:459485. Fleming TP, Warren PD, Chisholm JC, Johnson MH (1984): Trophectodermal processes regulate the expression of totipotency within the inner cell mass of the mouse expanding blastocyst. J Embrol Exp Morphol84:63-90. Hardy K, Handyside AH, Winston RML (1989): The human blastocyst: Cell number, death and allocation during late preimplantation development in vitro. Development 107:597-604. Johnson MH, Chakraborty J , Handyside AH, Wilson AH, Stern P (1979): The effect of prolonged decompaction on development of the preimplantation mouse embryo. J Embryo1 Exp Morphol 54:241261. Magnuson T, Demsey A, Stackpole CW (1977): Characterization of intercellular junctions in the preimplantation mouse embryo by freeze fracture and thin section electron microscopy. Dev Biol 61:252-261. Magnuson T, Jacobson JB, Stackpole CW (1978): Relationship between intercellular permeability and junction organization in the preimplantation mouse embryo. Dev Biol67:214-224. Manejwala FM, Schultz RM (1989):Blastocoel expansion in the preimplantation mouse embryo: Stimulation of sodium uptake by CAMP and possible involvement of CAMP-dependent protein kinase. Dev Biol 136560-563. Sathananthan H, Bongso A, Ng SC, Ho J, Mok H, Ratnam S (1990): Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells. Hum Reprod 5:309-318. Tesarik J (1989): Involvement of oocyte-codedmessage in cell differentiation control of early human embryos. Development 105:317-322. Wiley LM (1984): Cavitation in the mouse preimplantation embryo: NaiK-ATPase and the origin of the nascent blastocoelic fluid. Dev Biol 105:330-342. Wiley LM, Eglitis MA (1980): Effects of colcemid on cavitation during mouse blastocoele formation. Exp Cell Res 127:89-101. Wiley LM, Eglitis MA (1981):Cell surface and cytoskeletal elements: Cavitation in the mouse preimplantation embryo. Dev Biol86:493501.
Tight Junctions and Cavitation in the Human Pre-Embryo ROBERTO GUALTIERI, LUIGIA SANTELLA, AND BRIAN DALE Stazione Zoologica, Naples, Italy.
ABSTRACT In the human morula, tight junctions are found between all cell pairs, at all levels of cellular apposition, associated with underlying masses of microfilaments. In cavitating morula, lanthanum tracer gained access to the intercellular spaces, except at the intersections with nascent extracellular cavities, marking the first assembly of zonulae occludentes. Presumptive trophectoderm cells contained vacuoles and larger cavities often associated with secondary lysosome-likebodies. Since the vacuoles and intracellular and extracellular cavities contain electron-dense polygranules of about 23 nm diameter, they may have common origins. In trophectoderm cells of the early blastocyst, the large intracellular vacuoles and cavities were absent, and the zonulae occludentes were located apically. Mechanisms for nascent blastocoele formation are discussed. o 1992 Wiiey-ks, Inc. Key Words: Morula, Blastocoele, Zonulae occludentes
INTRODUCTION Compaction in the mammalian pre-embryo is a fundamental event that leads to the formation of the trophectoderm, the inner cell mass and the blastocoele cavity. Polarization of early blastomeres is thought to be at the basis of compaction, and much work has been dedicated to studying the mechanism of polarization particularly in the mouse (see review by Fleming and Johnson, 1988, for references). Cell polarity has been shown to be necessary for the formation of the nascent blastocoele (Wiley and Eglitis, 1981; Wiley, 19841, while compaction is essential for blastocoele expansion (Johnson et al., 1979). All three processes are spatially and temporally interdependent. Intercellular devices, in particular tight junctions and gap junctions (Ducibella and Anderson, 1975; Caveney, 1985; Fleming and Johnson, 1988), are considered fundamental elements in these cell-cell interactions. While there has been much work on the physiological mechanisms of blastocoele enlargement (Benos and Biggers, 1981; Wiley, 1984; Manejwala and Schultz, 1989), little is known about the initiation of cavitation in the mammalian embryo. It has been proposed that the early blastocoele is derived from the expansion of preexisting intercellular spaces (Calarco and Brown, 1969), while Wiley and Eglitis (1981) suggest that both mitochondria and lipid droplets are responsible for the origin of the nascent blastocoele fluid. We have carried 0 1992 WILEY-LISS, INC.
out a preliminary ultrastructural study on the mechanism of cavitation in the human pre-embryo by following the distribution of tight junctions and their assembly into zonulae occludentes.
MATERIALS AND METHODS Oocyte Collection and Embryo Culture Spare embryos were obtained from a n in vitro fertilization (IVF) programme in Naples, Italy, from consenting patients. The follicular stimulation protocol, described previously (Dale e t al., 1991), was Gn-rh analogue down-regulation (Buserelin, Hoechst) + human menopausal gonadotropin daily, from day 2 to day 9 of stimulation (Pergonal; Serono, Rome, Italy), followed by 5,000 IU human chorionic gonadotropin (hCG; Profasi; Serono) for ovulation induction. The oocytes were collected by transvaginal ultrasonography about 34 hr following hCG administration. Oocytes were cultured for 3-6 h r in minimum essential medium (Earle's; Gibco, Grand Island, NY) supplemented with 8% heat-inactivated human serum and maintained at 37°C in a gas mixture of 5% CO, in air. The same culture medium was used for preparing semen, which was used at a final concentration of 105/ml for insemination. Embryos were cultured in MEM + 16% HS, in 5% CO, in air, a t 37°C until use. Electron Microscopy A previously reported fixation method (Dale e t al., 1991) was modified to improve the preservation of membranes and microfilaments. Samples were processed at room temperature or at 4°C where indicated. Embryos were prefixed in 3% glutaraldehyde, 5 mM CaCl,, 0.1 M cacodylate buffer, pH 7.3, containing 0.05% OsO, (added 5 min before starting) for 10-15 rnin at 4°C in the dark. Fixation was then continued for 45 min in the same fixative without OsO,. After a 3 x 10 min, or overnight, wash at 4°C in the same buffer with CaCl,, embryos were postfixed in 1% OsO,, 0.8% K,Fe(CN)Gin buffer for 1h r at 4"C, washed 3 x 10 rnin in buffer at P C , treated with 0.15% tannic acid in buffer for 3 min, quickly washed in distilled water, and block stained in 1%uranyl acetate for 1hr. After dehydration, embryos were embedded in Epon 812. Received November 2,1991; accepted December 16,1991. Address reprint requests to Brian Dale, Stazione Zoologica, Villa Comunale, 80121 Naples, Italy.
82
R. GUALTIERI ET AL.
For studies with lanthanum tracer, embryos were fixed in 2% glutaraldehyde, 2% La(N0,)36H,0 in 0.1 M cacodylate buffer, pH 7.4, for 2 hr, washed overnight in the same medium without glutaraldehyde, postfixed in 1%OsO, in buffer plus lanthanum for 1hr, dehydrated for a total of 30 min at 4"C, and embedded in Epon 812. Semithin sections were stained with 1% toluidine blue in 1%sodium borate. Grey to white serial thin sections were cut with Diatome diamond knives on a Reichert Ultracut microtome, collected on thin bar grids, and stained with methanolic saturated uranyl acetate and Reynolds lead citrate. Thin sections were observed with a Philips EM 400 electron microscope at 80 kV. The embryos, assessed under a dissecting microscope, showed normal morphology and developmental progression. Cleavage-arrested and fragmented embryos were not used for experiments. From a total of 13 embryos fixed at days 4-5 after insemination and serially sectioned, three morula and two blastocysts were fixed conventionally, and five morula and two blastocysts were infiltrated with lanthanum.
RESULTS
resembling secondary lysosomes. The latter were often found associated or actually within vacuoles and intracellular cavities (Fig. 2a). Vacuoles and intracellular and extracellular cavities contained electron-dense polygranules of about 23 nm in diameter (Fig. 2a-e), morphologically indistinguishable from granules found intracellularly (Fig. 2e). At the nascent blastocyst stage, tight junctional regions were usually observed apically between trophectoderm (TE) cells (Figs. 2e, 31, and access to lanthanum was also blocked apically (Fig. 2f). The blastocoele was irregularly shaped, and TE cells were more attenuated than in the morula (Fig. 3, right inset). Normal-sized vacuoles and secondary lysosome-like bodies were observed in both the TE and the inner mass cells, while larger vacuoles and intracellular cavities were no longer present at this stage (Fig. 3). TE cells had numerous microvilli on their apical surfaces (Fig. 3). Tight junctional regions showed the same ultrastructural features as in the morula and were only rarely encountered in basal regions of the intercellular space between TE cells (Fig. 4). Points of tight membrane apposition were more frequently seen than in the earlier stages (Fig. 4b,c). Apically, they were sometimes found to delimit strongly electron-dense, amorphous material filling the intercellular space (Fig. 4b). This material may be a product of fixation; however, the nature and physiological meaning of this material were not investigated. At the early blastocyst stage, gap junctions were common (see also Dale et al., 1991) and sometimes annular (Fig. 4a).
At the morula stage, tight junctions were found at the apposed cell surfaces of polar, apolar, and polarapolar cell pairs. Since points of tight membrane apposition were found associated with underlying masses of microfilaments (Fig. l ) , the latter were used a s an indication for following tight junctional distribution at low magnifications. Small gap junctions were sometimes observed and were often intercalated with the tight junctional regions (Fig. 1). Gap junctions (gap = 2-3 DISCUSSION nm) were associated with a n electron-dense plaque, 4-7 In this study we have shown that in the human emnm thick, clearly different from the cytoplasmic aspect of the tight junctional region (see transitional zones in bryo assembly of tight junctions, ultrastructurally simFig. 1).Desmosome-like junctions, or fascia adherens- ilar to those observed in the mouse (Ducibella et al., like junctions, as they are often called, previously ob- 1975; Magnuson e t al., 1977; Fleming et al., 19841, is served a t six- to eight-cell stages (Dale et al., 1991), well advanced a t the morula stage. As was reported in a previous paper (Dale et al., 1991), assembly probably were not found at morula or blastocyst stages. In the five morulae fixed with lanthanum, the tracer starts at the six- to eight-cell stage in regions where gained access to the intercellular spaces, showing that fascia adherens-like junctions are present. The distrithe tight junctions were not zonular at this stage. Two bution of tight junctions in the morula a t all levels of of these morulae were found to have large cavities in cellular apposition is similar to that in the mouse successive sections. By serial sectioning, it was seen morula (Magnuson et al., 19771, a s is the observation that some of these cavities were intracellular and lo- that gap junctions are often found intercalated with cated in polar cells, while others, often appearing intra- tight junctional regions (Magnuson et al., 1977). In concellular in a n earlier section, were in fact extracellular. trast to the mouse embryo, however, in which the first For example, Figure 2a shows one of these cavitating gap junctions develop at compaction (late eight-cell morulae in a section in which a large extracellular cav- stage) (Ducibella and Anderson, 1975; Ducibella et al., ity is flanked by two other large cavities, which seem to 1975), we did not observe gap junctions prior to the be intracellular. The inset in Figure 2a shows a deeper morula stage. Since in a previous study (Dale et a]., section in which the cavity on the left disappeared, 1991) we did not detect spread of Lucifer yellow beconfirming its intracellular location, while that on the tween blastomeres until the blastocyst stage, the quesright became larger and was continuous with two inter- tion arises of whether the gap junctions found in the cellular spaces (one of which is shown in Fig. 2d). At morula are fully functional. The gap junctions reported this stage, lanthanum gained access to the intercellular by Tesarik (1989) to be present in the eight-cell human spaces, stopping only at the intersections with the ex- embryo are not dissimilar to the early tight junction tracellular cavities (two for each cavity) (Fig. 2a-d). included in a fascia adherens-likejunction in our prepThe cytoplasm of polar cells was filled by organelles arations (see Dale et al., 1991).
PRE-EMBRYO TIGHT JUNCTIONS AND CAVITATION
Fig. 1. a: Apposed cell surfaces of two polar cells at apical level in a human morula, showing an obliquely cut tight junctional region (TJR) (within two bars) adjacent to a gap junction (GI. x75,500. b Apposed cell surfaces of a polar and an apolar cell in a morula. A long tight junctional region (TJR),part of which is obliquely cut, with a n interca-
83
lated gap junction(G) is shown. Note the association of the tight junctional region with masses of microfilaments, which are substituted by a n electron-dense plaque a t the boundaries (bars) with the gap junction. Arrowheads point to sites of tight membrane contact. x 158,000.
84
R. GUALTIERI ET AL.
Fig. 2
PRE-EMBRYO TIGHT JUNCTIONS AND CAVITATION
85
Fig. 3. Micrographs showing a n early human blastocyst. Arrowheads point to apical tight junctional regions in TE cells. Note the polarized distribution of microvilli. V, vacuoles. SL, secondary lysosome-like bodies. ZP, zona pellucida. X3,lOO. Right inset: Thick section of the blastocyst cut at the edge between inner cell mass and blastocoele. X440. Left inset: Enlargement of an apical tight junction between TE cells. ~ 1 2 , 8 0 0 . Fig. 2. Cavitating human morula (a+) and midblastocyst (0 infiltrated with lanthanum. a:Three large cavities are evident: the one on the left (IC,) is intracellular,that in the middle (EC,) is extracellular, and that on the right (EC,) is also extracellular as seen in successive sections (thick section in inset). Smaller intracellular cavities (IC, and IC.J, one ofwhich (IC,) is filled by lysosomal material (SL),are closely apposed to EC,. ~ 2 , 8 0 0Inset: . x520. V, large vacuole. SL, secondary lysosome-like bodies. M, mitochondria. IC and EC, intra- and extracellular cavities. b,c: Micrographs showing the two intercellular spaces included in boxes in a. d Micrograph showing the region included in the box in inset in a. Arrowheads in b-d point to sites where lanthanum is arrested. b-d. x 16,000. e: High magnification of cytoplasmic and extracellular granules. x 36,000. fi Intercellular space with interdigitating microvilli between two TE cells in a mid blastocyst stage. Lanthanum is arrested apically (arrowhead). X 28,600.
The establishment of an intercellular high-resistance seal is a prerequisite for the development of the blastocoele (Magnuson et al., 1978; Wiley, 1984), although in the mouse the trophectoderm becomes a tight epithelium only a t blastocoele expansion (Wiley, 1984). Our experiments with lanthanum show that an intercellular permeability seal of unknown resistance is formed in the early cavitating human morula, at the intersection of the intercellular spaces with the early extracellular cavities. The complex geometry of extracellular cavities in the cavitating morula and the later assembly of apical zonulae occludentes in the TE of the
86
R. GUALTIERI ET AL.
Fig. 4. Micrographs showing appositional regions between TE cells in a n early human blastocyst. a: High magnification of the tight junctional region included in the box in the inset. Asterisks delimit the thickness of underlying masses of microfilaments. G, annular gap junction. x 103,000. Inset: Low magnification showing the distribution of tight junctional regions between TE cells. Asterisks mark the
basal position of some tight junctional regions. ~ 5 , 0 0 0b. Apical tight junction between TE cells. Arrowhead points to a site of tight membrane contact which delimits strongly electrondense amorphous material. ~90,000.c: Sites of tight membrane contact (arrowheads) in a tight junctional region more basally located than in b. x 146,000.
PRE-EMBRYO TIGHT JUNCTIONS AND CAVITATION blastocyst suggest that early zonulae occludentes lining the extracellular cavities are subject to assemblydisassembly in the human pre-embryo. For the human cavitating morula, we have shown that large vacuoles and intracellular and extracellular cavities contain granules indistinguishable in electron density, size, and arrangement from granules found in corresponding cavities in the mouse morula and shown to consist of glycogen (Aziz and Alexandre, 1991). Furthermore, secondary lysosomes are found in abundance in presumptive TE cells in association with the intrablastomeric vacuoles and cavities. In a n ultrastructural and cytochemical study on cavitation in the mouse, Aziz and Alexandre (1991) suggested that a lysosomemediated mechanism leads to the formation of intracellular vacuoles. These vacuoles coalesce, forming intracellular cavities, which, by a sort of exocytosis, form the initial extracellular cavities of the nascent blastocoele. Although our present studies are preliminary, the data are consistent with a n intrablastomeric fluid accumulation mechanism operating in the cavitating human morula. The absence of large vacuoles and intracellular cavities in the early human blastocyst lends support to the hypothesis that these transitory structures are involved in the formation of the blastocoele. The developmental heterogeneity in human pre-embryos (Hardy et al., 1989; Sathananthan et al., 19901, resulting in variability in cell numbers at the blastocyst stage, will lead to differences in the geometrical pattern of early extracellular cavities in the cavitating morula. Similarly, intrablastomeric cavities may be relatively large or small depending on cell number. In fact, experimental manipulation of the cavitating morula of the mouse with a variety of agents leads to the formation of large intrablastomeric vacuoles (Ducibella and Anderson, 1979; Johnson et al., 1979; Wiley and Eglitis, 1980). Nonetheless, since Tesarik (1989) has shown that the development of zonulae occludentes and the formation of a nascent blastocoele require the enhancement of embryonic transcription at the eightcell stage, these two parameters may be regarded as markers of “normal development” in the human.
ACKNOWLEDGMENTS This work was supported in part by a grant from Ares-Serono (to B.D.). REFERENCES Aziz M, Alexandre H (1991): The origin of the nascent blastocoele in preimplantation mouse embryos: Ultrastructural cytochemistry
87
and effect of cloroquine. Rouxs Arch Dev Biol200:77-85. Benos DJ, Biggers J D (1981): Blastocyst Fluid formation. In Mastroianni L J r , Biggers J D (eds):“Fertilization and Embryonic Development.” New York: Plenum. Calarco PG, Brown EH (1969): An ultrastructural and cytological study of preimplantation development of the mouse. J Exp Zoo1 171:253-284. Caveney S (1985):The role of gap junctions in development. Annu Rev Physiol47:319-335. Dale B, Gualtieri R, Talevi R, Tosti E, Santella L, Elder K (1991): Intercellular communication in the early human embryo. Mol Reprod Dev 29:22-28. Ducibella T, Albertini D, Anderson E, Biggers J D (1975):The preimplantation mammalian embryo: Charaderization of intercellular junctions and their appearance during development. Dev Biol 45231-250. Ducibella T, Anderson E (1975): Cell shape and membrane changes in the eight-cell mouse embryo: Prerequisites for morphogenesis of the blastocyst. Dev Biol47:45-58. Ducibella T, Anderson E (1979): The effects of calcium deficiency on the formation of the zonula occludens and blastocoel in the mouse embryo. Dev Biol73:46-58. Fleming TP, Johnson MH (1988): From egg to epithelium. Annu Rev Cell Biol4:459485. Fleming TP, Warren PD, Chisholm JC, Johnson MH (1984): Trophectodermal processes regulate the expression of totipotency within the inner cell mass of the mouse expanding blastocyst. J Embrol Exp Morphol84:63-90. Hardy K, Handyside AH, Winston RML (1989): The human blastocyst: Cell number, death and allocation during late preimplantation development in vitro. Development 107:597-604. Johnson MH, Chakraborty J , Handyside AH, Wilson AH, Stern P (1979): The effect of prolonged decompaction on development of the preimplantation mouse embryo. J Embryo1 Exp Morphol 54:241261. Magnuson T, Demsey A, Stackpole CW (1977): Characterization of intercellular junctions in the preimplantation mouse embryo by freeze fracture and thin section electron microscopy. Dev Biol 61:252-261. Magnuson T, Jacobson JB, Stackpole CW (1978): Relationship between intercellular permeability and junction organization in the preimplantation mouse embryo. Dev Biol67:214-224. Manejwala FM, Schultz RM (1989):Blastocoel expansion in the preimplantation mouse embryo: Stimulation of sodium uptake by CAMP and possible involvement of CAMP-dependent protein kinase. Dev Biol 136560-563. Sathananthan H, Bongso A, Ng SC, Ho J, Mok H, Ratnam S (1990): Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells. Hum Reprod 5:309-318. Tesarik J (1989): Involvement of oocyte-codedmessage in cell differentiation control of early human embryos. Development 105:317-322. Wiley LM (1984): Cavitation in the mouse preimplantation embryo: NaiK-ATPase and the origin of the nascent blastocoelic fluid. Dev Biol 105:330-342. Wiley LM, Eglitis MA (1980): Effects of colcemid on cavitation during mouse blastocoele formation. Exp Cell Res 127:89-101. Wiley LM, Eglitis MA (1981):Cell surface and cytoskeletal elements: Cavitation in the mouse preimplantation embryo. Dev Biol86:493501.
