Implantation of the blastocyst in mustelid carnivores Allen C Enders

1. Implantation of the blastocyst in mustelid carnivores Allen C Enders Department of Cell Biology and Human Anatomy University of California, Davis

2. Blastocyst stage of mustelid carnivores The blastocyst stage of implantation is particularly interesting in mustelids since some of the species exhibit delayed implantation, a period in which the blastocysts not only remain unattached but also have a reduced metabolic rate and RNA and DNA synthesis are reduced. Mead [1993] reported that 20 of the 60 species have short gestation periods without delay of implantation. Some have obligate delay of implantation and others, like the mink, have a longer gestation period when mating is at the beginning of the mating season. An activation stage, in which the blastocyst enlarges, follows diapause The ferret, however, does not have a delay of implantation. The blastocyst proceeds to enlarge and implantation without a period of diapause.

3. Multiple mink blastocysts. These blastocysts were flushed from the uterus during delay of implantation. Note the dark compact ICM. Mink blastocyst in plastic block. Lipid droplets darken the ICM and appear in mural trophoblast cells.

4. Mink blastocyst, delayed implantation. Lipid appears on the compact ICM, which still has a covering of polar trophoblast. Note that the zona pellucida is present during delay. A squamous layer of endoderm underlies both the mural trophoblast and the ICM. Mink blastocyst, early activation. Little lipid remains in the ICM, which is still compact. Higher magnification of the ICM. The polar trophoblast has been lost except for flanges at the margins, showing particularly clearly on the right. Note the complete endodermal layer underlying the ICM. Mink blastocyst, activated. The ICM is beginning to spread laterally. Note the dividing ICM cell on the left.

5. Blastocyst of the western spotted skunk during delayed implantation (diapause). Note the compact inner cell mass (ICM) on the left, and the intact zona pellucida. Skunk blastocyst in delay after osmium postfixation has darkened the ICM as well the lipid droplets in individual mural trophoblast cells. Section of a skunk blastocyst during delay. Note the abundant lipid in the compact ICM. Higher magnification of an ICM during delay. Lipid droplets are conspicuous.

6. Skunk blastocyst after activation. Note the flattened ICM, well-formed visceral endoderm layer, and the thin but persistent zona pellucida above. Electron micrograph of ICM during delay. The polar trophoblast (above) has less lipid than ICM cells. Higher magnification of the blastocyst above. The ICM cells are becoming columnar, and there is no polar trophoblast overlying the center of the ICM. Only a few small lipid droplets persist in the ICM cells.

7. Mink blastocyst, activation. Mink blastocysts were exposed to horseradish peroxidase prior to fixation. Note the peroxidase in micropinocytotic vesicles as well as internalized into a larger vacuole near the nucleus. TEM of the margin of an activated skunk blastocyst ICM. Note the absence of lipid in the ICM cells, the well-formed endodermal layer below, and the flange of the marginal polar trophoblast cell in the lower right.

8. ICM of a ferret blastocyst. Note that this ICM resembles the activated ICM of species which, unlike the ferret, have delayed implantation. Note also the thin zona pellucida still surrounding the enlarged blastocyst. Rough calculation shows an appreciable increase in volume in the zona from ovulation to implantation. Blastocyst of a weasel (Mustela frenata) after activation. The swelling of the blastocyst has produced a rounded chamber in the uterus. The thin mural trophoblast in this slightly shrunken specimen has collapsed into the lumen.

9. Comments on the blastocyst stage of mustelid carnivores. The disappearance of the numerous lipid droplets during activation is dramatic. It is tempting to say that metabolism of the lipid provides the energy for recovery from diapause. However other species with a short delayed implantation such as the rat and mouse or a long delay such as the armadillo do not have large amounts of lipid in blastocyst cells when they are in the diapause state. These animals also do not have a zona pellucida during delay, although there is a one day retardation in dissolution of the zona in the rat and mouse delay. Dissolution of the zona in mustelids, however, appears to be a responsibility of the trophoblastic plaques, since the zona persists for a short time abembryonically and for quite a while under the ICM. It would also be interesting to know how the zona pellucida increases in volume during expansion of the blastocyst.

10. Implantation in mustelid carnivores The ferret and spotted skunk are usually placed in different subfamilies of the family Mustelidae, or occasionally in the family Mustelidae and the family Mephistidae respectively. Like all carnivores, the blastocyst enlarges greatly before implantation, which is central. Eventually a zonary placenta and hemophagous region are formed. As will be seen in comparing these two species, implantation is generally similar with a few minor differences. Much of the material presented here was accumulated in preparation for three papers that appear in the bibliography. None of the color micrographs have appeared in press in this form, but some of the TEMs have.

11. Implantation in the ferret

12. The inner cell mass is antimesometrial but has residual uterine secretions and material from the zona pellucida between it and the underlying uterine epithelium. Day 12. Day 12. The uterine epithelium has been infiltrated in two locations. Although there is a plaque over a gland opening on the right, there is no attachment at this position. An unattached trophoblastic plaque. Note the thin remnants of the zona pellucida on the surface of the uterine luminal epithelium. Trophoblastic plaque near a gland opening. Note the squamous endodermal cells above the cytotrophoblast.

13. The syncytial trophoblast of a plaque is adhering to the apical ends of two uterine epithelial cells in the upper left. The trophoblast above is in close proximity to the microvilli of the uterine epithelial cells. There is still glycocalyx associated with the surface of the uterine cells.

14. Intrusion of syncytial trophoblast into the uterine luminal epithelium. Processes of the plaque reach the basement membrane of the uterine epithelium. There is interdigitation of trophoblast and uterine epithelial cell microvilli near the junctional complex between the uterine cells in the center. The area of the intrusion into the epithelium has been roughly outlined.

15. Interdigitation A tongue of trophoblast from a plaque extends from the upper left between uterine epithelial cells in the center of the micrograph. Note the endometrial capillaries under the luminal epithelium. The cytoplasm from a plaque has extended between uterine epithelial cells to the base of the epithelium where a capillary is present. Note the numerous vesicles and vacuoles in the dark cytoplasm of the syncytial trophoblast.

16. The intruding syncytial trophoblast in the center of the micrograph shows an ectoplasmic region where it is adherent to the uterine cell on the left. Note the numerous granules and vesicles within the trophoblast projection, and the dense material trapped between this projection and the uterine cell on the right. These ectoplasmic regions or adhesion plaques are frequently found in early ferret implantation stages, but were not seen in the skunk implantation sites.

17. Another example of an intrusion by syncytial trophoblast of a plaque into uterine epithelium. Note the pale cytotrophoblast above the syncytial trophoblast, and an endodermal cell superficial to the cytotrophoblast. The microvilli of the uterine epithelial cells on the right have retained their glycocalyx.

18. A single intrusion into the epithelium. Note that trophoblast to the right of the plaque is not adherent to the apical ends of the uterine epithelial cells. Two trophoblastic plaques have infiltrated into the uterine luminal epithelium, extending towards the basal lamina of the epithelium. Note that the plaque has not extended into the gland lumen. Wide intrusion in the luminal epithelium. One or two pale uterine epithelial cells have been trapped in the center of this syncytial mass. Note the continuous cuboidal layer of cytotrophoblast ICM at the stage of epithelial invasion. Note the residual zona pellucida between the epiblast and the uterine lumnal epithelium

19. As implantation proceeds the syncytial trophoblast of plaques fuse to become continuous. The underlying cytotrophoblast cells become more cuboidal and uterine cells are occasionally engulfed by the syncytial trophoblast. The area of syncytial trophoblast penetration into the endometrium is roughly outlined.

20. On the lower left a process from syncytial trophoblast has extended through the residual basal lamina of the uterine epithelium to contact the basal lamina of an endometrial capillary. Note the processes from the cytotrophoblast cells above the syncytial trophoblast and the area of adhesion between a processes from a cytotrophoblast cell and syncytial trophoblast.

21. Trophoblast adjacent to endometrial capillary. Note the abundant uniform folds between syncytial trophoblast and cytotrophoblast.

22. Trophoblast surrounding endometrial vessel Although syncytial trophoblast is wrapped around the endometrial capillary, the capillary endothelium is not yet hypertrophied, nor is there a substantial interstitial lamina.

23. Implantation in the western spotted skunk

24. Section through an early implantation site. Note the antimesometrial position of the ICM, and that it is not adherent to the underlying endometrium. Early implantation with a collapsed blastocyst. The abembryonic trophoblast, which was originally mesometrial, has ruptured and collapsed. Note that the trophoblast lateral to the ICM is held in place at its margins, where trophoblastic plaques are attached to the endometrium.

25. Higher magnification of the previous implantation site . Note that a single trophoblastic plaque has attached to the endometrium at the far left. Higher magnification of left side of the site. The trophoblastic plaque is attached to the endometrium. Note the lipid droplets in the syncytial trophoblast that forms the plaque. Trophoblast has invaded into the endometrial epithelium.

26. An isolated trophoblastic plaque. This plaque has not yet attached to the endometrium. An ICM from an early implantation. Note the presence of the dark-staining zona pellucida between the ICM and the endometrium. In other regions the zona has dissolved.

27. TEM of a trophoblast plaque. The plaque is adherent to two uterine luminal epithelial cells. Non-plaque trophoblast cells, although apposed to the underlying uterine epithelial cells, are not closely interdigitated or adherenc.

28. Trophoblastic plaques invading epithelium. Note that the trophoblast is not adherent to the epithelial cells between sites of invasion. Trophoblastic plaque extending to the basal lamina of the epithelium. Note that the implantation is on a ridge of uterine luminal epithlium.

29. Summary: Attachment and implantation Syncytial trophoblast plaques adhere to the endometrium before infiltrating between epithelial cells, with which they then share apical junctional complexes and adhesion junctions. It is not yet clear whether initial adhesion is principally at the junctions between uterine epithelial cells. The facts that there are multiple implantation sites in the ferret and numerous plaques from each blastocyst after initial attachment and that the timing of implantation is well known make this an ideal animal to study the difference in surface molecules on the developing plaques as apposed to the nonadhesive inter-plaque areas. Intrusion of syncytial trophoblast into uterine epithelium is a common feature in implantation in many mammals including most primates. It should be pointed out that intrusion of trophoblast is the opposite of erosion, since the intruding processes of syncytial trophoblast depend on healthy uterine luminal epithelial cells with which they adhere. The sharing of junctions keeps the luminal epithelium relatively intact and directs the intruding processes toward the underlying luminal epithelial basal lamina. (The ferret would be a highly suitable animal in which to study junction-sharing between uterus and synctial trophoblast during intrusion.) Although areas largely devoid of organelles and inclusions other than filaments are common in plaques of the ferret whereas lipid is more common in syncytial trophoblast of spotted skunk, the basic pattern of attachment and epithelial invasion is essentially similar.

30. TEM showing infiltration of trophoblast between epithelial cells. Two trophoblast processes are extending into the epithelium. The syncytial trophoblast of the plaque has lipid droplets and is closely adherent to the epithelial cells.

31. Trophoblast infiltration of endometrial epithelium. Note that the trophoblast cells in the lower right shares an apical junction with the endometrial epithelial cells in the upper left. Lipid droplets in the trophoblast and the larger mitochondria are helpful markers of trophoblast cytoplasm. High magnification of a junctional complex between a tongue of trophoblast on the right and a uterine epithelial cell on the left. Complemenary components of the junctional complex of the uterine cell, including intermediate filaments, are formed by the trophoblast cell.

32. Multiple trophoblast plaques have invaded. The plaque at the right is adherent but has not invaded into epithelium. The other plaques have invaded as far as the luminal epithelial basement membrane. Scanning electron micrograph of an implanting blastocyst. Processes from the trophoblast above extend into the uterine epithelium below. They are slightly stretched by folding back of trophoblast.

33. Implantation site with continuous cytotrophoblast. Note that the cytotrophoblast is now cuboidal, and that there are trophoblastic plaques bridging the openings of the two glands. Implantation site with continuous syncytial tropoblast. A few pale uterine epithelial cells are present on the left. On the right trophoblast has bridged a gland opening. Note the continuous layer of pale cytotrophoblast cells and the squamous endodermal layer above.

34. Extensive invasion site. The trophoblast of the plaque has extended between and around several uterine luminal epithelial cells. Note the endometrial capillary in the lower right.

35. Trophoblast invading luminal epithelium. Trophoblast has penetrated into the epithelium. Fragments of zone pellucida are trapped n a pocket between trophoblast and the uterine epithelial cells in the upper left.

36. TEM of a large region of trophoblast extending into epithelium. Note the cuboidal cytotrophoblast above the syncytial trophoblast and the microvilli between the two layers. As the region of trophoblast becomes larger there is more distortion of the uterine cells. the section may also be slightly tangential.

37. Implantation site with continuous cytotrophoblast and syncytial trophoblast. The syncytial trophoblast layer underlying the pale cytotrophoblast now rests on the residual basal lamina of the uterine luminal epithelium. Both the cytotrophoblast and the syncytial trophoblast hae increased in thickness. Note that several pale uterine epithelial cells appear to have been engulfed by the syncytial trophoblast.

38. TEM of basal lamina penetration. Higher magnification of one area of penetration. Note the arborization of the syncytial trophoblast processes in the space between trophoblast and the underlying maternal capillary.

39. Processes from syncytial trophoblast above extend between endothelial cells of the maternal capillaries on the right and left. Some of the terminal portions of the extensions are dilated and may or may not retain connection with overlying trophoblast. Note the small vesicles and granules in some of the processes.

40. Dark fragments of enclosed cells are presumably breakdown products of uterine epithelial cells within syncytial trophoblast. A tongue of trophoblast has reached and spread out on the residual basal lamina left by the uterine epithelium.

41. Summary: Elimination of uterine luminal epithelium After the initial invasion of the uterus, both the number of syncytial plaques and the area of individual syncytial plaques increase, in region that will become the zonary band of the definitive placenta. There is a dramatic increase in the amount of cytotrophoblast. The cytotrophoblast that was originally a squamous layer between plaques becomes a continuous cuboidal and in places even stratified layer. Syncytial trophoblast fuses and the uterine epithelium is eliminated with some of the epithelial cells being surrounded by syncytial trophoblast. Degradation of engulfed uterine epithelial cells occurs in multiple phagolysosomal structures. Whether or not some of the surrounded cells fuse with the syncytial trophoblast rather than undergoing degradation has not been fully investigated. Concomitant with the syncytial and cytotrophoblast forming confluent layers, processes of the syncytial trophoblast penetrate the residual basal lamina of the uterine luminal epithelium. Although this basal lamina is penetrated, that of the underlying endometrial capillaries is not. Is the difference in the basal lamina or are the penetrating processes only briefly lytic?

42. The thick layer of cytotrophoblast cells includes some darker cells apparently differentiating toward syncytial trophoblast.

43. Late implantation Formation of primary villi. Only at this stage in implantation does syncytial trophoblast followed by cytotrophoblast begin to invade endometrial gland openings, initiating formation of placental villi. Gland invasion. The dark syncytial trophoblast indented by cytotrophoblast can be followed into the opening of the gland overlying uterine symplasma. In the center of the micrograph, syncytial trophoblast with a core of cytotrophoblast has entered the opening of a uterine gland.

44. When the syncytial trophoblast replaces uterine epithelium, some individual cells such as that in the upper right and an occasional group of epithelial cells such as those in the upper left are trapped within syncytial trophoblast. Note that the endothelium of the maternal capillary below has begun to hypertrophy.

45. Yolk sac adjacent to trophoblast. Trophoblast is invading a gland in the lower right. Note the fetal vessel underlying the endoderm of the yolk sac (upper right). Trophoblast surrounding endometrial capillaries. The syncytial trophoblast has invaded endometrial stroma, surrounding maternal capillaries.

46. Secondary villi extend well into gland openings, and the vessels elongate, increasing depth of labyrinth. Note continued gland development. Low magnification of the yolk sac placental stage. Although trophoblast has surrounded endometrial capillaries, this region has not yet a well-formed labyrinth. Note the richly glandular endometrium below the area of trophoblast invasion.

47. Summary: Beginning of placenta formation Following substantial cytotrophoblast increase, the gland lumina become invaded for the first time. Syncytial trophoblast covers cytotrophoblast columns that begin to enter the glands. (Initial invasion of the endometrium was between glands.) Fetal mesenchyme follows the cytotrophoblast into the gland lumina as the first fetal placental villi develop. Only when these primary villi enter the glands does the gland epithelium begin to form some symplasma adjacent to the invading trophoblast. The uterine capillaries become completely surrounded by trophoblast. The endothelial cells of these capillaries begin to hypertrophy and the capillaries elongate into the trophoblast with endothelial cells showing both hypertrophy and hyperplasia. The stromal cells disappear and an interstitial membrane thickens between the syncytial trophoblast and the endometrial capillary endothelium. The labyrinth is thus formed both by intrusion of fetal villi into the endometrium which is initially important and especially by elongation of the endometrial capillaries into the trophoblast between villi subsequently. This pattern of placental labyrinth formation is the same in the spotted skunk, ferret and mink.

48. Bibliography: Enders AC, Schlafke S. Implantation in the ferret: epithelial penetration. Am J Anat 1972;133:291-326. Enders AC, Schlafke S, Hubbard NE, Mead RA. Morphologic changes in the blastocyst of the western spotted skunk during activation from delayed implantation. Biol Reprod 1986;334:423-37. Enders AC, Mead RA. Progression of trophoblast into the endometrium during implantation in the western spotted skunk. Anat Rec 1996;244:297-315.