Anatomic and cellular aspects of conceptus implantation in sheep
- Implantation mechanisms: insights from the sheep
Implantation in domestic ruminants (sheep, cattle and goats) takes place at the blastocyst stage. The blastocyst develops from the preceding morula stage embryo as the result of compaction and contains a blastocoele or central cavity surrounded by a cell monolayer or trophectoderm. The trophectoderm is involved in adhesive interactions with the endometrial epithelia that results in implantation. The structure of the endometrium has common features in all species. The endometrial mucosa is formed by a mono-or pseudostratified epithelium which is separated from the conjunctive stroma by a basal lamina. The stroma is highly vascularized and contains coiled and branched glands whose ducts open into the uterine lumen. The endometrial surface epithelium is composed of secretory cells with microvilli and ciliated cells, the latter being concentrated around the openings of the endometrial glands.
The timing of implantation differs among species and is not particularly related to the length of gestation (Guillomot et al. 1993). Differences among species arise from the length of the different implantation phases (hours in rodents to days in humans and domestic animals), the evolution of the cell–cell contacts, and the degree of endometrial invasion by the trophoblast.
In domestic ruminants and pigs, the blastocyst elongates during the latter stages of implantation, but this unique developmental event does not occur in laboratory rodents, horses, primates or humans (Guillomot et al. 1993, Allen & Stewart 2001). The polar trophectoderm over the inner cell mass (Rauber’s layer) is removed before elongation of the blastocyst. Before and perhaps during elongation of the blastocyst, the extraembryonic endoderm originates from the inner cell mass and migrates under the trophectoderm as the blastocoele expands. The mesoderm then originates from the inner cell mass and migrates between the endoderm and trophectoderm. This interposed mesoderm then cavitates; the outer layer forms the chorion with the trophectoderm, whereas the inner layer forms the yolk sac wall with endoderm. Therefore, the extraembryonic membranes form before implantation in domestic ruminants and pigs. It is tempting to speculate that the extraembryonic membranes are important for trophoblast elongation. In summary, the spherical blastocyst becomes tubular and then filamentous as it elongates and becomes a conceptus (embryo/fetus and associated extraembryonic membranes). In contrast, the blastocyst of laboratory rodents, primates and humans implants rapidly before expansion, and the extraembryonic membranes are formed after implantation (Renfree 1982, Guillomot et al. 1993, Carson et al. 2000). Furthermore, the polar trophectoderm does not disappear, but rather proliferates and gives rise to peripheral polyploid cells. However, the initial early stages of implantation are common to all species.
Based on a comparative implantation scheme proposed by Guillomot and colleagues (Guillomot et al. 1981, 1993, Guillomot 1995), the phases of implantation include: 1. shedding of the zona pellucida; 2. precontact and blastocyst orientation; 3. apposition; 4. adhesion; 5. endometrial invasion. In contrast to rodents and humans, true endometrial invasion does not occur in ruminants. Following is an anatomic and cellular description of the phases of implantation in sheep, which are illustrated in Figs 1 and 2.
Shedding of the zona pellucida (phase 1)
The morula (16–32 cells) stage embryo enters the uterus from the oviduct on day 4 after mating (day 0 = estrus/mating) (Fig. 1). The blastocyst is formed on day 6, and the zona pellucida is shed between days 8 and 9. Loss of the zona pellucida appears to be achieved by rupture and hatching after blastocyst growth or after enzymatic lysis by uterine and/or embryonic proteases. This stage can occur in blastocysts derived by in vitro maturation, fertilization and culture. In general, the zona pellucida is thought to prevent the trophoblast from contacting and attaching to the endometrial LE. The blastocyst is spherical on day 8, measures 200 µm in diameter and contains approximately 300 cells. By day 10, it measures 400–900 µm in diameter and contains approximately 3000 cells. After day 10, elongation of the blastocyst occurs, and it develops first into a tubular and then into a filamentous conceptus (Wintenberger-Torres & Flechon 1974).
Precontact and blastocyst orientation (phase 2)
Between days 9 and 14, no definitive cellular contacts are observed between the trophectoderm and the endometrial epithelium. The blastocyst appears to be positioned and immobilized in the uterus after loss of the zona pellucida. However, the blastocyst can be easily recovered from the uterus by lavage without causing structural damage. The nonrandom orientation of the blastocyst represents a biologic constant of a given species, and the blastocyst position in the uterine horn is central in species characterized by large expansion of the blastocyst, as in domestic animals.
Starting on day 11, the spherical or slightly tubular blastocyst begins to elongate until it reaches a length of 25 cm or more by day 17 and resembles a long filament composed mainly of extraembryonic trophoblast. By day 12, it has elongated markedly, reaching a length of 10–22 mm. At day 14, the filamentous conceptus is about 10 cm long. The primitive streak appears at this stage and somites soon thereafter. The conceptus, first located in the uterine horn ipsilateral to the corpus luteum, elongates into the contra-lateral horn on day 13 and may fill more than half of its length on day 17 when only one ovulation has occurred (Rowson & Moor 1966). Hatched blastocysts and trophoblastic vesicles are not able to elongate in vitro unless transferred into the uterus (Heyman et al. 1984, Flechon et al. 1986). Elongation of the blastocyst is critical for developmentally regulated production of interferon tau (IFN) (Farin et al. 1989, Guillomot et al. 1990, Gray et al. 2002), a type I IFN that is the signal for maternal recognition of pregnancy and acts in a paracrine manner on the endometrial epithelia to inhibit development of the luteolytic mechanism (Bazer 1992). The cellular and molecular mechanisms regulating blastocyst elongation are not well understood, but are hypothesized to require apposition and transient attachment of the trophoblast to the LE.
Apposition (phase 3)
Apposition of the conceptus involves the trophectoderm becoming closely associated with the endometrial LE followed by unstable adhesion. After day 14, the filamentous conceptus appears to be immobilized in the uterine lumen. The elongating blastocyst maintains close contact with the endometrial LE, which appears to imprint its rounded shape on the trophectoderm in fixed specimens (Guillomot et al. 1993). A close association of the apical membranes of both cell types is observed, although the conceptus can still be recovered intact from the uterus by lavage. In most species, the onset of apposition is accompanied by a reduction of the apical microvilli covering the trophectoderm, a reduction which occurs between days 13 and 15 on the sheep conceptus (Guillomot et al. 1981, 1993). In rodents, the endometrial epithelium undergoes the same modification, allowing a closer association with the trophoblast (Enders & Schlafke 1969); however, loss of apical microvilli on the uterine LE does not appear to occur in sheep (Guillomot et al. 1981, 1982). The permeability of uterine capillaries increases for pontamine blue at the same time (Boshier 1970). The apposition of the blastocyst is ensured by interdigitation of cytoplasmic projections of the trophectoderm cells and uterine epithelial microvilli (Guillomot et al. 1981, 1993). In sheep, apposition occurs first in the vicinity of the inner cell mass, that is, the embryo, and spreads toward the extremity of the elongated conceptus.
In ruminants, the uterine glands are also sites of apposition (Guillomot et al. 1981, Guillomot & Guay 1982). Between the caruncles, the trophoblast develops finger-like villi or papillae, which penetrate into the mouths of the superficial ducts of the uterine glands at days 15–18 (Guillomot et al. 1981, Wooding et al. 1982). During their short life (they vanish at day 20), these trophoblastic differentiations are hypothesized to anchor the periattachment conceptus and absorb the histotrophic secretions of the glands (Guillomot et al. 1981). Furthermore, the trophoblast papillae are hypothesized to facilitate the formation of more robust adhesive interactions between the trophoblast and endometrial LE (Wooding et al. 1982). Similar features were described in the cow conceptus from day 15 of pregnancy, but, curiously, the goat conceptus lacked trophoblast papillae.
The ovine uterine wall can be functionally divided into the endometrium and the myometrium. The normal adult ovine endometrium consists of LE, glandular epithelium (GE), several types of stroma (stratum compactum and stratum spongiosum), blood vessels and immune cells. In sheep, the endometrium has two distinct areas – aglandular caruncular and glandular intercaruncular. The caruncular areas have LE and compact stroma and are the sites of superficial implantation and placentation (Wimsatt 1950, Amoroso 1951). Synepitheliochorial placentation in sheep involves the fusion of placental cotyledons with endometrial caruncles to form placentomes, which serve a primary role in fetal–maternal gas exchange and derivation of nutrients by the placenta. The first changes in the endometrial LE begin on day 14 in both uterine horns (Guillomot et al. 1981). The caruncles become edematous with a folded and depressed surface. These modifications are progressive and do not occur simultaneously on all caruncles. Caruncular folding is perhaps the first step in the formation of crypts that constitute the maternal side of the future placentomes, which are structures that form with placental cotyledons (Wimsatt 1950). Dome-like cytoplasmic protrusions also appear on the caruncular epithelial cells, which have a convex apex. Similar protrusions, which are sites of endocytosis, are termed pinopods and have also been described on the uterine epithelium at the time of implantation in the mouse, rat, human and rabbit (Guillomot et al. 1981).
Adhesion (phase 4)
On day 16, the trophoblast begins to adhere firmly to the endometrial LE. Uterine lavage to recover the conceptus causes superficial structural damage at this time. The interdigitation of the trophectoderm and endometrial LE occurs in both the caruncular and intercaruncular areas of the endometrium. Adhesion of the trophectoderm to the endometrial LE progresses along the uterine horn and appears to be completed around day 22 (Boshier 1969, Guillomot et al. 1981). Interestingly, the arrest of IFN gene expression occurs in regions of the mononuclear trophectoderm which have established cellular contacts with the LE during the implantation process (Guillomot et al. 1990).
The trophoblast giant binucleate cells (BNC) have differentiated from the mononuclear trophoblast by day 16, but only mononuclear trophoblast cells are thought to adhere to the endometrial LE. The BNC of the ruminant placenta may be analogous in many respects to the trophoblast giant cells of the syncytiotrophoblast in humans (Hoffman & Wooding 1993). The BNC have at least two main functions: 1. to form a hybrid fetomaternal syncytium essential for successful implantation and subsequent placentomal growth; 2. to synthesize and secrete protein and steroid hormones, such as placental lactogen and progesterone, that regulate maternal physiology (Wooding 1992, Hoffman & Wooding 1993, Spencer et al. 2004). Trophoblast BNC are thought to arise from the mononuclear trophoblast stem cells by consecutive nuclear divisions without cytokinesis, migrate through the apical trophoblast tight junctions of the chorion, and flatten as they become apposed to the apical surface of the endometrial LE (Wimsatt 1951, Wooding 1984). The BNC then fuse apically with the endometrial LE and form syncytia of trinucleate cells, thereby assimilating and replacing the endometrial epithelium. Subsequently, the trinucleate cells enlarge by continued BNC migration and fusion to form syncytial plaques linked by tight junctions that appear to be limited in size in the ewe to 20–25 nuclei (Wooding 1984). The syncytial plaques eventually cover the caruncular surface and aid in formation of the placentome. Indeed, BNC migrate and fuse with the uterine epithelial cells or their derivatives throughout most of pregnancy. The uterine LE persists but is modified to a variable degree, depending on species, into a hybrid fetomaternal syncytium formed by the migration and fusion of the fetal BNC with those of the endometrial epithelium (Wooding 1992). The mature sheep placenta is defined as synepitheliochorial, being neither entirely syndesmochorial without uterine epithelium, nor completely epitheliochorial with two apposed cell layers whose only anatomic interaction is interdigitated microvilli, as in the pig.
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