Conceptus Elongation, Implantation, and Early Placental Development in Species with Central Implantation: Pigs, Sheep, and Cows

Abstract

Species have different strategies for implantation and placentation. Much can be learned about general molecular and cellular biology through the examination and comparison of these differences. To varying degrees, implantation in all species includes alterations in epithelial polarity, the transformation of the endometrial stroma, the differentiation of the trophoblast, cell-to-cell and tissue-to-tissue signaling through hormones, cytokines, and extracellular vesicles, and the alteration of the maternal immune system. This review focuses on implantation in pigs, sheep, and cows. These species share with mice/rats and humans/primates the key events of early embryonic development, pregnancy recognition, and the establishment of functional placentation. However, there are differences between the pregnancies of livestock and other species that make livestock unique biomedical models for the study of pregnancy and cell biology in general. Pig, sheep, and cow conceptuses (embryo/fetus and associated placental membranes) elongate prior to implantation, displaying central implantation, extended periods of conceptus attachment to the uterus, and epitheliochorial (pigs) and synepitheliochorial (sheep and cows) placentation. This review will discuss what is understood about how the trophoblast and extraembryonic endoderm of pig, sheep, and cow conceptuses elongate, and how a major goal of current in vitro models is to achieve conceptus elongation. It will then examine the adhesion cascade for conceptus implantation that initiates early placental development in pigs, sheep, and cows. Finally, it will conclude with a brief overview of early placental development in pigs, sheep, and cows, with a listing of some important "omics" studies that have been published.

Keywords: conceptus; cows; elongation; implantation; livestock; pigs; placentation; pregnancy; sheep.

Figure 1

Conceptus elongation in the pig. The top panels are Petri dishes in which uterine flushings from days 10, 11, and 15 of gestation contain conceptuses undergoing rapid morphological changes. The bottom panels graphically illustrate the migration of trophoblast and extraembryonic endoderm cells during conceptus elongation between days 10 and 15 of gestation. Electron micrographs suggest that the trophoblast and endoderm migrate towards the inner cell mass and then subsequently migrate outward in opposite directions to initiate elongation in pigs. Although pig conceptuses elongate to a greater extent than the conceptuses of sheep and cows, the process is believed to be mechanistically similar among the three species. For the conceptus in the bottom right corner the extraembryonic endoderm extends across the entire length of the trophoblast and the trophoblast layer is continuous, similar to the adjacent conceptuses. Both have been drafted in an abbreviated manner to simplify the drawing.

Figure 2

Pathways utilized by elongating conceptuses of pigs and sheep to metabolize the hexose sugars glucose and fructose. Shown are metabolic pathways confirmed to be active within intrauterine environment of pigs. Although active glutaminolysis pathway has yet to be established in sheep conceptuses, elongating sheep conceptuses are active in one-carbon metabolism, the pentose phosphate pathway, the polyol pathway (fructolysis), and the conversion of pyruvate into lactate. There is also evidence for hexosamine biosynthesis in pig and sheep conceptus trophoblasts.

Figure 3

Adapted from Tinning and collaborators (2023) [67]. Conceptus elongation is still not well understood in mechanistic detail because elongation has not been reproduced in vitro. This is an area of current intense interest for the field of early pregnancy in the livestock species. Shown is a recreation of a conceptualized macrofluidic system that could emulate local and systemic processes that occur during pregnancy. This system would be nourished via continuous culture media flow. The architecture of the tissue should be sustained by stromal scaffolds containing cultured endometrial spheroids. The apical surface would be populated with an endometrial LE monolayer. Above the endometrial LE, either trophoblast blastoids or trophoblast spheroids would be cultured in appropriate media, while still being exposed to products from the tissue cultured within the same well.

Figure 4

The peri-implantation period of pregnancy for pigs, sheep, and cows. Panel A illustrates pre-implantation events. (1) Histotroph is secreted from the endometrial luminal epithelium (LE) and glandular epithelium (GE) of pigs and GE of sheep and cows in response to progesterone and other factors. When conceptuses have elongated sufficiently, they secrete pregnancy recognition factors, primarily estrogen in the pig and interferon tau (IFNT) in sheep and cows. In addition, the conceptus IFNs, IFN gamma (IFNG) in the pig and IFNT in sheep and cows upregulate the expression of interferon stimulated genes (ISGs) in the endometrial stroma. Panel B illustrates the adhesion cascade for implantation. The elongating conceptuses of pigs, sheep, and cows orient the apical surface of the trophoblast to the apical surface of the endometrial LE. Removal of anti-adhesive mucins from the apical surface of the endometrial LE allows for close apposition and attachment of conceptus trophoblast. Initial juxtracrine interactions between the apical surface of the trophoblast and endometrial LE are mediated through carbohydrates, glycams, and galectins, supplied as histotroph, binding to carbohydrates, lectins, and glycans, present on the cell surfaces. Firm adhesion is later mediated through extracellular matrix (ECM) ligands, supplied as histotroph, binding to integrins on the cell surfaces. Pigs have true epitheliochorial placentation as the trophoblast does not breach the endometrial LE barrier to the underlying endometrial stroma. Panel C illustrates early stages of placentation in sheep and cows. The conceptuses of sheep and cows extend trophoblast papillae into the mouths of the endometrial GE perhaps for increased access to histotroph secreted by the GE. After adhesion, binucleate trophoblast giant cells (TGCs) begin to form and fuse with endometrial LE cells. These events result in significant disruption of the endometrial LE barrier to trophoblast interaction with the endometrial stroma.

Figure 5

Sites of conceptus implantation in a pig, sheep, and two cows. Shown are implantation sites from a day 20 pregnant (20P; H&E staining) pig, a day 20P sheep (H&E staining), a day 31P cow (H&E staining), and a day 26P cow (green = immunofluorescence staining for epithelial-cadherin (E-cadherin); red = immunofluorescence staining for pregnancy-associated glycoproteins (PAGs); blue = DAPI staining of nuclei for histological reference). True epitheliochorial placentation is illustrated for the pig. A syncytium forms at the uterine–placental interface of the sheep. The loss of regions of the endometrial luminal epithelium (LE) is observed in the D26P cow. GE, endometrial glandular epithelium; CAR, endometrial caruncle; ICAR, intercaruncular endometrium; Tr, trophoblast; MYO, myometrium.

Figure 6

(A) There are multiple cell types at the uterine–placental interface of pigs that express genes differentially. These include the tall columnar chorion cells at the top of the uterine–placental folds indicated by the asterisks (1). Proteins expressed exclusively within these cells of the folds include solute carrier family 2A8 (SLC2A8) [39], glutaminase (GLS) [31], and brain-type creatine kinase (CKB) [123]. (2) The low cuboidal chorion cells at the bottom of the uterine–placental folds. A protein expressed exclusively within these folds cells is glutamine synthetase (GLUL) [31]. (3) The tall columnar chorion cells of the areolae. Amongst others, proteins and genes expressed in these cells, but not the chorion cells of the uterine–placental folds include cathepsin L (CTSL1) [124], SLC2A8 protein [39], mRNA for SLC2A3 [36], aquaporin 5 (AQP5) protein [123], the beta 3 (β3) integrin subunit (ITGB3) protein [125], and claudin 4 (CLDN4) protein as illustrated in the section from day 84 of pregnancy (D84P) in the figure. (4) The low cuboidal endometrial luminal epithelial (LE) cells at the bottoms of the folds of the uterine–placental interface. The integrin receptor α2β1 (ITGA2B1) protein is shown to be expressed by these cells in the section from day 60P in the figure. (5) The squamous endometrial LE cells at the tops of the folds of the uterine–placental interface. SLC2A3 mRNA is expressed exclusively within these fold cells [36]. (6) The endometrial LE cells lining the surface of areolae near the openings of the endometrial glandular epithelium (GE). OPN mRNA and protein are expressed in all endometrial LE cells except for these cells [125]. (7) Endometrial GE. Many proteins are expressed by the endometrial GE, but acid phosphate 5, tartrate resistant (ACP5, commonly referred to as uteroferrin) is only expressed in this cell type within the uterine environment of pregnant pigs where it is secreted and delivered to areolae as a component of histotroph [126,127]. (B) There may be multiple ways that pregnancy-associated glycoprotein (PAG)-positive cells are incorporated into endometrial luminal epithelium (LE) during syncytialization in sheep [9,57,59]. (1) Illustrates the formation of PAG positive binucleate trophoblast giant cells (TGCs) in the trophoblast (1a), fusion with an endometrial LE cell (1b), formation of a trinucleate syncytial cell composed of trophoblast and endometrial LE (1c), and continued fusion of TGCs with growing syncytia to form syncytial plaques at the uterine–placental interface of sheep (1d). However, PAG positive cells have been detected at the uterine–placental interface that are not predicted by these events. (2) Mononucleate PAG positive cells that are common in the trophoblast layer (2a) and sometimes appear to be invading between endometrial LE cells (2b), and mononucleate PAG positive cells are present in the endometrial LE layer allowing for the possibility that PAG positive TGCs may fuse with PAG positive endometrial LE cells, and PAG positive mononucleate Tr cells may fuse with PAG positive endometrial cells (2c). (3) PAG positive TGCs sometimes appear to be invading between endometrial LE cells and not fusing with LE cells (3a) and they are observed within the endometrial LE layer (3b), allowing for another possible means of forming PAG positive trinucleate cells, i.e., mononucleated trophoblast cells fusing with TGCs within the endometrial LE (3c). (4) Finally, fusion and invasion of PAG positive cells into the endometrial LE can lead to disruption and destabilization of the endometrial LE barrier to trophoblast contact with endometrial stroma. Bottom panels are uterine–placental interface from sheep that have been immunofluorescence stained for PAGs (green) and E-cadherin (red).