Trophoblast differentiation, invasion and hormone secretion in a three-dimensional in vitro implantation model with rhesus monkey embryos

Abstract

Background: The initiation of primate embryo invasion into the endometrium and the formation of the placenta from trophoblasts, fetal mesenchyme, and vascular components are essential for the establishment of a successful pregnancy. The mechanisms which direct morphogenesis of the chorionic villi, and the interactions between trophectoderm-derived trophoblasts and the fetal mesenchyme to direct these processes during placentation are not well understood due to a dearth of systems to examine and manipulate real-time primate implantation. Here we describe an in vitro three-dimensional (3-D) model to study implantation which utilized IVF-generated rhesus monkey embryos cultured in a Matrigel explant system.

Methods: Blastocyst stage embryos were embedded in a 3-D microenvironment of a Matrigel carrier and co-cultured with a feeder layer of cells generating conditioned medium. Throughout the course of embryo co-culture embryo growth and secretions were monitored. Embedded embryos were then sectioned and stained for markers of trophoblast function and differentiation.

Results: Signs of implantation were observed including enlargement of the embryo mass, and invasion and proliferation of trophoblast outgrowths. Expression of chorionic gonadotropin defined by immunohistochemical staining, and secretion of chorionic gonadotropin and progesterone coincident with the appearance of trophoblast outgrowths, supported the conclusion that a trophoblast cell lineage formed from implanted embryos. Positive staining for selected markers including Ki67, MHC class I, NeuN, CD31, vonWillebrand Factor and Vimentin, suggest growth and differentiation of the embryo following embedding.

Conclusions: This 3-D in vitro system will facilitate further study of primate embryo biology, with potential to provide a platform for study of genes related to implantation defects and trophoblast differentiation.

Keywords: Embryo; Implantation; Non-human primate; Trophoblast.

Fig. 1

Growth of Rhesus Embryos in Matrigel Rafts. Embryos that failed to hatch (a) or that did not completely hatch (b), failed to develop and degenerated. Embryos that completed hatching (c) continued to develop (d), where within 15 days post embedding trophoblast protrusions and inner cavities formed. Scale bar = 100 μm

Fig. 2

Development of rhesus blastocysts embedded in Matrigel: (a) day 0 post embedding in Matrigel; (b) day 6: outgrowths and cystic structure shown; (c) day 14: extensive branch-like outgrowths extended from dense extraembryonic mass; (d) day 19; (e) day 26; (f) day 38. Over the course of development post-embedding, trophoblastic protrusions invaded into the Matrigel environment. Scale bar = 100 μm

Fig. 3

Hormone secretion from embryos embedded in Matrigel and co-cultured with BRL cells: (a) CG, n = 12 and (b) progesterone secretion, n = 9. Each individual colored line represents the secretion profile from an individual embryo, where the same color between (a) and (b) represent the same embryo

Fig. 4

Comparison of chorionic gonadotropin secretion in embryos derived from the same oocyte donor with less extensive development (a, b) or advanced development (c). Chorionic gonadotropin secretion profiles, illustrated in panel (d), from those three embryos showed higher CG secretion coincident with more advanced development of the embryo and trophoblastic shell. Scale bar = 100 μm

Fig. 5

Histological and immunohistochemical images of paraffin sections of embedded in vitro developed rhesus embryos: (a) bright field; (b-d) H&E; (e) bright field image of trophoblastic outgrowths; (f-h) Ki67; (i-k) cytokeratin (CytoK); (l) IgG negative control for cytokeratin; (m-o) CG; and (p) IgG negative control for CG. Results representative of 2 embryos. Scale bar = 500 μm

Fig. 6

Immunohistochemical staining for selected markers in Matrigel-embedded embryos: (a) HC10, (b) neuronal marker NeuN; (c-d) CD31, an endothelial and monkey extravillous trophoblast marker; (e-f) von Willebrand Factor (vWF), an endothelial marker (arrow); (g-h) Vimentin (VIM), a marker of mesenchymal cells. Results representative of 2 embryos. Scale bar = 100 μm