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The growth factors that drive the division and differentiation of stem cells during early human embryogenesis are unknown. The secretion of endorphins, progesterone (P4), human chorionic gonadotropin, 17β-estradiol, and gonadotropin-releasing hormone by trophoblasts that lie adjacent to the embryoblast in the blastocyst suggests that these pregnancy-associated factors may directly signal the growth and development of the embryoblast. To test this hypothesis, we treated embryoblast-derived human embryonic stem cells (hESCs) with ICI 174,864, a δ-opioid receptor antagonist, and RU-486 (mifepristone), a P4 receptor competitive antagonist. Both antagonists potently inhibited the differentiation of hESC into embryoid bodies, an in vitro structure akin to the blastocyst containing all three germ layers. Furthermore, these agents prevented the differentiation of hESC aggregates into columnar neuroectodermal cells and their organization into neural tube-like rosettes as determined morphologically. Immunoblot analyses confirmed the obligatory role of these hormones; both antagonists inhibited nestin expression, an early marker of neural precursor cells normally detected during rosette formation. Conversely, addition of P4 to hESC aggregates induced nestin expression and the formation of neuroectodermal rosettes. These results demonstrate that trophoblast-associated hormones induce blastulation and neurulation during early human embryogenesis.
Trophoblastic production of endorphins  suggests important, yet undefined, roles for this hormone during embryonic growth and development. Trophoblasts also have been reported to secrete an array of other pregnancy-associated hormones including progesterone (P4), human chorionic gonadotropin (hCG), 17β-estradiol, and gonadotropin-releasing hormone (GnRH) . Given the close spatial localization of trophoblasts to the embryoblast, it is conceivable that these pregnancy-associated factors may directly signal the growth and development of the embryoblast. Evidence supporting this notion includes the presence of placental opioid-enhancing factor in amniotic fluid and placenta . Likewise, trophoblastic and corpa luteal production of hCG/P4 is markedly elevated postconception and is obligatory for the maintenance of pregnancy . This pilot study was therefore undertaken to determine if modulating opioid receptor and P4 receptor (PR) signaling would affect early embryonic growth and development. Our results indicate that both opioid and progesterone signaling are vital for normal blastulation and neurulation.
Pluripotent H9 human embryonic stem cells (hESC; passage 29–33 XX karyotype; also known as WA09) obtained from WiCell Research Institute (Madison, WI, USA) were cultured on irradiated mouse embryonic fibroblast (MEF) feeder cells (BioVintage, CA, USA) plated on gelatin-coated wells and grown in Dulbecco's modified Eagle's medium (DMEM)-F12 supplemented with 20% Knockout™ Serum Replacement (KOSR), 1% nonessential amino acids (NEAAs), 1 mM l-glutamine, 4 ng/mL basic fibroblast growth factor (bFGF) (all from Invitrogen, Carlsbad, CA, USA) and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). For experiments examining the effects of P4, hESC were cultured in TESR1 media (lacking Li) and treated with P4 [2 μM; stock P4 was solubilized in dimethyl sulfoxide (DMSO) and then diluted into media] or control (the equivalent volume of DMSO) for 9 days and cells collected for the measurement of nestin expression by immunoblot analysis as previously described [4,5].
H9 hESC colonies were cultured for 4 days on an MEF feeder layer as described above. Intact colonies were enzymatically detached and cultured on an orbital shaker at 5% CO2 in medium containing Iscove's modified Dulbecco's medium and 20% fetal bovine serum (embryoid body media; both from Invitrogen) with or without ICI 174,864 (0.1 mM; Tocris Bioscience, Ellisville, MO, USA; stock ICI 174,864 was solubilized in water) and/or RU-486 (20 μM; Sigma Laboratories, St. Louis, MO, USA; stock RU-486 was solubilized in EtOH and then diluted into media) for another 10 days (when embryoid body formation occurs under normal conditions—day 14). Controls were treated with the equivalent volume of water or EtOH. Structures were then examined morphologically and the area of the structures quantitated using Image J Software (http://rsb.info.nih.gov/ij/).
hESC were differentiated into columnar neuroectodermal cells as described elsewhere  a protocol that mimics in vivo neuroectodermal development in terms of timing and morphology. In brief, hESC colonies were removed intact from the MEF layer after 4 days and were cultured in a special hESC growth medium (78.5% DMEM-F12, 20% KOSR, 1% NEAA, 1 mM l-glutamine, 0.1 mM 2-mercaptoethanol) for a further 4 days with daily replacement of media to form hESC aggregates. Colonies were then adhered to the culture surface where they form monolayer colonies in a chemically defined neural induction medium [32.6% F-12, 65.2% DMEM, 1% N2 supplement, 1% NEAA, 10 ng/mL bFGF (all from Invitrogen), 0.2% of 1 mg/mL Heparin (Sigma-Aldrich)]. Under this culture condition, columnar neuroectodermal cells appear in the center of each colony and organize into neural tube-like rosettes after a total of 9–10 d of differentiation culture. The neural induction media was replaced every other day. The neuroectodermal cells in the rosettes were selectively isolated through differential enzymatic treatment using dispase (0.5 mg/mL in DMEM-F12) and incubated for 2 h in neural induction medium to allow the nonneural cells to differentially attach to the flask. The floating cells (mostly aggregates of neuroectodermal cells) were transferred to new flasks where they rolled up to form round clusters. For treatments, cells were cultured in the neural induction media in the presence of RU-486 (20 μM) and/or ICI 174,864 (0.1 mM), and then examined morphologically and the cells collected for measurement of nestin expression by immunoblot analysis [4,5].
To investigate opioid signaling during early human embryogenesis, we utilized hESC derived from the inner cell mass of the blastocyst as a model of early embryogenesis [4,5]. Treatment of hESC colonies with the δ-opioid receptor–selective antagonist ICI 174,864 [7,8] for 10 days inhibited the formation of the embryoid body cystic structure (cavitation), and instead formed nonspherical structures that were ~40% the size of normal spheroidal embryoid bodies (Fig. 1A and B). ICI 174,864 also inhibited normal neuroectodermal rosette formation from hESC, inducing a more dense morphology compared with control rosettes (Fig. 1A). To confirm that blocking opioid signaling inhibits hESC differentiation into neuroectodermal cells, the treated hESC were collected for immunoblot analysis and probed with a monoclonal antibody against human nestin, an early marker of neural precursor cell formation. Two bands representative of nestin (205 and 220 kDa) were detected in control rosettes, but neither band was detected in cell aggregates treated with ICI 174,864 (Fig. 1C). These results indicate that ICI 174,864, in addition to disrupting the normal development of hESC into embryoid bodies, also inhibits neuroectodermal rosette formation demonstrating that δ-opioid receptors are required for normal human blastulation and neurulation.
δ-Opioid antagonists may function to inhibit embryogenesis by regulating hCG release  required for P4 production. Trophoblastic and corpa luteal production of hCG/P4 is markedly elevated postconception and is obligatory for the maintenance of pregnancy . Inhibition of P4 signaling using the PR antagonist RU-486 is known to result in endometrial decidual degeneration, trophoblast detachment and decreased hCG production from the syncytiotrophoblast, and P4 production from the corpus luteum . In addition, RU-486 induces cervical softening and dilatation, release of endogenous prostaglandins and an increase in myometrial sensitivity to the contractile effects of prostaglandins leading to the expulsion of the embryo/fetus. Although these abortive actions of RU-486 are well demonstrated, the effect of suppressing P4 production with RU-486 on the development of the early human embryo has not been studied.
Based on the above observations that opioid signaling is important for embryogenesis and the relationship between opioid signaling and P4 production , we tested the relevance of P4 signaling in early embryogenesis using RU-486. Treatment of hESC colonies with RU-486, like ICI 174,864, prevented the development of embryoid bodies (Fig. 1A); hESC colonies failed to form normal cystic structures after 10 days in culture, and instead formed solid irregular spheres that were ~20% the size of normal spheroidal embryoid bodies (Fig. 1B). Since P4 also is a known neurotrophic factor in the adult brain , we examined whether PR signaling is required for neurogenesis. RU-486 treatment of hESC colonies blocked the normal differentiation of hESC into neuroectodermal rosettes cultured in neural induction media containing P4 (Fig. 1A; RU-486 is a competitive receptor antagonist in the presence of P4). This was confirmed by the suppression of nestin expression in RU-486–compared to P–treated hESC aggregates (Fig. 1C). These results indicate the obligatory role of PR signaling in normal embryoid body and neuroectodermal rosette formation during early embryogenesis. To test whether P4 is directly required for neuroectodermal rosette formation, we treated hESC aggregates with P4 and measured nestin expression. P4 induced the expression of the 205- and 220-kDa forms of nestin (Fig. 2) confirming the requirement for P4 signaling in the formation of neuroectodermal rosettes. Together, these results suggest that the upregulation of trophoblastic P4 production following conception  is required not only for trophoblast attachment, but also for the normal development of the embryo. These results also reveal for the first time the basis for why neural induction media induces neural precursor cell formation; the N2 supplement used in the neural induction media contains P4.
The relative binding affinity of RU-486 for PR is twice that of P4 , and is used at a dose of 200–600 mg for the termination of pregnancies (this equates to ~6–19 μM, equivalent to that used in our study (20 μM; ref. 13)). Thus, blocking PR signaling with RU-486 during early pregnancy will block blastulation and neurulation.
Previous data has implicated P4 as acting in the arcuate nucleus and anteroventral periventricular nucleus through β-endorphin and dynorphin B neurons to affect preoptic area GnRH neurons and gonadotropin secretion [14,15]. Our results also suggest that there may be signaling crosstalk between P4 and opioids during early embryogenesis in the regulation of GnRH secretion, although the role of GnRH and gonadotropins during embryogenesis remains to be elucidated.
These data indicate for the first time that pluripotent hESC have an absolute requirement for steroid and opioid signaling during blastulation and neurulation. Furthermore, our data support a critical molecular signaling link between trophoblastic and/or maternal hormone production and early embryonic growth and development. The abortifacient effects of RU-486 therefore also extend to blocking normal embryonic growth and development.
The authors declare no conflict of interest. This is Geriatrics Research, Education, and Clinical Center VA article number 2008.08.