Amniotic fluid exerts a neurotrophic influence on fetal neurodevelopment via the ERK/GSK-3 pathway
© Jang et al. 2015
Received: 14 January 2015
Accepted: 13 July 2015
Published: 5 August 2015
The fetus is surrounded by the amniotic fluid (AF) contained by the amniotic sac of the pregnant female. The AF is directly conveyed to the fetus during pregnancy. Although AF has recently been reported as an untapped resource containing various substances, it remains unclear whether the AF could influence fetal neurodevelopment.
We used AF that was extracted from embryos at 16 days in pregnant SD rat and exposed the AF to the neural cells derived from the embryos of same rat. We found that the treatment of AF to cortical neurons increased the phosphorylation in ERK1/2 that is necessary for fetal neurodevelopment, which was inhibited by the treatment of MEK inhibitors. Moreover, we found the subsequent inhibition of glycogen synthase kinase-3 (GSK-3), which is an important determinant of cell fate in neural cells. Indeed, AF increased the neural clustering of cortical neurons, which revealed that the clustered cells were proliferating neural progenitor cells. Accordingly, we confirmed the ability of AF to increase the neural progenitor cells through neurosphere formation. Furthermore, we showed that the ERK/GSK-3 pathway was involved in AF-mediated neurosphere enlargement.
Although the placenta mainly supplies oxygenated blood, nutrient substances for fetal development, these findings further suggest that circulating-AF into the fetus could affect fetal neurodevelopment via MAP kinases-derived GSK-3 pathway during pregnancy. Moreover, we suggest that AF could be utilized as a valuable resource in the field of regenerative medicine.
In placental mammals, the placenta is physically connected with the developing embryos or fetus through the umbilical cord, which supplies oxygenated blood and nutrient substances for fetal development. Moreover, the amniotic fluid (AF), enveloped by the amniotic sac of a pregnant female, is a water-like fluid that is inhaled and exhaled by the fetus . Although AF contains many nutrients and potentially deleterious materials, its effects on fetal neurodevelopment are elusive.
Because the fetus is surrounded by the water-like AF, it was originally considered to function as a shock absorber to protect against external impacts. However, it has been recently reported that AF contains various proteins, carbohydrates, lipids, and urea, all of which circulated into fetus [1–3]. During the growth of the fetus, the volume of the AF increases faster than embryonic size, and is correlated positively with the development of the fetus . The amnion is an active membrane that closely covers the embryo, which metabolically secretes nutritious factors to maintain homeostasis of solutes and water in the AF [4, 5]. In fact, it has been revealed that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are present in human AF . Moreover, several factors in AF are also believed to be conveyed directly to the fetus, in response to physiological changes in the maternal body during pregnancy . In pregnancy, maternal stress or anxiety increases a stress hormone, cortisol levels in maternal plasma, which are also found correlatively in AF . It has also been reported that maternal obesity increases the level of inflammatory cytokines, such as TNF-α and interleukin-8 in AF . Taken together, these factors could potentially influence the development of embryonic neurons through neurotropic cascade signaling.
In developing neurons, the mitogen-activated protein (MAP) kinases pathway is a multi-functional signaling cascade that regulates neuronal proliferation, differentiation, and apoptosis responding to growth factors, neurotransmitters, neurotrophins, and hormones. These extracellular stimuli typically induce the activation of tyrosine receptor kinase receptors (TRKs) or G-protein-coupled receptors (GRCRs), which subsequently triggers the MAP kinases pathway. The MAP kinases have largely been classified into three major groups: extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAP kinase . In particular, ERK is a versatile protein kinase that has been implicated in the proliferation and differentiation of neural progenitor cells. Thus, we examined whether AF stimulates or inhibits the MAP kinases pathway in differentiated neurons and neural progenitor cells, and then investigated the underlying cellular signaling and AF’s effects in this study.
AF activated ERK, time- and dose-dependently, in cortical neurons
AF induced the formation of cell clustering in cortical neurons
AF increased the formation of neurospheres
AF-induced neurosphere enlargement was dependent on the MAP kinases and GSK-3 pathway
The present study shows that AF exhibits neurotrophic effects on fetal neurodevelopment during pregnancy. The treatment of embryonic cortical neurons with AF induced the MAP kinases pathway markedly, a key signaling pathway in neural development. Subsequently, we found the AF-induced MAP kinases activation has a suppressive effect on GSK-3 activity in cortical neurons. After the application of AF to cultured cortical neurons, we observed an increased neural clustering that resembled neural stem or progenitor cells. Indeed, we further showed that the AF-derived MAP Kinases/GSK-3 pathway was implicated in the proliferation of neural progenitor cells.
Recently, AF has been actively investigated for various functions beyond its role as a shock absorber. Interestingly, amniotic membrane and fluid-derived cells release neurotrophic factors required for neuron survival . In fact, implantation of human amniotic epithelial cells protects against the degeneration of dopaminergic neuron in a rat model of Parkinson’s disease . Moreover, medium conditioned by human amniotic epithelial cells improved the survival of rat retinal ganglion cells . Thus, it seems that AF contains numerous neurotrophic factors secreted by amniotic cells. However, most studies have investigated the protective effects against neuronal degeneration for therapeutic potential. Although AF is circulated into the fetus, its effects on the fetal brain are still unclear. In this study, we showed that AF increased the proliferative properties of fetal neural cells and associated cellular signaling.
Regarding the intracellular signaling underlying the effects of AF, several studies have supported roles for AF in the fetus and adult. In instance, human AF induces the proliferation of fetal and adult skin fibroblasts via ERK and the Akt signaling pathway . Moreover, AF stimulates the Nrf2/Keap1 pathway in forming and repairing epidermal barriers in utero . In this study, we further showed that AF stimulated the MAP kinases pathway, and, in turn, inhibited the activation of GSK-3 in developing neurons. This suppression of GSK-3 ultimately increased the proliferation of neural progenitor cells.
Amniotic fluid is a complex biological material that contains numerous proteins, lipids, even stem cells in the fluid. Using two-dimensional electrophoresis and mass spectrometry, proteome analysis identified 35 proteins in human AF . Moreover, proteomic comparison by gestational age showed large differences in the relative abundances of human AF proteins using two-dimensional electrophoresis . In addition to proteins, cell-free fetal nucleic acids were also detected at much greater concentrations in AF than in maternal plasma . During pregnancy, maternal anxiety caused an increase in stress hormone, corticosteroid levels in AF, as in maternal plasma. Thus, there are many kinds of proteins, lipids, and nucleic acids in AF, which tightly changes in response to various circumstances such as age, stress and diseases states. These materials initiate various intracellular signaling pathways via activation or inhibition of receptors and ion channels, influencing neural development [16, 26]. With numerous biological materials in AF, we should further determine the effective component with regard to neural proliferation in the fetus.
The AF is originally known to function as a shock absorber to protect against external impacts. The current study further suggest that circulating-AF in the fetus could affect neural progenitor cells via MAP kinases-derived GSK-3 pathway to support fetal brain development. Furthermore, stressful maternal behaviors such as drinking, smoking during pregnancy could increase harmful materials in the AF, which might negatively influence on fetal neurodevelopment.
Amniotic fluid preparation
According to guidelines issued by the Institutional Animal Care and Use Committee at Seoul National University, an embryonic day 16 pregnant Sprague–Dawley (SD) rat was sacrificed in a CO2 chamber. The uterus was removed quickly and placed into a 100 mm sterile Petri-dish containing cold Hank’s balanced salt solution (HBSS) on ice. The uterine walls were incised with maintaining the amnion. The amniotic sac was washed three times with cold HBSS, and then we obtained amniotic fluid (AF) in the tube, tearing the amnion using surgical scissors. After centrifuging the AF (5 rpm, 5 min, 4°C), we collected the supernatant fraction.
Cortical neuron culture
After extracting the AF, we used the embryos to culture cortical neurons. The collected cerebral cortex was transferred into Neurobasal® medium (Gibco), and triturated using a sterile Pasteur pipette. After passing through a 40 μm cell strainer, the cortical cells were cultured on the poly-l-ornithine-coated plates in Neurobasal® medium containing B-27® supplement (Gibco), penicillin/streptomycin (Gibco), and l-glutamine (Gibco) at 37°C in a 95% air/5% CO2 incubator.
To make neurospheres, we first collected the cerebral cortex, as above. The cerebral cortex was placed in DMEM/F12 medium (Gibco), and then triturated using a sterile Pasteur pipette. After passing through a 40 μm cell strainer, the cortical cells were incubated in an un-coated plate in DMEM/F12 medium containing 20 ng FGF, 20 ng FGF, and N2 supplement for 3 or 4 days at 37°C in a 95% air/5% CO2 incubator.
Cultured cortical neurons were lysed with RIPA cell lysis buffer (GenDEPOT) containing a protease inhibitor cocktail (Roche). The protein lysates were subjected to a 10% SDS-PAGE gel and transferred to PVDF membrane. The membranes were blocked for 1 h with TBS-T solution (20 mM Tris/HCl, 500 mM NaCl, 0.1% Tween 20) containing 3% skimmed milk powder and then incubated with primary antibodies against ERK (Cell Signaling), GSK-3 antibody sampler kit (Cell Signaling), α-tubulin (Millipore), Nestin (Millipore), GFAP (Sigma), BLBP (Abcam), and Tuj-1 (Abcam) overnight at 4°C on a rotary shaker. Membranes were washed three times in TBS-T solution for 30 min, incubated with secondary antibody for 1 h at RT, and then treated with WEST-ZOL® ECL solution (iNtRON Biotech). Blots were analyzed using an ImageQuant LAS 4000 chemiluminescence system (GE Healthcare).
Cortical neurons cultured on round coverslips were fixed with 4% paraformaldehyde. Briefly, primary antibodies raised against Nestin, or Tuj-1 were incubated overnight at 4°C on a rotary shaker. The primary antibody-treated cells were washed three times with phosphate buffer solution containing 0.5% Triton X-100, and then bathed for 1 h at RT with Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen).
Cortical neurons generated floating neurospheres for 5 days with or without AF. To measure the comparative size of the neurosphere, cells were run through a flow cytometry analyzer, a FACSCalibur (BD bioscience). The sizes of neurosphere were compared through frequencies of R1-gated cells, representing cell populations with FSC-H values above 600.
All results are expressed as means ± SEMs. Student’s t-test was used to determine statistical differences between two means. One-way ANOVA was used to perform multiple comparisons of means followed by the Tukey’s post hoc test. Statistical significance was accepted at p values of *<0.05, **<0.01, and ***<0.001, as indicated. All experiments repeated at least three times independent to raise accuracy.
YJ performed molecular and cellular experiments. EK conducted a flow cytometry analyzer. KS conducted additional experiments for the revised manuscript. WS and SK coordinated the study and revised the manuscript. All authors read and approved the final manuscript.
The authors thank Dr. Uhtaek Oh of the College of Pharmacy, Seoul National University for discussions regarding the experiments.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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