The effects of intra-amniotic administration of 1% l-glutamine (Gln) and 0.75% NaCl (NaCl) on jejunal enterocyte morphometric maturation before and after hatch were examined by visualizing cellular outlines by immunostaining of the adherens junction molecule E-Cadherin (E-cad), combined with the microvilli marker Villin and DAPI nuclear staining (Fig. 1). At embryonic day 19 (E19), enterocyte lengths in Gln-treated embryos exhibited a significant, 10% increase in comparison to Control embryos (P = 0.01), while no differences were found between NaCl-treated and Control embryos (Fig. 1A-C,G). At hatch, enterocyte lengths in all groups increased 2.2-fold, and remained 10% higher in Gln-treated chicks, compared to Control chicks (P = 0.01) (Fig. 1G). Enterocytes continued to elongate at D3 in all groups, and their lengths were significantly increased in both NaCl and Gln-treated chicks, compared to Control chicks, (by 14%, P = 0.01 and by 20%, P = 0.0002, respectively) (Fig. 1D-G). These effects did not persist at D7, in which both treatment groups did not differ from the Control group (Fig. 1G). Since enterocyte widths remained stable within groups at all pre- and post-hatch ages (Fig. 1G, bottom graph), the increases in their lengths generated a more columnar epithelium at an earlier age in the Gln group. We therefore conclude that intra-amniotic administration of Gln promoted early onset of enterocyte growth.
Villin immunostaining was barely detectable at pre-hatch ages. Post-hatch, Villin immunostaining was confined to enterocyte brush borders and portrayed stronger signal intensities in NaCl and Gln-treated chicks, compared to Control chicks (D3 representative images in Fig. 1D-F, bottom panel). This finding indicates a possible enhancement of microvilli development in these groups. We therefore visualized microvilli ultrastructure in all groups at pre- and post-hatch ages by scanning electron microscopy (SEM) and measured their lengths and widths (Fig. 2). Results showed that at E19, microvilli of both NaCl and Gln-treated embryos were more perpendicularly oriented to enterocyte apical surfaces, compared to microvilli of Control embryos (Fig. 2A-C). This indicates advanced brush border development, since pre-hatch microvilli straighten with age as a result of stabilization of the base of the microvilli via rootlet elongation and terminal web connection .
Additionally, a 20% increase in microvilli lengths at E19 was measured Gln-treated embryos, compared to Control embryos (P < 0.0001), while microvilli lengths in NaCl-treated embryos did not differ from Control embryos (Fig. 2G). Similarly, microvilli at hatch were 14% longer in Gln-treated embryos, compared to Control embryos (P = 0.009), and no differences were found between NaCl-treated and Control embryos (Fig. 2G). At D3, brush borders in both treatment groups portrayed advanced development in comparison to Control chicks, as microvilli of NaCl-treated chicks were 13% longer (P = 0.045), and microvilli of Gln-treated chicks were 22% longer (P = 0.018), reaching peak values of 2.2 and 2.4 µm, respectively. Furthermore, microvilli widths at D3 were significantly reduced in NaCl and Gln-treated chicks (by 10%, P = 0.029 and 20%, P = 0.0003, respectively) compared to Control chicks (Fig. 2D-G). Since microvilli organization was similar in all groups at this timepoint (Fig. 2D-F), a reduction in their widths enables higher microvillar density.
Based on our previous findings, microvilli reach maximal lengths at D3 and significantly amplify enterocyte apical surface areas through their dimensions and densities . Our current results indicate that intra-amniotic administration of Gln enhanced pre-hatch brush border developmental patterns and dramatically increased post-hatch enterocyte surface area expansion at D3.
At D7, microvilli followed the same pattern reported in our previous work , as their lengths decreased by 38, 32 and 44% in Control, NaCl and Gln treated chicks, respectively. However, microvilli of NaCl and Gln-treated chicks remained significantly longer (by 23%, P = 0.008 and 10%, P = 0.034, respectively) than microvilli of Control chicks at D7 (Fig. 2G).
This finding indicates possible long-term expansion of the brush border surface areas following Gln stimulation, thus significantly contributing to enterocyte absorptive capacities.
Since our results revealed distinct morphological maturation of microvilli, we evaluated the effects of intra-amniotic administration of Gln and NaCl on brush border nutrient transporter expression before and after hatch, as an indicative factor of enterocyte functionality. This was conducted by a Real-Time qPCR analysis of Peptide transporter 1 (PepT-1) and Sodium Glucose Transporter 1 (SGLT-1) (Fig. 3).
In the Control group, expression of both genes exhibited a dramatic 30-fold increases between pre- and post-hatch ages, in accordance with previous findings [33, 34, 35] (Fig. 3A,B). The surge in PepT-1 expression at hatch was 54% higher in Gln-treated chicks, compared to Control chicks (P = 0.021), while expression in NaCl-treated chicks did not differ significantly from Control chicks. At D3 and D7, PepT-1 expression decreased in all groups, in accordance with previous reports  and did not differ between Gln-treated chicks and Control chicks at these ages. However, NaCl-treated chicks exhibited a 63% decrease in PepT-1 expression at D7, compared to Control chicks (P = 0.036) (Fig. 3A).
In contrast, SGLT-1 expression was similar in all groups at hatch. However, Gln-treated chicks exhibited significant, 50 and 57% increases in SGLT-1 expression in comparison to Control chicks at D3 and D7, respectively (P = 0.042), while SGLT-1 expression in the NaCl group did not differ from the Control group at these timepoints. We conclude that intra-amniotic administration on Gln, but not NaCl, significantly improved the absorptive potential of di- and tri-peptides and glucose after hatch, corresponding to the enhancements in enterocyte and brush border morphometric parameters.
An additional crucial factor for intestinal maturation is the epithelial barrier, generated by tight junctions, which develop and mature before and after hatch . Additionally, tight junction formation is a key regulatory factor in brush border development [32, 37, 38]. We therefore examined the effects of intra-amniotic administration of Gln and NaCl on the expression of tight junction proteins 1 and 2 (TJP-1 and TJP-2, respectively), and the associated transmembrane protein Occludin . Since expression of these genes was stable within pre- and post-hatch ages, data are presented as fold change from Control in each group (Fig. 4).
Results showed that at E19, both TJP-1 and TJP-2 exhibited significant, 2.1-fold increases in expression in Gln-treated embryos, compared to Control embryos (P = 0.036 and P = 0.02, respectively). Post-hatch, tight junction protein expression remained higher Gln-treated chicks, compared to Control chicks: TJP-1 expression was 2.2-fold higher at D3 (P = 0.003) and 1.5-fold higher at D7 (P = 0.045), and TJP-2 expression was 1.79-fold higher (P = 0.031) at D3 (Fig. 4A,B). Occludin expression was also upregulated at D3 in Gln-treated chicks, compared to Control chicks, displaying a 3.3-fold increase in expression (P = 0.002) (Fig. 4C). No significant differences in TJP-1, TJP-2 or Occludin expression were found between NaCl-treated and Control embryos and chicks (Fig. 4C). These results indicate that Gln stimulation enhanced pre- and post-hatch tight junction development.
Taken together, intra-amniotic administration of Gln enhanced intestinal developmental dynamics by promoting enterocyte growth, nutrient transporter expression and tight junction formation, thus improving post-hatch nutrient absorption and intestinal integrity. These effects were restricted to the first week post-hatch, after which the small intestinal epithelium reaches maturity [4, 10].
In light of these results, we sought to determine the mechanism by which the administered Gln stimulated the developing small intestinal cells to initiate these effects. Various studies in mammals linked improved intestinal morphology, function and integrity to GLP-2 secretion by enteroendocrine L-cells [27, 28, 29, 30, 31], and Gln is a known stimulatory factor for GLP-2 secretion by L-cells [21, 22, 23]. We therefore hypothesized that the observed effects on enterocyte maturation were a result of Gln-mediated L-cell stimulation.
To test this hypothesis, we first identified and characterized jejunal L-cells at pre- and post-hatch ages. L-cells in mammals are most abundant in the ileum, and are distinguishable from enterocytes by their unique morphology and the presence of various secretory granules within their cytoplasm . In chicken, L-cells were identified in the ileum at D7 by Glucagon-like Peptide 2 (GLP-2) immunoreactivity through fluorescence and transmission electron microscopy (TEM) , and were also found with decreased abundancy along jejunum . We therefore visualized L-cells within our jejunal samples at pre- and post-hatch ages by GLP-2 immunostaining.
Though no L-cells were observed at E17, rare occurrences of L-cells were noted at E19 (≤ 1 per complete cross section, n = 6) (Fig. 5A). L-cells at E19 were restricted to villi bottoms and GLP-2 signal intensity was higher within the apical pole of the cell (Fig. 5A, right panel, arrowhead), outlining an oval cell structure. This is in contrast to adult ileal L-cells in chicken, which portray increased GLP-2 signal intensity within the basolateral pole, and possess a thin cytoplasmatic process extending into the intestinal lumen [41, 42]. At hatch, L-cells were found at upper crypt regions (Fig. 5B) as well as along the villus epithelium (Fig. 5B’). Their morphology resembled that of L-cells at D7 in the jejunum (Fig. 5C,C’) and ileum [41, 42] as well as adult mice ileal L-cells .
These results reveal that the jejunal L-cell population appears between E17 and E19, when the embryo ingests its amniotic fluid [1, 2, 12, 13], and fully matures after hatch, when exogenous feeding begins. This indicates synchronization between the development of enteroendocrine function in the small intestine and primary nutritional stimulation in chick embryos, as has been described in mammals .
We then proceeded to investigate the effects of amniotic enrichment with Gln and NaCl on L-cell stimulation by examining the expression of genes encoding key components of L-cell signaling. Genes examined were Proglucagon B (PCB), which gives rise to GLP-2 through tissue-specific alternative splicing in chicken [44, 45], GLP-2 Receptor (GLP-2R), Insulin-like Growth Factor 1 (IGF-1) and IGF-1 Receptor (IGF-1R). Results revealed a tendency of increased expression of PCB at hatch and D3 in Gln-treated chicks, but this effect was not statistically significant due to high variability between individual Gln-treated chicks (Fig. 6A). Similarly, previous studies in chicken reported that fasting and refeeding did not affect intestinal PCB expression . It may therefore be possible that the effects of intra-amniotic administration of Gln on GLP-2 synthesis occur at the post-translational level, or downstream.
However, expression of GLP-2R in the Gln group was higher by 75% (P = 0.072) and 62% (P = 0.006) than in the Control group at E19 and D3, respectively (Fig. 6B). Concurrently, IGF-1 expression at E19 increased by 47% at E19 in Gln-treated embryos, compared to Control embryos (P = 0.041) (Fig. 6C). These results may indicate activation of ISEMFs to synthesize and secrete IGF-1 in response to GLP-2 binding. The effects of IGF-1 on enterocytes are mediated by its receptor, IGF-1R, which showed a significant increase in expression in Gln-treated, compared to Control embryos and chicks at E19 (by 86%, P = 0.029), D3 (by 88%, P = 0.031) and D7 (by 58%, P = 0.01) (Fig. 6D). Expression of these genes in NaCl-treated embryos and chicks did not differ from the Control group at all ages (Fig. 6A-D).
Taken together, these results demonstrate pre- and post-hatch upregulation of key components in L-cell paracrine hormonal signaling, in response to pre-hatch luminal Gln, thus linking the effects of Gln on enterocyte maturation with L-cell stimulation.
Though intra-amniotic administration of NaCl elicited some beneficial effects on enterocyte maturation, these effects were limited to post-hatch morphometric parameters. Since amniotic enrichment with NaCl was previously shown to promote proliferation in the small intestinal epithelium through activation of ion-dependent nutrient transporters during amniotic fluid ingestion , It may be possible that NaCl promoted earlier morphological maturation of enterocytes, but did not stimulate L-cell signaling, and thus failed to enhance absorptive and barrier functions.
In conclusion, intra-amniotic administration of Gln stimulated L-cell paracrine hormonal signaling and resulted in enhanced enterocyte morphology, function and integrity during the critical first week post-hatch. Figure 7 provides a graphical representation of the major findings in this study and a proposed mechanism of action by which intra-amniotic administration of Gln induces enterocyte maturation through enteroendocrine stimulation.
This work is the first to provide a link between pre-hatch nutrient sensing and post-hatch enteroendocrine-driven effects on the developmental dynamics of small intestine of chicks. To date, studies of intestinal chemosensation in chicken have been limited to characterization and localization of enteroendocrine cells and their taste receptors [41, 42, 46, 47, 48]. Further investigation of chicken L-cell characteristics and function is needed in order to unravel the effects of intestinal nutrient sensing on absorption and satiety, as well as the gut-brain connection in chicken, which has been established in mammals [49, 50]. Nevertheless, our findings demonstrate that alterations in pre-hatch chemosensation is possible through intra-amniotic administration of specific nutrients and vastly influence intestinal morphology and function. We therefore provide a model for investigating the link between primary nutrient sensing and intestinal maturation, as well as an applicative method for shaping intestinal development in chicken.