At birth, the supply of nutrients to the offspring switches from a placental to an enteral source. In preparation for this transition, the fetal gastrointestinal tract undergoes a number of structural and functional changes near term (Trahair and Sangild, 1997; Buddington et al., 2012; Frazer and Good, 2022). The mucosa increases in size, vascularity and transporter expression to provide an effective secretory and absorptive surface area; gastrointestinal innervation and motility develops; and gastric acid, intrinsic factor and digestive enzymes are synthetised over the perinatal period. Overall, the offspring develops the capacity for the digestion of milk and absorption of nutrients at birth. Preterm infants commonly have impaired digestive function and are at increased risk of developing necrotising enterocolitis (NEC) and other gastrointestinal abnormalities (Frazer and Good, 2022).

Development of the gastrointestinal tract in utero is regulated by hormones and growth factors present in the circulation and in the amniotic fluid swallowed by the fetus (Trahair and Sangild, 1997). In fetal sheep and pigs, the prepartum rise in circulating cortisol promotes growth and maturation of the gastric glands and intestinal villus-crypt structure, and the expression of digestive enzymes in the intestinal brush border (Trahair et al., 1987a, 1987b; Sangild T. et al., 1994, 1995b). Indeed, antenatal treatment with synthetic glucocorticoids, that also bind to the glucocorticoid receptor, reduces the incidence of NEC and improves gastrointestinal outcomes in premature offspring (Jing et al., 2021).

Maturational events induced by cortisol in the fetus near term may be mediated, in part, by other endocrine systems (Fowden et al., 2016). In fetal pigs, cortisol increases the circulating concentration of the gut hormone gastrin (Sangild P. et al., 1994) which may act simply as a marker of gastrointestinal growth and development and/or may contribute to the maturational effects of glucocorticoids on the structure and function of the digestive system. Furthermore, in fetal sheep, cortisol stimulates the production of tri-iodothyronine (T3) from thyroxine (T4) via changes in deiodinase enzyme activities in fetal and placental tissues (Forhead et al., 2006). In turn, the prepartum rise in plasma T3 is known to contribute to the maturation of fetal organs, such as the lungs and liver, in preparation for birth (Forhead and Fowden, 2014).

Thyroid hormones are important for intestinal development in rodents around weaning and in amphibians during metamorphosis (Sirakov and Plateroti, 2011; Sirakov et al., 2014; Frau et al., 2017). In rat intestines, increments in type 1 deiodinase (D1), and decrements in type 3 deiodinase (D3), activities occur over the perinatal period to increase the production and decrease the clearance of T3, respectively, within the developing gastrointestinal tract (Bates et al., 1999). Moreover, in a variety of models studied postnatally, rodents deficient in circulating thyroid hormones or their receptors have abnormal intestinal morphology, motility and digestive enzyme expression (Yeh and Moog, 1977; Fraichard et al., 1997; Plateroti et al., 1999, 2001; 2006; Flamant et al., 2002). However, little is known about the role of thyroid hormones in gastrointestinal development before birth in a species that resembles the human fetus more closely in the timing of somatic growth and prepartum maturation.

At birth, lambs are considered to be pre-ruminants. The ruminant stomach consists of four compartments (the rumen, reticulum, omasum and abomasum) where, in the neonate, the abomasum is the largest and equivalent to the stomach of some mono-gastric species, such as humans and carnivores, in structure and function (Hill, 1968). An oesophageal groove delivers milk directly into the abomasum and, therefore, the developing digestive system of the ovine fetus and neonate is similar to that of the human. Ruminal digestion and microbial fermentation, however, are not initiated until the introduction of solid food.

This study investigated the effect of thyroid hormone deficiency on the structure of the gastrointestinal tract and the activity of brush border hydrolases in the ovine fetus in late gestation. It tested the hypothesis that hypothyroidism retards normal growth of the gastrointestinal mucosa and development of digestive enzyme activities near term.

Thyroid hormone deficiency in the ovine fetus impaired the structural and functional development of the gastrointestinal tract seen normally near term. Hypothyroidism caused growth retardation of the mucosa in the abomasum and small intestines, and reduced maltase and DPPIV enzyme activities in the ileum, in association with lower circulating concentrations of cortisol and gastrin. These effects on gastrointestinal development are likely to have consequences for digestive function in the neonatal period and later life.

The present findings are novel in a model relevant to the human fetus and are similar to those seen over the early postnatal period in more altricial rodent species. In rat pups thyroidectomised at 6 days after birth, intestinal villi and crypts showed little growth over the subsequent 2–3 weeks and the relative weight of the small intestines was reduced to 72% of that in the control animals (Yeh and Moog, 1977). Intestinal maltase and sucrase activities were also suppressed in the TX rat pups, and both the structural and digestive enzyme parameters were improved by treatment with glucocorticoids and T4 (Yeh and Moog, 1977). Over the same postnatal time-frame, the numbers of proliferating cells in the intestinal epithelium were reduced in mouse models of congenital hypothyroidism (Pax8^−/−, Flamant et al., 2002), deletion of the thyroid hormone receptor-α (THR-α^−/−, Plateroti et al., 2006) and pharmacological thyroid hormone deficiency (propylthiouracil and low iodine, Plateroti et al., 2001). In mouse pups, deletion of THR-α and pharmacological hypothyroidism were both associated with downregulation of key genes in the control of proliferation and differentiation pathways in the small intestines, such as Cyclin D1, β-Catenin, and Cdx1 and Cdx2 homeobox genes (Fraichard et al., 1997; Plateroti et al., 1999, 2001; 2006).

Before birth, hypothyroidism may influence the growth and development of the gastrointestinal tract by a variety of mechanisms, and the findings of the present study indicate important interactions between thyroid hormones, cortisol and gastrin in regulating these processes. Plasma cortisol concentration was reduced in the TX ovine fetus near term and this has been shown previously to be due to reductions in circulating adrenocorticotrophic hormone (ACTH), the size of the adrenal zona fasciculata and expression of steroidogenic enzymes (Camm et al., 2021). Cortisol has growth-promoting and maturational effects in the developing gastrointestinal tract and elevates circulating gastrin concentration in fetal pigs and sheep (Trahair et al., 1987a; Sangild T. et al., 1994, 1995b). Therefore, growth retardation and dysmaturation of the gastrointestinal tract may, in part, be attributed to the moderately lower circulating level of glucocorticoids in the TX fetus. In rats, hypothyroidism over the perinatal period also decreases the expression of components of the hypothalamic-pituitary-adrenal (HPA) axis and ACTH and glucocorticoid secretion (Dakine et al., 2000), although the relative contribution of suppressed glucocorticoid activity to the gastrointestinal abnormalities seen in models of fetal or neonatal hypothyroidism is unknown. Further investigations require the use of targeted thyroid hormone deficiency in the developing gastrointestinal tract without consequence for the HPA axis.

Gastrin-secreting G-cells are present primarily in the pyloric antrum and duodenum of sheep fetuses from mid-gestation, and antral and circulating gastrin concentrations increase with gestational age to peak at birth (Read et al., 1992; Franco et al., 1993). In the present study, the lack of rise in plasma gastrin near term induced by thyroid hormone deficiency suggests that T3 may regulate gestational and cortisol-induced increments in G-cell development and plasma gastrin concentration (Sangild T. et al., 1994). Fasting plasma gastrin, and responses to arginine injection, were lower in hypothyroid adult human patients and improved with thyroid hormone treatment, such that plasma gastrin correlated with T3 concentration (Seino et al., 1978; Sagara et al., 1983). Low plasma gastrin concentration in the TX ovine fetus may reflect the reduced size of the abomasal and duodenal mucosa and, in turn, may contribute to the impaired growth and development of the gastrointestinal tract. Removal of the abomasal antrum in fetal sheep at 90 dGA, which decreases plasma gastrin to less than half of the normal concentration, was associated with lower villi and crypt densities in the small intestine at 135 dGA (Avila et al., 1989). In this model of hypogastrinaemia, there were no changes in total gastrointestinal weight, thickness of the mucosa in the remaining abomasal fundus, or intestinal villus-crypt structure, although fetuses were not studied over the last 1–2 weeks of gestation when marked changes in gastrointestinal development take place (Avila et al., 1989; Buddington et al., 2012; Flores et al., 2018). The extent to which plasma gastrin plays a role in the maturation of the fetal gastrointestinal tract induced by glucocorticoids and thyroid hormones, or merely indicates G-cell number and secretory function, remains to be established.

Before birth, swallowing and passage of amniotic fluid, containing growth factors, hormones and nutrients, is critical for normal growth of the gastrointestinal tract, the activity of brush border enzymes and the development of nutrient uptake mechanisms (Kimble et al., 1999; Sangild et al., 2002; Cellini et al., 2006). Growth factor expression within the developing gastrointestinal tract may be impaired by hypothyroidism as observed previously in fetal ovine tissues such as the liver and skeletal muscle (Forhead et al., 2000, 2002). In fetal sheep, luminal infusion of IGF-I increased the weights of the whole stomach and intestines (Trahair et al., 1997), and in preterm piglets, subcutaneous IGF-I injections promoted intestinal growth, including villus height, and ApN and DPPIV activities, and reduced the appearance and severity of NEC lesions (Holgersen et al., 2020, 2022).

Although the size of the intestinal muscularis propria was unchanged in TX ovine fetuses, thyroid hormone deficiency may affect the innervation and contractility of smooth muscle in the gastrointestinal tract, and hence, luminal exposure to amniotic fluid along its length. Gastrointestinal hypomotility and prolonged transit time have been reported in newborn human infants with congenital hypothyroidism and in a case report of a child with a mutation in the THR-α gene (Smith et al., 1975; Bochukova et al., 2012). In colon segments taken from sheep fetuses treated with the synthetic glucocorticoid betamethasone, the muscle tension generated in response to a muscarinic agonist in vitro was greater when T4 was combined with the glucocorticoid (Ross et al., 1997). Similarly, fetal gut motility was accelerated by intra-amniotic injection of betamethasone and T4 in rhesus monkeys (Gilbert et al., 2001). Thyroid hormones are important for the development of both central and peripheral nervous systems (Williams, 2008; Prezioso et al., 2018), and have been shown to regulate proliferation and differentiation of progenitor cells of the enteric nervous system taken from neonatal mice and studied in vitro (Mohr et al., 2013). Furthermore, in zebrafish larvae, pharmacological hypothyroidism suppresses intestinal transit in association with a reduction in proliferating and total number of enteric neurones; these changes are restored to normal by T4 supplementation (Wang et al., 2020). Further experiments are needed to establish the extent to which the development of the enteric nervous system and control of gastrointestinal motility is influenced by hypothyroidism in the ovine fetus before birth with consequences for the maturation of the gastrointestinal tract.

In the present study, hypothyroidism in utero did not affect the normal ontogenic changes seen in enzyme activities in the duodenum or jejunum, some of which correlated with structural indices in these regions of the fetal small intestines. In the intact control fetuses, many of the enzymes were altered before the prepartum period and, therefore, appeared not related simply to growth of the intestinal mucosa or changes in circulating glucocorticoid or thyroid hormone concentrations. Ileal maltase and DPPIV activities, however, were suppressed in the TX compared to intact fetuses near term. For ileal maltase, the effects of hypothyroidism may have been mediated by relationships between enzyme activity and indices of villus-crypt architecture. In contrast, ileal ApN activity was increased by thyroid deficiency at both gestational ages studied which suggests that thyroid hormones may normally exert an inhibitory effect on ApN activity in the distal region of the small intestine. Regional and enzyme-specific responses to hypothyroidism may relate to differences in thyroid hormone signalling along the gastrointestinal tract which require further investigation. In rat pups studied at 3 weeks of postnatal age, pharmacological hypothyroidism suppressed intestinal maltase and sucrase activities and these were restored by T4 injections (Martin and Henning, 1982). Similarly, thyroidectomy further delayed the maturation of intestinal maltase, sucrase and lactase activities in adrenalectomized rat pups over the same postnatal period (Koldovský et al., 1975). Maternal T3 treatment in rats increased intestinal maltase and sucrase in the offspring, both before birth and during the suckling period (Jumawan et al., 1977; Koldovský et al., 1980; Vaucher et al., 1982).

The present study indicates the complexity of endocrine interactions in the development of the fetal gastrointestinal tract near term. Here, hypothyroidism in utero primarily impaired growth of the ovine gastrointestinal tract, whereas previous studies of cortisol treatment in fetal pigs have shown more marked effects on functional maturation of brush border enzymes and other digestive secretions (Sangild et al., 1994; 1995a). This suggests that the prepartum rises in both T3 and cortisol are important for the coordinated structural and functional maturation of the digestive system in the preparation for birth, as seen in other fetal tissues (Forhead and Fowden, 2014; Fowden et al., 2016). However, there may also be species differences in the nature and timeframe of development in the proximal and distal regions of the gastrointestinal tract in animals that will become either fore-gut (sheep) or hind-gut (pig) fermenters. Indeed, it will be intriguing to examine how hormones before birth prepare the environment for, and influence the profiles of, the microbiota that colonise the gut after the onset of enteral feeding.

Marked structural and functional changes in the gastrointestinal tract occurred in the intact control fetuses over the last quarter of gestation, similar to those reported previously in sheep and pigs (Trahair and Sangild, 1997; Buddington et al., 2012). These findings highlight the importance of this period for the onset of normal digestive function at birth and indicate the likely consequences for offspring born preterm. Growth retardation of the mucosa and villus-crypt structure, and altered digestive enzyme activities, in the TX ovine fetus will also influence the surface area and the secretory and absorptive capacity of the gastrointestinal tract and, in turn, the systemic acquisition of nutrients. However, the effects of prematurity and/or hypothyroidism on the expression of nutrient and other transporters, and on the secretory and uptake mechanisms in gastric cells and enterocytes, remain to be established in the ovine fetus. The findings of this study have implications for the understanding of gastrointestinal pathologies in infants with congenital hypothyroidism and those born preterm with low circulating thyroid hormone concentrations (Fisher, 2007). Use of antenatal synthetic glucocorticoids in preterm delivery promotes an earlier onset of enteral nutrition and reduces the risk of NEC in the premature human infant (Halac et al., 1990; Jing et al., 2021). In sheep fetuses, maternal dexamethasone treatment is associated with increased circulating T3 concentration which may, in part, contribute to the maturational effects of the synthetic glucocorticoids (Forhead et al., 2007). Thyroid hormones, therefore, have an important role in the coordinated control of growth and maturation of the fetal gastrointestinal tract near term and in the clinical management of prematurity.