Temporal Endocrine Consequences of In Utero Perturbation

While the pig is becoming increasingly recognized as a critical biomedical model, a basic understanding of the ontogenic trajectory of the porcine fetus remains underrepresented in the current literature. Additionally, while physiological disruptions during the in utero period are known to have a wide array of consequences, the temporal sensitivity and interaction between various fetal endocrine systems, as well as the persistence of endocrine disruption resulting from in utero perturbations, remains understudied in the porcine fetus and neonate. To address this, we first characterized the ontogeny of the porcine fetal insulin-like growth factor (IGF) system and local hepatic and renal renin-angiotensin systems (RAS) and then assessed the temporal impact of fetal hypothyroidism on these endocrine systems throughout gestation. Subsequently, we examined the postnatal consequences of intrauterine growth restriction (IUGR) on the piglet by assessing the potential persistence of resultant endocrine and hematological disruptions in the early postnatal period.

To assess the temporal impact of hypothyroidism on the fetal IGF system and RAS, we utilized a gestational-age dependent model of induced fetal hypothyroidism, as described in Chapters 2 and 3. Briefly, 24 pregnant gilts were evenly divided into two treatment groups, with n = 12 gilts receiving a daily oral dose of 5 mg/kg methimazole (MMI) to induce fetal hypothyroidism, and n = 12 gilts serving as a sham control (CON). Within each treatment group, gilts were further subdivided into four timepoints, with treatment of n = 3 gilts initiating at either gestational day 34, 45, 55, or 65 of the normal 114 day gestational period, and lasting for a total of 21 days. After 21 days of treatment, the gilts were humanely euthanized to allow for fetal sample collection. Fetal body weight was recorded, and fetal serum collected from a total of 335 fetuses. Subsequently, a subset of fetuses (n = 4/litter) were selected for more extensive sampling, including collection of the fetal thyroid (ROID), liver (LVR), and kidneys (KID). The ROID was fixed in 10% neutral buffered formalin, and portions of the LVR and KID snap frozen in liquid nitrogen for later assessment.

Fetuses from the MMI group were first confirmed hypothyroid by the presence of an enlarged ROID, which exhibited goitrous histology when assessed by standard brightfield microscopy. Subsequently, a fluorescent lectin staining method was developed for quantitative assessment of the severity of induced hypothyroidism, with results showing that MMI treatment induces abnormal ROID follicular morphology. Treatment with MMI was also found to result in reduced levels of triiodothyronine (T3) at the two later timepoints, with reduced thyroxine levels at all four timepoints providing further evidence of hypothyroidism in the MMI fetuses. The aforementioned methods were additionally utilized to assess ROID ontogeny in CON fetuses, revealing ontogenic increases in concentration of both major thyroid hormones throughout gestation, which occurred concurrently with increasing follicular size and decreasing follicular circularity.

Following confirmation of our experimental model, gene expression in the fetal LVR and KID was assessed in the previously selected fetal subset to first establish the ontogenic trajectory of key IGF and RAS genes in euthyroid fetuses, and then to determine the temporal effects of hypothyroidism on expression of these genes. Further, expression of key genes regulating thyroid hormone bioavailability, including those encoding thyroid hormone binding proteins and receptors, was also assessed in this manner. Of the 19 genes of interest assessed in the fetal LVR, 17 were found to be ontogenically regulated, with 9 of the 17 genes of interest assessed in the KID ontogenically regulated. Fetal hypothyroidism was found to have temporal effects on expression of many genes of interest, with 11 genes temporally dysregulated in the LVR, and 5 genes temporally dysregulated in the KID. In both euthyroid and hypothyroid fetuses, many of the changes observed in the LVR were discordant with that observed in the KID, suggesting tissue-specific regulation of these genes. In addition to increasing base knowledge of endocrine ontogeny in the porcine fetus, these results suggest a complex relationship between thyroid hormones and the porcine IGF system and RAS that is both temporal and tissue-specific.

To assess the potential longer-term impacts of in utero perturbation, we utilized a spontaneously occurring porcine model of IUGR to characterize the persistence of associated endocrine and hematological consequences within the early postnatal period, as described in Chapter 4. Briefly, n = 911 piglets were collected prior to suckling, weighed, and blood sampled, with rectal temperatures recorded two hours later. Weighing and blood sampling was repeated at 20 days of age (D20) from a total of n = 757 remaining piglets. Following sampling, a subset of biological extremes representing IUGR and large for gestational age (LGA) piglets was selected based on birth weight, with piglets below the 10^th percentile of birth weight selected to represent IUGR piglets (n = 92), and piglets above the 90^th percentile of birth weight selected to represent LGA piglets (n = 92).

Consistent with prior work, IUGR piglets exhibited reduced rectal temperatures and growth rates, with evidence of compensatory gain throughout the study period. At birth, whole blood analyses revealed that IUGR piglets had reduced hematocrit and blood glucose levels relative to LGA piglets, with plasma levels of total protein, circulating T3, and circulating T4 also reduced. While estradiol levels were unaffected in female IUGR piglets, IUGR males additionally exhibited reduced testosterone levels at birth. At D20, many of the observed endocrine and hematological dysregulations had normalized, with there no longer being any observed differences in hematocrit, blood glucose, or T4 levels. However, decreased plasma protein levels persisted at D20, with a marginal increase in T3 levels in IUGR piglets relative to LGA at this timepoint indicating a possible compensatory response to the low levels previously observed at birth. Collectively, these results provide insights into the pathogenesis of IUGR, effectively showing that many systemic dysregulations associated with this condition are acute, and do not persist throughout the early postnatal period. Further, they suggest that perturbations experienced in utero may have lasting consequences on postnatal physiology and phenotype, as IUGR piglets failed to reach an appropriate body weight by D20, and still experienced moderate systemic dysregulations at this timepoint.

In summary, our results regarding fetal hypothyroidism indicate temporal and tissue-specific interactions between critical endocrine systems in the porcine fetus. Additionally, our assessment of the systemic consequences of IUGR indicates that the impacts of in utero perturbation can persist into the early neonatal period. Collectively, this iterates the exquisite sensitivity of fetal life to perturbation, and the importance of this developmental period in determining postnatal physiology.