Mild Gestational Hypothyroidism in Mice Has Transient Developmental Effects and Long-Term Consequences on Neuroendocrine Systems

Abstract

Background: Thyroid hormones (TH) play a key role in fetal brain development. While severe thyroid dysfunction, has been shown to cause neurodevelopmental and reproductive disorders, the rising levels of TH-disruptors in the environment in the past few decades have increased the need to assess effects of subclinical (mild) TH insufficiency during gestation. Since embryos do not produce their own TH before mid-gestation, early development processes rely on maternal production. Notably, the reproductive network governed by gonadotropin-releasing hormone (GnRH) neurons develops during this critical period. Methods: The risk of mild maternal hypothyroidism on the development of GnRH neurons and long-term effect on neuroendocrine function in the offspring was investigated using a mouse model of gestational hypothyroidism induced by methimazole (MMI) treatment. Results: MMI treatment during gestation led to reduced litter size, consistent with increased miscarriages due to hypothyroidism. E12/13 embryos, collected from MMI-treated dams, had a decreased number of GnRH neurons, but the migration of the remaining GnRH neurons was normal. Cell proliferation was reduced in the vomeronasal organ (VNO), correlating with the reduced number of GnRH neurons detected in this region. Using a GnRH cell line confirmed attenuated proliferation in the absence of T3. Pups born from hypothyroid mothers had normal postweaning growth and estrus cycles, yet adult offspring had significantly more cells expressing estrogen receptor alpha in the arcuate nucleus. Notably, by adulthood, GnRH cell number and distribution was comparable with nontreated controls indicating that compensatory mechanisms occurred after E13. Conclusion: Overall, our work shows that mild TH disruption during gestation transiently affects proliferation of the pool of GnRH neurons within the VNO and has a long-term impact on neuroendocrine systems.

Keywords: GnRH neurons; embryonic development; maternal hypothyroidism; neuroendocrinology; reproduction; thyroid hormones.

FIG. 1.

Expression of thyroid hormone (TH) signaling pathway elements in gonadotropin-releasing hormone (GnRH) neurons. Nasal region of E11.5 embryos was dissected, cultured and subsequently used to generate microarray data from single GnRH cell cDNA libraries. (A) PCR gel showing expression of TH nuclear receptors (TRα1, TRα2, and TRβ), the two subunits of integrin αVβ3 membrane receptor (ItgαV and Itg3), and the deodinase 2 (Dio2) in three different primary GnRH neurons and in the Gn11 cell line. (B) Heat-map diagram showing expression of TH receptors, transporters, and deiodinases in single GnRH neurons isolated from nasal explants after 4 or 10 days in vitro (div), during which time the GnRH cells decrease migration and increase maturation.

FIG. 2.

Inducing hypothyroidism during gestation. (A) Schematic overview and timeline of the protocol for methimazole (MMI) administration in drinking water in dams. MMI was administered from gestational day 6 (GD6) to GD12, GD13, or birth. Embryos were collected at embryonic day 12 (E12) or E13 to study the effect of maternal hypothyroidism on GnRH proliferation and migration. To study the postnatal effects of gestational hypothyroidism, reproductive and metabolic factors were monitored in pups after weaning and brains were collected at postnatal day 57 (PN57). (B) Comparison of levels of T3 and T4 in serum and (C) litter size of controls (grey) and MMI-treated (orange) dams. *p < 0.05; ***p < 0.001.

FIG. 3.

Effect of gestational hypothyroidism on GnRH neuronal number and migration. (A) Schematic representation of the embryonic migratory path of GnRH neurons. GnRH neurons proliferate in the developing VNO and migrate along tracks before crossing the NFJ to reach the brain. (B) Comparison of the total number of GnRH neurons counted at E12 and E13 in embryos collected from control (grey) of MMI-treated (yellow) dams. (C) Comparative distribution of GnRH neurons in the different zones of the migratory path in E12 and E13 from control and MMI dams. (D) Time-course images of GnRH neurons recorded in live migration assay for 15, 30, 45 and 60 minutes after ± T3 (30 nM) treatment for 24 hours. The nucleus is used to track neuronal movement. The histogram shows the comparison of individual GnRH cell migration rates. Scale 10 µm. (E) Photograph (left) of a nasal explant stained for GnRH and thereafter template used to measure the cell migration of the GnRH neuronal population in explants ±T3 treatment. Graph (right) shows the percentage of GnRH neurons found in each migration zone in control and treated explants treated. Scale 250 µm. VNO, vomeronasal organ; NFJ, nasal forebrain junction; SFM, serum-free medium. *p < 0.05; **p < 0.01.

FIG. 4.

Effect of gestational hypothyroidism on GnRH cell proliferation. (A) Photograph of E12 stained for pHIS (brown) and gnRH (blue), showing the three zones sampled for proliferation: VNO, LGE, and MGE. Graphs (right) show the of number of pHIS+ cells in the VNO, LGE, and MGE of E12 embryos collected from control (grey) and MMI-treated (yellow) dams. Scale 250 µm. (B) Graph showing the correlation between the number of GnRH neurons and pHIS+ cells counted in the VNO of E12 and photographs showing the delineated VNO, stained for pHIS (brown) and GnRH (blue). Scale 100 µm. (C) Comparison of the normalized percentage of GnRH cells expressing pHIS in vitro with or without treatment for 24 hours with T3 (3 nM), T3+tetrac (100 nM) or T3 + 1–850 (1 µM) and photographs showing fluorescent staining of pHIS (green, Alexa488) and DAPI counterstaining. Scale 250 µm. LGE, lateral ganglionic eminence; MGE, median ganglionic eminence. *p < 0.5; **p < 0.01; ***p < 0.001.

FIG. 5.

Long-term effect of gestational hypothyroidism on offspring reproductive functions, cell proliferation, and hypothalamic neuroendocrine regulation. (A, left) Graph showing time-course of males (squares) and females (triangles) weight change in postnatal mice from control (gray) and treated (yellow) dams. (A, right) Graph indicating percentage of mice over pubertal period with vaginal opening (VO) from control (grey) or MMI-treated (yellow) dams. (B) Comparison between adult mice born from control or MMI-treated dams of (left) percentage of days spent in each estrus cycle stage [diestrus (D), proestrus (P), estrus (E)]; (middle) testis per body weight (BW) ratio; (right) total number of GnRH neurons. (C and D) Comparison between adult mice from control (gray) and MMI-treated (yellow) for (C, left) number of pHIS positive cells in subventricular zone (SVZ); (C, right) number of ERα-expressing cells in the arcuate nucleus (ARC) and the ventromedial hypothalamus ventrolateral (VMHvl); (D, left) number of RFRP-3 neurons; (D, right) corpus callosum conformation above subventricular zone (above) and hippocampus (bellow). Scale 500 µm. *p < 0.05.

FIG. 6.

Graphical summary of the developmental and long-term effect of maternal hypothyroidism during gestation in offspring.