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Endocrinology, doi:10.1210/en.2007-1617
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Endocrinology Vol. 149, No. 9 4396-4401
Copyright © 2008 by The Endocrine Society

Maternal Thyroid Hormone: A Strong Repressor of Neuronal Nitric Oxide Synthase in Rat Embryonic Neocortex

Rohit Anthony Sinha1, Amrita Pathak1, Vishwa Mohan, Sanghamitra Bandyopadhyay, Leena Rastogi and Madan M. Godbole

Department of Endocrinology (R.A.S., A.P., V.M., L.R., M.M.G.), Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, India; and Division of Developmental Toxicology (S.B.), Indian Institute of Toxicology Research, Lucknow 226 001, India

Address all correspondence and requests for reprints to: Professor Madan M. Godbole, Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226 014, India. E-mail: madangodbole{at}yahoo.co.in.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Understanding of how maternal thyroid inadequacy during early gestation poses a risk for developmental outcomes is still a challenge for the neuroendocrine community. Early neocortical neurogenesis is accompanied by maternal thyroid hormone (TH) transfer to fetal brain, appearance of TH receptors, and absence of antineurogenesis signals, followed by optimization of neuronal numbers through apoptosis. However, the effects of TH deprivation on neurogenesis and neuronal cell death before the onset of fetal thyroid are still not clear. We show that maternal TH deficiency during early gestational period causes massive premature elevation in the expression of neuronal nitric oxide synthase (nNOS) with an associated neuronal death in embryonic rat neocortex. Maternal hypothyroidism was induced by feeding methimazole (0.025% wt/vol) in the drinking water to pregnant Sprague Dawley rats from embryonic d 6. Cerebral cortices from fetuses were harvested at different embryonic stages (embryonic d 14, 16, and 18) of hypothyroid and euthyroid groups. Immunoblotting and real-time PCR results showed that both protein and RNA levels of nNOS were prematurely increased under maternal hypothyroidism, and showed reversibility upon T4 administration. Immunohistochemistry revealed an increased nNOS immunoreactivity in both the cortical plate and proliferative zone of neocortex along with a corroborative decrease in the microtubule associated protein-2 positive neurons under maternal TH insufficiency. Results combined, put forth nNOS as a novel target of maternal TH action in embryonic neocortex, and underscore the importance of prenatal screening and timely rectification of maternal TH insufficiency, even of a moderate degree.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOCRINOLOGISTS are still equivocal about thyroid screening in early pregnancy and treatment despite associated intelligence quotient loss seen in the offspring of pregnant women with hypothyroidism (1, 2, 3). The need for prevention of maternal thyroid hormone (TH) insufficiency before mid-pregnancy, however moderate, and whatever the underlying cause has been advocated (4). Unfortunately, despite increasing evidence (5, 6, 7, 8, 9, 10, 11) that TH action in brain occurs much before the onset of fetal thyroid function, we know very little about the commencement of its molecular action during brain development. Whether and how TH deprivation affects the developmental process such as cortical neurogenesis and neuronal apoptosis that coincides with TH receptor (TR) appearance (12) in brain is still not clear. Although TH insufficiency is known to be associated with defective expression of neuronal markers (8), the reasons for the same are still not clear. Both neurogenesis and neuronal apoptotic mechanisms are tightly regulated to ensure optimization of neuronal number during the course of development (13). One of the major players in this process is a neurotransmitter known as nitric oxide (NO) produced by an enzyme neuronal nitric oxide synthase (nNOS) expressed in newly generated neurons. Although nNOS expression is modulated by steroid and THs during postnatal development (14, 15), its modulation by TH during the embryonic period lacks definite evidence. Therefore, the present study has been performed to understand the effect of maternal TH deficiency on the expression of nNOS, and its consequent impact on generation and survival of neocortical neurons. We here uncover a novel role of maternal TH in repressing nNOS expression (an antineurogenic signal) during fetal neocortical development, and propose that its premature up-regulation even under moderate TH deficiency could have a detrimental bearing on fetal neurogenesis and neuronal survival during early gestation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Sprague Dawley rats were housed in a 12-h light, 12-h dark cycle environment with ad libitum availability of chow diet and tap water. The pregnant rats were divided into two groups, euthyroid and hypothyroid (n = 20 in each group). 2-Mercapto-1-methylimidazole (MMI) (0.025% wt/vol) was given to the pregnant rats in drinking water from gestational d 6 and continued until the animals were killed. The day of visualization of spermatozoa in vaginal smears was designated as embryonic d (ED) 0. Fetuses were separated on ice, fetal cortices quickly dissected, and five to seven brains from each litter were pooled. Cortices from three different litters were used for immunoblotting and real-time PCR analysis, and three to four sections from three different litters were analyzed in each group for immunohistochemistry. Cerebral cortices were collected at ED 14 (n = 20), 16 (n = 15), and 18 (n = 15). For the replacement group (hypothyroid plus T4), pregnant dams on an MMI regimen were injected daily with T4 (1.5 µg/100 g body weight) sc from ED 12–15, and fetal cortices were collected at ED 16 (n = 15). A hypothyroxinemic model was prepared as described by Auso et al. (4). In brief, MMI was administered to pregnant dams for 3 d from ED 12–15, and fetal cortices were collected at ED 16 (n = 15). Total T4 (TT4) and total T3 (TT3) were measured in the serum of killed dams by RIA using Diagnostic Products kits (Diagnostic Products Corp., New York, NY). All aforementioned animal procedures were approved by the Institutional Animal Ethics Committee as per International Guidelines for Animal Care and Research.

Western blotting
Cerebral cortices (in all groups) from three different litters were harvested at each developmental stage, snap frozen in liquid nitrogen, and stored at –80 C until further investigation. For preparation of tissue homogenates, cerebral cortices were washed once with PBS and suspended in 10 volumes of lysis buffer [10 mM Tris-Cl (pH 7.5), 50 mM sodium chloride, and 1% Triton X-100 containing phenylmethylsulfonyl fluoride (1 mM) and protease inhibitor cocktail (a mixture of 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, (2S,3S)-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-methylbutyl}carbamoyl)oxirane-2-carboxylic acid, bestatin, leupeptin, and aprotinin; Sigma-Aldrich, St. Louis, MO] and kept on ice for 10 min. The tissues were then homogenized using a Teflon homogenizer (DuPont Co., Wilmington, DE) and centrifuged at 12,000 x g for 15 min at 4 C, and the supernatant was collected. Protein concentration was determined in the aforementioned samples, in the tissue homogenate, using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Tissue homogenate proteins (50–100 µg) were subjected to 6–12% SDS-PAGE and electrotransferred onto nitrocellulose membrane. The membranes were incubated with anti-nNOS, anti-proliferating cell nuclear antigen (PCNA) (Abcam plc, Cambridge, UK), or poly ADP ribose polymerase (PARP) (Cell Signaling Technology, Inc., Danvers, MA) antibodies, respectively. The signals were detected using an enhanced chemiluminescence detection system (Amersham Biosciences, Little Chalfont, UK). Relative expression of each protein was determined by densitometric analysis using LabWorks 4.0 software (Ultra-Violet Products Ltd., Cambridge, UK).

RNA extraction and real-time PCR
Total RNA was isolated from the neocortex at ED 16 (from both euthyroid and hypothyroid groups) from three different litters following the single-step RNA isolation method using TRIZOL reagent (Molecular Research Center, Inc., Cincinnati, OH). Total RNA (2 µg) was reverse transcribed to cDNA using oligo-(deoxythymidine)16 primers with the Thermoscript RT-PCR kit (Invitrogen Corp., Carlsbad, CA) following the manufacturer’s instructions. Real-time analysis for nNOS, TR-{alpha}, and normalizing gene, glyceraldehyde-3-phosphate dehydrogenase, was performed using specific TaqMan Universal PCR Master mix assays [Rn00583793_m1 (nNOS), Rn00579692_m1(TR-{alpha}), Rn00576699_m1 (glyceraldehyde-3-phosphate dehydrogenase)] as per the manufacturer’s instruction (Applied Biosystems, Foster City, CA) on the ABI Prism 7500 Sequence Detection System (Applied Biosystems), and fold changes in gene expression were calculated using the 2–{Delta}{Delta}CT method (16).

Immunohistochemistry
Four percent paraformaldehyde fixed paraffin embedded (5 µm) brain sections were used. After deparaffinization and rehydration steps, the sections were boiled in a microwave oven using 10 mM citrate buffer (pH 6.0) for antigen retrieval. Sections were blocked with 10% normal sheep serum for 20 min, and were stained with polyclonal antibodies against nNOS, TR{alpha}, and microtubule associated protein (MAP)-2 using the Quick Universal ABC Kit (PK-8800; Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions, followed by peroxidase staining reaction with 3'3-diaminobenzidine HCl/H2O2 as substrate. In situ detection of apoptosis was performed by terminal deoxynucleotide-transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) as per the manufacturer’s instruction (Roche Diagnostics Corp., Indianapolis, IN). TUNEL-positive cells were counted in five randomly selected fields, spanning the cortical plate (CP). At least 10,000 cells in each section were scored. Relative TUNEL positivity was expressed as the number of TUNEL-positive cells per 100 nuclei (Hoechst stained). Image-Pro Plus 5.1 software (Media Cybernetics Inc., Silver Spring, MD) was used for image capturing and cell counting. Three to four sections from three different litters were analyzed in each group.

Statistical analysis
Statistical analysis was performed using SPSS software version 11 (SPSS, Inc., Chicago, IL). The data are presented as a mean ± SE. Significant differences between groups were compared by two-way ANOVA, the factors being developmental stages and experimental groups (euthyroid vs. hypothyroid). One-way ANOVA was then used to identify developmental stages affected by the hypothyroidism, followed by Turkey’s or Duncan post hoc test. P value less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Maternal TH insufficiency during early embryonic periods results in premature and increased expression of nNOS in developing cerebral cortex
Pregnant Sprague Dawley rats were rendered hypothyroid by administering MMI, and their thyroidal status was assessed by RIA. Total circulating T3 and T4 concentrations in MMI-treated dams, at ED 14, 16, and 18, were markedly reduced to less than 10% of those of the euthyroid animals. This model was further validated by Western blot analysis of glial fibrillary acidic protein (GFAP). As reported earlier (8), it is down-regulated under TH deficiency (Fig. 1AGo). In fetal neocortices obtained from euthyroid dams, nNOS expression is prominently detectable on Western blots only by ED 18, in agreement with earlier reports (17) (Fig. 1BGo). However, its massive premature expression under hypothyroidism is seen as early as ED 14, which remained elevated thereafter. This increase in the protein level under hypothyroidism follows a 37.3 x 103-fold increase in its RNA level (P < 0.001) measured by sensitive real-time PCR analysis at ED 16. Because THs are known to regulate gene expression through nuclear receptors, we, therefore, looked into the status of their receptors under maternal TH deficiency. Interestingly, the increase in levels of nNOS correlated with a 2.2 x 103-fold increase seen in the level of TR-{alpha} (P < 0.001) that is known to be expressed early during development (12). Relative spatial distribution of nNOS within the developing neocortex through immunohistochemical staining at ED 16 revealed its weak staining in the CP and near absence in ventricular zone under euthyroid condition (17, 18) (Fig. 2AGo). In contrast, we observed a significant (P < 0.001) increase in the levels of nNOS positive cells both in the proliferative zone and prominently in the CP of hypothyroid brain (Fig. 2Go, B–D). Similarly, TR-{alpha} levels were also increased both in the proliferative zone and CP under hypothyroidism (Fig. 2Go, E and F).


Figure 1
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  		FIG. 1. Maternal THs regulate nNOS expression in developing neocortex. A, Immunoblot showing the levels of GFAP in euthyroid and hypothyroid cerebral cortex at ED 16. B, Immunoblot showing 160-kDa immunoreactive band of nNOS. Relative density of nNOS in euthyroid and hypothyroid groups. Each bar represents the mean of the respective individual ratios ± SE (n = 3 rats at each developmental stage). Significant differences compared with age-matched euthyroid counterpart are indicated (*, P < 0.05, ANOVA).

 

Figure 2
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  		FIG. 2. Hypothyroidism induced nNOS and TR{alpha} up-regulation within both proliferative zone and CP of developing neocortex. nNOS immunoreactivity in euthyroid (A) and hypothyroid (B) neocortex at ED 16 (x40 magnification). Note the increase in the levels of nNOS in both the ventricular zone (VZ) (black arrows) and CP (black arrowheads) under hypothyroidism; both membrane and cytosolic staining is seen. C, An enlarged (x100 magnification) view showing nNOS immunoreactivity in the proliferative zone of developing cerebral cortex at ED 16 under hypothyroidism. Arrowheads denote nNOS positive cells. D, The number of nNOS labeled cells present in the CP and ventricular zone at ED 16 was determined in horizontal strips (400-µm length) spanning their entire thickness. Data (expressed as means of cell count ± SEM) were compared by ANOVA and post hoc t tests. *, P < 0.001, ANOVA. E, TR{alpha} immunoreactivity in euthyroid and hypothyroid neocortex at ED 16 (x40 magnification). F, An immunoblot showing TR{alpha} levels in euthyroid and hypothyroid neocortex at ED 16.

 
Increased nNOS expression correlates with decreased MAP-2 staining and increased cell death in the CP under hypothyroidism
Because nNOS is a negative regulator of neurogenesis (18, 19) and was up-regulated in the proliferative ventricular zone under maternal hypothyroidism, we next looked whether its increased expression associated with a defective neurogenesis under hypothyroidism. For this, we did immunohistochemistry with MAP-2 antibody (marker of differentiated neurons). Results showed a significant reduction in the number of MAP-2 positive cells in the CP, indicating reduced neurogenesis under hypothyroidism (Fig. 3Go, A–C). Moreover, the reversibility of MAP-2 staining on T4 supplementation demonstrated a correspondence with nNOS expression (Figs. 3Go, A–C, and 4A). NO inhibitory action on neurogenesis could involve either reduction in the proliferative potential of neural precursor cells (NPCs) (17) or a compromised survival of newly generated neurons after differentiation (13). To resolve this, we looked at the levels of PCNA (a proliferation marker) (Fig. 3CGo) and cleaved PARP (an apoptotic marker) (Fig. 3DGo). Results showed that maternal hypothyroidism does not alter proliferation of NPCs as indicated by PCNA levels. However, the cleavage of PARP was significantly increased under hypothyroidism. Because PARP cleavage is associated with increased neuronal death during development (20), these results provide evidence for increased cell death under TH deficiency, which was also confirmed by TUNEL (Fig. 3Go, E and F). However, the direct relationship suggested by the strong association between nNOS and poor survival of newly born neurons under TH deprivation still needs to be proved.


Figure 3
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  		FIG. 3. nNOS up-regulation is associated with increased cell death in developing cerebral cortex under hypothyroidism. A and B, MAP-2 immunoreactivity in euthyroid, hypothyroid (Hypo), and T4 supplemented groups at ED 16 in developing neocortex (x40 magnification). The number of MAP-2 labeled cells present in the CP was determined in horizontal strips (400-µm length) spanning their entire thickness. Data (expressed as means of cell count ± SEM) were compared by ANOVA and post hoc t tests. C and D, Immunoblots showing PCNA and cleaved PARP levels at ED 16. Significant differences compared with age-matched euthyroid counterpart are indicated (*, P < 0.05, ANOVA). E, Representative photomicrographs of coronal sections of developing cerebral cortex at ED 16 showing TUNEL-positive cells in the CP of the euthyroid and hypothyroid rat fetus. Note the increase in TUNEL-positive cells in the hypothyroid group compared with the euthyroid group (x60 magnification). F, Relative increase in TUNEL-positive cells in the CP in the euthyroid and hypothyroid rats. TUNEL-positive cells were counted in five different areas spanning the CP and expressed as the number of relative TUNEL-positive cells per 100 nuclei (Hoechst stained). Significant differences compared with age-matched euthyroid pups are indicated (*, P < 0.01). VZ, Ventricular zone; IZ, intermediate zone.

 
Decreased maternal T4 and not T3 is associated with the up-regulated nNOS levels in developing cerebral cortex
Recent studies by Morreale de Escobar et al. (3) have shown that most of the T3 available to the fetal brain before the onset of fetal thyroid function (around ED 17.5) is derived from deiodination of maternal T4. Therefore, to investigate the role of maternal T4/T3 alteration in regulating nNOS expression, we used a model of moderate and transient maternal TH deficiency or hypothyroxinemia, as described by Auso et al. (4). Results revealed that even a moderate and transient decrease in maternal T4 (with T3 levels still within the normal range) increased nNOS expression significantly, and this was reversible by T4 administration to hypothyroid pregnant dams (Fig. 4AGo). Therefore, our results support that the alterations in fetal brain are responsive to circulating maternal T4, and not T3.


Figure 4
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  		FIG. 4. Maternal T4 and not T3 regulates nNOS expression in fetal cerebral cortex. A, Immunoblot showing nNOS expression in euthyroid (TT4 = 35.0 ± 2.4 nM/liter and TT3 = 0.65 ± 0.065 nM/liter), hypothyroid (TT4 = 0.0 ± 0.5 nM/liter and TT3 = 0.05 ± 0.02 nM/liter), hypothyroxinemic (TT4 = 3.75 ± 1.5 nM/liter and TT3 = 0.6 ± 0.06 nM/liter), and T4 replacement group (TT4 = 30.0 ± 2.0 nM/liter and TT3 = 0.8 ± 0.05 nM/liter) at ED 16. Each bar represents the mean of the respective individual ratios ± SE (n = 3 rats at each developmental stage). Significant differences compared with age-matched euthyroid at ED 16 are indicated (*, P < 0.001, ANOVA).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During early pregnancy the fetus is totally dependent on maternal TH for normal brain development. Adequate dietary intake of iodine during pregnancy is essential for maternal TH production and later for thyroid function in the fetus (1, 2, 3). Several clinical studies in various countries have consistently documented a relationship between maternal thyroid deficiency during pregnancy and problems with neuropsychological development of the offspring (1, 2, 21, 22, 23). However, molecular explanations for these clinical findings have only recently been explored (5, 6, 7, 8, 9, 10, 11). In line with these attempts, we here report nNOS as a crucial target gene of maternal TH in developing fetal cerebral cortex. nNOS catalyzes the conversion of L-arginine to L-citrulline with production of NO. Besides its role in regulating synaptic plasticity in the adult brain (24, 25, 26), NO is known to regulate neurogenesis in both developing and adult brain (18, 19). The antineurogenic action of nNOS is perhaps justified by its ontogenic appearance observed during brain development, which marks the end of neurogenesis and a period of programmed cell death of postmitotic neurons (27). The precocious up-regulation of nNOS under maternal hypothyroidism and its association with decreased MAP-2 staining indicate defective neurogenesis in developing neocortex. Although NO inhibition of neurogenesis involves either mitotic arrest of NPCs (18) or increased death of the newly generated neurons (13, 19), the latter seems to be a more probable reason of decreased MAP-2 positivity under TH insufficiency, as shown by increased PARP cleavage and TUNEL positivity. However, whether this loss of MAP-2 immunoreactivity is also a contribution of a maturation defect in cortical neurons still needs to be investigated. Unfortunately, despite the huge increase in the nNOS expression, we still do not know the mechanism by which maternal hypothyroidism brings about this increase. A strong possibility exists that the unliganded TR-{alpha} regulates nNOS levels at certain neurodevelopmental stages (ED 14–18) under hypothyroidism. Regulation of brain development by unliganded TR is now well known (28, 29) and may have some implication in the premature onset of nNOS expression during neocortical development. However, whether such regulation of nNOS is purely genomic or also involves a nongenomic TH signaling as described for other NOS variants (30) will need further investigation. Moreover, whether TRs directly regulate nNOS expression or indirectly through repression of a putative nNOS transcriptional silencer also needs to be resolved. In this respect the appearance of nNOS at later stages under euthyroid condition could be due to the following possible reasons: 1) loss of this nNOS silencing mechanism during the progressive phase of neocortical development, 2) control of nNOS expression by other regulatory factors like neurotrophins, and 3) alteration in TRs and their gene regulatory potential through changes in the levels of heterodimerization partners and transacting modulators of transcription. Understanding and answering these questions will perhaps help us to resolve the paradox of whether TH deficiency per se or its unliganded receptors are detrimental for proper brain development (28, 29, 31) and whether the unliganded TRs act as "Dependence receptors," creating a state of dependence during developmental processes that may otherwise be independent of THs (32).

Recent studies (4, 23, 33, 34) show that even a modest maternal T4 insufficiency during neocorticogenesis may result in serious anatomical and cognitive insults, even if the maternal T3 and TSH are well maintained within the normal range. We extend support to these assertions and show that gene alteration in fetal brain from a hypothyroxinemic mother is as sensitive as that of a hypothyroid mother. Because maternal hypothyroxinemia is much more prevalent than hypothyroidism per se and is usually left undiagnosed in various societies (23, 35, 36), it warrants immediate attention, and our study adds to this view with molecular evidence showing a highly sensitive regulation of fetal nNOS by circulating maternal T4. To conclude, this work complements the growing evidence for the indispensable role of maternal T4 in fetal brain development and supplements a molecular insight into its action in part through repressing the ontogenic induction of nNOS during embryonic neocortical development.


   Acknowledgments
 
We thank Drs. P. J. Hanson (Aston University, Birmingham, UK) and K. P. Campbell (University of Iowa, Iowa City, IA) for providing specific antibodies against neuronal nitric oxide synthase, Dr. P. M. Yen (John Hopkins University, Baltimore, MD) for antibodies against thyroid hormone receptor-{alpha}, and Dr. B. Pettmann (Developmental Biology Institute of Marseille, Marseille, France) for microtubule associated protein-2 antibody.


   Footnotes
 
This work was supported by a grant from Department of Science & Technology, Government of India through Fund for Improvement of Science & Technology Infrastructure SR/SO/HS/17/2003 and SR/SO/HS/95/2007 (to M.M.G.) and Senior Research Fellowship from Council of Scientific and Industrial Research (to R.A.S.) [10-2(5 )/2003(II)-E.U.II] and University Grants Commission (to A.P.) [2-89/98(SA-I)] from the Government of India, New Delhi.

Disclosure Statement: The authors have nothing to declare.

First Published Online May 8, 2008

1 R.A.S. and A.P. contributed equally to this work. Back

Abbreviations: CP, Cortical plate; ED, embryonic d; GFAP, glial fibrillary acidic protein; MAP, microtubule associated protein; MMI, 2-mercapto-1-methylimidazole; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NPC, neural precursor cell; PARP, poly ADP ribose polymerase; PCNA, proliferating cell nuclear antigen; TH, thyroid hormone; TR, thyroid hormone receptor; TT4, total T4; TT3, total T3; TUNEL, deoxyuridine 5-triphosphate nick end labeling.

Received November 27, 2007.

Accepted for publication April 29, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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