2023 Volume 70 Issue 8 Pages 805-814
The secretion of several hypothalamic peptide hormones is activated during the preovulatory period. Hypothalamic thyrotropin-releasing hormone (TRH) is one such hormone with reproductive and/or metabolic significance. However, it remains unclear whether thyroid-stimulating hormone (TSH)-producing thyrotrophs are produced during the preovulatory period. We previously found a transient increase in the expression of the nuclear receptor NR4A3, a well-known immediate early gene, in the proestrus afternoon in the anterior pituitary glands of rats. To investigate the relationship between TRH secretion and pituitary NR4A3 expression during proestrus, we used proestrus and thyroidectomized rats to identify NR4A3-expressing cells and examined the regulation of Nr4a3 gene expression via the hypothalamus-pituitary-thyroid (HPT) axis. The percentage of NR4A3-expressing cells increased in thyrotrophs at 14:00 h of proestrus. Incubation of rat primary pituitary cells with TRH transiently stimulated Nr4a3 expression. Thyroidectomy to attenuate the negative feedback effects led to increased serum TSH levels and Nr4a3 gene expression in the anterior pituitary, whereas thyroxine (T4) administration conversely suppressed Nr4a3 expression. Additionally, the administration of T4 or TRH antibodies significantly suppressed the increase in Nr4a3 expression at 14:00 h of proestrus. These results demonstrate that pituitary NR4A3 expression is regulated by the HPT axis, and that TRH stimulates thyrotrophs and induces NR4A3 expression during the proestrus afternoon. This suggests the potential involvement of NR4A3 in the regulation of the HPT axis during pre- and post-ovulatory periods.
DURING THE PREOVULATORY PERIOD, hypothalamic activation is a response to high estrogen concentrations secreted by mature follicles in mammals [1]. Its main function is to promote the massive secretion of hypothalamic gonadotropin-releasing hormone (GnRH), commonly referred to as the GnRH surge, which induces the pituitary luteinizing hormone (LH) surge that triggers ovulation [2]. In rodents, hypothalamic activity occurs in the afternoon of proestrus during the preovulatory period and is integrated with circadian rhythms [3, 4]. This hypothalamic activity not only stimulates GnRH and LH, but also causes the secretion of other hypothalamic neuropeptides and pituitary hormones, such as thyrotropin-releasing hormone (TRH) [5] and prolactin (PRL) [6]. Such hypothalamus-pituitary system coordination may support ovulation or estrous cyclicity, oocyte quality, and the transition to the early stages of gestation.
TRH, a hypothalamic tripeptide hormone, is an upper regulator of the hypothalamus-pituitary-thyroid (HPT) axis that controls systemic metabolic regulation [7-9] and is also recognized as a PRL-releasing factor [10]. Evidence for the increase in TRH during proestrus is provided by experimental results that directly detected TRH in the pituitary portal blood [5] and revealed the suppression of the preovulatory PRL surge by the administration of TRH antiserum [11]. These reports suggest that an increase in TRH levels during the proestrus period may be partially responsible for PRL secretion. However, it remains unclear whether TRH is physiologically functional, as PRL secretion during proestrus is normal in TRH-knockout mice [12]. Moreover, only a 20% increase in blood thyroid-stimulating hormone (TSH) concentration was observed during the proestrus afternoon in rats [13]. Thus, there remains considerable uncertainty as to whether TRH in proestrus activates TSH-producing cell thyrotrophs to stimulate TSH secretion.
NR4A3, a member of the orphan nuclear receptor NR4A subfamily [14], is an immediate early response gene involved in the transcriptional regulation of various genes [15, 16]. All three members of the NR4A subfamily (NR4A1, NR4A2 and NR4A3) commonly bind to the nerve growth factor-induced clone B (NGFI-B) response element and Nur-response element (NurRE) [17] and can exert cooperative or antagonistic effects depending on the biological context [18]. In addition, the action of the NR4A subfamily is not ligand-dependent recruitment into the nucleus, but rather depends on its expression in response to various stimuli, such as hormonal signals [18]. NR4A3 is involved in the time- and cell-specific regulation of cellular functions, including proliferation or anti-proliferation [19, 20], differentiation [21-23], apoptotic or anti-apoptotic effects [24-26], oxidative metabolism [27], and secretion [28, 29]. Therefore, NR4A3 probably regulates the tissue- and cell-specific gene targets. Furthermore, NR4A3 has been reported to be highly expressed in the pituitary gland and cerebral cortex of rats [30]. However, the physiological expression patterns and significance of pituitary NR4A3 remain unclear.
We previously demonstrated that GnRH promotes Nr4a3 expression as an immediate early gene that regulates follicle-stimulating hormone beta gene expression in gonadotrophs [31]. We further found that the expression of Nr4a3 is transiently and significantly increased in the rat anterior pituitary from 14:00 to 17:00 h during proestrus [32]. These results suggest that NR4A3 may be involved in the regulation of secretory function in the pituitary gland during the preovulatory period. However, we confirmed that a GnRH antagonist significantly suppresses Nr4a3 expression by only 20–30% at 14:00 and 17:00 h in proestrus rats, and an LH surge occurs with a peak at 17:00 h, which is later than Nr4a3 expression [32]. Hence, the presence of other NR4A3-inducing factors as well as GnRH, has been suggested. It may also be possible that NR4A3 is induced in endocrine cells other than gonadotrophs in the pituitary gland. Therefore, we hypothesized that TRH was involved in pituitary NR4A3 expression during proestrus. To test this hypothesis, we confirmed the expression of NR4A3 in the thyrotrophs of proestrus rats and examined the regulation of NR4A3 by the HPT axis and its induction by TRH.
TRH and L-thyroxine (T4) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM, low glucose), antibiotic-antimycotic, MEM nonessential amino acids, fetal bovine serum (FBS), trypsin, and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) solution were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The NR4A3 antibody (anti-human NGFI-B gamma mouse monoclonal antibody) was obtained from Perseus Proteomics, Inc. (Tokyo, Japan). Two types of anti-TRH rabbit sera were prepared by the Peptide Institute Inc. (Osaka, Japan). The two antisera against TRH, denoted as aTRH#1 and aTRH#2, and normal rabbit serum as control were purified to IgG using Ab Spin Trap columns (GE Healthcare, Chicago, IL, USA), followed by replacement with PBS by dialysis. The TSH antibody used for immunohistochemistry was gifted by The Biosignal Research Centre Antibody Supply Program, Institute for Molecular and Cellular Regulation in Gunma University (Maebashi, Japan). Primary antibodies against TSH (NIDDK-anti-rat TSH-RIA-6, AFP329691Rb), purified rat TSH (rTSH-I-9, AFP-11542 B), and TSH (rTSH-RP-3, AFP-5512 B) for hormone measurements were provided by the National Hormone and Peptide Program, Harbor-UCLA Medical Center (Torrance, CA, USA). Anti-rabbit gamma-globulin goat serum, a secondary antibody for the immunoassays, was generated and purified by ammonium sulfate precipitation in our laboratory.
AnimalsFemale Wistar-Imamichi rats (200–300 g) were purchased from SLC Japan (Shizuoka, Japan). The rats were maintained under controlled temperature and light conditions (23 ± 3°C and 14-h/10-h light-dark cycle, respectively, with lights on at 5:00 h), with free access to laboratory chow and tap water. Vaginal smears were examined daily for at least two weeks before the start of the experiments. Female rats with regular 4-d estrous cycles were used in this study.
For RT-qPCR analysis and blood TSH assay, the animals were sacrificed by decapitation, and trunk blood samples were collected immediately. Pituitary tissues were collected immediately and frozen in liquid nitrogen. Perfusion fixation was also performed for immunohistochemical analysis of pituitary tissues. Under anesthesia with isoflurane, the rats were perfused with 4% paraformaldehyde in 50 mM phosphate buffer with a peristaltic pump, and then collected pituitary tissues were fixed overnight in 4% paraformaldehyde in 50 mM phosphate buffer at 4°C. All animal protocols were approved by the Animal Care and Use Committee of Kitasato University (approval no. 21-002).
Primary pituitary cell culturePrimary cultures of female rat anterior pituitary cells were prepared as previously described [33]. Briefly, normal female rat anterior pituitary glands were extracted under light isoflurane anesthesia, rapidly collected in a sterile manner, and dispersed in trypsin solution (0.25% trypsin, 10 mM EDTA, and 20 mM HEPES in DMEM). The dispersed pituitary cells were seeded at 1.5 × 105 cells/well in a 96-well plate in DMEM with 10% FBS, 1% MEM non-essential amino acids, and 1% antibiotic-antimycotic and cultured at 37°C with 5% CO2 for 48 h. After pre-incubation, the cells were cultured in fresh medium with 10–7 M TRH or without for 0, 1, 2, 4, and 8 h. The cells were also incubated with 10–7 M TRH or without containing the anti-serum against TRH (aTRH#1 or aTRH#2, 10 μg/mL) or Control IgG (10 μg/mL) for 1 h. The cells were collected using ISOGEN (Nippon Gene, Tokyo, Japan).
ThyroidectomyThyroidectomy was performed on the rats under anesthesia using a combination of anesthetics (MMB: 0.375 mg/kg medetomidine, 2.0 mg/kg midazolam, and 2.5 mg/kg butorphanol). The control rats were sham-operated without thyroidectomy. One week after the thyroidectomy or sham operation, pituitary tissues and blood samples were collected from the rats, as mentioned above, or during the administration experiment.
Administration of thyroxine (T4) and TRH antibodyT4 stock (20 mg/mL) in 0.5 N NaOH-70% ethanol was diluted with sterile saline to dose concentrations of 20 and 200 μg/mL. The solvent was diluted 100-fold with sterile saline as a vehicle control. T4 or vehicle was administered intraperitoneally at 1 mL/kg at 5:00 h to proestrus or to thyroidectomized rats.
Two antisera against TRH (aTRH#1 or aTRH#2, 1 mg/mL/head) or Control IgG (1 mg/mL/head) were administered into the tail veins at 5:00 h of proestrus rats. Pituitary tissues and blood samples were collected from rats treated with T4 or TRH antibodies, as described above.
ImmunohistochemistryThe fixed pituitary tissues were dehydrated using a graded ethanol series, cleared in xylene, embedded in paraffin, and sectioned at 4 μm with a microtome. The sections were dewaxed in xylene, rehydrated using graded ethanol, heated in a pressure pan cooker in TE buffer (10 mM Tris, 1 mM EDTA, pH 9.0) for 5 min, and subsequently allowed to cool for 60 min at room temperature. For the double immunofluorescence experiment, the sections were blocked with 5% normal goat serum for 60 min at 20°C, co-incubated with primary antibodies against NR4A3 (1:3,000 dilution) and TSHβ (1:100,000 dilution) overnight at 4°C. The sections were then labeled with fluorescent-labeled secondary antibodies (Alexa Fluor 488-labeled goat anti-mouse IgG, highly cross adsorbed, 1:1,000 dilution; A2066709, Thermo Fisher Scientific Inc., and Alexa Fluor 594-labeled goat anti-rabbit IgG, 1:1,000 dilution; A11011, Thermo Fisher Scientific Inc.) for 1 h at 20°C in the dark. Finally, the slides were mounted with Vectashield H1200 mounting medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) and images were captured using a confocal laser scanning microscope LSM710 (Carl Zeiss, Oberkochen, Germany). Using a 63× lens, we manually counted the number of nuclear NR4A3-positive cells in 20–40 randomly chosen images per animal. The area of the anterior pituitary was determined using a software (Zen 2008, Carl Zeiss), and the number of nuclear NR4A3-positive cells per area was calculated. Moreover, the percentage of nuclear NR4A3-positive cells among more than 30 TSH-producing cells containing nuclear structures was calculated.
Reverse transcription-quantitative PCR (RT-qPCR)Total RNA was extracted from the pituitary tissues and cells using ISOGEN and reverse transcribed to cDNA using a High-Capacity cDNA Synthesis Kit (Thermo Fisher Scientific Inc.). The primer sequences used for all RT-qPCR assays were as follows: Nr4a3, forward: 5'-AAA GAC GGA ACC TCC ACA GAA-3' and reverse: 5'-GTC GGG ATA GGC GAA GCA-3'; Tshb, forward: 5'-CAG CAT TAA CTC GCC AGT GC-3' and reverse: 5'-GAT GAC ACT TGC CCA CAA GC-3'; Rpl19, forward: 5'-GGA AGC CTG TGA CTG TCC AT-3' and reverse: 5'-ATC CTT CGC ATC CAG GTC AC-3'. qPCR was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). Relative gene expression levels were calculated using the ΔΔCT method, with ribosomal protein L19 (Rpl19) as the internal control for normalization.
Determination of TSH levelsPlasma TSH concentrations were measured by using a time-resolved fluorometric immunoassay (DELFIA System; PerkinElmer Inc., Waltham, MA, USA). Briefly, purified rat TSH (rTSH-I-9) was labeled with europium (Eu) using a DELFIA Eu-Labeling Kit (PerkinElmer Inc.). A 96-well plate (C96, Cert. Maxisorp, Nunc-Immuno Plate, Thermo Fisher Scientific Inc.) was coated with anti-rabbit gamma globulin goat serum and overlaid with anti-rat TSH serum (TSH-RIA-9, 1:125,000 dilution). Diluted plasma samples and TSH standards (rTSH-RP-3) were incubated at 4°C overnight and optimally diluted Eu-labeled TSH was added for 2 h at 20°C. After washing, the plates were treated with DELFIA Enhancement Solution (Perkin Elmer Inc.) and the specific fluorescence (excitation and emission wavelengths of 340 and 613 nm, respectively) was measured using a Multilabel Reader 2030 ARVO X4 (PerkinElmer Inc.). The intra- and inter-assay coefficients of variation for the TSH levels were 1.1% and 2.3%, respectively.
Statistical analysisThe results are presented as mean ± standard error of the mean (SEM). Differences were analyzed using Student’s t-test or one-way analysis of variance (ANOVA), followed by the Tukey-Kramer test. All statistical analyses were performed using JMP® Pro 17.0.0 (SAS Institute Inc., Cary, NC, USA).
We investigated NR4A3-expressing cells from the pituitary endocrine cells in proestrus rats using double-fluorescence immunohistochemistry. NR4A3 was observed in the nuclei of pituitary cells, and its fluorescence intensity increased at 14:00 h (Fig. 1A). Double-fluorescence immunostaining of TSHβ and NR4A indicated that approximately half of TSH-producing cells were NR4A3-positive cells. NR4A3-positive cells significantly increased within the TSH-producing cells at 14:00 h and then decreased at 17:00 h, albeit with little change per anterior pituitary area (Fig. 1B, C).
Expression of NR4A3 in thyrotroph cells of proestrus rat
Double-fluorescence immunohistochemistry of NR4A3 and TSHβ was performed on rat pituitary gland biopsies obtained at 11:00, 14:00, and 17:00 h during proestrus (A). NR4A3 (green), TSHβ (red), and nuclei (blue) were observed using confocal laser scanning microscopy. The white arrows indicate TSHβ-positive NR4A3-expressing cells. Scale bars indicate 10 μm. The number of NR4A3-positive cells per anterior pituitary area (B) and the percentage of NR4A3-positive cells among the TSHβ-positive cells (C) were determined. Data from individual rats are plotted and presented as the mean ± SEM (n = 3). Statistical analysis was performed using the Tukey-Kramer test; * p < 0.05.
We examined the direct effect of TRH on pituitary Nr4a3 expression in primary pituitary cell cultures. The treatment with TRH (10–7 M) to the dispersed pituitary cells resulted in a transiently increased Nr4a3 mRNA expression within an hour (Fig. 2A). In contrast, TRH administration had little effect on increasing Tshb mRNA expression (Fig. 2B). Prolonged incubation time also tended to decrease Nr4a3 and Tshb mRNA expression. Furthermore, the addition of an anti-TRH antibody (aTRH#1 or aTRH#2) significantly inhibited the induction of Nr4a3 expression by TRH (Fig. 2C).
Effect of TRH stimulation on Nr4a3 expression in the primary pituitary cells
Primary cultured rat anterior pituitary gland cells were treated with or without TRH (10–7 M) for 0–8 h. Nr4a3 (A) and Tshb (B) mRNA expression levels were measured. Temporal changes in mRNA levels are described relative to each other at 0 h in the control group without TRH (set to 1.0). (C) The cells were treated with TRH and TRH antibody (aTRH#1 or aTRH#2, 10 μg/mL) or normal rabbit IgG (Control IgG, 10 μg/mL) for 1 h. Changes in mRNA levels are described relative to those in the Control IgG group without TRH (set to 1.0). Data are presented as mean ± SEM (n = 5). Statistical analysis was performed using the Tukey-Kramer test; *** p < 0.001.
To determine whether TRH secretion increased pituitary NR4A3 expression, thyroidectomized rats with elevated TRH secretion from the hypothalamus were used. The pituitary glands of thyroidectomized rats showed a significant 2.07-fold increase in Nr4a3 mRNA expression (Fig. 3A). Tshb mRNA expression and blood TSH levels were significantly increased (Fig. 3B).
Effect of thyroidectomy on rat pituitary Nr4a3 expression
Thyroidectomized (THX) and sham-operated control (Sham) rats were established, and 7 days later, anterior pituitary tissues and blood samples were collected at 11:00 h. Nr4a3 and Tshb mRNA expression (A) and plasma TSH concentrations (B) were measured. Relative mRNA levels were compared with those of the sham-operated control (set to 1.0). Data are presented as mean ± SEM (n = 5). Statistical analysis was performed using Student’s t-test; * p < 0.05, ** p < 0.01.
Furthermore, exogenous T4 supplementation in thyroidectomized rats significantly suppressed the pituitary Nr4a3 mRNA expression in a concentration-dependent manner (Fig. 4A). T4 administration also significantly suppressed blood TSH levels but had no effect on Tshb mRNA expression (Fig. 4B, C).
Effect of T4 on pituitary Nr4a3 expression in thyroidectomized rats
Thyroidectomized rats were intraperitoneally injected with T4 (20 and 200 μg/kg) or the solvent only (vehicle group, Veh) at 5:00 h, and anterior pituitary tissues and blood samples were collected at 11:00 h. Nr4a3 (A) and Tshb (B) mRNA expression and plasma TSH concentration (C) were measured. Relative mRNA levels were compared to those in the vehicle group (set to 1.0). Data are presented as mean ± SEM (n = 5). Statistical analysis was performed using the Tukey-Kramer test; ** p < 0.01, *** p < 0.001.
Next, we observed the effect of T4 administration on pituitary Nr4a3 expression in proestrus rats. T4 administration significantly suppressed the marked increase in Nr4a3 mRNA expression at 14:00 h (Fig. 5A); however, this effect was no longer observed at 17:00 h. Blood TSH levels were also significantly suppressed by T4 administration compared to those in the vehicle group at 17:00 h (Fig. 5B). However, no significant changes were observed in the vehicle group at any of the time points.
Effect of T4 administration on the increase of Nr4a3 expression in proestrus rats
Proestrus rats were intraperitoneally injected with T4 (200 μg/kg) or solvent only (vehicle group, Veh) at 8:00 h, and anterior pituitary tissues and blood samples were collected at 11:00, 14:00, and 17:00 h. Nr4a3 mRNA expression (A) and plasma TSH concentrations (B) were measured. Relative mRNA levels were compared to those in the vehicle group at 11:00 h (set to 1.0). Data are presented as mean ± SEM (n = 5). Statistical analysis was performed using the Tukey-Kramer test; * p < 0.05, ** p < 0.01, *** p < 0.001.
Additionally, administration of the anti-TRH antibody (aTRH#1 or aTRH#2) to proestrus rats for direct blockade of TRH action significantly suppressed the increase in Nr4a3 expression at 14:00 h, but had no effect at 17:00 h (Fig. 6A). Furthermore, administration of the aTRH#2 antibody significantly reduced blood TSH levels at 17:00 h compared to those at 11:00 h, and administration of aTRH#1 also tended to reduce them (Fig. 6B).
Effect of antibody-mediated TRH neutralization on the increase of Nr4a3 expression in proestrus rats
Proestrus rats were intravenously injected with TRH antibody (aTRH#1 or aTRH#2, 1 mg/mL/head) or normal rabbit IgG (Control IgG, 1 mg/mL/head) at 8:00 h, and the anterior pituitary tissues and blood samples were collected at 11:00, 14:00, and 17:00 h. Nr4a3 mRNA expression (A) and plasma TSH concentrations (B) were measured. Relative mRNA levels were compared with those of the Control IgG group at 11:00 h (set to 1.0). Data are presented as mean ± SEM (n = 5). Statistical analysis was performed using the Tukey-Kramer test; * p < 0.05, ** p < 0.01, *** p < 0.001.
Little is known about the physiological significance of pituitary hormone-producing cells other than gonadotrophs during the preovulatory period in rats. Our study clearly demonstrated NR4A3 expression in the anterior pituitary thyrotrophs of rats and its induction by hypothalamic TRH in the afternoon of proestrus. These findings provide convincing evidence for TRH receptor activation in proestrus thyrotrophs.
Double-fluorescence immunostaining of the rat anterior pituitary revealed NR4A3 expression, especially in thyrotrophs, during the preovulatory period. Localization in a few gonadotrophs and lactotrophs was also observed but could not be defined in this study (data not shown). Our previous study revealed that GnRH was partially involved in pituitary Nr4a3 expression [32]. These observations suggested that multiple factors promote Nr4a3 expression in distinct cell types in the afternoon during the proestrus period. It is important to note that thyrotrophs account for less than 10% of all pituitary cells [34] and hence may underestimate the change in thyrotroph NR4A3 expression. It should be considered that the changes in anterior pituitary Nr4a3 gene expression validated by gene expression analysis may represent a stronger response in thyrotrophs.
TRH treatment quickly and transiently induces Nr4a3 expression in cultured primary pituitary cells, suggesting that NR4A3 plays a role in the early stages of transcriptional regulation under TRH receptor signaling. However, a major effect of TRH on increasing Tshb mRNA expression was not found in primary pituitary cells, thus rendering it difficult to determine the effect of TRH-induced Nr4a3 in our sequential in vitro experiments. Such reduced responsiveness to TRH stimulation often appears to occur in dispersed pituitary cells, suggesting an effect due to a lack of cell-cell interactions [35]. To date, this observation remains unresolved experimentally and it is understood that primary cultured cells are inadequate for the analysis of physiological TRH action. Previous studies on the intracellular mechanisms regulating TSH synthesis have been limited to partial analyses using cell lines with a minor thyrotroph function, such as the αTSH cell line [36], TSH-producing tumor tissue TtT-97 [37, 38], and TRH-responsive somatomammotroph cell line [39]. In the future, the molecular mechanism underlying NR4A3 expression and its action will need to be elucidated by integrating partial experiments with these cell lines.
Our in vivo data from thyroidectomy and T4 supplementation demonstrated that pituitary Nr4a3 expression was regulated by the HPT axis. Thyroidectomy decreases the concentrations of T4 and T3, which have negative feedback effects on the hypothalamus and pituitary and subsequently increases TRH and TSH secretion [40-42]. T4 produced in the thyroid gland is converted to T3 in the hypothalamus and pituitary gland, which suppresses TSH synthesis and release directly or indirectly via the suppression of TRH secretion [43]. In this study, T4 did not downregulate Tshb gene expression, possibly because of the short duration of T4 action. The treatment of T4 suppressed NR4A3 more rapidly and strikingly, at least compared to plasma TSH levels and Tshb mRNA expression, suggesting that NR4A3 is more sensitively modulated in response to changes in the regulatory system of the HPT axis.
T4 also specifically inhibited pituitary Nr4a3 expression at 14:00 h during proestrus. Furthermore, the administration of the two TRH antibodies had a similar inhibitory effect. These results clearly indicate that TRH is partially and transiently involved in the increased Nr4a3 expression during the proestrus period. These findings strongly suggest that TRH stimulation may affect thyrotroph function during the preovulatory period in rats.
Nevertheless, because plasma TSH concentrations between 14:00 and 17:00 h were not linked to elevated NR4A3 expression, it is difficult to assume that NR4A3 rapidly modulates thyrotroph function. The presence of NurRE, a common binding element of the NR4A subfamily, in the –1,091 bp to –1,083 bp region upstream of the mouse Tshb promoter has been reported but it is still unclear whether this element affects Tshb transcription [44]. Although in vitro experiments by Nakajima et al. suggested that NR4A1, a member of the NR4A subfamily, is recruited indirectly to the proximal region of Tshb promoter to facilitate Tshb transcription, it has also been shown that NR4A1 does not bind to NurRE at –1,091 bp to –1,083 bp [44]. Therefore, it is unlikely that members of the NR4A subfamily, including NR4A3, directly induce Tshb expression. However, our limited experiments during the proestrus period have not yet completely assessed the function of thyrotrophs. Whether NR4A3 expression in thyrotrophs indirectly or secondarily regulates Tshb expression or affects TRH receptor expression and other basic cellular functions requires more detailed and prolonged analysis.
Moreover, during the proestrus period in normal rats, blood TSH concentrations did not increase at 14:00 and 17:00 h, suggesting that TRH may fail to stimulate TSH release, at least during this period. Castro-Vazquez et al. reported that blood TSH levels increased by only approximately 20% between 10:00 and 13:00 h and then decreased by approximately 50% until 19:00 h in proestrus rats [13]. Moreover, shorter handling (40 s of cervical stimulation) at 10:00 h diminished these changes in TSH secretion. It is also possible that handling stress, such as reagent injection and vaginal smear sampling, affects the physiological TSH secretion patterns. However, even with this possibility, the responsiveness of TSH secretion is weaker than that of gonadotropin and prolactin surges during the proestrus period. Nevertheless, our findings regarding NR4A3 expression suggest that even apparently unvarying thyrotrophs undergo changes during the estrous cycle, which may ensure constitutive TSH secretion.
Healthy regulation of the HPT axis is essential for maintaining reproductive functions. Female animals with hypothyroidism have reproductive abnormalities such as delayed puberty, irregular ovulatory cycles, ovulation failure, and pregnancy complications [45-49]. Additionally, the risk of subclinical hypothyroidism (SCH) in humans is more likely apparent during pregnancy [50, 51]. In rats, SCH also leads to a reduced number of implantations and an increased number of aborted fetuses, comparable to the overt hypothyroidism model [52]. Although the mechanisms of these reproductive dysfunctions are not completely understood, it has been suggested that precise regulation of the HPT axis strongly influences reproductive success, at least during the periovulatory and early pregnancy periods. Our new findings on transient TRH signaling and NR4A3 expression in thyrotrophs in the afternoon of the proestrus period provide direct evidence for an intimate link between HPT and the hypothalamus-pituitary-ovarian axis and may have implications for the cyclic regulation of thyrotrophs and subsequent maintenance of thyroid function.
In conclusion, our study demonstrates that TRH action transiently induces NR4A3 expression in rat thyrotroph cells in the afternoon of proestrus, suggesting the possible involvement of NR4A3 in the regulation of the HPT axis during ovulation or early pregnancy. Our findings may be useful for further investigations of the mechanisms underlying thyroid hormone-related reproductive disorders.
We thank Hisae Kobayashi and Ken Sato (Gunma University) for generously providing anti-TSH antibodies under the joint/usage research program of the Institute for Molecular and Cellular Regulation, Gunma University (antibody ID: HAC-RT29-01RBP86). This work was supported by the Kitasato University Research Grant for Young Researchers.
None of the authors have any potential conflicts of interest associated with this research.
