Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12338 © 2015 British Society for Neuroendocrinology

ORIGINAL ARTICLE

Hypothalamic Effects of Tamoxifen on Oestrogen Regulation of Luteinising Hormone and Prolactin Secretion in Female Rats N. S. S. Aquino*, R. Araujo-Lopes*, I. A. R. Batista*, P. C. Henriques*, M. O. Poletini*, C. R. Franci†, A. M. Reis* and R. E. Szawka* *Departamento de Fisiologia e Biof
ısica, Instituto de Ci^encias Biol
ogicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. †Departamento de Fisiologia, Faculdade de Medicina de Ribeir~ao Preto, Universidade de S~ ao Paulo, Ribeir~ ao Preto, S~ ao Paulo, Brazil.

Journal of Neuroendocrinology

Correspondence to: Raphael E. Szawka, Departamento de Fisiologia e Biof
ısica, Instituto de Ci^encias Biol
ogicas, Universidade Federal de Minas Gerais, Avenida Ant^onio Carlos 6627, 31270-901, Belo Horizonte, MG, Brazil (e-mail: [email protected]).

Oestradiol (E2) acts in the hypothalamus to regulate luteinising hormone (LH) and prolactin (PRL) secretion. Tamoxifen (TX) has been extensively used as a selective oestrogen receptor modulator, although its neuroendocrine effects remain poorly understood. In the present study, we investigated the hypothalamic effects of TX in rats under low or high circulating E2 levels. Ovariectomised (OVX) rats treated with oil, E2 or TX, or E2 plus TX, were evaluated for hormonal secretion and immunohistochemical analyses in hypothalamic areas. Both E2 and TX reduced LH levels, whereas TX blocked the E2-induced surges of LH and PRL. TX prevented the E2-induced expression of progesterone receptor (PR) in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), although it did not alter PR expression in OVX rats. TX blocked the E2 induction of c-Fos in AVPV neurones, consistent with the suppression of LH surge. However, TX failed to prevent E2 inhibition of kisspeptin expression in the ARC. In association with the blockade of PRL surge, TX increased the phosphorylation of tyrosine hydroxylase (TH) in the median eminence of OVX, E2-treated rats. TX also precluded the E2-induced increase in TH expression in the ARC. In all immunohistochemical analyses, TX treatment in OVX rats caused no measurable effect on the hypothalamus. Thus, TX is able to prevent the positive- but not negative-feedback effect of E2 on the hypothalamus. TX also blocks the effects of E2 on tuberoinfundibular dopaminergic neurones and PRL secretion. These findings further characterise the anti-oestrogenic actions of TX in the hypothalamus and provide new information on the oestrogenic regulation of LH and PRL. Key words: tamoxifen, oestradiol, prolactin, luteinising hormone, hypothalamus, female

Luteinising hormone (LH) secretion is essential for fertility and reproduction in mammals. LH secretion is controlled by gonadotrophin-releasing hormone (GnRH) neurones projecting to the median eminence (ME) (1). In females, 17ß-oestradiol (E2) produced in the ovary acts centrally and at the pituitary to either suppress or stimulate LH secretion through the negative- and positive-feedback effects, respectively. In rodents, E2-induced surges of GnRH and LH occur on the afternoon of pro-oestrus and trigger ovulation on oestrus (2,3). Immunoneutralisation of E2 prevents the surge of LH (4), whereas E2 treatment in ovariectomised (OVX) rats reduces basal levels of LH and elicits afternoon surges of this hormone (5,6). GnRH neurones express oestrogen receptor (ER)-ß but not ERa or progestin receptor (PR) (7,8). However, both the negative- and positive-feedback effects of E2 have been shown to require ERa. The E2 positive-feedback

doi: 10.1111/jne.12338

appears to depend on classical ERa signalling in neurones, whereas nonclassical oestrogen response element (ERE)-independent ERa signalling appears to be sufficient for E2 negative-feedback (9,10). Kisspeptin–Kiss1r signalling is critical for the onset of puberty and fertility (11). Kisspeptin neurones express ERa and PR and are distributed in two distinct populations in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus of hypothalamus (ARC) (12,13). E2 increases kisspeptin mRNA and protein levels in the AVPV but suppresses their expression in the ARC (12–14). These effects are in line with the view that kisspeptin neurones in the AVPV are activated during the positive-feedback effect of E2, whereas the ARC population plays a role in negative-feedback. Accordingly, neurones in the AVPV express c-Fos synchronously with GnRH neurones by the time of the LH surge (15) and anti-oestrogen implants in the rostral

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preoptic area prevent the LH surge (16). The selective lesion of ARC kisspeptin neurones, in turn, prevents the rise in LH levels after ovariectomy but does not block the suppressive effect of E2 on LH release (17). Similar to LH secretion, E2 regulation of kisspeptin expression also depends on ERa (12,18), although the underlying mechanisms determining the opposite outcomes of inhibition or stimulation elicited by activation of ERa remain poorly understood. Prolactin (PRL) secreted from the pituitary is known for its main role in lactation (19). Nevertheless, PRL plays multiple roles in the organism, including the powerful modulation of fertility. Female mice with deletions of PRL receptors are infertile because of failure in luteal function and embryo implantation (20). On the other hand, hyperprolactinaemia is a prevalent cause of infertility in both males and females (21–23). PRL secretion is tonically inhibited by dopamine released in the ME, mainly produced by the tuberoinfundibular dopaminergic (TIDA) neurones located in the ARC (24). E2 regulates TIDA neurones and is a potent stimulator of PRL secretion (25). Accordingly, an E2-induced surge of PRL secretion occurs alongside the preovulatory LH surge on the afternoon of pro-oestrus (2), and OVX, E2-treated (OVX + E2) rats display daily afternoon surges of PRL, triggered by a reduction in the activity of TIDA neurones (26,27). TIDA neurones express ERa in male and female rats (28,29). However, little is know about the effects of ERa activation on the function of TIDA neurones. Tamoxifen (TX) is a selective oestrogen receptor modulator (SERM) that exerts tissue-selective mixed oestrogenic or anti-oestrogenic effects through binding to ERa and ERß (30). TX blocks the action of E2 in breast cells and is therefore widely used for the treatment and prevention of ER-positive breast cancer (31). By contrast, TX may exert oestrogenic actions in the bone and uterus (30). However, the nature of the effects of TX on the hypothalamus is still unclear. TX treatment has been shown to impair ovulation and reduce LH and PRL levels in pro-oestrous rats (32–34), although whether these effects are the result of central actions of TX is unresolved. Because of the unique pharmacological properties of a SERM, TX is a drug with multiple potentialities for both clinical practice and basic research, including its use in conditional transgenic animal models. Moreover, TX is known to cross the blood–brain barrier, which may be a useful property compared to other pure anti-oestrogens that do not (35). Thus, we investigated whether TX acts as an ER antagonist in the hypothalamus and whether its effects would depend on circulating E2 levels. In the present study, we used hormonal and immunohistochemical analyses to determine the oestrogenic and anti-oestrogenic effects of TX on hypothalamic areas related to gonadotrophin secretion. The results obtained demonstrate that TX blocks the hypothalamic effects of E2 causing stimulation of LH and PRL release but not the negative-feedback effects of E2 on kisspeptin expression and LH levels. Moreover, as determined by immunohistochemistry, TX by itself had no oestrogenic action in the hypothalamus of OVX rats, although it was able to reduce LH and slightly increase PRL secretion. These findings expand our knowledge about the effects of TX on the hypothalamus and the oestrogenic regulation of LH and PRL. © 2015 British Society for Neuroendocrinology

Materials and methods Animals Adult female Wistar rats weighing 220–280 g were grouped housed under a 12 : 12 h light/dark cycle (lights on 05.00 h) at 22
1 °C. Experimental protocols were approved by the Ethics Committee on the Use of Experimental Animals of the University of Minas Gerais. In all experiments, food and water were provided ad lib.

Experimental design To investigate the effects of TX on the hypothalamus, we used a 3-day treatment protocol because this is a time scale that allows evaluation of both negative- and positive-feedback effects and has the advantage of avoiding the misleading effects associated with long-term treatments. Rats showing at least three consecutive regular oestrous cycles were OVX and placed in individual cages 7 days before the experiment. OVX rats were treated with corn oil (OVX; 0.2 ml per rat; n = 6), E2 (OVX + E2; 10 lg/0.2 ml per rat, s.c.; n = 6), TX (OVX + TX; 3 mg/0.2 ml per rat; n = 5) or E2 plus TX (OVX + E2TX; n = 5) daily for three consecutive days, and the experiments were conducted on the fourth day. The jugular vein received a catheter 1 day before the experiment for serial blood sampling. Between 13.00 and 18.00 h, blood samples were withdrawn hourly through the jugular vein catheter for evaluation of plasma LH and PRL levels. Immediately after the last blood sample at 18.00 h, rats were anaesthetised and transcardially perfused. The uteri were weighed and the brains were processed for immunohistochemical labelling of PR, c-Fos, kisspeptin, tyrosine hydroxylase (TH) or Ser40-phosphorylated TH (S40pTH) in the AVPV, ARC and ME.

Surgeries and blood samples Ovariectomy and transcardial perfusion were performed under ketamine (80 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anaesthesia. Ovariectomy was performed through small bilateral incisions in the skin and muscle layers, and rats were treated with pentabiotic (Fort Dodge, Campinas, Brazil; 24 000 UI/kg, i.m.) and analgesic (flunixin meglumine; 2.5 mg/kg, s.c.) after surgery. For blood sampling, rats were anaesthetised with tribromoethanol (250 mg/kg body weight, i.p.) and a Silastic catheter (Dow Corning Corp., Midland, MI, USA) was inserted through the external jugular vein into the right atrium, as described previously (36). After this surgery, rats also received analgesic treatment with flunixin meglumine (2.5 mg/kg, s.c.). On the experimental day, a length of polyethylene tubing (PE-50) filled with heparinised saline (0.9% NaCl, 30 IU heparin/ml) was connected to the jugular catheter. Blood samples of 400 ll were withdrawn into plastic heparinised syringes. An equal volume of sterile 0.9% NaCl was replaced after removal of each blood sample. Plasma was separated by centrifugation at 1200 g for 20 min at 4 °C and stored at 20 °C until hormonal assay. We have reported previously that removal of similar blood volume did not alter the secretion of LH or PRL (36,37), nor the release of noradrenaline in the hypothalamus (6).

Hormonal and drug treatment E2 cypionate (10 lg/0.2 ml per rat, s.c.; Pfizer, Sao Paulo, Brazil) and TX (3 mg/0.2 ml per rat, s.c.; Sigma-Aldrich, St Louis, MO, USA) were diluted in corn oil. The dose of E2 used yields physiological concentrations of plasma E2 and generates preovulatory-like surges of LH (38). The dose of 3 mg of TX per rat in vivo has been reported to exert both oestrogenic and anti-oestrogenic effects on LH and PRL release from the rat pituitary (39,40).

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Immunohistochemistry

Radioimmunoassay

Rats were anaesthetised and transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde. Frontal sections of 30 lm were cut in four series throughout the rostrocaudal extension of the AVPV and ARC in accordance with a rat brain atlas (41) and stored at 20 °C in cryoprotectant solution. Immunoperoxidase immunohistochemistry was performed using a free-floating method as described previously (14). Sections of the AVPV were single-labelled for PR or c-Fos and sections of the ARC, for PR, kisspeptin, S40pTH or TH. All steps were performed at 22 °C, except for the incubation with the primary antibodies, performed at 4 °C for 40 h. Briefly, sections were incubated with one of the primary antibodies: rabbit anti-human PR (A0098; Dako, Glostrup, Denmark; dilution 1 : 400), rabbit anti-human c-Fos (Ab-5; PC38, Calbiochem, Darmstadt, Germany; dilution 1 : 20 000), rabbit anti-mouse kisspeptin-10 (A.C. 564; dilution 1 : 30 000), rabbit anti-rat S40pTH (36-8600 Zymed Laboratories; Invitrogen, Camarillo, CA, USA; dilution 1 : 25 000) or mouse anti-rat TH (T1299; Sigma-Aldrich; dilution 1 : 70 000). Sections were then incubated with biotinylated horse anti-rabbit IgG, or biotinylated horse anti-mouse IgG for TH labelling, at a dilution of 1 : 2000 for 2 h (Vector Laboratories, Burlingame, CA, USA), and avidin-biotin complex solution at a dilution of 1 : 400 for 1 h (Elite ABC kit, Vector Laboratories). A solution of nickel sulphate (Ni; 25 mg/ml), 3,30 -diaminobenzidineHCl (DAB; 0.2 mg/ml) and 0.03% H2O2 was used as chromogen. As controls, omission of the primary antibodies resulted in no labelling. The anti-PR antibody recognises both A and B isoforms of human PR and its specificity has been demonstrated previously by the absence of labelling after preadsorption with the immunising peptide (42,43). The immunoreactivity of the anti-kisspeptin antibody is also abolished by the preadsorption with kisspeptin-10 (14). The anti-c-Fos antibody was raised against the N-terminal sequence encompassing residues 4–17 of human c-Fos. The specificity of this antibody has been confirmed in previous studies reporting a lack of immunoreactivity after preadsorption with the immunising peptide (44,45). The mouse anti-TH has been extensively used and the labelling of this antibody reported in the present study and in other studies from our group (29) corresponds to previous reports of TH staining in the rat brain (24). Although preadsorption tests have not been conducted for the anti-S40pTH antibody, this antibody has been shown to stain only a single band in western blots of murine brains in previous studies (46,47). Moreover, consistent with these previous reports (46,47), our present results show that the functional responses of S40pTH labelling differ from those of TH labelling, further attesting for the specificity of the anti-S40pTH antibody. Brain sections were analysed under a light microscope with an image analysis system by an experimenter who was unaware of experimental groups. In the AVPV, the number of PR-immunoreactive (IR) and c-Fos-IR neurones was quantified bilaterally in three sections per rat between +0.12 and 0.24 from bregma. Boxes delimited the area in which neurones were counted (width 100 lm from third ventricle; length 160, 240 or 360 lm) (6). In the ARC, the number of PR, kisspeptin, TH and S40pTH single-labelled neurones was quantified bilaterally in six sections per rat between approximately 2.4 and 3.96 mm from bregma. For the measurement of the optical density of S40pTH and TH labelling, the brain sections of all rats were analysed in the same immunohistochemistry and revealed during the same time in Ni-DAB. The optical density was measured in the external layer of the ME in three sections per rat from the same rostrocaudal levels using IMAGE J (National Institutes of Health, Bethesda, MD, USA). Images were captured using a 9 40 objective and converted to a 1-bit image using the same threshold for all sections. Boxes (width 160 lm; length 80 lm) were used to delimit the region of interest in which analyses were performed. The pixels corresponding to S40pTH and TH immunoreactivity were expressed as a percentage of the total area analysed.

PRL and LH were assayed by double-antibody radioimmunoassay with kits provided by the National Hormone and Peptide Program (Harbor-UCLA, St Torrance, CA, USA). The antiserum and reference preparation for PRL were anti-rat PRL-S9 and PRL-RP3, respectively. The antiserum and reference preparation for LH were anti-rat LH-S10 and LH-RP3, respectively. Iodination was carried out by incubation with chloramine-T (1 mg/ml in 0.05 M phosphate buffer) for 1 min. The 125I-hormones were eluted in a Sephadex-G75 column with 0.01 M phosphate buffer (pH 7.4). All samples of the experiment were assayed in the same radioimmunoassay. The lower limits of detection were 0.7 and 0.16 ng/ml for PRL and LH, respectively. The intraassay coefficients of variation were 2.5% and 1.8% for PRL and LH, respectively.

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Statistical analysis Data are presented as the mean
SEM. For hormonal data, comparisons among time points within the same experimental group were made by oneway ANOVA for repeated measures and comparisons between groups were performed using two-way ANOVA. Integrated hormonal responses were expressed as the area under curve (AUC). Immunohistochemical data and uterine weight were analysed by two-way ANOVA. In all analyses, ANOVA was followed by the Bonferroni post-hoc test. P < 0.05 was considered statistically significant.

Results Treatment with TX in cycling rats has been reported to preclude ovulation and reduce circulating levels of LH and PRL on the afternoon of proestrus (32–34). Similarly, TX has also been shown to decrease secretion of LH and PRL in the experimental model of OVX + E2 rats (33,48). In the present study, we determined the hypothalamic effects of TX on the regulation of LH and PRL secretion in rats under conditions of either low (OVX) or high (OVX + E2) circulating E2 levels. Accordingly, the uterine weight was significantly higher in OVX + E2 rats than in OVX animals (P < 0.01). TX treatment, in turn, reduced the weight of uterus in OVX + E2TX rats compared to OVX + E2 (P < 0.01), whereas it had no effect in OVX + TX animals (OVX + E2: 390.0
64.1; OVX: 161.7
9.1; OVX + E2TX: 154.0
13.6; OVX + TX: 150.0
10.0; mg/100 g b.w.). Figure 1 shows the effects of TX treatment on LH secretion in OVX and OVX + E2 rats. As a result of the time-dependent and opposite effects of E2 on LH secretion, the AUC of LH levels was analysed separately in two different periods: from 13.00 to 15.00 h, a period reflecting the negative-feedback effect, and from 15.00 to 18.00 h, the time of the positive-feedback effect. Two-way-ANOVA identified a main effect for TX on plasma LH levels in both OVX (P < 0.001) and OVX + E2 (P < 0.01) rats. LH levels were lower in OVX + TX than in OVX rats, reflecting an oestrogenic inhibitory effect of TX on LH release in the absence of E2 (Fig. 1A). Although the Bonferroni post-hoc test did not detect differences between OVX and OVX + TX for individual time points, the AUC of LH was significantly lower in the OVX + TX group at the two periods evaluated (P < 0.05) (Fig. 1C,D). OVX + E2 rats displayed a LH surge between 16.00 and 18.00 h (P < 0.01), which was blocked by TX treatment in OVX + E2TX rats (P < 0.05) (Fig. 1B). The AUC of LH © 2015 British Society for Neuroendocrinology

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Fig. 1. Effect of tamoxifen (TX) on luteinising hormone (LH) secretion in female rats under low or high oestradiol (E2) levels. Ovariectomised (OVX) rats were treated with oil (OVX; n = 6), E2 (OVX + E2; n = 6), TX (OVX + TX; n = 5) or E2 plus TX (OVX + E2TX; n = 5) daily for 3 days, and the experiments were conducted on the fourth day. Blood samples were withdrawn hourly between 13.00 and 18.00 h. (A, B) Plasma LH levels in OVX and OVX + TX rats (A) and in OVX + E2 and OVX + E2TX rats (B). *P < 0.05 and **P < 0.001 compared to 13.00 h; #P < 0.05 compared to OVX + E2TX. (C, D) Area under the curve (AUC) of plasma LH levels from 13.00 h to 15.00 h (C) and from 15.00 h to 18.00 h (D), periods identified by dotted lines in the graphs (A, B). *P < 0.001 OVX versus OVX + TX; **P < 0.01 OVX + E2 versus OVX + E2TX; #P < 0.05 compared to OVX. Data are shown as the mean
SEM.

shows that TX did not interfere with the negative-feedback effect of E2 between 13.00 and 15.00 h (Fig. 1C) but prevented the positive-feedback between 16.00 and 18.00 h (Fig. 1D). E2 is a powerful regulator of PRL secretion and induces daily afternoon surges of PRL in OVX + E2 rats (26). Figure 2 shows the effect of TX on PRL release in OVX and OVX + E2 rats. As determined by two-way-ANOVA, TX had a main effect on plasma PRL levels in both OVX (P < 0.001) and OVX + E2 (P < 0.001) rats. PRL levels were slightly higher in OVX + TX compared to OVX rats, which was statistically significant only at 18.00 h (P < 0.05) (Fig. 2A). This response indicated a minor oestrogenic effect of TX on PRL release in the absence of E2. On the other hand, OVX + E2 rats displayed the E2-induced PRL surge between 15.00 and 18.00 h (P < 0.001), which, similar to the LH surge, was completely blocked in OVX + E2TX rats (P < 0.001) (Fig. 2B,C). The AVPV and ARC are recognised as main brain areas implicated in the positive- and negative-feedback effects of E2 on the control of LH secretion, respectively (11). The ARC also contains the perikarya of TIDA neurones, the major population of dopaminergic neurones regulating PRL release (25). Because E2 induces PR expression © 2015 British Society for Neuroendocrinology

in the hypothalamus (49), we evaluated the effect of TX on PR levels in the AVPV and ARC. Accordingly, the number of PR-IR neurones in the AVPV was markedly increased in OVX + E2 rats compared to the OVX group (P < 0.001) and TX prevented this increase in OVX + E2TX rats (P < 0.05), whereas it had no effect in OVX + TX rats (Fig. 3). The same response was found in the ARC, where TX did not affect PR levels in OVX + TX rats but potently suppressed E2-induced PR expression in the OVX + E2TX group (P < 0.01) (Fig. 4). These results demonstrate that, although TX by itself does not exert oestrogenic actions in the AVPV or ARC, it is able to block the effects of E2 on the hypothalamus. Because the activation of AVPV neurones is a critical step in the E2 positivefeedback for generating the LH surge (9,15), we evaluated the effect of TX on c-Fos expression in the AVPV. Associated with the occurrence of the LH surge, OVX + E2 rats displayed an increased number of c-Fos-IR neurones in the AVPV (P < 0.01) and this neuronal activation was blocked in OVX + E2TX rats (P < 0.05). Conversely, c-Fos expression was equally low in OVX and OVX + TX rats (Fig. 5). Kisspeptin neurones located in the AVPV and ARC are powerful stimulators of GnRH/LH secretion (11). Although we have not deterJournal of Neuroendocrinology, 2016, 28, 10.1111/jne.12338

Suppression of the effects of oestrogen by tamoxifen

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Fig. 2. Effect of tamoxifen (TX) on prolactin (PRL) secretion in female rats under low or high oestradiol (E2) levels. Ovariectomised (OVX) rats were treated with oil (OVX; n = 6), E2 (OVX + E2; n = 6), TX (OVX + TX; n = 5) or E2 plus TX (OVX + E2TX; n = 5) daily for 3 days, and the experiments were conducted on the fourth day. Blood samples were withdrawn hourly between 13.00 and 18.00 h. (A, B) Plasma PRL levels in OVX and OVX + TX rats (A) and in OVX + E2 and OVX + E2TX rats (B). *P < 0.05 and **P < 0.01, ***P < 0.001 compared to 13.00 h; #P < 0.05 compared to OVX; ##P < 0.01 and ###P < 0.001 compared to OVX + E2TX. (C) Area under the curve (AUC) of plasma PRL levels from 13.00–18.00 h. ***P < 0.001 OVX + E2 versus OVX + E2TX; ###P < 0.001 compared to OVX. Data are shown as the mean
SEM.

mined the phenotype of c-Fos-expressing neurones in the AVPV, it is reasonable to assume that they comprise kisspeptin neurones, which are activated during the positive-feedback (13). On the other hand, E2 is known to suppress ARC kisspeptin, which appears to be implicated in the negative-feedback effect on LH secretion (12,14). We therefore evaluated the effect of TX on kisspeptin immunoreactivity in the ARC (Fig. 6). The number of kisspeptin-IR neurones in the ARC was 54% lower in OVX + E2 than in OVX rats (P < 0.001). Kisspeptin levels in OVX + E2TX rats were slightly higher than in OVX + E2 rats (P < 0.05) but still 32% lower than in OVX and OVX + TX rats (P < 0.05) (Fig. 6E). Thus, TX only partially restored the E2-induced suppression of ARC kisspeptin, which was not sufficient to prevent the negative-feedback effect on LH secretion in OVX + E2TX rats (Fig. 1C). Additionally, the lack of difference in kisspeptin expression between OVX + TX and OVX rats is in contrast to the lower levels of LH in the OVX + TX group (Fig. 1A,C), suggesting that this inhibitory effect of TX on LH release is not a result of its action in kisspeptin neurones. We also evaluated whether TX action in TIDA neurones would explain its effects on PRL secretion. TH, the rate-limiting enzyme in Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12338

catecholamine biosynthesis, is stimulated by phosphorylation of serine residues, including S40 (46,50). Figure 7 shows the effect of TX on S40pTH immunoreactivity in the ARC and ME, corresponding to the cell bodies and terminals of TIDA neurones, respectively. The expression of S40pTH in the ME, as determined by the optical density of S40pTH staining, was significantly higher in OVX + E2TX compared to OVX + E2 rats (P < 0.05), suggesting a greater TH activity in the TIDA nerve terminals of OVX + E2TX rats (Fig. 7E). Because decreases in TH phosphorylation and dopamine release in the ME are associated with the E2-induced PRL surge (27,46), this response is consistent with the blockade of PRL surge caused by TX in OVX + E2TX rats (Fig. 2). Additionally, TX alone had no effect on S40pTH expression in the ME, nor was there any effect of E2 or TX on S40pTH levels in the ARC (Fig 7E,F). Figure 8 shows the effect of TX on TH expression in the ARC and ME. Neither E2, nor TX affected TH immunoreactivity in the ME (Fig. 8E). In the ARC, in turn, OVX + E2 rats displayed an increased number of TH-IR neurones compared to OVX rats (P < 0.01) and this effect of E2 was blocked in OVX + E2TX rats (P < 0.05) (Fig. 8F). Thus, TX is able to antagonise the effects of E2 on phosphorylation and long-term expression © 2015 British Society for Neuroendocrinology

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Fig. 3. Effect of tamoxifen (TX) on the expression of progesterone receptor (PR) in the anteroventral periventricular nucleus (AVPV). Ovariectomised (OVX) rats were treated with oil (OVX; n = 6), oestradiol (OVX + E2; n = 6), TX (OVX + TX; n = 5) or oestradiol plus TX (OVX + E2TX; n = 5) daily for 3 days. Blood samples were withdrawn hourly between 13.00 and 18.00 h and rats were perfused at 18.00 h. (A) Schematic diagram representing the area analysed for PR expression in the AVPV (black rectangles). (B–E) Photomicrographs illustrating PR immunoreactivity in the AVPV of OVX (B), OVX + TX (C), OVX + E2 (D) and OVX + E2TX (E) rats. (F) Mean
SEM number of PR-immunoreactive (-IR) neurones/section. *P < 0.05 OVX + E2 versus OVX + E2TX; ###P < 0.001 compared to OVX. och, optic chiasm; 3V, third ventricle. Scale bar = 100 lm.

of TH in TIDA neurones, which are associated with the blockade of E2-induced increase in PRL secretion.

Discussion In the present study, we determined the effects of TX on the hypothalamus of rats under low or high circulating E2 levels, focusing on the oestrogenic regulation of LH and PRL secretion. The results demonstrate that TX exerts anti-oestrogenic actions in neurones of the AVPV and ARC. Although oestrogenic effects might also be expected from a SERM, no agonist effects of TX were detected on the hypothalamus. TX prevented the E2-induced increase in PR and c-Fos expression in the AVPV, which was associated with the blockade of the preovulatory-like LH surge. TX neutralised the inhibitory effect of E2 on the enzymatic activity of TIDA nerve terminals, as determined by S40pTH immunoreactivity in the ME, which was associated with suppression of the E2-induced PRL surge. On the other hand, TX failed to prevent the E2-induced inhi© 2015 British Society for Neuroendocrinology

bition of kisspeptin expression in the ARC. These findings demonstrate that TX effectively precludes the positive- but not negativefeedback effect of E2 on the hypothalamus. Additionally, TX treatment in OVX rats resulted in lower LH and slightly increased PRL levels. These oestrogenic effects, however, were not linked to measurable changes in the hypothalamus, suggesting they are a result of the nonhypothalamic actions of TX, probably at the pituitary level. Although we cannot exclude the possibility of oestrogenic effects of TX on the hypothalamus, which may have not been detected in our immunohistochemical analyses, our findings suggest that TX exerts mainly (if not only) anti-oestrogenic actions in the hypothalamus. Thus, the results reported here further characterise the anti-oestrogenic actions of TX in the hypothalamus and provide new information on the oestrogenic regulation of LH and PRL. The uterine weight in OVX + E2 + TX rats was lower than in OVX + E2 and similar to OVX rats. This confirmed the efficacy of TX treatment and is consistent with previous reports in cycling and Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12338

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Fig. 4. Effect of tamoxifen (TX) on the expression of progesterone receptor (PR) in the arcuate nucleus (ARC). Ovariectomised (OVX) rats were treated with oil (OVX; n = 6), oestradiol (OVX + E2; n = 6), TX (OVX + TX; n = 5) or oestradiol plus TX (OVX + E2TX; n = 5) daily for 3 days. Blood samples were withdrawn hourly between 13.00 and 18.00 h and rats were perfused at 18.00 h. (A) Schematic diagram representing the area analysed for PR expression in the ARC (black squares). (B–E) Representative photomicrographs of PR expression in the ARC of OVX (B), OVX + TX (C), OVX + E2 (D) and OVX + E2TX (E) rats. (F) Mean
SEM number of PR-immunoreactive (-IR) neurones/section. **P < 0.01 OVX + E2 versus OVX + E2TX; ###P < 0.001 compared to OVX. 3V, third ventricle; ME, median eminence. Scale bar = 100 lm.

OVX rats (39,48). With the regimen of TX treatment used (3 mg/rat daily for 3 days), we have not found an oestrogenic action of TX in the uterus of OVX rats, which would probably require either higher doses or longer periods of TX treatment (48,51). TX was also efficient in suppressing the E2-induced expression of PR in the AVPV and ARC. These results demonstrate that systemically administered TX is able to act in hypothalamic areas related to the control of LH and PRL secretion and the results are also consistent with previous reports of TX attenuation of E2-induced expression of PR in the medial preoptic area and ventromedial hypothalamic nucleus (35,52). The suppression of the E2-induced LH surge by TX is consistent with previous reports of TX blockade of the LH surge in pro-oestrous and OVX + E2 rats (32–34,48). Furthermore, our results demonstrate that TX suppression of PR expression and neuronal activation in the AVPV are the possible underlying causes of the blockade of the LH surge. AVPV neurones express ER and are essential for the generation of LH surges in females (9,15). E2 induces PR expression in ER-containing neurones of the AVPV (53) and PR is required for the occurrence of the LH surge in rodents (54). On the other hand, TX was not able to counteract E2 inhibition of either basal LH levels, as seen in the period before the LH surge (13.00– Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12338

15.00 h), or kisspeptin expression in the ARC, which is considered to be a hypothalamic landmark of the E2 negative-feedback (11). These results demonstrate that TX does not interfere with the negative-feedback of E2. Although, ERa is required for both positiveand negative-feedback effects, the positive-feedback depends on classical ERa signalling, whereas an ERE-independent ERa signalling appears to be sufficient for the negative-feedback (10). Thus, TX appears to effectively block the classical ERa signalling required for activation of AVPV neurones during the positive-feedback but not the ERE-independent signalling responsible for the negative-feedback effect on ARC kisspeptin (18). ERa is a ligand-inducible transcription factor that contains two transcriptional activation functions: the AF1 located in the N-terminal region and AF2 located in the C-terminal portion of the receptor. The mixed anti-oestrogenic and oestrogenic effects of TX appear to depend on tissue selective modulation of the activity of AF-1 and AF-2 (55,56). Interestingly, TX not only failed to prevent the E2 inhibition of ARC kisspeptin, but also had no effect on kisspeptin expression when administered to OVX rats in the absence of E2. Thus, these results provide additional pharmacological evidence that E2 regulates kisspeptin expression in the ARC in an EREindependent way, which does not require the transcriptional activa© 2015 British Society for Neuroendocrinology

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##

80

60

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Fig. 5. Effect of tamoxifen (TX) on the activity of anteroventral periventricular (AVPV) neurones. Ovariectomised (OVX) rats were treated with oil (OVX; n = 6), oestradiol (OVX + E2; n = 6), TX (OVX + TX; n = 5) or oestradiol plus TX (OVX + E2TX; n = 5) daily for 3 days. Blood samples were withdrawn hourly between 13.00 and 18.00 h and rats were perfused at 18.00 h. (A–D) Photomicrographs illustrating c-Fos immunoreactivity in the AVPV of OVX (A), OVX + TX (B), OVX + E2 (C) and OVX + E2TX (D) rats. (E) Mean
SEM number of c-Fos-immunoreactive (-IR) neurones/section. *P < 0.05 OVX + E2 versus OVX + E2TX; ## P < 0.01 compared to OVX. 3V, third ventricle. Scale bar = 100 lm.

tion functions of ERa, and therefore cannot be modulated by TX. Nevertheless, although not linked to an effect on hypothalamic kisspeptin, treatment with TX in OVX rats did reduce plasma LH levels. This effect of TX is in agreement with previous studies in rats and mice and may result from an agonist action of TX at the pituitary © 2015 British Society for Neuroendocrinology

level (39,40), which requires the integrity of AF-2 in ERa (56). Similarly, the slight increase in PRL levels found in OVX + TX rats is also probably a result of a weak oestrogenic action of TX at the pituitary. Accordingly, similar to E2, TX acts in anterior pituitary cells to increase GnRH self-priming, expression of progesterone receptor, Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12338

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25 *

20

# 15 ### 10 5 0

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Fig. 6. Effect of tamoxifen (TX) on the expression of kisspeptin (Kp) in the arcuate nucleus (ARC). Ovariectomised (OVX) rats were treated with oil (OVX; n = 6), oestradiol (OVX + E2; n = 6), TX (OVX + TX; n = 5) or oestradiol plus TX (OVX + E2TX; n = 5) daily for 3 days. Blood samples were withdrawn hourly between 13.00 and 18.00 h and rats were perfused at 18.00 h. (A–D) Kp expression in the ARC of OVX (A), OVX + TX (B), OVX + E2 (C) and OVX + E2TX (D) rats. (E) Mean
SEM number of Kp-immunoreactive (-IR) neurones/section. *P < 0.05 OVX + E2 versus OVX + E2TX; ###P < 0.001 compared to OVX; #P < 0.05 compared to OVX + TX. 3V, third ventricle. Scale bar = 100 lm.

as well as PRL secretion (39,40). Interestingly, our findings are consistent with the recent reports showing that E2 was still able to reduce LH in OVX rats bearing selective lesion of ARC kisspeptin neurones (17) and that the suppression of Kiss1 mRNA in the ARC was not required for the E2 suppression of LH secretion in kisspeptin specific ERa-knockout mice (57). Nevertheless, the negativefeedback effect of E2 appears to require the involvement of ARC neurones because it has been recently demonstrated that chronic E2 fails to suppress LH secretion in mice bearing depletion of ERa in the ARC (58). An interesting question that emerges in this new Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12338

scenario is which population of ARC neurones, apart from the kisspeptin one, is implicated in the negative-feedback. Thus, it is important to investigate the phenotypes of ARC neurones responsible for the negative-feedback and whether TX can modulate their activity. An E2-induced surge of PRL secretion occurs on the afternoon of pro-oestrus in rodents, and this surge can be replicated in the model of OVX + E2 rat (26). TX efficiently blocked the E2-induced surge of PRL, which is consistent with previous studies showing that TX reduced PRL levels in both pro-oestrous and OVX + E2 rats © 2015 British Society for Neuroendocrinology

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S40pTH-IR area (%)

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50

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TX

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Fig. 7. Effect of tamoxifen (TX) on phosphorylation of tyrosine hydroxylase (TH) in the arcuate nucleus (ARC) and median eminence (ME). Ovariectomised (OVX) rats were treated with oil (OVX; n = 6), oestradiol (OVX + E2; n = 6), TX (OVX + TX; n = 5) or oestradiol plus TX (OVX + E2TX; n = 5) daily for 3 days. Blood samples were withdrawn hourly between 13.00 and 18.00 h and rats were perfused at 18.00 h. (A–D) Immunoreactivity to TH phosphorylated at Ser40 (S40pTH) in the ARC and ME of OVX (A), OVX + TX (B), OVX + E2 (C) and OVX + E2TX (D) rats. White rectangles represent the area analysed for optical density of S40pTH labelling in the ME. (E, F) Mean
SEM percentage of S40pTH-immunoreactive (-IR) area in the ME (E) and the number of S40pTH-IR neurones/section in the ARC (F). *P < 0.05 OVX + E2 versus OVX + E2TX. 3V, third ventricle. Scale bar, 100 lm.

(32–34). Our current findings demonstrate that TX interferes with the enzymatic activity of TIDA neurones, as determined by S40pTH immunoreactivity in the ME, and this is likely to cause the suppression of PRL surge. The activity of TH, the rate-limiting enzyme in dopamine biosynthesis, is activated in vivo by phosphorylation of serine residues S19, S31 and S40 in its N-terminal domain (46,50). The expression of S40pTH in TIDA nerve terminals in the ME was markedly higher in OVX + E2TX rats than in OVX + E2 rats receiving vehicle, indicating an increased activity of TH in the ME of TX-treated rats and, presumably, higher dopamine released in the portal blood to inhibit PRL secretion. The E2-induced PRL surge is triggered © 2015 British Society for Neuroendocrinology

by the withdrawn of the inhibitory dopamine tonus. This is verified by the gradual decrease in dopamine synthesis and release in the ME of OVX + E2 rats, with lower levels in the afternoon compared to the morning (27). A similar pattern of reduction is seen for S40pTH expression by western blotting in the ME of pro-oestrous rats (46). Thus, although we did not evaluate the morning period in the present study, it is reasonable to assume that the S40pTH expression found at 18.00 h in OVX + E2 rats was probably reduced compared to morning levels in the same group, and this reduction was prevented by TX. Indeed, unpublished data (R. Araujo-Lopes and R.E. Szawka) from ongoing studies in our Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12338

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OVX

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80 60 40 20 0

OVX

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*

ARC 60

Oil

50

TX

##

40 30 20 10 0

OVX

OVX + E2

Fig. 8. Effect of tamoxifen (TX) on the expression tyrosine hydroxylase (TH) in the arcuate nucleus (ARC) and median eminence (ME). Ovariectomised (OVX) rats were treated with oil (OVX; n = 6), oestradiol (OVX + E2; n = 6), TX (OVX + TX; n = 5) or oestradiol plus TX (OVX + E2TX; n = 5) daily for 3 days. Blood samples were withdrawn hourly between 13.00 and 18.00 h and rats were perfused at 18.00 h. (A–D) Photomicrographs illustrating TH expression in the ARC of OVX (A), OVX + TX (B), OVX + E2 (C) and OVX + E2TX (D) rats. (E, F) Mean
SEM percentage of TH-immunoreactive (-IR) area in the ME (E) and number of TH-IR neurones/section in the ARC (F). *P < 0.05 OVX + E2 versus OVX + E2TX; ##P < 0.01 compared to OVX. 3V, third ventricle. Scale bar = 100 lm.

laboratory showed higher levels of S40pTH immunoreactivity in the ME of OVX + E2 rats in the morning compared to the afternoon (approximately 70% and 50% of S40pTH-IR area, respectively). Additionally, the already lower activity of TIDA neurones in OVX rats (29) explains the lack of difference in S40pTH expression between OVX and OVX + E2 rats. Unlike S40pTH, TH expression in the ME was not found to change among the experimental groups. This reinforces the efficacy of S40pTH staining in reflecting TH activity in nerve terminals of TIDA neurones and is in line with the results of a previous study reporting TH and S40pTH labelling in the ME of lactating mice (47). The TIDA neurones express ERa (28,29) and there is practically no expression of ERß in the ARC (59). Thus, Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12338

taken together with these previous reports, our findings suggest that ERa activation is required for E2-induced down regulation of TH activity in the ME to generate the PRL surge. However, although we have obtained consistent results measuring the optical density of immunohistochemical labellings, this is conceivably not an ideal method of quantification. Future experiments using western blotting may thus confirm the findings reported in the present study. TX prevented the E2-induced increase in both PR and TH expression in the ARC, suggesting that these hypothalamic effects of E2 also depend on classical ER signalling. The higher expression of PR in the ARC of OVX + E2 rats is in agreement with a previous report of PR induction in TIDA neurones by E2 (60). Conversely, as far as © 2015 British Society for Neuroendocrinology

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we are aware, this is the first report showing that E2 increases the number of TH-IR neurones in the ARC. In a previous study, we did not find such effect when OVX and OVX + E2 rats were perfused during the morning period (37). Thus, it appears that the E2induced increase in TH expression in the ARC may be linked to the time of the day, as is the PRL surge. However, more studies are required to further characterise this effect of E2 and determine its relevance in the control of PRL secretion. In the present study, we characterised the nature of the effects of TX on the hypothalamus of female rats under conditions of low or high circulating levels of E2 and determined the impact of these effects on LH and PRL secretion. TX precludes the positive- but not negative-feedback effect of E2 on hypothalamic neurones involved with LH control. TX also prevents E2 effects on TIDA neurones and PRL release. These findings expand our knowledge of the hypothalamic effects of TX and are of possible relevance to the fields of both clinical and basic research.

Acknowledgements We thank Dr Alain Caraty (Universit
e de Tours, Nouzilly, France) for the generous gift of anti-kisspeptin antibody. This work was supported by Fundacß~ao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), Pr
o-Reitoria de Pesquisa Universidade Federal de Minas Gerais (PRPq-UFMG), Fundacß~ao de Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cient
ıfico e Tecnol
ogico (CNPq). The authors declare that they have no conflicts of interest.

Received 18 August 2015, revised 14 October 2015, accepted 6 November 2015

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