Aixa R. Bello

Identification of Neurotensin-lmmunoreactive Cells in the Anterior Pituitary of Normal and Castrated Rats

The localization of neurotensin (NT) has been studied in the rat pituitary by means of immunocytochemistry at the light- and electron-microscopic levels. Cells immunoreactive for NT (NT-IR) were obser

Tamoxifen but Not Other Selective Estrogen Receptor Modulators Antagonizes Estrogen Actions on Luteinizing Hormone Secretion while Inducing Gonadotropin-Releasing Hormone Self-Priming in the Rat

Selective estrogen receptor modulators (SERMs) are compounds which may function as agonists or antagonists depending upon the target tissue. This study compares the actions of different SERMs on luteinizing hormone (LH) secretion, and on gonadotropin-releasing hormone (GnRH) self-priming in the rat. To do this, 4-day cyclic rats were injected twice, on day 2 (metestrus) and day 3 of the estrous cycle, with one of the following SERMs: 0.25 mg ICI 182,780, 3 mg tamoxifen (TX), LY139481-HCl or LY117018-HCl, or 0.5 mg RU58668. Control rats were given subcutaneous injections of 0.2 ml oil. On the morning of day 4 (proestrus in controls), rats from each group were either injected intraperitoneally with pentobarbital (40 mg/kg) for in vivo study or decapitated and their pituitaries collected for incubation (in vitro study). Additionally, pituitaries taken on each day of the estrous cycle from control rats as well as on day 4 from SERM-treated rats were processed for immunohistochemical determination of the estrogen receptor-α (ERα) gonadotrope. The plasma concentration or accumulation of LH in the medium was determined after 1 h (basal secretion). Thereafter, an intravenous bolus of GnRH (50 ng/0.5 ml/100 g BW) or 10–8M GnRH was injected or added to the medium, respectively. After 1 h of GnRH exposure, blood or medium were taken, and another challenge of GnRH was made. At the end of the 3rd h of the experiment, blood or medium samples were taken again and the LH plasma concentration or accumulation in the medium were determined. All SERM treatments reduced uterus weight and decreased basal and stimulated LH secretion. Also, on day 4, rats treated with any SERM other than TX showed vaginal smears infiltrated by leukocytes and a reduction in GnRH self-priming. TX-treated rats exhibited cornified vaginal smears and an estrogenic effect on GnRH self-priming. Moreover, 15-min exposure to two consecutive GnRH (10–8M) challenges 1 h apart in incubated pituitaries with estradiol (E2, 10–8M), TX (10–7M), E2 + TX, or medium alone form ovariectomized rats injected for 3 days with estradiol benzoate (25 µg), TX (3 mg), estradiol benzoate + TX, or 0.2 ml oil, respectively, showed that TX increased GnRH self-priming, as did E2, whereas it reduced the E2-sensitizing effect on GnRH-stimulated LH secretion and cancelled the E2-dependent GnRH self-priming. All SERMs prevented the physiological nucleocytoplasmic shuttling of ERα exhibited during proestrus in control rats, and TX, in addition, induced a significantly larger number of gonadotropes displaying strong cytosolic immunosignals corresponding to ERα than the rest of the experimental groups. Overall, data from this study indicated that, in contrast to the general antagonistic effect of the tested SERMs, TX seemed to display both selective agonist and antagonist activity at the gonadotrope level and on GnRH self-priming of LH secretion respectively.

Role of the Non-Canonical Notch Ligand Delta-Like Protein 1 in Hormone-Producing Cells of the Adult Male Mouse Pituitary

To better understand the role of the non‐canonical Notch ligand delta‐like protein 1 (DLK1), in hormone‐producing cells, we studied the cell distribution and subcellular localisation of DLK1 in the pituitary of male adult 129/SvJ mice, and analysed the variations in the hormone‐producing cells associated with the lack of this gene in Dlk1 knockout mice. The results obtained showed the presence of DLK1‐immunoreactive (ir) cells in all hormone‐producing cells of the anterior pituitary. Immunoelectron microscopy showed DLK1‐ir in the rough endoplasmic reticulum and inside secretory vesicles, suggesting that DLK1 is released together with pituitary hormones. Moreover, we found that prolactin (PRL)‐DLK1‐ir cells are in intimate contact with follicle‐stimulating hormone (FSH)‐ir‐DLK1‐negative cells. In Dlk1 knockout mice, we detected a significantly lower number of gowth hormone (GH)‐ir cells, a reduction in the FSH and PRL immunostaining intensity, and a significant decrease in FSH mRNA expression compared to wild‐type mice. An increase in pituitary GH mRNA expression and serum leptin levels was also found. These findings provide evidence supporting several regulatory functions of DLK1 in the pituitary gland.

Developmental Expression of Neurotensin in Thyrotropes and Gonadotropes of Male and Female Rats

Besides its potential roles as a central neuromodulator or a hypothalamic neurohormone, neurotensin (NT) may also have endocrine function in the anterior pituitary of mammals. We previously found that NT immunoreactivity is present in the secretory granules of gonadotropes and thyrotropes in both male and female rats, where its levels of expression are under the control of sex steroids. In this work, using immunocytochemistry and in situ hybridization, we have studied the postnatal development of NT-like immunoreactivity (NTir) and the mRNA encoding NT (mRNA-NT) in specific anterior pituitary cells of both male and female rats. NT expression starts after birth and displays an identical pattern in both sexes until sexual maturity, with mRNA-NT being detected from day 2 of postnatal life in thyrotropes localized in the central portion of the anterior lobe. This pattern of expression develops progressively throughout the 2nd and 3rd weeks in both sexes. By the beginning of the 3rd week, mRNA-NT can also be detected in gonadotropes localized in the periphery of the gland coinciding with a rise in serum estradiol concentrations in both sexes, and by day 21, mRNA-NT is extensively present in both the periphery and the central region. NTir is observed from days 5–6 in thyrotropes predominantly localized in the central portion of the anterior lobe, and by day 21, NTir is also detected in gonadotropes localized in the periphery of the gland. This pattern remains similar in both sexes until the time of puberty, when female rats start displaying plastic changes in NT expression according to the stage of the estrous cycle. These findings indicate that NT expression in the rat anterior pituitary is cell specific, and develops from birth to adulthood under the control of sex steroid hormones. In addition, preliminary data showing the presence of NT receptors in rat pituitary cells support the hypothesis of a paracrine or an autocrine role for this peptide within the pituitary.

Expression of Neurotensin and its Receptors in Pituitary Adenomas

The neurotensin (NT) produced in the hypothalamus and in pituitary gonadotrophs and thyrotrophs participates in neuroendocrine regulation. Recently, the involvement of this peptide in normal and neoplastic cell proliferation has been postulated. In the present study, we evaluated the expression of NT and its receptors (NTR1, 2 and 3) in a series of 50 pituitary adenomas [11 growth hormone (GH)‐, eight prolactin (PRL)‐, four adrenocorticotrophic hormone (ACTH)‐ and 27 nonfunctioning adenomas]. NT mRNA expression was significantly higher in functioning compared to nonfunctioning adenomas and with normal pituitary. Nonfunctioning pituitary adenomas showed lower expression of NT mRNA than normal pituitary. In the immunohistochemical study of functioning adenomas, NT was colocalised with GH, PRL and ACTH secreting cells. In nonfunctioning adenomas, the NT immunoreactivity intensity was variable among the samples. NTR3 mRNA expression was observed in all examined samples and was higher in the adenomas, both functioning and nonfunctioning, compared to normal pituitary. By contrast, NTR1 and NTR2 mRNA were not detected in either pituitary adenomas or normal tissue. The higher expression of NTR3, as well as the expression of NT by tumoural corticotrophs, lactotrophs and somatotrophs, which are cells types that do not express this peptide in the normal pituitary, suggests that NT autocrine and/or paracrine stimulation mediated by NTR3 may be a mechanism associated with the tumourigenesis of functioning adenomas.

Sex steroids modulate luteinizing hormone-releasing hormone secretion in a cholinergic cell line from the basal forebrain.

The function of a particular neuronal population is in part determined by its neurotransmitter phenotype. We have found that a neuronal-derived septal cell line (SN56), known for its cholinergic properties, also synthesizes and releases luteinizing hormone-releasing hormone. In addition, these cells express the messenger RNAs encoding estrogen and progesterone receptors. The activation of these receptors by their respective ligands cooperatively modulates the depolarization-induced release of luteinizing hormone-releasing hormone in these cells. We have also found that a number of septal neurons in postnatal (1-week-old) mice are immunoreactive to both choline acetyltransferase and luteinizing hormone-releasing hormone. These results indicate that both neurotransmitters, acetylcholine and luteinizing hormone-releasing hormone, may co-exist in septal neurons of the CNS and that they could be modulated by gonadal hormones, and suggest that luteinizing hormone-releasing hormone could be involved in some of the actions of sex steroids on cholinergic neurotransmission.

Tyrosine hydroxylase activity in peripherally denervated rat pineal gland.

The presence of tyrosine hydroxylase (TH) in the rat pineal gland was studied using a combination of immunochemical and biochemical methods. In superior cervical ganglionectomized (SCGx) animals and in isolated pineals incubated for 72 h, both TH immunoreactive (TH-IR) fibers and TH biochemical activity were still present but reduced. Conversely, in dispersed pinealocytes incubated for only 24 h we were unable to detect either TH activity or TH-positive cells. Since in the pineal gland of intact rats total 3-methoxy-4-hydroxy phenylglycol (MHPG) was undetectable, and only traces of norepinephrine (NE) were present in the pineal of ganglionectomized animals, the results suggest a central pinealopetal catecholaminergic pathway which could use dopamine as a neurotransmitter.

Differentially regulated expression of neurokinin B (NKB)/NK3 receptor system in uterine leiomyomata

STUDY QUESTION Are the vasoactive peptide neurokinin B (NKB) and its preferred NK3 receptor (NK3R) differentially expressed in leiomyomas compared with normal myometrium? SUMMARY ANSWER In leiomyomas, NKB is up-regulated and delocalized, while its preferred NK3R is also differentially regulated. WHAT IS KNOWN ALREADY The expression of NKB/NK3R in the central nervous system is essential for proper function of the human reproductive axis. Additionally, this system is also widely expressed throughout the female genital tract. Leiomyomas impair fertility and are a major source of abnormal uterine bleeding. The aberrant synthesis of local factors can contribute to the pathological symptoms observed in women with leiomyomata. NKB could be one of these factors, since a vasoactive role of this peptide at a peripheral level has been observed in different systems and species, including humans. NK3R is strongly regulated by estrogens and its activation leads to nuclear translocation affecting chromatin structure and gene expression. STUDY DESIGN, SIZE, DURATION Samples were obtained between 2006 and 2012 from 28 women of reproductive age at different stages of the menstrual cycle by hysterectomy. Leiomyomas and matched macroscopically normal myometrium from each woman were analysed in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS RT-PCR, quantitative real time, immunohistochemistry and in situ hybridization were used to investigate the pattern of expression of NKB/NK3R in tissue samples. MAIN RESULTS AND THE ROLE OF CHANCE Expression of the gene encoding NKB (TAC3) was up-regulated 20-fold in leiomyomas, compared with matched myometrium (P = 0.0008). In tumour tissue, not only connective cells, but also myometrial, endothelial and vascular smooth muscle cells express TAC3 mRNA. Immunoreactivity to NKB was preferentially located in the smooth muscle cell nuclei from normal myometrium in the secretory phase, unlike matched leiomyoma, which showed a predominant cytoplasmic expression pattern. In the normal myometrium, TACR3 mRNA showed variable expression throughout the menstrual phases, with samples showing strong, reduced or no amplification. In leiomyoma, TACR3 was significantly up-regulated compared with matched myometrium (P = 0.0349). LIMITATIONS, REASONS FOR CAUTION This study is descriptive and although we observed clear differential regulation of the NKB/NK3R system at mRNA and immunohistochemical staining levels in leiomyoma, future functional studies are needed to determine the precise role of NKB in the myometrium in normal and pathological conditions. In addition, further analysis (e.g. in cell culture models) will be required to determine the role of NKB in the nucleus of normal smooth muscle cells, whether nuclear translocation is mediated by NK3R and the consequences of the cytoplasmic expression of NKB in tumour cells. WIDER IMPLICATIONS OF THE FINDINGS The NKB/NK3R system dysregulation observed in leiomyoma may contribute to the pathological symptoms observed in women with leiomyomata.

Neurotensin and Neurotensin Receptor 1 Expression in Human Myometrium and Uterine Leiomyomas

Leiomyomas or fibroids are the most frequently diagnosed tumors of the female genital tract, and their growth seems to be steroid-hormone dependent by a yet undetermined cellular and molecular mechanism. Sexual hormones induce the secretion of growth factor peptides and the expression of their receptors, stimulating cell proliferation. One of these factors is neurotensin, and increasing evidence suggests that it can promote growth of different cancer cells. Since there are no data on neurotensin expression in normal and tumoral uterine tissue, we have analyzed the expression of NTS and NTSR1 receptor using immunohistochemistry for protein detection, in situ hybridization to detect cells expressing NTS mRNA, and RT-PCR to detect NTSR1 transcript as well as any of the alternative splice variants recently described for this receptor. We found that NTS and NTSR1 are expressed in connective cells of normal myometrium. In leiomyomas, immunoreactivity for NTS and NTSR1 receptor is colocalized in the smooth muscle cells that are also transcribing NTS. Women receiving high doses of steroids for in vitro fertilization showed tumor growth and increased immunoreactivity for neurotensin and NTSR1 receptor. Interestingly, alternative splice variants of NTSR1 receptor were detected only in tumoral tissue. These findings suggest a role of steroid hormones inducing neurotensin expression in leiomyoma smooth muscle cells. In these cells, NTS could act autocrinally through NTSR1 receptor, promoting their proliferation.

Sexual Dimorphism in the Distribution of a-Neoendorphin-Like Immunoreactivity in the Anterior Pituitary of the Rat

The localization of the opioid peptide α‐neoendorphin (α‐Neo‐E) was studied in the anterior pituitary of normal and castrated male and normal female rats, Immunoreactive (ir) cells were noted in both sexes. These α‐Neo‐E‐ir cells were further characterized using double immunostaining with an elution‐restaining procedure. It was seen that in males, α‐Neo‐E‐ir cells corresponded mainly to luteinizing hormone/follicle‐stimulating hormone cells and a few thyroid‐stimulating hormone (TSH) cells, whereas in females, virtually all α‐Neo‐E‐ir cells corresponded to TSH cells.