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.
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.
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 observed in the anterior lobe of rats of both sexes. Following an elution-restaining procedure, it was found that most of these NT-IR cells correspond to cells also reacting to β-LH or FSH antisera. However, a few NT-IR cells did not stain for β-LH nor FSH, but for TSH. The subcellular localization of NT was studied at the electron-microscopic level by means of the immunogold procedure. The immunoreactivity was always localized over the secretory granules. By using two sizes of gold particles, it was shown that NT- and β-LH-IR were colocalized in part of the cell granules. Castration caused a progressive decrease of the NT-IR in the β-LH- or FSH-positive cells, while the number of NR-IR, TSH-IR cells increased. After 2 weeks, NT-IR was mainly observed in this latter cell type. The present work demonstrates that NT-IR is localized within the secretory ganules of the gonadotrophs and a few thyrotrophs, indicating that NT could be released together with gonadotrophins and TSH. Moreover, since gonad-ectomy results in typical qualitative and quantitative changes of NT-IR gonadotrophs, the intervention of the pituitary NT in the regulation of the hypo-physogonadal axis is strongly suggested.
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.
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