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PURPOSE: The endocrine secretion of TSH is a finely orchestrated process controlled by the thyrotropin-releasing hormone (TRH). Its homeostasis and signaling rely on many calcium-binding proteins belonging to the "EF-hand" protein family. The Ca2+/calmodulin (CaM) complex is associated with Ca2+/CaM-dependent kinases (Ca2+/CaMK). We have investigated Ca2+/CaMK expression and regulation in the rat pituitary. METHODS: The expression of CaMKII and CaMKIV in rat anterior pituitary cells was shown by immunohistochemistry. Cultured anterior pituitary cells were stimulated by TRH in the presence and absence of KN93, the pharmacological inhibitor of CaMKII and CaMKIV. Western blotting was then used to measure the expression of these kinases and of the cAMP response element-binding protein (CREB). TSH production was measured by RIA after time-dependent stimulation with TRH. Cells were infected with a lentiviral construct coding for CaMKIV followed by measurement of CREB phosphorylation and TSH. RESULTS: Our study shows that two CaM kinases, CaMKII and CaMKII, are expressed in rat pituitary cells and their phosphorylation in response to TRH occurs at different time points, with CaMKIV being activated earlier than CaMKII. TRH induces CREB phosphorylation through the activity of both CaMKII and CaMKIV. The activation of CREB increases TSH gene expression. CaMKIV induces CREB phosphorylation while its dominant negative and KN93 exert the opposite effects. CONCLUSION: Our data indicate that the expression of Ca2+/CaMK in rat anterior pituitary are correlated to the role of CREB in the genetic regulation of TSH, and that TRH stimulation activates CaMKIV, which in turn phosphorylates CREB. This phosphorylation is linked to the production of thyrotropin.

The aim of the study was to investigate the effects of chronic nandrolone decanoate treatment and/or swimming training on immunohistomorphometric parameters on rat pituitary gonadotropic cells. Male Wistar albino rats, 10 weeks old, were classified into four groups: control (T−N−), nandrolone (T−N+), swimming training (T+N−), and swimming training with nandrolone (T+N+). The T+ groups swam for 4 weeks, 1 h/day, 5 days/week. The N+ groups received nandrolone decanoate (20 mg/kg) once per week for 4 weeks. Pituitary tissue sections were processed and stained for immunohistochemical analysis and immunofluorescence. The volume density of luteinizing hormone (LH) cells was decreased by 48% in T−N+ and for 35% in the T+N+ group. The volume density of follicle-stimulating hormone (FSH) cells was decreased by 39% in T−N+ and for 30% in T+N+ compared to the control. Nandrolone alone, or combined with swimming training, decreased the number of LH/FSH cells compared to the control. The levels of the immunofluorescent signal of LH/FSH cells were increased in all experimental groups. Nandrolone alone decreased the serum level of LH by 17%, whereas swimming training alone increased FSH levels by 11% compared to the control. Serum levels of testosterone were increased in all experimental groups. Nandrolone alone, or combined with swimming training, decreased immunohistomorphometric parameters of gonadotropic cells, whereas the levels of immunofluorescent signal were increased.

Di-n-butylphthalate (DnBP) exhibits alarming thyroid disrupting activities. However, the toxic mechanism of DnBP is not completely understood. In this study, we investigated the mechanism of DnBP in thyroid disruption. Rat pituitary tumor cell lines (GH3) were treated with DnBP in different scenarios, and cell viabilities, target gene transcriptions and protein levels were measured accordingly. The results showed that after treatment with DnBP (20 μmol/L), cell proliferation increased to 114.69% (p

Phytoestrogens are a diverse group of steroid–like compounds that occur naturally in many plants. There are various types of phytoestrogens, including the best-researched isoflavones which are commonly found in soy. The consumption of soy products has many health benefits, including protection against breast cancer, prostate cancer, menopausal symptoms, heart disease and osteoporosis. In contrast, use of hormonally active compounds-isoflavones may unfortunately interfere with the endocrine system and can have far-reaching consequences. Genistein, the most abundant soy-bean derived isoflavone, possesses a ring system similar to estrogens and acts through an estrogen receptor (ER)-mediated mechanism, by increasing or decreasing the transcription of ER-dependent target genes. Also, genistein can act on cells through ER non-dependent mechanisms, such as tyrosine kinase inhibitor. The neuroendocrine systems are responsible for the control of homeostatic processes in the body, including reproduction, growth, metabolism and energy balance, and stress responsiveness. It is well known, that estrogen is important for development of the neuroendocrine system in both sexes. At the pituitary level, estrogen is known to affect the regulation of all hormone producing (HP) cells, by direct and/or indirect mechanisms. Due to structural and functional resemblance to estrogen, the question may arise of whether and how genistein affects the morphofunctional features of pituitary HP cells. This review deals with the consequences of genistein’s effects on morphological, stereological and hormonal features of HP cells within the anterior pituitary gland. Transparency on this issue is needed because isoflavones are presently highly consumed. Inter alia, genistein as well as other isoflavones, are present in various dietary supplements and generally promoted as an accepted alternative to estrogen replacement therapy. Potential isoflavone biomedical exploitation is not only limited to estrogen replacement therapy, so it should be treated in a wider context of different ageing symptoms remediation.

Orexin A and B, are neuropeptides that are mainly produced in the hypothalamus. Orexins play important roles in food intake and in regulation of sleep and arousal. In the peripheral tissues, orexins also regulate various endocrine activities including the hypothalamic-pituitary-adrenal and -gonadal axes and growth hormone control. However, the effects of orexin on prolactin synthesis have not been clarified. Here, we focused on the role of bone morphogenetic protein (BMP)-4, which is involved in promoting PRL productivity and tumorigenesis of pituitary prolactinomas. We previously reported that BMP-4 induces prolactin secretion by rat lacto-somatotrope GH3 cells. In the present study, we studied the effects of orexin A and the interaction of orexin and BMP system on the regulatory role of prolactin synthesis by using rat lactotrope GH3 cells. Orexin type 1 receptor (OX1R), but not type 2 receptor (OX2R), was expressed in GH3 cells. Orexin A suppressed forskolin-induced, but not basal, prolactin mRNA expression without reducing cAMP levels. Of note, orexin A suppressed BMP-4-induced prolactin mRNA and cAMP synthesis. Interestingly, BMP-receptor signaling inhibitor reduced prolactin mRNA levels, which suggested that endogenous BMP action was to be involved in the activation of prolactin synthesis by GH3 cells. Orexin A suppressed Smad1/5/9 phosphorylation and Id-1 transcription induced by BMP-4, which was reversed by dual orexin-receptor antagonist, suggesting that the inhibitory effect of orexin A occurred via OX1R. Moreover, orexin A reduced BMP type I receptor, ALK-3 expression but increased inhibitory Smad6/7 expression, while BMP-4 treatment downregulated OX1R expression. These results indicated that orexin A has an inhibitory role in prolactin synthesis through suppression of endogenous BMP activity, suggesting that a new functional role of the interaction between orexin A and BMP-4 is modulation of prolactin levels in lactotrope cells.

Circular RNAs (circRNAs) are a new class of RNA that have a stable structure characterized by covalently closed circular molecules and are involved in invasive pituitary adenomas and recurrent clinically nonfunctioning pituitary adenomas. However, information on circRNAs in the normal pituitary, especially in rats, is limited. In this study, we identified 4123 circRNAs in the immature (D15) and mature (D120) rat anterior pituitary using the Illumina platform, and 32 differentially expressed circRNAs were found. A total of 150 Gene Ontology terms were significantly enriched, and 16 KEGG pathways were found to contain differentially expressed genes. Moreover, we randomly selected eight highly expressed circRNAs and detected their relative expression levels in the mature and immature rat pituitary by qPCR. In addition, we predicted 90 interactions between 53 circRNAs and 57 miRNAs using miRanda. Notably, circ_0000964 and circ_0001303 are potential miRNA sponges that may regulate theFshbgene. The expression profile of circRNAs in the immature and mature rat anterior pituitary may provide more information about the roles of circRNAs in the development and reproduction in mammals.

The growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis has recently been recognized as an important factor related to the longevity of many organisms. MicroRNAs (miRNAs or miRs) could also participate in diverse biological processes. However, the role of miRNAs in the decline of pituitary GH during the growth process remains unclear. To better characterize the effects of miRNAs on the pituitary, we used a miRNA microarray to investigate the miRNA profile in the rat pituitary from postnatal development throughout the growth process. Then, in vitro experiments were conducted to analyze the miRNAs&rsquo, potential roles related to GH regulation. Taken together, the microarray results indicated that there were 22 miRNAs differentially expressed during pituitary development. The bioinformatics analysis suggested that the most differentially expressed miRNAs may participate in multiple pathways associated with the pituitary function. Furthermore, the in vitro findings demonstrated that miR-141-3p was involved in GH regulation.

In areas with moderate continental climate, increased average ambient temperature during the summer represents a stressogenic factor that affects the hypothalamo-pituitaryadrenocortical axis in mammals. Therefore, we wanted to examine the effects of 4 days of constant exposure to moderately elevated ambient temperature (35 ± 1oC) on the histomorphometric and immunofl uorescent characteristics, as well as on the hormonal secretion of pituitary corticotropes (ACTH) cells in adult male rats. In comparison with the controls kept at 20 ± 2oC, a signifi cant increase (p

The protein expressions of steroidogenic factor l (SF-l) and pituitary-specific transcription factor 1 (Pit-1) were investigated immunohistochemically for 53 spontaneous pituitary adenomas of the pars distalis from male Crl:CD(SD) rats. Luteinizing hormone (LH)-positive/prolactin (PRL)-negative and LH-negative/PRL-positive adenomas showed that the expression of SF-1 and Pit-1 was exclusively related to the immunoreactivity of LH and PRL, respectively. All double-positive adenomas (positive for both LH and PRL) were positive for Pit-1 and were supposed to be derived from PRL cells, although some of them also showed SF-1 immunoreactivity. In addition, all null cell adenomas (negative for all anterior pituitary hormones) were positive for SF-1 and negative for Pit-1, indicating that they originated from the gonadotroph cell lineage. This is the first report focusing on the application of transcription factors for the classification of rat pituitary adenomas.

Estradiol and human chorionic gonadotropin (hCG) are very important in controlling the secretory activity of hormone producing cells in the female rat pituitary glands. The aim of the present study was to examine the morphometric parameters of immunohistochemically la be led ACTH cells in juvenile (16th day) female rat pituitaries after treatment with five doses of estradiol dipropionate (EDP) and two doses of hCG during the neonatal period of life. The controls were treated on the same schedule with an equivalent volume of vehicle. All animals were sacrificed 24 h after the last treatment. ACTH-producing cells were studied using the peroxidase-antiperoxidase immunohistochemical procedure. The absolute and relative pituitary weights were increased (p0.05) changes neither in the volume of ACTH cells nor in the volume of their nuclei, in comparison with the controls. On the basis of the results obtained in our study, it can be concluded that EDP, injected into female rats during the neonatal period of life, has an inhibitory effect on the immunohistomorphometric parameters of ACTH cells, but such an effect is not clearly expressed after treatment with hCG. Estradiol i humani horionski gonadotropin (hCG) veoma su važni u kontroli sekretorne aktivnosti ćelija hipofize koje proizvode hormone. Cilj ove studije je bio ispitati morfometrijske parametre imunohistohemijski obeleženih ACTH ćelija hipofize ženki pacova, u juvenilnom periodu (16. dan), nakon tretmana sa pet doza estradiol-dipropionata (EDP) ili dve doze humanog horionskog gonadotropina (hCG). Kontrole su bile tretirane na istovetan način sa odgovarajućom količinom rastvarača. Sve životinje su bile žrtvovane 24 h nakon poslednjeg tretmana. ACTH ćelije su bile imunohistohemijski obeležene metodom peroksidaza-antiperoksidaza (PAP). Dobijeni rezultati ukazuju na značajno povećanje (p0,05) promenjeni u odnosu na kontrolu. Na osnovu dobijenih rezultata možemo zaključiti da EDP, injeciran ženkama pacova tokom neonatalnog perioda života, ima inhibitorni efekat na imuno histomorfometrijske parametre ACTH će lija, što nije tako jasno izraženo nakon tretmana hCG-om. Projekat ministarstva br. 173009

OBJECTIVE The study was aimed to observe the effect of amlodipine on rat pituitary gonadotropins after amlodipine administration and withdrawal. METHODS It was an experimental study done at Army Medical College, National University of Sciences and Technology, Islamabad, Pakistan from 2009-2010. Sixty adult male rats were divided into groups A and B. Each group was further subdivided into two subgroups of 15 rats each; A1 (control), A2 (control recovery), B1 (amlodipine-treated) and B2 (amlodipine recovery). Amlodipine, 0.04 mg/kg body weight daily for fifty days was given by means of gavage to groups B1 and B2. Groups A1 and A2 were given vehicle (0.5 ml distilled water). After 50 days, rats in groups A1 and B1 were sacrificed and their serum LH and FSH levels were measured by Enzyme Immunoassay method. Vehicle and amlodipine were withdrawn in groups A2 and B2, respectively, and the rats were left for recovery to take place for another fifty days. The above procedure was adopted for the measurement of LH and FSH levels in the recovered rats. RESULTS Amlodipine administration for 50 days resulted in a significant rise in serum LH (p < 0.01) whereas serum FSH remained unchanged (p ≥ 0.05). Serum LH in amlodipine-treated rats returned to normal after amlodipine withdrawal (p ≥ 0.05). CONCLUSION Amlodipine causes a reversible increase in serum LH but it has no effect on serum FSH (Fig. 2, Ref. 17).

In this study, dioscin was isolated from Dioscoreae Rhizoma (DR), which is the rhizome of Dioscorea batatas D(ECNE). that inhabits broad areas of Korea and Japan. To determine whether dioscin induced growth hormone (GH) release, we evaluated its induction effects on GH release both in vitro and in vivo. The 70% methanol extract of DR, and its n-hexane and n-BuOH fractions, induced rat GH (rGH) release in rat pituitary cells 10-fold, 8-fold, and 5- fold higher than the control (0.36 +/- 0.02 nM), respectively (p < 0.05 each). The dioscin-induced rGH release of the cells was concentration-dependent and its ED(50) was 1.14 x 10(-5) M. Within 90 minutes after intravenous administration of 10 microg/kg (p < 0.05 at t(max)), dioscin caused the greatest increase in rGH concentration (C(max)) in the rat plasma (34.16 +/- 14.10 ng/ml) (n = 4), which was twice as high as the control group (12.88 +/- 3.29 ng/ml) (n = 27).

Kampo medicine (Chinese herbal medicine) has been reported to be effective in the treatment of pituitary-ovarian dysfunction in young women and in the treatment of undefined symptoms in perimenopausal women. It has been considered that both the diagnosis and treatment of ovulatory failure in adolescent girls or young women are necessary for the sake of future fertile capacity. However, treatment for the above patients is not easy as steroid hormones and strong stimulators of ovulation should not be recommended because of the several side-effects it causes. Furthermore, young women, especially teenagers, seem to be reluctant to visit gynecology clinics. A number of herbal medicines have been used for many centuries in China and Japan for the treatment of menstrual disorders and infertility. In general, the traditional Chinese herbal prescriptions are rather inexpensive and safe with little side-effects, and have properties for normalizing biological balances. Unkei-to is known to stimulate the synthesis and release of gonadotropins in the rat pituitary. In humans, Unkei-to is known to be effective in various menstrual disorders, abnormal uterine bleeding, and infertility. In the treatment of ovulatory failure, recent dynamic studies have revealed the mechanism by which Unkei-to stimulates the diencephalon-pituitary-ovarian axis in humans. In this paper, we critically review the clinical data available (to date) in order to assess the efficacy of Unkei-to in inducing ovulation in several types of ovulatory disturbances. (Reprod Med Biol 2003; 2: 45-61).

Bioceramic materials are used for the reconstruction or replacement of the damaged parts of the human body. In this study an improved procedure is described for producing ceramic scaffolds with controlled porosity. Bioinert alumina ceramic was used to make porous scaffolds by using indirect fused deposition modeling (FDM), a commercially available rapid prototyping (RP) technique. Porous alumina samples were coated with hydroxyapatite (HAp) to increase the biocompatibility of the scaffolds. Initial biological responses of the porous alumina scaffolds were assessed in vitro using rat pituitary tumor cells (PR1). Both porous alumina and HAp coated alumina ceramics provided favorable sites for cell attachments in a physiological solution at 37 °C, which suggests that these materials would promote good bonding while used as bone implants in vivo. Based on these preliminary studies, similar tests were performed with human osteosarcoma cells. Cell proliferation studies show that both the ceramic materials can potentially provide a non-toxic surface for bone bonding when implanted in vivo.

SUMMARY There has been considerable interest in the relationship between hormonesecreting endocrine cells and their microvessels in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). This study was designed to visualize and to reveal the relationship between hormone-secreting endocrine cells and their microvessel environment in 3D, using rat pituitary glands under various (hyper/hypo) experimental conditions by confocal laser scanning microscopy (CLSM). Female adult Wistar rats were used after bilateral adrenalectomy or ACTH administration for 2 weeks. Clear 3D reconstructed images of ACTH cells, the microvessel network and counterstained nuclei were obtained at a maximal focus depth of 1 mm by CLSM without any background noise. In the hyperfunctional state, slender cytoplasmic processes of hypertrophic stellate ACTH cells frequently extended to the microvessels. In the hypofunctional state, ACTH cells appeared atrophic and round with scanty cytoplasm, and cytoplasmic adhesions to microvessel network patterns were inconspicuous. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways. (J Histochem Cytochem 48:569‐577, 2000)

Sapecin B is an antibacterial protein isolated and purified from culture medium of the embryonic cell line derived from the flesh fly (Sarcophaga peregrina). It has structural simil

Whole cell patch-clamp techniques were used on clonal pituitary cells (GH3) and primary cultures of somatotrophs and lactotrophs to study currents that would be active at or below voltages for the threshold for action potential generation. When GH3 cells were held at -60 mV and pulsed to -120 mV, a slow-activating sustained inward current was observed (-16.5 +/- 1.5 pA in physiological baths, n = 72; approximately 1 s to half-maximal activation, voltage for 50% activation - 101 mV). The current was insensitive to bath application of 10 mM tetraethylammonium, 10 mM 4-aminopyridine, and 1 mM barium but was completely blocked by 3 mM cesium. The current was found to be a mixed cation current with a sodium permeability of 0.29 relative to potassium. These properties indicate that the current belongs to the hyperpolarization-activated cation current (Ih), or I(f), family of currents. However, the current was not altered by the addition of adenosine 3',5'-cyclic monophosphate (cAMP) to the pipette or forskolin to the bath. A similar but smaller current was observed in 15 of 16 somatotrophs but in only 1 of 9 lactotrophs. Application of cesium to spontaneously spiking GH3 cells or somatotrophs had no effect. However, cesium did block an inward holding current observed at -80 mV. These results demonstrate that the I(h) in pituitary cells does not serve as a pacemaking current but suggest that it may influence membrane potential responses when somatotrophs become hyperpolarized.

The present study was designed to characterize bihormonal cells in rat pituitary cells which secrete PRL and gonadotropins. This was done by using sandwich cell immunoblot assay (CIBA) and reverse hemolytic plaque assay (RHPA) in combination with immunocytochemistry (ICC) and by measuring the intracellular free calcium concentration ([Ca2+]i). The result of the experiment with sandwich CIBA revealed that the populations of LH- and FSH-secreting cells in the PRL-secreting cells were 6.23% and 5.91%, respectively, and the populations of the PRL secreting cells in the LH- and FSH-secreting cells were 18.4% and 15.5%, respectively. Additional studies by the combined techniques of RHPA with ICC revealed that the populations of LH- and FSH-containing cells in the PRL secreting cells were 4.43% and 2.40%, respectively, which were consistent with the results of Sandwich CIBA. Some of the PRL-secreting cells determined by RHPA showed responsiveness to TRH and GnRH in [Ca2+]i. These results suggest that bihormonal cells which secrete both PRL and LH, or both PRL and FSH are present in the normal rat pituitaries.

Due to a typesetting error, there was an error in the name of the sixth author. The correct author name is: Ettore C. degli Uberti

Numerous studies have established a link between estrogen levels and activity of the hypothalamic pituitary adrenal (HPA) system. Considering the ``weak estrogenic{''} effect of soy isoflavones, this study was designed to evaluate the influence of soy extract treatment on some morphofunctional parameters of rat pituitary corticotropes in vivo. Adult male orchidectomized Wistar rats received estradiol-dipropionate or soy extract in oil/ethanol solvent subcutaneously for 3 weeks. Both controls, sham-operated (So) and orchidectomized (Orx) rats, were injected with solvent, in the same regime. Changes in pituitary volume, total volume, total number and volume of individual corticotropes were evaluated stereologically, while ACTH levels were determined biochemically. In comparison with So rats, estradiol treatment provoked increases (p < 0.05) of ACTH level (166\%), pituitary weight (167\%) and volume (102\%), total volume (20\%) and total number of corticotropes (18\%). In comparison to Orx, following estradiol treatment elevation (p < 0.05) of ADE level (69\%), pituitary weight (131\%) and volume (82\%), total (30\%) and individual volume (29\%) of corticotropes was observed. Soy extract treatment led to enhancement (p < 0.05) of: ACTH level (28\%), total (25\%) and individual volume (20\%) of corticotropes. It can be concluded that soy extract acts in a similar way to estradiol, but the increased activity of corticotropes was weaker. (C) 2013 Elsevier GmbH. All rights reserved. Ministry of Education and Science of Serbia {[}173009]

We have examined statistically the actions of thyrotropin-releasing hormone (TRH) and Bay K 8644, an L-type Ca(2+)-channel agonist, on the frequency and shape of cytosolic Ca2+ spikes in individual GH4C1 rat pituitary cells. TRH induced a brief (0-40 s) suppression of Ca2+ spikes followed by a period (40-200 s) of increased spike frequency. TRH treatment reduced the rate of rise and amplitude of Ca2+ spikes, and increased the rate of fall, relative to spontaneous spikes before treatment. TRH had no significant effect on the correlation between spike amplitude and the spike decay time constant tau, suggesting that the increased rate of fall was due to enhanced Ca2+ extrusion and not to decreased Ca(2+)-induced Ca2+ release. Bay K rapidly (t1/2 = 9-13 s) induced a 2-fold increase in the rate of rise of spikes with no change in the total rise time, leading to an increase in spike amplitude. It increased by 2-fold the fall time of spikes, as predicted solely by the previously observed relationship between spike amplitude and fall time. Bay K therefore appeared to increase the number of Ca2+ channels participating in each spike event without altering the kinetics of channel activation or deactivation, and without influencing Ca2+ extrusion. After addition of Bay K, the interval between spikes gradually (t1/2 approximately 100 s) increased, whereas the rate of rise remained constant and maximal. To explain these actions of TRH and Bay K, we postulate that a fraction of L-type Ca2+ channels are inactivated during each spike and must be re-activated in order to participate in a subsequent spike. We conclude further that the changes in spike frequency and profiles induced by these secretagogues are most consistent with a model in which TRH induces increases in both Ca2+ influx and efflux while Bay K induces a large increase in Ca2+ influx but has little effect on efflux.

The activity of GnSI/AF was measured in pig follicular fluid (pFF) from 58 individual follicles of various sizes, by bioassay using rat pituitary cells, to investigate the relationship between gonadotrophin surge inhibiting/attenuating factor (GnSI/AF) activity and follicular development. In addition, the correlation between GnSI/AF and inhibin activities and the content of sex steroids (oestradiol, progesterone and testosterone) of follicles was examined. The activity of GnSI/AF in pFF varied significantly (0.155-1.69 U microliter-1) with size of the follicle. The activities (mean +/- SEM) were intermediate and constant in follicles with diameters from 3 to 5 mm (0.583 +/- 0.080 U microliter-1, n = 24), were higher and reached the highest value in follicles with diameters between 6 and 8 mm (0.863 +/- 0.068 U microliter-1, n = 21), and were lower, reaching the lowest value in follicles with diameters of 9 and 10 mm (0.401 +/- 0.089 U microliter-1, n = 13). In contrast, inhibin activity was almost constant during the development of follicles, although individual values varied from 0.9 to 2.5 U microliters-1. For follicles with diameters of 4-8 mm, inhibin activity was 1.754 +/- 0.042 U microliters-1 (n = 39); activity was higher in the smallest follicles with diameters of 3 mm (2.063 +/- 0.015 U microliters-1, n = 6) and was lower in follicles with diameter of 9 mm, reaching the lowest value in follicles with diameter of 10 mm (1.176 +/- 0.068 U microliters-1, n = 7); inhibin activity was not significantly correlated with GnSI/AF activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The following rat pituitary cell culture experiments were carried out to find an explanation for the increased premature LH surges in stimulation protocols using clomiphene citrate (CC) and subsequent gonadotrophin with mid-cycle administration of the GnRH antagonist cetrorelix (Cetrotide(R)) in IVF cycles. Rat pituitary cells were pretreated with CC, then stimulated with GnRH with and without co-administration of different concentrations of GnRH antagonist and compared with non-CC-pretreated controls. While lower concentrations of cetrorelix suppressed LH in CC-non-pretreated cells, but not in CC-pretreated cells, higher concentrations of cetrorelix suppressed LH in both groups. CC pretreatment increased the sensitivity of the pituitary for GnRH and reduced the efficacy of subsequent GnRH antagonist. Thus, the elevated risk of premature LH surges in clinical protocols using CC, gonadotrophin and GnRH antagonist could be overcome by increasing the antagonist dose.

The direct effects of human GH and IGF-I on PRL secretion and cell proliferation were studied on PRL secreting rat pituitary tumor 7315 b cells in vitro , as well as the effects in vivo of human GH administration on body weight, IGF-I levels and tumor size in rats bearing this transplantable tumor. In the in vitro studies IGF-I levels above 5 nM stimulated PRL release in a dose-dependent manner while GH, in concentrations of 0.23–45 nM , did not affect PRL release. Cell proliferation was stimulated by IGF-I in a dose-dependent manner from 0.5 nM onwards, while GH did not have an effect. The in vivo studies showed that 1 mg GH/rat/day prevented tumor-induced cachexia and normalized the suppressed IGF-I levels without stimulating tumor growth. It is concluded that tumor-induced cachexia can be prevented by exogenous GH administration without an increase in tumor mass, even if a tumor model is used whose cultured tumor cells respond to exposure to IGF-I with a mitotic response.

For simultaneous measurement of multiple hormones in blood samples from small experimental animals, multiple radioimmunoassay (Multi-RIA) was developed for rat pituitary hormones (LH, FSH, TSH, PRL, and GH) and epidermal growth factor (EGF) using double antibody method.Parallel RIA, where 125I-and 131I-labelled hormones were employed together, could measure 2 hormones at a time, and their assay values correlated well with those obtained by ordinary single assays. Smaller assay values were obtained with TSH, PRL, and GH. However, the lower slope of the regression line in TSH assay could be overcome by changing the anti-TSH serum for another batch of antiserum, indicating that this problem may be due to the antibodies used. Assay results of parallel RIA for female rat serum LH and FSH with samples obtained on proestrus day agreed well with those by single RIA.Serial RIA, where 2 hormones were assayed successively, also produced assay values which correlated well with those from single assays. The lower binding of hormones in the second assay of serial RIA was improved by increasing the concentration of normal rabbit serum in the dilutant for the antiserum without affecting assay sensitivity.Multi-RIA, which was developed by combining parallel RIA and serial RIA, could measure up to 4 hormones at a time. Application of multi-RIA to serum samples from castrated and cannulated male rats gave results which suggested synchronous secretion of LH, FSH, and PRL, indicating the usefulness of this assay method.

To clarify the acute and chronic effects of GnRH agonists on pituitary gonadotropes, changes in the ultrastructure of male rat gonadotropes were examined immunocytochemically and morphometrically after the administration of a one-month depot formulation of the GnRH agonist, leuprorelin. Immediately after the depot administration, the relative amounts of secretory granules drastically decreased in gonadotropes concomitantly with a marked increase in the plasma LH level. After the acute hyperstimulated phase, secretory granules in gonadotropes were gradually restored although the newly synthesized granules were less densely immunolabeled for LHbeta; their relative amounts and sizes were still significantly smaller than the controls after depot treatment for 28 days. Eighty-four days after the leuprorelin depot administration, however, the ultrastructural characteristics of pituitary gonadotropes appeared to recover as observed in controls: there were no significant differences in the relative amounts, sizes, and labeling densities for LHbeta of secretory granules, and the amounts of chromogranin A (CgA) and secretogranin II (SgII) were restored in secretory granules to control levels. When the rats were repeatedly treated with the leuprorelin depot at intervals of 4 weeks, the expression and intracellular storage levels of gonadotropins remained highly suppressed, judging from the labeling density for LHbeta. These findings suggest that the depot formulation of the GnRH agonist could suppress both the biosynthesis and release of gonadotropins for a month by synergistically depleting the intracellular storage of secretory granules at the onset of the treatment and by inducing the subsequent desensitization of the GnRH receptor signaling.

A key step in the intrinsic apoptotic pathway is the assembly of the apoptosome complex. The apoptosome components are well known; however, the physiology of the assembly of the apoptosome complex at the cellular level is still poorly defined. The aim of this work was to study the subcellular distribution of the apoptosome scaffold protein apoptotic protease-activating factor 1 (Apaf-1) before and after triggering apoptosis in single somatotrophs. Somatotrophs are the subject of extensive pituitary tissue remodeling in different physiological situations in which the quality and the number of pituitary cells are determined by cell proliferation and apoptosis. We show herein that 2 h after triggering apoptosis with rotenone, Apaf-1 redistributed to the proximity of mitochondria. In addition, the degree of colocalization between Apaf-1 and fluorescently labeled caspase-9 significantly increased during the same period. Furthermore, we show herein for the first time in single cells that the colocalization between Apaf-1 and cytochrome c increases only transiently, indicating a transient interaction between cytochrome c and Apaf-1 during the activation of apoptosis in these cells.

The pituitary gland is a heterogeneous tissue comprised of several hormone-secreting cells most of which are targeted by sex steroids. Our long-term studies were concentrated on the response of rat pituitary TSH cells to gonadal steroids applied to animals of different age. With this goal, we examined immunoreactive and morphometric, as well as subcellular organization of pituitary TSH cells in rats of both sexes after neonatal and perinatal estradiol-dipropionate (EDP) treatment. The results undoubtedly indicated persistent EDP-related inhibitory changes of tyrotrophs up to the adulthood. At the subcellular level, a delayed differentiation of TSH cells was noticed. Besides, a special attention has been paid to the changes in the structure of immunoreactive TSH cells of middle-aged (14-month-old) rat females, chronically treated with EDP, calcium (Ca) or a combination of EDP and Ca. Based on our results it can be concluded that both EDP and Ca act inhibiting the thyrotrophs under the applied experimental conditions. Hipofiza je heterogeno tkivo sastavljeno od nekoliko vrsta ćelija koje luče hormone, a na većinu od njih deluju polni hormoni. Naša višegodišnja istraživanja su bila usmerena na proučavanje odgovora hipofiznih TSH ćelija pacova različite starosti na polne steroide. U tom cilju morfometrijski je ispitivana imunoreaktivnost i subćelijska organizacija TSH ćelija hipofize pacova oba pola posle tretiranja estradiol dipropionatom tokom neonatalnog ili perinatalnog perioda života. Dobijeni rezultati nesumnjivo ukazuju na trajne inhibitorne promene tirotropnih ćelija izazvane estradiolom. Te promene se zapažaju sve do zrelosti. Na subćelijskom nivou je uočena odložena diferencijacija TSH ćelija. Osim toga, posebna pažnja je posvećena strukturnim promenama imunoreaktivnih TSH ćelija menopauzalnih ženki pacova (14 meseci), hronično tretiranih estradiolom, ili kalcijumom, ili kombinacijom estradiola i kalcijuma. Na osnovu naših rezultata se može zaključiti da pri primenjenim eskperimentalnim uslovima i estradiol i kalcijum inhibiraju tirotropne ćelije hipofize.

We used modified immunocytochemical conditions to quantify a membrane form of estrogen receptor-α (mERα) in a rat pituitary tumor cell line, GH3/B6/F10. We studied the regulation of mERα vs. levels of intracellular ERα (iERα) using our 96-well plate immunoassay. The anti-ERα antibody C542 was used to label the ERα (via conjugated alkaline phosphatase) in fixed permeabilized (for iERα) vs. nonpermeabilized cells (for mERα). Expression of mERα was highest at low cell densities (