Induction of PER1 mRNA expression in immortalized gonadotropes by gonadotropin‐releasing hormone (GnRH): Involvement of protein kinase C and MAP kinase signaling

The initiation and maintenance of reproductive function in mammals is critically dependent on the pulsatile secretion of gonadotropin‐releasing hormone (GnRH). This peptide drives the pulsatile release of FSH and LH from the pituitary pars distalis via signaling pathways that are activated by the type I GnRH receptor (GnRH‐R). Recently, a microarray analysis study reported that a number of genes, including mPer1, are induced by GnRH in immortalized gonadotrope cells. In view of these data, we have begun to analyze in detail the signaling pathways mediating the action of GnRH on mPer1 expression in these cells. Using quantitative real‐time polymprose cho read (PCR), we could confirm that exposure of immortalized gonadotropes (LβT2 cells) to the GnRH analog, buserelin, markedly induces mPer1 (but not mPer2) expression. Consistent with GnRH receptor signaling via the protein kinase (PK)‐C pathway, exposure of the cells to phorbol 12,13‐dibutyrate rapidly elevates both mPer1 and LHβ subunit mRNA levels, while pharmacological inhibition of PKC prevents the mPer1 and LHβ response to buserelin. As GnRH is known to regulate gonadotropin synthesis via activation of p42/44 mitogen‐activated protein kinase (MAPK) signaling pathways, we then examined the involvement of this pathway in regulating mPer1 expression in gonadotropes. Our data reveal that GnRH‐induced mPer1 expression is blocked following acute exposure to a MAPK kinase inhibitor. Although the involvement of this signaling mechanism in the regulation of mPer1 is known in neurons, e.g., in the suprachiasmatic nuclei, the induction of mPer1 in gonadotropes represents a novel mechanism of GnRH signaling, whose functional significance is still under investigation.     

Assessment of “activation” of estrogen receptors in lactating mammary gland using DNA-cellulose binding

To circumvent the need for isolated nuclei in studies on activation of estrogen-receptor complexes in mammary gland of the rat, a DNA-cellulose binding assay was employed using a cell-free system. Incubation at 28°C for 30 min of receptors previously charged with [³H]estradiol markedly enhanced their association with DNA-cellulose. Once activated, estrogen-receptor complexes bound maximally to DNA-cellulose within 20–30 min. The temperature optimum for activation was 28 ± 2°C using cytosol preparations. The temperature-induced activation required the presence of both steroid and cytosolic receptors simultaneously. Density gradient centrifugation revealed that, unlike those of uterus, both activated and charged estrogen-receptor complexes of lactating mammary tissue sedimented as a 4 S species in sucrose gradients containing 0.4 M KCl.     

Paracoccidioidomycosis in the region of Botucatu (state of São Paulo,Brazil). Evaluation of serum thyroxine (T4) and triiodothyronine (T3) levels and of the response to thyrotropin releasing hormone (TRH)

T₄, T₃ and TSH serum levels were measured in 25 patients with paracoccidioidomycosis. Thyroid T₃ reserves were measured on the basis of the increase in T₃ (T₃) 2 h after intravenous injection of 200 g TRH, and pituitary TSH reserves were measured on the basis of TSH increase (TSH) 20 min after the same injection. Twenty healthy volunteers with no history of thyroid disease were used as controls. When the two groups were compared, the following results were obtained: (a) there was no significant difference in mean T₄, T₃, TSH between groups; (b) reduced T₃ levels were detected more frequently in patients with paracoccidioidomycosis, especially among those with the acute form of the disease or with the severely disseminated chronic form. The results suggest the occurrence of a reduction in peripheral conversion of T₄ to T₃, but do not indicate the occurrence of hypothyroidism in any of its forms (thyroid, pituitary or hypothalamic).     

Gonadotropin releasing hormone stimulation of pituitary cell 3′–5′ cyclic AMP in a carp(Cyprinus carpio) is dependent on extracellular calcium

Pituitaries were collected from a common carp,yprinss carpi, belonging to vitellogenic phase and cells were disaggregated by using 0.3% collagenase and 0.05% tsypsin. Enzymatically dispersed cells were incubatedin vitro in Ca²⁺-free medium to observe the effect ofCanna punctatus GnRH (cGnRH) and Ca²⁺ on pituitary cell cAMP accumulation. Addition of cGnRH (20 Big) to pituitary cell incubation (6 × 10⁴ cells/well) containing 4 mM theophylline, a phosphodiesterase inhibitor, caused two-fold increase of cAMP accumulation in comparison to control, Addition of Ca²⁺ (2 mM) to cGnRH further augmented cAMP accumulation, i.e., four-fold as compared to control. Increasing concentrations of cGnRH in the presence of Ca²⁺ resulted in a dose-dependent increase in cAMP accumulation. To examine the specificity of Ca²⁺ augmentory effect on cGnRH-stimulated pituitary cell cAMP accumulation, a specific Ca²⁺-channel blocker, verapamil was used, At 3 μM dose verapamil completely waived Ca²⁺-augmentation of cGnRH stimulatory effect on cAMP. Interestingly, verapamil also significantly inhibited cGnRH stimulation of cAMP in the Ca²⁺-free medium. Extent of Ca²⁺ plus cGnRH stimulatory effect on cAMP was further increased by the addition of 25 pmol of calmodulin, a Ca²⁺-carrier protein, Addition of verapamil to this system strongly inhibited Ca²⁺ and ealmodulin augnientory effect on cGnRH. Reduced level of cAMP in the pituitary cell due to verapamil was even lower than that of cGnRH plus ealmodulin incubation. Data indicates a contamination of Ca²⁺ in an apparently Ca²⁺-free medium, Results suggest that in lower vertebrate, i.e., fish, GnRH stimulation of pituitary cell cAMP is dependent on extracellulnr Ca²⁺ and incubation of pituitary cell in Ca²⁺-free medium is truly not free of Ca²⁺.     

Immunocytochemistry of a “private” luteinizing-hormone-releasing hormone system in the pituitary

Summary Immunocytochemistry of paraffin sections of Bouin-fixed rat pituitaries with antiserum to luteinizinghormone-releasing hormone (LHRH) revealed two types of cells. Type I cells exhibit granular staining throughout their cytoplasm. The immunoreactivity of type II cells is confined to a much smaller area of the cytoplasm. Type I cells are located in the ventral margin of the pars intermedia, the region between the pars intermedia and the pars distalis, and the pars distalis adjacent to this region. Type II cells have a broader distribution in the pars distalis, but tend to concentrate in the region of the pars distalis near the pars intermedia. Type I cells are distinct from gonadotropes. Type II cells appear to comprise a subgroup of gonadotropes. Staining in type I, but not type II, cells in pituitary explants, maintained in serum-free media for seven days, is as intense as that in normal pituitary tissue. The data suggest that the type I cells are producing an intrinsic LHRH-like material and may be responsible, in part, for the regulation of some gonadotropes.Supported by NIH grants HD12932, NS15843 and NS15809 (LAS), National Science Foundation grant BNS 82-05643 (LAS), and a grant from the Phillippe Foundation (JYL)     

Serum progesterone concentrations,follicular development and time of ovulation using a new progesterone releasing device (DICO®) in sheep

Four experiments were conducted to evaluate the effectiveness of a new controlled drug releasing device containing 0.3 g progesterone (DICO^®) on ovarian control in sheep. In experiment 1, serum progesterone concentrations induced by a 14 days treatment of DICO^® (n = 9) and CIDR-G^® (n = 9) were compared in ovariectomized ewes. Both devices induced similar responses and no differences were recorded. In experiment 2, the onset of oestrus and the time of ovulation obtained after 14 days treatment with DICO^® (n = 8) and CIDR-G^® (n = 7) were compared in cyclic ewes. Both devices induced oestrus and ovulation in all of the ewes. The onset of oestrus (34.5 ± 2.8 and 30.0 ± 7.7 h), the time of ovulation (60.0 ± 9.1 and 54.9 ± 6.4 h), the ovulation rate (1.3 ± 0.5 and 1.4 ± 0.5), the follicular diameter at ovulation (7.0 ± 0.8 and 7.3 ± 1.1 mm), and the lifespan of the ovulatory follicles (8.6 ± 2.2 and 10.0 ± 2.9 days) were similar for the DICO^® and CIDR-G^® devices, respectively. In Experiment 3, the re-utilization of DICO^® devices inserted for 6 days (i.e. short-term protocol) was evaluated in ovariectomized ewes. The females received a re-used (previously used for 6 days; n = 11) or a new DICO^® (n = 11) for a period of 6 days. The re-used DICO^® devices induced a lower serum progesterone concentration than the new devices (P < 0.05). However, the re-used DICO^® device maintained serum progesterone concentrations above 7.1 nmol/L (i.e. >2 ng/ml) throughout treatment. In Experiment 4, the administration of eCG treatment at DICO^® withdrawal was evaluated in cyclic ewes. The short-term protocol using DICO^® devices for 6 days was applied with (n = 8) or without (n = 7) 300 IU eCG at the time of device withdrawal. The administration of eCG advanced ovarian follicular development, synchronizing the onset of oestrus at 36 h and the time of ovulation at 60 h from device withdrawal. In conclusion, data from these experiments show the use of DICO^® or CIDR-G^® devices containing 0.3 g of progesterone to have a similar efficiency in controlling serum progesterone concentrations, follicular development and the time of ovulation in sheep. The re-use of the devices, associated with the short-term protocol for 6 days is possible, although further studies on induced fertility rates are warranted.     

Molecular characterization of gonadotropin subunits (fshβ, lhβ and cgα) of largemouth bass (micropterus salmoides) and their expression in response to luteinizing hormone-releasing hormone analogue and dopamine antagonists

Gonadotropins (GtHs), including follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are important hormones involved in gametogenesis, gonadal steroidogenesis, and maturation. In this study, GtH subunits (FSHβ, LHβ and CGα) of largemouth bass (Micropterus salmoides) were cloned and characterized, and their regulation by luteinizing hormone-releasing hormone analog (LHRHa2) and dopamine antagonist (DOM) treatment in vivo was investigated. The full-length cDNA sequences of FSHβ, LHβ, and CGα were 683, 576, and 685bp, encoding 120, 152, and 132 amino acids, respectively. The deduced amino acid sequence analysis revealed that GtH subunits contain conserved cysteine residues and potential N-linked glycosylation sites and showed high homology with the corresponding subunit sequences from other Perciformes by phylogenetic analysis. In the primary growth cortical alveoli stage of largemouth bass, the three GtH subunits were highly expressed in the pituitary, brain and ovary, but weakly in other tissues. After LHRHa2 and DOM injection, mRNA levels of FSHβ, LHβ, and CGα in the pituitary were significantly increased at 12 or 24 hr (p <.05), but significantly decreased at 48 hr; plasma FSH and LH levels showed a consistent trend. Histological analysis classified ovaries from LHRHa2- and DOM-treated fish into the early oocyte maturation stage and late vitellogenesis stage, while those from the control group fish were classified into the early vitellogenesis stage. These results suggested that both LHRHa2 and DOM stimulated GtH subunit expression, increased plasma FSH and LH and accelerated ovary development of largemouth bass, providing a framework for a better understanding of the mechanisms of hormone-mediated reproduction control in teleosts.     

Pharmacokinetics of recombinant human growth hormone administered by cool.click™ 2, a new needle-free device,compared with subcutaneous administration using a conventional syringe and needle

Background

Growth hormone (GH) is used to treat growth hormone deficiency (GHD, adult and paediatric), short bowel syndrome in patients on a specialized diet, HIV-associated wasting and, in children, growth failure due to a number of disorders including Turner's syndrome and chronic renal failure, and in children born small for gestational age. Different brands and generic forms of recombinant human growth hormone (r-hGH) are approved for varying indications in different countries. New ways of administering GH are required because the use of a needle and syringe or a device where a patient still has to insert the needle manually into the skin on a daily basis can lead to low adherence and sub-optimal treatment outcomes. The objective of this study was to assess the relative bioavailability of r-hGH (Saizen^®, Merck Serono) administered by a new needle-free device, cool.click? 2, and a standard needle and syringe.

Methods

The study was performed with 38 healthy volunteers who underwent pituitary somatotrope cell down-regulation using somatostatin, according to a randomized, two-period, two-sequence crossover design. Following subcutaneous administration of r-hGH using cool.click? 2 or needle and syringe, pharmacokinetic parameters were analysed by non-compartmental methods. Bioequivalence was assessed based on log-transformed AUC and C_max values.

Results

The 90% confidence intervals for test/reference mean ratio of the plasma pharmacokinetic variables C_max and AUC_0-inf were 103.7–118.3 and 97.1–110.0, respectively, which is within the accepted bioequivalence range of 80–125%. r-hGH administered by cool.click? 2 is, therefore, bioequivalent to administration by needle and syringe with respect to the rate and extent of GH exposure. Treatment using cool.click? 2 was found to be well tolerated. With cool.click? 2 the t_max was less (3.0 hours) than for needle and syringe delivery (4.5 hours), p = 0.002 (Friedman test), although this is unlikely to have any clinical implications.

Conclusion

These results demonstrate that cool.click? 2 delivers subcutaneous r-hGH exposure that is bioequivalent to the conventional mode of injection. The new device has the additional advantage of being needle-free, and should help to increase patient adherence and achieve good therapeutic outcomes from r-hGH treatment.

     

Antimüllerian hormone (AMH) not only a marker for prediction of ovarian reserve

The main characteristics of the Antimüllerian hormone from the points of view of biochemistry, molecular genetics, physiological functions and importance for diagnostics in reproductive endocrinology and related biomedical fields are reviewed. The role of the hormone in male and female development, its participation in oocyte maturation including selection of a dominant follicle are summarized, as well as its changes under various pathological situations in both sexes. The physiological changes of serum AMH leves in the life span in both sexes and their alterations under various pathological conditions are provided, too.     

Temporal relationships of a pulse of prolactin (PRL) to a pulse of a metabolite of PGF2α in mares

Hourly blood samples were collected from 10 mares during 24 h of each of the preluteolytic, luteolytic, and postluteolytic periods. The autocorrelation function of the R program was used to detect pulse rhythmicity, and the intra-assay CV was used to locate and characterize pulses of prolactin (PRL) and a metabolite of prostaglandin F2α (PGFM). Rhythmicity of PRL and PGFM concentrations was detected in 67% and 89% of mares, respectively. Combined for the three periods (no difference among periods), the PRL pulses were 5.2 ± 0.4 h (mean ± SEM) at the base, 7.5 ± 1.5 h between nadirs of adjacent pulses, and 12.3 ± 1.5 h from peak to peak. The peaks of PRL pulses were greater (P < 0.05) during the luteolytic period (46 ± 14 ng/mL) and postluteolytic period (52 ± 15 ng/mL) than during the preluteolytic period (17 ± 3 ng/mL). Concentrations of PRL during hours of a PGFM pulse were different (P < 0.003) within the luteolytic period and postluteolytic period and were greatest at the PGFM peak; PRL concentrations during a PGFM pulse were not different during the preluteolytic period. The frequency of the peak of PRL and PGFM pulses occurring at the same hour (synchrony) was greater for the luteolytic period (65%, P < 0.01) and postluteolytic period (50%, P < 0.001) than for the preluteolytic period (17%). This is the first report in mares on characterization and rhythmicity of PRL pulses, synchrony between PRL and PGFM pulses, and greater PRL activity during the luteolytic and postluteolytic periods than during the preluteolytic period.     

Evaluation of canopy transpiration rate by applying a plant hormone “abscisic acid”

A method for evaluation of temporal changes in canopy transpiration rate and stomatal conductance in crop fields by using a plant hormone abscisic acid (ABA) has recently been developed. The method was applied to a corn canopy at different growth stages in the upper Yellow River basin, China. Diurnal changes in the canopy transpiration rate and stomatal conductance were evaluated at the initial stage with a leaf area index (LAI) of 0.37 on June 7 and the crop development stage with an LAI of 4.39 on July 15, 2005. The proportions of the accumulated transpiration rate during daytime to the accumulated evapotranspiration were 24% and 74% at the initial and crop development stages, respectively. Stomatal conductance varied in parallel with transpiration rate in the initial stage of the crop. However, in the crop development stage with low soil water content, stomatal conductance reached the maximum value at 10:00 a.m. and thereafter decreased rapidly at around noon with high evaporative demand to corn canopy. This shows the midday stomatal closure was caused by excessive water stress to corn canopy in the crop development stage. Thus, the proposed method with ABA application is useful for evaluation of temporal changes in transpiration rate and stomatal conductance, and hence, can detect the plant water stress.     

An attempt to construct a (general) mathematical framework to model biological “context-dependence”

Context-dependent nature of biological phenomena is well documented in every branch of biology. While there have been few previous attempts to (implicitly) model various (particular) facets of biological context-dependence, a formal and general mathematical construct to model the wide spectrum of context-dependence, eludes the students of biology. Such an objective model, from both ‘bottom-up’ as well as ‘top-down’ perspective, is proposed here to serve as the template to describe the various kinds of context-dependence that we encounter in different branches of biology. Interactions between biological contexts was found to be transitive but non-commutative. It is found that a hierarchical nature of dependence among the biological contexts models the emergent biological properties efficiently. Reasons for these findings are provided in a general model to describe biological reality. Scheme to algorithmically implement the hierarchic structure of organization of biological contexts was proposed with a construct named ‘Context tree’. A ‘Context tree’ based analysis of context interactions among biophysical factors influencing protein structure was performed.     

“Broadbeam” irradiation of mammalian cells using a vertical microbeam facility

A “broadbeam” facility is demonstrated for the vertical microbeam at Surrey’s Ion Beam Centre, validating the new technique used by Barazzuol et al. (Radiat Res 177:651–662, 2012). Here, droplets with a diameter of about 4 mm of 15,000 mammalian cells in suspension were pipetted onto defined locations on a 42-mm-diameter cell dish with each droplet individually irradiated in “broadbeam” mode with 2 MeV protons and 4 MeV alpha particles and assayed for clonogenicity. This method enables multiple experimental data points to be rapidly collected from the same cell dish. Initially, the Surrey vertical beamline was designed for the targeted irradiation of single cells with single counted ions. Here, the benefits of both targeted single-cell and broadbeam irradiations being available at the same facility are discussed: in particular, high-throughput cell irradiation experiments can be conducted on the same system as time-intensive focused-beam experiments with the added benefits of fluorescent microscopy, cell recognition and time-lapse capabilities. The limitations of the system based on a 2 MV tandem accelerator are also discussed, including the uncertainties associated with particle Poisson counting statistics, spread of linear energy transfer in the nucleus and a timed dose delivery. These uncertainties are calculated with Monte Carlo methods. An analysis of how this uncertainty affects relative biological effect measurements is made and discussed.     

Designing a new biosensor “DNA ELISA” to detect Escherichia coli using genomic DNA and comparison of this method to PCR-ELISA

In this experiment, DNA-ELISA biosensor was introduced, bearing the ability to detect specific bacteria in about 4?h. This is a more rapid system in comparison to conventional methods, like colony counting method. Moreover, this method does not require any amplification and directly detects genomic DNA of bacteria, giving a lower limit to the sensitivity of 40,000 bacteria. In this study, two specific probes capture (biotin labelled) and detector (dig labelled), were used against special regions of 16s rRNA gene of Escherichia coli ATCC 25922. The capture probe has the ability to trap the target bacterial DNA from a pool of other kinds of bacteria under specific conditions. The detector probe then was used to hybridize to the genome of trapped bacteria. The detection proceeds by adding HRP-anti dig enzyme and its substrate, ABTS to emit light. Light absorbance is measured for verifying the detection.     

Constitutive cholesterol-dependent endocytosis of melanocortin-4 receptor (MC4R) is essential to maintain receptor responsiveness to α-melanocyte-stimulating hormone (α-MSH)

Melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor expressed in the hypothalamus where it controls feeding behavior. MC4R cycles constitutively and is internalized at the same rate in the presence or absence of stimulation by the agonist, melanocyte-stimulating hormone (α-MSH). This is different from other G-protein-coupled receptors, such as β(2)-adrenergic receptor (β(2)AR), which internalizes more rapidly in response to agonist stimulation. Here, it is found that in immortalized neuronal Neuro2A cells expressing exogenous receptors, constitutive endocytosis of MC4R and agonist-dependent internalization of β(2)AR were equally sensitive to clathrin depletion. Inhibition of MC4R endocytosis by clathrin depletion decreased the number of receptors at the cell surface that were responsive to the agonist, α-MSH, by 75%. Mild membrane cholesterol depletion also inhibited constitutive endocytosis of MC4R by ～5-fold, while not affecting recycling of MC4R or agonist-dependent internalization of β(2)AR. Reduced cholesterol did not change the MC4R dose-response curve to α-MSH, but it decreased the amount of cAMP generated per receptor number indicating that a population of MC4R at the cell surface becomes nonfunctional. The loss of MC4R function increased over time (25-50%) and was partially reversed by mutations at putative phosphorylation sites (T312A and S329A). This was reproduced in hypothalamic GT1-7 cells expressing endogenous MC4R. The data indicate that constitutive endocytosis of MC4R is clathrin- and cholesterol-dependent. MC4R endocytosis is required to maintain MC4R responsiveness to α-MSH by constantly eliminating from the plasma membrane a pool of receptors modified at Thr-312 and Ser-329 that have to be cycled to the endosomal compartment to regain function.     

Reversible switching of coordination modes of nickel(ΙΙ) complexes using a hemilabile 4,7-diazadecanediamide ligand

The reaction of NiCl₂·6H₂O with a hemilabile diaminodiamide ligand 4,7-diazadecanediamide gave a pale blue compound [Ni(C₈H₁₈N₄O₂)(H₂O)₂]·2Cl (1). In basic solution, 1 was transformed to a yellow species [Ni(C₈H₁₆N₄O₂)]·3H₂O (2) by the Ni-O to Ni-N bond rearrangement at two amide sites of the complex. Structures of 1 and 2 were characterized by single-crystal X-ray diffraction analysis. Structural data for 1 indicated that the nickel atom adopts a six-coordinated N₂O₄ environment and gives an octahedral geometry. The structure of 2 showed that the nickel atom adopts a four-coordinated N₄ environment, giving a square-planar geometry. Compounds 1 and 2 are structurally switchable in response to pH in an aqueous solution. The results presented could be useful in the design of new Ni(II)-based switching materials.