Hubertus Jarry

Hippocampal Synapses Depend on Hippocampal Estrogen Synthesis

Estrogens have been described to induce synaptogenesis in principal neurons of the hippocampus and have been shown to be synthesized and released by exactly these neurons. Here, we have focused on the significance of local estrogen synthesis on spine synapse formation and the synthesis of synaptic proteins. To this end, we reduced hippocampal estrogen synthesis in vitro with letrozole, a reversible nonsteroidal aromatase inhibitor. In hippocampal slice cultures, letrozole treatment resulted in a dose-dependent decrease of 17β-estradiol as quantified by RIA. This was accompanied by a significant decrease in the density of spine synapses and in the number of presynaptic boutons. Quantitative immunohistochemistry revealed a downregulation of spinophilin, a marker of dendritic spines, and synaptophysin, a protein of presynaptic vesicles, in response to letrozole. Surprisingly, no increase in the density of spines, boutons, and synapses and in spinophilin expression was seen after application of estradiol to the medium of cultures that had not been treated with letrozole. However, synaptophysin expression was upregulated under these conditions. Our results point to an essential role of endogenous hippocampal estrogen synthesis in the maintenance of hippocampal spine synapses.

Chaste tree (Vitex agnus-castus)--pharmacology and clinical indications.

Extracts of the fruits of chaste tree (Vitex agnus castus = AC) are widely used to treat premenstrual symptoms. Double-blind placebo-controlled studies indicate that one of the most common premenstrual symptoms, i.e. premenstrual mastodynia (mastalgia) is beneficially influenced by an AC extract. In addition, numerous less rigidly controlled studies indicate that AC extracts have also beneficial effects on other psychic and somatic symptoms of the PMS. Premenstrual mastodynia is most likely due to a latent hyperprolactinemia, i.e. patients release more than physiologic amounts of prolactin in response to stressful situations and during deep sleep phases which appear to stimulate the mammary gland. Premenstrually this unphysiological prolactin release is so high that the serum prolactin levels often approach heights which are misinterpreted as prolactinomas. Since AC extracts were shown to have beneficial effects on premenstrual mastodynia serum prolactin levels in such patients were also studied in one double-blind, placebo-controlled clinical study. Serum prolactin levels were indeed reduced in the patients treated with the extract. The search for the prolactin-suppressive principle(s) yielded a number of compounds with dopaminergic properties: they bound to recombinant DA2-receptor protein and suppressed prolactin release from cultivated lactotrophs as well as in animal experiments. The search for the chemical identity of the dopaminergic compounds resulted in isolation of a number of diterpenes of which some clerodadienols were most important for the prolactin-suppressive effects. They were almost identical in their prolactin-suppressive properties than dopamine itself. Hence, it is concluded that dopaminergic compounds present in Vitex agnus castus are clinically the important compounds which improve premenstrual mastodynia and possibly also other symptoms of the premenstrual syndrome.

miR-212 and miR-132 are required for epithelial stromal interactions necessary for mouse mammary gland development

MicroRNAs are small noncoding RNAs that carry out post-transcriptional regulation of the expression of their target genes. However, their roles in mammalian organogenesis are only beginning to be understood. Here we show that the microRNA-212/132 family (which comprises miR-212 and miR-132) is indispensable during the development of the mammary glands in mice, particulary for the regulation of the outgrowth of the epithelial ducts. Mammary transplantation experiments revealed that the function of the miR-212/132 family is required in the stroma but not in the epithelia. Both miR-212 and miR-132 are expressed exclusively in mammary stroma and directly target the matrix metalloproteinase MMP-9. In glands that lack miR-212 and miR-132, MMP-9 expression increases and accumulates around the ducts. This may interfere with collagen deposition and lead to hyperactivation of the tumor growth factor-β signaling pathway, thereby impairing ductal outgrowth. Our results identify the miR-212/132 family as one of the main regulators of the epithelial-stromal interactions that are required for proper pubertal development of the mammary gland.

Phytoestrogens: endocrine disrupters or replacement for hormone replacement therapy?

OBJECTIVES This review presents findings with clear statements from the literature as well as own results of effects of soy, red clover and their isoflavones as well as of the Cimicifuga racemosa extract BNO 1055. Experimental and clinical effects on climacteric complaints, osteoprotective effects, activity in the urogenital tract, and risks concerning cardiovascular diseases and mammary and endometrial tissue will be compared, also in comparison to classical hormone preparations. The question whether soy and red clover products and/or Cimicifuga racemosa (CR) preparations are endocrine disrupters or may fulfill the criteria of the so-called phyto-SERMs will be discussed. METHODS Review of selected publications since 1980 and summary of unpublished own results of the authors. RESULTS Experimental and clinical evidences suggest that soy/red clover and their isoflavones do not fulfill the criteria of an ideal SERM. They appear to have mild osteoprotective effects but do not improve climacteric complaints. Furthermore, they seem to stimulate uterine growth and mammary epithelial proliferation. In ovariectomized rats, the CR extract BNO 1055 showed many of the beneficial effects of 17beta-estradiol, including effects in the brain/hypothalamus to reduce serum LH levels, effects in the bone to prevent osteoporosis and estrogenic effects in the urinary bladder. The CR extract BNO 1055 had no uterotrophic effect. CONCLUSION If clinical studies confirm these results, the Cimicifuga racemosa preparation BNO 1055 would appear as an ideal SERM and may therefore be an alternative to hormone replacement therapy.

Amino Acid Neurotransmitter Release in the Preoptic Area of Rats during the Positive Feedback Actions of Estradiol on LH Release

To investigate the role of amino acid neurotransmitters in the regulation of LH secretion in ovariectomized (ovx) rats with or without estrogen substitution, we measured the release rates of gamma-aminobutyric acid (GABA), taurine, glycine, aspartate, glutamate, homocysteic acid, and also of the neurally inactive amino acids serine and glutamine in push-pull perfusate samples of the preoptic/anterior hypothalamic area (PO/AH) collected at 30-min intervals. To achieve this we had to develop a highly sensitive assay utilizing phenylisothiocyanate prederivatization which was followed by HPLC chromatography. In confirmation of our earlier results we observed again a conspicuous drop of preoptic GABA release prior to and during the time of estrogen-induced LH surge. In addition, the release rates of the excitatory amino acid neurotransmitters aspartate and glutamate in the PO/AH increased during this time. Interestingly, also secretion of taurine and glycine was increased during the LH surge, whereas preoptic release rates of serine and glutamine and of homocysteic acid, the putative endogenous ligand of the so-called N-methyl-D-aspartate receptor, remained unchanged. No such changes of amino acid neurotransmitters release rates were observed in ovx rats. This finding underlines that the changes of amino acid secretion in ovx estrogen-primed rats are likely due to the influence of the steroid rather than due to a diurnal rhythm. We conclude that GnRH neurons are under a tonic inhibitory tone exerted by GABA which is relieved during the time of the estrogen-induced LH surge. During this time, aspartate and glutamate may have additional stimulatory effects on GnRH neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin-releasing hormone regulates spine density via its regulatory role in hippocampal estrogen synthesis

Spine density in the hippocampus changes during the estrus cycle and is dependent on the activity of local aromatase, the final enzyme in estrogen synthesis. In view of the abundant gonadotropin-releasing hormone receptor (GnRH-R) messenger RNA expression in the hippocampus and the direct effect of GnRH on estradiol (E2) synthesis in gonadal cells, we asked whether GnRH serves as a regulator of hippocampal E2 synthesis. In hippocampal cultures, E2 synthesis, spine synapse density, and immunoreactivity of spinophilin, a reliable spine marker, are consistently up-regulated in a dose-dependent manner at low doses of GnRH but decrease at higher doses. GnRH is ineffective in the presence of GnRH antagonists or aromatase inhibitors. Conversely, GnRH-R expression increases after inhibition of hippocampal aromatase. As we found estrus cyclicity of spine density in the hippocampus but not in the neocortex and GnRH-R expression to be fivefold higher in the hippocampus compared with the neocortex, our data strongly suggest that estrus cycle–dependent synaptogenesis in the female hippocampus results from cyclic release of GnRH.

In vitro effects of the Cimicifuga racemosa extract BNO 1055.

OBJECTIVES Extracts of Black cohosh (Cimicifuga racemosa or CR) have been used for the treatment of climacteric complaints since decades. Efficacy, particularly concerning neurovegetative and psychic symptoms, has been proven in clinical trials. As active principle yet unknown substances with selective estrogen receptor modulator (SERM) activity are assumed. Recently, evidence arose that CR may also contain dopaminergic compounds, which may contribute to the therapeutic activity of the extract. METHODS Two subtypes of the estrogen receptor (ERalpha and ERbeta) are known. To examine, whether active substances of CR extract BNO 1055 (which is contained in Klimadynon and Menofem) bind to either of the two estrogen receptors, subtype-specific estrogen receptor ligand-binding assays with recombinant ERalpha or ERbeta were conducted. A ligand-binding assay with recombinant dopamine D(2)-receptor protein was employed to assess possible dopaminergic activity in the CR extract BNO 1055. RESULTS While a displacement of radiolabeled estradiol from binding sites of a cytosol preparation from procine and human endometrium by CR extract BNO 1055 was shown no such displacement was achieved when either ERalpha or ERbeta protein was used as ligands for tracer. Dopaminergic activity in the CR extract BNO 1055 could be demonstrated with the D(2)-receptor assay. A countercurrent chromatography resulted in a separation of estrogenic and dopaminergic activity in two distinct fractions. CONCLUSIONS It is suggested that not yet identified substances in the CR extract BNO 1055 bind to a yet unknown estrogen-binding site in the endometrium. Also, yet unknown dopaminergic compounds may contribute to the pharmacological profile of CR extract BNO 1055.

Proliferation and apoptosis of hippocampal granule cells require local oestrogen synthesis

Ovarian oestrogens have been demonstrated to influence neurogenesis in the dentate gyrus. As considerable amounts of oestrogens are synthesized in hippocampal neurones, we focused on the role of hippocampus‐derived estradiol on proliferation and apoptosis of granule cells in vitro. We used hippocampal dispersion cultures, which allowed for cultivation of the cells under steroid‐ and serum‐free conditions and monitoring of oestrogen synthesis. To address the influence of hippocampus‐derived estradiol on neurogenesis, we inhibited oestrogen synthesis by treatment of hippocampal cell cultures with letrozole, a specific aromatase inhibitor. Alternatively, we used siRNA against steroidogenic acute regulatory protein (StAR). The number of proliferative cells decreased whereas the number of apoptotic cells increased dose‐dependently, in response to reduced estradiol release into the medium after treatment with letrozole. This also held true for siRNA against StAR transfected cell cultures. Application of estradiol to the medium had no effect on proliferation and apoptosis whereas the anti‐proliferative and pro‐apoptotic effects of StAR knock‐down and letrozole treatment were restored by treatment of the cultures with estradiol. Our findings suggest that neurogenesis and apoptosis in the hippocampus require a defined range of estradiol concentrations that is physiologically provided by hippocampal cells but not by gonads.

Gamma-Aminobutyric Acid Neurons in the Preoptic/Anterior Hypothalamic Area Synchronize the Phasic Activity of the Gonadotropin-Releasing Hormone Pulse Generator in Ovariectomized Rats

To achieve a bolus-type release of gonadotropin-releasing hormone (GnRH) into the portal vessels it is required that GnRH neurons exert phasic and synchronous activity. The activity of GnRH neurons appears to be under an inhibitory influence of the amino acid neurotransmitter gamma-aminobutyric acid (GABA). Preoptic GABA concentrations in ovariectomized (OVX) rats decrease prior to a luteinizing hormone (LH) episode. This reduction of GABAergic activity in the preoptic/anterior hypothalamic area (PO/AH) may be the synchronizing signal for the simultaneous release of GnRH from the hypothalamus. To further study the role of GABA in controlling the GnRH pulse generator we applied GABA, 3-mercaptopropionic acid (MPA) or bicuculline (BIC) locally into the PO/AH by means of push-pull cannulae (PPC). PPC were implanted into the PO/AH of OVX rats and the contralateral, not PPC-implanted PO/AH was lesioned electrochemically. The effects of GABA, MPA or BIC on the GnRH pulse generator were determined by measuring LH levels in blood samples collected in 5-min intervals. Local application of GABA into the PO/AH caused a pronounced reduction of average LH secretion and abolished LH pulsatility. This inhibitory effect was completely reversible. Results of intrapreoptic MPA application on GABA secretion were variable. In only 45% of treated rats MPA caused a reduction of GABA secretion which was associated with a cessation of pulsatile LH release. A pronounced reduction of LH secretion and pulsatility was observed upon local application of the GABA antagonist BIC. Based on these data we propose that oscillating GABA levels in the PO/AH may be the synchronizing signal which triggers bolus release of GnRH into the portal vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine disruptors and the thyroid gland--a combined in vitro and in vivo analysis of potential new biomarkers.

Background There is growing evidence that, in addition to the reproductive system, the hypothalamic–pituitary–thyroid axis is a target of endocrine-disrupting compounds (EDCs). However, this is not reflected adequately in current screening and assessment procedures for endocrine activity that to date determine only general parameters of thyroid function. Objective and Methods We used several in vitro and ex vivo assays in an attempt to identify suitable biomarkers for antithyroid action testing a selected panel of putative EDCs. Results In vitro we detected stimulation or inhibition of iodide uptake into FRTL-5 rat thyroid cells, inhibition of thyroid hormone binding to transthyretin, agonistic or antagonistic effects in a thyroid hormone receptor–dependent reporter assay, and inhibition of thyroid peroxidase using a novel assay system based on human recombinant thyroperoxidase that might be suitable for routine screening for potential EDCs. In rats, chronic application of several EDCs led to changes in thyroid morphology, alterations of thyrotropin and thyroid hormone serum levels as well as alterations in peripheral thyroid hormone–regulated end points such as malic enzyme and type I 5′-deiodinase activity. Conclusions As the effects of EDCs do not reflect classic mechanisms of hormone-dependent regulation and feedback, we believe multitarget and multimodal actions of EDCs affect the hypothalamic–pituitary–thyroid axis. These complex effects require a diverse approach for screening, evaluation, and risk assessment of potential antithyroid compounds. This approach involves novel in vitro or cell-based screening assays in order to assess thyroid hormone synthesis, transport, metabolism, and action as well as in vivo assays to measure thyroid hormone–regulated tissue-specific and developmental end points in animals.