Uwe Wehrenberg

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.

Steroidogenic factor-1 expression in marmoset and rat hippocampus: co-localization with StAR and aromatase

Steroidogenic factor‐1 (SF‐1), an orphan nuclear receptor, was studied with respect to the expression of steroidogenic enzymes in the hippocampus of rat and marmoset, since SF‐1 is a regulator of steroid biosynthesis in the gonads. We used the steroidogenic acute regulatory protein (StAR) as a marker of the first step in the cascade of oestrogen synthesis and aromatase as a marker of the last. StAR transports cholesterol to the inner mitochondrial membrane where it is converted by the cytochrome P‐450 enzyme complex. This is the rate‐limiting step in steroid biosynthesis. Aromatase metabolizes testosterone to oestrogen. Using an anti‐SF‐1 antibody we show that SF‐1 is highly expressed in neuronal cells of the pyramidal layer (CA1–CA3) and in the dentate gyrus of rat and marmoset hippocampi. Binding of the antibody was seen in more than 60% of all cells in the pyramidal layer and in the fascia dentata. In situ hybridization studies revealed the same expression pattern for StAR and aromatase. StAR and aromatase‐positive cells were strictly correlated with SF‐1 as shown by computer‐assisted confocal microscopy in double labelling experiments (immunohistochemistry and in situ hybridization). This coexpression may imply SF‐1 as a possible regulator of steroidogenesis in the hippocampus. However, a few interneurones express solely SF‐1 and aromatase but are negative for StAR. Since the expression of StAR represents the first step in steroidogenesis its expression is suggestive for a de novo synthesis of steroids. A small population of interneurones must import precursors for oestrogen synthesis from other sources. Responsive cells, as evidenced by the presence of oestrogen receptor transcripts, were also found in the pyramidal layer and dentate gyrus. In conclusion, (1) SF‐1 could play a regulatory role in steroidogenesis in the hippocampus of marmoset and rat and (2) with respect to the capacity of steroidogenesis two populations of hippocampal neurones coexist.

Estrogen up-regulates estrogen receptor α and synaptophysin in slice cultures of rat hippocampus

Previous studies have shown that estrogen application increases the density of synaptic input and the number of spines on CA1 pyramidal neurons. Here, we have investigated whether Schaffer collaterals to CA1 pyramidal cells are involved in this estrogen-induced synaptogenesis on CA1 pyramidal neurons. To this end, we studied estrogen-induced expression of both estrogen receptor (ER) subtypes (ERalpha and ERbeta) together with the presynaptic marker synaptophysin in the rat hippocampus. In tissue sections as well as in slice cultures mRNA expression of ERalpha, ERbeta and synaptophysin was higher in CA3 than in CA1, and mRNA expression and immunoreactivity for both ER subtypes were found in both principal cells and interneurons. By using quantitative image analysis we found stronger nuclear immunoreactivity for ERalpha in CA3 than in CA1. In slice cultures, supplementation of the medium with 10(-8) M estradiol led to an increase of nuclear immunoreactivity for ERalpha, but not for ERbeta, which was accompanied by a dramatic up-regulation of synaptophysin immunoreactivity in stratum radiatum of CA1. Together these findings indicate that estrogen effects on hippocampal neurons are more pronounced in CA3 than in CA1 and that ER activation in CA3 neurons leads to an up-regulation of a presynaptic marker protein in the axons of these cells, the Schaffer collaterals. We conclude that estradiol-induced spine formation on CA1 pyramidal cells may be mediated presynaptically, very likely by activation of ERalpha in CA3 pyramidal cells, followed by an increase in Schaffer collateral synapses.

Role of Tumor Necrosis Factor in Preovulatory Follicles of Swine

Abstract The effects of tumor necrosis factor (TNF) on cultured porcine granulosa cells that were obtained from preovulatory follicles were studied with regard to following parameters: 1) TNF receptor type I expression, 2) progesterone receptor and transforming growth factor β receptor type II (TβR II) as markers of luteinization, 3) proliferation, and 4) apoptosis. For comparative purposes the effects of TNF were also studied on insulin/forskolin-treated cells, as this treatment is well established to induce luteinization. Cytochemical methods followed by semiquantitative analysis were used. Our data show that TNF treatment upregulates TNF receptor type I expression in granulosa cells. TNF downregulates the expression of TβR II of insulin/forskolin-stimulated and of unstimulated cells. The progesterone receptor is also downregulated by the cytokine after insulin/forskolin-induced luteinization. Supplementation of the medium with TNF leads to increased proliferation and at the same time it induces apoptosis. Our results indicate that TNF exerts an inhibitory influence on luteinization and that TNF influences the balance between follicular growth (proliferation) and atresia (apoptosis).

Mapping of the Bovine Oxytocin Gene Control Region: Identification of Binding Sites for Luteal Nuclear Proteins in the 5′ Non‐Coding Region of the Gene

In view of the small number of hormone‐producing cells, the factors regulating oxytocin gene expression in the classic site of synthesis, in the magnocellular neurons of the hypothalamus, have not yet been characterized. In the early bovine corpus luteum there is a tissue‐specific oxytocin expression involving many more cells. This tissue therefore was chosen as a experimental system to identify deoxyribonucleic acid elements and nuclear proteins involved in the regulation of oxytocin gene expression. 3.2 kb from the 5’non‐coding region of the bovine oxytocin gene have been sequenced and subcloned fragments used as probes for gel retardation and footprinting experiments. Binding sites for luteal as well as more ubiquitous proteins were detected in the oxytocin promoter region and in an artiodactyl‐specific dispersed repeated deoxyribonucleic acid element. A binding site in the promoter region with a superficial similarity to an estrogen‐responsive element (‐159 to ‐152) was shown not to bind this steroid hormone receptor but to bind two nuclear proteins alternatively. One is a luteal protein, the other a more general transcription factor belonging to the steroid hormone receptor superfamily and similar, if not identical to the COUP protein. This alternative binding of a tissue‐ and phase‐specifically expressed protein or an ubiquitous factor to the same site in the oxytocin promoter suggests a role for these two proteins in the transient up‐regulation and subsequent down‐regulation of the oxytocin gene during the differentiation of the bovine corpus luteum.

The COUP transcription factor (COUP-TF) is directly involved in the regulation of oxytocin gene expression in luteinizing bovine granulosa cells

Competition with specific oligonucleotides in DNA-binding experiments, polyacrylamide gel electrophoresis, and recognition by specific antibodies have identified the ubiquitous transcription factor COUP as one of the nuclear proteins binding to the promoter region of the bovine oxytocin gene in endogenously expressing bovine granulosa cells. PCR cloning of partial cDNA sequences for bovine COUP-TF I and II and development of RNase protection assays demonstrated the up-regulation of COUP-TF in bovine granulosa cells and corpus luteum under conditions where the oxytocin gene is switched off. These experimental results from in vitro and in vivo studies point to the direct involvement of COUP-TF in oxytocin gene down-regulation during luteinization of bovine granulosa cells.

The Orphan Receptor SF-1 Binds to the COUP-Like Element in the Promoter of the Actively Transcribed Oxytocin Gene

The factors regulating oxytocin expression have not yet been characterized in detail. Although direct control by ligand‐dependent binding of nuclear hormone receptors to the oxytocin promoter has been suggested, the presence of these receptors in the tissues expressing oxytocin has not been shown consistently. We have analyzed nuclear proteins from preovulatory bovine granulosa cells and corpus luteum, tissues actively expressing the oxytocin gene, and describe here the characterization of a tissuespecific factor binding to the conserved element in the oxytocin promoter that has been implicated in the control of this gene. This factor is the bovine homologue of SF‐1, an orphan receptor expressed specifically in steroidogenic tissues. It is suggested that SF‐1 binds to the oxytocin promoter in vivo and is involved in control of oxytocin gene expression possibly by interaction with other factors.

Possible involvement of transforming growth factor-β1 and transforming growth factor-β receptor type II during luteinization in the marmoset ovary

Abstract The expression of transforming growth factor-β1 (TGF-β1), and transforming growth factor-β receptor type II (TβR-II), were evaluated in periovulatory marmoset ovaries. Histochemical methods were used, in particular double-labelling techniques, in order to correlate growth factor/receptor expression with proliferation (Ki 67), apoptosis (TUNEL method) and luteinization (3β-hydroxysteroid dehydrogenase (3β-HSD)). The latter was used as a luteinization marker. Periovulatory ovaries are especially suited for studying all aspects since they typically consist of small non-luteinized follicles, large luteinizing follicles and corpora lutea accessoria (Clas), which have developed from large luteinizing follicles. TGF-β1 and TβR-II expression was found in luteinizing theca cells of large periovulatory follicles and in all luteal cells of Clas. Non-luteinized theca cells, including those of small follicles were always devoid of any immunostaining. Granulosa cells of small follicles were immunopositive for TβR-II. Large follicles with granulosa cell immunoreactivity of both antibodies coexisted with non-reactive follicles of comparable size. The highest activity of the luteal marker enzyme 3β-HSD was co-localized in the same cells that expressed TGF-β1 and TβR-II. The double-labelling experiments revealed that TGF-β1 and TβR-II expression is not correlated with proliferation or apoptosis of follicular cells. Our results indicate that TGF-β1 and TβR-II participate in differentiation processes, i.e. luteinization, rather than proliferation. In particular, the dynamics of TβR-II expression appear highly related to the process of luteinization.

The expression of sf-1/Ad4BP is related to the process of luteinization in the marmoset (Callithrix jacchus) ovary.

Abstract Expression of the nuclear receptor, steroidogenic factor-1 (SF-1/Ad4BP), was studied in a primate (marmoset) ovary using immunohistochemical, RT-PCR, and immunoblot techniques. The periovulatory phase was compared with the luteal phase. With PCR we found a marmoset homolog of SF-1/Ad4BP to be expressed in ovarian and other steroidogenic tissues. Characteristically, the periovulatory ovaries consisted of growing (non-luteinized) small follicles together with large luteinizing follicles and many corpora lutea accessoria (Clas), which had developed from atretic large follicles. During the luteal phase, true corpora lutea (Cls) were additionally found. In general, we found that small follicles were devoid of any immunoreactivity of SF-1/Ad4BP. In large follicles, the luteinizing theca and granulosa cells express SF-1/Ad4BP. All luteal cells of Clas showed a nuclear staining in both ovary types. In Cls, only a few luteal cells were positive. Large follicles of different sizes showed no differences in expression level, as evidenced by immunoblot analysis. Our results indicate that SF-1/Ad4BP participates in the activation of gene transcription during the onset of luteinization and that Clas are essential for ovarian luteal function.