Regulating Mechanisms of Gonadal and Pituitary Tumorigenesis in Mice Producing Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are structurally and functionally similar glycoprotein hormones acting through the same luteinizing hormone chorionic gonadotropin receptor (LHCGR). The functions of LH in reproduction and hCG in pregnancy are well known. Recently, the expression of LHCGR has been found in many nongonadal tissues and cancers, and this has raised the question of whether LH/hCG could affect the function or tumorigenesis of these nongonadal tissues. We have also previously generated an hCG expressing mouse model presenting nongonadal phenotypes. Using this model it is possible to improve our understanding of nongonadal action of highly elevated LH/hCG. In the current study, we analyzed the effect of moderately and highly elevated hCG levels on male reproductive development and function. The main finding was the appearance of fetal Leydig cell (FLC) adenomas in prepubertal males. However, the development and differentiation of FLCs were not significantly affected. We also show that the function of hCG is different in FLCs and in adult Leydig cells (ALC), because in the latter cells hCG was not able to induce tumorigenesis. In FLCs, LHCGR is not desensitized or downregulated upon ligand binding. In this study, we found that the testicular expression of two G protein-coupled receptor kinases responsible for receptor desensitization or downregulation is increased in adult testis. Results suggest that the lack of LHCGR desensitization or downregulation in FLCs protect testosterone (Te) synthesis, but also predispose FLCs for LH/hCG induced adenomas. However, all the hCG induced nongonadal changes observed in male mice were possible to explain by the elevated Te level found in these males. Our findings indicate that the direct nongonadal effects of elevated LH/hCG in males are not pathophysiologically significant. In female mice, we showed that an elevated hCG level was able to induce gonadal tumorigenesis. hCG also induced the formation of pituitary adenomas (PA), but the mechanism was indirect. Furthermore, we found two new potential risk factors and a novel hormonally induced mechanism for PAs. Increased progesterone (P) levels in the presence of physiological estradiol (E2) levels induced the formation of PAs in female mice. E2 and P induced the expression and nuclear localization of a known cell-cycle regulator, cyclin D1. A calorie restricted diet was also able to prevent the formation of PAs, suggesting that obesity is able to promote the formation of PAs. Hormone replacement therapy after gonadectomy and hormone antagonist therapy showed that the nongonadal phenotypes observed in hCG expressing female mice were due to ovarian hyperstimulation. A slight adrenal phenotype was evident even after gonadectomy in hCG expressing females, but E2 and P replacement was able to induce a similar phenotype in WT females without elevated LH/hCG action. In conclusion, we showed that the direct effects of elevated hCG/LH action are limited only to the gonads of both sexes. The nongonadal phenotypes observed in hCG expressing mice were due to the indirect, gonadal hormone mediated effects of elevated hCG. Therefore, the gonads are the only physiologically significant direct targets of LHCGR signalling.

The effects of prolonged exposure to elevated temperatures and elevated CO2 levels on the growth, yield and dry matter partitioning of field-sown meadow fescue

Selostus: Kohotetun lämpötilan ja kohotetun CO2-pitoisuuden vaikutukset peltoon kylvetyn nurminadan kasvuun, satoon ja kuiva-aineen jakautumiseen

Arrangement of experiments for simulating the effects of elevated temperatures and elevated CO2 levels on field-sown crops in Finland

Selostus: Koejärjestelyt kohonneen lämpötilan ja CO2-tason vaikutusten simuloimiseksi peltokasveilla Suomessa

Feeding levels during the growing phase affect the production of primiparous Finnish Landrace ewes

Selostus: Ruokinnan voimakkuuden vaikutus siitosuuhikaritsan tuotantoon

Peat properties and vegetation along different trophic levels on an afforested, fertilised mire

Tiivistelmä: Turpeen ominaisuudet ja kasvillisuus metsitetyn ja lannoitetun avosuon eri trofiatasoilla

Pain-evoked alterations on gingival blood flow and gingival crevicular fluid (GCF) neuropeptide SP and collagenase-2 (MMP-8) levels

Previous studies have demonstrated that clinical pulpal pain can induce the expression of pro-inflammatory neuropeptides in the adjacent gingival crevice fluid (GCF). Vasoactive agents such as substance P (SP) are known to contribute to the inflammatory type of pain and are associated with increased blood flow. More recent animal studies have shown that application of capsaicin on alveolar mucosa provokes pain and neurogenic vasodilatation in the adjacent gingiva. Pain-associated inflammatory reactions may initiate expression of several pro- and anti-inflammatory mediators. Collagenase-2 (MMP-8) has been considered to be the major destructive protease, especially in the periodontitis-affected gingival crevice fluid (GCF). MMP-8 originates mostly from neutrophil leukocytes, the first line of defence cells that exist abundantly in GCF, especially in inflammation. With this background, we wished to clarify the spatial extensions and differences between tooth-pain stimulation and capsaicin-induced neurogenic vasodilatation in human gingiva. Experiments were carried out to study whether tooth stimulation and capsaicin stimulation of alveolar mucosa would induce changes in GCF MMP-8 levels and whether tooth stimulation would release neuropeptide SP in GCF. The experiments were carried out on healthy human volunteers. During the experiments, moderate and high intensity painful tooth stimulation was performed by a constant current tooth stimulator. Moderate tooth stimulation activates A-delta fibres, while high stimulation also activates C-fibres. Painful stimulation of the gingiva was achieved by topical application of capsaicin-moistened filter paper on the mucosal surface. Capsaicin is known to activate selectively nociceptive C-fibres of stimulated tissue. Pain-evoked vasoactive changes in gingivomucosal tissues were mapped by laser Doppler imaging (LDI), which is a sophisticated and non-invasive method for studying e.g. spatial and temporal characteristics of pain- and inflammation-evoked blood flow changes in gingivomucosal tissues. Pain-evoked release of MMP-8 in GCF samples was studied by immunofluorometric assay (IFMA) and Western immunoblotting. The SP levels in GCF were analysed by Enzyme immunoassay (EIA). During the experiments, subjective stimulus-evoked pain responses were determined by a visual analogue pain scale. Unilateral stimulation of alveolar mucosa and attached gingiva by capsaicin evoked a distinct neurogenic vasodilatation in the ipsilateral gingiva, which attenuated rapidly at the midline. Capsaicin stimulation of alveolar mucosa provoked clear inflammatory reactions. In contrast to capsaicin stimuli, tooth stimulation produced symmetrical vasodilatations bilaterally in the gingiva. The ipsilateral responses were significantly smaller during tooth stimulation than during capsaicin stimuli. The current finding – that tooth stimulation evokes bilateral vasodilatation while capsaicin stimulation of the gingiva mainly produces unilateral vasodilatation – emphasises the usefulness of LDI in clarifying spatial features of neurogenic vasoactive changes in the intra-oral tissues. Capsaicin stimulation of the alveolar mucosa induced significant elevations in MMP-8 levels and activation in GCF of the adjacent teeth. During the experiments, no marked changes occurred in MMP-8 levels in the GCF of distantly located teeth. Painful stimulation of the upper incisor provoked elevations in GCF MMP-8 and SP levels of the stimulated tooth. The GCF MMP-8 and SP levels of the non-stimulated teeth were not changed. These results suggest that capsaicin-induced inflammatory reactions in gingivomucosal tissues do not cross the midline in the anterior maxilla. The enhanced reaction found during stimulation of alveolar mucosa indicates that alveolar mucosa is more sensitive to chemical irritants than the attached gingiva. Analysis of these data suggests that capsaicin-evoked neurogenic inflammation in the gingiva can trigger the expression and activation of MMP-8 in GCF of the adjacent teeth. In this study, it is concluded that experimental tooth pain at C-fibre intensity can induce local elevations in MMP-8 and SP levels in GCF. Depending on the role of MMP-8 in inflammation, in addition to surrogated tissue destruction, the elevated MMP-8 in GCF may also reflect accelerated local defensive and anti-inflammatory reactions.

Novel Roles of HCG/LH Signalling in the Mouse Mammary Gland and its Tumorigenesis.

Breast cancer is the most common cancer in women, and its development is intimately related to hormonal factors, but how hormones affect breast physiology and tumorigenesis is not sufficiently known. Pregnancy elicits long-term protection from breast cancer, but during the first ten years after pregnancy, breast cancer risk is increased. In previous studies, there has been conflicting data on the role of human chorionic gonadotropin (HCG) and the functionality of its receptor in extragonadal tissues. The aim of this study was to elucidate the role of chronically elevated HCG in mouse physiology. We have created a transgenic (TG) mouse model that overexpresses HCG. HCG is similar to lutenizing hormone (LH), but is secreted almost solely by the placenta during pregnancy. HCG and LH both bind to the LH receptor (LHR). In the current study, mammary gland tumors were observed in HCG TG mice. We elucidated the role of HCG in mammary gland signalling and the effects of LHR mediated signalling in mouse mammary gland gene expression. We also studied the effects of HCG in human breast epithelial cell cultures. Several endocrine disturbances were observed in HCGβ TG female mice, resulting in precocious puberty, infertility, obesity and pituitary and mammary gland tumors. The histology of the mammary gland tumors of HCGβ TG females resembled those observed in mouse models with activated Wnt/β-catenin signalling pathway. Wnts are involved in stem cell regulation and tumorigenesis, and are hormonally regulated in the mammary gland. We observed activated β-catenin signalling and elevated expression of Wnt5b and Wnt7b in TG tumors and mammary glands. Furthermore, we discovered that HCG directly regulates the expression of Wnt5b and Wnt7b in the mouse mammary gland. Pharmacological treatment with HCG also caused upregulation of several Wnt-pathway target genes in ovariectomized wild type (WT) mice in the presence of physiological concentrations of estradiol and progesterone. In addition, differential expression of several metabolic genes was observed, suggesting that HCG affects adipocyte function or glucose metabolism. When WT mice were transplanted with LHR deficient or wild type WT mammary epithelium, differential expression of several genes affecting the Wnt-signalling pathway was observed in microarray analysis. Diminished expression of several genes associated with LHR function in other tissues, such as the ovary, was observed in mammary glands deficient of epithelial LHR. In cultured human mammary epithelial cells HCG upregulated the expression of WNT5B, WNT7B similar to mouse, suggesting that the observations found are relevant in human physiology. These studies suggest that HCG/LHR signalling affects gene expression in non-gonadal tissues, and that Wnt-signalling is regulated by HCG/LH in human and mouse mammary glands.

Factors affecting the usability of aspen (Populus tremula) wood dried at different temperature levels

In this dissertation the main aim was to study the usability of aspen wood in the mechanical wood processes, especially the factors that affect the usability of aspen wood, which is dried at different temperature levels. The problematic of varying temperatures from level to another seem to have significant effects on distortions. According to the studies conducted, many practical solutions for drying of aspen timber can be made. European aspen sawn timber should be dried in relatively narrow widths, short lengths, edged and sawn pith free. The results achieved give an interesting view for the distortions of aspen wood between HT drying and heat treatment. The most significant result is that cupping seems to decrease when increasing temperature level from HT drying to heat treatment phase. The difference is significant. Other very obvious result is that bow and crook are increasing between these two temperature levels, bow significantly. Also the modelling gives a good background for this result since cupping is reduced by higher temperatures. It can also be assumed that these distortions can be affected for example by sorting out the timber used to different moisture levels before drying. This could be a very simple solution for practical purposes. From practical point of view, it was also noted during the drying tests performed that the discoloration seem to be no problem for aspen boards dried in temperatures under 150 °C. Altogether, this dissertation covers many interesting points of view of factors affecting distortions of wood in different temperature stages. Aspen as species in use of mechanical wood industries seems to be a species suitable for component production. According to the results, the best yield from aspen timber can be achieved in industry that can utilize relatively short components of sawn wood. Results achieved give a significant indication about the factors affecting distortions of wood at different temperature stages of drying, especially considering European aspen.

Behavioral Responses in Mice Selectively Bred for High and Low Levels of Open-Field Thigmotaxis

In animal psychology, the open-field (OF) test is a traditional method for studying different aspects of rodent behavior, with thigmotaxis (i.e., wallseeking behavior) being one of the best validated OF parameters employed to measure emotionality. The main purpose of the present study was to investigate the selection response in mice selectively bred for high and low levels of OF thigmotaxis (the HOFT and LOFT lines, respectively). The mice (N = 2048) were selected for 23 generations, resulting in bidirectional phenotypic divergence between the two lines; that is, the HOFT mice were more thigmotactic (i.e., more emotional) than the LOFT mice across the different generations. The origin of the line difference in thigmotaxis was further investigated by using the crossfostering paradigm, with the results suggesting that the divergence between the two lines was primarily innate in origin and not influenced by differing maternal behavior. The stability of the selection trait was examined by testing the animals at different ages as well as in varying conditions. The results indicated that the line difference in thigmotaxis was not affected by age at the time of testing, and it also persisted in the different OF testing situations as well as during pregnancy and lactation. The examination of a possible coselection of other characteristics revealed that the more thigmotactic HOFT mice lived longer than the less thigmotactic LOFT mice. In addition, the HOFT mice tended to rear and explore less than the LOFT mice, supporting the general assumption that emotionality and exploration are inversely related. The two lines did not generally differ in ambulation and defecation, that is, in the traditional OF indexes of emotionality, conforming to the suggestion that emotionality is a multidimensional construct. The effects of sex on different OF parameters were also assessed, with the results suggesting that among the HOFT and LOFT lines, the female mice were more emotional than the male mice. The examination of the temporal changes in the HOFT and LOFT lines’ OF behavior revealed some contradictory findings that also partially conflicted with general assumptions. Although this study did not show prominent differences in maternal responsiveness between the HOFT and LOFT mothers, the results suggested that the line divergence in emotionality was more pronounced in the presence of a pup after parturition than during pregnancy. The present study clearly demonstrates that OF thigmotaxis is a strong characteristic for producing two diverging lines of mice. The difference in thigmotaxis between the selectively bred HOFT and LOFT mice seemed to be a stable and robust feature of these animals, and it appeared to stem from a genetic background.