Regulation of Spermatogenesis: Differentiation of GFP-labeled Stem Cells and the Function of Cytoplasmic Bridges

During spermatogenesis, different genes are expressed in a strictly coordinated fashion providing an excellent model to study cell differentiation. Recent identification of testis specific genes and the development of green fluorescence protein (GFP) transgene technology and an in vivo system for studying the differentiation of transplanted male germ cells in infertile testis has opened new possibilities for studying the male germ cell differentiation at molecular level. We have employed these techniques in combination with transillumination based stage recognition (Parvinen and Vanha-Perttula, 1972) and squash preparation techniques (Parvinen and Hecht, 1981) to study the regulation of male germ cell differentiation. By using transgenic mice expressing enhanced-(E)GFP as a marker we have studied the expression and hormonal regulation of beta-actin and acrosin proteins in the developmentally different living male germ cells. Beta-actin was demonstrated in all male germ cells, whereas acrosin was expressed only in late meiotic and in postmeiotic cells. Follicle stimulating hormone stimulated b-actin-EGFP expression at stages I-VI and enhanced the formation of microtubules in spermatids and this way reduced the size of the acrosomic system. When EGFP expressing spermatogonial stem cells were transplanted into infertile mouse testis differentiation and the synchronized development of male germ cells could be observed during six months observation time. Each colony developed independently and maintained typical stage-dependent cell associations. Furthermore, if more than two colonies were fused, each of them was adjusted to one stage and synchronized. By studying living spermatids we were able to demonstrate novel functions for Golgi complex and chromatoid body in material sharing between neighbor spermatids. Immunosytochemical analyses revealed a transport of haploid cell specific proteins in spermatids (TRA54 and Shippo1) and through the intercellular bridges (TRA54). Cytoskeleton inhibitor (nocodazole) demonstrated the importance of microtubules in material sharing between spermatids and in preserving the integrity of the chromatoid body. Golgi complex inhibitor, brefeldin A, revealed the great importance of Golgi complex i) in acrosomic system formation ii) TRA54 translation and in iii) granule trafficking between spermatids.

Highly functional binding surface for miniaturised solid-phase immunoassays. - A spot story -

Several bioaffinity assays are based on the detection of an analyte which is bound on a solid substrate via biochemical interaction. These so called solid phase assays are based on the adhesion of the primary binding partner on a solid surface, which then binds the analyte to be detected. In this thesis work a novel solid phase based assay technology, known as spot technology, was developed. The spot technology is based on combination of high-capacity solid phases, concentrated in a spot format, utilising modified streptavidin molecules and recombinant antibody fragments. The reduction of the solid phase binding surface to a size of a spot enabled denser binding of the target molecules, providing improved signal intensities and signal-to-background ratio when applied in different solid phase immunoassays. Streptavidin-biotin interactions are commonly utilised in numerous different bioaffinity assays and the ultimate nature of streptavidin to bind biotin is among the strongest non-covalent interaction reported between two biomolecules. In this study native core streptavidin was chemically modified to provide polymerised streptavidin molecules with altered adsorption properties. These streptavidin conjugates, when coated onto polystyrene surface, provided enhanced biotin binding capacity and surface stability when compared to a reference coating constructed with native streptavidin. Furthermore, the combination of chemically modified streptavidin, sitespecifically biotinylated antibody fragments and the spot coating technology provided highly dense solid phase coating with improved binding properties. The performance of the spot assay technology was further demonstrated in different immunoassay configurations. Human thyroid stimulating hormone (TSH) and human cardiac troponin I (cTnI) were used as model analytes to show the applicability of the highly sensitive spot-based solid-phase immunoassay for detection of very low levels of analytes. It was demonstrated that the spot technology provided an assay concept with enhanced sensitivity and short turn-around times, characteristics that are highly suitable for point-of-care applications.

Identification and characterization of new genes and pathways regulating spermatogonial cell fate

Spermatogenesis, i.e sperm production in the seminiferous tubules of the testis, is a complex process that takes over one month to complete. Life-long ability of sperm production ultimately lies in a small population of undifferentiated cells, called spermatogonial stem cells (SSCs). These cells give rise to differentiating spermatogonia, which are committed to mature into spermatozoa. SSCs represent a heterogeneous population of cells and many aspects of their basic biology are still unknown. Understanding the mechanisms behind the cell fate decision of these cells is important to gain more insights into the causes of infertility and testis cancer. In addition, an interesting new aspect is the use of testis-derived stem cells in regenerative medicine. Our data demonstrated that adult mouse testis houses a population of Nanog-expressing spermatogonia. Based on mRNA and protein analysis these cells are enriched in stage XII of the mouse seminiferous epithelial cycle. The cells derived from this stage have the highest capacity to give rise to ES cell-like cells which express Oct4 and Nanog. These cells are under tight non- GDNF regulation but their fate can be dictated by activating p21 signalling. Comparative studies suggested that these cells are regulated like ES cells. Taken together these data imply that pluripotent cells are present in the adult mammalian testis. CIP2A (cancerous inhibitor of PP2A) has been associated with tumour aggressiveness and poor prognosis. In the testis it is expressed by the descendants of stem cells, i.e. the spermatogonial progenitor cells. Our data suggest that CIP2A acts upstream of PLZF and is needed for quantitatively normal spermatogenesis. Classification of CIP2A as a cancer/testis gene makes it an attractive target for cancer therapy. Study on the CIP2A deficient mouse model demonstrates that systemic inhibition of CIP2A does not severely interfere with growth and development or tissue or organ function, except for the spermatogenic output. These data demonstrate that CIP2A is required for quantitatively normal spermatogenesis. Hedgehog (Hh) signalling is involved in the development and maintenance of many different tissues and organs. According to our data, Hh signalling is active at many different levels during rat spermatogenesis: in spermatogonia, spermatocytes and late elongating spermatids. Localization of Suppressor of Fused (SuFu), the negative regulator of the pathway, specifically in early elongating spermatids suggests that Hh signalling needs to be shut down in these cells. Introduction of Hh signalling inhibitor resulted in an increase in germ cell apoptosis. Follicle-stimulating hormone (FSH) and inhibition of receptor tyrosine kinases resulted in down-regulation of Hh signalling. These data show that Hh signalling is under endocrine and paracrine control and it promotes germ cell survival.

Upconverting diagnostics: Multiplex assays utilizing upconversion luminescence technology

Conventional diagnostics tests and technologies typically allow only a single analysis and result per test. The aim of this study was to propose robust and multiplex array-inwell test platforms based on oligonucleotide and protein arrays combining the advantages of simple instrumentation and upconverting phosphor (UCP) reporter technology. The UCPs are luminescent lanthanide-doped crystals that have a unique capability to convert infrared radiation into visible light. No autofluorescence is produced from the sample under infrared excitation enabling the development of highly sensitive assays. In this study, an oligonucleotide array-in-well hybridization assay was developed for the detection and genotyping of human adenoviruses. The study provided a verification of the advantages and potential of the UCP-based reporter technology in multiplex assays as well as anti-Stokes photoluminescence detection with a new anti- Stokes photoluminescence imager. The developed assay was technically improved and used to detect and genotype adenovirus types from clinical specimens. Based on the results of the epidemiological study, an outbreak of adenovirus type B03 was observed in the autumn of 2010. A quantitative array-in-well immunoassay was developed for three target analytes (prostate specific antigen, thyroid stimulating hormone, and luteinizing hormone). In this study, quantitative results were obtained for each analyte and the analytical sensitivities in buffer were in clinically relevant range. Another protein-based array-inwell assay was developed for multiplex serodiagnostics. The developed assay was able to detect parvovirus B19 IgG and adenovirus IgG antibodies simultaneously from serum samples according to reference assays. The study demonstrated that the UCPtechnology is a robust detection method for diverse multiplex imaging-based array-inwell assays.

Sleep and Menopause. Hormone Therapy and Sleep Deprivation

This study evaluated the effect of menopause, hormone therapy (HT) and aging on sleep. Further, the mechanisms behind these effects were examined by studying the associations between sleep and the nocturnal profiles of sleep-related hormones. Crosssectional study protocols were used to evaluate sleep in normal conditions and during recovery from sleep deprivation. The effect of initiation of HT on sleep and sleeprelated hormones was studied in a prospective controlled trial. Young, premenopausal and postmenopausal women were studied, and the methods included polysomnography, 24-h blood sampling, questionnaires and cognitive tests of attention. Postmenopausal women were less satisfied with their sleep quality than premenopausal women, but this was not reflected in sleepiness or attention. The objective sleep quality was mainly similar in pre- and postmenopausal women, but differed from young women. The recovery mechanisms from sleep deprivation were relatively well-preserved after menopause. HT offered no advantage to sleep after sleep deprivation or under normal conditions. The decreased growth hormone (GH) and prolactin (PRL) levels after menopause were reversible with HT. Neither menopause nor HT had any effect on cortisol levels. In premenopausal women, HT had only minor effects on PRL and cortisol levels. The temporal link between GH and slow wave sleep (SWS) was weaker after menopause. PRL levels were temporally associated with sleep stages, and higher levels were seen during SWS and lower during rapid-eye-movement (REM) sleep. Sleep quality after menopause is better determined by age than by menopausal state. Although HT restores the decreased levels of GH and PRL after menopause, it offers no advantage to sleep quality under normal conditions or after sleep deprivation.

Targeted ablation of gonadotropin dependent endocrine tumors in transgenic mice through their luteinizing hormone receptor (LHR)

Gonadal somatic cell and adrenocortical endocrine tumors are rare. The incidence of adrenocortical carcinomas is only 1-2/1000000 a year. However, they are aggressive, especially in adulthood and currently surgery is the only curative treatment. Cytotoxic agents are in use in advanced cancers, but side effects and multidrug resistance are often problems. Thus there is a need for novel curative treatment methods. In contrast, ovarian granulosa cell tumors and testicular Leydig cell tumors are usually benign, especially at a younger age. The aim of the present thesis was to study a novel targeted treatment method through luteinizing hormone/chorionic gonadotropin receptor (LHCGR) in a transgenic mouse tumor model. The cytotoxic agent was lytic peptide Hecate-CGbeta conjugate where 23 amino acid Hecate, a synthetic form of honeybee venom melittin, was conjugated to 15 amino acid fragment of human chorionic gonadotropin β subunit. Lytic peptides are known to act only on negatively charged cells, such as bacteria and cancer cells and hereby, due to hCGbeta fragment, the conjugate is able to bind directly to LHCGR bearing cancer cells, saving the healthy ones. The experiments were carried out in inhibin-alpha-Simian Virus 40-T-antigen transgenic mice that are known to express LHCGR-bearing gonadal tumors, namely Leydig and granulosa cell tumors by 100% penetrance. If the mice are gonadectomized prepubertally they form adrenocortical tumors instead. Transgenic and wild type mice were treated for three consecutive weeks with control vehicle, Hecate or Hecate-CGbeta conjugate. GnRH antagonist or estradiol was given to a group of mice with or without Hecate-CGbeta conjugate to analyze the additive role of gonadotropin blockage in adrenocortical tumor treatment efficacy. Hecate-CGbeta conjugate was able to diminish the gonadal and adrenal tumor size effectively in males. No treatment related side effects were found. Gonadotropin blockage through GnRH antagonist was the best treatment in female adrenal tumors. The mode of cell death by Hecate-CGbeta conjugate was proven to be through necrosis. LHCGR and GATA-4 were co-expressed in tumors, where the treatment down-regulated their expression simultaneously, suggesting their possible use as tumor markers. In conclusion, the present thesis showed that Hecate-CGbeta conjugate targets its action selectively through LHCGR and selectively kills the LHCGR bearing tumor cells. It works both in gonadal somatic and in ectopic LHCGR bearing adrenal tumors. These results establish a more general principle that receptors expressed ectopically in malignant cells can be exploited in targeted cytotoxic therapies without affecting the normal healthy cells.

Building organizational creativity – a multitheory and multilevel approach for understanding and stimulating organizational creativity

Organizational creativity is increasingly important for organizations aiming to survive and thrive in complex and unexpectedly changing environments. It is precondition of innovation and a driver of an organization’s performance success. Whereas innovation research increasingly promotes high-involvement and participatory innovation, the models of organizational creativity are still mainly based on an individual-creativity view. Likewise, the definitions of organizational creativity and innovation are somewhat equal, and they are used as interchangeable constructs, while on the other hand they are seen as different constructs. Creativity is seen as generation of novel and useful ideas, whereas innovation is seen as the implementation of these ideas. The research streams of innovation and organizational creativity seem to be advancing somewhat separately, although together they could provide many synergy advantages. Thereby, this study addresses three main research gaps. First, as the knowledge and knowing is being increasingly expertized and distributed in organizations, the conceptualization of organizational creativity needs to face that perspective, rather than relying on the individual-creativity view. Thus, the conceptualization of organizational creativity needs clarification, especially as an organizational-level phenomenon (i.e., creativity by an organization). Second, approaches to consciously build organizational creativity to increase the capacity of an organization to demonstrate novelty in its knowledgeable actions are rare. The current creativity techniques are mainly based on individual-creativity views, and they mainly focus on the occasional problem-solving cases among a limited number of individuals, whereas, the development of collective creativity and creativity by the organization lacks approaches. Third, in terms of organizational creativity as a collective phenomenon, the engagement, contributions, and participation of organizational members into activities of common meaning creation are more important than the individualcreativity skills. Therefore, the development approaches to foster creativity as social, emerging, embodied, and collective creativity are needed to complement the current creativity techniques. To address these gaps, the study takes a multiparadigm perspective to face the following three objectives. The first objective of this study is to clarify and extend the conceptualization of organizational creativity. The second is to study the development of organizational creativity. The third is to explore how an improvisational theater based approach fosters organizational creativity. The study consists of two parts comprising the introductory part (part I) and six publications (part II). Each publication addresses the research questions of the thesis through detailed subquestions. The study makes three main contributions to the research of organizational creativity. First, it contributes toward the conceptualization of organizational creativity by extending the current view of organizational creativity. This study views organizational creativity as a multilevel construct constituting both of individual and collective (group and organizational) creativity. In contrast to current views of organizational creativity, this study bases on organizational (collective) knowledge that is based on and demonstrated through the knowledgeable actions of an organization as a whole. The study defines organizational creativity as an overall ability of an organization to demonstrate novelty in its knowledgeable actions (through what it does and how it does what it does).Second, this study contributes toward the development of organizational creativity as multi-level phenomena, introducing developmental approaches that face two or more of these levels simultaneously. More specifically, the study presents the cross-level approaches to building organizational creativity, by using an approach based in improvisational theater and considering assessment of organizational renewal capability. Third, the study contributes on development of organizational creativity using an improvisational theater based approach as twofold meaning. First, it fosters individual and collective creativity simultaneously and builds space for creativity to occur. Second, it models collective and distributed creativity processes, thereby, contributing to the conceptualization of organizational creativity.

Alpha- and gamma-melanocyte stimulating hormones in obesity. A study of the key central areas of metabolic regulation

The melanocortin system is an important regulator of feeding, energy metabolism,and cardiovascular function and it consists of the pro-opiomelanocortin (POMC) derived melanocyte stimulating hormones (α-, β- and γ-MSH) and their endogenous melanocortin receptors, MC1R to MC5R. In the hypothalamus, α-MSH reduces food intake, and increases energy expenditure and sympathetic tone by binding to MC4R. Mutations affecting the MC4R gene lead to obesity in mammals. On the other hand, the metabolic effects of MC3R stimulation using agonists such as the endogenously expressed γ-MSH have been less extensively explored. The main objective of this study was to investigate the long-term effects of increased melanocortin tone in key areas of metabolic regulation in the central nervous system (CNS) in order to investigate the sitespecific roles of both α-MSH and γ-MSH. The aim was to stereotaxically induce local overexpression of single melanocortin peptides using lentiviral vectors expressing α-MSH (LVi-α-MSH-EGFP) and γ-MSH (LVi-γ-MSH-EGFP). The lentiviral vectors were shown to produce a long-term overexpression and biologically active peptides in cell-based assays. The LVi-α-MSHEGFP was targeted to the arcuate nucleus in the hypothalamus of diet induced obese mice where it reduced weight gain and adiposity independently of food intake. When the nucleus tractus solitarus in the brainstem was targeted, the LVi-α-MSH-EGFP treatment was shown to cause a small decrease in adiposity, which did not impact weight development. However, the α-MSH treatment increased heart rate, which was attenuated by adrenergic receptor blockade indicative of increased sympathetic activity. The LVi-γ-MSH-EGFP was targeted to the hypothalamus where it decreased fat mass in mice eating the standard diet, but the effect was abated if animals consumed a high-fat Western type diet. When the diet induced obese mice were subjected again to the standard diet, the LVi-γ-MSH-EGFP treated animals displayed increased weight loss and reduced adiposity. These results indicate that the long-term central anti-obesity effects of α-MSH are independent of food intake. In addition, overexpression of α-MSH in the brain stem efficiently blocked the development of adiposity, but increased sympathetic tone. The evidence presented in this thesis also indicates that selective MC3R agonists such as γ-MSH could be potential therapeutics in combination with low fat diets.