Marine and freshwater environments support numerous species of teleost fish with a wide and diverse range of reproductive strategies. Despite the considerable interest in fish reproduction, our knowledge regarding ejaculate traits and factors affecting them is limited. Using computer-assisted sperm analysis (CASA) I measured ejaculate traits (sperm swimming speed, motility, path trajectory, longevity and concentration) from sexually mature chinook salmon (Oncorhynchus tshawytscha) activated in freshwater and ovarian fluid. I also looked at these ejaculate traits in relation to measures of male quality (body condition) and investment into reproduction (relative testes mass). Furthermore, I determined the chemical composition of seminal and ovarian fluid and looked at the effect these fluids have on sperm behaviour. A considerable amount of intraspecific variation existed in all ejaculate traits measured, and investment into reproduction (relative testes mass) was dependent on male body condition, as males in better condition had relatively larger testes. However, these males did not have superior quality ejaculates or ejaculates with a higher density of spermatozoa; hence the potential reproductive advantage of having larger relative testes in this species remains unknown and requires further investigation. In addition, a positive relation between sperm longevity and sperm swimming speed was observed defying the expected trade-off between ejaculate traits according to theory. There was also a weak negative trend in our data between body condition and sperm swimming speed, linearity, and longevity. All sperm traits measured were greatly enhanced when activated in a solution containing ovarian fluid (a viscous fluid which is excreted with the egg batch during spawning) from female chinook salmon. Interestingly, sperm swimming speed activated in fresh water only accounted for < 12% of the observed variation in mean sperm swimming speed in ovarian fluid. This result suggests the sperm traits measured in fresh water are not relevant to those same traits measured in ovarian fluid, so caution should be applied when comparing the potential for individual males to fertilize ova when sperm traits are activated in water, especially in studies of sperm competition in an externally fertilising species. Sperm competition between males is known to strongly influence sperm and ejaculate traits, but less is known about female sperm choice after copulation via a process called cryptic female choice (CFC). In CFC, females may have the ability to favour the sperm of one male over another and bias fertilisation accordingly. To test whether ovarian fluid could act as a mechanism of CFC in an externally fertilising fish species, I measured sperm traits from each male activated in the ovarian fluids from different females. I found that mean sperm swimming speed, longevity, and path trajectory differed significantly among males, but most importantly, the pattern of within-male variation in these traits also varied significantly among males in response to different females’ ovarian fluids. This result suggests that ovarian fluid may be a potential mechanism of CFC whereby females differentially enhance the swimming speed of sperm from different males. In addition, I found that sperm longevity was negatively correlated with variation in [Ca²⁺] and [Mg²⁺] concentration in the ovarian fluid, while percent motility increased with increasing concentration of [Mg²⁺]. These observations provide a possible chemical basis for cryptic female choice whereby female ovarian fluid differentially influences the behaviour of sperm from different males and thus their fertilisation success. This finding is particularly exciting, as we may have uncovered a potential mechanism of CFC in an externally fertilising species, which is poorly understood. In addition, results from this study suggest new directions for genetic studies to provide direct evidence for CFC. For example, does sperm selection via ovarian fluid promote favoured genetic combinations that enhance male reproductive success?

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

This study investigated several, previously little-known, aspects of reproductive physiology and endocrinology in the substrate-spawning cÌchlid Tilapia zillii; a tilapia that is becoming increasingly popular in world aquaculture. Studies were undertaken in controlled laboratory aquaria, thereby reducing the potential influence of environmental variation evident in many previous field studies of this species. Analysis involved two strains of T. zillii: strain 'A' (T. zillii) and strain 'B' (formerly known as Tilapia tholloni). Spawning periodicity and total fecundity generally increased with fish size. Egg size varied within a narrow window and did not generally increase with fish size though fish weighing 100 - 200g tended to produce the largest eggs. The best estimate of spawning periodicity was considered to be 'mean days elapsed/spawn' as this figure was based upon both spawning and non-spawning fish in an experimental group. Mean days elapsed/spawn increased with increasing fish size and averaged 61.4 days and 37.5 days in strains 'A' and 'B' respectively. The shortest reproductive cycles observed were just 7 days and 6 days for strains 'A' and 'B' respectively. Total fecundity ranged from 461 - 11640 eggs/clutch. Mean total fecundity was 3606+/-280 in strain 'A' and 3560+/-243 in strain 'B'. Mean egg diameter was 1.5+/-0.04mm and 1.4+/-0.08mm in strains 'A' and 'B' respectively. Fecundity and egg size also varied over successive spawns in serial-spawning females but these variations did not appear to be related to spawning periodicity. Regression analysis revealed strong relationships between fish size (weight and length) and total fecundity, relative fecundity and total egg volume. Relationships between fish size and egg size were generally much weaker. Fecundity and egg size were related to the length of the preceding inter-spawn-interval (ISI) in fish of certain weight categories but not others, providing limited evidence that length of ISI may in par, control fecundity and egg size in this species. Ovarian recrudescence was classified into ten distinct developmental stages based upon oocyte size, biochemical properties and structure. This classification scheme was comparable to classification schemes developed for other teleosts but represents the first detailed description of oocyte growth in a substrate-spawning tilapia. Radioimmunoassay and stereological analysis provided valuable and novel data concerning the dynamics of ovarian development in this species. Levels of 17ßoestradiol (E2) and testosterone (T) peaked within 6 days of spawning, suggesting that vitellogenesis began as early as day 2 or 3 post-spawning. By day 8, ovaries were dominated by large late-vitellogenic/maturing oocytes (stages 6 & 7) occupying 60 - 70% of the ovary. Gonadosomatic index (GSI) reached maximal levels by day 14. Since the proportion of stage 6/7 oocytes exhibited little change from day 8 onwards, it is suggested that pre-vitellogenic oocytes are recruited into vitellogenic growth immediately after spawning and complete vitellogenesis as early as day 8 postspawning. Analysis of serial-spawning fish found that initial post-spawn E2 and T peaks (on days 2 - 6) were much lower after the second spawning. Sex steroid levels were also found to be suppressed in confined T. zillii (i.e. where stocking densities were > lOkg/m3). Confined females failed to spawn but displayed a marked tendency to do so after transfer to individual aquaria. Serum E2 and T were suppressed during confinement but increased rapidly following transfer to individual aquaria (coincident with resumed spawning activity). It is suggested that levels of E2 and T under confinement are not sufficient to allow completion of vitellogenic growth and are most probably suppressed via a pheromonal mechanism. Finally, the present study investigated the effect of prolonged food restriction on various aspects of reproduction. T. zillii were rationed from first feeding and throughout the following 17 months. Despite very large differences in fish size, no significant differences were detected in total fecundity, egg diameter nor total egg volume once data had been adjusted for differences in fish size. These data suggest that despite very large differences in food availability throughout the periods of sexual differentiation and on-growing, investment in reproduction remained relatively consistent. It appeared that during food restriction, T. zillii sacrificed body weight and growth so as to maintain reproductive investment. In summary, this study provides valuable and novel information regarding the reproductive physiology and endocrinology of female T. zillii and suggests that this species may be a suitable 'model' species for future work on fecundity and ovarian development.

Baigiamojo darbo tikslas buvo ištirti 3 metų amžiaus jūrų kiaulyčių kiaušidžių folikulų ir jų ovocitų morfometrinius ypatumus. LSMU Patologijos centre buvo atliktas trijų jūrų kiaulyčių preparavimas ir paimta tyrimams reikalinga medžiaga. Anatomijos ir Fiziologijos katedroje buvo atliktas histologinių preparatų paruošimas. Preparatai buvo naudojami atlikti mikroskopines nuotraukas naudojant histologinių vaizdų analizatorių (mikroskopą) „Olympus DP72 BX63“. Naudojant UTHSCSA Image Tool for Windows Version 3 programą buvo atlikta 3 jūrų kiaulyčių, kurių amžius 3 metai, užuomazginių, pirminių ir antrinių kiaušidės folikulų, bei juose esančių ovocitų, ovocitų branduolių, folikulinių ląstelių bei pamatinės membranos morfometriniai matavimai. Statistinė duomenų analizė atlikta naudojant statistinę programą ANOVA (Statistica Version 10, StatSoft inc.). Mūsų tyrimo metu buvo nustatyta, kad 3 metų amžiaus jūrų kiaulyčių kiaušidžių užuomazginių folikulų diametras svyravo nuo 23,22±2,62 µm iki 26,16±3,58 µm, ovocitų diametras svyravo nuo 6,26±1,13 µm iki 7,37±0,51 µm. Pirminių kiaušidžių folikulų diametras svyravo nuo 48,68±1,47 µm iki 61,78±4,33 µm, ovocitų dydis siekė nuo 13,56±5,48 µm iki 14,61±2,79 µm. Antrinių kiaušidžių folikulų diametras svyravo nuo 149,34±29,97 µm iki 209,46±52,54 µm, ovocitų diametras svyravo nuo 41,6±8,35 µm iki 63,59±4,67 µm. Visų tirtų jūrų kiaulyčių užuomazginių kiaušidžių folikulų diametras, plotas, ovocitų diametras ir plotas, ovocitų branduolių... [toliau žr. visą tekstą] / Master ending work includes 43 pages, 35 pictures, to write the work was used 32 literary sources. The aim of this work was to investigate morphometric features of different stages ovarian follicles and oocytes in 3 years old three guinea pigs. 3 guinea pigs‘ preparation was performed in the LSMU Pathology center, was taken required material for the research. Histological preparations laying in were performed in the Anatomy and Physiology department. Preparations were used perform microscopic images using histological image analyzer (microscope) „Olympus DP72 BX63“. Using UTHSCSA Image Tool for Windows Version 3 program was performed 3 guinea pigs, with age 3 years, primordial, primary and secondary ovarian follicles’, oocytes’, oocyte nucleis’, follicular cells’ and basement membranes’ morphometric measurements. Statistical analysis of the data performed using the statistical program ANOVA (Statistical Version 10, Stat Soft inc.). Our research showed, that 3 years old guinea pigs‘ primordial ovarian follicle diameter ranged from 23,22±2,62 µm till 26,16±3,58 µm, oocyte diameter ranged from 6,26±1,13 µm till 7,37±0,51 µm. Primary ovarian follicle diameter ranged from 48,68±1,47 µm till 61,78±4,33 µm, oocyte size was from 13,56±5,48 µm till 14,61±2,79 µm. Secondary ovarian follicle diameter ranged from 149,34±29,97 µm till 209,46±52,54 µm, oocyte diameter ranged from 41,6±8,35 µm till 63,59±4,67 µm. All guinea pigs primordial follicles’ diameter and area, oocytes’ diameter... [to full text]

Tiriamojo darbo sritis – histologiniai kačių kiaušidžių tyrimai. Darbo tikslas buvo ištirti keturių kačių nuo 7 mėn. iki 1,8 metų įvairių stadijų kiaušidžių folikulus ir juos įvertinti.Kačių kiaušidės buvo paimtos ovariohisterektomijos metu.Kiaušidės supjaustytos į 5µm storio pjūvius, pjūviai dažyti hematoksilino-eozino dažais. Folikulai suskirstyti pagal išsivystymą ir matmenis į užuomazginius, pirminius, ankstyvuosius antrinius. Atlikti matavimai, išmatuoti užuomazginių, pirminių, antrinių folikulų diametrai, plotai, folikulų ovocitų diametrai, plotai, jų branduolių diametrai, plotai, folikulinės ląstelės – jų aukštis ir plotis, pamatinė membrana, antrinių folikulų skaidrioji zona. Ištirta, jog užuomazginių, pirminių bei antrinių folikulų, ovocitų ir jų branduolių diametras, plotas, pamatinės membranos storis ir antrinių folikulų ovocitų skaidriosios zonos storis yra panašūs. Užuomazginių folikulų ovocitą gaubia vienas sluoksnis plokščiųjų folikulinių ląsteliu – joms būdinga tai, jog, jų plotis didesnis už aukštį, o pirminių folikulų ovocitą gaubia kūbiškosios ląstelės, kurių tiek aukštis, tiek plotis panašūs.Antrinių ankstyvųjų folikulų ovocitų paviršių gaubia 2-3 sluoksnių folikulinių ląstelių, kurių plotis ir aukštis yra panašūs. Statistinių duomenų patikimumas 95 % arba p<0,05. / Range of investigation is histological analysis of feline ovary. The purpose of this scientific research is to explore various stages ovarian follicles in four cats (age 7 months- 1,8 years).Ovaries were cut into 5 µm sections, than sections were stained by eosin-hematoxylin. Folicles were classified regarding to their development and dimensions into primordial, primary and early secoundary follicles. Were performed measurements.Were measured diameters and areas of primordial, primary and early secoundary follicles, diameters and areas of oocytes and nucleus of oocytes, heigt and width of folliculas cells, thickness of basal lamina .Also were measured zona pellucida of secoundary follicles. Results revealed that diameter and area of primordial, primary and early secoundary follicles, oocytes and nucleus were comparable.Basal lamina thickness were simmilar too, the same thing is about zona pellucida in secoundary follicles. Primordial follicle is surrounded by one layer of squamous cells - basic feature of these cells is that their width is much bigger than height meanwhile primary follicle is surrounded by cuboidal one layer cells with almost equal width and height. Cells of early secoundary follicle are surrounding oocyte at least with two layers and their width and height are almost equal. Calculated statistical data reliability is 95 % or p<0,05.

Ovarian cancer, with the highest mortality rate amongst gynaecological malignancies in Australia, is the eighth most common cancer and the fifth cause of cancer-related deaths in women. Currently, five-year survival for women diagnosed with ovarian cancer is only 40 % and despite many patients experiencing remission, approximately 80 % of them will relapse due to residual micrometastasis. The limited impact of standard therapies on the prognosis for recurrent chemotherapy-resistant disease and the need to identify less toxic alternatives has motivated the development of strategies to combat the aggressive and life-threatening burden of ovarian cancer. A novel therapy against cancer utilises dendritic cells (DC), potent antigen presenting cells, to deliver tumour antigens to the immune system for the stimulation of cytotoxic T-lymphocyte (CTL) responses. DC immunotherapy has been used for the treatment of patients with ovarian cancer; however, clinical responses after the injection of antigen-loaded DC have been disappointing. Therefore, the identification of additional tumour associated antigens (TAA) is required. A TAA highly expressed in ovarian cancer cells, CA125, is a candidate target for DC-based immunotherapy. Initially, CTL responses to CA125 were studied in the context of HLA-A*0201. CD8+ T-cell responses specific for CA125 peptides (with high affinity for the MHC class I) were generated from cultures initiated with peptide-loaded monocyte-derived DC (Mo-DC). To expand the evaluation of T-cell recognition of CA125 to non-HLA-A*0201 individuals, messenger RNA (mRNA) was investigated as an antigen-loading vehicle. RNA encodes for the repertoire of epitopes presented by the TAA, potentially inducing immune responses in the context of multiple MHC class I and II molecules to known/unknown antigens. One focus of this study was to investigate a novel mRNA transfection system utilising mannan for the delivery of mRNA into DC. Initially the immunomodulating effect of mannan was examined in terms of DC activation. Mannan induced the phenotypic and functional maturation of immature Mo-DC in vitro. Next, the ability of oxidised mannan (OxM) linked to mRNA was investigated for its capacity to deliver enhanced green fluorescent protein (EGFP) mRNA into DC. We observed high transfection efficiencies in the murine and in human DC systems using low mRNA concentrations, in the absence of significant cell viability impairment. Interestingly, upon mRNA delivery via the OxM-PEI complex, DC maturation was induced to considerably higher levels as compared with that achieved with electroporation and non-transfected controls, this was measured by phenotype (CD83) and IL-12 secretion. Within this study, OxM-PEI did not deliver TAA encoding mRNA into DC for the stimulation of CTL. In summary, mannan is a novel strategy to deliver mRNA and a strong maturation signal simultaneously to human Mo-DC. The functional capacity of this system requires further investigation before it can be considered for clinical use. Electroporation has evolved as a superior method for mRNA delivery into DC as reported in the literature. Therefore, a comprehensive study was performed encompassing the critical issues associated with transfection efficiency, in order to standardise an electroporation protocol for use in DC immunotherapy schedules. EGFP was used as a model antigen to optimise mRNA uptake by Mo-DC by monitoring the expression of the reporter gene by FACS analysis. Influenza matrix protein 1 mRNA was, then, utilised as a model antigen for MHC class I restricted antigen presentation, for confirmation of the optimised loading parameters. The efficiency of this delivery system was assessed using CA125 mRNA in stimulating antigen-specific T-cell responses in PBMC of healthy individuals. CD4+ and CD8+ antigen-specific T-cell responses were generated recognising CA125 mRNA loaded Mo-DC and also ovarian cancer cell lines endogenously expressing CA125. This study has identified CA125 specific T-cell responses in healthy donors, allowing further investigation into the potential for its use as a candidate TAA in ovarian cancer immunotherapy. Furthermore, the use of Mo-DC transfected with mRNA encoding TAA is a promising strategy for the delivery of TAA in the generation of antigen-specific T-cell responses. In summary, the results gained from this PhD thesis should be taken into consideration when designing future DC immunotherapy strategies to combat one of the leading causes of cancer mortality in women, ovarian cancer.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 6 uppsatser.