Blood coagulation factors, factor XII (FXII), plasma kallikrein (PK), factor XI (FXI), and high-molecular-weight kininogen (HMWK), are four proteins that are involved in the plasma contact activation system (CAS) and kallikrein-kinin system (KKS). FXII is the initiation point of the intrinsic pathway of blood coagulation and is also the link between procoagulant and proinflammatory reactions. A number of inhibitors for blood coagulation factors have been approved for clinical usage. These inhibitors not only show inhibitory activities against thrombosis, but also disrupt haemostasis causing excessive bleeding. Contrasted with other coagulation proteases, the deficiency in FXII, instead of causing excessive bleeding, only reduces thrombus formation in blood coagulation. This unique property has made FXII a potential target for the treatments of thrombosis. FXII inhibitors such as aptamers, antibodies and peptides have been reported. Due to their limitations, some inhibitors have poor selectivity or weak binding affinity. Some inhibitors (biopolymers) have pharmaceutical problems. A chemically synthesised FXII inhibitor, with high selectivity is of great importance. The current lack of small molecule FXII inhibitors is limiting efforts to fully understand and validate FXII as a drug target. This project aims to design and synthesise a novel class of 1, 2, 5-tri-substituted benzene urea compounds as potent FXIIa inhibitors. Using Lossen rearrangement, a total of 134 urea compounds were chemically synthesised in this project. All compounds were biologically tested against activated factor XII (FXIIa). Detailed structure-activity relationships (SAR) information was obtained by functional enzyme assays (FXIIa). The range of activity of urea lead compounds in series is between 5.7 μM and 182.0 μM. The urea compound (127) bearing a benzamidine functional group (4-((2-(3-(2-(Pyrrolidin-1-yl)-5-(trifluoromethyl)phenyl)ureido)ethyl)sulfonamido)benzamidine) had the most potent inhibition against FXIIa (α-FXIIa: 5.7 ± 0.1 μM; β-FXIIa: 5.9 ± 0.2 μM). The top 25 most potent inhibitors were selected and were biologically tested for their selectivity between FXIIa and activated factor X (FXa). There are four out of 25 compounds showed their activities against FXa in a range between 1.9 ± 0.6 μM and 141.7 ± 16.5 μM. The selectivity between FXa and FXIIa was rationalised by serine protease-ligand crystal structure (FXa-rivaroxaban complex) and computational modelling simulation (FXIIa-hybrid model). In conclusion, potent FXIIa inhibitors were identified. The present study provides a rationale for further development of FXIIa inhibitors for the treatment of thrombosis and the investigation of selectivity between FXIIa and factor Xa (FXa).

Magnesium plays several physiological roles, including the formation of bones and teeth, muscle contraction, and cardiovascular function. Magnesium deficiency affects cardiovascular health through the modulation of endothelial cell function, particularly by having a negative impact on endothelial cell proliferation, increasing monocyte adhesion, inhibiting cell migration and markedly altering endothelial cell gene expression. With the recognition of the protective role of magnesium in endothelial cells, the aim of this study was to investigate the effects of magnesium sulphate on human umbilical vein endothelial cells (HUVECs) proliferation, gene expression, and the pro-inflammatory response caused by a bacterial endotoxin lipopolysaccharide (LPS). After culture for 120 hours in low (0.1mM) and high (5mM) magnesium, the viability of HUVECs decreased in the low magnesium (P=0.024), whilst the proliferation of HUVECs increased in the high magnesium (P=0.016). Moreover, exposing the cells to LPS lowered viability in the culture with low magnesium (P=0.002), but high magnesium protected the HUVECs from LPS-induced cell death (P=0.037). Magnesium dose-dependent gene expression profiles of HUVECs were investigated via microarray, revealing extensive effects of low and high magnesium on the transcriptome (2930 up-regulated genes and 2828 down-regulated genes). Magnesium deficiency in endothelial cells activated inflammatory pathways through the transcription factor nuclear factor κB (NF-κB), which plays an important role in the early pathology of atherosclerosis. LPS-treated HUVECs cultured in low magnesium showed up-regulation of mRNA expression for pro-inflammatory factors, such as ICAM-1, VCAM-1, IL-8, and MCP-1, and the expression of cytokine proteins, including IL-2, IL-3, IL-8, IL-15, MCP-1, GRO, and GROa. In contrast, high magnesium decreased the expression of IL-6 and MCP-1 mRNA and the protein concentrations of IL-2 and IL-6. The study found that LPS activates the NF-κB pathway through the TLR2 and TLR4 receptors and this pathway was inhibited by high magnesium concentration. Overall, the work presented demonstrates that inflammatory processes in vascular endothelial cells are sensitive to magnesium. This suggests that magnesium nutrition may be an overlooked factor in CVD and is worthy of further investigation.

Contemporary reports showing negative temporal trends in human sperm quality have provided a weight of evidence, in spite of recognised methodological weaknesses of the early research, strongly indicative of declining male fertility over the past six decades. Other aspects of human male reproductive development, such as hypospadias, cryptorchidism and testis cancer are also showing negative trends in incidence and geographic variation in prevalence. Together these negative changes in parameters of fertility, which have comorbidity, are termed Testicular Dysgenesis Syndrome (TDS). The relative short time period of the trend data and the spatial heterogeneity of these factors suggest an environmental aetiology. Two neighboring and ethno-linguistic similar countries, Denmark and Finland, are used as evidence of this as Denmark has significantly decreased sperm quality as well as an increased incidence of other aspects of TDS when compared to neighboring Finland. Additionally, exposure to persistent environmental chemical pollutants has been associated with decreased sperm quality and increases in other components of TDS. The suggested underlying cause of TDS is perturbed foetal/embryonal programming and reproductive development. In spite of overall poor epidemiological information and a paucity of empirical research data from companion animals there are suggestions that environmental pollutants are affecting the health of cats and dogs. Indeed, the dog may be a relevant research model as it shares our environment and ill health associated with it, such as human TDS. Based on the evidence of environmental pollutants affecting human and animal reproductive development and fertility, this PhD project was developed in order to determine if man’s closest companion, and the animal which wholly shares our environment, demonstrates any parallel reproductive impairment. Specifically, this thesis aims to investigate negative temporal trends related to canine fertility, determine the presence of and geographic variation in tissue concentrations of exemplar environmental pollutants and examine testes tissue and sperm cells, via culture and histology, for association between chemical concentrations and quantitative assessments. Canine testes were collected from three countries; UK (n = 58), Finland (Helsinki region) (n = 20), Denmark (Copenhagen region) (n = 10). These samples were tested for concentrations of three representative chemical groups: PCBs, PBDEs, DEHP. Samples of commercial dog food (n = 28), and a range of canine body fluid was also analysed for these same chemical groups. A retrospective analysis of database information from a population of service dogs in the UK was used to determine temporal trends in canine sperm quality and cryptorchidism over a 26 year period. The concentrations of the three chemical pollutant types were additionally analysed against land use with a Geographic Information System (GIS) and two CORINE land use categories: Artificial and Agricultural. These tissues were assessed histologically using histomorphometric scores, CYP11A1 area stained. Canine testes tissue and sperm were cultured with PCB153 and DEHP to determine direct effects of short term exposures to environmental pollutants by assessing testosterone production (testes explant culture), motility (CASA), sperm viability (hypo osmotic swelling test, chromatin integrity, live:dead ratio). Database analysis of service dog fertility reported two components of TDS: negative temporal trends in sperm quality and a concurrent increased incidence of cryptorchidism. Samples of canine testes tissue contained measurable concentrations of three exemplar pollutants (PCBs, PBDEs, DEHP). These pollutants were also largely present in commercial dog food and some were also present in prostatic fluid, full ejaculate and bitches milk. The profile of EDs and concentrations of specific chemicals showed significant variation across three regions of the UK (Southeast, East Midlands, and West Midlands). Notably, concentrations of legacy industrial chemicals (PCBs, PBDEs) were highest in the West Midlands and phthalate (DEHP) was highest in the southeast. The concentrations of DEHP was significantly higher in Artificial land categories compared to tissue collected in Agricultural land use areas (P < 0.05). Canine testis samples collected from three international regions contained concentrations of PCBs and DEHP which were significantly higher in the UK (P < 0.05) and PBDEs which were significantly higher in Finland (P < 0.05). The histomorphometric assessment of testes showed significant variations across the three countries. The quantification of immunohistochemical testis area stained for CYP11A1 from the three countries showed a significant variation (P=0.0001), and tissue from Denmark showed a significant association between area stained and concentrations of DEHP (P < 0.05). Tissue exposed, via testes explant culture, to PCB153 and DEHP at two concentrations showed no significant blunting of LH stimulated testosterone secretion. Sperm exposed, via culturing with two environmentally relevant concentrations of PCB153 and DEHP, exhibited significantly increased (P < 0.05) and decreased (P < 0.05) measures of motility with these chemicals respectively. Additionally sperm viability, measured using the hypo osmotic swelling assay, was significantly reduced in the presence of PCB153 (P < 0.05) and a significant increased when cultured with DEHP (P < 0.01). Both chemicals significantly increased sperm DNA fragmentation. Additionally, short term exposure to both chemicals significantly reduced the Live:Dead ratio of sperm (P < 0.05). Robust, determinative evidence of negative trends in canine sperm quality and incidence of cryptorchidism indicate that canine and human male fertility are decreasing in parallel. An environmental aetiology is supported by the presence of environmental chemicals in canine diet, testes tissue, prostatic fluid and full canine ejaculate. Geographic differences (National and International) in testis chemical profiles and concentrations may equate with reported regional differences in human TDS. In support of this hypothesis, specific canine testis chemical concentrations were associated with histomorphometric scores from the same cohort of samples. Direct effects of PCB153 and DEHP on sperm motility and viability may be indicative of an alternative acute negative effect on male fertility. These data suggest that the dog, while living in a shared environment with humans, exhibits some parameters of TDS and that canine, as well human, male fertility is declining due to exposure to environmental chemicals.

In vertebrates, muscle precursor cells delaminate and migrate from the somites into the limb, where they proliferate and differentiate to muscle masses. Many transcription factors, including Pax and the myogenic regulatory factors (Mrfs) play a vital role in regulating limb muscle growth and development. In the limb, the progenitors begin the process of myogenesis by expressing the myogenic determination factors Myf5 and MyoD, and differentiation factors Myog and Mrf4 then eventually form dorsal and ventral skeletal muscle masses. However, the role of insulin-like growth factors (IGFs) in regulating the early processes of limb myogenesis, and the functional relevance of IGF-1 and IGF-2 in myogenic determination remains poorly defined. By whole-mount in situ hybridisation (WISH), the present study first characterised the expression of both Mrfs and components of the IGF in chick limbs at precise stages during embryonic development. The analysis of the spatial and temporal expression patterns of both Mrfs and IGFs allowed further interventions to be precisely targeted. Therefore, to determine the mechanism of how these factors interact, application of IGF-soaked beads into chick limb during the development was followed by analysis of muscle specific marker genes. Both Pax3 and MyoD were selected as markers for undifferentiated and differentiated myogenic cells respectively. First, the present study demonstrated both IGF-1 and IGF-2 as novel players in early limb myogenesis in vivo by stimulating the activation of Pax3 and MyoD expression. IGF-1 was also shown to increase numbers of both Pax3+ve and mitotic cells, but not the numbers of mitotic Pax3+ve cells, suggesting the increase in Pax3 expression was not simply via increased proliferation. IGF signalling was then examined using picropodophyllotoxin (or PPP) as a specific inhibitor of the IGF-1 receptor. The present data showed that blocking of IGF-1R with PPP inhibited both IGF-1 and IGF-2 induced expression of both Pax3 and MyoD. To confirm the mechanisms for the effects of the IGFs, the current study also used a panel of pharmacological inhibitors, such as U0126 and LY294002 to block the mitogen activated protein kinase (MAPK) and AKT signalling pathways, respectively as well as Nocodazole to inhibit cell proliferation. Results showed that the effects of both IGF-1 and IGF-2 on Pax3 and MyoD expression were mediated by the MAPK pathway. In contrast, inhibition of the AKT signalling pathway inhibited IGF-2 (but not IGF-1) stimulated Pax3 expression, as well as IGF-1 (but not IGF-2) stimulated MyoD expression. Inhibition of cell proliferation with Nocodazole had no effect on IGF-1 stimulated Pax3 or MyoD expression. Similarly Nocodazole had no effect on IGF-2 stimulated Pax3 expression, whereas it inhibited IGF-2 stimulated MyoD expression. SU5402, an inhibitor of FGF receptor, showed no effect on IGF-1 and IGF-2 stimulated Pax3 expression whereas it inhibited their stimulation of MyoD. These findings led to the proposal of a model where IGF signalling directs the timing of the early steps of myogenic cells by stimulating Pax3 expression, whereas the upregulation in MyoD expression is indirectly controlled via Fgf. Together, these results give new insights into the early embryonic development of limb muscle in birds. Additionally, a deeper knowledge of a specific interaction between IGFs and Mrfs may well provide a molecular basis for preventing the loss of muscle and muscle diseases in animals and humans. The present study may well also shed a light to improve the production of chicken meat.

Silica is an essential trace element in human nutrition and dietary deficiency leads to abnormal bone formation in experimental animal studies (Carlisle, 1972, Schwartz 1972). Silica-containing glasses and glass ceramics, within a certain range, are bioactive, forming a strong bond with bone and soft tissue when they are used as bone replacement materials. The aim of this work was to investigate the effect of silica on the osteoblast in vitro with a view to its eventual incorporation into biomaterials to improve bone-bonding properties. Two distinct approaches were used. The first involved the analysis of silica as a nutrient, by supplementing osteoblast growth medium with sodium metasilicate, and evaluating the osteoblast response in terms of cell growth, mineralisation and cytotoxicity. The second approach examined the response of osteoblasts to silica as a biomaterial. A silica gel was used to isolate the effects of silica on the osteoblast in vitro without the effects of the other ions present in bioactive glass. The biocompatibility of patterned silicon wafers was investigated to evaluate the potential use of these materials in the field of biomaterials. Finally, the bioactivity and osteoblast response to a novel silicon/polymer composites as assesseda s a potential biomaterial. The results of nutrition studies showed that in some cases low levels (1-100ppm) of silicate appeared to have a beneficial effect on bone formation in terms of nodule formation and mineralisation. Higher levels of silicate supplementation (>300ppm) caused rapid apoptosis in osteoblasts, fibroblasts and macrophages and affected cell spreading. The biomaterial studies showed that the silica gel surface was bioactive and osteoblasts responded favourably demonstrating enhanced, earlier nodule formation. Bioactive surfaces formed a calcium phosphate (CaPi) layer and released silicic acid when incubated in a simulated body fluid (SBF). Bulk silicon wafers (Si) supported osteoblast growth however, the removal of the oxide layer by wet etching (ESi) imparted bioactive properties to the wafer. Patterned Si/Esi surfaces supported the formation of a CaPi layer over the entire surface and demonstrated osteoblast preferences for bioactive surfaces. The incorporation of silica particles into a bioabsorbable polymer matrix rendered the composite bioactive and supported osteoblast growth. The results of this work demonstrate the importance of silica in bone mineralisation, osteoblast apoptosis and particularly the potential benefits of the use of silica and silicon to improve bone bonding in non-bioactive biomaterials and biosensors.

The aim of this work was to enhance the immune responses to ovalbumin (OVA) following its oral administration, by the association of the protein with colloidal carriers, which may protect the protein from degradation in the gastrointestinal tract and/or facilitate its uptake across the intestine. An enzyme linked immunosorbent assay (ELISA) was established for the determination of rat anti-OVA antibodies and an immunisation protocol was established to induce a statistically significant salivary antibody response to OVA in the rat. A radioimmunoassay for the determination of rat anti-OVA antibodies was also established, to confirm the ELISA results. Methods were established to determine the extent of incorporation or adsorption of OVA into or onto the colloidal carrier formulations. OVA was incorporated into liposomes and polyacrylamide microparticles, and adsorbed to poly 2-butylcyanoacrylate particles, and gastrically intubated into separate groups of experimental rats. The primary and memory immune responses, both sera and saliva, were compared for each formulation with suitable control and blank groups. All colloidal carriers induced enhanced immune responses to OVA following oral administration in the rat, when compared with the respective control group responses. However, the enhancement for the liposomal group was not statistically significant when assessed in an Unpaired Student 't' test. The effect of particle size on the immune responses was assessed by the oral administration of 100 nm and 3pm poly 2-butylcyanoacrylate particles with adsorbed OVA. An electron microscopy study was undertaken with gold labelled poly 2-butylcyanoacrylate particles in an attempt to demonstrate the uptake of particles by M-cells overlying the Peyers' patches in the rat intestine.

Over the last three decades, there has been increasing concern over environmental effects on human male reproductive health. Both temporal and regional trends in semen quality, testicular cancer and malformations at birth have been associated with changes or differences in exposure to chemicals present within the environment. These abnormalities are typically classified under one entity, Testicular Dysgenesis Syndrome [TDS]. Since temporal trends in sperm quality have also been reported in the dog, it was proposed that this may reflect a common cross-species environmental aetiology and that the dog is a sentinel for human exposure to ECs. The overarching hypothesis of this thesis is that the dog may exhibit regional differences symptomatic of TDS and may respond to environmental influences in a similar manner to the human. Experimental studies designed to test this hypothesis focused on (1) the sensitivity of sperm to environmental influences, (2) canine sentinel testicular chemical profiles and pathological features of testes from specific geographical regions and (3) possible environmental influences impacting on cryptorchidism in dogs. Humans and animals are not directly exposed to single chemicals but to a mixture of environmental toxicants present within the environment. Chapter 3 initiated investigations into mixture effects of ECs by utilising a novel full factorial chemical model of two chemicals known to be present in reproductive tissues. Concentrations of di (2-ethylhexyl) phthalate [DEHP] and polychlorinated biphenyl congener 153 [PCB-153] at environmentally relevant levels, as determined by testicular chemical profiling of dog testes, and their effects upon parameters of sperm quality, were tested in vitro. While subtle differences in motility were observed between species, DNA fragmentation was increased similarly in both the human and dog following EC exposure. Although this applied to individual and mixed chemicals, the effects of one chemical impacted on the activity of the other dependent on the concentration ratio. Interestingly, for DNA fragmentation, data presented suggests that PCB-153 is the driver behind increased sperm DNA damage in both species. Since the data alluded to above support the concept of utilising the domestic dog as a sentinel for human exposure to ECs, the dog was used to investigate regional variation upon testicular developmental, morphological and histopathological features. The regions selected for in this component of the thesis display different degrees of industrialisation and thus variation in exposure to environmental contaminants. Data presented demonstrate significant regional variation in chemical profiles, testicular developmental markers and histopathological features indicative of TDS. Specifically, testicular DEHP and PCB-153 with known geographical variation, were found to be positively associated with markers of proliferation and spermatogenesis. Interestingly, a further chemical present in dog testis, poly-brominated diphenyl ether congener 47 [PBDE-47], was negatively correlated with these markers. Furthermore, a novel system developed to assess and score histopathological abnormalities in testes, revealed a higher range of atypical features in testes from the UK compared to those collected from Scandinavia. A further novel element of this thesis was the development of a survey to assess environmental influences on cryptorchidism across several breeds of dog. Uniquely, a higher prevalence of cryptorchidism was observed in deerhounds originating from the East Midlands. Of the range of environmental influences investigated, a key observation was that some bitches of cryptorchid pups were fed a specific brand of feed previously reported to contain ECs. Assessment of further environmental factors covered by the survey such as exposure to pesticides, cigarette smoke and air fresheners provided preliminary information pending the further repeat release of the survey to the same breeders in future years. These data provide preliminary evidence into possible environmental factors that could influence canine and human reproductive health. In conclusion, the results presented in this thesis are significant since they add considerable weight to the paradigm that environmental factors impact directly on male reproductive function. Unique data presented within this thesis emphasises that specific chemical types perturb sperm function and these chemicals vary by region. Furthermore, the work presented here consolidate the suitability of the domestic dog as a sentinel for human exposure to contaminants thus providing the added benefit of enabling access to reproductive tissues from different regions as an index of human reproductive health.

Congenital heart defects are a leading cause of postnatal loss; they could genetically or environmentally induced. Herbal remedies are often used during the early stages of pregnancy, being considered ‘harmless’ and ‘natural'. To alleviate pregnancy-induced symptoms, women frequently use herbal medicines such as ginger to relieve nausea and vomiting - ‘morning sickness’, gingko biloba and ginseng as dietary supplements or tonics to boost body energy and blood circulation, particularly to the brain. Also, chamomile and holy basil are recommended to promote calmness and reduce stress, often related to planned or unplanned pregnancy. These easily available and accessible medicinal herbs could be possible causes of congenital malformations. Additionally, diabetes mellitus in gestation is a considerable medical challenge, since it is related to ‎augmented dangerous morbidity and mortality for both the fetus ‎and the pregnant woman. Two in vitro methods were utilised; chick embryonic heart micromass (MM) and mouse embryonic D3 stem cells (ESD3). The potential effects of the tested herbal components in both in vitro systems were evaluated by monitoring the alteration in several endpoints. These include contractile activity (morphological scoring system), cell activity, total protein content, ROS production, DNA damage, transmembrane proteins expression (connexin43, integrin β1) and stem cell migration. In MM, 6-gingerol decreased contractility, cell activity and protein content. It caused an increase in ROS production and DNA damage and a decrease in transmembrane protein expression (connexin43, integrin β1) at high concentrations. In ESD3, 6-gingerol severely affected differentiation into cardiomyocytes cell activity and protein content at both low and high concentrations. With regards to ginkgolide A and ginkgolide B, there were alterations for few endpoints in both systems at moderate to high concentrations. G-Rg1, from ginseng, decreased contractile activity, cell activity, protein content and elevated ROS production in both systems only at high concentrations. α-bisabolol (chamomile) showed no immediate effects on all end points at low concentrations, but several disturbances occurred at high concentrations. Eugenol (holy basil) at moderate to high concentrations, significantly decreased contractility, cell activity and protein content. The diabetic formula used showed an increase in DNA damage and a decline in cell migration in mouse embryonic stem cells. Molecular endpoints indicate a role for reactive oxygen species and changes in cell membrane proteins. To summarise, these data indicate that some herbal remedies used in the first trimester of pregnancy might not be safe for fetal development. Also care needs to be taken to ensure good glycaemic control in diabetic pregnancy.

The heart is the first functioning organ to develop during embryogenesis to maintain the growing embryo with oxygen and nutrients. However, cardiogenesis is a complex and highly coordinated biological process, and any perturbation to this process can result in detrimental defects to the heart. Haemodynamics is known to play an important role in cardiac growth and vasculature remodelling. Congenital heart defects (CHDs) accounts for 0.4-1.3% of all live birth, whereas cardiomyopathy accounts for 8-11% of cardiovascular disease diagnoses detected in utero. Although the heart defects and cardiomyopathies are known to be attributed by genetic mutations, most cases have unknown etiology. Hence, OFT-banding model was employed to alter the haemodynamic loading via pressure overloading. Upon alteration of haemodynamics, enlargement of the heart with a spectrum of cardiac anomalies were found (e.g ventricular septal defects, thickened epicardium and dysmorphic atrioventricular valves) upon morphological and stereological analysis. A study of global differential expression of OFT-banded hearts by RNA sequencing revealed a number of differentially expressed genes and they were associated with cardioprotection, metabolism, shear stress and valve development; further, a reduction of apoptosis was seen in these banded hearts as well. One of the cardiac phenotypes seen upon OFT-banding, the abnormal primordial atrioventricular valve, was further characterized to provide an insight how the atrioventricular valve is affected upon alteration of haemodynamics. Aberrant expressions of extracellular matrix (ECM) genes such as TBX20, Aggrecan and Periostin alongside with the shear stress responsive genes (KLF2 and EDN1) were found, and a decrease in apoptosis was seen. Moreover, dysregulation of ECM proteins such as fibrillin-2, type III collagen and tenascin were further demonstrated in more mature primordial AV leaflets at HH35, with a concomitant decrease of ECM cross-linking enzyme, transglutaminase-2. In addition, for many years sarcomeric proteins have been associated with a range of cardiomyopathies, but only in recent years they have been linked to congenital heart defects (CHDs). To date, cardiac troponin T (TNNT2) has been associated with cardiomyopathies but not with isolated CHDs. TNNT2 encodes for cTnT regulatory proteins of the thin filament of the sarcomere and is vital for muscle contraction and force generation within cardiomyocytes. To investigate a role of TNNT2 in the early developing heart, targeted manipulation of TNNT2 was performed in embryonic chick to reduce the protein levels of cTNT (protein product of TNNT2) in ovo via translational block. Abnormal atrial septal growth, reduced ventricular trabeculation and ventricular diverticula were found upon TNNT2 morpholino treatment. The abnormal phenotype observed in the TNNT2 morpholino-treated groups was potentially suggested by differential expression of shear stress responsive gene, NOS3 gene.

The peptide hormone Insulin-like peptide 3 (INSL3) shows important roles in the reproductive system. In the male, the testicular Leydig cell peptide INSL3 is involved in fetal testis descent and sperm maturation during the adulthood. In females of reproductive age INSL3 is mainly produced by the ovarian follicular theca interna cells. The current project aims to explore the detailed actions of INSL3 in the female reproductive system. Firstly, the secretion pattern of the INSL3 peptide in the serum of young women and women attending an infertility clinic was assessed. The serum levels of INSL3 peptide appeared to increase during the follicular phase, decrease during the luteal phase, and drop to a nadir at menses. The secretion pattern corresponded to the growth of antral follicles within a follicular wave. Pathological conditions leading to an alteration of the antral follicle count, such as polycystic ovarian syndrome and low ovarian reserve, were accompanied by an increase or decrease in the serum INSL3 level, respectively. The bovine system was adopted and validated as a model for the human in regard to the follicular production of INSL3, as the secretion pattern for INSL3 in bovine serum through estrous cycles was similar to that in women. Primary bovine theca interna cells were then used to study the regulation of the INSL3 gene and its secreted peptide products. At both peptide and mRNA levels INSL3 production could be up-regulated by progesterone receptor signalling. Together with the analysis of the transcriptional activity of the bovine INSL3 gene promoter, it could be shown that estradiol (E2) – estrogen receptor alpha (ERα) signalling stimulated and E2-ERß signalling decreased INSL3 production. Dihydrotestosterone (DHT) acting through the androgen receptor (AR) and androstenedione (A4) probably acting through ERα and/or ERß both contribute to regulation of INSL3 production. Moreover, luteinising hormone (LH) from the anterior pituitary also influenced the INSL3 production, with the downstream pathway from low level LH stimulation probably involving synergy between cAMP-PKA and ER signalling. The potential target of INSL3 was also identified in the female bovine reproductive system; full-length transcripts for the INSL3 receptor, RXFP2, were detectable in ovarian theca interna cells, in oocytes, as well as in the myometrium. Meanwhile, full-length transcripts for the closely related receptor, RXFP1, were detected in theca interna cells, endometrium and in myometrium, although the gene encoding the ligand for this receptor, relaxin, has been deleted during ruminant evolution. When transfecting DNA expression plasmids encoding the individual RXFP2 and RXFP1 receptors into HEK293T cells, the expressed bovine RXFP2 could be activated by bovine and human INSL3 (EC50 = 0.7 & 0.6nM) and porcine and human relaxin (EC50 = 10.5 & 27.3nM). The expressed bovine RXFP1 could be activated by porcine and human relaxin (EC50 = 10.8 & 48.7nM) and human relaxin 3 (EC50 = 97.9nM). Functional analysis showed that bovine myometrial cells were able to respond to both exogenous relaxin and INSL3, whereas theca interna cells could respond to INSL3 only. Together, however, these experiments support the view that endogenous RXFP2 and RXFP1 are both fully functional in the bovine even though the primary ligand for the latter is missing. The results showed that for the female INSL3 is mainly produced by the theca interna cells of antral follicles, and is thus a potential biomarker of ovarian functionality. The expression of INSL3 can be regulated by sex steroid hormones in an auto/paracrine manner, as well as by LH in an endocrine manner. In conclusion, the INSL3-RXFP2 system acts in follicles and probably also the uterus to enable and orchestrate female reproductive physiology.